4 n Year 2023, Vol. 70, No. 2 ActaChimicaSlovenica ActaChimicaSlovenica ActaChimicaSlovenica ActaChimicaSlovenica SlovenicaActaChim A cta C him ica Slovenica 70/2023 Pages 173–294 Pages 173–294 n Year 2023, Vol. 70, No. 2 http://acta.chem-soc.si 2 70/2023 2 ISSN 1580-3155 Kynurenic acid is metabolite of tryptophane that can be found in different foods, also in honey. The content of kynurenic acid was determined in more than 100 samples of different botanical sources. Results have shown that chestnut honey has much higher concentration of kynurenic acid than other honeys. EDITOR-IN-CHIEF EDITORIAL BOARD ADVISORY EDITORIAL BOARD ASSOCIATE EDITORS Alen Albreht, National Institute of Chemistry, Slovenia Aleš Berlec, Jožef Stefan Institute, Slovenia Janez Cerkovnik, University of Ljubljana, Slovenia Mirela Dragomir, Jožef Stefan Institute, Slovenia Krištof Kranjc, University of Ljubljana, Slovenia Matjaž Kristl, University of Maribor, Slovenia Maja Leitgeb, University of Maribor, Slovenia Helena Prosen, University of Ljubljana, Slovenia Aleš Ručigaj, University of Ljubljana, Slovenia Jernej Stare, National Institute of Chemistry, Slovenia Irena Vovk, National Institute of Chemistry, Slovenia ADMINISTRATIVE ASSISTANT Eva Mihalinec, Slovenian Chemical society, Slovenia Wolfgang Buchberger, Johannes Kepler University, Austria Alojz Demšar, University of Ljubljana, Slovenia Stanislav Gobec, University of Ljubljana, Slovenia Marko Goličnik, University of Ljubljana, Slovenia Günter Grampp, Graz University of Technology, Austria Wojciech Grochala, University of Warsaw, Poland Danijel Kikelj, University of Ljubljana Janez Košmrlj, University of Ljubljana, Slovenia Mahesh K. Lakshman, The City College and The City University of New York, USA Blaž Likozar, National Institute of Chemistry, Slovenia Janez Mavri, National Institute of Chemistry, Slovenia Jiři Pinkas, Masaryk University Brno, Czech Republic Friedrich Srienc, University of Minnesota, USA Walter Steiner, Graz University of Technology, Austria Jurij Svete, University of Ljubljana, Slovenia David Šarlah, University of Illinois at Urbana-Champaign, USA; Università degli Studi di Pavia, Italy Ivan Švancara, University of Pardubice, Czech Republic Gašper Tavčar, Jožef Stefan Institute, Slovenia Ennio Zangrando, University of Trieste, Italy Polona Žnidaršič Plazl, University of Ljubljana, Slovenia Chairman Branko Stanovnik, Slovenia Members Udo A. Th. Brinkman, The Netherlands Attilio Cesaro, Italy Vida Hudnik, Slovenia Venčeslav Kaučič, Slovenia Željko Knez, Slovenia Radovan Komel, Slovenia Stane Pejovnik, Slovenia Anton Perdih, Slovenia Slavko Pečar, Slovenia Andrej Petrič, Slovenia Boris Pihlar, Slovenia Milan Randić, Des Moines, USA Jože Škerjanc, Slovenia Đurđa Vasić-Rački, Croatia Marjan Veber, Slovenia Gorazd Vesnaver, Slovenia Jure Zupan, Slovenia Boris Žemva, Slovenia Majda Žigon, Slovenia FRAnC PERDIH University of Ljubjana, Facuty of Chemstry and Chemical Technology, Večna pot 113, SI-1000 Ljubljana, Slovenija E-mail: ACSi@fkkt.uni-lj.si, Telephone: (+386)-1-479-8514 Izdaja – Published by: SLOVENSKO KEMIJSKO DRUŠTVO – SLOVENIAN CHEMICAL SOCIETY Naslov redakcije in uprave – Address of the Editorial Board and Administration Hajdrihova 19, SI-1000 Ljubljana, Slovenija Tel.: (+386)-1-476-0252; Fax: (+386)-1-476-0300; E-mail: chem.soc@ki.si Izdajanje sofinancirajo – Financially supported by: National Institute of Chemistry, Ljubljana, Slovenia Jožef Stefan Institute, Ljubljana, Slovenia Faculty of Chemistry and Chemical Technology, University of Ljubljana, Slovenia Faculty of Chemistry and Chemical Engineering, University of Maribor, Slovenia University of Nova Gorica, Slovenia Acta Chimica Slovenica izhaja štirikrat letno v elektronski obliki na spletni strani http://acta.chem-soc.si. 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Acta Chimica Slovenica is indexed in: Academic Search Complete, Central & Eastern European Academic Source, Chemical Abstracts Plus, Chemical Engineering Collection (India), Chemistry Citation Index Expanded, Current Contents (Physical, Chemical and Earth Sciences), Digitalna knjižnica Slovenije (dLib.si), DOAJ, ISI Alerting Services, PubMed, Science Citation Index Expanded, SciFinder (CAS), Scopus and Web of Science. Impact factor for 2021 is IF = 1.524. Articles in this journal are published under the   Creative Commons Attribution 4.0 International License – Graphical Contents Graphical Contents ActaChimicaSlovenica ActaChimicaSlovenica SlovenicaActaChimica Year 2023, Vol. 70, No. 2 173–183 Chemical, biochemical and environmental engineering A Critical Review on Corrosion and Fouling of Water in Water Distribution Networks and Their Control Andi Muhammad Anshar, Bulkis Musa, Muhammad Ayaz, Syahruddin Kasim, Indah Raya, Andrés Alexis Ramírez-Coronel, Shakhawat Chowdhury, Rahman S. Zabibah, Rosario Mireya Romero-Parra, Luis Andres Barboza-Arenas, Yasser Fakri Mustafa and Ali Hussein Demin Al-Khafaji 184–195 Chemical education Uncovering Students’ Genuine Misconceptions: Evidence to Inform the Teaching of Chemical Kinetics Habiddin Habiddin and Elizabeth Mary Page REVIEW ARTICLE SCIENTIFIC PAPER 196–203 Analytical chemistry Potential Biochemical Properties of Endemic Onosma mutabilis Pelin Eroglu, Mehmet Ulas Civaner, Selda Dogan Calhan, Mahmut Ulger and Riza Binzet Graphical Contents 226–230 Biomedical applications Effect of Ozone on Oxygen Transport and Pro-Oxidant- Antioxidant Balance of Red Blood Cell Suspension Victor Zinchuk and Elena Biletskaya 218–225 Biochemistry and molecular biology Phytochemical Profile, Antioxidant and Antimicrobial Potency of Aerial Parts of Salvia Tomentosa Miller Şehnaz Balkır, Ömer Hazman, Laçine Aksoy, Mustafa Abdullah Yılmaz, Oguz Cakir, Recep Kara and İbrahim Erol 204–217 Biomedical applications Design, Development and Optimization of Carmustine- Loaded Freeze-Dried nanoliposomal Formulation Using 32 Factorial Design Approach Sandip M. Honmane, Manoj S. Charde and Riyaz Ali M. Osmani 231–239 Chemical, biochemical and environmental engineering Chemical and Antioxidant Profile of Hydroalcoholic Extracts of Stachys Officinalis L., Stachys Palustris L., Stachys Sylvatica L. from Romania George Florian Apostolescu, Diana Ionela (Stegarus) Popescu, Oana Botoran, Daniela Sandru, Nicoleta Anca Şuţan and Johny Neamtu 240–246 Organic chemistry Synthesis, Spectroscopic Characterization, Crystal Structures and Antibacterial Activity of Benzohydrazones Derived from 4-Pyridinecarboxaldehyde with Various Benzohydrazides Yi-Xuan Zhou, Wei Li and Zhonglu You Graphical Contents 274–280 analytical chemistry Direct Determination of Kynurenic Acid with HPLC- MS/MS Method in Honey Anže Pavlin, Matevž Pompe and Drago Kočar 261–273 Organic chemistry novel 5,6,7,8-tetrahydrobenzo[b]pyran Derivatives: Synthesis and Anticancer Activity Amira E. M. Abdallah, Rafat M. Mohareb, Maher H. E. Helal and Mariam M. Abd Elkader 247–260 applied chemistry new Bis-1,3,4-Thiadiazoles Based on Fumaric Acid: Preparation, Structure Elucidation, Antibacterial Activities, and Quantum-Chemical Studies Halit Muğlu, Hasan Yakan, Ghaith Alabed Ibrayke Elefkhakry, Ergin Murat Altuner and M. Serdar Çavuş 281–293 Organic chemistry Synthesis, SC XRD Based Structure Elucidation, Supramolecular Assembly Exploration Via Hirshfeld Surface Analysis, Computational and QTAIM Study ... Muhammad Nawaz Tahir, Muhammad Ashfaq, Akbar Ali, Chin Hung Lai, Bojja Rajeshwar Rao, Khurram Shahzad Munawar and Irshad Ali Shahid 173Acta Chim. Slov. 2023, 70, 173–183 Anshar et al.: A Critical Review on Corrosion and Fouling of Water ... DOI: 10.17344/acsi.2022.7939 Review article A Critical Review on Corrosion and Fouling of Water in Water Distribution Networks and Their Control Andi Muhammad Anshar1, Bulkis Musa1*, Muhammad Ayaz2, Syahruddin Kasim1, Indah Raya1*, Andrés Alexis Ramírez-Coronel3, Shakhawat Chowdhury4, Rahman S. Zabibah5, Rosario Mireya Romero-Parra6, Luis Andres Barboza-Arenas7, Yasser Fakri Mustafa8 and Ali Hussein Demin Al-Khafaji9 1 Department of Chemistry, Faculty of Mathematics and Natural Science, Hasanuddin University Makassar 90245, Indonesia 2 Sensor Networks and Cellular Systems (SNCS) research center, University of Tabuk, Tabuk, Saudi Arabia 3 Research group in educational statistics, National University of Education (UNAE), Azogues, Ecuador 4 Department of Civil and Environmental Engineering, King Fahd University of Petroleum & Minerals, Dhahran, Saudi Arabia 5 Medical Laboratory Technology Department, College of Medical Technology, The Islamic University, Najaf, Iraq 6 Universidad Continental, Lima, Perú 7 Universidad Tecnológica del Perú. Lima, Perú 8 Department of Pharmaceutical Chemistry, College of Pharmacy, University of Mosul, Mosul-41001, Iraq 9 Department of Laboratories Techniques, Al-Mustaqbal University College, Babylon, Hillah, Iraq * Corresponding author: E-mail: bulkismusa@unhas.ac.id & indahraya@unhas.ac.id Received: 12-10-2022 Abstract Corrosion and scaling are among the problems that may arise when storing water in tanks. Several factors affect the cor- rosion and scaling of water, which include pH, temperature, alkalinity, Ca2+ hardness, TDS, concentrations of Cl–, SO42–, CO32–, and HCO3–. Also, several indices can be measured using these properties such as the Langelier saturation index, Ryznar index, Aggressive index, Larson, Scold index, water quality index, and Puckorius index. These indicators deter- mine the degree of corrosiveness and sedimentation of water. The purpose of this review article was to study the impact of various factors on the corrosiveness and sedimentation of water. To this end, different sources of water in different countries were studied and the impact of physical and chemical parameters on their corrosiveness was investigated. Also, the reaction mechanism of water corrosion inside the pipe was studied. Finally, practical and constructive suggestions were presented to solve the problems of corrosion and sedimentation of desalination water. Keywords: Corrosion, Fouling, Langelier Saturation Index, Ryznar Index, Inhibitors, Water 1. Introduction Access to clean and unpolluted water resources is one of the basic human needs for a healthy and sustainable society.1,2 Development of industrial and agricultural ac- tivities have led to the spread of environmental pollu- tion.3–6 The natural cycle of life on earth is menaced by the introduction of harmful chemicals. The water transmis- sion and distribution network is responsible for storing and transporting water.7–10 One of the problems that may arise when storing water in tanks is the corrosiveness of the water, resulting in corrosion of facilities (eg, pipes, tanks, etc.). Corrosion of drinking water transmission equipment is a global problem.11,12 Corrosion is a physi- cal-chemical reaction between a substance and its sur- roundings.13 Determining water corrosion indices is one 174 Acta Chim. Slov. 2023, 70, 173–183 Anshar et al.: A Critical Review on Corrosion and Fouling of Water ... of the effective ways to manage drinking water resources. Corrosion of water can cause economic damage, reduce the useful life of water supply facilities, and illness in con- sumers.14–16 Corrosion in water distribution networks not only destroys equipment, but also reduces the quality of drinking water because of chemical and biological reac- tions that occur in the water distribution system. Corro- sion processes in drinking water pipes depend on the type of pipes, water quality and hydraulic conditions. Qualita- tive parameters related to water corrosion include pH, al- kalinity, degree of buffering, dissolved oxygen, natural or- ganic matter, microorganisms, temperature, inhibitors if used, etc.17–20 Durowaye et al. (2014) surveyed the impact of water pH on the corrosion of mild steel and realized that the corrosion rate decreases with enhancing pH from 7.2 to 11.2.21 Generally, pipe corrosion is prevented by con- trolling the chemical composition of water and using in- hibitors. Due to its chemical properties, desalinated water is known as very corrosive water.22,23 Sedimentation is another fundamental problem in water distribution systems.24 A thin layer of scaling is use- ful because it can prevent corrosion on the metal surface. However, if the thickness of the deposit on the pipe surface increases, it can cause a lot of damage, including pressure drop, reduced water flow, and reduced heat transfer rate, which leads to an increase in the energy required for pumping. This action reduces thermal conductivity and increases energy consumption.25,26 Generally, various factors affect water corrosion, in- cluding pH, CO2 concentration, hardness, alkalinity, tem- perature, speed of water, TDS, dissolved oxygen, residual chlorine, fatigue, tension and other factors like cavita- tion.23,27 Prevention of equipment corrosion is usually done by controlling the chemical composition of water and using inhibitors. Inhibitors are chemicals that, when added in low concentrations to a corrosive environment, decline or prevent the reaction between metals and the en- vironment. Vinyl acetate-methacrylic acid and vinyl ace- tate-acrylic acid have shown high antifouling efficiency at lower pHs and temperatures.28 The corrosion of equip- ment increases with enhancing water pH. High concentra- tion of sodium and chlorine in water increases the water conductivity, resulting in an increase in water corrosion. According to WHO standards for drinking water, the per- missible limit of calcium and TDS is 75 mg/L and 500 mg/L, respectively. Also, the standard value of pH is be- tween 7–8.5. Moreover, the permissible limit of CaCO3 alkalinity should be between 30-500 mg/L based on WHO standard.25 The Langelier index, along with the Ryznar and Puckorius indices, determines the corrosive or sedimenta- tion status of water in the distribution network. The best case is when the water does not cause corrosion and de- posits in the pipes of the water distribution network be- cause both cases reduce the life of the pipes in the water transmission network and are costly. In distribution net- work management, by measuring the Langelier, Ryznar and Puckorius indices, they try to prevent the corrosion or clogging of the pipes by improving the water quality, which is called water stabilization. Water instability occurs when water quality characteristics such as hardness, alkalinity, temperature, TDS and pH are not balanced.29,30 In general, physical, chemical and microbial factors are the three main causes of corrosion. Dissolved oxygen concentra- tion, TDS, alkalinity, pH, CO2 concentration and residual chlorine are chemical factors in corrosion. Also, tempera- ture, fluid speed, and metal composition of pipes are phys- ical factors31 and biological factors include 1) iron bacteria such as Gallionella and Chronotropic32 and 2) sulfate-re- ducing bacteria such as Desulfovibrio and De Sulfuric Ans.33 Therefore, considering the problems and damages caused by corrosion and sedimentation in the water trans- mission and distribution system, it is necessary to reduce its effects by monitoring and controlling the factors affect- ing this phenomenon. The purpose of this review paper is to investigate various factors on corrosion and fouling in water distribution networks. To this end, the chemical and physical features of water were studied. Then, effective fac- tors on corrosion and fouling were fully investigated. Also, the corrosiveness and sedimentation of water for various water distribution systems were fully studied by several in- dices such as Langelier, Ryznar, Larson-Scold, Aggressive, and Puckorius. Finally, practical suggestions were present- ed to improve or fix these problems. 2. Effective Factors on Corrosion and Scaling Sedimentation in water pipes occurs when divalent metal ions or hardness factors in water are combined with other ions dissolved in water and deposited on the inner wall of the pipe. The main forms of sediments consist of calcium sulfate, magnesium carbonate, calcium carbonate, and magnesium chloride. Based on previous studies, more than 60% of corrosion in pipes and water transmission networks is due to chemical factors, and 40% is caused by biological parameters. The rate of fouling enhances with increasing temperature and salt concentration.34 Sedimen- tation reduces the amount of water flow inside pipes, re- duces heat transfer, increases pressure drop and energy required for pumping, clogs pipes and increases the cost of operation and maintenance of water supply facilities. The effective factors in the occurrence of deposits and clogging of pipes are a function of temperature, pH and concentra- tion of dissolved solids.35,36 If the water is corrosive or sed- imentary, it will cause many problems in the water trans- mission and distribution pipes and reduce the life of the facilities. The best pH value of water for preventing corro- sion is 7. In fact, water with pH values below 6.5 or above 7.5 will be corrosive. Also, the corrosion rate triplicates 175Acta Chim. Slov. 2023, 70, 173–183 Anshar et al.: A Critical Review on Corrosion and Fouling of Water ... with enhancing temperature from 15.55 to 60 °C.37 More- over, the presence of some gases such as H2S can enhance the corrosion of metals. Water containing magnesium or calcium salts (hard water) is less likely to cause corrosion because the minerals coat the inside of the pipe and pro- tect them. Soft water containing sodium salts doesn’t cover the pipe and is therefore more corrosive.25,27 Figure 1 illus- trates various factors affecting the water corrosion and the appropriate amounts for preventing corrosion. Figure 1. Effective factors and their conditions for preventing water corrosion and fouling Corrosion is a physical-chemical reaction between a material and water, which changes the substance features. Excessive hardness of water causes corrosion and serious damage to facilities.38 Stable water is water that does not cause corrosion in contact with metals and prevents sedi- mentation inside water pipes. Two important standards in the water supply network against corrosion are ISO1885 and EN12502.39 Corrosiveness and scaling of water can be deter- mined by Langelier, Ryznar, Aggressive, and Puckorius in- dices. The Langelier index indicates the state of water in terms of corrosiveness and sedimentation, which depends on various factors such as water acidity, TDS, carbonate concentration, bicarbonate concentration, water tempera- ture and alkalinity. The following relationship is utilized to measure the Langelier saturation index (LSI): (1) Where, pHs is the saturation pH. There are different ways to calculate pHs. In the first method, the pHs can be determined through Figure 2. As shown, pHs can be easily determined by having the water temperature, calcium ion concentration and alkalinity by referring to the graph.38 Figure 2. Determination of pHs in terms of Ca hardness, alkalinity, and pH parameters38 In the second method, the following relationship is utilized to calculate pHs: (2) (3) (4) (5) (6) Where, TDS and T are in terms of mg/l, and kelvin, respectively. For LSI= 0, water is not corrosive or sedimen- tation. For LSI<0 and LSI>0, water tends to dissolve CaCO3 and precipitate CaCO3, respectively. When the index is negative, the water has corrosion potential.40,41 Table 1 pre- sents the different modes of the Langelier index and based on that, the desired suggestions are presented to fix them. Also, the Ryznar index is used as a basis for measur- ing sediment thickness in urban water supply systems in order to predict the chemical influence of water. The Ryz- nar stability index (RSI) can be employed to compute the corrosiveness and sedimentation potential of water. The RSI value can be determined using Equation 7. At RSI>7 and RSI<6, water will be corrosive and precipitator, respec- tively. Also, for RSI between 6–7, the water will be stable.42 (7) The table below presents the degrees of water sedi- mentation based on RSI values. As reported, an RSI value 176 Acta Chim. Slov. 2023, 70, 173–183 Anshar et al.: A Critical Review on Corrosion and Fouling of Water ... Furthermore, Larson-Scold index (LRI) is used to show the degree of water corrosiveness for the steel metal surface. LRI is determined through the following equa- tion: (10) where, C is the concentration of each ion (meq/L). If LRI<0.8, the water is not corrosive. Also, for 0.81.2, the water will be very corrosive.44 Finally, the Aggressive index (AI) is used to monitor corrosion in asbestos pipes and can be used as an indicator of water corrosion. This index is calculated using the actu- al water pH, calcium hardness, and total alkalinity. In ad- dition, this index is simpler and easier than the Langelier index because it is not affected by temperature or TDS. The following relationship is utilized to calculate AI: (11) where, A and H are alkalinity and total hardness, respec- tively. If the AI index is equal to or greater than 12, the water will be non-corrosive. It is worth noting that for AI smaller than 10, water is highly corrosive and in values be- tween 10 and 12, it will be moderately corrosive. Water quality index (WQI) is another significant pa- rameter for determining water quality. WQI indicates the combined impact of various water quality variables. To calculate WQI, each physicochemical factor is assigned a weight (wi). The value of wi depends on the impact of each Table 1. Different values of the Langelier index and desired sugges- tions LSI Situation Suggestion value –5 Intense corrosion Needs purification –4 Intense corrosion Needs purification –3 Moderate corrosion Needs purification –2 Moderate corrosion Purification might be required –1 Weak corrosion Purification might be required –0.5 Very weak corrosion Maybe no need for purification 0 Stable No need for purification 0.5 Weak sedimentation Maybe no need for treatment 1 Gentle sedimentation Purification might be required 2 Gentle to moderate Purification might be required sedimentation 3 Moderate sedimentation Purification is recommended 4 Intense sedimentation Purification is recommended Figure 3. RSI versus pH and temperature43 between 6–7 is considered the best amount, so that water leads to little scale or corrosion. Also, Figure 3 indicates the RSI parameter in terms of temperature and pH. As shown, RSI at a certain amount of temperature declines with increasing pH. Also, at constant pH, the value of RSI decreases with enhancing temperature. Considering that the best value of RSI should be between 6 and 7, therefore water under high values of pH and temperature will form severe fouling. Also, water at very low pH and temperature will be corrosive.43 Table 2. RSI values and their interpretations42 RSI value Case 4–5 Severe fouling 5–6 light fouling 6–7 Little fouling and corrosion 7–7.5 Moderate corrosion 7.5–9 Severe corrosion >9 Unbearable corrosion Moreover, the Puckorius index (PI) presents a rela- tionship between scale formation and saturation state. High calcium concentration and low alkalinity of water result in a high level of calcite saturation. PI can be deter- mined as follows:24 (8) where, pHs and pHeq are the water pH at saturation state, and the water pH at equilibrium state, respectively. pHeq can be calculated as follows: (9) where, alkalinity is in terms of mg/L. If PI > 6 and PI < 6, the water tends to corrode and sediment, respectively. 177Acta Chim. Slov. 2023, 70, 173–183 Anshar et al.: A Critical Review on Corrosion and Fouling of Water ... factor on health or its importance on the quality of water. Then, the relative weight (Wi) for n variables can be calcu- lated as follows: (12) Also, quality rating can be determined as follows: (13) Where, Ci and Si are concentration of each chemical factor (mg/L) and the standard value for each factor except for pH (mg/L), respectively. Moreover, the sub-index of the ith factor (SIi) and WQI are calculated through Eqs. 14 and 15:38 (14) Table 3 presents the water quality in various WQI amounts. As demonstrated, the WQI values below 100 are suitable for water quality. Table 3. WQI values for drinking water and their cases45 WQI amount Description 0–50 Great 50–100 Good 100–200 Poor 200–300 Very poor >300 Improper 3. Reaction Mechanism Iron is the most extensively used element in pipes in potable water distribution networks that are utilized to transport drinking water.46,47 It is estimated that iron- based pipes make up a large portion of the drinking water distribution system around the world. For example, 67.2% of water distribution pipes in Italy, 56.6% in the US, 75.5% in China, 93% in Innsbruck (Austria), and 91% in Warsaw (Poland) are made of iron.47 Corrosion and fouling easily occur in pipes, which involve complex reactions between the pipe surface and the passing water. In the corrosion process in water, iron and oxidants act as anode and cath- ode (electron acceptor), respectively. Chlorine, dissolved oxygen, hypochlorite ions, and hypochlorous acid are the most common oxidants in water distribution systems.46–48 These oxidants can quickly react with zero-valent iron (Fe0) inside the pipe’s wall. Iron hydroxides (i.e., goethite, ferric hydroxide, maghemite) and iron oxides are the main corrosion products that can gently deposit on the pipe wall. The cumulation of corrosion on the pipes increases corrosion resistance and creates an obstacle between the transferred water and the metal pipe, leading to a decrease in the rate of corrosion. The following reactions can be oc- curred:47 (15) (16) (17) (18) The corrosion process may occur non-uniformly in different locations. Pit corrosion, galvanic, and crevice corrosion are common types of non-uniform corrosion. With the beginning of corrosion in a pipe, corrosion prod- ucts are produced and accumulate on the pipe surface, which gradually lead to the formation of scales. Figure 4 shows the impact of various factors on the formation of corrosion on the pipe wall. Figure 4. Different factors affecting the formation of corrosion on the pipe wall Figure 5. Schematic of corrosion and different layers inside the pipe 178 Acta Chim. Slov. 2023, 70, 173–183 Anshar et al.: A Critical Review on Corrosion and Fouling of Water ... Also, the following figure illustrates different lay- ers inside the pipe. As shown, there are 4 layers in iron pipes, which include corroded layer, porous layer, shell-like layer, and top layer. The corroded layer con- tains zero-valent iron. Also, the porous layer is com- posed of iron components like ferric hydroxide (Fe(OH)3), ferrous hydroxide (Fe(OH2)), goethite (α-FeOOH), siderite, hematite (Fe2O3), and lepido- crocite (γ-FeOOH). Moreover, the shell-like layer contains the porous core and finally, the top layer is a heterogeneous layer containing silicates, carbonates, phosphates, goethite, iron hydroxide, and lepidocrocite. The corrosion composition depends on various varia- bles like the quality of water, the operation time, and pipe material.47 4. Previous Studies Corrosion is a physical-chemical reaction between a material and its surrounding, which leads to changing the properties of that material. By attacking the inner wall of the pipe, the corrosive water dissolves the materials of the pipes and causes many problems. Economic losses, the formation of by-products, problems with taste, smell, color, staining and increasing turbidity are among the most important problems related to corrosiveness. Langeli- er, Ryznar, and Puckorius indices are important factors for determining the corrosiveness of water. Many studies have been done on the water’s corrosiveness, which are reported in Table 4. Hasani et al. (2021) suggested that pH, Cl– concen- tration, dissolved oxygen, and sulfate should be continu- ously checked and controlled to prevent water corro- sion.50 Also, Bouderbala (2021) measured different properties of water such as pH, EC, Cl−, TDS, COD, BOD, Na+, K+, Mg2+, SO42−, Ca2+, HCO3−, NH4+, NO3−, NO2–, and PO43− to determine water quality for irriga- tion and industrial applications. The outcomes indicated that the WQI is in the range of 50–100, which shows the proper quality of water for irrigation.51 In other studies, other characteristics such as pH, water temperature, hardness, TDS, electrical conductivity, alkalinity, HCO3–, CO32–, Ca2+, Mg2+, Na+, Cl–, and SO42– were calculated to determine the rate of corrosion and fouling.44, 52 In addition to these variables, García-Ávila et al. measured other factors such as sulfate, phosphate, and nitrite to predict and control corrosion.55 Eslami et al. (2020) showed that the concentration of Cl– and SO42– has a greater effect on the corrosion and fouling potential of water than other parameters. They proposed that the wa- ter pH must be controlled to avoid water corrosion and scaling.52 The morphology and composition of sediments are strongly related to the electrochemical features of water, and with the increase in corrosion and sedimen- tation, the corrosion current density decreases continu- ously. By adding sodium hypochlorite disinfection to the groundwater, the water pH increases and results in the formation of calcium carbonate. Calcium carbonate strongly affects the corrosion potential.62 Also, con- trolling water quality before entering the water distribu- tion network is an effective way to prevent corrosion and fouling.36 Shahmohammadi et al. (2018) surveyed the corro- sion and sedimentation potential of 46 water supply sources in Sarvabad County (Iran). The Langelier index in some water sources indicated that water tends to dis- solve calcium carbonate and in other areas, water tends to form calcium carbonate scale. According to the Ryznar index, the tendency of water to corrode steel pipes in- creases. The Aggressive index (11.6) showed that the cor- rosion potential of water is moderate. The corrosion po- tential of water was also detected by the Puckorius index (7.03).56 Therefore, several indicators simultaneously in- dicate the corrosiveness of water. Furthermore, Maeng et al. surveyed the corrosion potential of river water in Ko- rea. The amount of LSI (–2.97), RSI (12.8), and AI (9.26) showed severe corrosiveness of river water. Also, their results indicated that pH and alkalinity reduce in the rainy season, while calcium hardness has little change throughout the year.58 Davoudi et al. (2016) suggested that stabilization of water before entering the distribu- tion network can prevent water corrosiveness.63 Accord- ing to the study done by Akter et al. in Bangladesh, the water quality index in some cities such as Kurigram Sa- dar and Rangabali was lower than 50, which indicates the water has excellent quality. However, some cities such as Anwara and Kamalganj had WQI values above 250, indi- cating very poor water quality. These results indicate that the WQI amount of water below 100 can be suitable for drinking.45 Hoseinzadeh et al. (2013) studied the water corro- sion and fouling in the water treatment network in Tak- ab city during ten months. According to their results, the amount of LSI was 0.22, which indicates that the water was slightly scale-forming and corrosive. Also, the amount of RSI (7.6) showed that the water is corro- sive. Moreover, the AI value (12.63) indicated that the water is non-aggressive. They suggested that corrosion and fouling can be controlled by adjusting the pH and temperature of the water.59 Moreover, the LSI value in water sources of Tafila (Jordan) was in the range of –0.39 and –1.5, and the RSI value was in the range of 8.7 and 9.8, which indicates that the water is corrosive. Al- so, microbiological experiments indicated that three water samples were contaminated with faecal coliform bacteria.61 In general, it can be concluded that RSI, PI, AI, LSI, and WQI are critical indices for determining water corro- siveness and fouling, which have been widely utilized in previous researches. 179Acta Chim. Slov. 2023, 70, 173–183 Anshar et al.: A Critical Review on Corrosion and Fouling of Water ... Table 4. Characterization of different water sources, problems, and suggestions for solving their problems City/Country Effective factors Description Their suggestions Ref. Thanjavur/India LSI = 0.13, AI = 12.09, Scaling and corrosiveness 30 RSI = 7.92, PI = 8.02, LRI = 1.08 Bangladesh WQI for Sadar = 11.79 Excellent quality of water – 45 WQI for Rangabali = 40.05 Bangladesh WQI for Alfadanga = 169, Poor quality of water – 45 WQI for Kendua = 142.5, WQI for Shajahanpur = 135.6, WQI for Debhata = 113, WQI for Bijoynagar = 111.8 Bangladesh WQI for Rupsha = 92.14 Good quality of water – 45 WQI for Patharghata = 75.35 Bangladesh WQI for Anwara = 253.29 Very poor quality – 45 WQI for Kamalganj = 258.36 Bangladesh WQI for Shibchar = 371.5 Unsuitable for drinking – 45 Juja/Kenya WQI = 131–151 The water quality is very poor – 49 Ardebil/Iran LSI = –1.34 RSI = 10.03 Water is corrosive and has Water pH, Cl–, dissolved 50 a high fouling capability oxygen and sulfate should be monitored. Oued Fodda dam/ Algeria WQI = 50–100 Desirable suitable – 51 water quality Oued Fodda dam/ Algeria RSI>7.5 from November to June Heavy corrosion – 51 Oued Fodda dam/ Algeria RSI<7.5 from July to October Little corrosion and – 51 sedimentation Kerman/Iran LSI<0, RSI>7.5, PI> 6, Water is corrosive Controlling pH 52 and 103.0.CO;2-M 30. R. Justi, J. K. Gilbert, Sci. Educ. 1999, 8, 287–307. DOI:10.1023/A:1008645714002 31. M. Orgill, K. Crippen, J. Coll. Sci. Teach. 2010, 40, 78–84. 32. N. Glazer, Stud. Sci. Educ. 2011, 47, 183–210. DOI:10.1080/03057267.2011.605307 33. M. Sozbilir, T. Pinarbasi, N. Canpolat, Eurasia J. Math. Sci. Technol. Educ. 2010, 6, 111–121. 34. S. Chairam, E. Somsook, R. K. Coll, Res. Sci. Technol. Educ. 2009, 27, 95–115. DOI:10.1080/02635140802658933 35. J. G. Rodriguez, S. Santos-Diaz, K. Bain, M. H. Towns, J. Chem. Educ. 2018, 95, 2114. DOI:10.1021/acs.jchemed.8b00584 36. H. D. Barke, A. Hazari, S. Yitbarek, Misconceptions in Chem- istry: Addressing Perceptions in Chemical Education, Spring- er Berlin Heidelberg, 2008. 37. M. S. Cracolice, J. C. Deming, B. Ehlert, J. Chem. Educ. 2008, 85, 873–878. DOI:10.1021/ed085p873 38. G. M. Bowen, W. M. Roth, Res. Sci. Educ. 2002, 32, 303–327. DOI:10.1023/A:1020833231966 39. D. Dolničar, B. Boh Podgornik, V. Ferk Savec, Acta Chim. Slov. 2022, 69, 167–186. 40. T. Thayban, H. Habiddin, Y. Utomo, M. Muarifin, Acta Chim. Slov. 2021, 68, 736–743. DOI:10.17344/acsi.2021.6836 41. D. Gabel, J. Chem. Educ. 1999, 76, 548–554. DOI:10.1021/ed076p548 42. P. J. Garnett, P. J. Garnett, M. W. Hackling, Stud. Sci. Educ. 1995, 25, 69–96. DOI:10.1080/03057269508560050 43. M. Rusek, K. Vojíř, I. Bártová, M. Klečková, V. Sirotek, J. Štro- fová, Acta Chim. Slov. 2022, 69, 371–377. DOI:10.17344/acsi.2021.7250 44. R. A. Hoban, O. E. Finlayson, B. C. Nolan, Int. J. Math. Educ. Sci. Technol. 2013, 44, 14–35. DOI:10.1080/0020739X.2012.690895 45. G. Önal Karakoyun, E. Asiltürk, Acta Chim. Slov. 2021, 68, 645–657. DOI:10.17344/acsi.2021.6666 46. S. Agung, M. S. Schwartz, Int. J. Sci. Educ. 2007, 29, 1679– 1702. DOI:10.1080/09500690601089927 Except when otherwise noted, articles in this journal are published under the terms and conditions of the  Creative Commons Attribution 4.0 International License Povzetek Namen študije je raziskati napačno razumevanje študentov prvega letnika univerzitetnega študija o kemijski kinetiki z analizo podatkov, pridobljenih s štiristopenjskim diagnostičnim pristopom za kemijsko kinetiko (FTDICK). V tej študiji je sodelovalo 335 študentov prvega letnika kemije z dveh indonezijskih in ene britanske univerze. Opisani postopek je prvi te vrste, ki zagotavlja, da so ta napačna razumevanja resnična. Med študenti prvega letnika študija kemije so se razkrila številna napačna razumevanja na področju kemijske kinetike. Čeprav se mnoga od ugotovljenih napačnih razumevanj ujemajo z rezultati, ki so bili predhodno objavljeni z uporabo drugih pristopov, je bilo odkritih tudi nekaj novih ugotovitev. Ta napačna razumevanja je mogoče pripisati različnim dejavnikom, vključno z matematično šibkostjo, neprevidnostjo in težavami pri razlagi in pridobivanju informacij iz diagramov, grafov in drugih nebesedilnih informacij. Na podlagi rezultatov te študije podajamo nekaj priporočil za izboljšanje učinkovitosti poučevanja kemijske kinetike na tej stopnji. 196 Acta Chim. Slov. 2023, 70, 196–203 Eroglu et al.: Potential Biochemical Properties of Endemic Onosma mutabilis DOI: 10.17344/acsi.2023.7998 Scientific paper Potential Biochemical Properties of Endemic  Onosma mutabilis Pelin Eroglu,1,* Mehmet Ulas Civaner,1 Selda Dogan Calhan,2 Mahmut Ulger3 and Riza Binzet4 1 Department of Chemistry, Faculty of Science, Mersin University, 33343, Mersin, Turkey 2 Department of Pharmaceutical Biotechnology, Faculty of Pharmacy, Mersin University, 33169, Mersin, Turkey 3 Department of Pharmaceutical Microbiology, Faculty of Pharmacy, Mersin University, 33169, Mersin, Turkey 4 Department of Biology, Faculty of Science, Mersin University, 33343, Mersin, Turkey * Corresponding author: E-mail: pelineroglu@mersin.edu.tr Phone: +90 324 3610001/14560 Received: 01-04-2023 Abstract The Onosma L. (Lithospermae, Boraginaceae) genus contains many plant species with therapeutic properties due to its rich phytochemicals. Onosma mutabilis Boiss. & Hausskn. ex Boiss. (O. mutabilis) is the species for which there is not enough information on its characteristics. Objective: The total phenolic content, antioxidant activity, possible bioactive compounds, and antibacterial activities of ethanolic extracts of leaf, stem, root, and flower parts of endemic O. mutabilis were investigated. Conclusions: The total phenolic content of all O. mutabilis extracts was in the range of 9.2–31 mg gallic acid equivalent (GAE)/g of extract. According to the results of antioxidant activity, the IC50 antioxidant capacity values determined by the 1,1-diphenyl-2-picrylhydrazyl (DPPH) method were between 4.39–29 µg/mL, while the equivalent trolox antioxi- dant activity determined by the cupric reducing antioxidant values (CUPRAC) was 0.45–0.78 mmol of trolox equivalents (TE)/g of extract. Bioactive compounds have been analysed using gas chromatography coupled with mass spectrometry (GC/MS) and were found to contain 29 different chemical components. All plant extracts tested showed effective anti- bacterial activity against A. baumannii (ATCC 02026) (62.5 µg/mL MIC value) when compared to the reference drug Ampicillin (125 µg/mL). Keywords: Onosma mutabilis, phenolic compounds, antioxidant activity, antibacterial activity. 1. Introduction Onosma L. (1762: 196) (Lithospermae, Boraginace- ae) is a large genus in the world. It is distributed from the northwest of Africa to Europe and Asia and mainly in Tur- key and Iran1,2. The total number of Onosma species known from Turkey is 103 3,4. When the high rate of ende- mism in Turkey (57.84%) was taken into account, it was seen that Turkey was the centre of diversity of the Onosma genus. Onosma species is widely used worldwide in tradi- tional medicine. The various parts of Onosma species are known to be used for the treatment of various disorders such as bronchitis, hemorrhoids, tonsillitis, pain relief, and relief of blood disorders in Turkey.5,6 On the other hand, antioxidant enzymes produced by our body’s defence system are critical to maintain the oxidant-antioxidant balance. In addition, plant-derived antioxidant substances have been reported to be effective against degenerative diseases caused by oxidative stress.7 For this reason, the determination of the effects of thera- peutically effective plants on free radical-induced oxida- tive damage attracts the attention of many researchers. Despite the unique bioactive composition of plants, the phytochemical content of approximately 15% was investi- gated and the biological activity of 6% was screened.8 An- timicrobial compounds isolated from medicinal plants are effective against different bacteria.9,10 In addition, the emergence of multidrug-resistant pathogens in recent times has had adverse effects on public health. This en- courages new research and the development of more effec- tive drugs to replenish therapeutic drug reservoirs.11 How- 197Acta Chim. Slov. 2023, 70, 196–203 Eroglu et al.: Potential Biochemical Properties of Endemic Onosma mutabilis ever, the chemical components and antioxidant and antimicrobial properties of endemic plants grown in vari- ous countries and used for medicinal purposes still need to be discovered. Therefore, active research on plants is nec- essary to identify potential candidates as safer and more effective agents in the future. To our knowledge, only one study has been conducted to determine the phytochemical content of O. mutabilis. In the study performed by Jabbar et al.12 different extraction solvents were used, and it was reported that 18 different bi- oactive species were detected. Cytotoxicity studies have been carried out on different cell lines, but no studies have been carried out on the antibacterial activities of the plant. In this study, we have identified the phenolic com- pound, chemical composition, antioxidant and antibacte- rial activity of the ethanolic extract obtained from roots, stems, flowers, and leaves of endemic O. mutabilis. 2. Experimental 2. 1. Chemicals and Instruments The Folin-Ciocalteu reagent and ethanol (99%) were supplied from Merck (Darmstadt, Germany), gallic acid (3,4,5-trihydroxybenzoic acid, abbreviated as GA), anhy- drous sodium carbonate (Na2CO3) was obtained from Flu- ka (USA). DPPH (1,1-diphenyl-2-picrylhydrazyl radical), BHT (2,6-Di-tert-butyl-4-methylphenol), and Muel- ler-Hinton broth (Sigma 70192) and Resazurin dye (Sigma R7017) were obtained from Sigma-Aldrich (St. Louis, MO). The soxhlet apparatus was supplied by Isolab (Wertheim, Germany). A Rotary evaporator (Buchi B-491, Germany), UV-1601 spectrophotometer (UV-1601, Shi- madzu, Japan), GC/MS (GC: 7890 A, MS: 5975 C, Agilent, USA) were used throughout this work. 2. 2. Plant Materials The samples of O. mutabilis were identified and col- lected by Dr. Riza Binzet from Mersin (Location: C5 Mersin, Mersin-Gözne, around Darısekisi, rocky slopes and scrub, 36°58’10.91”N 34°34’11.79”E, 780 m) (Fig. 1). 2. 3. Preparation of Plant Extracts Fresh leaves, roots, stem, and flower samples of O. mutabilis were air dried in the shade at room temperature (25 °C) for three weeks. Then the leaves, roots, stems and flowers samples were reduced to powder separately with a blender (Blender 8011ES Model HGB2WTS3, 400 W) and kept in glass bottles at room temperature. Ten grams of powdered samples were extracted in 300 mL of ethanol solvent using the Soxhlet extraction method for 6 hours. Ethanol was evaporated at 50–60 °C using a Rotary Evap- orator with bath water. Stock solutions were prepared at concentrations of 1 mg/mL of each part of the plant. Ex- tracts were kept before analysis in a sealed vial at +4 °C. 2. 4. Determination of the Total Phenolic Content The content of phenolic compounds in extracts ob- tained from different parts of the plant analysis according to the Folin-Ciocalteau method.13 1 mL of Folin-Ciocal- teau reagent was added to 1 mL of ethanolic plant solution (1 mg/mL). The sample was kept in the dark for five min- utes. Then 2 mL of Na2CO3 solution (20%, (w/v)) and 2 mL of water were added to the reaction medium. After incubation at room temperature for 30 minutes in the dark, the absorbance was measured at 714 nm. The total polyphenol content was calculated using the gallic acid calibration curve and reported as mg of gallic acid equiva- lent per gram of extract (mg GAE/g E). 2. 5. DPPH Radical Scavenging Assay The free radical scavenging of the ethanolic extracts obtained from different parts of O. mutabilis by the DPPH· test according to the method established by Ilokiassanga et al.14 First, a stock solution of dried plant extracts was pre- pared with ethanol at a concentration of 1 mg/mL. The solutions of ethanolic extracts of O. mutabilis prepared in each concentration range (100–1000 µg/mL) were ana- lysed. 100 µL of the extract solutions were mixed with 100 µL of freshly prepared DPPH (0.2 mM). The mixture ob- a) b) Fig. 1 (a) Habitus and (b) map of the distribution of O. mutabilis. 198 Acta Chim. Slov. 2023, 70, 196–203 Eroglu et al.: Potential Biochemical Properties of Endemic Onosma mutabilis tained was slightly shaken and incubated at room temper- ature for 30 min in the dark. BHT was used as a reference. The absorbance values of the sample solutions and BHT were measured at 517 nm using ELISA (Thermo Scientific TM Multiskan TM FC). The tests were repeated 3 times. The percent inhibition of the DPPH free radical scavenging ac- tivity was calculated with Eq. (1): % Inhibition = [(Abs control–Abs sample) / Abs control] x 100 (1) Radical scavenging activity was indicated as IC50, which shows the concentration of plant extracts required to inhibit 50% of the free radicals DPPH. 2. 6. Cupric Reducing Antioxidant Capacity (CUPRAC) Assay CUPRAC assay,15 and the results were expressed as Trolox equivalents. In detail, 1 mL of 1.0 × 10–2 M copper chloride solution, 7.5 × 10–3 M neocuproin solution and 1 M (pH = 7) ammonium acetate buffer are added to the test tube, respectively. Then Trolox and distilled water are add- ed. Solutions prepared with a total volume of 4.1 mL are kept closed for 30 minutes under room conditions. At the end of this period, the absorbance values are measured at 450 nm against the reference solution without a sample. 2. 7. Antibacterial activity The antibacterial activity and minimum inhibitory concentration (MIC) values of extracts obtained from dif- ferent parts of the plant were tested using REMA.16 The following five bacteria were tested in this study: Staphylo- coccus aureus ATCC 25925, Bacillus subtilis ATCC 6633, Aeromonas hydrophila ATCC 95080, Escherichia coli ATCC 25923, Acinetobacter baumannii ATCC 020226. 2. 8. Determination of MIC Values for O. mutabilis Antibacterial activity was evaluated using the mi- crodilution assay in 96-well sterile polystyrene microplates. Extract at concentrations of 1000 μg/mL was prepared by dissolving in DMSO and filtered through a 0.22 μm micro- porous filter. Each well in the microplate was filled with 100 µL of Mueller-Hinton broth (Sigma 70192). The work- ing solutions of the extracts with serial twofold dilutions were adjusted to 500–0.24 μg/mL. Ampicillin was used as the standard drug in the study and the dilution of the standard drug was carried out in the same way. The bacte- rial suspension was prepared from standard bacterial strains at 0.5 McFarland density. This suspension was then diluted with sterile distilled water (1/20). 10 µl of this sus- pension was added to the corresponding wells. Thus, the final bacterial density in the wells was adjusted to 5x105 CFU/mL (CLSI 2012). The working solution of Resazurin (resazurin sodium salt, Sigma R7017) was prepared in 0.01% (w/v) distilled water and sterilised by passing through a 0.22 μm membrane filter. 10 µL of sterilised re- sazurin was added to the wells. Plates were covered with a plastic film (ThermoFisher Scientific MicroAmp® optical adhesive film, 4360954) to prevent evaporation. The plates were then incubated at 37 °C for 24 hours. At the end of the period, the colour change in the plates was visually evaluated. The change in resazurin from blue to pink or colourless was considered bacterial growth. The MIC val- ue was determined as the lowest concentration of plant samples that prevents the growth of bacteria that prevent- ed resazurin from turning blue to pink or colourless. All antibacterial activity assays were repeated three times. 2. 9. Determination of Bioactive Compounds The essential compounds of O. mutabilis were analysed with a7890A GC system with an inert MSD of 5975C and a capillary column [Agilent Technologies 19091S‐433-HP5- MS]. The injection temperature was 285 °C. The volume of injection was 2 µL. The GC temperature programme was used as follows: At 40 °C, holding there for 5 min, 40 to 220 °C at a rate of 4°C/min and holding at 220 °C for 5 min, and then increased from 220 to 280 °C at a rate of 5°C/min and holding there for 15 min, from 280 to 300 °C at a rate of 15 °C/min and holding there for 5 min. Spectra were obtained in the range of 50–550 m/z. Helium gas was used as the car- rier gas with a flow rate of 1 mL/min. The maximum temper- ature was 325 °C. Total analysis time: 82.5 min. The chemical components of the extract were identified by matching the retention times and mass spectral fragmentation patterns with those of the compounds resulting from data from the NIST/EPA/NIH mass spectral library (NIST05a.L). 3. Results and Discussion 3. 1. Total Phenolic Content Analysis Composed of an aromatic hydroxyl core, plant phe- nolics are one of the most important groups of compounds that work as primary antioxidants and free radical scaven- gers. Spectrophotometric measurements were performed based on the blue colour of the phosphomolybdic-phos- photungstic-phenol complex formed in the Folin-Ciocal- teu method,17 which is widely used in the determination of the total phenolic content. The total phenolic content of the O. mutabilis extracts, expressed as gallic acid equiva- lents, are shown below in Fig. 2 In this study, the findings we obtained for the phenol content of O. mutabilis are compatible with the literature. In a study with O. mutabilis grown in Iran, the total phe- nolic content of the methanolic extract of this plant species was determined to be 37.24 mg equivalent rutin equiva- lents/g extract.12 Sarikurkcu et al.18,19 determined the total 199Acta Chim. Slov. 2023, 70, 196–203 Eroglu et al.: Potential Biochemical Properties of Endemic Onosma mutabilis phenolic content of the methanolic extracts of O. gigantea and O. rascheyana to be 9.12 μmol GAEs/g and 31.55 mg GAE / g, respectively. Furthermore, Kirkan et al.20 Onosma tauricum var. tauricum species and showed that the total phenolic content of this plant is 16.20 μmol GAEs/g. In a study with another Onosma species (Onosma chlorotri- cum) the total phenolic content was determined as 56.10 mg GAE / g of dry extract.21 Emsen et al.,22 analysed the ethyl acetate extracts of O. bozakmanii and determined the total phenolic content as 36.29 µg GAE/mg extract. 3. 2. Antioxidant Activity The antioxidant activity of the ethanolic extracts of O. mutabilis was evaluated using DPPH and CUPRAC methods. The DPPH method is widely used because its as- say is reliable, simple, fast, and sensitive and determines the antioxidant activity in vitro of several natural bioactive compounds.23,24 Basically, in this method, there is a de- crease in the strong absorbance of DPPH at 517 nm due to the reaction of proton transfer to the DPPH free radical by the antioxidant.25 Table 1 shows the IC50 values of the ethanolic extract of different parts of O. mutabilis. Free radical scavenging ability is expressed as the IC50 value. The IC50 value is the amount of antioxidant required to reduce 50% of the ini- tial concentration of DPPH.26 A low IC50 value means high free radical scavenging activity.27 The IC50 values of the roots, stems and flowers were the highest with 5.37, 4.39 and 8.02 μg/mL, respectively. The phenolic content of the plant is proportional to the concentration of the extract and indicates that it has very high antioxidant activity.28 While the CUPRAC method shows the ability of the extract to reduce Cu metal, the results are proportional to the total amount of copper reduced by antioxidant com- pounds through electron transfer. The CUPRAC assays were expressed as mmol of Trolox equivalent/g of extract. The extracts of O. mutabilis gave CUPRAC values with a total antioxidant capacity ranging between 0.45 and 0.78 as mmol Trolox equivalent/g. The highest antioxidant capacity determined by the CUPRAC analysis was record- ed for the root extract. This is followed by flower, leaf, and stem extracts, respectively (Table 1). The highest reduction potential determined by the CUPRAC assay was also ob- served in the root extract. Our results support previous studies on the antioxi- dant capacities of other medicinal plants in the Boragi- naceae family. Researchers used methanol extracts, unlike us, in the research carried out on different types of this plant. Jabbar12 investigated the antioxidant activity of O. mutabilis methanol extracts using the DPPH method and reported an IC50 value of 3.54 mg/mL, respectively. Saravanakumar et al.29 investigated the free radical scavenging activity of the methanolic extract of O. bracteo- sa plant with the DPPH test and showed that the IC50 value was 4.58 mg/mL. Furthermore, Sarikurkcu et al.,30 deter- mined the antioxidant activities of methanolic extracts of O. frutescens with DPPH and CUPRAC tests as 1.14 and 0.53 mg/mL, respectively, and showed that they have high antioxidant potential. Kumar et al.,31 recorded the DPPH IC50 value of methanolic O. hispidum root extract as 2.73 µg/mL. Kirkan et al32., determined that methanol extracts of O. cappadocica showed high activity based on the DPPH scavenging test and the CUPRAC test. In addition, another study by Kirkan et al.20 showed that methanolic extracts of O. tauricum var. tauricum exhibited a high antioxidant po- tential when tested with the DPPH and CUPRAC methods. It can be said from the results that the ethanolic ex- tracts of O. mutabilis, especially the roots and stem, have quite high antiradical activities, with radical scavenging values close to that of the standard. The difference in free radical scavenging activity in various parts of O. mutabilis, such as the root, stem, flower, and leaves, may be related to its chemical composition. It is difficult to compare the re- sults of different methods used to determine antioxidant activity, such as CUPRAC and DPPH.33 Therefore, the re- sults are not given comparatively. 3. 3. Antibacterial Activity The MIC values of the extracts, compared to the standard bacterial strains used in the study, were deter- mined to be in the range of 250–31.25 μg/mL. The test- Fig. 2 Total phenolic contents of the ethanolic extracts of different parts of O. mutabilis. The values presented represent the mean of three experiments ± SD. Table 1. Antioxidant activities (DPPH, and CUPRAC) of ethanolic extracts from different parts of O. mutabilis*. Sample DPPH assay CUPRAC (IC50 µg/mL) (mmol TE/g extract) Root 5.37±0.23 0.78±0.22 Stem 4.39±0.29 0.45±0.26 Flower 8.02±0.57 0.52±0.34 Leaf 29±0.63 0.67±0.36 BHT 1.75±0.18 0.64±0.28 * The values presented are the mean of three experiments ± SD. 200 Acta Chim. Slov. 2023, 70, 196–203 Eroglu et al.: Potential Biochemical Properties of Endemic Onosma mutabilis ed extracts had higher antibacterial activity against A. baumannii with a 62.5 μg/mL MIC value, compared to the reference drug ampicillin with a 125 μg/mL MIC value. Based on the results, it was determined that the MIC values of the extracts against A. hydrophila were 62.5 μg/ mL. It was determined that the results showed a lower an- tibacterial effect compared to the reference drug, but a re- sult close to the MIC value (31.25 μg/mL) of ampicillin. Although the MIC results of the plant extracts (31.25 μg/ mL) for B. subtilis, a Gr (+) bacterium, were higher than the MIC results of the other four bacteria, the activity was found to be lower when compared to the MIC value of am- picillin (0.9 μg/mL). The plant extracts were defined to show low activity against standard bacterial strains of S. aureus and E. coli (Table 2). acid and derivatives of fatty acids such as ethyl linoleate and hexanamide have been found. Butanoic acid was found only in the leaf part of the plant; hexadecanoic acid was found in all parts of the plant, but in different concentrations. Relative rates of hexadeca- noic acid, which has a strong antimicrobial and anti-in- flammatory36,37 effect, are mainly flower, leaves, roots, and stem, respectively. Hexanamide and 14-pentadecanoic acid were found only in the root of the plant and linoleic acid was detected only in the flower of the plant. Furthermore, ethyl linoleate was found in the flower and root part of the plant; 9,12,15-octadecatrienoic acid was detected in the flower and leaves of the plant. Octadecanoic acid was found in all parts of the plant. Fatty acids are compounds with important structural functions. Studies have shown that fatty acids such as stearic acid, oleic acid, and linoleic acid Table 2. MIC (μg/mL) values of extracts and reference drugs tested against standard bacterial strains. S. aureus E. coli A. baumannii B. subtilis A. hydrophila (ATCC25925) (ATCC25923) (ATCC02026) (ATCC6633) (ATCC95080) Root 125 125 62.5 31.25 62.5 Stem 125 125 62.5 31.25 62.5 Flower 250 125 62.5 31.25 62.5 Leaf 125 125 62.5 31.25 62.5 Ampicillin 31.25 15.62 125 0.9 31.25 A lot of research is focused on studying the antimi- crobial activity of various parts of plants of the family Bor- aginaceae. In various studies, root extracts from different species of Onosma have been shown to be effective against Gr (+) bacteria.34 In our study, the MIC value of root ex- tracts of O. mutabilis against S. aureus was 125 µg/mL and against B. subtilis was 31.25 µg/mL. In other studies, the MIC values of O. dichroanthum root extracts against Gr (+) bacteria were in the range of 0.156–0.312 mg/mL.34 Dousti and Nabipor21 showed that by the MIC assay O. chlorotricum Boiss methanol extracts showed higher anti- bacterial activity against Gr (+) bacteria than Gr (-) bacte- ria. Halim et al.,35 reported that O. Bracteatum extracts inhibited Gr (+) bacteria more than G (-) bacteria. 3. 4. Chemical Composition Analysis Using GC-MS analysis of O. mutabilis flower, leaf, stem, and root extracts, a total of 29 compounds with high-quality peaks were detected (Table 3). The results showed that there are different compounds in the flower, leaf, stem, and root parts of the plant, and that the rates of these compounds varied by their peak areas. In our study, based on the results of the GC-MS analysis of O. mutabilis flower, leaf, stem and root extracts, fatty acids such as bu- tanoic acid, hexadecanoic acid, 14-pentadecanoic acid, li- noleic acid, 9,12,15-octadecatrienoic acid, octadecanoic reduce inflammation due to their antioxidant properties, albeit indirectly, in vascular endothelial cells. Therefore, it has been suggested that treatment using these parts of the plant could reduce the risk of atherosclerosis and cardio- vascular disease.38 Phytol is an important diterpene with antimicrobial, antioxidant and anticancer activities.36,39,40 Neophytadiene, another important bioactive compound found in the flower and stem parts of O. mutabilis, has an- algesic, antipyretic, anti-inflammatory, antimicrobial and antioxidant effects.41 The compound 14ß-Pregnane, found in the root part of O. mutabilis at a concentration of 1%, has a steroid structure and is a defence chemical with preven- tive and therapeutic effects against diabetic retinopathy.42 Another bioactive compound detected based on GC-MS results is β-Sitosterol, commonly known as phytosterol. Phytosterols, found in plant cell membranes, are chemical- ly similar to mammalian cell-derived cholesterol. It has been shown in many in vitro and in vivo studies that β-si- tosterol has various biological effects, including anxiolytic and sedative effects, analgesic, immunomodulatory, anti- microbial, anticancer, anti-inflammatory, and lipid-lower- ing effects; it is also hepatoprotective and showed a protec- tive effect against nonalcoholic fatty liver disease.43 Hydrocarbons, another important group of organic com- pounds, are found in the flower, leaf, stem and root extracts of O. mutabilis. Hexadecane, tri-tetracontane, heptadecane, octadecane, nonadecane, tricosane, hexacosane, tetra- 201Acta Chim. Slov. 2023, 70, 196–203 Eroglu et al.: Potential Biochemical Properties of Endemic Onosma mutabilis cosane, eicosane, heneicosane, heptacosane, docosane, and octacosane are among the hydrocarbons detected based on GC-MS results. Among these compounds, eicosane is in- teresting for its antibacterial activity,44 heneicosane for its antimicrobial effect,45 and tetracosane, heptadecane, hexa- decane for its antioxidant and antimicrobial proper- ties.44,46,47 Decaborane, which is found in the leaf part of O. mutabilis, attracts attention due to its toxic and volatile properties.48 1H-Indole, an aromatic organic compound, was detected in all parts of the plant, including flowers, leaves, stems, and roots. In the study by Jabbar in 2021 that evaluated the phytochemical content, antioxidant proper- ties and toxicity of O. mutabilis, the plant was examined as a whole and the contents of flowers, leaves, stems, and roots of the plant were not compared in terms of phytochemicals. However, according to our results, the parts of the flower, leaves, stem and root of the plant contain different bioactive species at different rates. On the contrary, among the 29 compounds found in our study, unlike Jabbar’s previous re- port, many different compounds have been detected, main- ly phytol, neophytadiene, 14ß-Pregnane, 1H-Indole, lin- oleic acid, ethyl linoleate, 14-pentadecenoic acid, 9,12,15-octadecatrienoic acid, octadecanoic acid, tricosane, hexacosane, tetracosane, heneicosane, heptacosane, docosane, and octacosane. Therefore, endemic O. mutabilis can be considered as a bioactive agent with superior poten- tial for pharmacological and chemical applications. 4. Conclusion Ethanolic extracts obtained from different parts of O. mutabilis collected from the Mersin region of Turkey have antioxidant and antibacterial effects due to the large num- ber of bioactive compounds (hexadecanoic acid and β-si- tosterol, etc.). Our results show that there is a positive cor- relation between the amount of phenolic substances and free radical scavenging activities. In our study, the root and stem showed the highest antioxidant activity, respectively. Furthermore, it was found that the root, stem, leaf, and flower extracts of the plant were effective against A. bau- mannii bacteria known as a nosocomial infection agent. Due to the limited information on the anticancer, anti-in- flammatory, antifungal, and many other molecular-level properties of the plant, more studies are needed for its pharmaceutical and industrial use. Table 3. Phytochemical contents of flower, leaf, stem, and root samples of O. mutabilis analyzed by GC-MS. Compound Chemical Flower Leaf Stem Root tR (min) CAS NO Formula % Butanoic acid C4H8O2 – 2.65 – – 9.67 016844-99-8 1H-Indole C8H7N 3.40 1.24 1.24 2.99 18.21 000120-72-9 Decaborane B10H14 – 5.37 – – 29.1 017702-41-9 Hexadecane C16H34 – – – 0.43 35.74 000638-36-8 Tri-tetracontane C43H88 – – – 0.20 35.85 007098-21-7 Heptadecane C17H36 – – – 1.35 37.23 000629-78-7 Octadecane C18H38 – – – 4.06 39.60 000593-45-3 Neophytadiene C20H38 2.45 6.84 – – 41.03 000504-96-1 Hexanamide C6H13NO – – – 1.57 43.51 998195-79-6 Hexadecanoic acid C16H32O2 10.5 4.07 0.85 8.69 44.18 000057-10-3 14ß-Pregnane C21H36 – – – 1.00 44.55 998433-89-7 1,7-Dimethyl Phenanthrene C16H14 – – – 8.63 45.85 000483-87-4 Phytol C20H40O 4.63 – – 47.51 000150-86-7 Azaperone C19H22FN3O – – – 3.51 48.08 001649-18-9 Linoleic acid C18H32O2 2.06 – – – 48.23 998405-19-4 Ethyl linoleate C20H36O2 4.81 – – 5.23 48.55 000544-35-4 14-Pentadecenoic acid C15H28O2 – – – 7.52 48.66 017351-34-7 9,12,15- Octadecatrienoic acid C18H36O2 14.0 3.97 – – 48.73 001191-41-9 Octadecanoic acid C18H36O2 4.71 17.1 17.1 10.04 49.22 000111-61-5 Hexacosane C26H54 – – 3.12 – 51.42 000630-01-3 Tetracosane C24H50 – – 4.40 – 54.14 000646-31-1 Nonadecane C19H40 4.04 1.73 5.75 0.74 56.83 000629-92-5 Tricosane C23H48 2.63 1.32 0.57 – 57.65 000638-67-5 Heneıcosane C21H44 – – 5.82 – 58.08 000629-94-7 Docosane C22H46 – – 9.82 – 58.48 000629-97-0 Heptacosane C27H56 5.45 – – – 59.72 000593-49-7 Octacosane C28H58 – – 8.51 – 60.73 000630-02-4 Eicosane C20H42 0.68 7.31 4.63 – 61.78 000112-95-8 β-Sitosterol C29H50O 1.49 – – – 68.91 000083-46-5 202 Acta Chim. Slov. 2023, 70, 196–203 Eroglu et al.: Potential Biochemical Properties of Endemic Onosma mutabilis Funding: The authors would like to thank to Mersin University Scientific Research Project Unit for financial support (Pro- ject no: 2017-2-TP2-2516). 5. Reference 1. L. Cecchi, A. Coppi, F. Selvi. Phytotaxa. 2016, 288, 201–213. DOI:10.11646/phytotaxa.288.3.1 2. M. Firat, R. Binzet. Adansonia 2021, 43, 185-195. DOI:10.5252/adansonia2021v43a16 3. R. Binzet, Ö. Eren. Phytotaxa 2018, 356, 117-130. DOI:10.11646/phytotaxa.356.2.2 4. R. Binzet. Turk J Bot 2016, 40, 194-200. DOI:10.3906/bot-1410-23 5. A. Tosun, K. E. Akkol, O. Bahadir, E. Yeşilada. J. Ethnophar- macol. 2008, 120, 378-381. DOI:10.1016/j.jep.2008.09.007 6. U. Ozgen, M. Ikbal, A. Hacimuftuoglu, P. J. Houghton, F. Gocer et al. J. Ethnopharmacol. 2006, 104, 100-103. DOI:10.1016/j.jep.2005.08.052 7. V. López, S. Akerreta, E. Casanova, J. M. García-Mina, R. Y. Cavero et al. Plant Food Hum. Nutr. 2007, 62, 151-155. DOI:10.1007/s11130-007-0056-6 8. R. Verpoorte. J. Pharm. Pharmacol. 2000, 52, 253-262. DOI:10.1211/0022357001773931 9. L. Yarmolinsky, M. Bronstein, J. Gorelick. Isr. J. Plant Sci. 2015, 62, 294-297. DOI:10.1080/07929978.2015.1067076 10. Z. Wang, Y. Zhou, X. Shi, X. Xia, Y. He et al. Food Biosci. 2021, 42, 101206. DOI:10.1016/j.fbio.2021.101206 11. N. Aelhidar, A. Nafis, A. Kasrati, A. Goehler, J. A. Bohnert et al. Ind. Crop. Prod. 2019, 130, 310-315. DOI:10.1016/j.indcrop.2018.12.097 12. A. A. Jabbar. Food Sci. Nutr. 2021, 9: 5755-5764. DOI:10.1002/fsn3.2544 13. P. Ersan, Ö. Sönmez, B. Gözmen. J. Iran. Chem. Soc. 2020, 17, 871-879. DOI:10.1007/s13738-019-01824-x 14. S. B. Iloki-Assanga, L. M. Lewisluján, C. L. Laraespinoza, A. A. Gilsalido, D. Fernandezangulo et al. BMC Res. Notes 2015, 8: 1-14. DOI:10.1186/s13104-015-1388-1 15. R. Apak, K. Güçlü, M. Özyürek, S. Çelik. E. Microchim. Acta 2008, 160, 413-419. DOI:10.1007/s00604-007-0777-0 16. A. W. Bauer, W. M. Kirby, J. C. Sherris, M. Turck. Am. J. Clin. Pathol. 1966, 45, 493-496. DOI:10.1093/ajcp/45.4_ts.493 17. V. L. Singleton, R. Orthofer, R. M. Lamuela-Raventos. Meth- ods Enzymol. 1999, 299, 152-178. DOI:10.1016/S0076-6879(99)99017-1 18. C. Sarikurkcu, B. Kirkan, M. S. Ozer, O. Ceylan, N. Atilgan, M. Cengiz, B. Tepe. Ind. Crop. Prod. 2018, 115, 323-329. DOI:10.1016/j.indcrop.2018.02.040 19. C. Sarikurkcu, E. Demir, M. S. Ozer, R. Binzet. Int. J. Plant Pharm. 2022, 2, 128-135. 20. B. Kirkan, C. Sarikurkcu, M. S. Ozer, M. Cengiz, N. Atılgan, O. Ceylan, B. Tepe. Ind. Crop. Prod. 2018, 125, 549-555. DOI:10.1016/j.indcrop.2018.09.043 21. B. Doustı, F. Nabıpor. J. Biotechnol. Comp. Biol. Bionanotech- nol. 2021, 102, 377-386. DOI:10.5114/bta.2021.111095 22. B. Emsen, B. Surmen, H. S. Karapinar. Plant Biosyst. 2022, 1-10. DOI:10.1080/11263504.2023.2165561 23. H. Koraqi, FC Ajazi, K. Kimete Lluga-Rizani, S. Kazlauskaite. Croat. J. Food Sci. Techno. l 2022, 14: 1-8. DOI:10.17508/CJFST.2022.14.1.12 24. M. M. Ebulue. ASEAN J. Sci. Eng. 2022, 3, 69-78. DOI: 10.17509/ajse.v3i1.45017 25. J. R. Soares, T. C. P. Dins, A. P. Cunha, L. M. Ameida. Free Rad. Res. 1997, 26, 469-478. DOI:10.3109/10715769709084484 26. T. H. Lee, C. H. Lee, P. Y. Ong, S. L. Wong, N. Hamdan et al. S. Afr. J. Bot. 2022, 148: 170-179. DOI:10.1016/j.sajb.2022.04.026 27. F. B. Mukeba, J. B. Mukoko, M. M. Mayangi et al. Eur. J. Med. Plants 2020, 31: 33-47. DOI:10.9734/ejmp/2020/v31i2030355 28. H. Kikuzaki, M. Hisamoto, K. Hirose, K. Akiyama, H. Tani- guchi. J. Agric. Food Chem. 2002, 50: 2161-8. DOI:10.1021/jf011348w 29. K. Saravanakumar, C. Sarikurkcu, R. T. Sarikurkcu, M. H. Wanga. Ind. Crop. Prod. 2019, 142, 111878. DOI:10.1016/j.indcrop.2019.111878 30. C. Sarikurkcu, S. S. Sahinler, B. Tepe. Ind. Crop. Prod. 2020, 154, 11263. DOI:10.1016/j.indcrop.2020.112633 31. N. Kumar, A. Singh, D. K. Sharma, K. Kishore. Int. J. Pharm. Biol. Sci. 2017, 7 (3), 30-35. 32. B. Kirkan, C. Sarikurkcu, A. S. Tepe. Biointerface Res. Appl. Chem. 2023, 13 (1): 1-10. DOI:10.33263/BRIAC131.088 33. M. Rafi, L. Wulansari, D. A. Septaningsih, T. F. Purnomo, R. Auliatifani, K. Khaydanur et al. J. Trop. Life Sci. 2021, 11, 375- 382. DOI:10.11594/jtls.11.03.14 34. P. Z. Moghaddam, M. Mazandarani, M. R. Zolfaghari, M. T. Badelehand, E. A. Ghaemi E. Afr. J. Microbiol. Res. 2012, 6, 1776-1781. DOI:10.5897/AJMR11.1225 35. A. Halim,  M. A. Zeb, M. Sajid, T. U. Rahman, K. F. Khattak, S. Ullah, et al. J. Microbiol. Exp. 2015, 3: 00074. DOI:10.15406/jmen.2015.02.00074 36. M. Saha, P. K. Bandyopadhyay. Microb. Pathog. 2020, 141: 103977. DOI:10.1016/j.micpath.2020.103977 37. V. Aparna, K. V. Dileep, P. K. Mandal, P. Karthe, C. Sadasivan et al. Chem. Biol. Drug Des. 2012, 80, 434-439. DOI:10.1111/j.1747-0285.2012.01418.x 38. D. Richard, K. Kefi, U. Barbe, P. Bausero, F. Visioli. Pharma- col. Res. 2008, 57, 451-455. DOI:10.1016/j.phrs.2008.05.002 39. C. C. M. P. Santos, M. S. Salvadori, V. G. Mota, L. M. Costa, A. A. C. Almedia et al. J. Neurosci. 2013, 949452. DOI:10.1155/2013/949452 40. Y. Song, S. K. Cho. Biochem. Anal. Biochem. 2015, 4: 1-7. DOI:10.4172/2161-1009.1000211 41. S. Vats, T. Gupta. Physiol. Mol. Biol. Plants 2017, 23, 239-248. DOI:10.1007/s12298-016-0407-6 42. D. Durak, Y. Kalender. Folia Biol. 2007, 55, 133-141. DOI:10.3409/173491607781492551 43. S. Babu, S. Jayaraman. Biomed. Pharmacother. 2020, 131, 110702. DOI:10.1016/j.biopha.2020.110702 203Acta Chim. Slov. 2023, 70, 196–203 Eroglu et al.: Potential Biochemical Properties of Endemic Onosma mutabilis 44. O. Boussaada, S. Ammar, D. Saidana, J. Chriaa, I. Chraif et al. Microbiol. Res. 2008, 163, 87-95. DOI:10.1016/j.micres.2007.02.010 45. V. Vanitha, S. Vijayakumar, M. Nilavukkarasi, V. N. Puni- tha, E. Vidhya, P. K. Praseetha. Ind. Crop. Prod. 2020, 154, 112748. DOI:10.1016/j.indcrop.2020.112748 46. T. Rhetso, R. Shubharani, M. S. Roopa, V. Sivaram. Future J. Pharm. Sci. 2020, 6, 1-9. DOI:10.1186/s43094-020-00100-7 47. S. Yogeswari, S. Ramalakshmi, R. Neelavathy, J. Muthumary. Glob. J. Pharmacol. 2012, 6, 65-71. DOI:10.1080/00431672.2012.666178 48. G. B. Dunks, K. Palmer-Ordonez, E. Hedaya, P. Keller, P. Wunz. Inorg. Synth. 1984, 22, 202-207. DOI:10.1002/9780470132531.ch46 Except when otherwise noted, articles in this journal are published under the terms and conditions of the  Creative Commons Attribution 4.0 International License Povzetek Rod Onosma L. (Lithospermae, Boraginaceae) vsebuje številne rastlinske vrste, ki imajo zaradi številnih fitokemikalij terapevtske lastnosti. Onosma mutabilis Boiss. & Hausskn. ex Boiss. (Onosma mutabilis) je vrsta, za katero ni dovolj podatkov o njenih značilnostih. Cilj: Raziskali smo skupno vsebnost fenolov, antioksidativno aktivnost, možne bioaktivne spojine in antibakterijske akti- vnosti etanolnih izvlečkov listov, stebla, korenin in cvetnih delov endemične O. mutabilis. Zaključki: Skupna vsebnost fenolov v vseh ekstraktih O. mutabilis se je gibala med 9,2 in 31 mg ekvivalentov galne kisline (GAE)/g ekstrakta. Glede na rezultate antioksidativne aktivnosti so bile vrednosti antioksidativne kapacitete IC50, do- ločene z metodo 1,1-difenil-2-pikrilhidrazil (DPPH), med 4,39 in 29 µg/ml, medtem ko je bila ekvivalentna antioksidati- vna aktivnost trolox, določena z merjenjem reducirajoče antioksidativne vrednosti bakrovih ionv (CUPRAC), 0,45–0,78 mmol trolox ekvivallentov (TE)/g ekstrakta. Bioaktivne spojine so bile analizirane z uporabo plinske kromatografije v povezavi z masno spektrometrijo (GC/MS) in ugotovljeno je bilo, da vsebujejo 29 različnih kemičnih sestavin. Vsi tes- tirani rastlinski izvlečki so pokazali učinkovito antibakterijsko delovanje proti A. baumannii (ATCC 02026) (vrednost minimalne inhibitorne koncentracije (MIC) 62,5 µg/ml) v primerjavi z referenčnim zdravilom ampicilin (125 µg/ml). 204 Acta Chim. Slov. 2023, 70, 204–217 Honmane et al.: Design, Development and Optimization of Carmustine- ... DOI: 10.17344/acsi.2023.8002 Scientific paper Design, Development and Optimization of Carmustine- Loaded Freeze-Dried Nanoliposomal Formulation Using 32 Factorial Design Approach Sandip M. Honmane1,2*, Manoj S. Charde2 and Riyaz Ali M. Osmani3 1 Department of Pharmaceutics, Annasaheb Dange College of B. Pharmacy, Ashta, Shivaji University, Kolhapur 416301, Maharashtra, India. 2 Department of Pharmaceutical Chemistry, Government College of Pharmacy, Karad, Shivaji University, Kolhapur 415124, Maharashtra, India. 3 Department of Pharmaceutics, JSS College of Pharmacy, JSS Academy of Higher Education and Research, Mysuru 570015, Karnataka, India * Corresponding author: E-mail: sandiphonmane@gmail.com Mob: +918600392878 Received: 01-05-2023 Abstract The objective of the current study was to develop and optimize a novel lyophilized liposomal formulation of anticancer agent carmustine, or bis-chloroethyl nitrosourea (BCNU) for prolonged release that could overcome the dose-depend- ent side effects and improve its bioavailability at the site of action. The optimization was done using a 32 factorial design approach wherein soya phosphatidylcholine (SPC) and cholesterol (CH) as independent variables. The optimized for- mulation (F4) exhibited high entrapment efficiency (81.57%) with an average vesicle size of 141.7 nm and a −22.6 mV Zeta potential. In-vitro drug release studies from all formulations revealed that the BCNU was released for up to 36 hours following the Higuchi matrix release model. The TEM, FTIR, DSC, PXRD, and SEM analyses confirm the formation of liposomes. BCNU-loaded nanoliposomal formulation demonstrated prolonged release, suggesting that it could be used to supplement cancer therapy efficiently with a reduction in dose-dependent side effects. Keywords: Carmustine; Nanoliposomes; 32 Factorial design; Release kinetics; Freeze-drying. 1. Introduction Despite the fact that cancer has been the second leading cause of mortality in the 21st century (besides car- diac ailments), it is plausibly the most complex disease and a serious health threat to people.1,2 Currently, to treat can- cer, physicians  use chemotherapy, hormone treatment, gene therapy, surgery, and radiation therapy. Usually, can- cer is treated with chemotherapy. On the other hand, high doses of chemotherapy drugs have undesired side effects and can be harmful to the body.3 In comparison to con- ventional chemotherapy, the nanocarrier-targeted drug delivery system offers the advantage that it reduces drug exposure to healthy tissues and the risk of organ and tissue damage, which reduces the development of multi-drug re- sistance and improves bioavailability.4–7 Moreover, a nano- carrier drug delivery system can also reduce toxicity and chemotherapy costs while achieving a long biological half- life and controlled drug release of chemotherapeutic drugs. Over time, a variety of nanocarriers have been de- veloped for the delivery of tumor-specific drugs, including micelles, liposomes, inorganic nanoparticles, polymeric nanoparticles, nanorods, and others.8,9 Liposomes might be one of the most promising drug delivery systems. It consists of one or more concentric phospholipid bilayers formed from synthetic or natural phospholipids that surround an aqueous core. They can include both hydrophilic and lipophilic molecules while yet being dispersed in water as a result of a phospholipid bilayer. These features make liposomes a special nano-car- rier for the delivery of biological therapeutics.10,11 Further- more, because liposomes are comprised of naturally oc- 205Acta Chim. Slov. 2023, 70, 204–217 Honmane et al.: Design, Development and Optimization of Carmustine- ... curring substances found in biological membranes, they offer the advantages of being biodegradable and non-toxic. Currently, liposomes are a desirable delivery system be- cause of their flexibility, structure, and colloidal size.12 Li- posomes have been produced using a variety of manufac- turing techniques and lipid compositions in sizes ranging from nanometres to micrometres. More flexible liposomes can be created by altering the bilayer elements, which pro- duce hard, impermeable, or porous and stable vesicles.13 For their improved solubility, precise drug targeting, and controlled release of different formulations, liposomes are widely preferred nowadays.14 According to the results of numerous experimental studies, cancer cells prefer nano- particles up to 500 nm due to their enhanced penetration and retention effects (EPR). Nanoparticles as small as 500 nm can extravasate because the blood arteries in tumor cells are more permeable than those in healthy tissue.15,16 The sole FDA-approved chemotherapeutic drug to treat high-grade gliomas (HGG) is carmustine or BCNU.17 It is a non-specific, alkylating antineoplastic drug that is used to treat many malignant neoplasms, including brain tumors.18 Multiple pathways are used by BCNU to cause tumor cytotoxicity, and it frequently disrupts DNA tran- scription and replication.19 In addition, BCNU binds to and alters (carbamoylates) glutathione reductase enzyme leading to cell death.20 BCNU’s short half-life of about 15 to 30 minutes and high toxic side effects (lung fibrosis and bone marrow sup- pression) limit its efficacy in treating glioma; these are among its most significant disadvantages. Furthermore, it has poor bioavailability due to hepatic metabolism.21–23 Therefore, an advanced novel prolonged-release formula- tion is needed for the efficient delivery of BCNU to the brain and other related malignancies, which may help re- duce the dose as well as any dose-related side effects. Therefore, the current work sought to evaluate the effects of polymer concentration and other process varia- bles to create and optimize a nanoliposomal formulation with the desired size range, high entrapment efficiency, and prolonged release of BCNU, an anticancer drug. 2. Materials and Methods 2. 1. Materials BCNU was received as a gift sample from Emcure Phar- maceuticals Ltd., Pune, Maharashtra, India. SPC was provided by the German company lipoid GmbH as a gift sample. CH, chloroform, and methanol were purchased from Loba Che- mie Pvt. Ltd. Mumbai, Maharashtra, India. The other solvents and materials employed were of an analytical standard. 2. 2. Optimization of the Solvent System The solvent system for the lipid phase was optimized using several combinations of organic solvents, specifical- ly, methanol and chloroform and the homogeneity of the film was assessed as depicted in Table 1. Table 1. Optimization of the solvent system Trial Chloroform Methanol Observation (mL) (mL) 1 3 0 Uniform, transparent film 2 3 1 Non-uniform sticky flocks 3 3 2 Non-uniform sticky flocks 4 3 3 Non-uniform sticky flocks 5 0 3 Non-uniform sticky flocks 2. 3. Optimization of Process Parameters for Preparation of Liposomes Using chloroform as an organic solvent, preliminary optimization of the speed of rotation and hydration medi- um for uniform film formation and maximal drug entrap- ment efficiency of liposomes was investigated. To create a thin and uniform film, which controls the liposomal preparation process’s result, the speed of rotation was changed from 30 revolutions per minute (rpm) to 90 rpm during film deposition under vacuum as depicted in Table 2. The drug’s ability to become entrapped in liposomes de- pends on the pH of the phosphate buffer. Entrapment effi- ciency was calculated after the pH of the hydration buffer was changed to levels closer to the drug’s pKa using phos- phate buffer saline (PBS) solution pH 5.0, 6.8, and 7.4 as depicted in Table 2. Table 2. Optimization of process parameters Parameters Variable Observation Speed of (rpm) 30 A thin and uniform film rotary 60 A non-uniform film with flocks at the evaporator centre of round bottom flask (RBF) 90 A non-uniform film with flocks at the centre of RBF The pH of 5.0 F4 (38.48 %) Hydrating 6.8 F4 (57.59 %) medium 7.4 F4 (81.57 %) 2. 4. Preparation of Liposomes A small modification to the thin film hydration process was used to produce blank and BCNU-loaded liposomes. SPC and CH were dissolved in chloroform as an organic phase at various molar ratios, along with BCNU (5 mg), to obtain a 60 mg/mL lipid phase concentration in a 250 mL rotary flask. The flask was attached to a rotary evaporator (Aditya Scientific, Hyderabad) that revolved at 30 rpm while immersed in a water bath that was maintained at 40 °C tem- perature and vacuumed for an hour to form the film.10,11 Ta- ble 3 depicts the components of the liposomal formulation. 206 Acta Chim. Slov. 2023, 70, 204–217 Honmane et al.: Design, Development and Optimization of Carmustine- ... After the organic phase had evaporated, the flask was placed in a desiccator overnight to remove any remaining organic solvent residues from the film. The following day, a liposome with a 10 mg/mL lipid concentration was pro- duced by thoroughly hydrating the thin film with PBS solution, pH 7.4, for one hour at a constant rotation of 160 rpm. To transform the produced liposomes from multila- mellar to unilamellar vesicles, they were subjected to Ultra Turrax (IKA T25) at 7000 rpm for 15 min. Then they were passed through a high-pressure homogenizer (HPH) (GEA Lab, Panda PLUS 1000) at 200 bar pressure for 50 cycles to reduce particle size and obtain uniform sized- li- posomes at the required nanometre size. The produced nanoliposomes were stored at 4 °C for further use. Table 3. Optimization of BCNU-loaded liposomal formulation us- ing a 32-factorial design Formulation Factors SPC:CH Lipid: Drug code [A:B] (mg) Molar Ratio ratio (mg) F1 60(−1):20(−1) 1:0.67 16:1 F2 60(−1):40(0) 1:1.33 20:1 F3 60(−1):60(+1) 1:2 24:1 F4 70(0):20(−1) 1:0.57 18:1 F5 70(0):40(0) 1:1.14 22:1 F6 70(0):60(+1) 1:1.71 26:1 F7 80(+1):20(−1) 2:1 20:1 F8 80(+1):40(0) 1:1 24:1 F9 80(+1):60(+1) 1:1.5 28:1 2. 5. Full Factorial Design The BCNU-loaded liposomes were developed using a 32 factorial design. In this approach, the quantities of SPC (A) and CH (B) were evaluated as independent varia- bles. The fixed responses used were vesicle size (Y1) and percent drug entrapment (PDE) (Y2). By taking each con- trol variable at three distinct levels nine alternative combi- nations were made, as depicted in Table 3. Later, the best- fit model derived from fit summary and analysis of variance (ANOVA) was used to examine the impact of various control variables on dependent variables. De- sign-Expert® software point prediction method was used to achieve the predicted formulation and verify optimiza- tion. 2. 6. Characterization of BCNU Loaded Liposome 2. 6. 1. Particle Size The mean vesicle size and size distribution of blank and BCNU-loaded liposomes were measured using a de- vice based on the dynamic light scattering method (HOR- IBA scientific SZ-100). The liposomal dispersion was di- luted with distilled water (1:100 v/v ratio, dispersant viscosity 0.896 mPa.s) using an ultrasonicator for 15 min- utes to obtain a stable suspension. A portion of the suspen- sion was transferred to a quartz cuvette (four openings). Size analysis was performed using a 90° angle of detection for 120 seconds at room temperature. Analysis was per- formed in triplicates.3 2. 6. 2. Zeta Potential Using Zetasizer (HORIBA scientific SZ-100), the surface charge of liposomes was measured. Before being positioned in measuring cells (cuvette with the carbon electrode, 6 mm), all compositions were diluted with dis- tilled water (1:100 v/v). The measurement of average zeta potential and charge on the liposomes was done by sub- jecting the formulation for 60 seconds run time. Analysis was performed in triplicates.3 2. 6. 3. Entrapment Efficiency To calculate the total quantity of drug (A) present in the formulation, 2 mL of the liposomal formulation was suspended in 2 mL of methanol to break up the liposomal matrix. This mixture was then centrifuged at 10,000 rpm at 1 °C temperature using a cooling centrifuge (REMI CM-12 Plus)  for 30 minutes. The produced pellet was rinsed by overtaxing with a 1 mL PBS solution (pH 7.4) to remove the free drug deposited on the liposome’s surface. The re- sultant dispersion was mixed with 10 ml of PBS solution (pH 7.4) and filtered using a 0.2-micron microsyringe fil- ter. Using a UV/visible spectrophotometer (Shimadzu 1800, Japan), the absorbance was measured at 229 nm to determine the quantity of BCNU in the filtrate.3 For the determination of free drug concentration (B), 2 mL of a drug-loaded liposomal mixture was centrifuged at 10,000 rpm at 1 °C for 30 minutes using a cooling centrifuge. The supernatant was discarded and diluted it with 10 mL of PBS solution (pH 7.4). The resultant solution was filtered through a microsyringe filter (0.2 µm), and absorbance was measured at 229 nm using a UV/visible spectropho- tometer.3 The entrapment efficiency was calculated by us- ing a formula- Where ‘A’ is the total amount of drug and ‘B’ is the free drug concentration. 2. 6. 4. Transmission Electron Microscopy TEM images were used to examine the structural in- tegrity of BCNU-loaded liposomes (using Hitachi S-7500). A few drops of diluted liposomal dispersion were applied to a 200-mesh carbon-coated copper grid and photo- graphed at 30,000x magnification and 100 kV.10 207Acta Chim. Slov. 2023, 70, 204–217 Honmane et al.: Design, Development and Optimization of Carmustine- ... 2. 6. 5. In-vitro Drug Release Study An in-vitro drug release study of optimized liposo- mal formulation (F4) and pure drug (BCNU) was carried out by the diffusion method using a dialysis bag. The treat- ed cellophane membrane (molecular weight cutoff [MW- CO] 12 kDa, Thermo Fisher Scientific) was tied at both ends after filling the liposomal sample (equivalent to 5mg of BCNU) in it and placed into the 100 mL beaker contain- ing 50 mL of PBS solution pH 7.4 as a dissolution medium. A magnetic stirrer was used to agitate the dissolving media at 100 rpm while maintaining the temperature at 37 ± 1 °C. 2 mL samples were taken from the receiver at periodic in- tervals up to 36 h and replaced with equal quantities of fresh dissolving liquid. Using a UV/Visible spectropho- tometer, a spectrometric analysis was performed at 229 nm to obtain drug content. Three separate recordings of each reading were taken.24 2. 6. 6. Kinetic Modeling of Release Profiles Several kinetic models, including zero order, first or- der, Higuchi matrix, Korsmeyer-Peppas, and Hix- son-Crowell, were used to fit the data from in-vitro drug release studies of liposomal formulations. The best-suited model was chosen, based on the correlation coefficient with the highest value.3 2. 6. 7. Physical Stability of Liposomal Formulation As per the ICH guidelines, stability experiments were carried out for the optimized formulation (F4) to evaluate the physical stability. The liposomal formulation (F4) was stored at room temperature (25±2 °C / 60±5 %RH) and in the refrigerator (4±2 °C) for three months.  The samples were collected at predetermined intervals of ini- tial, 30, 60, and 90 days to assess their physical appearance, mean vesicle sizes, size distributions, and amounts of drug entrapment as previously mentioned.10,25 2. 6. 8. Optimization of Cryoprotectant and Freeze-Drying Process The cryoprotectant concentration and formulation parameters that are most likely to affect the freeze-drying cycle and the quality of the finished product were studied. A drug-loaded liposomal sample (F4) was centrifuged for 30 minutes at 10,000 rpm (REMI CM-12 Plus). The super- natant was discarded after centrifugation, and the sedi- ment was collected in glass vials for freeze-drying. Along with the liposomal formulations, the cryoprotectant man- nitol was used in various concentrations (lipid: mannitol 1:0w/w, 1:5w/w, 1:10w/w, and 1:15w/w). To produce ho- mogenous ice nucleation, the above mixture was frozen overnight at –50 °C (1 °C/min) in a deep freezer. After that, it was freeze-dried using Christ, Alpha 1-2 LDplus. The aqueous solvent was then sublimated by maintaining the sample at –50 °C and 0.011 mBar for 12 h. The temper- ature and pressure were then raised to –20 °C (1 °C/min) and 1.0 mBar for 6 h. Secondary drying was done to re- move bound water. For this, the shelf temperature was raised by 1°C every minute and maintained at 20°C and 1.6 mBar for almost 3 h. After the process was completed, the vials were sealed with rubber caps and kept at 4 °C for further analysis.26 2. 6. 9. Moisture Content The Karl Fisher method was used to calculate the re- maining moisture (RM) in the freeze-dried cake. 0.1 g of the sample was transferred to the titration cell. The water content was determined using a Metrohm 870 KF Titrino plus KF titrator. 2. 6. 10. Compatibility Studies Using an FTIR spectrometer (Bruker Alpha II), the FTIR spectra of pure BCNU, physical mixtures, and freeze- dried formulation were recorded and analyzed between the wavelengths of 4000 and 650 cm–1. 2. 6. 11. Differential Scanning Calorimetry Using the Mettler Toledo DSC 822e instrument, DSC analysis of pure BCNU and a freeze-dried formula- tion were carried out to check the compatibility. Zinc and indium were used as standards to calibrate the tem- perature and enthalpy scales. Samples were heated in hermetically sealed aluminium containers at a constant rate of 10 °C/min from –60 to 200 °C. Liquid nitrogen was used at a flow rate of 40 mL/min to create an inert atmosphere. 2. 6. 12. Powder X-ray Diffraction PXRD is a crucial method for determining whether a substance is crystalline or amorphous. Using a powder X-ray diffractometer (AXS D8 Advances, Bruker Ltd., Germany) diffractograms of a pure drug and formulation were obtained with tube anode Cr spanning the range of 10–70°/2θ employing copper as the X-ray target and a 1.54 Å wavelength. 2. 6. 13. Scanning Electron Microscopy A scanning electron microscope (JSM-6360, Jeol In- struments, Japan) was used to examine the surface mor- phology of the BCNU-loaded freeze-dried liposomal for- mulation. With a 15 kV accelerating voltage, photomicro- graphs were taken of the sample while it was mounted on a double-faced gold-coated adhesive tape.27 208 Acta Chim. Slov. 2023, 70, 204–217 Honmane et al.: Design, Development and Optimization of Carmustine- ... 3. Results and Discussion 3. 1. Development of the Solvent System This system used organic solvents to dissolve the li- pid phase and form a thin, uniform, and non-sticky film. Since the nature of the film affects the liposomal size and entrapment efficiency. Different compositions of chloro- form and methanol were assessed for film formation. From the blend of organic solvents, a thick and sticky film was observed at the centre of the RBF, while chloroform alone produced a thin, uniform, and non-sticky film at the sides of the RBF. The results are depicted in Table 1. 3. 2. Optimization of Process Parameters for Preparation of Liposomes For the preparation of liposomes, process parame- ters like the speed of rotation and pH of the hydrating me- dium were studied for thin, uniform non-sticky film for- mation and entrapment efficiency, respectively. From the observations, it was found that at slow speed, RBF (30 rpm) produced a uniform non-sticky film at the sides, while at high speed (60 rpm and 90 rpm), lipid phase ag- gregated at the centre, possibly due to a high central force. The effect of hydrating buffer pH on entrapment efficiency was studied as the pH of hydrating buffer effect on entrap- ment of the drug into the lipid phase. Entrapment efficien- cy was varied at different pH values (5.0, 6.8, and 7.4). High entrapment efficiency was observed at a pH of 7.4 as the drug (BCNU) is unionized in aqueous fluid at that pH and more soluble in the lipidic phase while more ionized form at less pH and decreases entrapment into the lipid phase.18 The results are depicted in Table 2. 3. 3. Full Factorial Design When compared to unsaturated phospholipids, hy- drogenated SPC is more stable and biocompatible. Based on earlier research, SPC and CH concentrations were cho- sen to produce stable liposomes devoid of any aggregation or fusion, with small vesicles and higher drug entrapment efficiencies. This reveals that the amount of SPC and CH is the more important element in liposome production. Op- timized concentrations of SPC (60–80 mg) and CH (20-60 mg) were adequate to synthesize liposomes with small ves- icle sizes, excellent drug entrapment, and no aggregation or sedimentation. A full factorial design was employed to investigate the factors systematically. Using DESIGN EX- PERT® (version 8.0) software, the impact of different inde- pendent variables such as SPC (A) and CH (B) was exam- ined by response surface plots. Figure 1 displays the response to the impacts of independent factors for liposo- mal vesicle size (Y1) and PDE (Y2). The following equa- tions were produced, via regression and graphical analysis of data obtained from the experimental runs, where F ra- tios were statistically significant (p < 0.05), and Adj-R2 val- ues ranged from 0.8 to 1. The data was well-fit by these model equations. The effect on vesicle size (Y1) and PDE (Y2) was ob- served to be significant by ANOVA and the linear equation was found as follows: (1) (2) The response surface plots and regression equations mentioned above make it clear that the SPC and CH, at varying concentrations, produce a positive association concerning the vesicle size of BCNU-encapsulated lipos- omes. An increase in lipid concentration within the bilayer led to an increase in size. The level of CH was found to be closely correlated with a slight but substantial (p < 0.05) decline in entrapment efficiency. Similar outcomes for sev- eral lipophilic medications, such as alpha-tocopherol,28 ciprofloxacin,29 and triamcinolone acetonide,30 have pre- viously been observed. In the liposomal bilayer, CH mole- cules are positioned between the nearby phospholipid molecules. As a result, they take up some area and com- pete with BCNU for inclusion in the bilayer. Moreover, CH makes the bilayer stiffer, making it more challenging to incorporate drug molecules. The adjusted determination coefficient (R2= 0.8948 and 0.8873 for Y1 and Y2, respectively) and predicted de- termination coefficient (R2 = 0.8217 and 0.8227 for Y1 and Y2, respectively) values were comparable and showed the high significance of the model. By rejecting the null hy- pothesis, these “p” values of 0.05 (Prob > F) show that the model terms are significant. The “p” values for vesicle size and PDE were 0.0005 and 0.0006, respectively. For 32 fac- torial design model, the sum of the “p” values and the “ad- justed R2” values reveals a substantial synergistic associa- tion between both independent variables at P < 0.05. 3. 4. Characterization of BCNU Loaded Liposome 3. 4. 1. Particle Size The mean vesicle size of the various drug-loaded li- posomal formulations, which had 20–60 mg CH and 60– 80 mg SPC, was found to be between 141.0 and 170.9 nm. For drug-loaded liposomes, the polydispersity index ranged from 0.31 to 0.53, indicating narrow vesicle size dispersion shown in Table 4. A slightly small range of size distribution was present in every liposomal  formulation. The amount of SPC and CH present was significantly relat- ed to the size of the drug-loaded liposomes. Rather than the lipid content in the liposomal dispersion, the CH en- hances the stiffness of the membrane. Figure 2 shows a typical particle size distribution profile obtained for the optimized formulation (F4). 209Acta Chim. Slov. 2023, 70, 204–217 Honmane et al.: Design, Development and Optimization of Carmustine- ... 3. 4. 2. Zeta Potential Zeta potential measurements provide information on particle charge and the stability of the dispersion. Zeta potential shows the degree of repulsion between the charged particles in the dispersion. High zeta poten- tial indicates highly charged particles, which avoids particle aggregation owing to electrostatic repulsion. If the zeta potential is low, attraction overcomes repulsion and the dispersion forms aggregates. A zeta potential value of +30 mV to −30 mV is thought to be optimal for good stabilization.31 High zeta potential values, be- tween ±20 and ±40 mV, offer system stability and are less prone to agglomeration formation or particle size growth. However, it should be noted that zeta potential Figure 1. Linear plots (A, C) and Surface response plots (B, D) for particle size and % entrapment efficiency respectively Figure 2. A typical particle size distribution curve of optimized for- mulation (F4) 210 Acta Chim. Slov. 2023, 70, 204–217 Honmane et al.: Design, Development and Optimization of Carmustine- ... values are not an absolute measure of nanoparticle sta- bility.31 The zeta potential of freshly prepared liposomes ranged from −18.9 mV to −32.7 mV revealing that they had enough charge and mobility to prevent vesicle aggre- gation (Table 4). The Zeta potential of the optimized for- mulation (F4) was depicted in Figure 3. Figure 3. Zeta potential of optimized formulation (F4) 3. 4. 3. Percentage Drug Entrapment PDE is measured as the drug retention in liposomes as a percentage of the total drug. Percent entrapment effi- ciency for all formulations was found to be between 48.58% – 81.57 % depicted in Table 4. The amount of SPC and CH optimized for liposomal formulation by consider- ing the small vesicle size and maximum entrapment effi- ciency because these characteristics predominantly affect the encapsulation of the drug. Furthermore, smaller vesi- cle size offers better uptake by the cells and augmented drug deposition. Entrapment of the drug may be directly related to the overall surface area, as there are a higher number of vesicles more quantity of the drug will be en- trapped. As the particle size decreases, the surface area in- creases that subsequently results in an increase in drug encapsulation. PDE in liposomes demonstrates that drug entrapment efficiency in the liposomes decreases with de- creasing SPC concentrations. This is because the lipid bi- layer is saturated with respect to the drug and has a re- stricted capacity for entrapment due to its low SPC content. Figure 4. HR-TEM (A) and SAED (B) images of optimized formu- lation (F4) Table 4. Vesicle size, PDI, Zeta potential, and PDE of different batches of liposomal formulations Formulation Before HPH After HPH Zeta Potential PDE codes– Vesicle size (nm) PDI Vesicle size (nm) PDI (mV) Blank 131.2±0.34 0.453±0.04 95.1±0.42 0.207±0.06 −21.4±0.32 – F1 215.9±0.42 0.703±0.06 141.0±0.38 0.331±0.08 −11.5±0.42 64.64±0.43 F2 213.2±0.16 0.474±0.02 145.2±0.23 0.472±0.04 −23.2±0.12 58.94±0.74 F3 256.2±0.26 0.416±0.07 149.0±0.52 0.422±0.02 −36.1±0.26 48.58±0.63 F4 219.8±0.46 0.487±0.04 141.7±0.24 0.251±0.03 −22.6±0.36 81.57±0.92 F5 223.2±0.24 0.400±0.06 156.8±0.68 0.382±0.09 −32.6±0.35 61.36±0.34 F6 248.2±0.57 0.290±0.07 158.5±0.44 0.385±0.06 −25.8±0.48 54.60±0.64 F7 254.4±0.46 0.494±0.09 154.7±0.26 0.531±0.04 −18.4±0.16 75.61±0.83 F8 262.5±0.63 0.396±0.09 160.4±0.44 0.315±0.06 −28.9±0.23 62.22±0.93 F9 275.0±0.28 0.951±0.11 170.9±0.34 0.381±0.07 −30.2±0.28 57.60±0.46 Each value represents Mean ± SD, n = 3. 211Acta Chim. Slov. 2023, 70, 204–217 Honmane et al.: Design, Development and Optimization of Carmustine- ... Based on the PDE data, it was revealed that when CH con- centration increased, it provided rigidity to the bilayer and decreased PDE. Due to the high drug entrapment efficien- cy and small vesicle size of the F4 formulation, it was de- termined to be pertinent. 3. 4. 4. TEM Analysis The TEM image of the optimized formulation (F4) showed spherical liposomes with a small vesicle size with an average particle size of 141.7nm (Figure 4A). Figure 4B showed the selected area electron diffraction (SAED) pat- tern of liposomes that confirms the formation of lipos- omes. This supports the results of particle size. 3. 4. 5. In-Vitro Drug Release Studies The in-vitro drug release from the liposomal formu- lations and the pure BCNU was assessed using a PBS solu- tion with a pH of 7.4. All formulations showed drug re- lease up to 36 h, except the pure BCNU solution, which was released in less than 2 h. All formulations showed more than 90 % drug release within the 36 h (Figure 5). Formulation F4 showed a 96.64 % drug release over 36 h. which indicate controlled release of drug over a prolonged period of time. Figure 5. Cumulative % drug release from BCNU liposomes, and pure BCNU 3. 4. 6. Release Kinetic The data obtained from the in-vitro drug release in- vestigation of developed liposomes was fitted into kinetic models to identify the drug release mechanism. For the optimal fitting, the correlation coefficient value (R2) was used. The values of R2 for formulations ranged from 0.887 to 0.989. The correlation data for various models for all formulations are displayed in Table 5. According to the measured R2 values, the Higuchi matrix kinetic model best describes the in vitro drug releases from BCNU liposomes. It demonstrates that a diffusion process was adopted to re- lease the drug from the liposomes. 3. 4. 7. Physical Stability of Liposomal Formulation The stability of the liposomal formulation is a further essential factor in the development of an effective drug de- livery system. As a result, we tested the durability of the improved liposomal formulation in various settings, in- cluding room temperature (25 °C / 60 %RH) and the re- frigerator (4 °C). At initial, 30, 60, and 90-day intervals, all liposomal formulations were assessed and determined to be stable. At various storage conditions, caking and discol- oration were not seen. As a function of temperature, the mean particle size and formulation entrapment percentage were assessed. The results were depicted in Table 6 and a graphical rep- resentation of the change in particle size and entrapment efficiency is shown in Figure 6. Liposomes stored at 4 °C and 25 °C do not differ significantly in mean particle size. The entrapment efficiency showed a little decline, indicat- ing a considerable loss of BCNU from the formulation over time when held at 25 °C. Therefore, based on the find- ings of the stability study, it is advised that the liposomal formulation be kept in a refrigerator for better stability. 3. 5. Optimization of Cryoprotectant and Freeze-Drying Process Optimization of the cryoprotectant concentration used in the formulation is essential, along with careful consideration of the process parameter, to enable efficient stability of the liposomes with retaining formulation prop- erties. We need to maintain the product’s primary drying temperature below either the glass transition temperature (Tg´) or the somewhat higher collapse temperature (Tc) per guidelines for pharmaceutical freeze-drying. Typically, Tc and Tg´ can be used interchangeably because they are 1 to 2 °C apart. According to earlier research, the liposomal formulation with mannitol has a Tg´ of between –30 and –32 °C. Considering these values, the shelf temperature during primary drying was kept at –50 °C.26 Table 5. Mathematical models in drug release kinetics of liposomal formulations Formu- Zero First Higuchi Hixon Korsmeyer- lation order order Matrix Crowell Peppas codes (R2) (R2) (R2) (R2) (R2) F1 0.887 0.974 0.980 0.977 0.939 F2 0.900 0.977 0.984 0.981 0.933 F3 0.893 0.978 0.981 0.972 0.899 F4 0.893 0.949 0.981 0.977 0.941 F5 0.913 0.987 0.989 0.986 0.963 F6 0.917 0.973 0.986 0.980 0.920 F7 0.893 0.924 0.984 0.978 0.959 F8 0.905 0.959 0.987 0.978 0.954 F9 0.901 0.972 0.983 0.976 0.901 212 Acta Chim. Slov. 2023, 70, 204–217 Honmane et al.: Design, Development and Optimization of Carmustine- ... In the first section of the investigation, we explored how freeze-dried liposome stability was affected by man- nitol content. This was accomplished by lyophilizing lipo- somal suspension in the presence of mannitol while vary- ing the weight ratio of lipids to carbohydrates from 1:0 to 1:15. The stability of liposomes during freeze-drying was evaluated by measuring the proportion of the drug that was retained in the liposomes and comparing the size and PDI before and after freeze-drying. Since the drug retained after freeze-drying is closely correlated to the lipid phase transition and the aggregation of particles, it is considered the most sensitive measure that reflects all the harm caused by freeze-drying. The physicochemical properties of the liposomes were examined before freeze-drying. The liposomes were 141.7 nm in size with a 0.251 PDI, indicating a nar- row size distribution displayed in Table 4. In the case of non-cryoprotected liposomes, vesicle aggregation/fu- sion occurred during freeze-drying was evidenced by the size and PDI of the liposomes obtained after rehy- dration being significantly higher when freeze-dried without a cryoprotectant (control). It reveals that the freeze-drying process without cryoprotectant affects the integrity of the liposomes. Most of the drug that was en- capsulated leaked during the process. In contrast, ly- ophilized formulations with cryoprotectant content demonstrated increased stability as evidenced by narrow size distribution with controlled vesicle size, and less amount of drug leakage shown in Table 7. However, the stability of the liposomes was significantly impacted by the cryoprotectant concentration. A Lipid: mannitol weight ratio of 1:15 during freeze-drying of liposomes produced vesicles that were two times larger than those of the fresh liposomes. The distribution of population sizes within a given sample is essentially represented by PDI. The PDI’s nu- merical value range is 0.0 (uniform or monodisperse) to 1.0. (Polydisperse). A PDI of 0.3 and below is thought to be acceptable in drug delivery applications using li- pid-based carriers, such as liposome and nanoliposome formulations, and it denotes a homogenous (narrow) dis- tribution of phospholipid vesicles.32 Table 7 findings show that the freeze-drying procedure did not affect the PDI of rehydrated liposomes that included cryoprotectant in a different weight ratio, with the liposomes having a similar PDI to liposomes before the freeze-drying process (below 0.3). The size distribution of the liposomes was relatively wide, having a value of 0.661 at high lipid-to-mannitol ra- tios, 1:15, indicating that aggregation/fusion occurs dur- ing the processing. Over a limited range, the weight ratio of carbohydrate to lipid increased while the percentage of drug entrapment was reduced when more carbohydrate was added. The liposome membrane integrity was found to be best preserved at an intermediate ratio of 1:10 (li- pid-to-mannitol). Previous literature has reported similar outcomes.33 Uniform cakes have been seen for all samples with an RM ≤ 5%. For all samples, the secondary drying pro- cess eliminated unfrozen water rather slowly, especially when it was done at a temperature of 20 °C. Table 7 dis- plays the RM of cakes and the rehydrated liposomes’ char- acteristics. Table 6. The average particle size and PDE of the formulation (F4) stored at various temperatures Storage temperature 4±2 °C 25 ±2 °C (60±5 %RH) Parameter Vesicle size (nm) PDE Zeta Potential (mV) Vesicle size (nm) PDE Zeta Potential (mV) Initial 141.7±0.24 81.57±0.92 −22.6±0.36 141.7±0.24 81.57±0.92 −22.6±0.36 30 days 150.4±0.32 80.16±0.42 −22.0±0.18 151.4±0.38 78.74±0.23 −24.6±0.12 60 days 156.9±0.25 79.68±0.12 −25.1±0.27 170.3±0.54 74.46±0.32 −25.3±0.24 90 days 167.1±0.37 78.72±0.14 −26.6±0.14 189.4±0.24 72.39±0.42 −25.5±0.18 Each value represents Mean ± SD, n = 3. Figure 6. Physical stability of liposomes (F4) stored at different stor- age conditions; particle size (A) and % drug entrapment (B) 213Acta Chim. Slov. 2023, 70, 204–217 Honmane et al.: Design, Development and Optimization of Carmustine- ... 3. 6. Compatibility Studies Utilizing FTIR tests, it was assessed whether the drug was compatible with other excipients and formula- tions. Using an ATR-FTIR spectrometer, the infrared spectrum of a pure drug (BCNU) and a physical mixture of a BCNU with excipients and formulation were recorded (Figure 7). The distinctive peaks of the BCNU FTIR spec- trum may be seen to correspond to COO– groups at 1278 and 1456 cm–1 and to the double bond C=H at 1134 cm–1. Additionally, peaks at 626, 1318, 1354, and 1432cm–1 were observed, which, respectively, corresponded to aromatic CH bending, C-N stretch, aliphatic CH bending, and CH2 bending. Typical characteristic peaks of the BCNU were also seen in the FTIR spectrum of the physical mixture and formulation with no obvious change from the spectra of the individual drug and excipients. This demonstrated that mannitol, the drug, lipids, and cholesterol did not in- teract chemically. These findings are consistent with previ- ous research.21 3. 7. DSC study DSC studies were used to analyse the thermal behav- iour of pure BCNU, physical mixture and its formulation. The results are displayed in Figure 8. The DSC of pure BC- NU shows a prominent endothermic peak at 31  °C and –141.4 J/g, indicating the melting of pure BCNU. The SPC endothermic peak was observed at 48.0 °C (the tempera- ture of the phase transition) and had an enthalpy of –0.551 J/g, whereas the mannitol endothermic peak was observed at 161.0 °C and had an enthalpy of –157.4 J/g. At 148 °C, the typical cholesterol peak was discovered. It might be ac- counted for by the lipids’ nanocrystalline structure in li- posomes. The absence of an endothermic peak for BCNU in the formulation indicated that the lipid matrix had completely dissolved BCNU. The retention of the charac- teristic endothermic peak of mannitol in the formulation suggested its crystallinity and did not interact chemically. The conclusions of the DSC analysis were further support- ed by the PXRD data. Table 7. The effects of mannitol concentration on vesicle size, PDI, PDE, Zeta potential, and RM of a freeze-dried liposomal formulation (F4). Parameters Lipid: mannitol weight ratio 1:0 (Control) 1:5 1:10 1:15 Vesicle size (nm) 416.6±0.46 237.3±0.62 191.4±0.46 218.5±0.54 PDI 0.933±0.07 0.299±0.08 0.226±0.06 0.661±0.04 PDE 56.40±0.57 72.60±0.53 80.48±0.68 74.67±0.56 Zeta potential (mV) –26.7±0.38 –27.1±0.64 –28.5±0.49 –23.8±0.32 RM (%) 2.72±0.34 2.20±0.23 2.42±0.32 2.90±0.15 Each value represents Mean ± SD, n = 3. Figure 7. FTIR spectra of the pure drug (BCNU), physical mixture, and optimized formulation (F4) 214 Acta Chim. Slov. 2023, 70, 204–217 Honmane et al.: Design, Development and Optimization of Carmustine- ... Figure 8. DSC thermogram of the pure drug (BCNU) (A), physical mixture (B), and freeze-dried formulation (F4) (C) Figure 9. PXRD of the pure drug (BCNU), mannitol, and freeze-dried formulation (F4) 215Acta Chim. Slov. 2023, 70, 204–217 Honmane et al.: Design, Development and Optimization of Carmustine- ... 3. 8. PXRD Figure 9 displays the PXRD of a lyophilized formula- tion (F4) and pure BCNU. Typical diffraction peaks re- markably at 2θ diffraction angles of 8.58°, 18.66°, 21.18°, 23.88°, 28.60°, 29.52°, 33.11°, and 34.90° were used to identify the crystalline nature of BCNU. The pure BCNU exhibits an intense crystalline peak between 5° and 50°. However, the peak of the pure drug (BCNU) in the ly- ophilized liposomal formulation (F4) was reduced; indi- cating a decrease in crystallinity. It was anticipated that BCNU was dispersed as a molecule in the thin lipid film layer. While the intense peak in the formulation might be due to the crystalline nature of mannitol. 3. 9. SEM The microstructure of the product can be directly observed and the impact of the freeze-drying procedure on cake morphology can be determined by performing a microscopic examination of the freeze-dried cake. Figure 10 depicts a crystalline, porous matrix at a 200-fold mag- nification. Previous literature has reported similar out- comes.33 The conclusions of the SEM analysis were further supported by the PXRD data. Figure 10. SEM images of freeze-dried formulation (F4) 4. Conclusions For improving the characteristics and performance of nanoliposomal formulation of the anticancer drug (BC- NU), we have assessed and examined the impact of various process parameters on formulation properties such as ves- icle size, entrapment efficiency, zeta potential, and drug leakage after freeze-drying, to list a few. SPC and CH were investigated in various compositions using a 32-factorial design to fabricate nanoliposomes for targeted drug deliv- ery. Surface response plots and regression equations showed a positive association between the vesicle size of BCNU-loaded liposomes and the SPC and CH at various ratios. A higher lipid content led to an increase in the size and stiffness of the liposomal bilayer. In vitro drug release and release kinetics investigations of BCNU-loaded lipos- omes revealed that the drug is released through a diffusion mechanism and the Higuchi matrix model is followed over a prolonged period. Stability studies showed that lipid compositions are stable under refrigerated storage (4 °C) conditions. FTIR and DSC analysis data demonstrated that mannitol as a cryoprotectant protects the liposomal structure at an optimum concentration during freeze-dry- ing. In contrast, SEM microscopy revealed that the manni- tol leads to the porous microstructure of the final product at an optimum concentration with some extent of crystal- linity. The crystalline nature of mannitol in the final lyo- phile provided mechanical strength to the final cake. In order to maintain mannitol in a crystalline state in the final product, it is necessary to ensure that mannitol does not crystallize when the system is in the glassy state (T < Tg’). As a result, it is evident from the out- comes of testing the parameters for the BCNU nanoli- posomal formulation that it may minimize the dosing frequency and effectively be targeted at the site of ac- tion. Moreover, it will reduce the adverse effects brought on by the anticancer agent BCNU’s high dose and non-targeted distribution. The pharmacokinetics and 216 Acta Chim. Slov. 2023, 70, 204–217 Honmane et al.: Design, Development and Optimization of Carmustine- ... pharmacodynamics properties of these formulations can be explored through in vivo bioavailability studies to develop an efficient drug delivery system for aug- mented anticancer therapy. 5. Future Prospects The utilization of organic solvents in liposome-based pharmaceuticals has certain limitations. These solvents must be eliminated during the drug production process, which requires adhering to strict safety and regulatory standards. As a result, there is a rise in production expens- es due to the need for further purification and waste man- agement procedures. There are various techniques availa- ble to decrease the size and distribution of the initial hetero-dispersed liposome suspensions. Among these techniques, homogenization is widely employed as it is ap- plicable for large-scale production and yields a desirable size reduction and distribution. This involves pumping the hetero-dispersed liposome preparation through a small re- action tank under high pressure in a cyclic manner until the desired average liposome size is attained. To decrease the size of liposomes, another technique is to subject them to sonication or ultrasonic irradiation, which generates shear forces during the process. Another effective size re- duction method involves extruding the liposomes through membranes with uniform pore sizes to achieve uniform liposome preparation. While the thin film hydration technique is a useful method for synthesizing liposomes, there are certain drawbacks that must be addressed. These include the need to use and completely remove organic solvents, the formation of multilamellar vesicles, and a broad distribu- tion of particle sizes. Further investigation into the pro- cess design and preparation of liposomes through thin film hydration with homogenization techniques on an industrial scale is crucial. This is due to the current lack of continuous production at high levels and the draw- backs linked with the utilization of organic solvents. Nonetheless, before proceeding with large-scale lipos- ome production, it is necessary to thoroughly examine the impact of each parameter of the thin film hydra- tion-assisted process. Author contribution statement All authors listed have significantly contributed to the development and the writing of this article. Funding statement A grant from Shivaji University, Kolhapur, 416 0004 Maharashtra, India under the Research Initiation Scheme (SU/C. & U. D. Section/97/170 Dated: 31/08/2021), sup- ported this work. Acknowledgement We gratefully acknowledge lipoid GmbH, Germany for providing a gift sample of phospholipid. The authors also would like to thank the Principal and management of Annasaheb Dange College of B. Pharmacy, Ashta, Mahar- ashtra, India for providing the facility to complete this re- search. Declarations Conflict of Interest The authors declare that they have no conflict of in- terest. 6. References 1. M. Fang, C. W. Peng, D. W. Pang, Cancer Biol Med. 2012, 9 (3), 151–163. DOI:10.7497/j.issn.2095-3941.2012.03.001. 2. D. Bikiaris, Biochem. Pharmacol. 2012, 1, 1–5. DOI:10.4172/2167-0501.1000e122 3. S. M. Honmane, S. M. Chimane, S. A. Bandgar, S. S. Patil, Indian J. Pharm. Educ. Res. 2020, 54 (2), 376–384. DOI:10.5530/ijper.54.2.43 4. Y. H. Bae, and K. Park, J. Control. Release. 2011, 153, 198– 205. DOI:10.1016/j.jconrel.2011.06.001 5. J. L. Markman, A. Rekechenetskiy, E. Holler, Adv. Drug Deliv. Rev. 2013, 65, 1866–1879. DOI:10.1016/j.addr.2013.09.019 6. L. S. Jabr-Milane, L. E. Vlerken, S. Yadav, Cancer Treat. Rev. 2008, 34, 592–602. DOI:10.1016/j.ctrv.2008.04.003 7. D. S. Spencer, A. S. Puranik, N. A. Peppas, Curr. Opin. Chem. Eng. 2015, 7, 84–92. DOI:10.1016/j.coche.2014.12.003 8. M. Kanapathipillai, A. Brock, D. E. Ingber, Adv. Drug Deliv. Rev. 2014, 79–80, 107–118. DOI:10.1016/j.addr.2014.05.005 9. A. Jhaveri, P. Deshpande, V. Torchilin, J. Control. Release. 2014, 190, 352–370. DOI:10.1016/j.jconrel.2014.05.002 10. S. Honmane, S. Salunkhe, A. Hajare, N. Bhatia, S. Mali, Int. Res. J. Pharm. 2014, 5 (2), 70–74. DOI:10.7897/2230-8407.050214 11. S. Honmane, A. Hajare, H. More, S. Salunkhe, J. Liposome Res. 2019, 23, 1–11. DOI:10.1080/08982104.2018.1531022 12. T. A. Elbayoumi, V. P. Torchilin, Methods Mol. Biol. 2010, 605, 1–27. DOI:10.1007/978-1-60327-360-2_1 13. A. Akbarzadeh, R. Rezaei-Sadabady, S. Davaran, Nanoscale Res. Lett. 2013, 8, 102. DOI:10.1186/1556-276X-8-102 14. A. Khan, K. S. Allemailem, S. A. Almatroodi, A. Almatroudi, A. H. Rahmani, 3 Biotech. 2020, 10 (4), 163. DOI:10.1007/s13205-020-2144-3 15. L. Brannon-Peppas, and J. O. Blanchette, Adv Drug Deliv. Rev. 2004, 56, 1649–1659. DOI:10.1016/j.addr.2004.02.014 16. S. Feng, and S. Chien, Chem. Eng. Sci. 2003, 58 (18), 4087– 4114. DOI:10.1016/S0009-2509(03)00234-3 17. C. G. Hadjipanayis, and W. Stummer, J Neurooncol. 2019, 141, 479–486. DOI:10.1007/s11060-019-03098-y 18. Carmustine. [(Accessed on 25 August 2020)]; Available on- line: https://go.drugbank.com/drugs/DB00262. 217Acta Chim. Slov. 2023, 70, 204–217 Honmane et al.: Design, Development and Optimization of Carmustine- ... Povzetek Cilj raziskave je bil razviti in optimizirati novo liofilizirano liposomsko formulacijo protirakave učinkovine karmustin, ali bis-kloretil nitrosourea (BCNU), za podaljšano sproščanje, s čimer bi lahko odpravili od odmerka odvisne stranske učinke in izboljšali biološko uporabnost na mestu delovanja. Optimizacija je bila izvedena z uporabo 32-faktorskega pris- topa, pri čemer sta bila sojin fosfatidilholin (SPC) in holesterol (CH) neodvisni spremenljivki. Optimizirana formulacija (F4) je pokazala visoko učinkovitost vključevanja (81,57 %) s povprečno velikostjo veziklov 141,7 nm in zeta potencia- lom -22,6 mV. In vitro študije sproščanja učinkovine iz vseh formulacij so pokazale, da se BCNU sprošča do 36 ur po Higuchijevem modelu matričnega sproščanja. Analize TEM, FTIR, DSC, PXRD in SEM potrjujejo nastanek liposomov. Nanoliposomska formulacija z BCNU je izkazovala podaljšano sproščanje, kar kaže, da bi jo lahko učinkovito uporabili za dopolnilno zdravljenja raka z zmanjšanjem od odmerka odvisnih stranskih učinkov. Except when otherwise noted, articles in this journal are published under the terms and conditions of the  Creative Commons Attribution 4.0 International License 19. S. H. Lin, and L. R. Kleinberg, Expert Rev. Anticancer Ther. 2008, 8 (3), 343–359. DOI:10.1586/14737140.8.3.343 20. N. Doroshenko, and P. Doroshenko, Eur. J. Pharmacol. 2004, 497 (1), 17–24. DOI:10.1016/j.ejphar.2004.06.043 21. S. Yi, F. Yang, C. Jie, G. Zhang, Artif. Cells Nanomed. Biotech- nol. 2019, 47, 3438–3447. DOI:10.1080/21691401.2019.1652628 22. V. T. De Vita, P. P. Carbone, A. H. Jr. Owens, G. L. Gold, M. J. Krant, J. Edmonson, Cancer Res. 1965, 25, 1876–1881. 23. B. R. O’Driscoll, S. Kalra, H. R. Gattamaneni, A. A. Wood- cock, Chest. 1995, 107, 1355–1357. DOI:10.1378/chest.107.5.1355 24. D. Bi, L. Zhao, H. Li, Y. Guo, X. Wang, M. Han, Int. J. Pharm. 2019, 559, 76–85. DOI:10.1016/j.ijpharm.2019.01.033 25. H. Ola, S. A. Yahiya, O. N. El-Gazayerly, Saudi Pharm J. 2010, 18 (4), 217–224. DOI:10.1016/j.jsps.2010.07.003 26. Z. Hua, B. Li, Z. Liu, D. Sun, Drying Technology. 2003, 21 (8), 1491–1505. DOI:10.1081/DRT-120024489 27. S. Honmane, A. Kadam, S. Choudhari, R. Patil, S. A. Ansari, V. Gaikwad, J. Drug Deliv. Sci. Technol. 2021, 64, 102578. DOI:10.1016/j.jddst.2021.102578 28. H. Tabandeh, and S. A. Mortazavi, Iran J Pharm Res. 2013, 12 (Suppl), 21–30. 29. K. M. Hosny, AAPS PharmSciTech. 2010, 11 (1), 241–246. DOI:10.1208/s12249-009-9373-4 30. B. Clares, V. Gallardo, M. M. Medina, M. A. Ruiz, J. Liposome Res. 2009, 19 (3), 197–206. DOI:10.1080/08982100902736571 31. C. Freitas, R. H. Müller, Int J Pharm. 1998, 168 (2)221–229. DOI:10.1016/S0378-5173(98)00092-1 32. I. Khan, R. Needham, S. Yousaf, C. Houacine, Y. Islam, R. Bn- yan, S. K. Sadozai, M. A. Elrayess, A. Elhissi, J. Drug Deliv. Sci. Technol. 2021, 66, 102822. DOI:10.1016/j.jddst.2021.102822 33. B. Sylvester, A. Porfire, P. J. Van Bockstal, S. Porav, M. Achim, T. Beer, I. Tomuţă, J Pharm Sci. 2018, 107 (1), 139–148. DOI:10.1016/j.xphs.2017.05.024 218 Acta Chim. Slov. 2023, 70, 218–225 Balkır et al.: Phytochemical Profile, Antioxidant and Antimicrobial ... DOI: 10.17344/acsi.2023.8008 Scientific paper Phytochemical Profile, Antioxidant and Antimicrobial Potency of Aerial Parts of Salvia Tomentosa Miller Şehnaz Balkır1, Ömer Hazman1, Laçine Aksoy1*, Mustafa Abdullah Yılmaz2, Oguz Cakir2, Recep Kara3 and İbrahim Erol1 1 Department of Chemistry, Faculty of Science and Arts, Afyon Kocatepe University, 03200, Afyonkarahisar, Turkey 2 Dicle University Science and Technology Research and Application Center, 21280, Diyarbakir, Turkey 3 Afyon Kocatepe University, Faculty of Veterinary Medicine, Department of Food Hygiene and Technology, Afyonkarahisar * Corresponding author: E-mail: sehnazblkr@hotmail.com Received: 01-11-2023 Abstract Antioxidant activity, antimicrobial potency and components of the aerial parts (leaf, stem, flower and mixture) of Salvia tomentosa Miller were determined qualitatively and quantitatively in this study. Aqueous extracts of Salvia tomentosa (ST) were prepared by using the flower, leaf and stem parts and all the above-ground parts of the plant (flower-leaf- stem mixture) for this purpose. The radical scavenging activity, total antioxidant/oxidant status, antimicrobial potential, phenolic substances and qualitative/quantitative analyzes of the components in the extracts were determined. ST-stem phenolic acid amount (599 ± 34 mg gallic acid equivalent (GAE)/g extract) was found to be close to the standard sub- stance caffeic acid (651 ±3 1 mg GAE/g extract). Total antioxidant status of ST-mix (3.4 ± 0.1 mmol Trolox Equiv./L) and ST-stem (3.4 ± 0.1 mmol Trolox Equiv./L) and natural antioxidant Vitamin C (3.6 ± 0.1 mmol Trolox Equiv./L) were not statistically different. The extract produced by using S. tomentosa aerial parts (flower-stem-leaf) showed stronger antioxidant and antimicrobial activity than the aqueous extracts obtained separately from the flower, stem and leaf of the plant. However, it was determined that the components of the separately prepared flower, stem and leaf extracts and the extract components obtained from the aerial parts were largely similar. At the same time, it was observed that there were significant differences in the presence of these components. Keywords: Salvia tomentosa Miller, antimicrobial, antioxidant, LC-MS/MS 1. Introduction Salvia tomentosa is a perennial semi-shrub with whi- te or lilac-purple flowers up to 55 cm tall. It blooms betwe- en April and September. It grows in forests of Pinus brutia, Pinus nigra, Quercus pubescens and on limestone and vol- canic slopes. The leaves are used as herbal tea.1–3 Salvia species contain many bioactive compounds that can be classified as monoterpenes, diterpenes, triterpenes and phenolic compounds. The most common monoterpenes are; α-thujone and β-thujone, 1,8-cineole and camphor, diterpenes; carnosol, carnosic acid, rosmadial and mano- ol, triterpenes; oleanolic and ursolic acids. Phenolic com- ponents are phenolic acids such as caffeic, vanillic, ferulic and rosmarinic acids, and flavonoids such as luteolin, api- genin and quercetin.4–6 Sage taxa grown in Turkey were classified by Başer7 (2002) according to the main compo- nents of essential oil. According to this classification, Sal- via tomentosa belongs to the pinene group; the plant which contains 0.6–1.3% essential oil, contains 6–29% α-pinene and 5–33% β-pinene.7–9 The use of herbal medicines for therapeutic purpo- ses, the fact that fragrant plants constitute the main raw material of perfumery, food and cosmetics industry, and the emergence of new areas of use increases the demand for medicinal and aromatic plants day by day and causes the industrial sector to consume these plants as raw mate- rials intensively.10, 11 Like many other medicinal plants, S. tomentosa was extensively collected from its natural habi- tat, and this careless collection has caused some plants to become extinct. Therefore, these plants are grown in order to promote sustainable and standard agricultural produc- tion.12 It is stated that the essential oil of Salvia tomentosa aerial parts significantly inhibits the growth of Gram-posi- 219Acta Chim. Slov. 2023, 70, 218–225 Balkır et al.: Phytochemical Profile, Antioxidant and Antimicrobial ... tive and Gram-negative bacteria tested except Pseudomo- nas aeruginosa in the literature.13 In another study, it is stated that the plant has antioxidative effects.14 In this study, the biological activity and phytochemical content of the parts of Salvia tomentosa consumed as tea are present- ed in detail. 2. Materials and Methods 2. 1. Plant Material and Extraction Protocol Salvia tomentosa is a perennial plant. The stem of the plant consists of upright, four-sided, usually branched, hairy and sessile glands extending up to 1 m. Leaves sim- ple, narrowly oblong to ovate, 2–11 x 0.8–5 cm, rounded to cordate at base, entire to oblong-cubic. The petiole is 1.7–5.5 cm. Verticillasters 4–10 flowered, distant or dense at the top. The crowns are lilac, purple or white. They usu- ally grow in the forests of P. brutia and P. nigra, on lime- stone or magmatic slopes, at an altitude of 90–2000 m. Salvia tomentosa was collected in 2020 from Ardıçlı vil- lage, Keçiborlu, Isparta, Turkey (37° 48´ 8.9928” and 30° 12´ 13.4676”). Separate aqueous extracts were prepared by using the flower (ST-flower), leaf (ST-leaf) and stem (ST-stem) parts of the plant and the total aerial parts of the plant (flower-leaf-stem mixture, ST-mix). The plant, which was allowed to dry well in the shade. Each separat- ed part was crushed into powder with the help of a special blender (Waring 32BL80, Connecticut, USA). The water extract from the samples was obtained using the macera- tion method. Water was placed in glass bottles and heated in a water bath until boiling. The powder sample was add- ed to glass bottles. The plant-water suspension in glass bottles was incubated in the dark at room temperature for 24 hours. At the end of the incubation, the mixture in the glass bottle was passed through filter paper (Whatman, Grade 589/1) and the plant particles were separated from the filtrate. In order to remove the solvent in the filtrate, the filtrate was placed in the balloon of the rotary evapo- rator (Heidolph, 562-00000-00-0, Germany) under vacu- um. Solutions containing dense extract and poured into glass petri dishes were kept in dark at room temperature until the solvent evaporated completely (2–3 days). Ex- tracts obtained in dry form were stored at +4 °C to be used in qualitative, quantitative and biological activity analyses.15 2. 2. Qualitative and Quantitative Analysis of Phytochemicals in Extracts Determination of Total Phenolic Acid amount Total phenolic acid content was measured by the modified Folin-Ciocalteu method. The reference material caffeic acid was used in order to compare the amounts of phenolic substances in the extracts in the analysis. A standard curve of gallic acid was created for this purpose. The standard solutions of gallic acid at different concentra- tions (100, 200, 400, 600, 800 µg/mL) were prepared. Fo- lin-Ciocalteu reagent was added to the prepared solutions of plant extract, caffeic acid and standards in the analyses. Sodium carbonate was added and incubated for 2 hours at room temperature. The absorbance of the mixture was measured spectrophotometrically at 760 nm against water. The total amount of phenolic acid in 1 mg of the extracts was calculated as gallic acid equivalent (µg GAE/mg ex- tract) using the absorbance obtained as a result of the anal- ysis of the plant extracts and the straight equation obtained from the gallic acid standard curve.16 Determination of Components in Extracts and Concentrations Qualitative and quantitative determination of the components in the extracts were made by LC-MS/MS sys- tem in the laboratories of Dicle University Science and Technology Application and Research Center. The reverse phase UHPLC system used in the LC-MS/MS system pre- ferred in the analysis; It consisted of an autosampler (SIL- 30AC), a column furnace (CTO-10ASvp), a gradient pump system (LC-30AD) and a degaser (DGU-20A3R). Chromatographic separation was performed using a col- umn (Agilent Poroshell 120 EC-C18) (150 mm×2.1mm, 2.7 µm). The column temperature was set to 40 °C. The elution gradient was composed of mobile phase A (ultra pure water+5 mM ammonium formate+0.1% formic acid) and mobile phase B (ultrapure water +5 mM ammonium formate+0.1% formic acid).15 The gradient elution profile used was as follows: 20– 100% B (0–25 min), 100% B (25–35 min), 20% B (35–45 min). In addition, the mobile phase flow rate and injection volume were determined as 0.5mL/min and 5 µL, respec- tively. For mass spectrometry detection of the LC-MS/MS system used, a Shimadzu LCMS-8040 model sequential mass spectrometer equipped with an electrospray ioniza- tion source operating in both positive and negative modes was used. LC-ESI-MS/MS data was acquired and pro- cessed with LabSolutions software (Shimadzu). MRM (multiple reaction monitoring) mode was used for the quantification of phytochemicals. The MRM method has been optimized for the selective detection and quantifica- tion of phytochemicals based on the screening of specific major ion-fragmentation ion transitions. Collision ener- gies (CE) are optimized to achieve optimum phytochemi- cal fragmentation and maximal migration of desired frag- mentation ions. Applied MS operating conditions: drying gas (N2) flow, 15 L/min; nebulizer gas (N2) flow, 3 L/min; DL temperature, 250 °C; The heat block temperature was determined as 400 °C and the interface temperature as 350 °C. The amounts of phenolic acid species whose amounts were determined in the extracts were expressed as mg-an- alyte/g-extract.17 220 Acta Chim. Slov. 2023, 70, 218–225 Balkır et al.: Phytochemical Profile, Antioxidant and Antimicrobial ... 2. 3. Determination of Biological Activity of Extracts Free Radical Scavenging Activity Free radical scavenging activity of S. tomentosa and standard antioxidant substances (Butylated Hydroxy Tolu- ene-BHT and Vitamin C) used in the study were deter- mined by DPPH (1,1-diphenyl-2-picrylhydrazyl) radical. A calibration curve was created using different concentra- tions of DPPH solution. Using the curve equation obtained from the calibration chart and sample absorbances, it was determined how much DPPH radical each sample inhibit- ed. Extracts and standards were added to the solution con- taining DPPH radical and their absorbance was measured at 517 nm. Concentrations from the measured absorbanc- es were determined by the calibration curve. % inhibitions were determined using the formula below.15 Inhibition rate (%) = [(Absorbancecontrol – Absorbancesample) /Absorbancecontrol)] × 100 Total Antioxidant Status (TAS), Total Oxidant Status (TOS) Levels Total Antioxidant Status (TAS) levels were measured using spectrophotometric commercial kits (Rel Assay, Ga- ziantep, Turkey). In order to determine TAS levels, 0.5–2 mmol/L Trolox was used as a standard. TAS levels were determined as mmol Trolox Equivalent/L according to the calibration graph taken from the ELISA reader using three standards (0.5 mmol/L Trolox, 1mmol/L Trolox and 2 mmol/L Trolox) according to the kit protocol. TOS levels were determined at 540 nm by spectro- photometric method using commercial kits (Rel Assay, Gaziantep, Turkey). To determine TOS levels, a calibration curve was created using three H2O2 standards (5 µmol/L, 10 µmol/L and 20 µmol/L) according to the kit protocol. The results were determined as µmol H2O2 Equiv./L. Oxi- dative stress index (OSI) levels were determined by divid- ing the TOS level of each sample by the TAS level.18 Evaluation of Antimicrobial Efficacy of Extracts Microdilution method was used to determine the antimicrobial activity of extracts of Salvia tomentosa. Stock solutions were prepared from all extracts at a con- centration of 60 mg/mL. 1/2, 1/4, 1/8, 1/16, 1/32 and 1/64 dilutions were prepared from these stock solutions, re- spectively. 107 CFU/mL of 1% (v/v) bacterial solutions were added to the same volume of extract solutions from the dilutions and incubated at 37ºC for 24 hours. Mini- mum inhibitory concentration (MIC) is inhibited value after incubation; The lowest extract concentrations at which bacterial growth was inhibited were evaluated by both measuring at 450 nm and inoculation into the me- dium.19 2. 4. Statistical analysis The extracts used in the study were prepared in trip- licate, and the measured results were expressed as mean ± standard deviation (mean ± SD). SPSS 20 package program was used in the statistical analysis of the data in this study. Differences between groups were determined by one-way analysis of variance (one-way ANOVA). The difference be- tween which groups was determined at p < 0.05 signifi- cance value according to Duncan’s multiple range test. 3. Results Plants have many activities such as antioxidant, anti- microbial and anticarcinogenic effects due to the second- ary metabolites they synthesize and possess.20 The amount of phenolic acid, which is one of the secondary metabolites in S. tomentosa, was determined by modifying the meth- od.16 The amounts of phenolic substances expressed as mg gallic acid equivalent (GAE)/g-extract in the samples were interpreted by comparing the amount of phenolic sub- stance in the extracts with the amount of phenolic sub- stance in the caffeic acid. The data obtained are presented in Figure 1. It was determined that the total amount of phenolic substance was the least in the aqueous extract ob- tained from the flower part of the plant. The extracts of the stem (ST-stem), leaf (ST-leaf), flower (ST-flower) and aerial parts of the plant (stem-leaf-flower; ST-mix) have high total phenolic content close to caffeic acid were deter- mined. These data show that especially the stem and leaf of the plant are rich in phenolic content. The amount of phe- nolic acid in ST-flower was found to be statistically signif- icantly lower (p < 0.05) than the others. Figure 1. The total amount of phenolic substances detected in the extracts and the standard. ST-Stem; Aqueous extract of stem of Sal- via tomentosa, ST-Flower; Aqueous extract of flower of Salvia to- mentosa, ST-Leaf; Aqueous extract of leaf of Salvia tomentosa, ST- Mix; Aqueous extract of a mixture of stem, leaf and flower of Salvia tomentosa. (a, b) The difference between the means labelled with different letters is statistically significant (p < 0.05). LC-MS/MS system was used to determine the phyto- chemicals contained in Salvia tomentosa. The components 221Acta Chim. Slov. 2023, 70, 218–225 Balkır et al.: Phytochemical Profile, Antioxidant and Antimicrobial ... of the aerial parts of the plant are shown separately in Ta- ble 1. When the amount of components in the extracts is examined in general, it can be said that especially three components are more intense than S. tomentosa extracts compared to other components. These components are rosmarinic acid, quinic acid and fumaric acid, respectively. When the extracts were analyzed separately, the presence and concentrations of 16 components in ST-stem extract, 15 components in ST-flower extract, and 17 components in ST-leaf and ST-mix extracts were determined. Hesperi- din, quinic acid, fumaric acid, aconitic acid, protocatechu- ic acid, gentisic acid, protocatechuic aldehyde, chlorogenic acid, caffeic acid, salicylic acid, apigenin, o-coumaric acid, rosmeric acid, cosmosiin, luteolin are chemicals found in all parts of the plant. Table 1. Types and amounts of some phytochemicals in Salvia to- mentosa extract Concentration (mg analyte/g extract) Plant parts ST-Stem ST-Flower ST-Leaf ST-Mix Hesperidin 5.507 0.081 0.718 2.060 Quinic acid 25.628 29.217 32.247 37.750 Fumaric aid 10.888 0.923 7.592 11.155 Aconitic acid 0.525 0.677 0.373 0.391 Protocatechuic acid 0.851 1.829 0.694 0.568 Gentisic acid 0.231 0.546 0.209 0.168 Protocatechuic 0.883 0.712 1.046 0.844 aldehyde Chlorogenic acid 1.632 1.242 1.429 1.757 Caffeic acid 2.247 0.560 2.245 2.339 Salicylic acid 1.596 2.102 1.473 0.799 Apigenin 0.003 0.006 0.057 0.035 o-Coumaric acid 0.042 0.037 0.056 0.032 Rosmarinic acid 50.235 61.590 89.892 83.625 Cosmosiin 0.077 0.931 0.793 0.560 Luteolin 0.017 0.035 0.348 0.210 Hesperetin 0.071 ND 0.025 0.077 Naringenin ND ND 0.011 0.011 ND: Not detected. ST-Stem; Aqueous extract of stem of Salvia to- mentosa, ST-Flower; Aqueous extract of flower of Salvia tomentosa, ST-Leaf; Aqueous extract of leaf of Salvia tomentosa, ST-Mix; Aque- ous extract of a mixture of stem, leaf and flower of Salvia tomentosa It was determined that the major component deter- mined in all of the extracts obtained from S. tomentosa was rosmarinic acid. The amount of rosmarinic acid is quite high when compared to other ingredients. One of the commonly used methods to determine the antiradical activity of a sample is the method using the DPPH radical. In this method, sample (extract) at certain concentrations is added to the DPPH radical dissolved at a certain concentration in a suitable solvent. The percenta- ges of inhibition of DPPH radical by using the solutions of extracts and standards (BHT and Vitamin C) of S. tomen- tosa obtained at the same concentrations are presented in Figure 2. When the data are examined, it is seen that the ST-leaf extract is statistically significantly (p < 0.05) lower than all other extracts. It can be mentioned that the other extracts (ST-stem, ST-flower and ST-mix) have close radi- cal scavenging effects, such as BHT and Vitamin C, which are antioxidants used for comparison. Figure 2. DPPH radical inhibition ratios of the extracts. ST-Stem; Aqueous extract of stem of Salvia tomentosa, ST-Flower; Aqueous extract of flower of Salvia tomentosa, ST-Leaf; Aqueous extract of leaf of Salvia tomentosa, ST-Mix; Aqueous extract of a mixture of stem, leaf and flower of Salvia tomentosa. BHT; Butylated Hydroxy Toluene. (a,b) The difference between the means labelled with dif- ferent letters is statistically significant (p < 0.05). Spectrophotometric method were used to determine the antioxidant and oxidant capacity of extracts of S. to- mentosa. Oxidative stress indexes of the samples were de- termined using these antioxidant and oxidative capacity datas. It was evaluated that the antioxidant activity of the extracts with low oxidative stress index data was higher. Antioxidant and oxidant capacity, oxidative stress indexes values were shown in Figure 3. As a result of the analysis, it was determined that the extract with the highest antioxidant capacity among the extracts of S. tomentosa belonged to ST-stem and ST-mix. TAS levels of ST-flower and ST-leaf extract were found to be statistically significantly (p < 0.05) lower than other extracts and Vitamin C. When TOS levels were examined, it was determined that the TOS level of ST-stem extract was the lowest, while the TOS level of ST-flower extract was the highest. OSI values obtained by dividing the TOS levels of the extracts of S. tomentosa by the TAS levels, exp- ress the capacity of a plant to create oxidative stress in the organism from which it is taken. The OSI values of the ST- stem extract, which were determined to have the highest TAS levels and the lowest TOS levels, were found to be the lowest. It was observed that the extract with the highest OSI value was ST-leaf. When the analyzes made on behalf of the antioxi- dant activity of the extracts were examined in general, the phenolic substance amounts, DPPH scavenging effects and TAS levels of the ST-stem and ST-mix extracts of the 222 Acta Chim. Slov. 2023, 70, 218–225 Balkır et al.: Phytochemical Profile, Antioxidant and Antimicrobial ... plant were found to be higher than the other leaf and flow- er extracts. The antimicrobial activity of extracts of S. tomentosa was determined by microdilution method and given in Table 2. When the data were examined, it was found that the lowest 1/16 dilution of ST-flower extract was effective on Escherichia coli O157:H7, Staphylococcus aureus and Candida albicans; The lowest 1/8 dilution of ST-leaf ex- tracts was effective on Listeria monocytogenes, Salmonella typhimurium and Candida albicans; The lowest 1/16 dilu- tion of ST-Stem extracts was effective on all agents (L. monocytogenes, S. typhimurium, S. aureus, C. albicans) except E. coli O157:H7; All dilutions of ST-mix extract were found to be effective on L. monocytogene and C. albi- cans. Based on the data obtained from Table 2, consider- ing the doses corresponding to each dilution ratio, the MIC values of the extracts are presented in Table 3. It was determined that ST-mix was the most effective inhibitory extract for all microorganisms used. This data shows that Table 2. Antimicrobial activity of Salvia tomentosa extracts Extract Dilution Rate E. coli L. monocytogenes S typhimurium S. aureus C. albicans ST-Flower 1/2 + + + + + 1/4 + + + + + 1/8 + + + + + 1/16 + – – + + 1/32  – – – – – 1/64 – – – – – ST-Leaf 1/2 + + + – + 1/4 + + + – + 1/8 – + + – + 1/16 – – – – + 1/32  – – – – – 1/64 – – – – – ST-Stem 1/2 + + + + + 1/4 + + + + + 1/8 + + + + + 1/16 – + + + + 1/32  – – – – – 1/64 – – – – – ST-Mix 1/2 + + + + + 1/4 + + + + + 1/8 + + + + + 1/16 + + + – + 1/32  – + – – + 1/64 – + – – + ST-Stem; Aqueous extract of stem of Salvia tomentosa, ST-Flower; Aqueous extract of flower of Salvia tomentosa, ST-Leaf; Aqueous extract of leaf of Salvia tomentosa, ST-Mix; Aqueous extract of a mixture of stem, leaf and flow- er of Salvia tomentosa Figure 3. A: Total antioxidant capacities of extracts of Salvia tomentosa. B: Total oxidant capacities of extracts of Salvia tomentosa (a, b, c) The diffe- rence between the means labelled with different letters is statistically significant (p < 0.05) 223Acta Chim. Slov. 2023, 70, 218–225 Balkır et al.: Phytochemical Profile, Antioxidant and Antimicrobial ... the use of the aerial part of the plant together is more ben- eficial in terms of providing antimicrobial activity. Table 3. MIC values of extracts Microorganisms ST-Flower ST-Leaf ST-Stem ST-Mix E. coli 3.75 15 7.5 1.875 L. monocytogenes 7.5 7.5 3.75 0.9375 S. typhimurium 7.5 7.5 3.75 1.875 S. aureus 3.75 – 3.75 7.5 C. albicans 3.75 3.75 3.75 0.9375 ST-Stem; Aqueous extract of stem of Salvia tomentosa, ST-Flower; Aqueous extract of flower of Salvia tomentosa, ST-Leaf; Aqueous extract of leaf of Salvia tomentosa, ST-Mix; Aqueous extract of a mixture of stem, leaf and flower of Salvia tomentosa. MIC; Mini- mum concentration at which microorganism growth is inhibited 4. Discussion Metabolites consist of intermediate products formed by the effect of metabolic activities. Metabolites have functions such as energy generation, building blocks, stimulating enzymes and additionally inhibiting them, being under the influence of catalyst, defense and other organisms, giving odor. Primary metabolites are directly involved in processes involved in normal growth, devel- opment and reproduction. Although secondary metabo- lite species are not directly related to these processes, they contribute to the maintenance of these processes by the plant. Secondary metabolites may contribute to the adap- tation of the species to its specific conditions. These com- pounds, which enable plants to have their unique color, taste, aroma and texture, also play an important role in many metabolic events that occur in the plant.21, 22 In ad- dition to contributing to the resistance mechanism in the body at the time of disease in plants, they are produced as a defense mechanism for plants and increases as stress in- creases. Phenolic compounds are synthesized during the normal development of plants and when the plant is sick and injured. In addition, phenolic production is depend- ent on environmental conditions and are synthesized when exposed to UV rays, at low temperatures and during periods when nitrogen, phosphate and iron content are low.21, 22 In a study, the phenolic composition and antioxidant properties of wild and cultured S. tomentosa were investi- gated. Total phenolics of S. tomentosa were found between 49.27 and 66.15 mg GAE/dw. The total phenolic content of the grown samples was determined to be higher than that of the wild samples. 17 different phenolic compounds con- taining 7 phenolic acids and 10 flavonoids were identified and quantified in S. tomentosa. Rosmarinic acid was meas- ured as the main component of S. tomentosa. It is followed by caffeic acid, morin, p-coumaric acid and myricetin.23 In the present study, the phenolic acid amounts of plant parts ranged from 386.48 to 599.24 mg GAE/g extract. Identi- fied and quantified phenolic substances are respectively, rosmarinic acid (50.235 mg/g extract), quinic acid (25.628 mg/g extract), fumaric acid (10.888 mg/g extract), hes- peridin (5.507 mg/g extract), caffeic acid (2.247 mg/g ex- tract), chlorogenic acid (1.632 mg/g extract) and salicylic acid (1.596 mg/g extract). Rosmarinic acid is one of the most abundant phenol- ic substances in all S. tomentosa extracts. In many studies, it is stated that rosmarinic acid has antiviral, antibacterial, anti-inflammatory and antioxidant effects.24 In a study, bi- otechnological production of rosmarinic acid, which is found in high amounts in Salvia officinalis, has been sug- gested.25 A high amount of rosmarinic acid is also expect- ed in S. tomentosa, another species from the Salvia family. It can be said that rosmarinic acid, the major component of S. tomentosa, has an important role in the formation of antioxidant and antimicrobial activity of the extracts. Fumaric acid and its derivatives are among the well- known antioxidants that provide various health benefits due to their potent free radical scavenging properties, an- ti-inflammatory and immunomodulatory effects.26,27 These data in the literature may be related to the wound healing activity of Salvia tomentosa due to its high fumaric acid content. When the analysis made on behalf of the antioxidant activity of the extracts are examined in general, the phe- nolic substance amounts, DPPH scavenging effects and TAS levels of the ST-stem and ST-mix extracts of the plant were found to be higher than the other ST-leaf and ST-flower extracts. It is seen that the antioxidant active substance levels of hesperidin and fumaric acid in ST-stem extract, and hesperidin, fumaric acid and rosmarinic acid in ST-mix extract are higher than other extracts. Among these components, besides the antioxidant activities of fu- maric acid and hesperidin, antimicrobial activities are prominent in the literatüre.28–30 In addition, the antioxidative properties of ST-flow- er extract (except for DPPH radical scavenging activity) were found to be lower than other extracts, while TOS levels were found to be high. This may be due to the fact that the levels of hesperidin, fumaric acid and caffeic acid in the flower extract are lower than in other extracts. While the antioxidative property of ST-flower extract was low, DPPH radical scavenging activity was found to be high. Some antioxidants act by preventing the formation of free radicals, while other antioxidants act by scaveng- ing existing radicals. Therefore, there may be more com- ponents in the flower extract that increase the radical scavenging activity of the extracts. It is seen that the amounts of some components (aconitic acid, protocate- chuic acid, gentisic acid, salicylic acid and cosmosiin) are higher in the flower extract. The second component, which was determined to be higher in ST-stem and ST-mix extracts compared to other extracts, is hesperidin. Hesperidin, which has a 224 Acta Chim. Slov. 2023, 70, 218–225 Balkır et al.: Phytochemical Profile, Antioxidant and Antimicrobial ... flavonoid structure, protects the organism against oxi- dants with its strong antioxidant property in biological systems31, and also protects the body against infections with its antiviral and antibacterial activity.32 Recent studies on wound healing show that hesperidin is very effective in healing wounds that develop due to various diseases or that may occur in daily life25. For thousands of years, extract of St. John’s Wort (Hypericum perfora- tum) flour obtained with olive oil have been used tradi- tionally in the treatment of burns and open wounds and in the removal of their scars. Considering that one of the major components of St. John’s wort is hesperidin33, the effectiveness of hesperidin can be better understood in terms of wound healing activity. From this point, it is expected that the extract of Salvia tomentosa, which contain plenty of hesperidin, prepared with water, have antimicrobial effects. It was determined that the ST-mix extract showed stronger antioxidant and antimicrobial activity than those obtained from parts of the plant. However, it was deter- mined that the components of the ST-flower, ST-stem and ST-leaf extracts and the components of the ST-mix extract were largely similar. On the other hand, it was observed that there were significant differences in the proportion of the same type of components. This probably caused the different antimicrobial and antioxidant activities of each extract in the components they contained in different pro- portions. 5. Conclusions The phenolic and flavonoid contents of the aqueous extracts of the ST-flower, ST-stem, ST-leaf and ST-mix parts of S. tomentosa were determined qualitatively and quantitatively. The phenolic content of the fractions was found to be quite high except for ST-flower. Rosmarinic acid, quinic acid and fumaric acid are phenolic substances found in high amounts in all parts of the plant. Total anti- oxidant levels of ST-stem and ST-mix are quite high. It has been determined that plant parts are very effective radical scavengers expect for ST-leaf. In addition, it was deter- mined that the most effective extracts from S. tomentosa in terms of antibacterial activities were ST-stem and ST-mix. It has been interpreted that the antimicrobial and antioxi- dant activities between the extracts may be due to the component differences in their contents. It is thought that the obtained data will contribute to the literature in order to explain the phytotherapeutic activity of Salvia tomento- sa Miller. Acknowledgements This work is supported by the Scientific Research Project Fund of Afyon Kocatepe University (AKU-BAP) under the Project number 19.FEN.BİL.36. 6. References 1. V. Papageorgiou, C. Gardeli, A. Mallouchos, M. Papaioan- nou, M. Komaitis, J. Agr. Food Chem. 2008, 56, 7254–7264. DOI:10.1021/jf800802t 2. M.Z. Haznedaroglu, N.U. Karabay, U. Fitoterapia 2001, 72, 829–831. DOI:10.1016/S0367-326X(01)00335-5 3. Y. Lu, L. Foo, Phytochemistry 2002, 59, 117–140. DOI:10.1016/S0031-9422(01)00415-0 4. N. Tan,  S.T. Yazıcı,  Y.C. Yeşil,  B. Demirci,  Tan E. Rec. Nat. Prod. 2017, 11, 456–461. DOI:10.25135/rnp.57.16.12.086 5. M. Jakovljević, S. Jokić, M. Molnar, M. Jašić, J. Babić, H. Jukić, I. Banjari, Plants 2019, 8, 1–30. DOI:10.3390/plants8030055 6. E. Hanlidou, R. Karousou, D. Lazari, Chem. Biodivers. 2014, 11, 1205–1215. DOI:10.1002/cbdv.201300408 7. K.H.C. Başer, Pure Appl. Chem. 2002, 74, 527–545. DOI:10.1351/pac200274040527 8. T. Aşkun, K.H.C. Başer, G. Tümen, M. Kürkçüoğlu, Turk. J. Biol. 2010, 34, 89–95. DOI:10.3906/biy-0809-2 9. G. Ozek, T. Ozek, K.H.C. Baser, E. Hamzaoglu, A. Duran, Chem. Nat. Compd. 2007. 43, 667–671. DOI:10.1007/s10600-007-0224-9 10. Z. Dumanoğlu, S. Mokhtarzadeh. TURKJANS 2020, 7, 596– 602. DOI:10.30910/turkjans.663891 11. S. Mokhtarzadeh, B. Demirci, H.G. Ağalar, K.M. Khawar, N. Kırımer, Rec. Nat. Prod. 2019, 13, 121–128. DOI:10.25135/rnp.86.18.04.105 12. I. Erdogan-Orhan, E. Baki, S. Senol, G. Yilmaz, J. Serb. Chem. Soc. 2010, 75, 1491–1501. DOI:10.2298/JSC100322115E 13. M.Z. Haznedaroglu, N.U. Karabay, U. Zeybek. Fitoterapia 2001, 72, 829–831. DOI:10.1016/S0367-326X(01)00335-5 14. B. Tepe, D. Daferera, A. Sokmen, M. Sokmen, M. Polissiou, Food Chem. 2005, 90, 333–340. DOI:10.1016/j.foodchem.2003.09.013 15. Ö. Hazman, L. Aksoy, A. Büyükben, R. Kara, M. Kargıoğlu, İ.H. Cigerci, M.A. Yılmaz, Indian J. Exp. Biol. 2022, 60, 743– 752. DOI:10.56042/ijeb.v60i10.55070 16. K. Slinkard, V.L. Singleton, Am. J. Enol. Vitic. 1977, 28, 49–55. DOI:10.5344/ajev.1974.28.1.49 17. M.A. Yılmaz, Ind. Crops Prod. 2020, 149, 112347. DOI:10.1016/j.indcrop.2020.112347 18. L. Aksoy, İ. Güzey, M. Düz, Turk. J. Pharm. Sci. 2022, 19, 76–83. DOI:10.4274/tjps.galenos.2021.86422 19. S.S. Shaikh, A.S. Bawazir, B.A. Yahya, Turk. J. Pharm. Sci. 2022, 19, 145–152. DOI:10.4274/tjps.galenos.2021.02058 20. B. Tepe, D. Daferera, A. Sokmen, M. Sokmen, M. Polissiou, Food Chem. 2005, 90,333–340. DOI:10.1016/j.foodchem.2003.09.013 21. S. Neugart, S. Baldermann, F.S. Hanschen, R. Klopsch, M. Wiesner-Reinhold, M. Schreiner, Scient. Hortic. 2018, 233, 460–478. DOI:10.1016/j.scienta.2017.12.038 22. M. Wink, Nat. Prod. Commun. 2008, 3, 1205–1216. DOI:10.1177/1934578X0800300801 23. C. Dinçer, I. Tontul, I. B. Çam, K.S. Özdemir, A. Topuz, H.Ş. Nadeem, S.T. Ay, R. S. Göktürk. Turk. J. Agric. For. 2013, 37, 561–567. DOI:10.3906/tar-1211-72 225Acta Chim. Slov. 2023, 70, 218–225 Balkır et al.: Phytochemical Profile, Antioxidant and Antimicrobial ... 24. H. Guan, W. Luo, B. Bao, Y. Cao, F. Cheng, S. Yu, Q. Fan, L. Zhang, Q. Wu, M. Shan. Molecules 2022, 27, 3292. DOI:10.3390/molecules27103292 25. M. Petersen, M.S. Simmonds, Phytochemistry 2003, 62, 121– 125. DOI:10.1016/S0031-9422(02)00513-7 26. S.A. Scuderi, A. Ardizzone, I. Paterniti, E. Esposito, M. Cam- polo. Antioxidants 2020, 9, 630. DOI:10.3390/antiox9070630 27. A. Shakya, G.K. Singh, S.S. Chatterjee, V. Kumar, J. Intercult. Ethnopharmacol. 2014, 3, 173–178. DOI:10.5455/jice.20140912021115 28. U. Wollina, Indian Dermatol. Online J. 2011, 2, 111. DOI:10.4103/2229-5178.86007 29. M. Vabeiryureilai, K. Lalrinzuali, G.C. Jagetia, Burns. 2022, 48, 132–145. DOI:10.1016/j.burns.2021.04.016 30. W. Li, A.D. Kandhare, A.A. Mukherjee, S.L. Bodhankar, Exc- li. J. 2018, 17, 399–419. DOI: 10.17179/excli2018-1036 31. P.K. Wilmsen, D.S. Spada, M. Salvador, J. Agric. Food Chem. 2005, 53, 4757–4761. DOI:10.1021/jf0502000 32. H.W. Kim, H.J. Woo, J.Y. Yang, J.B. Kim, S.H. Kim, Int. J. Mol. Sci. 2021, 17, 10035. DOI:10.3390/ijms221810035 33. C. Sarikurkcu, M. Locatelli, A. Tartaglia, V. Ferrone, A.M. Juszczak, M.S. Ozer, B. Tepe, M. Tomczyk, Molecules 2020, 6, 1202. DOI:10.3390/molecules25051202 Except when otherwise noted, articles in this journal are published under the terms and conditions of the  Creative Commons Attribution 4.0 International License Povzetek V tej študiji smo kvalitativno in kvantitativno določili antioksidativno aktivnost, protimikrobno učinkovitost in sestavine nadzemnih delov (listov, stebla, cvetov in mešanice) rastline Salvia tomentosa Miller. Vodne izvlečke S. tomentosa (ST) smo pripravili z uporabo cvetov, listov in stebel ter vseh nadzemnih delov rastline (mešanica cvetov, listov in steblo). Določili smo aktivnost odstranjevanja radikalov, skupni antioksidantivni/oksidativni status, protimikrobni potencial, fenolne snovi in kvalitativne/kvantitativne analize sestavin v ekstraktih. Ugotovljeno je bilo, da je količina fenolnih kislin v ST-steblu (599 ± 34 mg ekvivalenta galne kisline (GAE)/g ekstrakta) blizu količini standardne snovi kofeinske kisline (651 ± 31 mg GAE/g ekstrakta). Skupni antioksidativni statusi mešanice ST (3,4 ± 0,1 mmol ekvivalenta troloxa /L), stebla ST (3,4 ± 0,1 mmol ekvivalenta troloxa /L) in naravnega antioksidanta vitamina C (3,6 ± 0,1 mmol ekvivalenta troloxa /L) se statistično niso razlikovali. Izvleček, pridobljen z uporabo nadzemnih delov S. tomentosa (cvet-steblo-listi), je pokazal močnejše antioksidativno in protimikrobno delovanje kot vodni izvlečki, pridobljeni ločeno iz cvetov, stebla in listov rastline. Ugotovljeno je bilo, da so si sestavine ločeno pripravljenih izvlečkov cvetov, stebla in listov ter sestavine izvlečka, pridobljenega iz nadzemnih delov, v veliki meri podobne. Hkrati pa je bilo ugotovljeno, da obstajajo pomembne razlike v prisotnosti teh sestavin. 226 Acta Chim. Slov. 2023, 70, 226–230 Zinchuk and Biletskaya: Effect of Ozone on Oxygen Transport and Pro-Oxidant-Antioxidant ... DOI: 10.17344/acsi.2023.8032 Scientific paper Effect of Ozone on Oxygen Transport and Pro-Oxidant- Antioxidant Balance of Red Blood Cell Suspension Victor Zinchuk* and Elena Biletskaya* Grodno State Medical University, Department of Normal Physiology, 230009, Gorkogo str, 80, Grodno, Belarus * Corresponding author: E-mail: zinchuk@grsmu.by; Tel.: +37529-7859027 biletskaya.e@inbox.ru; Tel.: +37529-2689339 Received: 01-31-2023 Abstract Introduction: Ozone affects blood oxygen transport and the pro-oxidant-antioxidant balance. However, the role of blood formed elements and gas transmitters in these processes still remains unclear. The aim of the present study was to investigate the effect of ozone on oxygen transport and the pro-oxidant-antioxidant balance in a red cell suspension. Methods: The red cell suspension was incubated with ozone at a concentration of 6 mg/l and substances affecting the synthesis of gas transmitters (nitroglycerin, sodium hydrosulfide). Parameters of blood oxygen transport and pro-oxi- dant-antioxidant balance were determined. Results: The effect of ozone on blood oxygen transport was found which was manifested in an increase in oxygen partial pressure and the degree of oxygenation. The index of hemoglobin –oxygen affinity p50 actual was raised and a shift of the oxyhemoglobin dissociation curve rightwards was noticed. The addition of the gas transmitter donor, nitric oxide, en- hanced the effect of this gas on the parameters of oxygen transport in the erythrocyte suspension Conclusion: We found that ozone induced a change in oxygen-binding properties of the erythrocyte suspension which is a mechanism of action of the above gas on the adaptive processes in the body realized directly at the level of erythrocytes. Keywords: Ozone, red blood cells, gas transmitter, hemoglobin oxygen affinity, nitric oxide, hydrogen sulfide 1. Introduction Ozone (O3) exerts various physiologic effects on the organism: increases the rate of erythrocyte glycolysis, im- proving oxygen delivery to tissues, and activates the enzy- matic link of the antioxidant system (glutathione, peroxi- dase, catalase and superoxide dismutase).1 Our earlier studies demonstrated the effect of O3 on blood oxygen transport, which was manifested by a distinct shift of the oxyhemoglobin dissociation curve (ODC) rightwards and elevated concentrations of the gas transmitters hydrogen sulfide (H2S) and nitric oxide (NO).2 An effect of ozone on hemoglobin – oxygen affinity (HOA) due to activation of the gas transmitter system is suggested.3 Red blood cells possess their own mechanisms of NO synthesis and can serve as an essential source of NO under hypoxia.4 Moreo- ver, erythrocytes were shown to contain 3-mercaptopyru- vate sulfotransferase, contributing to H2S production.5 However, NO-synthase activity is not only inherent to red blood cells; it is also a characteristic of leukocytes and thrombocytes. Thrombocytes were demonstrated to contain two isoforms of NO-synthase (inducible and en- dothelial), and the application of flow cytometry allowed Mahaj et al. to detect inducible NO-synthase in leuko- cytes.6,7 Therefore, it was necessary to investigate the abili- ty of red blood cells to respond to the action of ozone. The purpose of this work was to study the effect of ozone on oxygen transport in a red blood cell suspension. 2. Materials and Methods 2. 1. Materials and Study Design Venous blood was drawn with a heparin-pretreated syringe (50 U/ml). This study was conducted, using a suspension of red cells from the blood of albino Sprague Dawley male rats fed a standard laboratory diet. The experimental protocol was approved by the Ethical Committee of Grodno State University (approval No1 of January 14, 2019). To separate blood plasma and erythrocytes, blood samples were centrifuged at 3000 r.p.m. over 10 min and washed twice with a cold isotonic solution. Thereafter the 227Acta Chim. Slov. 2023, 70, 226–230 Zinchuk and Biletskaya: Effect of Ozone on Oxygen Transport and Pro-Oxidant-Antioxidant ... obtained red blood cell suspension was divided into 4 groups of samples of ten 1.2 ml samples in each group (the hematocrit level was 40%) (Figure 1). An isotonic solution of sodium chloride was enriched with an ozone-oxygen mixture for 4–5 minutes using an ozone generator UOTA-60-01 (Medozon, Russia), which makes it possible to measure ozone concentrations by an optical method in the ultraviolet range, which is provided by the technical capabilities of the device. Figure 1: Study design Group 1 samples (control) contained the erythrocyte suspension (1.2 ml) + isotonic solution of sodium chloride (1.1 ml). The contents of Group 2 samples were as follows: the red blood cell suspension (1.2 ml) + 1 ml of the ozonized isotonic solution of sodium chloride (O3 concen- tration was 6 mg/l) + 0.1 ml of the isotonic solution of so- dium chloride. The samples of Group 3 were composed of the red blood cell suspension (1.2 ml) + 1 ml of the ozonized isotonic solution of sodium chloride (O3 concen- tration was 6 mg/l) + 1 ml of the solution containing the gas transmitter nitroglycerin (SchwarzPharma AG) at the final concentration of 0.05 mmol/l. Group 4 samples con- tained the red blood cell suspension (1.2 ml) + 1 ml of the ozonized isotonic solution of sodium chloride (O3 concen- tration was 6 mg/l) + 0.1 ml of the solution containing the gas transmitter sodium hydrosulfide (Sigma-Aldrich) at the final concentration of 0.38 mmol/l. The contents of each sample were mixed. The incubation time was 60 min. Thus, we had control without ozonation (Group 1), the ozonized red blood cell suspension (Group 2) and the red blood cell- and gas-transmitter-containing suspensions (Groups 3 and 4). 2. 2. Methods 2. 2. 1. Blood Oxygen Transport (Hemoglobin- Oxygen Affinity) Parameters of blood oxygen transport and acid-base status were measured using a Stat-Profile pHOx plus L gas analyzer at 37ºC. Partial pressures of oxygen (pO2) and carbon dioxide (pCO2), the degree of oxygenation (SO2), standard bicarbonate (SBC), actual base excess/standard base excess (ABE/SBE), hydrogen carbonate (HCO3–), pH and total plasma carbonic acid (TCO2) were determined. Hemoglobin-oxygen affinity was assessed spectrophoto- metrically by p50actual (pO2 corresponding to 50% Hb sat- uration with oxygen). The Severinghaus formulas were used to calculate p50standard and ODC position.8 2. 2. 2 Pro-Oxidant/Antioxidant System The activity of free radical processes was evaluated by the contents of primary (diene conjugates, DC) and inter- mediate (malondialdehyde, MDA) products of lipid perox- idation (LPO) in the red blood cell suspension. The level of DC was measured spectrofluorimetrically (an SM 2203 spectrofluorimeter, SOLAR) by a method based on the in- tensity of absorption of diene structures of lipid hydroper- oxides at 233 nm, in comparison with the blank samples in which the biological material was substituted by distilled water.9 The DC content was expressed as U/ml. The con- centration of MDA (TBARS) was assessed by the interac- tion with 2’-thiobarbituric acid (TBA) which, when heated in acidic medium, causes the formation of a pink trime- thine complex.10 The intensity of color was determined spectrophotometrically at a wavelength of 55 nm with a PV12 51 SOLAR spectrophotometer and compared to con- trol. The MDA concentration was expressed as µmol/l. To determine catalase activity in hemolysates, we used the method of Koroliuk based on spectrophotometri- cal recording of the amount of the colored product of the reaction of H2O2 with ammonium molybdate having a maximum absorption at a wavelength of 410 nm.11 The ac- tivity of catalase was expressed as mmol H2O2/min/g Hb. The amount of the enzyme catalyzing the formation of 1 mmol of the product per 1 min under the experimental conditions was taken as the unit of activity. 2. 2. 3. Statistical Analysis The correspondence of the study data to the normal distribution law was tested by the Shapiro-Wilk test. With consideration for this criterion, non-parametric statistics with application of Statistica 10.0 software (StatSoft Inc., Tulsa, Oklahoma, USA) was used. Three and more inde- pendent groups were compared by the Kruskal-Wallis one-way analysis-of-variance-by ranks test. Allowing for the small sample and multiple comparisons, the signifi- cance of the data obtained was evaluated using the Mann-Whitney U-test. The Wilcoxon signed ranks test was applied for paired in-group comparisons of the indices levels using repeated measures ANOVA. The results were presented as a median (Me), the interquartile range be- tween the 25th and the 75th percentile. The data were con- sidered significant at the level of P < 0.05. 228 Acta Chim. Slov. 2023, 70, 226–230 Zinchuk and Biletskaya: Effect of Ozone on Oxygen Transport and Pro-Oxidant-Antioxidant ... 3. Results and Discussion The O3 treatment of the red blood cell suspension resulted in an increase in the fundamental indices of blood oxygen transport: SO2 by 121.8 % (P < 0.05), pO2 by 74.1% (P < 0.05) (Table 1). Under these conditions, the value of HOA parameter p50actual was increased by 21.4% (P < 0.05) and the ODC was shifted rightwards (Figure 2) com- pared to the control group. The value of p 50standard was al- so observed to rise. No significant changes were found when analyzing the acid-base balance parameters. Nitro- glycerin enhanced the effect of ozone on the oxygen trans- port in the red blood cell suspension. The values of SO2 and pO2 increased by 12.5% and 21.0 (P < 0.05), respec- tively, in comparison with the samples pretreated with the ozonized isotonic solution of sodium chloride. Under these conditions, the HOA parameter p50actual increased by 7.5% (P < 0.05) and the ODC was shifted rightwards more distinctly (Figure 2). The H2S donor (sodium hydro- sulfide) did not exert a similar effect. The treatment of the red blood cell suspension with the ozonized saline solution caused an 85.3% elevation of the MDA content in the red blood cell suspension and the concentration of DC was increased by 77.4% (P < 0.05) in comparison with the control group (Table 2), whereas cat- alase activity was decreased by 44.5% (P < 0.05). The addi- tion of the gas transmitter donors, nitroglycerine and sodi- um hydrosulfide, did not cause any changes in the LPO indices. However, it raised catalase activity by 46.1% (P < 0.05) and 43.8% (P <0 .05), respectively compared to the group of samples containing the ozone –treated red blood cell suspension. In comparison with other blood formed elements, red blood cells are an important target for the action of ozone. Due to the hexose monophosphate shunt, ozone promotes activation of 2,3-diphosphoglycerate mutase (DPGM), which finally results in conversion of erythro- cyte1,3-DPGM to 2,3-DPGM that, binding to the hemo- globin β-chain, may bring about an ODC shift right- Table 1: Effect of ozone on oxygen transport in red blood cell suspension (mеdian [25th; 75th percentile]) Parameter Control Red blood cell suspension Red blood cell suspension Red blood cell suspension + (n = 10) + ozone (n = 10) + ozone + NO (n = 10) ozone + H2S (n = 10) SO2, % 26.96 59.80 67.30 58.10 [26.00; 32.10] [56.30; 62.20]* [62.70; 67.70]*# [57.90; 58.70]*Ψ pO2, mmHg. 18.15 31.60 38.25 31.05 [17.40; 19,60] [30.10; 34.50]* [37.40; 39.20]*# [28.70; 31.30]*Ψ рН, units 7,305 7.331 7.313 7.318 [7.287; 7.356] [7.321; 7.352] [7.293; 7.315]# [7.312;7.353] pСO2, mmHg. 5.25 4.70 6.45 6.45 [4.70; 5.90] [4.50; 5.10] [4.30; 8.70] [5.20; 8.20]# НСO3–, mmol/L 2.70 2.45 3.25 3.30 [2.30; 3.30] [2.30; 2.70] [2.30; 4.20] [2.80;4.18]# ТСO2, mmol/L 2.85 2.60 3.5 3.45 [2.40; 5.90] [2.40; 2.90] [2.40; 4.50] [2.83;4.50]# ABE, mmol/L –23.60 –23.65 –23.30 –23.20 [–24.60; –20,40] [–23.80;–23.60] [–24.10; –22.40] [–23.48;–22.30]# SBE, mmol/L –19.90 –20.60 –20.05 –20.80 [–21.00; –17.10] [–20.70; –20.30] [–20.30; –19.50]# [–21.15; –20.53]Ψ SBC, mmol/L 9.00 8.60 8.85 8.75 [8.50; 10.80] [8.60; 8.90] [8.40; 9.40] [8.40;9.38] р50 actual, mmHg 22.96 27.88 29.99 27.55 [22.40; 23.97] [27.49; 27.92]* [29.79; 31.13]*# [27.49; 27.85]*Ψ р50standard, mmHg 20.63 25.40 28.35 23.40 [20.20; 21.60] [23.00; 26.90]* [27.40; 29.00]*# [23.40; 23.60]*Ψ Note: changes compared to control (*), red blood cell suspension + ozone (#), Red blood cell suspension + ozone + NO (Ψ). Figure 2: Effect of ozone on the position of the oxyhemoglobin dis- sociation curve at real pH and рСО2 values. ■ – control;  – red blood cell suspension + ozone; ◆ – red blood cell suspension +ozone +NO. pO2, mmHg 229Acta Chim. Slov. 2023, 70, 226–230 Zinchuk and Biletskaya: Effect of Ozone on Oxygen Transport and Pro-Oxidant-Antioxidant ... wards.12 We believe that in addition to the above mechanism, other mechanisms can be involved in this process, in particular those mediated through gas trans- mitters. Red blood cells contain the constitutive isoform of NO synthase, which produces NO.13 Blood plasma shows nitrites/nitrates, but during inhibition of erythrocyte NO synthase, their concentration considerably decreases, which proves that red blood cell NO is exported via anion exchanger 1 in the form of secondary nitrogen species to which hemoglobin is unlikely to bind, thus providing for a free NO pool.14 Our findings show that the addition of the exogenous donor nitric oxide (nitroglycerin) increases the effect of O3 on blood oxygen transport in the red blood cell suspension. However, the hydrogen sulfide donor (sodium hydrosulfide) does not have this effect. It is known that NO is capable of changing HOA.15 Its release from red blood cells is controlled by the blood pO2 level, whereas the O3 treatment promotes an increase in this parameter.16 Ac- cording to our findings, red blood cells directly respond to the effect of ozone, and this response is manifested with involvement of the gas transmitters, independently of leu- kocytes and thrombocytes. Exposure of blood to O3 leads to production of reac- tive oxygen species, inducing activation of lipid peroxida- tion in cell membranes and the development of oxidative stress.17 Continuous exposure of red blood cells to the multitude of different oxidants contributes to the forma- tion of their potent intracellular antioxidant defense sys- tem, with gas transmitter mechanisms occupying a special place in the hierarchy of these processes.18 Due to its oxi- dative activity, ozone stimulates the antioxidant system of erythrocyte defense and improves cell deformability.19 Re- active oxygen species are neutralized to give hydrogen per- oxide, which finally results in an increase in catalase activ- ity.20 However, in our studies, the activity of the enzyme decreased, thus providing evidence for an imbalance be- tween antioxidants and free radicals. In the membrane fraction of red blood cells, ozone, as a source of oxygen, reacts with NO to form the powerful oxidant peroxini- trite.21 Subsequent oxidation of methemoglobin by perox- initrite may induce synthesis of globin radicals, which en- hance pro-oxidant activity in red blood cells.22 In turn, NO and hydrogen sulfide are also capable of affecting the me- tabolism of antioxidants due to the gas transmitter en- zymes and their reaction products and this feature was noticed in the groups of samples with nitroglycerin and hydrogen sulfide which showed elevated catalase activity compared to the group containing the ozone-treated red blood cell suspension.23 Thus, we found ozone-induced changes in parame- ters of blood oxygen transport of the erythrocyte suspen- sion, which is one of the mechanisms of the action of ozone on adaptive processes in the body which are directly realized by red blood cells. 4. Conclusion 1. The exposure of the red blood cell suspension to ozone improved its oxygen transport indices: the in- crease in pO2 and SO2 as well as the ODC shift right- wards were found. 2. Although the addition of nitroglycerin enhanced the effect of ozone on the oxygen transport in the red blood cell suspension under the above experimental conditions (the distinct ODC shift rightwards), sodi- um hydrosulfide did not exert a similar effect. 3. Ozone induced an elevation of the DC and MDA con- centrations as well as a decrease of catalase activity. The gas transmitter donors did not enhance oxidative stress, but activated catalase. Acknowledgments Funding This study was financially supported by the National Anti-Doping Laboratory, Republic of Belarus, Draft State Programs for Scientific Research No. 30-24/549-21. Author contributions VZ designed, organized, and wrote the article; de- signed the outline; solved queries related to scientific pub- lications from the journals. EB performed Pubmed search- es, aided in writing, and critiqued the literature. All authors have read and approved the manuscript provided. Table 2. Effect of ozone on indices of pro-oxidant-antioxidant balance of red blood cell suspension (mеdian [25th; 75th percentile]) Parameter Control Red blood cell suspension Red blood cell suspension Red blood cell suspension + (n = 10) + ozone (n = 10) + ozone + NO (n = 10) ozone + H2S (n = 10) MDA, μmol /L 8.73 16.18 14.60 15.55 [7.63; 9.21] [11.84; 17.57]* [11.57; 17.84]* [14.73; 16.31]* DK, U/mL 16.62 29.48 28.48 28.50 [15.50; 18.23] [17.54; 34.87]* [25.29; 29.26]* [27.12; 29.89]* Catalase, mmol 13.53 7.51 10.97 10.80 H2O2/min/g Hb [12.19; 14.02] [6.18; 9.88]* [10.23; 11.47]*# [10.04; 11.60]*# Note: changes compared to control (*), red blood cell suspension + ozone (#). 230 Acta Chim. Slov. 2023, 70, 226–230 Zinchuk and Biletskaya: Effect of Ozone on Oxygen Transport and Pro-Oxidant-Antioxidant ... Institutional Review Board Statement The study was conducted according to the guidelines of the Declaration of Helsinki, and Ethical Committee of the Grodno State Medical University (approval No. 1) ap- proved the study on January 14, 2019. 5. References 1. K. Re, J. Gandhi, R. Liang, S. Patel, G. Joshi, N. L Smith, I. Reid, S. A. Khan, Med. Gas Res. 2023, 13, 1-6. DOI:10.4103/2045-9912.351890 2. V. V. Zinchuk, E. S. Biletskaya, Biophysics. 2020, 65, 779-783. DOI:10.1134/S0006350920050231 3. V. V. Zinchuk, D. D. Zhadko, Nitric Oxide 2019, 84, 45-49. DOI:10.1016/j.niox.2019.01.007 4. V. Kuhn, L. Diederich, T. C. S. Keller, C. M. Kramer, W. Lück- städt, C. Panknin, T. Suvorava, B. E. Isakson, M. Kelm, M. M. Cortese-Krott. Antioxid. Redox Signal. 2017, 26, 718-742. DOI:10.1089/ars.2016.6954 5. M. M. Cortese-Krott Red blood cells as a “central hub” for sulfide bioactivity: Scavenging, metabolism, transport, and cross-talk with nitric oxide. Antioxid. Redox Signal. 2020; 33, 1332-1349. DOI:10.1089/ars.2020.8171 6. E. Gkaliagkousi, J. Ritter, A. Ferro Platelet-derived nitric ox- ide signaling and regulation. Circ Res. 2007, 101, 654-662 DOI:10.1161/CIRCRESAHA.107.158410 7. S. Maharaj, K. D. Lu, S. Radom-Aizik, F. Zaldivar, F. Haddad, H.W. Shin, S.Y. Leu, E. Nussbaum, I. Randhawa, D. M. Coop- er. Nitric Oxide 2018, 72, 41-45. DOI:10.1016/j.niox.2017.11.002 8. J. W. Severinghaus, J Appl Physiol. 1966, 21, 1108-1116. DOI:10.1152/jappl.1966.21.3.1108 9. R. Mendes, C. Cardoso, C. Pestana, Food Chem. 2009, 112, 1038-1045. DOI:10.1016/j.foodchem.2008.06.052 10. A. T. Diplock, M. C. R. Symons, C. A. Rice-Evans Techniques in free radical research: (Laboratory techniques in biochem- istry and molecular biology, volume 22), Elsevier, Amster- dam, 1991. 11. M. A. Koroliuk, L. I. Ivanova, I. G. Maĭorova, V. E. Tokarev, Lab. Delo 1988, 1, 16-19. 12. V. Bocci, Free Radic. Res. 2012, 46, 1068-1075. DOI:10.3109/10715762.2012.693609 13. S. Kishimoto, T. Maruhashi, M. Kajikawa, S. Matsui, H. Hashimoto, Y. Takaeko, T. Harada, T. Yamaji, Y. Han, Y. Ki- hara, K. Chayama, C. Goto, F. M. Yusoff, A. Nakashima, Y. Higashi, Sci. Rep. 2020, 10, 11467-1-9. DOI:10.1038/s41598-020-68319-1 14. K. J. Richardson, L. Kuck, M. J. Simmonds, Am. J. Physiol. Heart Circ. Physiol. 2020, 319, 866-872. DOI:10.1152/ajpheart.00441.2020 15. A. G. Tsai, M. Intaglietta, H. Sakai, E. Delpy, C. D. La Ro- chelle, M. Rousselot, F. Zal, Curr. Drug Discov. Technol. 2012, 9, 166-172. DOI:10.2174/157016312802650814 16. S. Zheng, N. A. Krump, M. M. McKenna, Y. H. Li, A. Han- nemann, L. J. Garrett, J. S. Gibson, D. M Bodine., P. S Low. J. Biol. Chem. 2019, 294, 2519-2528. DOI:10.1074/jbc.RA118.006393 17. C. H. Wiegman, F. Li, B. Ryffel, D. Togbe, K. F. Chung, Front. Immunol. 2020, 11, 1957-1-9. DOI:10.3389/fimmu.2020.01957 18. R. Franco, G. Navarro, E. Martínez-Pinilla, Antioxidants, 2019, 8, 46-1-10. DOI:10.3390/antiox8020046 19. I. H. Akbudak, V. Kucukatay, O. Kilic-Erkek, Y. Ozdemir, M. Bor-Kucukatay, Clin. Hemorheol. Microcirc. 2019, 71, 365- 372. DOI:10.3233/CH-180417 20. F. Dei Zotti, R. Verdoy, D. Brusa, I. I. Lobysheva, J. L. Balli- gand, Redox Biol. 2020, 34, 101399-1-10. DOI:10.1016/j.redox.2019.101399 21. F. Dei Zotti, I. I. Lobysheva, J. L. Balligand, PLoS One 2018, 13, e0200352-1-20. DOI:10.1371/journal.pone.0200352 22. J. M. Rifkind, E. Nagababu. Antioxid. Redox Signal. 2013, 18, 2274-2283. DOI:10.1089/ars.2012.4867 23. D. D. Guerra, K. J. Hurt, Biol. Reprod. 2019, 101, 4-25. DOI:10.1093/biolre/ioz038 Except when otherwise noted, articles in this journal are published under the terms and conditions of the  Creative Commons Attribution 4.0 International License Povzetek Uvod: Ozon vpliva na prenos kisika v krvi in na prooksidativno-antioksidativno ravnovesje, vendar vloga sestavin kri in plinskih prenašalcev pri teh procesih še vedno ostaja nejasna. Namen te raziskave je bil proučiti vpliv ozona na prenos kisika in prooksidativno-antioksidativno ravnovesje v suspenziji rdečih krvničk. Metode: Suspenzijo eritrocitov smo inkubirali z ozonom v koncentraciji 6 mg/l in snovmi, ki vplivajo na sintezo plinskih prenašalcev (nitroglicerin, natrijev hidrosulfid). Določeni so bili parametri prenosa kisika v krvi in prooksidativno-an- tioksidativno ravnovesje. Rezultati: Ugotovili smo vpliv ozona na prenos kisika v krvi, ki se je kazal v povečanju delnega tlaka kisika in stopnje oksigenacije. Indeks dejanske afinitete hemoglobina do kisika p50 se je povečal, opazen pa je bil tudi premik disociacijske krivulje oksihemoglobina v desno. Dodatek donorskega plinskega prenašalca, dušikovega oksida, je povečal učinek tega plina na parametre prenosa kisika v suspenziji eritrocitov. Zaključek: Ugotovili smo, da je ozon povzročil spremembo lastnosti vezave kisika v suspenziji eritrocitov, kar predstavlja mehanizem delovanja omenjenega plina na prilagoditvene procese v telesu, ki se dogajajo neposredno na ravni eritroc- itov. 231Acta Chim. Slov. 2023, 70, 231–239 Apostolescu et al.: Chemical and Antioxidant Profile of Hydroalcoholic ... DOI: 10.17344/acsi.2023.8046 Scientific paper Chemical and Antioxidant Profile of Hydroalcoholic Extracts of Stachys Officinalis L., Stachys Palustris L., Stachys Sylvatica L. from Romania George Florian Apostolescu1, Diana Ionela (Stegarus) Popescu2, Oana Botoran2, Daniela Sandru3, Nicoleta Anca Şuţan4,* and Johny Neamtu5 1 Doctoral school, Faculty of Pharmacy, University of Medicine and Pharmacy of Craiova, 2 Petru Rareş Street, 200349 Craiova, Romania 2 National Research and Development Institute for Cryogenics and Isotopic Technologies–ICSI Ramnicu Valcea, 4th Uzinei Street, 240050 Ramnicu Valcea, Romania 3 Department of Agricultural Sciences and Food Engineering, Lucian Blaga University of Sibiu, Doctor Ion Rațiu 7, 550012 Sibiu, Romania 4 Department of Natural Sciences, University of Pitesti, Targul din Vale 1, 110040 Pitesti, Romania 5 Faculty of Pharmacy, University of Medicine and Pharmacy of Craiova, 2-4 Petru Rares Str., 200349 Craiova, Romania * Corresponding author: E-mail: anca.sutan@upit.ro Tel.: +40 348 453 260 Received: 02-04-2023 Abstract Stachys officinalis L., Stachys palustris L., Stachys sylvatica L. (Lamiaceae) are widely used as herbal remedies. In this study, comparative assessment of the phenolic acids, flavonoids, anthocyanin, and tannins content, together with antioxidant activity of the extracts obtained from flowers, leaves and stems was performed. Phenolic acids determined by the HPLC method reached highest values in flower extract of S. palustris, stem extract of S. officinalis, and leaf extracts of S. sylvati- ca. Flavonoids were found at values exceeding 100 mg quercetin equivalents (QE)/g dry weights in all three species, based on the spectrophotometric method. Anthocyanins were detectable only in extracts from flowers. S. officinalis stood out for the highest content of anthocyanins and tannins. Antioxidant activity was present in all three species studied, with S. palustris standing out for the most intense ferric reducing antioxidant power. The results obtained lead to the validation of applicability of these plants for curative and food purposes, given their variety and richness in bioactive compounds and antioxidants. Keywords: Stachys; Phenolic Compounds; Flavonoids; Anthocyanin; Tannins; Antioxidant 1. Introduction Many plants are known for their therapeutical effects in the treatment of certain diseases, but more and more are being discovered, and nowadays there is an ever-increas- ing return to nature and what it has to offer. Advanced or classical extraction technologies of valuable components lead to the completion of information in this field, the re- sults being visible both in the scientific and commercial areas. One of the often refferened families in folk medicine is Lamiaceae, with genera and species identified world- wide, most of them presenting exceptional curative prop- erties. The genus Stachys is represented by 300–400 spe- cies, native or acclimatized, natural or ornamental, their importance and complex chemical composition being val- idated by the increasingly varied research that is being car- ried out and the possibility of superior exploitation of their bioactive potential.1,2 Recent studies revealed antioxidant, enzyme inhibi- tion, antidiabetic, anti-cholinesterase and anti-tyrosinase properties of Stachys cretica  subsp.  mersinaea (Boiss.) Rech.f., cytotoxic and antifungal activities of Stachys parv- 232 Acta Chim. Slov. 2023, 70, 231–239 Apostolescu et al.: Chemical and Antioxidant Profile of Hydroalcoholic ... iflora L., Stachys cretica subsp. bulgarica Rech.f. (SC), Stachys byzantina K. Koch (SB), Stachys thirkei K. Koch, antibacterial activity against Gram-positive microorgan- isms of Stachys byzantina K.Koch, S. officinalis and S. syl- vatica, nephroprotective, anti-inflammatory, hepatopro- tective and anticancer properties of Stachys pilifera Benth, antiphlogistic effects of S. alpina, S. germanica, S. officinalis and S. recta antidepressant activity and apoptotic effect of Stachys pilifera Benth.3–11 Antioxidant activities were men- tioned for all the above species. Nutritional value was also showed by a number of studies for species such as Stachys affinis Bunge, Stachys lavandulifolia Vahl. var. lavandulifo- lia, Stachys sieboldii Miq. 12–14 The chemical composition of the extracts differs de- pending on the species,6,9 on the solvent, on the different parts of plants used for extraction15 and the geographical area that the plants grow, 16,17 and so are the antioxidant and antimicrobial properties.16,18 In the central area of Romania (Sibiu County), seven species of Stachys genus have been identified so far: Stachys alpina L., present on the valleys and slopes of the Cibin and Făgăraș mountains; Stachys annua L. found on the montan hills at altitudes between 300 m and 700 m; Sta- chys germanica L. found on hills and montan hills at altitu- des of 320–550 m; Stachys officinalis L. identified in hi- lly-mountain areas at altitudes between 330–1250 m; Stachys palustris L. growing sporadically at high altitudes between 300 m and 900 m; Stachys recta L. present in the hilly-mountainous area at altitudes between 260–800 m; Stachys sylvatica L. present frequent on mountain hills at high altitudes comprised 340–1470 m.19 Considering the therapeutic and nutritional potenti- al of the species of the genus Stachys, this study provides a comprehensive and comparative evaluation of polyphe- nols and antioxidant profile of extracts obtained from flowers, leaves and stems of the three species grown in the central area of Romania (Sibiu County): S. officinalis, S. pa- lustris and S. sylvatica. Although other reports include chemical profile of Stachys sp., this is the first study that shows the chemical and antioxidant profile differentiated according to the aerial part of the plant and provide im- portant clues regarding the optimal exploitation of plants, through the use of plant organs with abundant bioactive compounds. 2. Experimental 2. 1. Plant Samples and Description of the Area of Interest Plant samples: Stachys officinalis L. (hemicrypto- phyte, Eurasia), Stachys palustris L. (hemicryptophyte, cir- cumpolar), and Stachys sylvatica L. (hemicryptophyte, Eurasia) were collected in July 2022, in the maximum flowering period from depression Mărginimii groups. The area that was studied is located between coordinates: 45°45'23"N 23°55'28"E and 45°45'58"N 23°54'29"E, at an altitude between 560 m and 610 m that covers the media between villages Fântânele (Cacova) and Sibiel from Mărginimea Sibiului. Depression Mărginimii groups is located at the foot- hills of Mountains Cindrel and is formed by two depres- sions, one of Sibiu and the other of Săliște, separated by Măgura Beleuța with an altitude of 630 m. Depression is characterized by gradually hill Miocene aged at the foot- hills of mountains, meadows, and terraces, attributes that frame it in the contact area. The climate is distinguished according to the landscape, with the depression area show- ing warm sides, rich in precipitation, and more significant in winter. The solar radiation exceeds 115 kcal/cm2 /year overall. Air temperature oscillates depending on the land- scape, depression area presenting an annual average tem- perature of 9 °C and northwest winds. Rainfall totals over 600 mm, with summer showers. Woody and herbaceous species are specific to the foothill area. The xerophiles meadows from the Depression Mărginimii (of Săliște) stand out through boreal plant diversity, dominated by plants original from Eurasia, followed by those Europeans and Central-European. Floristic species from this area were botanically researched with results that led to a very thorough and complete inventor.19 Plant samples of S. officinalis, S. palustris, and S. syl- vatica were recorded within the CCBIA from L. Blaga Uni- versity, Sibiu, Romania under no. 314/1, 314/2, and 314/3 respectively. 2. 2. Chemicals and Reagents The chemicals and reagents used in the process were sodium nitrite (NaNO2) 5%, aluminum chloride hexa-hy- drate (AlCl3 . 6H2O) 10%, sodium hydroxide (NaOH) 1M, quercetin, potassium chloride (KCl) 0.025M, sodium ace- tate (CH3COONa) 0.4M, hydrochloric acid (HCl), cyani- din-3-glucoside, reagent Folin-Ciocâlteu, sodium car- bonate (Na2CO3) 20%, tannic acid, casein, 0.5% formic acid in H2O, methanol (CH3OH), ferric-tripyridyltriazine (Fe3+-TPTZ), Trolox (6-hydroxy-2,5,7,8-tetramethylchro- man-2-carboxylic acid) from Fluka (Germany) and Sig- ma-Aldrich (Germany). The HPLC standards were caffeic acid, chlorogenic acid, p-coumaric acid, ferulic acid, m-coumaric acid, sinapic acid, trans-cinnamic acid, ben- zoic acid, ellagic acid, gallic acid, p- hydroxybenzoic acid, rosemarinic acid, syringic acid, and vanillic acid from Sig- ma-Aldrich (Germany). 2. 3. Preparation of Stachys sp. Extracts Flowers, stems, and leaves of S. officinalis, S. palus- tris, and S. sylvatica were dried separately at a temperature of 40 °C until the constant mass. Each 50 g of shredded dried material was soaked in a 500 mL solution of aqueous 233Acta Chim. Slov. 2023, 70, 231–239 Apostolescu et al.: Chemical and Antioxidant Profile of Hydroalcoholic ... 80% methanol for 3 days at a temperature of 18 °C in a covered container. The samples were decanted, filtered with a Buchner vacuum pump (Whatman filter paper No. 1001 090), and concentrated in a rotary evaporator. The dry extracts were resuspended in distilled water to a con- centration of 1:1 mg/ml. 2. 4. Determination of Phenolic Acids (PAs) PAs were quantified through the HPLC method pro- posed by Baczek et al.20 slightly modified, and by consult- ing other methods that were already applied on plant ex- tracts.21,22 Phenolic acids were identified following the HPLC system Smartline, KNAUER GmbH (Berlin, Ger- many), equipped with a quaternary pump, automatic in- jection and DAD detector, set to the following λ wave- lengths: 280 nm, 320 nm, 360 nm. Briefly, C18 columns (Zorbax SB – Aq: 250 mm × 4.6 mm i.d., 5.0 μm p.s) were used. For the mobile phase, a solution of deionized H2O and phosphoric acid (pH 3.5) was used as eluent A, and acetonitrile (pH 3.5) as eluent B, with the follows ratio: 0.00 min – 20% B; 0.45 min – 20% B; 5.50 min – 30% B; 5.55 min – 90% B; 6.50 min – 95% B; 6.51 min – 20% B; 15.00 min – STOP. A volume of 2 µL extract was injected into the column for chromatographic analysis, and the flow rate was 1 mL/min, at the temperature of 35 °C and 15 min total time of analysis. The identification and quantifi- cation of phenolic acids was achieved by comparison with selected standards, using calibration curves for each indi- vidual compound. The experiments were performed in triplicate and the results were expressed in µg/g extract.24 2. 5. Determination of Total Flavonoid Content (TFC) Flavonoids were determined based on the spectro- photometric method described by Popescu et al.23 The aqueous extracts (5 mL) were homogenized with 5% Na- NO2 solution (0.3 mL) and incubated for 5 minutes. Later a solution of AlCl3 . 6H2O 10% (0.5 mL) was added and the mixture was left to react in darkness. After 15 minutes of reaction 2 mL of 1M NaOH solution was added and made up to 10 mL with distilled water. The samples were read with UV-1900 SHIMADZU spectrophotometer (Shimad- zu Corporation, Kyoto, Japan) at a wavelength of 510 nm. The TFC was expressed in mg quercetin equivalents /gram of dry weight (mg QE/g DW). 2. 6. Determination of Total Monomeric Anthocyanin Pigment Content The colorimetric method based on the difference of absorbance of anthocyanins at a change in pH (pH 1 and pH 4.5) was applied for determination of total monomeric anthocyanin  pigment  (MAPC) content.24  Depending on their concentration, the difference in the absorbance of MAPC was read at a wavelength of 520 nm, respectively 700 nm, using UV-1900 SHIMADZU spectrophotometer (Shimadzu Corporation, Kyoto, Japan). Results obtained in mg/L cyanidin-3-glucoside were converted into mg/g.24 2. 7. Determination of Total Tannin Content (TTC) For the assessment of TTC, comparative quantifica- tion of total polyphenols determined through Folin-Ci- ocâlteu method and express the results in µg tannic acid equivalents/ml (µg TAE/ml) and polyphenols residuals in casein was applied. The difference between the total level of polyphenols and polyphenols residuals represents the TTC expressed in mg tannic acid equivalents /g dry weight (mg TAE/g DW).25,26 2. 8. Determination of Ferric Reducing Antioxidant Power (FRAP) Antioxidant properties of the extracts were evaluat- ed based on the reduction of Fe3+ -TPTZ in Fe2+-TPTZ by antioxidants ingredients from the samples. FRAP was monitored using the spectrophotometric method de- scribed by Lachowicz-Wisniewska et al.27 Briefly, 1 mL of each aqueous extracts was homogenized with 3 mL Fe3+ -TPTZ, absorbance being read at a wavelength of 593 nm with UV-1900 SHIMADZU spectrophotometer (Shimad- zu Corporation, Kyoto, Japan), after 10 minutes of incuba- tion. The results are expressed in mg Trolox equivalents/g of dry weight (mg TE/g DW). 2. 9. Multivariate Analysis In order to explain the significant correlations be- tween quality parameters (phenolic acids data), principal component analysis (PCA) was the main approach of mul- tivariate statistical analysis. In order to display data as sin- gle point for each variable and to reveal the correspond- ence between the principal component and the direction of maximum variance, the data were mean-centered. Pear- son correlations (p < 0.05 and p < 0.01) were used to iden- tify correlations between all variables included in the data- set. All statistical analyzes were performed using Addinsoft XLSTAT software, version 2014.5.03 (Addinsoft Inc., New York, NY, USA). 3. Results and Discussions 3. 1. Phenolic Acids in Stachys Extracts Through their anti-cancer, anti-inflammatory and antimicrobial action5,28–31 or through their positive effects on curing neurodegenerative diseases such as Alzhei- mer’s,3 phenolic acids represent bioactive plants secondary metabolites with important preventive and curative acti- 234 Acta Chim. Slov. 2023, 70, 231–239 Apostolescu et al.: Chemical and Antioxidant Profile of Hydroalcoholic ... ons. Seven hydroxybenzoic acids and eight hydroxycinna- mic acids were identified in Stachys flower extracts, with very low (0.01 µg/g trans-cinnamic acid) or generous val- ues (27970.53 µg/g benzoic acid). The results presented in Table 1 indicate that benzoic acid accumulates significantly especially in flower of S. syl- vatica (19071.32 µg/g) and S. palustris (27970.53 µg/g) and leaves of S. officinalis (4564.43 µg/g). The lowest values of benzoic acid were observed in extracts of stems, varying between a minimum of 282.28 µg/g in S. palustris and a maximum of 1270.16 µg/g in S. officinalis. At a significant- ly lower detected concentration (3080 μg/g extract), ben- zoic acid was indicated as one of the most abundant phe- nolic compound of S. cretica subsp. Mersinaea.3 The ellagic acid has been identified in the flowers ex- tracts in quantities between 18.02 µg/g for S. palustris and 32.01 µg/g for S. sylvatica, the obtained values for the stems extracts being below 7 µg/g, and those for the leaves ex- tracts reaching a maximum of 21.12 µg/g in S. sylvatica. Uneven amounts of gallic acid were found in the studied extracts. Gallic acid was found in values below 10 µg/g in flower extracts and it was undetected in stems. In compar- ison, higher content of 16.59 mg gallic acid equiv./g dry matter in Stachys lavandulifolia Vahl.32 or 900.61±0.06 mg gallic acid equivalent /100 g in dried herb in Stachys aleur- ites Boiss. & Heldr. was reported.33 The p-hydroxybenzoic acid was identified at significant values in the flower ex- tracts of S. sylvatica (83.15 µg/g) and in the leaves extracts of S. officinalis (73.43 µg/g). Salicylic acid was found in trace, with amounts between 0.22µg/g – 9.42 µg/g in flow- ers extracts, and with subunit values in extracts of stems and leaves (0.27 µg/g – 0.96 µg/g), irrespectively of the spe- cies. Significantly lower amount of p-hydroxybenzoic acid (0.006 mg g−1 DW) and significantly higher amount of sa- licylic acid (0.168 mg g−1  DW) were found in methanol extracts of leaves of S. byzantina, in comparison with leaves and flower extracts in our study.34 These results sug- gest a species-specific phenolic acid pattern. Syringic acid was fluctuated in flower extracts between 389.41 µg/g in S. officinalis and 569.78 µg/g in S. palustris, in stems extracts between 11.24 µg/g in S. palustris and 126.32 µg/g in S. sylvatica, and in leaf extracts between 9.29 µg/g in S. palustris and 111.11 µg/g in S. sylvatica. A syringic acid derivative was found in ethanol extract of dried roots of Stachys geobombycis C.Y.Wu.35 Vanillic acid was not detected in the stems and leaves of studied extracts, and was identified only in the flower extracts at values between 266.78 µg/g (S. officinalis) and 343.21 µg/g (S. palustris). Among the hydroxycinnamic acids identified, the most significant amounts were found in the case of chloro- genic acid with values varying in the flower extracts be- tween 1011.78 µg/g (S. officinalis) and 7132.29 µg/g (S. palustris). Chlorogenic acid was also identified in stems (125.37 µg/g – 333.25 µg/g) and in leaves (113.48 µg/g – 452.65 µg/g) in all three species. Chlorogenic acid and vanillic acid were predominant in aerial parts extracts of Stachys cretica L. subsp. vacillans Rech. Fil.30 Syringic acid and vanillic acid were identified in Stachys sp. aff. Schim- peri whole plant extract.36 Caffeic acid was identified in the flower extracts at values over 100 µg/g, but in leaf and stems extracts the val- ues were only subunit, or undetectable in the stems ex- tracts of S. officinalis. Caffeic acid was found as major phe- nolic compound for Stachys tmolea Boiss.37 The p-coumaric acid was detected in all Stachys extracts, values being sig- nificantly identified in the flower extracts (35.66 µg/g – Table 1. Phenolic acids identified and quantified in extracts obtained from flowers, stems and leaves of S. officinalis, S. palustris, S. sylvatica Phenolic acid S. officinalis (µg/g) S. palustris (µg/g) S. sylvatica (µg/g) Flowers Stems Leaves Flowers Stems Leaves Flowers Stems Leaves Hydroxybenzoic acid Benzoic acid 12464.34 1270.16 4564.43 27970.53 282.28 2225.44 19071.32 347.79 3447.22 Ellagic acid 24.25 4.56 12.34 18.02 1.27 2.97 32.01 6.96 21.12 Gallic acid 7.33 n.d 0.27 2.48 n.d 0.22 9.34 n.d n.d P-hydroxybenzoic acid 25.39 2.11 73.43 49.27 8.54 57.14 83.15 n.d 4.04 Salicylic acid 5.23 0.96 0.35 9.42 0.27 0.22 1.22 0.29 0.77 Syringic acid 389.41 34.12 22.44 569.78 11.24 9.29 487.76 126.32 111.11 Vanillic acid 266.78 n.d n.d 343.21 n.d n.d 312.22 n.d n.d Hydroxycinamic acid Caffeic acid 102.56 n.d 0.01 176.53 0.02 0.15 149.28 0.04 0.11 Chlorogenic acid 1011.78 125.37 452.65 7132.29 234.23 217.02 2119.69 333.25 113.38 P- coumaric acid 35.66 10.21 12.92 46.22 9.22 16.78 55.19 22.33 24.21 Ferulic acid 821.32 247.77 293.99 916.16 196.78 241.39 441.02 133.44 188.07 M-coumaric acid 5.66 1.21 2.92 4.22 n.d n.d 5.19 2.33 4.21 Rosmarinic acid 9.56 n.d n.d 4.93 n.d n.d 4.55 n.d n.d Sinapic acid 7.99 n.d n.d 3.23 n.d 0.03 7.13 n.d 0.05 Trans-cinnamic acid 0.01 n.d n.d n.d n.d n.d n.d n.d n.d Total 15177.27 1696.47 5435.75 37246.29 743.85 2770.65 22779.07 972.75 3914.29 Values are expressed as mean (n = 3), n.d = not detected 235Acta Chim. Slov. 2023, 70, 231–239 Apostolescu et al.: Chemical and Antioxidant Profile of Hydroalcoholic ... 55.19 µg/g), and lower in stems and leaves (9.22 µg/g – 24.21 µg/g). In all assessed extracts a significant amount of ferulic acid were quantified. The values determined in the flower extracts varied between 441.02 µg/g and 916.16 µg/g, and in the stem extracts up to a maximum of 247.77 µg/g. Ferulic acid were identified in other species, such as Stachys germanica L.,38 Stachys pumila Banks & Sol.,39 S. byzantine34 and in Stachys thirkei K. Koch was found as major phenolic compounds along with chlorogenic acid, caffeic acid and rosmarinic acid.37 In the extracts obtained from flowers have been de- tected m-coumaric acids (4.22 µg/g – 5.66 µg/g), ros- marinic acid (4.55 µg/g – 9.56 µg/g), sinapic acid (3.23 µg/g – 7.29 µg/g), trans-cinnamic acid (0.01 µg/g for Stachys Officinalis L). Other authors analyzed phenolics compounds, respectively PAs from various Stachys ex- tracts, results being noted in the case of species S. officina- lis, 20,40,41,42 S. palustris,12,41 S. sylvatica,12,38 Stachys cretica ssp. anatolica Rech. Fil.,31 Stachys lavandulifolia Vahl.,43 Stachys tmolea Boiss.44 3. 2. Total Flavonoid Content in Flower, Stem and Leaf Extracts Flavonoids are important bioactive compounds identified in all extracts, irrespective of plant species or or- gan used. As noted in table 2, the highest value of 51.66 mg QE/g DW were identified in flower extract of S. palustris, followed by flower extracts of S. officinalis (45.36 mg QE/g DW) and S. sylvatica (39.48 mg QE/g DW). TFC was lower in the extracts obtained from the stems and leaves regard- less of the species. Similar or lower TFC was identified by other authors in Stachys species. Sarikurkcu et al. reported a TFC between 39.24 mg Re/g extract (routine equiva- lents) and 47.70 mg Re/g for S. byzantina extract,45 and Ahmadvand et al. referenced a TFC of 17.09 mg QE/g ex- tract and 31.18 mg QE/g for Stachys inflata Benth extract.46 3. 3. Anthocyanins Content in Flower, Stem and Leaf Extracts Anthocyanins are water-soluble, colored and bioac- tive compounds, associated with the red color of the flower petals of the three studied species. In this study, anthocya- nins were identified at an average value of 32.61 mg/g ex- tract in Stachys officinalis flowers, 19.88 mg/g extract in Stachys palustris flowers and 27.72 mg/g extract in Stachys sylvatica flowers. Table 2 shows the lack of anthocyanins in the extracts from stems and leaves. Anthocyanins were al- so detected by Lachowicz-Wisniewska et al.27 in the flowers of the species Stachys palustris at an average amount of 20 mg/100 g d.m. or by Bursal et al.47 in the extracts of Stachys annua at an average value of 34.3 μg/g, but also by other authors who highlighted their antioxidant and anti-in- flammatory qualities.48 Table 2. Flavonoids, anthocyanins, tannins and antioxidant activity of S. officinalis, S. palustris, S. sylvatica Species Aerial Total Antho- Total FRAP part flavonoids cyanins tannins (mgQE/g (mg/g (mg TAE/ (mg TE/ DW) DW) g DW) g DW) S. officinalis Flowers 45.36 32.61 87.55 71.34 Stems 39.45 n.d 77.39 56.38 Leaves 31.22 n.d 84.27 83.22 Total 38.67 10.87 83.07 70.31 S. palustris Flowers 51.66 19.88 75.54 93.76 Stems 19.78 n.d 44.97 66.09 Leaves 32.67 n.d 71.76 76.21 Total 34.70 19.88 64.09 78.68 S. sylvatica Flowers 39.48 27.72 101.33 87.54 Stems 29.07 n.d 56.39 63.27 Leaves 31.63 n.d 51.15 75.77 Total 34.70 27.72 69.62 75.52 3. 4. Determination of Total Tannin Content Tannins are phenolic compounds produced as sec- ondary metabolites by terrestrial and aquatic plants.49 Ta- ble 2 stands out the fact that tannins vary in the flower extracts from 75.54 mg TAE/g DW to 101.33 mg TAE/g DW, the significantly higher value being attributed to S. sylvatica. In the extracts derived from stems, TTC was quantified to a value of 44.97 mg TAE/g DW and 77.39 mg TAE/g DW, the lowest value being defining for the species S. palustris. Lachowicz-Wisniewska et al. identified 36 hy- drolysable tannins in S. palustris flower extracts, 32 in stem extracts and 31 in leaf extracts.27 TTC varied between the level of 1.72% and 2.91% pyrogallol equivalent in S. officinalis, depending of the vegetative stage of plant devel- opment.20 3. 5. Principal Component Analysis of Flowers, Stems and Leaves Sample of S. Officinalis, S. Palustris and S. Sylvatica The results obtained through HPLC method were analyzed and interpreted to explain and to identify the re- lationships and the patterns of chemical compounds char- acteristic of S. officinalis, S. palustris and S. sylvatica flow- ers, leaves and stems. The first principal component (PC1) corresponds to 68% of the total variation, while the second principal component (PC2) explains only approximately 11% (Figure 1). The analysis of the PCA from flowers, leaves and stems showed a separation of the samples de- pending on their chemical composition, leaves and stems of S. sylvatica, stems of S. officinalis being located on the negative semiaxes. The location of the S. officinalis flower sample in quadrant II, away from the other samples, sug- gests the highest content of anthocyanins, flavonoids, ros- marinic acid, M-coumaric acid, etc. A similar content of 236 Acta Chim. Slov. 2023, 70, 231–239 Apostolescu et al.: Chemical and Antioxidant Profile of Hydroalcoholic ... PAs are indicated by the close location of samples from flowers of S. palustris and S. sylavtica in the first quadrant, on the one hand, and samples from leaves of S. palustris and S. officinalis in the IV quadrant, on the other side (Fig- ure 1a). The main components of positive side of PC1 were p-coumaric acid, flavonoids, ellagic acid, rosmarinic acid, anthocyanin, salicylic acid, syringic acid, sinapic acid and trans-cinnamic acid, while the positive side of the PC2 is identified with vanillic acid, tannins, caffeic acid, ferulic acid, p-hydroxybenzoic acid (Figure 1b). The results are confirmed by the Pearson correlation coefficient. In the heatmap presented in Figure 2, a signifi- cant positive correlation can be observed between the chemical variables identified in the analyzed samples, very rarely being identified a weak negative correlation between the chemical components, such as between trans-cinnam- ic acid and P-hydroxybenzoic acid or FRAP. nua L. that FRAP values varied between 334.5 mg TE/g extract and 1409.5 mg TE/g extract.50 Other studies re- vealed that FRAP values of Stachys thirkei K. Koch. and Stachys turcomanica Trautv. extracts varied depending on the solvent type and on the concentration of the solvent used for extraction, respectively.5, 51 4. Conclusions Extracts obtained from flowers, leaves and stems of S. officinalis, S. palustris, S. sylvatica have a chemical com- position rich in phenolic compounds. The comparative analysis has completed the literature data with new and comprehensive information about the phenolic and anti- oxidant profile. Compared to stems and leaves, these bio- active compounds are more abundant in flowers, but to- (a) (b) 3. 6. FRAP of Flower, Stem and Leaves Extracts In the flower, stem and leaf extracts of S. officinalis, S. palustris, S. sylvatica, FRAP values varied between 56.38 mg TE/g DW and 93.76 mg TE/g DW (Table 2). It is noted that this activity is more significant for flower extracts ob- tained from S. palustris, followed by S. sylvatica, and then by S. officinalis. Regarding the leaf extracts, a more pro- nounced activity is on S. officinalis (83.22 mg TE/g ex- tract), followed by S. palustris (76.21 mg TE/g extract) and S. sylvatica (75.77 mg TE/g extract). Significant values were also obtained on Stachys cretica L. extract (12.98±0.11 mg TE/g extract, 236.44±2.96 mg TE/g extract, 254.40±8.58 mg TE/g extract, 127.20 mg TE/g extract).3,29,30,31,47 Cüce et al. established for micropropagated plants of Stachys an- Figure 1. Differentiation of flowers, leaves and stems sources based on the compositional profile; (a) PCA score plot illustrating differentiation of flowers, leaves and stems sources based on the compositional profile. Colored symbols correspond to the flowers, leaves and stems of the three spe- cies addressed in this study (So – S. officinalis, Sp – S. plustris and Ss – S. sylvatica). The first two principal axes explained approximately 79% of the variance; (b) PCA loading plot showing the multivariate variation among the flowers, leaves and stems of the three species in terms of chemical compositional variables gether they create a generous profile. Flavonoids, ant- hocyanins and tannins are found in the most significant amounts in S. officinalis, followed by S. palustris and S. syl- vatica. The PCA analysis revealed significant differences in chemical composition of flowers, leaves and stems. Valua- ble elements such as hydroxybenzoic or hydroxycinnamic acids, the plenteous load of natural antioxidants in the as- sessed extracts, place them in the recommended list for their further use in pharmaceutical, cosmetic and food industries. Funding This work was supported by the grant POCU/993/6/13 -153178, co-financed by the European Social Fund within 237Acta Chim. Slov. 2023, 70, 231–239 Apostolescu et al.: Chemical and Antioxidant Profile of Hydroalcoholic ... the Sectorial Operational Program Human Capital 2014 – 2020, PN 23150301 (The implementation of integrated is- otopic-chemical-nuclear analytical method-ologies for the authentication of traditional Romanian food product- s).N.A.Ş. gratefully acknowledges the support obtained through project number PN-III-P4-ID-PCE-2020-0620, within PNCDI III, a grant from the Romanian Ministry of of Education and Research, CNCS–UEFISCDI. 5. References 1. M.S. Kocak, M.C. Uren, M. Calapoglu, A. Sihoglu Tepe, A. Mocan, K.R.R. Rengasamy, C. Sarikurkcu, S. Afr. J. Bot. 2017, 113, 128–1321. DOI:10.1016/j.sajb.2017.08.005 2. E.M.Tomou, C. Barda, H. Skaltsa, Medicines 2020, 7, 63. DOI:10.3390/medicines7100063 3. M.B. Bahadori, B. Kirkan, C.Sarikurkcu, Ind. Crop Prod. 2019, 127, 82–87. DOI:10.1016/j.indcrop.2018.10.066 4. A.Shakeri, G. D’Urso, S.F. Taghizadeh, S. Piacente, S.Norouzi, V.Soheili, J.Asili, D. Salarbashi, J. Pharm. Biomed. Anal. 2019, 168, 209–216. DOI:10.1016/j.jpba.2019.02.018 5. G. Gülsoy Toplan, T. Tașkın, E. Mataracı Kara, G.E. Genç., Istanbul J. Pharm. 2021, 51, 341–347. DOI:10.26650/IstanbulJPharm.2021.974035 6. D.I. Stegăruș, E. Lengyel, G.F. Apostolescu, O.R. Botoran, C. Tanase, Plants 2021, 10, 2710. DOI:10.3390/plants10122710 7. H. Sadeghi, D. Rostamzadeh, E. Panahi Kokhdan, A. As- faram, A.H. Doustimotlagh, N. Hamidi, S. Hossein, Evid. Based Complement. Alternat. Med. 2022, 7621599. DOI:10.1155/2022/7621599 8. H. Sadeghi, M. Mansourian, E.P. Kokhdan, Z. Salehpour, I. Sadati, K. Abbaszadeh-Goudarzi, A. Asfaram, AH. Dousti- motlagh, J. Food Biochem. 2020, 44, e13190. DOI:10.1111/jfbc.13190 9. E. Háznagy-Radnai, Á. Balogh, S. Czigle, I. Máthé, J. Hoh- mann, G. Blazsó, Phytother. Res. 2012, 26, 505–509. DOI:10.1002/ptr.3582 10. R. Jahani, D. Khaledyan, A. Jahani, E. Jamshidi, M. Kamali- nejad, M. Khoramjouy, M. Faizi, Res. Pharm. Sci. 2019, 14, 544–553. DOI:10.4103/1735-5362.272563 11. E. Panahi Kokhdan, H. Sadeghi, H. Ghafoori, H. Sadeghi, N. Danaei, S. Salaminia, M.R. Aghamaali, Armaghane Danesh 2019, 24, 17–30. Figure 2. Heatmap of Pearson correlation coefficient obtained from chemical compositional variables analyzed from flowers, leaves and stems of S. officinalis, S. palustris and S. sylvatica 238 Acta Chim. Slov. 2023, 70, 231–239 Apostolescu et al.: Chemical and Antioxidant Profile of Hydroalcoholic ... 12. A. Venditti, C. Frezza, D. Celona, A. Bianco, M. Serafini, K. Cianfaglione, D. Fiorini, S. Ferraro, F. Maggi, A.R. Lizzi, G. Celenza, 2017, Food Chem. 221, 473–481. DOI:10.1016/j.foodchem.2016.10.096 13. M. Tuncturk, R. Tuncturk, U. Karik, T. Eryigit, Int. J. Agric. Environ. Food Sci. 2019, 3, 5–8. DOI:10.31015/jaefs.2019.1.2 14. J.K. Lee, J.-J. Lee, Y.-K. Kim, Y. Lee, J.-H. Ha, Nutrients 2020, 12, 2063. DOI:10.3390/nu12072063 15. K. Namvar, E.A. Salehi, N. Mokhtarian, Bioscience Journal 2018, 34, 1349–1356. DOI:10.14393/BJ-v34n5a2018-41517 16. C.Georgescu, A. Frum, L.I. Virchea, A. Sumacheva, M. Shamtsyan, F.G. Gligor, N.K. Olah, E. Mathe, M. Mironescu, Molecules 2022, 27, 4986. DOI:10.3390/molecules27154986 17. V.B. Vundać, A.H. Brantner, M. Plazibat, Food Chem. 2007, 104,1277–1281. DOI:10.1016/j.foodchem.2007.01.036 18. C. Popescu, C. Popescu, B. Popescu, D. Daas, C. Morgovan, N.K. Olah, Farmacia 2014, 62, 743–752. DOI:10.1016/j.foodchem.2007.01.036 19. C. Drăgulescu (Ed.), „Lucian Blaga” University Sibiu, 2010, pp.450–452. 20. K. Bączek, O. Kosakowska, J.L. Przybył, Z. Węglar, Herba Pol. 2016, 62, 7–16. DOI:10.1515/hepo-2016-0007 21. F.G. Gligor, A. Frum, L.G. Vicas, M. Totan, C. Roman-Filip, C.M. Dobrea, Anal. Lett. 2020, 53, 1391–1406. DOI:10.1080/00032719.2019.1708373 22. V.I. Craciun, F.G. Gligor, A.M. Juncan, A.A. Chis, L.L. Rus, Rev. Chim. 2019, 70, 3202–3205. DOI:10.37358/RC.19.9.7516 23. D.I. Popescu, E. Lengyel, F.G. Apostolescu, L.C. Sun, O.R. Bo- toran, N.A. Şuțan, Horticulturae 2022, 8, 952. DOI:10.3390/horticulturae8100952 24. J. Lee, R.W. Durst, R.E. Wrolstad, J.  AOAC Int. 2005, 88, 1269–1278. DOI:10.1093/jaoac/88.5.1269 25. E.L.C. Amorim, J.E. Nascimento, J.M. Monteiro, T.J.S.P. So- brinho, T.A.S. Araujo, U.P. Albuquerque, Functional Ecosys- tems and Communities 2008, 88–94. 26. C.L. Gomes, C.C.A.R. Silva, C.G. De Melo, M.R.A. Ferreira, L.A.L. Soares, R.M.F. Da Silva, L.A. Rolim, P.J. Rolim Neto, An. Acad. Bras. Cienc. 2021, 93, e20190373. DOI:10.1590/0001-3765202120190373 27. S. Lachowicz-Wisniewska, A. Pratap-Singh, I. Kapusta, A. Kruszynska, A. Rapak, I. Ochmian, T. Cebulak, W. Zu- kiewicz-Sobczak, P. Rubinski, Pharmaceuticals 2022, 15, 785. DOI:10.3390/ph15070785 28. V. Amalan, V. Natesan, I. Dhananjayan, R. Arumugam, Bio- med. Pharmacother. 2016, 84, 230–236. DOI:10.1016/j.biopha.2016.09.039 29. M.B. Bahadori, B. Kirkan, C. Sarikurkcu, O. Ceylan, Ind. Crops Prod. 2019, 131, 85–89. DOI:10.1016/j.indcrop.2019.01.038 30. B. Kirkan, C. Sarikurkcu, O. Ceylan, Ind. Crop and Prod. 2019, 131, 85–89. DOI:10.1016/j.indcrop.2019.01.038 31. I. Carev, C. Sarikurkcu, Plants 2021, 10, 1054. DOI:10.3390/plants10061054 32. S. Rahimi Khoigani, A. Rajaei, S.A.Goli, Nat Prod. Res. 2017, 31, 355–358. DOI:10.1080/14786419.2016.1233410 33. G. Ozkan, R.S. Gokturk, O. Unal, S. Celik, Chem. Nat. Comp. 2006, 42, 172–174. DOI:10.1007/s10600-006-0070-1 34. O. Sytar, I. Hemmerich, M. Zivcak, C. Rauh, M. Brestic, Saudi J Biol. 2018, 25(4), 631–641. DOI:10.1016/j.sjbs.2016.01.036 35. X. Zhou, S. Huang, P. Wang, Q. Luo, X. Huang, Q. Xu, X. Qin, J. Qin, C. Liang, X. Chen, Nat. Prod. Res. 2017, 1–6. DOI:10.1080/14786419.2017.1405413 36. M. Abdel-Mogib, H.S.M. Al-Zahrani, JKAU: Sci. 2005, 17, 77–82. DOI:10.4197/Sci.17-1.8 37. T. Askun, E.M. Tekwu, F. Satil, S. Modanlioglu, H. Aydeniz, BMC Complement. Altern. Med. 2013, 13, 365. DOI:10.1186/1472-6882-13-365 38. S.S. Mitic, M. Stojkovic, J.L. Pavlović, M. Mitić, B.T. Stojano- vić, Oxid. Commun. 2012, 35, 1011–1021. 39. R.A. Kepekçi, S. Polat, G. Çoșkun, A. Çelik, A.S. Bozkurt, Ö. Yumrutaș, M. Pehlivan, J.  Food Biochem. 2017, 41, 12286. DOI:10.1111/jfbc.12286 40. I. Šliumpaite, P. Venskutonis, M. Murkovic, O. Ragažinskie- ne, Ind. Crop Prod. 2013, 50, 715–722. DOI:10.1016/j.indcrop.2013.08.024 41. V. B. Vundać, 2019, Plants (Basel), 8, 32. DOI:10.3390/plants8020032 42. J.S. Lazarević, A.S. Ðorđević, D.V. Kitić, B.K. Zlatković, G.S. Stojanović, Chem. Biodivers. 2013, 10, 1335–1349. DOI:10.1002/cbdv.201200332 43. M.N. Bingol, E. Bursal, Int. Lett. Nat. Sci. 2018, 72, 28–36. DOI:10.18052/www.scipress.com/ILNS.72.28 44. W. Elfalleh, B. Kirkan, C. Sarikurkcu, Ind. Crop Prod. 2019, 127, 212–216. DOI:10.1016/j.indcrop.2018.10.078 45. C. Sarikurkcu, M.S. Kocak, M.C. Uren, M. Calapoglu, A.S. Tepe, Eur. J. Integr. Med. 2016, 8, 631–637. DOI:10.1016/j.eujim.2016.04.010 46. H. Ahmadvand, S. Farajollahi, H. Amiri, A. Amiri, Herb. Med. J. 2017, 2, 97–104. DOI: 10.22087/hmj.v0i0.623 47. S.E. Bursal, P. Taslimi, A.C. Gören, I. Gülçin, Biocatal. Agric. Biotechnol. 2020, 28, 101711. DOI:10.1016/j.bcab.2020.101711 48. G. Paun, E. Neagu, V. Moroeanu, C. Albu, T.-M. Ursu, A. Zanfirescu, S. Negres, C. Chirita, G.L. Radu, Rev. Bras. Far- macogn. 2018, 28, 57–64. DOI:10.1016/j.bjp.2017.10.008 49. M. Fraga-Corral, P. Otero, J. Echave, P. Garcia-Oliveira, M. Carpena, A. Jarboui, B. Nuñez-Estevez, J. Simal-Gandara, M.A. Prieto, Foods 2021, 10, 137. DOI:10.3390/foods10010137 50. M. Khanavi, M. Hajimahmoodi, M. Cheraghi-Niroomand, Z. Kargar, Y. Ajani, A. Hadjiakhoondi, M. R. Oveisi, Afr. J. Biotechnol. 2009, 8, 1143–1147. DOI:10.5897/AJB2009.000-9182 51. M. Cüce, T. Bekircan, A.H. Laghari, M. Sökmen, A. Sökmen, E.Ö. Uçar, A.O. Kılıç, Indian J. Tradit. Knowl. 2017, 16, 407– 416. 52. K. Namvar, A. Mohammadi, E.A. Salehi, P. Feyzi, Pharm. Sci. 2017, 23, 244–248. DOI:10.15171/PS.2017.36 239Acta Chim. Slov. 2023, 70, 231–239 Apostolescu et al.: Chemical and Antioxidant Profile of Hydroalcoholic ... Except when otherwise noted, articles in this journal are published under the terms and conditions of the  Creative Commons Attribution 4.0 International License Povzetek Stachys officinalis L., Stachys palustris L., Stachys sylvatica L. (Lamiaceae) se pogosto uporabljajo kot zdravila rastlinskega izvora. V tej raziskavi je bila opravljena primerjalna ocena vsebnosti fenolnih kislin, flavonoidov, antocianinov in taninov ter antioksidativne aktivnosti izvlečkov, pridobljenih iz cvetov, listov in stebel. Fenolne kisline, določene z metodo HPLC, so dosegle najvišje vrednosti v izvlečku cvetov S. palustris, izvlečku stebla S. officinalis in izvlečku listov S. sylvatica. Na podlagi spektrofotometrične metode so bile pri vseh treh vrstah ugotovljene vrednosti flavonoidov, ki so presegale 100 mg ekvivalentov kvercetina (QE)/g suhe snovi. Antocianini so bili zaznani le v izvlečkih iz cvetov. S. officinalis se je od- likoval z najvišjo vsebnostjo antocianinov in taninov. Antioksidativna aktivnost je bila prisotna pri vseh treh proučevanih vrstah, pri čemer se je vrsta S. palustris odlikovala z najintenzivnejšo antioksidativno sposobnostjo reduciranja železovih ionov. Dobljeni rezultati so zaradi raznolikosti in bogastva bioaktivnih spojin in antioksidantov pripeljali do potrditve uporabnosti teh rastlin v zdravilne in prehrambene namene. 240 Acta Chim. Slov. 2023, 70, 240–246 Zhou1 et al.: Synthesis, Spectroscopic Characterization, Crystal Structures ... DOI: 10.17344/acsi.2023.8123 Scientific paper Synthesis, Spectroscopic Characterization, Crystal Structures and Antibacterial Activity of Benzohydrazones Derived from 4-Pyridinecarboxaldehyde with Various Benzohydrazides Yi-Xuan Zhou1, Wei Li1,*and Zhonglu You2 1 Department of Radiology, The Second Hospital of Dalian Medical University, Dalian 116023, P.R. China 2 Department of Chemistry and Chemical Engineering, Liaoning Normal University, Dalian 116029, P.R. China * Corresponding author: E-mail: liwei_dlmu@126.com Received: 03-08-2023 Abstract Reaction of 4-pyridinecarboxaldehyde with 3-hydroxy-4-methoxybenzohydrazide, 4-bromobenzohydrazide and 4-dimethylaminobenzohydrazide, respectively in methanol afforded three new benzohydrazones. They are 3-hy- droxy-4-methoxy-N’-(pyridin-4-ylmethylene)benzohydrazide (1), 4-bromo-N’-(pyridin-4-ylmethylene)benzohy- drazide (2), and 4-(dimethylamino)-N’-(pyridin-4-ylmethylene)benzohydrazide (3). The compounds have been char- acterized by elemental analysis, 1H and 13C NMR and IR spectroscopy, as well as single crystal X-ray diffraction. The antibacterial activities of the compounds against E. coli, P. aeruginosa, B. subtilis, and S. aureus were investigated and gave interesting results. Keywords: Benzohydrazones; 4-pyridinecarboxaldehyde; synthesis; crystal structure; antibacterial activity. 1. Introduction Hydrazones are a class of compounds containing –C(O)–NH–N=CH– groups, which can be facile synthe- sized from the condensation reactions of aldehydes with hydrazides. The compounds have wide application in bio- logical fields like antibacterial,1 antifungal,2 antitumor,3 anti-inflammatory,4 and cytotoxic.5 It was reported that the compounds containing halide groups on the aromatic rings usually show improved biological activities especial- ly the antibacterial and antifungal activities.6 Rai and co-workers reported a series of fluoro, chloro, bromo, and iodo-substituted compounds, and found that they have significant antimicrobial activities.7 In addition, nicotino- hydrazide has remarkable antituberculotic activity. As a continuation of work on the exploration of novel antibac- terial drugs, in the present paper, three new benzohydra- zones, 3-hydroxy-4-methoxy-N’-(pyridin-4-ylmethylene) benzohydrazide (1), 4-bromo-N’-(pyridin-4-ylmethylene) benzohydrazide (2), and 4-(dimethylamino)-N’-(pyri- din-4-ylmethylene)benzohydrazide (3) were prepared and evaluated for their antibacterial activities. Scheme 1. The benzohydrazones 241Acta Chim. Slov. 2023, 70, 240–246 Zhou1 et al.: Synthesis, Spectroscopic Characterization, Crystal Structures ... 2. Experimental 2. 1. Materials and Measurements Commercially available 4-pyridinecarboxaldehyde, 3-hydroxy-4-methoxybenzohydrazide, 4-bromobenzohy- drazide and 4-dimethylaminobenzohydrazide were pur- chased from Sigma-Aldrich and used without further pu- rification. Other solvents and reagents were made in China and used as received. C, H and N elemental analyses were performed with a Perkin-Elmer elemental analyzer. Infra- red spectra were recorded on a Nicolet AVATAR 360 spec- trometer as KBr pellets in the 4000–400 cm–1 region. 1H NMR spectra were recorded on a Bruker 500 MHz instru- ment. Single crystal X-ray diffraction was carried out on a Bruker D8 VENTURE PHOTON diffractometer equipped with MoKα radiation. 2. 2. Synthesis of the Compounds The three compounds were synthesized according to the same method as described. 4-Pyridinecarboxaldehyde (1.0 mmol, 11 mg) dissolved in methanol (20 mL) was added to the methanolic solution (20 mL) of 3-hy- droxy-4-methoxybenzohydrazide (1.0 mmol, 18 mg), 4-bromobenzohydrazide or 4-dimethylaminobenzohy- drazide aroylhydrazine, and were stirred at room temper- ature for 30 min to give clear solution. X-ray quality single crystals were formed by slow evaporation of the solution in air for a few days. 2. 2. 1. 3-Hydroxy-4-methoxy-N’-(pyridin-4- ylmethylene)benzohydrazide (1) Colorless crystals. Yield: 76%. Anal. calcd. for C29H32N6O8: C, 58.78; H, 5.44; N, 14.18; found C, 58.63; H, 5.55; N, 14.06%. Characteristic IR data (cm–1): 3484 (w), 3409 (w), 3205 (w), 1657 (s), 1604 (s), 1560 (m), 1510 (s), 1445 (w), 1361 (w), 1289 (s), 1221 (m), 1141 (m), 1069 (m), 1018 (w), 947 (w), 853 (m), 752 (w), 532 (m). 1H NMR (500 MHz, d6-DMSO): δ: 11.87 (s, 1H, NH), 9.76 (s, 1H, OH), 8.65 (s, 2H, PyH), 8.43 (s, 1H, CH=N), 7.65 (s, 2H, PyH), 7.50 (s, 1H, ArH), 7.47 (d, 1H, ArH), 6.90 (d, 1H, ArH), 3.85 (s, 3H, CH3). 13C NMR (126 MHz, d6-DM- SO): δ: 162.91, 150.38, 150.19, 147.29, 144.36, 141.64, 123.72, 121.56, 120.85, 114.96, 111.79, 55.73. 2. 2. 2. 4-Bromo-N’-(pyridin-4-ylmethylene) benzohydrazide (2) Colorless crystals. Yield: 82%. 1-173 °C. Anal. calcd. for C15H18N4O2: C, 62.92; H, 6.34; N, 19.57; found C, 63.11; H, 6.26; N, 19.43%. Characteristic IR data (cm–1): 3188 (w), 1645 (s), 1602 (s), 1517 (s), 1353 (w), 1281 (s), 1196 (m), 1128 (m), 1065 (w), 938 (w), 820 (w), 756 (w), 672 (w), 506 (w). 1H NMR (500 MHz, d6-DMSO): δ: 11.93 (s, 1H, NH), 8.65 (s, 2H, PyH), 8.45 (s, 1H, CH=N), 7.72 (s, 2H, PyH), 7.81 (d, 2H, ArH), 7.72 (d, 2H, ArH). 13C NMR (126 MHz, d6-DMSO): δ: 162.83, 150.17, 146.85, 144.22, 132.33, 131.53, 128.76, 125.83, 120.12. 2. 2. 3. 4-(Dimethylamino)-N’-(pyridin-4- ylmethylene)benzohydrazide (3) Colorless crystals. Yield: 73%. Anal. calcd. for C13H10BrN3O: C, 51.34; H, 3.31; N, 13.82; found C, 51.19; H, 3.22; N, 13.75%. Characteristic IR data (cm–1): 3396 (w), 3243 (w), 1650 (s), 1603 (s), 1590 (w), 1539 (s), 1466 (m), 1436 (w), 1342 (w), 1280 (m), 1141 (m), 1073 (w), 1062 (w), 1011 (w), 957 (w), 915 (m), 851 (w), 780 (w), 745 (w), 658 (w), 618 (w), 526 (w). 1H NMR (500 MHz, d6-DMSO): δ: 12.16 (s, 1H, NH), 8.65 (s, 2H, PyH), 8.45 (s, 1H, CH=N), 7.88 (d, 2H, PyH), 7.76 (d, 2H, ArH), 7.67 (d, 2H, ArH), 3.13 (s, 6H, CH3). 13C NMR (126 MHz, d6-DM- SO): δ: 162.43, 153.73, 150.23, 145.65, 141.33, 132.08, 131.52, 129.76, 120.95, 42.7. 2. 3. Single Crystal X-ray Crystallography Diffraction intensities for the compounds were collect- ed at 298(2) K using a Bruker D8 VENTURE PHOTON dif- fractometer with MoKa radiation (l = 0.71073 Å). The col- lected data were reduced using the SAINT program,8 and multi-scan absorption corrections were performed using the SADABS program.9 The structures were solved by direct method, and refined against F2 by full-matrix least-squares method using the SHELXTL.10 All of the non-hydrogen at- oms were refined anisotropically. The water and amino hy- drogen atoms were located from difference Fourier maps and refined isotropically, with O–H, H···H and N–H distanc- es restrained to 0.85(1), 1.37(2) and 0.90(1) Å, respectively. All other hydrogen atoms were placed in idealized positions and constrained to ride on their parent atoms. The crystallo- graphic data for the complexes are summarized in Table 1. 3. Results and Discussion 3. 1. Chemistry The benzohydrazones were prepared by the conden- sation reaction of equimolar quantities of 4-pyridinecar- boxaldehyde with 3-hydroxy-4-methoxybenzohydrazide, 4-bromobenzohydrazide and 4-dimethylaminobenzohy- drazide, respectively, in methanol. The compounds have been characterized by elemental analysis, IR, 1H and 13C NMR spectra. Structures of the compounds were further confirmed by single crystal X-ray diffraction. The three compounds were crystallized as well- shaped single crystals. They are soluble in MeOH, EtOH, MeCN, CHCl3, DMF and DMSO. The characteristic in- tense bands in the range 1645–1657 cm–1 are generated by the ν(C=O) vibrations, whereas the bands in the range 1602–1604 cm–1 are assigned to the ν(C=N) vibrations.11 242 Acta Chim. Slov. 2023, 70, 240–246 Zhou1 et al.: Synthesis, Spectroscopic Characterization, Crystal Structures ... In the spectrum of 1, there are broad absorptions centered at 3484 and 3409 cm–1, which can be attributed to the hy- drogen-bonded phenol and hydroxyl groups. The sharp and weak bands in the range 3188–3243 cm–1 are assigned to the ν(N–H) vibrations. The C, H, N analyses were in accordance with the chemical formulae proposed by the single crystal X-ray crystallography. 3. 2. Crystal Structure Description The molecular structures of compounds 1–3 are shown in Figures 1–3, respectively. Selected bond lengths are listed in Table 2. The asymmetric unit of compound 1 contains two benzohydrazone molecules, one methanol molecule and one water molecule. The asymmetric unit of compound 2 contains one benzohydrazone molecule and one water molecule. There is only one benzohydrazone molecule in compound 3. The benzohydrazone molecules of the compounds adopt E configuration with respect to the methylidene units. The distances (1.270–1.273 Å) of the methylidene bonds in the compounds are comparable to each other, which confirm them as typical double bonds. The shorter distances of the C–N bonds and the longer dis- tances of the C=O bonds for the –C(O)–NH– units than usual, suggest the presence of conjugation effects in the molecules. All the bond lengths in the three compounds are within normal values.12 The dihedral angles between the benzene ring and the pyridine ring in the benzohydra- zone molecules are 17.3(2) and 22.8(2)º for 1, 44.8(2)º for 2, and 19.1(3)º for 3. In the crystal structure of compound 1, the benzo- hydrazone molecules are linked by water molecules through O–H···O hydrogen bonds to form a dimer. The dimers are further linked through O–H···N hydrogen bonds to form chains. The methanol molecules are linked to the benzohydrazone molecules through O–H···N and O–H···O hydrogen bonds (Table 3, Figure 4). In the crys- tal structure of compound 2, the benzohydrazone mole- cules are linked by water molecules through O–H···O, O–H···N, N–H···O and C–H···O hydrogen bonds to form a three-dimensional network (Figure 5). In the crystal structure of compound 3, the benzohydrazone molecules are linked through N–H···O hydrogen bonds to form chains (Figure 6). Table 2. Selected bond distances (Å) for the compounds 1 · 0.5MeOH·0.5H2O N1–C6 1.273(2) N1–N2 1.3683(17) N2–C7 1.358(2) N4–C20 1.270(2) N4–N5 1.3727(17) N5–C21 1.356(2) O1–C7 1.2236(19) O4–C21 1.2242(19) 2 · H2O N1–C6 1.270(3) N1–N2 1.379(2) N2–C7 1.366(3) O1–C7 1.226(3) 3 N1–C7 1.359(3) N1–N2 1.376(3) N2–C8 1.272(3) O1–C7 1.229(3) Table 1. Crystallographic data and refinement parameters for the compounds 1·0.5MeOH·0.5H2O 2·H2O 3 Chemical formula C29H32N6O8 C15H18N4O2 C13H10BrN3O Mr 592.60 286.33 304.15 Crystal color, habit Colorless, block Colorless, block Colorless, block Crystal system Monoclinic Orthorhombic Orthorhombic Space group P21/n P212121 Pbca Unit cell parameters a (Å) 12.2635(15) 7.2634(5) 10.183(2) b (Å) 12.3785(15) 11.7864(9) 7.949(1) c (Å) 19.569(2) 17.1102(12) 30.863(2) β (°) 97.977(1) V (Å3) 2942.0(6) 1464.8(2) 2498.3(5) Z 4 4 8 Dcalc (g cm-3) 1.338 1.298 1.617 μ (mm-1) 0.099 0.089 3.281 F(000) 1248 608 1216 Collected data 17255 10810 13785 Number of unique data 5469 3625 2330 Number of observed data [I > 2σ(I)] 4119 2839 1759 Number of parameters 406 201 167 Number of restraints 5 4 1 R1, wR2 [I > 2σ(I)] 0.0415, 0.1028 0.0423, 0.0902 0.0355, 0.0758 R1, wR2 (all data) 0.0586, 0.1165 0.0611, 0.1015 0.0563, 0.0840 Goodness of fit on F2 1.013 1.035 1.019 243Acta Chim. Slov. 2023, 70, 240–246 Zhou1 et al.: Synthesis, Spectroscopic Characterization, Crystal Structures ... Table 3. Hydrogen bond distances (Å) and bond angles (°) for the compounds D–H∙∙∙A d(D–H), d(H∙∙∙A), d(D∙∙∙A), Angle Å Å Å (D–H∙∙∙A),° 1·0.5MeOH·0.5H2O O2–H2B∙∙∙N6i 0.82 1.96 2.727(2) 156(3) O5–H5B∙∙∙N3ii 0.82 1.91 2.687(2) 157(3) O8–H8∙∙∙O4iii 0.82 2.27 2.985(2) 146(3) O8–H8∙∙∙N4iii 0.82 2.38 3.079(2) 144(3) N2–H2∙∙∙O7 0.90(1) 1.95(1) 2.846(2) 171(2) N5–H5∙∙∙O8 0.90(1) 1.96(1) 2.839(2) 167(2) O7–H7A∙∙∙O1iv 0.85(1) 2.01(1) 2.823(2) 158(2) O7–H7A∙∙∙N1iv 0.85(1) 2.66(2) 3.282(2) 131(2) O7–H7B∙∙∙O5 0.85(1) 1.98(1) 2.826(2) 170(2) C28–H28A∙∙∙O3v 0.96 2.58(2) 3.371(3) 140(3) 2·H2O N2–H2∙∙∙O2vi 0.90(1) 1.98(2) 2.844(2) 159(3) O2–H2A∙∙∙N3vii 0.85(1) 2.02(1) 2.848(3) 164(3) O2–H2B∙∙∙O1 0.85(1) 2.05(2) 2.854(2) 159(3) O2–H2B∙∙∙N1 0.85(1) 2.53(2) 3.146(3) 131(2) C6–H6∙∙∙O2vi 0.93 2.59(2) 3.226(3) 126(3) C9–H9∙∙∙O2vi 0.93 2.52(2) 3.427(3) 166(3) 3 N1–H1∙∙∙O1viii 0.90(1) 1.98(1) 2.877(3) 174(4) Symmetry codes: i) 2 – x, – y, 1 – z; ii) – x, – y, – z; iii) ½ – x, ½ + y, ½ – z; iv) 3/2 – x, ½ + y, ½ – z; (v) –3/2 + x, ½ – y, –½ + z; vi) 1 – x, ½ + y, ½ – z; vii) –½ + x, ½ – y, 1 – z; viii) ½ – x, ½ + y, z. Figure 1. ORTEP plot of the crystal structure of 1. Displacement ellip- soids of non-hydrogen atoms are drawn at the 30% probability level. Figure 2. ORTEP plot of the crystal structure of 2. Displacement ellip- soids of non-hydrogen atoms are drawn at the 30% probability level. Figure 3. ORTEP plot of the crystal structure of 3. Displacement ellipsoids of non-hydrogen atoms are drawn at the 30% probability level. Figure 4. Molecular packing diagram of 1. Viewed along the b axis. Hydrogen atoms not related to hydrogen bonding are omitted. Hy- drogen bonds are shown as dashed lines. Figure 5. Molecular packing diagram of 2. Viewed along the a axis. Hydrogen atoms not related to hydrogen bonding are omitted. Hy- drogen bonds are shown as dashed lines. Figure 6. Molecular packing diagram of 3. Viewed along the a axis. Hydrogen atoms not related to hydrogen bonding are omitted. Hy- drogen bonds are shown as dashed lines. 244 Acta Chim. Slov. 2023, 70, 240–246 Zhou1 et al.: Synthesis, Spectroscopic Characterization, Crystal Structures ... 3. 3. Antibacterial Activity The antibacterial assay was performed according to the literature method.13 Penicillin G was used as a stand- ard drug. DMSO was used as solvent and the solutions were further diluted by distilled water. The DMSO at the tested concentration has no activity on the bacteria. The zone of inhibition for the 5000 μg mL–1 test solutions on the four bacteria Escherichia coli, Pseudomonas aerugino- sa, Salmonella typhi and Staphylococcus aureus is given in Table 4. The MIC values are given in Table 5. The results indicated that the compounds have from weak to strong activities against the four bacteria. Compound 1 has medi- um activity on E. coli and P. aeruginosa, and weak activity on B. subtilis and S. aureus. Compound 2 has strong activ- ity on E. coli and P. aeruginosa, and medium activity on B. subtilis and S. aureus. Compound 3 has medium activity on E. coli and P. aeruginosa, strong activity on B. subtilis, and weak activity on S. aureus. Among the compounds, compound 2 has the most activity on E. coli and P. aerugi- nosa with MIC values of 3.13 μg mL–1, which is even com- parable to Penicillin G. The four compounds have better activities against E. coli, B. subtilis and S. aureus than the pyrroles bearing thiazole moiety.14 Compounds 2 and 3 have stronger activity against E. coli, weak activity against B. subtilis, and similar activity against S. aureus when com- pared with the fluoro-substituted aroylhydrazones.15 After careful comparison we noticed that the Br sub- stituent group in compound 2 might contribute to the ac- tivity on E. coli and P. aeruginosa, and the NMe2 group in compound 3 may contribute to the activity on B. subtilis. Table 4 Antibacterial screening results Compound Zone of inhibition (mm) E. coli P. aeruginosa B. subtilis S. aureus 1 17 ± 1.9 14 ± 1.7 5.3 ± 1.4 7.2 ± 1.6 2 25 ± 2.8 23 ± 2.5 18 ± 2.6 15 ± 1.7 3 21 ± 2.3 18 ± 2.0 22 ± 2.5 11 ± 1.4 Penicillin G 30 ± 2.8 26 ± 3.1 30 ± 3.2 24 ± 2.9 Table 5 Antibacterial activities as MIC values (μg mL–1) Compound E. coli P. aeruginosa B. subtilis S. aureus 1 12.5 12.5 25 25 2 3.13 3.13 6.25 6.25 3 6.25 6.25 3.13 12.5 Penicillin G 3.13 6.25 1.56 6.25 4. Conclusions In summary, three new benzohydrazone compounds were prepared and structurally characterized. The antibac- terial activities against the bacteria E. coli, P. aeruginosa, B. subtilis, and S. aureus were evaluated. Among the com- pounds, 4-bromo-N’-(pyridin-4-ylmethylene)benzohy- drazide has strong activity on E. coli and P. aeruginosa, and 4-(dimethylamino)-N’-(pyridin-4-ylmethylene)ben- zohydrazide has strong activity on B. subtilis, with MIC values of 3.13 μg mL–-1. The compounds could be useful as templates for future development through modification to explore more effective antibacterial agents. Supplementary Material CCDC–2246870 (1), 2246872 (2), and 2246873 (3) contain the supplementary crystallographic data for this paper. These data can be obtained free of charge at http:// www.ccdc.cam.ac.uk/const/retrieving.html or from the Cambridge Crystallographic Data Centre (CCDC), 12 Un- ion Road, Cambridge CB2 1EZ, UK; fax: +44(0)1223- 336033 or e-mail: deposit@ccdc.cam.ac.uk. 5. References 1. (a) S. Verma, S. Lal, R. Narang, K. Sudhakar, ChemMedChem 2023, DOI:10.1002/cmdc.202200571 (b) M. A. Shah, A. Uddin, M. R. Shah, I. Ali, R. Ullah, P A. Hannan, H. Hussain, Molecules 2022, 27, 6770; (c) M. Nabizadeh, M. R. Naimi-Jamal, M. Rohani, P. Azerang, A. Tahghighi, Lett. Appl. Microbiol. 2022, 75, 667–679 DOI:10.1111/lam.13692 DOI:10.3390/molecules27196770 (d) G.-X. He, L.-W. Xue, Acta Chim. Slov. 2021, 68, 567–574 DOI:10.17344/acsi.2020.6333 (e) K. Pyta, A. Janas, M. Szukowska, P. Pecyna, M. Jaworska, M. Gajecka, F. Bartl, P. Przybylski, Eur. J. Med. Chem. 2019, 167, 96–104 DOI:10.1016/j.ejmech.2019.02.009 (f) I. Shabeeb, L. Al-Essa, M. Shtaiwi, E. Al-Shalabi, E. You- nes, R. Okasha, M. Abu Sini, Lett. Org. Chem. 2019, 16, 430– 436. DOI:10.2174/1570178616666181227122326 2. (a) M. Aydin, A. Ozturk, T. Duran, U. O. Ozmen, E. Sumlu, E. B. Ayan, E. N. Korucu, J. Mycologie Medicale 2023, 33, 101327 DOI:10.1016/j.mycmed.2022.101327 (b) I. R. Silva, T. Kronenberger, E. C. L. Gomes, I. C. Cesar, R. B. Oliveira, V. G. Maltarollo, Eur. J. Pharm. Sci. 2021, 156, 105575 DOI:10.1016/j.ejps.2020.105575 (c) I. A. Khodja, H. Boulebd, C. Bensouici, A. Belfaitah, J. Mol. Struct. 2020, 1218, 128527 DOI:10.1016/j.molstruc.2020.128527 (d) A. E. Dascalu, A. Ghinet, E. Lipka, C. Furman, B. Rigo, A. Fayeulle, M. Billamboz, Bioorg. Med. Chem. Lett. 2020, 30, 127220 DOI:10.1016/j.bmcl.2020.127220 (e) N. R. Appna, R. K. Nagiri, R. B. Korupolu, S. Kanugala, G. K. Chityal, G. Thipparapu, N. Banda, Med. Chem. Res. 2019, 28, 1509–1528 DOI:10.1007/s00044-019-02390-w (f) A. Erguc, M. D. Altintop, O. Atli, B. Sever, G. Iscan, G. Gormus, A. Ozdemir, Lett. Drug Des. Discov. 2018, 15, 193– 202; DOI:10.2174/1570180814666171003145227 (g) N. J. P. Subhashini, P. Janaki, B. Bhadraiah, Russ. J. Gen. 245Acta Chim. Slov. 2023, 70, 240–246 Zhou1 et al.: Synthesis, Spectroscopic Characterization, Crystal Structures ... Chem. 2017, 87, 2021–2026. DOI:10.1134/S1070363217090183 3. (a) M. S. A. Abdelrahman, F. M. Omar, A. A. Saleh, M. A. El- ghamry, J. Mol. Struct. 2022, 1251, 131947 DOI:10.1016/j.molstruc.2021.131947 (b) B. Ay, O. Sahin, B. S. Demir, Y. Saygideger, J. M. Lopez-de- Luzuriaga, G. Mahmoudi, D. A. Safin, New J. Chem. 2020, 44, 9064–9072 DOI:10.1039/D0NJ00921K (c) E. M. Gungor, M. D. Altintop, B. Sever, G. A. Ciftci, Lett. Drug Des. Discov. 2020, 17, 1380–1392 DOI:10.2174/1570180817999200618163507 (d) H. M. A. Abumelha, J. Heterocycl. Chem. 2018, 55, 1738– 1745 DOI:10.1002/jhet.3211 (e) H. F. He, X. Y. Wang, L. Q. Shi, W. Y. Yin, Z. W. Yang, H. W. He, Y. Liang, Bioorg. Med. Chem. Lett. 2016, 26, 3263–3270. DOI:10.1016/j.bmcl.2016.05.059 4. (a) M. A. M. B. Medeiros, M. G. E. Silva, J. D. Barbosa, E. M. de Lavor, T. F. Ribeiro, C. A. F. Macedo, L. A. M. D. Duarte- Filho, T. A. Feitosa, J. D. Silva, H. H. Fokoue, C. R. M. Araujo, A. D. Gonsalves, L. A. D. Ribeiro, J. R. G. D. Almeida, Plos One 2021, 16, e0258094 DOI:10.1371/journal.pone.0258094 (b) M. X. Song, B. Liu, S. W. Yu, S. H. He, Y. Q. Liang, S. F. Li, Q. Y. Chen, X. Q. Deng, Lett. Drug. Des. Discov. 2020, 17, 502–511 DOI:10.2174/1570180816666190731113441 (c) U. Debnath, S. Mukherjee, N. Joardar, S. P. S. Babu, K. Jana, A. K. Misra, Eur. J. Pharm. Sci. 2019, 134, 102–115 DOI:10.1016/j.ejps.2019.04.016 (d) U. Kendur, G. H. Chimmalagi, S. M. Patil, K. B. Gudasi, C. S. Frampton, C. V. Mangannavar, I. S. Muchchandi, J. Mol. Struct. 2018, 1153, 299–310 DOI:10.1016/j.molstruc.2017.10.022 (e) V. Gorantla, R. Gundla, S. S. Jadav, S. R. Anugu, J. Chi- makurthy, S. K. Nidasanametla, R. Korupolu, New J. Chem. 2017, 41, 13516–13532 DOI:10.1039/C7NJ03332J (f) M. A. Abdelgawad, M. B. Labib, M. Abdel-Latif, Bioorg. Chem. 2017, 74, 212–220. DOI:10.1016/j.bioorg.2017.08.014 5. (a) E. Aydin, A. M. Senturk, H. B. Kucuk, M. Guzel, Molecules 2022, 27, 7309 (b) M. A. Shah, A. Uddin, M. R. Shah, I. Ali, R. Ullah, P. A. Hannan, H. Hussain, Molecules 2022, 27, 6770 DOI:10.3390/molecules27196770 (c) F. Beygi, A. Mostoufi, A. Mojaddami, Chem. Biodivers. 2022, 19, e202100754 DOI:10.1002/cbdv.202100754 (d) Z. Ozdemir, N. Basak-Turkmen, I. Ayhan, O. Ciftci, M. Uysal, Pharm. Chem. J. 2019, 52, 923–929 DOI:10.1007/s11094-019-01927-y (e) J. C. Coa, W. Castrillon, W. Cardona, M. Carda, V. Ospina, J. A. Munoz, I. D. Velez, S. M. Robledo, Eur. J. Med. Chem. 2015, 101, 746–753. DOI:10.1016/j.ejmech.2015.07.018 6. (a) L. C. Felton, J. H. Brewer, Science 1947, 105, 409–410 DOI:10.1126/science.105.2729.409 (b) M. Gopalakrishnan, J. Thanusu, V. Kanagarajan, R. Go- vindaraju, J. Enzym. Inhib. Med. Chem. 2009, 24, 52–58 DOI:10.1080/14756360801906632 (c) L. Shi, H.-M. Ge, S.-H. Tan, H.-Q. Li, Y.-C. Song, H.-L. Zhu, R.-X. Tan, Eur. J. Med. Chem. 2007, 42, 558–564 DOI:10.1016/j.ejmech.2006.11.010 (d) M. Zhang, D.-M. Xian, H.-H. Li, J.-C. Zhang, Z.-L. You, Aust. J. Chem. 2012, 65, 343–350. DOI:10.1071/CH11424 7. (a) N. P. Rai, V. K. Narayanaswamy, T. Govender, B. K. Manu- prasad, S. Shashikanth, P. N. Arunachalam, Eur. J. Med. Chem. 2010, 45, 2677–2682 DOI:10.1016/j.ejmech.2010.02.021 (b) N. P. Rai, V. K. Narayanaswamy, S. Shashikanth, P. N. Arunachalam, Eur. J. Med. Chem. 2009, 44, 4522–4527. 8. Bruker, SMART and SAINT, Bruker AXS Inc., Madison, Wis- consin, USA, 2012. 9. G. M. Sheldrick, SADABS Program for Empirical Absorption Correction of Area Detector, University of Göttingen, Ger- many, 1996. 10. G. M. Sheldrick, Acta Crystallogr. 2008, A64, 112–122. DOI:10.1107/S0108767307043930 11. W. J. Geary, Coord. Chem. Rev. 1971, 7, 81–122. DOI:10.1016/S0010-8545(00)80009-0 12. (a) M. Kuriakose, M. R. P. Kurup, E. Suresh, Spectrochim. Acta A 2007, 66, 353–358 DOI:10.1016/j.saa.2006.03.003 (b) L.-W. Xue, S.-T. Li, Y.-J. Han, X.-Q. Luo, Acta Chim. Slov. 2022, 69, 385–392 DOI:10.17344/acsi.2021.7252 (c) G.-X. He, L.-W. Xue, Acta Chim. Slov. 2021, 68, 567–574. DOI:10.17344/acsi.2020.6333 13. (a) Y. M. Chumakov, B. Y. Antosyak, V. I. Tsapkov, N. M. Samus, J. Struct. Chem. 2001, 42, 335–339 DOI:10.1023/A:1010587923496 (b) Y.-J. Han, X.-Y. Guo, L.-W. Xue, Acta Chim. Slov. 2022, 69, 928–936 DOI:10.17344/acsi.2022.7817 (c) H.-Y. Zhu, Acta Chim. Slov. 2021, 68, 65–71. DOI:10.17344/acsi2020.6138 14. M. A. Salem, S. Y. Abbas, M. A. M. Sh. El-Sharief, M. H. Helal, M. A. Gouda, M. A. Assiri, T. E. Ali, Acta Chim. Slov. 2021, 68, 990–996. DOI:10.17344/acsi.2021.6980 15. F.-M. Wang, L.-J. Li, G.-W. Zang, T.-T. Deng, Z.-L. You, Acta Chim. Slov. 2021, 68, 541–547. DOI:10.17344/acsi.2020.6051 246 Acta Chim. Slov. 2023, 70, 240–246 Zhou1 et al.: Synthesis, Spectroscopic Characterization, Crystal Structures ... Except when otherwise noted, articles in this journal are published under the terms and conditions of the  Creative Commons Attribution 4.0 International License Povzetek Reakcija 4-piridinkarboksaldehida s 3-hidroksi-4-metoksibenzohidrazidom, 4-bromobenzohidrazidom in 4-dimetila- minobenzohidrazidom v metanolu je dala tri nove benzohidrazone. To so 3-hidroksi-4-metoksi-N’-(piridin-4-ilmetilen) benzohidrazid (1), 4-bromo-N’-(piridin-4-ilmetilen)benzohidrazid (2) in 4-(dimetilamino)-N’-(piridin-4-ilmetilen) benzohidrazid (3). Spojine smo okarakterizirali z elementno analizo, 1H in 13C NMR in IR spektroskopijo ter monokris- talno rentgensko difrakcijo. Preučevali smo antibakterijske aktivnosti spojin proti E. coli, P. aeruginosa, B. subtilis in S. aureus ter dobili zanimive rezultate. 247Acta Chim. Slov. 2023, 70, 247–260 Muğlu et al.: New Bis-1,3,4-Thiadiazoles Based on Fumaric Acid: ... DOI: 10.17344/acsi.2022.7822 Scientific paper New Bis-1,3,4-Thiadiazoles Based on Fumaric Acid: Preparation, Structure Elucidation, Antibacterial Activities, and Quantum-Chemical Studies Halit Muğlu,1,* Hasan Yakan,2 Ghaith Alabed Ibrayke Elefkhakry,1 Ergin Murat Altuner,3,* and M. Serdar Çavuş4 1 Department of Chemistry, Kastamonu University, Kastamonu, Turkey 2 Department of Chemistry Education, Ondokuz Mayıs University, Samsun, Turkey 3 Department of Biology, Kastamonu University, Kastamonu, Turkey 4 Biomedical Engineering Department, Kastamonu University, Kastamonu, Turkey * Corresponding author: E-mail: hmuglu@kastamonu.edu.tr ergin.murat.altuner@gmail.com Received: 09-24-2022 Abstract New bis-1,3,4-thiadiazoles 1–7 were obtained by the reaction of fumaric acid and N-(alkyl/aryl/cyclic)thiosemicarba- zides in the presence of phosphorous oxychloride. The structures of all compounds were elucidated by FT-IR, 1H NMR, and 13C NMR and elemental analysis. Antibacterial activity of the compounds was studied for eight selected bacteria. Compounds 2–7 exhibited effect on Klebsiella pneumoniae. However, none of the compounds effect on Pseudomonas aeruginosa, Staphylococcus epidermidis, Salmonella enterica serovar Kentucky, Serratia marcescens. Self-consistent reac- tion force (SCRF) calculations were performed in DMSO medium to examine solvent energies using CPCM and SMD models. 6-31G(d) and 6-311++G(2d,2p) basis sets were used for DFT calculations. Besides electronic parameters such as electronegativity, electrophilicity and spectroscopic examinations of the compounds, QTAIM, local electron affinities, and Fukui analyses were also performed. Theoretical approaches supporting the experimental observations revealed that compounds containing aromatic and cyclic groups exhibit stronger antibacterial behavior than compounds containing aliphatic groups. Keywords: Bis-1,3,4-thiadiazoles; fumaric acid; antibacterial activity; spectroscopic methods; DFT calculations. 1. Introduction Heterocyclic compounds represent a very significant part of organic chemistry. They have an extensive scope of medicinal, biological, and synthetic applications.1 Thiadi- azoles have both one sulphur and two nitrogen atoms which form aromatic five-membered heterocyclic ring compounds. Thiadiazoles have four isomeric forms, 1,3,4-thiadiazole is one of them, being thermally the most stable among these isomers.1h 1,3,4-Thiadiazoles exhibit various applications in medicinal, biological, agricultural, and materials chem- istry such as antioxidant,2 antiviral,3 antifungal,4 anti- microbial,5 analgecis,6 antidepressant,7 antileishmanial,8 anticonvulsant,9 anti-inflammatory,10 antitubercular,11 an- ticancer,12 and antiproliferative activities.13 Despite the fact that an extensive number of chem- otherapeutics and antibiotics exist, the appearance of new and old antibiotic-resistant bacterial strains have shown a need to consider both the synthesis and exploration of novel more harmless, powerful, and confident antimicro- bial agents.14 In the paper, new bis-1,3,4-thiadiazole deriva- tives were obtained from the reaction of fumaric acid, N-(alkyl/aryl/cyclic)thiosemicarbazides, and phospho- rous oxychloride (POCl3). The structures of the com- pounds were determined by using FTIR, 1H NMR, and 13C NMR spectroscopic methods and elemental analysis. The antibacterial activity of the compounds was inves- tigated against several Gram-positive bacteria (Bacillus subtilis, Staphylococcus epidermidis, and Enterococcus 248 Acta Chim. Slov. 2023, 70, 247–260 Muğlu et al.: New Bis-1,3,4-Thiadiazoles Based on Fumaric Acid: ... faecium) and Gram-negative bacteria (Escherichia coli, Salmonella enterica serovar Kentucky, Serratia marc- escens, Klebsiella pneumoniae, and Pseudomonas aerug- inosa) by minimum inhibition concentration (MIC) and minimum bactericidal concentration (MBC) tests. Moreover, optimized molecular structures and spatial conformations of the compounds were investigated by theoretical studies using the DFT method; experimental spectroscopic data were also supported by the calcula- tion results. Some electronic parameters of the com- pounds were calculated and used to establish the struc- ture-activity relationship. 2. Experimental 2. 1. Measurement and Reagents Melting points were determined on a Stuart SMP 30 electrothermal apparatus. Eurovector EA3000-Single device was utilized for elemental analysis. A Bruker Al- pha FT-IR spectrophotometer were used to obtain Fouri- er transform infrared (FT-IR) spectra. 1H NMR and 13C NMR spectra were taken in DMSO-d6 on a Bruker Avance DPX-400 spectrophotometer (400 MHz and 101 MHz, respectively) spectrometer using tetramethylsilane as an internal standard. The splitting patterns are indicated as Scheme 1. Structures of compounds 1–7 and synthetic route to them. 249Acta Chim. Slov. 2023, 70, 247–260 Muğlu et al.: New Bis-1,3,4-Thiadiazoles Based on Fumaric Acid: ... s (singlet), d (doublet), dd (doublet of doublet), t (triplet), dt (doublet of triplet), td (triplet of doublet) and m (multi- plet). All chemical reagents were purchased from Aldrich, Carlo Erba, Acros Organics, or Merck Chemical Company and used without further purification. 2. 2. Synthesis of the Compounds 1–7 The mixture of fumaric acid (n mol) and N-(alkyl/ aryl/cyclic)thiosemicarbazide derivatives (2n mol) was chilled in a refrigerator and phosphorous oxychloride (3n mol) was added drop-wise during stirring. Then, reflux- ing was continued at 90 οC for 3–5 h. After completion of the reaction, the mixture was cooled to room temperature, poured into ice-cold water with stirring, and then neutral- ized with ammonia. The precipitated product was filtered, washed with water, and crystallized in a suitable solvent. These new bis-1,3,4-thiadiazoles were prepared according to the procedure described in the literature.1b They were obtained in good yield (61–93%) as shown in Scheme 1. 2. 3. Antibacterial Activity Testing Bacteria Strains The antibacterial activities of the synthesized com- pounds were determined against three Gram-positive (Bacillus subtilis DSMZ 1971, Enterococcus faecium (food isolate), and Staphylococcus epidermidis DSMZ 20044) bacteria, and five Gram-negative (Escherichia coli (food isolate), Klebsiella pneumoniae (food isolate), Pseu- domonas aeruginosa DSMZ 50071, Salmonella enterica serovar Kentucky (food isolate), and Serratia marcescens (clinical isolate)). All the bacteria were obtained from the culture collection of Kastamonu University, Department of Biology, Microbiology Laboratory. Preparation of Chemical Compounds The stock solutions were prepared by dissolving 24 mg of each compound 1–7 in 1 mL of dimethyl sulfoxide (DMSO) (Merck). Since DMSO is toxic for living cells, the in-test DMSO concentration was adjusted as 1%.15 Preparation of the Inocula The bacteria used in the study were incubated at 37 °C for 24 hours and similar colonies were collected by a sterile loop, transferred into 0.9% sterile saline solution, and the turbidities were adjusted to 0.5 McFarland stand- ard.16 Minimum Inhibition Concentration (MIC) Test MIC test was applied as a broth dilution test as previ- ously defined in previous studies.17 Serial 2-fold dilutions were done in a 96-well plate and a concentration range of 0.234–120 µg/mL was obtained. The MIC value was de- termined as the lowest concentration of extract inhibiting any visible bacterial growth.18 All tests were studied in triplicate. Minimum Bactericidal Concentration (MBC) Test The MBC test is complementary to the MIC test, where the MIC test demonstrates the lowest level of an- timicrobial agent that inhibits growth, and the MBC demonstrates the lowest level of antimicrobial agent that causes microbial death. The MBC values were determined by sub-culturing the contents of non-turbid MIC test wells to agar plates. The MBC is identified as the lowest concen- tration of antibacterial agent that reduces the viability of the initial bacterial inoculum by ≥99.9%.19 Controls 1% DMSO was used as a negative control, where gentamicin (GEN), tobramycin (TOB), and ciprofloxacin (CPFX) were used as positive controls. 2. 4. Computational Procedure The molecular structure of the compound in the ground state (in vacuum) was obtained from the op- timization calculations performed using the B3LY- P/6-311++g(2d,2p) level of theory by the Kohn–Sham density functional theory (KS-DFT).20 All calculations were performed using Gaussian 09 software21 without any symmetry restrictions. The optimized state geometries of the compounds with minimum energy correspond to the global minimum energy points on the potential energy surface, that is, no imaginary frequencies are present in the IR calculations. Moreover, solvent effects were studied using solvation model based on density (SMD) and con- ductor-like polarizable continuum model (CPCM), at the same level of theory, to calculate the properties of com- pounds in solution. 1H and 13C NMR chemical shifts calculations were performed using Gauge-independent atomic orbital (GIAO) method in dimethyl sulfoxide (DMSO) phase, in accordance with the experiments. Relative chemical shift values were obtained by subtracting (31.8149 and 183.737 ppm for 1H and 13C NMR, respectively) from the absolute chemical shielding of tetramethylsilane (TMS), which was also calculated at B3LYP/6-311++g(2d,2p) level of theory. The electronic parameters of the compounds were obtained from the calculations performed at the level of B3LYP/6-311++g(2d,2p) in the gas phase. Global chem- ical reactivity parameters such as HOMO-LUMO energy gap (Eg), Chemical hardness (η), electronegativity (χ), and electrophilic index (ω) were calculated using frontier molecular orbital (FMO) energy eigenvalues. The calcula- tions of the Fukui functions were performed at the B3LY- 250 Acta Chim. Slov. 2023, 70, 247–260 Muğlu et al.: New Bis-1,3,4-Thiadiazoles Based on Fumaric Acid: ... P/6-31g(d) level, and the electrophilic and nucleophilic attack regions were analyzed by visualizing the data. In addition, the reactivity and behavior of the compounds in different environments were estimated by FMO calcu- lations and prominent descriptors were reported. Besides the population analysis of natural bond orbitals (NBOs), intramolecular interactions were studied through topo- logical properties using Bader’s quantum theory of atoms in molecules (QTAIM) approach22 and were also used to calculate electron charge distributions on compounds. Ring critical points (RCPs) of charge density distribution and bond critical points (BCPs) of bonded atoms were de- termined by QTAIM analyses performed using Multiwfn software,23 and interaction region indicator (IRI) calcula- tions were performed to visualize intramolecular interac- tions. 3. Results and Discussion 3. 1. Physical Data All the compounds are new. In Table 1, the physical data, melting points, yields, and elemental analysis of these compounds are presented. Table 1. Physical data and elemental analysis results of the compounds Calculated/Found Comp. Compound’s Names M. P. Yields% Colour C% H% N% (οC) (Mass, g) 1 (E)-N-methy-5-(2-(5-(methylamino)-1,3,4-thiadiazol- 65 Brown 37.78/ 3.96/ 33.04/ 2-yl)vinyl)-1,3,4-thiadiazol-2-amine 240–241 (0.206) 37.69 4.05 32.89 2 (E)-N-ethyl-5-(2-(5-(ethylamino)-1,3,4-thiadiazol-2- >300 61 Dark Brown 42.53/ 5.00/ 29.76/ yl)vinyl)-1,3,4-thiadiazol-2-amine (0.296) 42.45 4.90 29.67 3 (E)-N-allyl-5-(2-(5-(allylamino)-1,3,4-thiadiazol-2-yl) >300 73 Cream 47.04/ 4.61/ 27.43/ vinyl)-1,3,4-thiadiazol-2-amine (0.289) 46.94 4.71 27.35 4 (E)-N-(2-methoxyphenyl)-5-(2- (5- 175–176 87 Dark Yellow 54.78/ 4.14/ 19.16/ (2-methoxyphenylamino)-1,3,4-thiadiazol-2-yl)vinyl)- (0.404) 54.72 4.08 19.21 1,3,4-tiadiazol-2-amine 5 (E)-N-(3-chlorophenyl)-5-(2-(5-(3-chlorophenylamino)- 168–169 75 Dark Cream 48.33/ 2.70/ 18.79 1,3,4-thiadiazol-2-yl)vinyl)-1,3,4-thiadiazol-2-amine (0.289) 48.26 2.75 18.64 6 (E)-5-(2-(5-(p-toluidin)-1,3,4-thiadiazol-2-yl)vinyl)-N- 198-199 88 Dark Yellow 59.09/ 4.46/ 20.67/ p-tolyl-1,3,4-thiadiazol-2-amine (0.462) 59.17 4.41 20.88 7 (E)-N-cyclohexyl-5-(2-(5-(cyclohexylamino)-1,3,4- >300 93 Cream 55.35/ 6.71/ 21.52/ thiadiazol-2-yl)vinyl)-1,3,4-tihadiazol-2-amine (0.457) 55.43 6.59 21.46 Figure 1. IR spectrum of compound 2 251Acta Chim. Slov. 2023, 70, 247–260 Muğlu et al.: New Bis-1,3,4-Thiadiazoles Based on Fumaric Acid: ... 3. 2. Vibrational Frequencies In the FT-IR spectrum of the synthesized compounds, the fumaric acid (–COOH) signal of the starting material was not observed near 3500–2700 cm−1. Furthermore, the asymmetric and symmetric stretching bands of the amino group (–NH2) were not observed at 3450–3200 cm−1. These results pointed out a successful reaction, as expected. For all compounds, the peaks of the amino group (–NH) were showed between at 3248–3195 cm−1; the –C=N stretching vibrations of thiadiazole ring were observed between at 1635 and 1518 cm–1; the –C–N stretching vibrations were shown between 1174–1083 cm–1; the –C–S signals were observed between 721–634 cm−1. For compound 2, the band of the amino group (–NH) was shown at 3248 cm−1 as shown in Figure 1. The aliphatic CH stretching vibra- tions were observed at 2929–2853 cm−1. The –C=N stretch- ing vibrations of thiadiazole ring appeared at 1518 cm–1. The –C–N stretching vibration appeared at 1131 cm–1; the –C–S signal was observed at 696 cm−1. These values pro- vided significant proofs for the products formation. These observations are consistent with values published previous- ly for similar compounds.1b,5b,24 IR vibrations for the syn- thesized compounds are presented in Table 2. 3. 3. 1H NMR Spectral Interpretations The 1H NMR spectra of the synthesized compounds were measured in DMSO-d6 as the solvent and the chemi- Table 2. Experimental and calculated FT-IR values of the compounds 1–7 (cm–1). C. –NH Ar. CH C=N C–N C–S Spec. Vib. 1 3199 – 1586–1555 1153 634 CH(*): 2924–2847 2 3248 – 1518 1131 696 CH(*): 2929–2853 3 3219 – 1591–1532 1142 697 CH(*): 2928–2871 4 3248 3075–3002 1635–1601 1174 654 CH(*): 2939–2837 -C-O : 1114 5 3247 3092–3061 1631–1585 1170 721 -C-Cl : 994 6 3195 3061–2992 1635–1604 1083 714 CH(*): 2920–2874 7 3230 – 1611–1560 1120 696 CH(*): 2926–2852 1 3645.81 – 1518.64, 1510.03 1040.41 685.41 CH(*): 3140.07–3030.96 2 3622.25 – 1511.22, 1506.33 1060.03 685.91 CH(*): 3113.31–3016.33 3 3619.58 – 1508.94, 1498.25 1054.43 686.96 CH(*): 3222.01–3019.94 4 3622.59 3239.37–3195.42 1513.02, 1500.08 1266.40 689.77 CH(*): 3142.48–3024.14 -C-O : 1051.00 5 3642.73 3250.32–3166.74 1513.73, 1500.88 1256.08 688.46 -C-Cl : 907.16 6 3642.51 3239.59–3151.08 1512.38, 1501.75 1252.28 687.50 CH(*): 3106.15–3028.97 7 3625.36 – 1511.35, 1509.87 1087.40 684.87 CH(*): 3097.13–3011.82 C.: Compounds, (*) : Aliphatic CH. Ex pe ri m en ta l C al cu la te d Figure 2. 1H NMR spectrum of compound 2. 252 Acta Chim. Slov. 2023, 70, 247–260 Muğlu et al.: New Bis-1,3,4-Thiadiazoles Based on Fumaric Acid: ... cal shifts are shown in Table 3. For compound 2, the proton signal of –NH coupled to the CH2 proton was detected as a triplet at 8.11–8.09 ppm. The alkyenic (CH=C) H1 proton was shown as a singlet at 7.14 ppm. The H2 (CH2) proton coupled to the H3 and NH protons was observed as a pen- tet at 3.37–3.30 ppm. The H3 (CH3) proton coupled to the H2 protons was observed as a triplet at 1.21–1.17 ppm as shown in Figure 2. DMSO-d6 and water in DMSO (HOD, H2O) signals are shown around at 2.00, 2.50 (quintet) and 3.30 (variable, based on the solvent and its concentration) ppm, respectively.25 These data are consistent with the val- ues of those reported earlier for similar compounds.5b,26 3. 4. 13C NMR Spectral Interpretations The 13C NMR spectra of all compounds were meas- ured in DMSO-d6 and the chemical shifts are shown in Table 4. The 13C NMR spectrum of the compound 2 showed 5 different resonances in good agreement with the proposed structure as shown in Figure 3. In compound 2, the carbon signals of thiadiazole ring (C2 and C3) were detected at 155.4 and 169.0 ppm, respectively. The C3 car- bon atom is shifted down-field (high values, δ) due to the presence of electron-negative nitrogen atom (NH group). The alkyenic C1 carbon atom (CH=C) was observed at 124.6 ppm. While the C4 (CH2) carbon atom was detected at 40.1 ppm, the C5 (CH3) resonated at 14.6 ppm. The C4 (CH2) carbon atom is shifted down-field (high values, δ) as does the C3 carbon atom due to the presence of electron- egative nitrogen atom (NH group). For compounds 1–7, the alkyenic C1 carbon atom (CH=C) was observed between 125.7 and 120.8 ppm. While the C2 carbon signals of thiadiazole ring resonated a between 157.2 and 152.4 ppm, the C3 resonated between 169.6 and 164.3 ppm. For compound 3, the C4 carbon atom (CH2) was observed at 47.8 ppm. The alkyenic C5 and C6 carbon atoms (CH=CH2) were detected at 134.6 and 117.3 ppm, respectively. In compounds 4–6, the aromatic car- bons (C4–C9) were observed at 149.1–111.7, 142.0–116.7, and 138.8–117.9 ppm, respectively. The methoxy (–OCH3) signal was detected at 56.4 ppm for compound 4, the me- thyl (CH3) group was observed at 20.8 ppm for compound 6. In compound 7, the cyclic carbons (C4–C9) were ob- served between 53.9 and 24.7 ppm. These spectroscopic data are consistent with the values reported previously for similar compounds in the literature.5b,26a,b,27 3. 5. Antibacterial Activity Assessments In vitro antibacterial activity results for the synthe- sized 1,3,4-thiadiazole compounds are given in Table 5. The antibacterial activities of bis-1,3,4-thiadiazole com- pounds 1–7 were determined against three Gram-positive (S. epidermidis, B. subtilis, and E. faecium) and Gram-neg- ative (S. enterica serovar Kentucky, E. coli, K. pneumoniae, S. macrescens, and P. aeruginosa) bacteria by two sequen- Table 3. Experimental and calculated 1H NMR values of the compounds (δ, ppm, in DMSO–d6) C. NH H1 H2 H3 H4 H5 H6 1 8.06–8.03 (q) 7.15 (s) 2.93–2.92 (d) – – – – 2 8.11–8.09 (t) 7.14 (s) 3.97–3.28 (p) 1.21–1.17 (t) – – – 3 8.33–8.30 (t) 7.14 (s) 3.97–3.88 (t) 5.97–5.87 (m) 5.29–5.15 (dd) – – 4 10.05 (s) 7.36 (s) OCH3: 3.89 (s) 8.31–8.29 (m) 7.09–7.04 (m) 7.01–6.97 (m) 5 10.52 (s) 7.50–7.48 (d) 7.94 (s) – 7.41–7.37 (dd) 7.10–7.08 (d) 6 10.22 (s) 7.46 (s) 6.84–6.80 (d) 7.14–6.99 (d) CH3: 2.51 (s) 7.14–6.99 (d) 6.84–6.80 (d) 7 7.93–7.91 (d) 6.74 (s) 1.95–1.03, (m), Cyclic 11H 1 5.09 7.76 3.29–3.02 – – – – 2 4.79 7.76 3.68–3.41 1.41–1.26 – – – 3 4.66 7.79 4.69, 3.79 6.51 5.82–5.63 – – 4 8.19 7.99 4.38, 3.97 7.30 7.46 7.42 9.03 5 7.37 8.05 9.18 – 7.38 7.71 7.16 6 7.25 7.98 8.94 7.61 CH3: 2.63–2.15 7.70 7.25 7 4.66 7.72 2.14–1.29, Cyclic 11H C.: Compounds, s (singlet), d (doublet), dd (doublet of doublet), t (triplet), td (triplet of doublet) and m (multiplet). Ex pe ri m en ta l C al cu la te d 253Acta Chim. Slov. 2023, 70, 247–260 Muğlu et al.: New Bis-1,3,4-Thiadiazoles Based on Fumaric Acid: ... tial tests, the first being the minimum inhibitory concen- tration (MIC) and the second the minimum bactericidal concentration (MBC) test. The results of the antibacterial screening given in Table 5 show that none of the compounds 1–7 had ac- tivity against P. aeruginosa, S. epidermidis, S. enterica se- rovar Kentucky, and S. macrescens. All compounds except 1 showed an effect on Gram-negative K. pneumoniae. Compounds 2 and 3 showed antibacterial activity at MIC concentrations of 2.75 mg/L. The rest of compounds 4, 5, 6, and 7 presented antibacterial activity with MIC concen- trations of 1.375 mg/L. Table 4. Experimental and calculated 13C NMR values of the compounds (δ, ppm, in DMSO–d6) Comp. C1 C2 C3 C4 C5 C6 C7 C8 C9 R1 1 124.9 155.7 169.6 32.2 – – – – – – 2 124.6 155.4 169.0 40.1 14.6 – – – – – 3 124.9 155.8 168.5 47.8 134.6 117.3 – – – – 4 125.2 157.1 165.3 129.7 149.1 111.7 123.7 121.0 120.0 56.4 5 125.7 157.2 164.3 142.0 117.6 133.9 122.5 131.2 116.7 – 6 121.2 154.8 165.4 138.8 117.9 129.9 130.2 130.2 117.9 20.8 7 120.8 152.4 169.4 53.9 32.5 24.7 25.7 24.7 32.5 – 1 122.7 164.4 178.7 32.6 – – – – – – 2 122.8 164.3 178.2 44.1 14.8 – – – – – 3 123.1 164.9 178.3 52.6 143.9 125.6 – – – – 4 123.3 165.0 173.3 134.9 154.0 113.4 127.2 125.4 121.8 57.6 5 123.7 165.7 173.8 147.6 122.0 149.4 127.5 136.2 120.6 – 6 123.1 164.8 173.5 144.1 121.9 136.2 140.0 135.6 122.5 22.3 7 122.1 163.6 177.6 53.6 29.0 23.8 22.5 23.6 33.2 – Ex pe ri m en ta l C al cu la te d Figure 3. 13C NMR spectrum of compound 2. 254 Acta Chim. Slov. 2023, 70, 247–260 Muğlu et al.: New Bis-1,3,4-Thiadiazoles Based on Fumaric Acid: ... The activity type of any compound is decided by looking at the MBC/MIC ratio. If the MBC/MIC ratio is lower than 4 the activity of this compound is accepted as bactericidal, otherwise the activity is decided to be bacte- riostatic.28 The MBC test applied after the MIC test for K. pneu- moniae shows that compounds 2 and 3 presented a bacte- ricidal activity because MBC/MIC is lower than 4. Com- pounds 4, 5, 6 and 7 showed bacteriostatic activity because MBC/MIC is equal to 4. MIC test results demonstrated that compounds 3 and 7 have antibacterial activity on Gram-negative E. coli at concentrations of 5.5 mg/L. According to the MBC test, compound 3 exhibited a bactericidal activity, but it is not possible to conclude whether the activity of compound 7 is bactericidal or bacteriostatic. Because the MBC of the compound could not be determined, thus MBC/MIC ratio cannot be calculated. Compounds 4, 6, and 7 show antibacterial activity on Gram-negative B. subtilis at 2.75 mg/L, and 5.5 mg/L for compound 5. MBC test shows that compounds 4 and 7 possess bactericidal activity because MBC/MIC ratio is lower than 4. On the other hand, since the MBC of com- pounds 5 and 6 could not be determined, it is not possible to conclude whether the activities of these two compounds are bactericidal or bacteriostatic. Compounds 3, 5, 6, and 7 exhibited antibacterial ac- tivity on Gram-negative E. faecium at concentrations of 5.5 mg/L for each. MBC test shows that compounds 5 and 6 have bactericidal activities because MBC/MIC ratios are lower than 4. On the other hand, since the MBC of com- pounds 3 and 7 could not be determined, it is not possible to conclude whether the activities of these two compounds are bactericidal or bacteriostatic. Rauckyte et al.29 synthesized some 1,3,4-thiadiazole derivatives and investigated 12.5, 25.0, 50.0, and 100 g/L concentrations of these compounds for their antibacterial activity against selected bacterial strains. As a result, they observed that the MIC value against E. coli for 2-aceta- mide-1,3,4-thiadiazol-5-sulfonamide was 12.5 g/L. In our study, 1,3,4-thiadiazole derivatives (compounds 3 and 7) showed antibacterial activity against E. coli with MIC val- ues of 0.0055 g/L. It was seen that our compounds showed higher antibacterial activity against E. coli, compared to the compounds synthesized by Rauckyte et al..29 But these two results cannot be compared since the E. coli strains were not the same. The MIC value against E. faecium for 2-aceta- mide-1,3,4-thiadiazol-5-sulfonamide was 12.5 g/L as de- termind by Rauckyte et al.29 In our study, compounds 3, 5, 6, and 7 demonstrated antibacterial activities with MIC values of 0.0055 g/L. This shows that our compounds have higher antibacterial activity against E. faecium, but since the strains used in these studies were different it is not suit- able to compare these results. The MIC value against B. subtilis for 2-aceta- mide-1,3,4-thiadiazol-5-sulfonamide has shown no activ- ity as determined by Rauckyte et al.29 In the study con- ducted for B. subtilis, compounds 4, 5, 6, and 7 showed antibacterial activity with MIC values either 0.00275 or 0.0055 g/L. This indicates that the compounds show high- er antibacterial activity against B. subtilis, again it is not convenient to compare the results as the B. subtilis strains used in these studies were different. Table 5. MIC and MBC values of the synthesized compounds (mg/L). Bacteria Activity 1 2 3 4 5 6 7 GEN TOB CPFX S. enterica serovar Kentucky MIC – – – – – – – MBC – – – – – – – 2.500 2.500 0.078 E. coli MIC– – – 5.5 – – – 5.5 MBC – – 5.5 – – – – 10.00 10.00 – K. pneumoniae MIC – 2.75 2.75 1.375 1.375 1.375 1.375 MBC 5.5 5.5 5.5 5.5 5.5 5.5 0.078 0.078 – S. macrescens MIC – – – – – – – MBC – – – – – – – – – – P. aeruginosa MIC – – – – – – – MBC – – – – – – – 10.00 10.00 1.250 S. epidermidis MIC – – – – – – – MBC – – – – – – – – – – B. subtilis MIC – – – 2.75 5.5 2.75 2.75 MBC – – – 5.5 – 5.5 1.250 2.500 – E. faecium MIC – – 5.5 – 5.5 5.5 5.5 MBC – – – – 5.5 5.5 – – 0.625 0.156 GEN: gentamycin, TOB: tobramycin, CPFX: ciprofloxacin. 255Acta Chim. Slov. 2023, 70, 247–260 Muğlu et al.: New Bis-1,3,4-Thiadiazoles Based on Fumaric Acid: ... Moshafi et al.30 synthesized 5-nitro-2-furyl and 5-ni- tro-2-imidazolyl derivatives of 1,3,4-thiadiazole and test- ed their antibacterial activity against S. epidermidis PTCC 1114, B. subtilis PTCC 1023, Streptococcus pyogenes PTCC 1447, Micrococcus luteus PTCC 1110, E. coli PTCC 1330, P. aeruginosa PTCC 1074, K. pneumoniae PTCC 1053, S. marcescens PTCC 1621 with a concentration range of 0.5, 1, 2, 4, 8, 16, 32 and 64 mg/L. As a result, they observed that the activity of the compounds were ranging between 0.50 and 64.0 mg/L. For example, the MIC for 2-(1-methyl-5- nitro-H1-imidazol-2-yl)-5-(n-pentylsulfinyl)-1,3,4-thiad- iazole against K. pneumoniae was obtained to be 0.50–64.0 mg/L In addition, the same MIC value was observed for 2-(5-nitro-2-furyl)-5-(n-butylthio)-1,3,4-thiadiazole against E. coli. Also, 2-(5-nitro-2-furyl)-5-(n-butylsulfon- yl)-1,3,4-thiadiazole presented a MIC value of 0.50–16.0 mg/L against B. subtilis. In our study, compounds 2, 3, 4, 5, 6, and 7 showed antibacterial activity against K. pneu- moniae with MIC values of 2.75 and 1.375 mg/L. Again, compounds 3 and 7 presented MIC values of 5.5 mg/L against E. coli. In addition, compounds 4, 5, 6, and 7 showed antibacterial activity with MIC values either 2.75 or 5.5 g/L against B. subtilis. These results indicate that our compounds usually showed higher antibacterial activity against K. pneumoniae, E. coli, and B. subtilis. However, these differences may be due to the use of different strains. 3. 6. Theoretical Analysis Although electronic parameters such as the kinetic stability or chemical hardness of a molecule, hence its Eg, have a role on the probability of a chemical reaction to take place, the unpredictability of the steps of the reac- tions with living organisms such as bacteria causes these electronic parameters to be interpreted with hypothet- ical approaches specific to the relevant experiment in a way that supports the experimental data. It is known that HOMOs are the parameters that determine the capacity to donate electrons and LUMOs to accept electrons. The HOMO-LUMO gap represents the range in the binding energy spectrum in which the most probable excitations can occur, and indeed there is an inverse relationship be- tween the HOMO-LUMO energy gap and the probabil- ity of excitations, i.e. the smaller the Eg means that the excitation will occur more easily. Furthermore, Eg can be a useful tool to study (or predict) the stability and chem- ical reactivity of a molecule, taking into account its sen- sitive dependence on many factors such as environment (reaction medium), molecular conformation, tempera- ture, conjugation effects, intra- and intermolecular in- teractions. The small HOMO-LUMO gap of a molecule results in a lower kinetic stability, meaning higher chemi- cal reactivity or lower chemical hardness. In this context, although not a very strong approximation, among syn- thesized compounds, compounds 4–7 can be expected to exhibit higher chemical reactivity than compounds 1–3 (see Figure 4b). The calculations show that the com- pounds can be classified in three groups: the first group, compounds 1–3 containing aliphatic structures attached to –NH; the second group, compounds 4–6 containing ar- omatic structures attached to –NH; and the third group, compound 7 with cyclic structure. The antibacterial ac- Figure 4. (a) HOMO-ESP and LUMO maps of compound 5; (b) HOMO-LUMO energy gap values of the compounds calculated in gas and DMSO phase; (c) Polarizability values of the compounds calculated in gas phase and DMSO phase (by 6-311++g(2d,2p) basis set). 256 Acta Chim. Slov. 2023, 70, 247–260 Muğlu et al.: New Bis-1,3,4-Thiadiazoles Based on Fumaric Acid: ... tivities of the first group compounds appear to be weak, where compound 1 shows no activity, but in this group, the antibacterial activity increases partially as the aliphatic component grows, but aliphatic components do not seem to have a significant effect on the Eg values of the com- pounds (see Figure 4b). It can be said that aromatic and cyclic structures reduce the Eg values of the compounds and increase the antibacterial activity. On the other hand, the fact that compound 7 has a higher Eg value (3.409 eV) than the compounds in the second group (3.143, 3.273, and 3.164 eV for compounds 4–6, respectively), but is more sensitive to bacterial diversity, strengthens the opin- ion that cyclic group is a stronger factor for antibacterial activity than aromatic structures. In addition, the fact that the Eg values obtained from the calculations in the DMSO phase are lower than those in the gas phase (see Figure 4b) leads to the expectation that the compounds will show higher activity in the solution. At this point, the solubili- ty of the compounds in DMSO is another parameter that affects the reactions. As it is known, the solvation energy is the difference of the total energies of the compound in the solvent and vacuum environment and depends on the nature of both the solvent and the compound (and the substituted groups). Solvation energies of the first group compounds were calculated to be lower than for those in the other groups (–16.40, –17.54, and –16.42 kcal/mol for compounds 1–3, respectively), which indicates that the first group compounds have poor solubility in DMSO. It was observed that the antibacterial properties of the second and third group compounds with high solubility or high solvation energies (between –18.76 kcal/mol and –22.82 kcal/mol) were higher (see Supplementary file, Ta- bles S1, S2 for all calculated electronic data). The interactions of the compounds with bacteria occur electrostatically in the first place, and in the lat- er stages, the intramolecular interactions are involved in determining the coordination of the reaction. At this point, although molecular polarizability is defined as the response of electron distribution on a molecule to an external static electric field, it is an important pa- rameter because the intermolecular interaction energy can be expressed in terms of polarization and dispersion contributions. The polarizability of the first group com- pounds to which the aliphatic groups are attached was calculated to be lower (230.778, 262.483, and 294.367 a.u. for compounds 1–3, respectively) than that of the other compounds containing aromatic (441.604, 423.492, and 433.128 a.u. for compounds 4–6, respectively) and cy- clic (361.137 a.u. for compound 7) structures (see Figure 4c). In addition, compound 1, which does not exhibit antibacterial properties, has the lowest polarizability. It was observed that the compounds with the highest po- larizability were those with aromatic rings. Calculations performed in the DMSO phase show that the polarizibity values of the compounds increase proportionally in this phase. Although the dipole moments and polarizabilities of the compounds contribute to the activation of reactive sites in intermolecular interactions, the conformational degrees of freedom of the compounds are another strong factor. π-Conjugation between thiadiazole groups reduc- es the conformational degrees of freedom of the com- pounds, and the reduction of the effect of intramolecular interactions on conformations increases the probability of the compounds to be exposed to steric effects during the reaction. Moreover, intramolecular interactions of al- iphatic, aromatic or cyclic structures become a variable of the degree of freedom as a limiting parameter of the conformational orientations of the compounds. Since in- tramolecular interactions change the electron density dis- tributions of the reactive sites, they can cause the reaction to occur easily and quickly, and vice versa. The average root mean square (RMS) errors of the superimpositions of the gas and DMSO phase conformations of the com- pounds were 0.057, 0.042, and 0.044 Å for the first group compounds, respectively; 0.019, 0.016, and 0.019 Å for the second group of compounds, respectively; for com- Figure 5. IRI surfaces of compound 5 and QTAIM data: Electron density (e/bohr3) in BCP and RCP; RCP: Ring critical point, BCP: Bond critical point. 257Acta Chim. Slov. 2023, 70, 247–260 Muğlu et al.: New Bis-1,3,4-Thiadiazoles Based on Fumaric Acid: ... pound 7 with a cyclic group, it was calculated as 0.175 Å. Cyclohexyl group does not show resonance effects due to its absence of π-conjugation, and the larger RMS error of interaction with DMSO than that of other compounds indicates a higher conformational freedom than in the other compounds. This property of cyclohexyl ring may mean that compound 7 can perform a high electrophilic attack as well as less exposure to the steric effect. More- over, the cyclohexyl group pumps electrons to the thidi- azole region, and in addition to being exposed to nucleop- hilic attacks by molecular structures (or atoms) with high electronegativity, it also supports the electrophilic attack of the thiadiazole region. The IRI surfaces and QTAIM data of compound 4 are given in Figure 5 as a quantitative visualization of intramolecular interactions (see Supple- mentary Figure S19 for all compounds). The growth of electron donor aliphatic groups in the first group compounds caused an increase in electron density towards the central thiadiazole structures. In addi- tion, in terms of the change of electron distribution on the compounds, both the conformational and electronic prop- erties of aliphatic, aromatic and cyclic groups are among the factors that determine the degree of electrophilic at- tack (or nucleophilic attack) of the compounds. Figure 6 shows the electrophilic and nucleophilic attack region maps of compound 6 (see Supplementary Figure S20 for all compounds). The methyl group pumps electrons to the phenyl ring inductively and exhibits ortho-para direct- ing behavior. An increase in electron density is observed both on the carbon atom to which it is attached and on the para carbon atom of the phenyl ring. A similar situ- ation is observed in the methoxy substituted compound 4 exhibiting both strong mesomeric and weak inductive effect. In compound 5, which has an electron-withdraw- ing chlorine substituent from the ring with an inductive effect, a result close to the effect of the methoxy group was observed. In addition, intramolecular interaction of –NH with phenolic groups and electron donation to the phenyl ring with strong mesomeric effect strengthens the electro- philic attacks of compounds 4–6. In this context, it can be said that the second group compounds can perform elec- trophilic attack, which is partially stronger than the cyclic compound 7, but much stronger than the first group com- pounds. The nucleophilic attack regions of the compounds are predominantly concentrated around the alkenylic group (–C=C–) and the thiadiazole carbon atom to which this group is attached. Considering whether the compounds show anti- bacterial action properties in terms of electrophilic and nucleophilic attacks, we can say that the reaction with K. pneumoniae, B. subtilis, and E. faecium bacteria occurs through electrophilic attacks, because for all of the com- pounds, the nucleophilic attack sites are on the thiadi- azole and alkenylic groups; if the reactions had occurred through nucleophilic attacks, similar reaction rates would be expected for each bacterial species, but this was not ob- served for other bacterial species. At this point, the inabili- ty of the nucleophilic attack regions to perform the desired reaction may be due to the π-conjugation of these regions forcing the molecular structure to be planar and the ali- phatic, aromatic and cyclic groups creating a steric effect. Moreover, the inability of the compounds to show activity on S. enterica serovar Kentucky, S. macrescens, P. aerugino- sa, and S. epidermidis species may be due to the fact that these bacterial species react with nucleophilic rather than electrophilic attacks. Figure 6. Electrophilic and nucleophilic attack sites maps of compound 6 (by 6-31g(d) basis set). 258 Acta Chim. Slov. 2023, 70, 247–260 Muğlu et al.: New Bis-1,3,4-Thiadiazoles Based on Fumaric Acid: ... 4. Conclusions In this study, seven new bis-1,3,4-thiadiazoles 1–7 were obtained in excellent yields (61–93%) starting from fumaric acid. The synthesized compounds were charac- terized with FT-IR, 1H NMR and 13C NMR spectroscopic methods and elemental analysis. The conformations of the compounds and some electronic parameters were calcu- lated. Experimental spectroscopic data were supported by DFT calculations. As a result of experimental MIC procedures, thiad- iazoles were found to have antimicrobial activity against K. pneumoniae, E. coli, B. subtilis, and E. faecium. We also evaluated the antibacterial activity of newly synthesized compounds with the MBC test. As a result of the MBC test, it was observed that the activity of compounds 2–7 on K. pneumoniae, compounds 5 and 6 on E. faecium, com- pounds 4 and 7 on B. subtilis, and compound 3 on E. coli were bactericidal. None of the compounds was found to have activity against P. aeruginosa, S. epidermidis, S. enter- ica serovar Kentucky, and S. macrescens. Although the calculations cannot give definite an- swers in determining the antibacterial properties of the compounds, the fact that the antibacterial experiments give very close results each time reveals the existence of dominant variables that determine the results of the re- actions. In this study, it was investigated whether there is a relationship between the antibacterial properties of the compounds and electronic data, electrophilic and nu- cleophilic attack sites. Although the HOMO-LUMO gap is a quantity related to the reactivity of the compounds, single molecular calculations cannot determine the con- formational orientations caused by intermolecular inter- actions in the reaction medium of the compounds and the changes in electronic data caused by these orienta- tions, that is, the Eg values change directly depending on the intermolecular interactions (hence their confor- mation) of the compounds during the reaction process, which prevents Eg from being a quantitative determinant of the reactions. However, it is also possible to make some predictions, albeit rough, for compounds that are similar derivatives of each other and in which only the substit- uents change. Especially in molecules with less confor- mational freedom, the effect of substituted groups can be analyzed with more accurate results. In this context, electronic data such as Eg, polarizability, solvent effects and Fukui function maps calculated for compounds 1–7 can be helpful in determining the reactivity and possible electrophilic/nucleophilic attack properties of the com- pounds, albeit partially. Although it is undeniable that more data is needed for the results of unknown variables in the reaction environment with bacteria, it was also observed in calculations that compounds with aromat- ic and cyclic groups are more reactive than compounds containing aliphatic groups, in accordance with experi- mental data. Acknowledgements We are grateful to the Scientific Research Centre for Industrial and Technological Applications and Research Centre (Gubitam) and Dr. Ömer Faruk Ensari for taking the NMR spectra. This study’ synthesis, characteriza- tion, and antibacterial parts are belong to Ghaith Alabed Ibrayke Elefkhakry’ master thesis. Author contribution statement Halit Muğlu: Supervision, Methodology, Synthesis, Writing-Review. Hasan Yakan: Conceptualization, Meth- odology, Structure Characterization, Writing-Review. Ghaith Alabed Ibrayke Elefkhakry: Synthesis, Investiga- tion, Methodology. Ergin Murat Altuner: Antibacterial Activities Assay, Methodology, Writing-Review. M. Serdar Çavuş: Theoretical Calculations, Writing–Review. Declaration of competing interest The authors declare that they have no conflict of in- terest. This study was not supported by any organization . Supplementary Material All spectra (FT-IR, 1H NMR and 13C NMR ) of the compounds are presented in the supporting information. Data availability The datasets generated and/or analyzed during the current study are available from the corresponding author on reasonable request. 5. References 1. (a) K. Gowda, H. A. Swarup, S. C. Nagarakere, S. Rangappa, R. S. Kanchugarkoppal, M. Kempegowda, Synth. Commun. 2020, 50, 1528–1544; DOI:10.1080/00397911.2020.1745843 (b) H. Muğlu, N. Şener, H. A. M. Emsaed, S. Özkınalı, O. E. Özkan, M. Gür, J. Mol. Struct. 2018, 1174, 151–159; DOI:10.1016/j.molstruc.2018.03.116 (c) H. Tahtaci, M. Er, T. Karakurt, K. Sancak, Tetrahedron 2017, 73, 4418–4425; DOI:10.1016/j.tet.2017.06.006 (d) A. Al-Mulla, Der Pharma Chem. 2017, 9, 141–147; (e) M. A. Al-Omair, A. R. Sayed, M. M. Youssef, Molecules 2015, 20, 2591–2610; DOI:10.3390/molecules20022591 (f) J. K. Gupta, R. K. Yadav, R. Dudhe, P. K. Sharma, Int. J. Pharmtech Res. 2010, 2, 1493–1507; (g) M. Hanif, M. Saleem, M. T. Hussain, N. H. Rama, S. Zaib, M. A. M. Aslam, P. G. Jones, J. Iqbal, J. Braz. Chem. Soc. 2012, 23, 854–860; (h) K. Shrivastava, S. Purohit, S. Singhal, Asian J. Biomed. Pharm. Sci 2013, 3, 6–23; (i) B. Ardan, Y. Slyvka, E. Goreshnik, M. Mys’kiv, Acta Chim. Slov. 2013, 60, 484–490. 259Acta Chim. Slov. 2023, 70, 247–260 Muğlu et al.: New Bis-1,3,4-Thiadiazoles Based on Fumaric Acid: ... 2. I. Khan, S. Ali, S. Hameed, N. H. Rama, M. T. Hussain, A. Wa- dood, R. Uddin, Z. Ul-Haq, A. Khan, S. Ali, Eur. J. Med. Chem. 2010, 45, 5200–5207. DOI:10.1016/j.ejmech.2010.08.034 3. L. Yu, X. Gan, D. Zhou, F. He, S. Zeng, D. Hu, Molecules 2017, 22, 658. 4. X. Wang, W.-G. Duan, G.-S. Lin, M. Chen, F.-H. Lei, Res. Chem. Intermed. 2021, 47, 4029–4049. DOI:10.1007/s11164-021-04510-x 5. (a) W. S. Hamama, M. E. Ibrahim, H. A. Raoof, H. H. Zoorob, J. Heterocycl. Chem. 2017, 54, 2360–2366; DOI:10.1002/jhet.2826 (b) H. Muğlu, H. Yakan, H. A. Shouaib, J. Mol. Struct. 2020, 1203, 127470; DOI:10.1016/j.molstruc.2019.127470 (c) A. H. Moustafa, D. H. Ahmed, M. T. El-Wassimy, M. F. Mohamed, Synth. Commun. 2021, 51, 570–584; DOI:10.1080/00397911.2020.1843179 (d) Y. Liu, G. Liang, D. Yin, Res. Chem. Intermed. 2015, 41, 2019–2024; DOI:10.1007/s11164-013-1328-4 (e) A. Aly, R. El-Sayed, Chem. Pap. 2006, 60, 56–60. DOI:10.1016/S8756-5005(08)70230-0 6. S. Schenone, C. Brullo, O. Bruno, F. Bondavalli, A. Ranise, W. Filippelli, B. Rinaldi, A. Capuano, G. Falcone, Biorg. Med. Chem. 2006, 14, 1698–1705. DOI:10.1016/j.bmc.2005.10.064 7. N. Siddiqui, S. B. Andalip, R. Ali, O. Afzal, M. J. Akhtar, B. Azad, R. Kumar, J. Pharm. Bioallied Sci. 2011, 3, 194–212. DOI:10.4103/0975-7406.80765 8. F. Poorrajab, S. K. Ardestani, S. Emami, M. Behrouzi-Fard- moghadam, A. Shafiee, A. Foroumadi, Eur. J. Med. Chem. 2009, 44, 1758–1762. DOI:10.1016/j.ejmech.2008.03.039 9. J. J. Luszczki, M. Karpińska, J. Matysiak, A. Niewiadomy, Pharmacol. Rep. 2015, 67, 588–592. DOI:10.1016/j.pharep.2014.12.008 10. S. M. Gomha, Z. A. Muhammad, H. M. Gaber, M. M. Amin, J. Heterocycl. Chem. 2017, 54, 2708–2716. DOI:10.1002/jhet.2872 11. B. Kasetti Ashok, I. Singhvi, N. Ravindra, A. B. Shaik, Rev. Roum. Chim 2020, 65, 771–776. DOI:10.33224/rrch.2020.65.9.01 12. (a) I. Singh, L. H. Al-Wahaibi, R. Srivastava, O. Prasad, S. K. Pathak, S. Kumar, S. Parveen, M. Banerjee, A. A. El-Emam, L. Sinha, ACS Omega 2020, 5, 30073–30087; DOI:10.1021/acsomega.0c04474 (b) U. A. Çevik, D. Osmaniye, S. Levent, B. N. Sağlik, B. K. Çavuşoğlu, A. B. Karaduman, Y. Özkay, Z. A. Kaplancikli, Acta Pharm. 2020, 70, 499–513. DOI:10.2478/acph-2020-0034 13. M. M. Wassel, Y. A. Ammar, G. A. E. Ali, A. Belal, A. B. Meh- any, A. Ragab, Bioorg. Chem. 2021, 110, 104794. DOI:10.1016/j.bioorg.2021.104794 14. F. Prestinaci, P. Pezzotti, A. Pantosti, Pathog. Glob. Health 2015, 109, 309–318. DOI:10.1179/2047773215Y.0000000030 15. P. Cos, A. J. Vlietinck, D. V. Berghe, L. Maes, J. Ethnophar- macol. 2006, 106, 290–302. DOI:10.1016/j.jep.2006.04.003 16. K. Canli, Ö. Şimşek, A. Yetgin, E. M. Altuner, Bangladesh J. Pharmacol. 2017, 12, 463–469. DOI:10.3329/bjp.v12i4.33652 17. (a) I. Wiegand, K. Hilpert, R. E. Hancock, Nat. Protoc. 2008, 3, 163–175; DOI:10.1038/nprot.2007.521 (b) B. Baldas, E. M. Altuner, Communications Faculty of Sciences University of Ankara Series C Biology 2018, 27, 1–10. 18. (a) A. Vollekova, D. Košťálová, R. Sochorova, Folia Microbio- logica 2001, 46, 107–111; DOI:10.1007/BF02873586 (b) H. Usman, A. Haruna, I. Akpulu, M. Ilyas, A. Ahmadu, Y. Musa, J. Trop. Biosci 2005, 5, 72–76. 19. E. M. Altuner, B. Çetin, Kastamonu Üniversitesi Orman Fakültesi Dergisi 2018, 18, 126–137. DOI:10.17475/kastorman.315779 20. (a) P. Hohenberg, W. Kohn, Phys. Rev. 1964, 136, B864–B871; DOI:10.1103/PhysRev.136.B864 (b) W. Kohn, L. J. Sham, Phys. Rev. 1965, 140, A1133–A1138; DOI:10.1103/PhysRev.140.A1133 (c) N. A. Sánchez-Bojorge, L. M. Rodríguez-Valdez, N. Flores-Holguín, J. Mol. Model. 2013, 19, 3537–3542. DOI:10.1007/s00894-013-1878-9 21. M. J. Frisch, G. W. Trucks, H. B. Schlegel, G. E. Scuseria, M. A. Robb, J. R. Cheeseman, G. Scalmani, V. Barone, B. Men- nucci, G. A. Petersson, H. Nakatsuji, M. Caricato, X. Li, H. P. Hratchian, A. F. Izmaylov, J. Bloino, G. Zheng, J. L. Sonnen- berg, M. Hada, M. Ehara, K. Toyota, R. Fukuda, J. Hasega- wa, M. Ishida, T. Nakajima, Y. Honda, O. Kitao, H. Nakai, T. Vreven, J. A. Montgomery, Jr., J. E. Peralta, F. Ogliaro, M. Bearpark, J. J. Heyd, E. Brothers, K. N. Kudin, V. N. Starover- ov, R. Kobayashi, J. Normand, K. Raghavachari, A. Rendell, J. C. Burant, S. S. Iyengar, J. Tomasi, M. Cossi, N. Rega, J. M. Millam, M. Klene, J. E. Knox, J. B. Cross, V. Bakken, C. Ada- mo, J. Jaramillo, R. Gomperts, R. E. Stratmann, O. Yazyev, A. J. Austin, R. Cammi, C. Pomelli, J. W. Ochterski, R. L. Martin, K. Morokuma, V. G. Zakrzewski, G. A. Voth, P. Salvador, J. J. Dannenberg, S. Dapprich, A. D. Daniels, Ö. Farkas, J. B. Foresman, J. V. Ortiz, J. Cioslowski, D. J. Fox, M. J. Frisch, G. W. Trucks, H. B. Schlegel, G. E. Scuseria, M. A. Robb, J. R. Cheeseman, G. Scalmani, V. Barone, B. Mennucci, G. A. Petersson, H. Nakatsuji, M. Caricato, X. Li, H. P. Hratchi- an, A. F. Izmaylov, J. Bloino, G. Zheng, J. L. Sonnenberg, M. Hada, M. Ehara, K. Toyota, R. Fukuda, J. Hasegawa, M. Ishi- da, T. Nakajima, Y. Honda, O. Kitao, H. Nakai, T. Vreven, J. A. Montgomery, Jr., J. E. Peralta, F. Ogliaro, M. Bearpark, J. J. Heyd, E. Brothers, K. N. Kudin, V. N. Staroverov, R. Kobayas- hi, J. Normand, K. Raghavachari, A. Rendell, J. C. Burant, S. S. Iyengar, J. Tomasi, M. Cossi, N. Rega, J. M. Millam, M. Klene, J. E. Knox, J. B. Cross, V. Bakken, C. Adamo, J. Jaramillo, R. Gomperts, R. E. Stratmann, O. Yazyev, A. J. Austin, R. Cam- mi, C. Pomelli, J. W. Ochterski, R. L. Martin, K. Morokuma, V. G. Zakrzewski, G. A. Voth, P. Salvador, J. J. Dannenberg, S. Dapprich, A. D. Daniels, Ö. Farkas, J. B. Foresman, J. V. Ortiz, J. Cioslowski, D. J. Fox, Gaussian 09, Revision B.01, Gaussian, Inc., Wallingford CT, 2009. 22. (a) R. F. Bader, Acc. Chem. Res. 1985, 18, 9–15; DOI:10.1021/ar00109a003 (b) R. F. Bader, Chem. Rev. 1991, 91, 893–928; DOI:10.1021/cr00005a013 260 Acta Chim. Slov. 2023, 70, 247–260 Muğlu et al.: New Bis-1,3,4-Thiadiazoles Based on Fumaric Acid: ... (c) V. S. Duarte, G. D. D’Oliveira, J. M. Custodio, S. S. Ol- iveira, C. N. Perez, H. B. Napolitano, J. Mol. Model. 2019, 25, 1–14; DOI:10.1007/s00894-019-4082-8 (d) A. S. Abo Dena, Z. A. Muhammad, W. M. Hassan, Chem. Pap. 2019, 73, 2803–2812. DOI:10.1007/s11696-019-00833-7 23. T. Lu, F. Chen, J. Comput. Chem. 2012, 33, 580–592. DOI:10.1002/jcc.22885 24. (a) A. Ç. Karaburun, U. Acar Çevik, D. Osmaniye, B. N. Sağlık, B. Kaya Çavuşoğlu, S. Levent, Y. Özkay, A. S. Koparal, M. Behçet, Z. A. Kaplancıklı, Molecules 2018, 23, 3129; DOI:10.3390/molecules23123129 (b) M. Gür, N. Şener, H. Muğlu, M. S. Çavuş, O. E. Özkan, F. Kandemirli, İ. Şener, J. Mol. Struct. 2017, 1139, 111–118; DOI:10.1016/j.molstruc.2017.03.019 (c) Y.-T. Liu, L. Feng, D.-W. Yin, B.-J. Su, Res. Chem. Intermed. 2014, 40, 1607–1612. DOI:10.1007/s11164-013-1065-8 25. W. Kemp, Nuclear Magnetic Resonance Spectroscopy. In: Or- ganic Spectroscopy, Palgrave, London, 1991, pp. 101–241. DOI:10.1007/978-1-349-15203-2_3 26. (a) S. Ningaiah, U. K. Bhadraiah, A. Sobha, D. Shridevi, Poly- cycl. Aromat. Compd. 2020, 1–11; (b) N. Kerru, L. Gummidi, S. V. Bhaskaruni, S. N. Maddila, S. B. Jonnalagadda, Monatsh. Chem. 2020, 151, 981–990; DOI:10.1007/s00706-020-02625-2 (c) H. Muğlu, M. Akın, M. S. Çavuş, H. Yakan, N. Şaki, E. Güzel, Comput. Biol. Chem. 2022, 96, 107618. DOI:10.1016/j.compbiolchem.2021.107618 27. H. Yakan, El-Cezeri Journal of Science and Engineering 2021, 8, 155–163. 28. T. R. Keepers, M. Gomez, C. Celeri, W. W. Nichols, K. M. Krause, Antimicrob. Agents Chemother. 2014, 58, 5297–5305. DOI:10.1128/AAC.02894-14 29. T. Rauckyte-Żak, B. Szejniuk, Ecol. Chem. Eng., A 2011, 18, 1691–1704. 30. M. H. Moshafi, A. Peymani, A. Foroumadi, M. R. Zabihi, F. Doostishoar, Intern. Med. Med. Investig. J. 2019, 4, 1–8. DOI:10.24200/imminv.v4i2.213 Povzetek Serijo novih bis-1,3,4-tiadiazolov 1–7 smo pripravili z reakcijo med fumarno kislino in N-(alkil/aril/ciklo)tiosemikar- bazidi v prisotnosti fosforjevega oksiklorida. Strukture vseh spojin smo določili z FT-IR, 1H NMR in 13C NMR ter z elementno analizo. Antibakterijsko aktivnost vseh spojin smo proučili na osmih izbranih bakterijah. Spojine 2–7 iz- kazujejo učinek na Klebsiella pneumoniae. Vendar pa nobena izmed spojin ni učinkovala na Pseudomonas aeruginosa, Staphylococcus epidermidis, Salmonella enterica serovar Kentucky, in Serratia marcescens. SCRF (“self-consistent reaction force”) izračune smo izvajali v DMSO kot mediju z namenom ugotoviti energije solvatacij s pomočjo CPCM in SMD modelov. Pri DFT izračunih smo uporabili bazne sete 6-31G(d) and 6-311++G(2d,2p). Poleg elektronskih parametrov smo raziskali tudi elektronegativnost, elektrofilnost in spektroskopske lastnosti spojin, QTAIM, lokalne elektronske afi- nitete ter izvedli Fukuijevo analizo. Teoretični rezultati podpirajo eksperimentalna opažanja ter nakazujejo, da spojine, ki vsebujejo aromatske ali ciklične skupine, izkazujejo močnejše antibakterijsko delovanje kot spojine, ki vsebujejo alifatske skupine. Except when otherwise noted, articles in this journal are published under the terms and conditions of the  Creative Commons Attribution 4.0 International License 261Acta Chim. Slov. 2023, 70, 261–273 Abdallah et al.: Novel 5,6,7,8-tetrahydrobenzo[b]pyran Derivatives: ... DOI: 10.17344/acsi.2022.7893 Scientific paper Novel 5,6,7,8-tetrahydrobenzo[b]pyran Derivatives: Synthesis and Anticancer Activity Amira E. M. Abdallah,1* Rafat M. Mohareb,2 Maher H. E. Helal1 and Mariam M. Abd Elkader1 1 Department of Chemistry, Faculty of Science, Helwan University, Ain Helwan, Cairo, 11795, A. R. Egypt 2 Department of Chemistry, Faculty of Science, Cairo University, Giza 12614, A. R. Egypt * Corresponding author: E-mail: amiraelsayed135@yahoo.com Tel: +2 01091769838 Received: 11-19-2022 Abstract Many new cyclized pyran systems with a potential anti-cancer activity were designed and prepared. Pyran systems showed high reactivity to various chemical reagents. 24 products of the prepared compounds were chosen and tested in (mM) as respectable anticancer factors. The findings revealed that compounds 3b, 6b, and 8 were the widely effective compounds against the three cancer cell lines including A-549 (lung carcinoma), HC-29 (colorectal adenocarcinoma), and MKN-45 (gastric cancer) compared to the standard reference control foretinib. Keywords: Tetrahydrobenzo[b]pyran, thiophene, pyridine, anti-proliferative activity. 1. Introduction Pyran as a six-membered heterocyclic ring sys- tem was considered one of the most important rings in the synthesis of numerous bioactive fused systems with carbocyclic or heterocyclic ring systems. Figure 1 dis- plays some important pyran-based synthetic marketed drugs. Moreover, Figure 2 shows some natural product compounds which have the pyran ring in their struc- tures and are found in different food sources such as fruits, trees, and olive oil in addition to pigments in leaves.1 Due to the continuous need to prepare novel poly- functionalized heterocyclic compounds used in a variety of applications in industry and medicine, terahydroben- zo[b]pyrans were selected as important bioactive scaffolds widely utilized in such fields. For drug and pharmaceutical applications, tetrahydrobenzo[b]pyran derivatives were used as antiviral,2,3 antioxidant,4 anticancer,5–7 anticoagu- lant,8 diuretic,9,10 antimicrobial,11–13 anti-inflammatory,14 and anti-anaphylactic agents.15 In the area of industrial application, they were used as a raw material in laser dyes, food additives, hand soaps, detergents, lotions  and per- fume. Figure 1. Some important pyran-based synthetic marketed drugs. 262 Acta Chim. Slov. 2023, 70, 261–273 Abdallah et al.: Novel 5,6,7,8-tetrahydrobenzo[b]pyran Derivatives: ... Multi-component reactions were widely used to pre- pare tetrahydrobenzo[b]pyran systems in the presence of different catalysts,16–20 and reaction conditions such as mi- crowave, ultrasonic, or electrochemical synthesis conditions in deep eutectic solvents.21–25 These methods mostly intro- duce green synthesis for the production of tetrahydroben- zo[b]pyrans obtained in pure form with good yields and shorter reaction times than the other traditional methods. The current study constitutes a further thread in green organic chemistry and one-pot multi-component reactions (MCRs); it aims to improve the synthetic proce- dures of various prepared compounds.26–30 Herein prepa- rations of many 5,6,7,8-tetrahydrobenzo[b]pyrans are de- scribed through multi-component reactions and via other simple methods. The obtained products were tested on three human tumor cell lines, including A-549 (lung carci- noma), HC-29 (colorectal adenocarcinoma), and MKN- 45 (gastric cancer). 2. Experimental Section On a digital thermoelectric melting point instru- ment, the melting points were measured and are not cali- brated. By using Pye Unicam SP-1000 spectrophotometer, the infrared spectra (KBr disc) were determined. The Var- ian Gemini-300 (300 MHz) (Cairo University) instrument was utilized in the measurement of the 1H NMR spectra by using DMSO-d6 as solvent and TMS as the internal standard; chemical shifts (δ) are given in ppm. The mass spectrometry was carried out using a GCMS-QP2010 Shi- madzu instrument. The analytical data were recorded at Cairo University, by using a Vario El III Elemental CHNS analyzer. 2. 1. Synthetic Procedures 2. 1. 1. General Method for the Preparation of 2-Amino-4-phenyl-5,6,7,8-tetrahydro-4H- chromene-3-carbonitrile Derivatives 1a,b To a solution of compound cyclohexanone (0.98 g, 0.01 mol) in absolute ethanol (25 mL), either benzalde- hyde (1.06 g, 0.01 mol) or para-methoxybenzaldehyde (1.08 g, 0.01 mol) was added with malononitrile (0.66 g, 0.01 mol) in triethylamine (0.50 mL). Under reflux, the re- action was heated for 1 h. The resultant products were treated by adding them onto ice/water mixture with a few drops of HCl added. The precipitated product was collect- ed by filtration, and recrystallize from ethanol. 2-Amino-4-phenyl-5,6,7,8-tetrahydro-4H-chromene- 3-carbonitrile (1a). Brown crystals, yield: 1.66 g (66%), m.p. 257–260 °C. IR (ν, cm–1): 3417, 3340 (NH2), 3031 (CH-aromatic), 2932–2831 (CH2), 2209 (CN), 1645, 1599 (C=C). 1H NMR (δ, ppm): 1.66–1.71 (m, 4H, 2CH2 ), 2.16–2.81 (m, 4H, 2CH2 ), 5.73 (s, 1H, CH pyran), 7.14–7.89 (m, 7H, C6H5, NH2). 13C NMR (δ, ppm): 21.0, 24.9, 27.0, 42.9, 112.4, 116.2, 126.9, 128.6, 128.8, 128.9, 129.3, 132.4, 134.6, 143.5. Anal. Calcd for C16H16N2O (252.31): C, 76.16; H, 6.39; N, 11.10. Found: C, 76.21; H, 6.40; N, 11.12 2-Amino-4-(4-methoxyphenyl)-5,6,7,8-tetrahydro-4H- chromene-3-carbonitrile (1b). Pale brown crystals, yield: 1.98 g (70%), m.p. 269–272 °C. IR (ν, cm–1): 3419, 3340 (NH2), 3013 (CH aromatic), 2943–2836 (CH2, CH3), 2211 (CN), 1645, 1602 (C=C). 1H NMR (δ, ppm): 1.46–1.48 (m, 4H, 2CH2), 2.16–2.51 (m, 4H, 2CH2), 3.87 (s, 3H, OCH3), 5.72 (s, 1H, CH pyran), 6.99–7.89 (m, 6H, C6H4, NH2). Anal. Calcd for C17H18N2O2 (282.34): C, 72.32; H, 6.43; N, 9.92. Found: C, 72.55; H, 6.65; N, 10.29. 2. 1. 2. General Method for the Preparation of Ethyl N-(3-cyano-4-phenyl-5,6,7,8,- tetrahydro-4H-chromen-2-yl)formimidate Derivatives 2a,b To form a mixture of an equimolar amount of 1a (2.52 g, 0.01 mol) or 1b (2.82 g, 0.01 mol) in acetic acid (20 mL), triethyl orthoformate (1.45 g, 0.01 mol) was added. The reaction was refluxed for 2 h and then added to a mix- ture of ice/water with a few drops of HCl added. The ob- tained products were filtered and recrystallized by using acetic acid. Figure 2. Pyran-containing natural product compounds. 263Acta Chim. Slov. 2023, 70, 261–273 Abdallah et al.: Novel 5,6,7,8-tetrahydrobenzo[b]pyran Derivatives: ... Ethyl N-(3-cyano-4-phenyl-5,6,7,8,-tetrahydro-4H- chro men-2-yl)formimidate (2a). Green crystals, yield: 2.00 g (65%), m.p. 212–215 °C. IR (ν, cm–1): 3061 (CH-ar- omatic), 2937, 2868 (CH2, CH3), 2191 (CN), 1639, 1491 (C=C), 1580 (C=N). 1H NMR (δ, ppm): 1.20 (t, 3H, CH3), 1.56–1.91 (m, 4H, 2CH2), 2.49–2.51 (m, 4H, 2CH2), 4.25 (q, 2H, CH2), 6.35 (s, 1H, CH-pyran), 6.95 (s, 1H, CH), 7.19–7.52 (m, 5H, C6H5). MS m/z (%): 310 [M+ + 2] (1.51), 309 [M+ + 1] (1.51), 308 [M+] (1.28), 275 (100.00), 77 [C6H5]+ (14.10). Anal. Calcd for C19H20N2O2 (308.37): C, 74.00; H, 6.54; N, 9.08. Found: C, 74.29; H, 6.67; N, 9.40. Ethyl N-(3-cyano-4-(4-methoxyphenyl)-5,6,7,8,-tet- rahydro-4H-chromen-2-yl)formimidate (2b). Redish brown crystals, yield: 2.40 g (71%), m.p. 82–85 °C. IR (ν, cm–1): 3010 (CH-aromatic), 2935 (CH, CH2, CH3), 2200 (CN), 1637, 1510 (C=C), 1602 (C=N). 1H NMR (δ, ppm): 1.10 (t, 3H, CH3), 1.66–1.91 (m, 4H, 2CH2), 2.49–2.50 (m, 4H, 2CH2), 3.81 (s, 3H, OCH3), 4.30 (q, 2H, CH2), 5.43 (s, 1H, CH-pyran), 6.85 (s, 1H, CH), 6.88–7.89 (m, 4H, C6H4). 13C NMR (δ, ppm): 21.4, 22.0, 22.4, 24.9, 26.6, 55.1, 55.4, 113.8, 114.1, 141.5, 118.0, 129.3, 129.7, 131.8, 146.7, 149.2, 158.6, 159.2, 159.7. Anal. Calcd for C20H22N2O3 (338.40): C, 70.99; H, 6.55; N, 8.28. Found: C, 71.32; H, 6.90; N, 8.49. 2. 1. 3. General Method for the Preparation of N"-(3-Cyano-4-phenyl-5,6,7,8-tetrahydro- 4H-chromen-2-yl)formimidohydrazide derivatives 3a–d To an equimolar amount of 2a (3.08 g, 0.01 mol) or 2b (3.38 g, 0.01 mol) in absolute ethanol (25 mL), hydra- zine hydrate (0.50 g, 0.01 mol) or phenyl hydrazine (1.08 g, 0.01 mol) were added. By using the reflux heating, the re- action lasted for 3 h. The resultant products were treated by adding them to ice/water mixture with a few HC1 drops added. The resultant products were collected and filtered; then ethanol was used to recrystallize them. N"-(3-Cyano-4-phenyl-5,6,7,8-tetrahydro-4H-chro men- 2-yl)formimidohydrazide (3a). Yellow crystals, yield: 2.79 g (95%), m.p. 157–160 °C. IR (ν, cm–1): 3444, 3355 (NH2), 3243 (NH), 3070 (CH-aromatic), 2934, 2865 (CH2), 2197 (CN), 1639, 1448 (C=C), 1596 (C=N). 1H NMR (δ, ppm): 1.58–1.74 (m, 4H, 2CH2), 2.13–2.15 (m, 4H, 2CH2), 6.40 (s, 1H, CH-pyran), 6.96 (s, 1H, CH), 7.20–7.54 (m, 7H, C6H5, NH2), 10.80 (s, 1H, NH). 13C NMR (δ, ppm): 22.0, 22.3, 24.9, 26.7, 45.7, 95.4, 115.4, 115.7, 124.6, 127.6, 128.2, 128.5, 128.6, 143.6, 146.8, 150.1, 150.7. Anal. Calcd for C17H18N4O (294.35): C, 69.37; H, 6.16; N, 19.03. Found: C, 69.71; H, 6.30; N, 19.12. MS m/z (%): 295 [M+ + 1] (27.04), 294 [M+] (66.09), 293 [M+ – 1] (100.00). N"’-(3-Cyano-4-phenyl-5,6,7,8-tetrahydro-4H-chro- men-2-yl)-N’-phenylformimidohydrazide (3b). Dark brown crystals, yield: 2.82 g (75%), m.p. 107–110 °C. IR (ν, cm–1): 3442–3244 (2NH), 3075 (CH-aromatic), 2936, 2865 (CH2), 2211(CN), 1638, 1492 (C=C), 1598 (C=N). 1H NMR (δ, ppm): 1.56–1.74 (m, 4H, 2CH2), 2.13–2.17 (m, 4H, 2CH2), 6.37 (s, 1H, CH-pyran), 6.60 (s, 1H, CH), 7.19–7.86 (m, 10H, 2C6H5), 10.30 (s, 1H, NH), 10.90 (s, 1H, NH). MS m/z (%): 378 [M+ + 2] (4.24), 377 [M+ + 1] (6.47), 376 [M+] (8.13), 273 (100.00), 77 [C6H5]+ (20.56). Anal. Calcd for C23H28N4O (376.49): C, 69.37; H, 6.16; N, 19.03. Found: C, 69.39; H, 6.20; N, 19.04. N"-(3-Cyano-4-(4-methoxyphenyl)-5,6,7,8-tetrahydro- 4H-chromen-2-yl)formimidohydrazide (3c). Redish brown crystals, yield: 2.76 g (85%), m.p. 97–100 °C. IR (ν, cm–1): 3437, 3348 (NH2), 3225 (NH), 3080 (CH-aromat- ic), 2934, 2862 (CH2), 2197 (CN), 1639, 1511 (C=C), 1605 (C=N). 1H NMR (δ, ppm): 1.63–1.70 (m, 4H, 2CH2), 2.16– 2.20 (m, 4H, 2CH2), 3.60 (s, 3H, OCH3), 6.30 (s, 1H, CH- pyran), 6.86 (s, 1H, CH), 6.89–7.32 (m, 6H, C6H4, NH2), 10.90 (s, 1H, NH). 13C NMR (δ, ppm): 22.3, 24.9, 25.6, 26.6, 44.7, 55.1, 113.9, 114.1, 115.9, 119.0, 129.6, 129.7, 130.5, 149.2, 158.6. MS m/z (%): 322 [M+ – 2] (6.18), 305 (100.00), 76 [C6H4]+ (4.89). Anal. Calcd for C18H20N4O2 (324.38): C, 66.65; H, 6.21; N, 17.27. Found: C, 70.01; H, 6.30; N, 17.29. N"-(3-Cyano-4-(4-methoxyphenyl)-5,6,7,8-tetrahydro- 4H-chromen-2-yl)formimidohydrazide (3d). Dark brown crystals, yield: 4.04 g (99%), m.p. 82–85 °C. IR (ν, cm–1): 3435–3225 (NH), 3005 (CH-aromatic), 2861, 2838 (CH, CH2, CH3), 2207 (CN), 1639, 1510 (C=C), 1602 (C=N). 1H NMR (δ, ppm): 1.56–1.71 (m, 4H, 2CH2), 2.16– 2.20 (m, 4H, 2CH2), 3.86 (s, 3H, OCH3), 6.31 (s, 1H, CH- pyran), 6.96 (s, 1H, CH), 7.03–7.88 (m, 9H, C6H4, C6H5), 9.87 (s, 1H, NH), 10.10 (s, 1H, NH). MS m/z (%): 408 [M+ + 2] (38.22), 407 [M+ + 1] (35.80), 406 [M+] (26.39), 405 [M+ – 1] (12.85), 404 [M+ – 2] (7.92), 303 (100.00), 77 [C6H5]+ (35.65), 76 [C6H4]+ (5.20). Anal. Calcd for C24H30N4O2 (406.52): C, 71.98; H, 6.04; N, 13.99. Found: C, 72.12; H, 6.30; N, 13.99. 2. 1. 4. Synthesis of N’-(3-Cyano-4-phenyl -5,6,7,8-tetrahydro-4H-chromen-2-yl)-N- phenylformimidamide (4). For an equimolar amount of 2a (3.08 g, 0.01 mol) in absolute ethanol (25 mL), aniline (0.93 g, 0.01 mol) was added. The reaction was refluxed for 3 h and then the mix- ture was added to an ice/water mixture with a few HC1 drops added. The obtained product was filtered and recrys- tallized by using ethanol. Brown crystals, yield: 2.84 g (80%), m.p. 97–100 °C. IR (ν, cm–1): 3417–3242 (NH), 3058 (CH-aromatic), 2935, 2862 (CH, CH2), 2210 (CN), 1640, 1495 (C=C), 1596 (C=N). 1H NMR (δ, ppm): 1.54–1.79 (m, 4H, CH2), 2.13– 2.39 (m, 4H, CH2), 5.73 (s, 1H, CH-pyran), 6.80–7.58 (m, 264 Acta Chim. Slov. 2023, 70, 261–273 Abdallah et al.: Novel 5,6,7,8-tetrahydrobenzo[b]pyran Derivatives: ... 11H, 2C6H5, CH), 10.01 (s, 1H, NH). 13C NMR (δ, ppm): 21.4, 22.0, 24.9, 26.9, 42.9, 66.4, 112.6, 115.4, 115.8, 116.2, 124.3, 128.0, 128.2, 128.3, 128.5, 128.6, 129.3, 129.4, 137.3, 143.6, 146.9, 150.1, 150.7. MS m/z (%): 357 [M+ + 2] (31.33), 356 [M+ + 1] (40.77), 355 [M+] (23.18), 300 (100.00), 77 [C6H5]+ (36.91). Anal. Calcd for C23H21N3O (355.43): C, 77.72; H, 5.96; N, 11.82. Found: C, 77.94; H, 6.17; N, 12.20. 2. 1. 5. General Method for the Preparation of 2-(((3-Cyano-4-phenyl-5,6,7,8-tetrahydro- 4H-chromen-2-yl)imino)methyl) malononitrile derivatives 5a–d To compound 2a (3.08 g, 0.01 mol) or 2b (3.38 g, 0.01 mol) in absolute ethanol (25 mL), malononitrile (0.66 g, 0.01 mol) and ethyl cyanoacetate (1.13 g, 0.01 mol) were added. On the reflux system, the reaction was heated for 3 h. The resultant products were poured onto the ice/water mixture with a few drops of HCI added. The precipitated products were collect by filtration and then recrystallized from ethanol. 2-(((3-Cyano-4-phenyl-5,6,7,8-tetrahydro-4H-chro- men-2-yl)imino)methyl)malononitrile (5a). Yellow crystals, yield: 3.12 g (95%), m.p. 95–98 °C. IR (ν, cm–1): 3100 (CH-aromatic), 2936, 2865 (CH, CH2), 2260, 2220, 2208 (3CN), 1639, 1448 (C=C), 1597 (C=N). 1H NMR (δ, ppm): 1.49–1.72 (m, 4H, CH2), 2.13–2.18 (m, 4H, CH2), 5.80 (s, 1H, CH-pyran), 6.37, 6.93, (2d, 2H, 2CH), 7.10– 7.53 (m, 5H, C6H5). 13C NMR (δ, ppm): 21.1, 22.4, 24.4, 24.9, 26.9, 66.4, 112.4, 115.4, 115.8, 116.2, 124.3, 127.9, 128.2, 128.5, 128.6, 137.3, 143.6, 146.7, 150.1, 150.7. MS m/z (%): 330 [M+ + 2] (17.26), 329 [M+ + 1] (13.72), 328 [M+] (17.92), 327 [M+ – 1] (12.17), 300 (100.00), 77 [C6H5]+ (19.91). Anal. Calcd for C20H16N4O (328.42): C, 73.15; H, 4.91; N, 17.06. Found: C, 73.34; H, 4.95; N, 17.28. Ethyl-2-cyano-3-((3-cyano-4-phenyl-5,6,7,8-tetrahy- dro-4H-chromen-2-yl)imino)propanoate (5b). Brown crystals, yield: 2.48 g (66%), m.p. 147–150 °C. IR (ν, cm–1): 3061 (CH-aromatic), 2937, 2865 (CH, CH2, CH3), 2260, 2213 (2CN), 1697 (C=O), 1644, 1448 (C=C), 1595 (C=N). 1H NMR (δ, ppm): 1.19–1.21 (t, 3H, CH3), 1.54–1.74 (m, 4H, 2CH2), 2.13–2.17 (m, 4H, 2CH2), 4.19–4.21 (q, 2H, CH2), 5.75 (s, 1H, CH-pyran), 6.37, 6.80 (2d, 2H, 2CH), 7.20–7.53 (m, 5H, C6H5). MS m/z (%): 377 [M+ + 2] (4.15), 375 [M+] (2.60), 273 (100.00), 77 [C6H5]+ (9.84). Anal. Calcd for C22H21N3O3 (375.42): C, 70.38; H, 5.64; N, 11.19. Found: C, 70.58; H, 5.67; N, 11.29. 2-(((3-Cyano-4-(4-methoxyphenyl)-5,6,7,8-tetrahydro- 4H-chromen-2-yl)imino)methyl)malononitrile (5c). Red crystals, yield: 1.97 g (55%), m.p. 82–85 °C. IR (ν, cm–1): 3006 (CH-aromatic), 2936 (CH, CH2, CH3), 2260, 2203, 2190 (3CN), 1639, 1450 (C=C), 1604 (C=N). 1H NMR (δ, ppm): 1.46–1.73 (m, 4H, 2CH2), 2.16–2.25 (m, 4H, 2CH2), 3.84 (s, 3H, OCH3), 5.03 (s, 1H, CH-pyran), 6.32–6.70 (2d, 2H, 2CH), 6.85–7.99 (m, 4H, C6H4). Anal. Calcd for C21H18N4O2 (358.39): C, 70.38; H, 5.06; N, 15.63. Found: C, 70.39; H, 5.17; N, 15.70. Ethyl-2-cyano-3-(3-cyano-4-(4-methoxyphenyl)- 5,6,7,8-tetrahydro-4H-chromen-2-yl)imino)pro- panoate (5d). Yellow crystals, yield: 2.03 g (50%), m.p. 102–105 °C. IR (ν, cm–1): 3100 (CH-aromatic), 2935, 2862 (CH, CH2, CH3), 2260, 2202 (2CN), 1701 (C=O), 1639, 1451 (C=C), 1600 (C=N). 1H NMR (δ, ppm): 1.06–1.08 (t, 3H, CH3), 1.67–1.70 (m, 4H, 2CH2), 2.18–2.22 (m, 4H, 2CH2), 3.84 (s, 3H, OCH3), 4.22–4.24 (q, 2H, CH2), 5.70 (s, 1H, CH-pyran), 6.30, 6.67 (2d, 2H, 2CH), 6.86–7.32 (m, 4H, C6H4). MS m/z (%): 407 [M+ + 2] (4.56), 406 [M+ + 1] (4.27), 405 [M+] (2.99), 404 [M+ – 1] (2.63), 305 (100.00), 76 [C6H4]+ (6.13). Anal. Calcd for C23H23N3O4 (405.45): C, 68.13; H, 5.72; N, 10.36. Found: C, 68.33; H, 5.78; N, 10.69. 2. 1. 6. General Method for the Preparation of 2-Cyano-N-(3-cyano-4-phenyl-5,6,7,8- tetrahydro-4H-chromen-2-yl)acetamide derivatives 6a,b To form a mixture of equimolar amounts of 1a (2.52 g, 0.01 mol) or 1b (2.82 g, 0.01 mol) in N,N-dimethylfor- mamide (15 mL), ethyl cyanoacetate (1.13 g, 0.01 mol) was added. The chemical reaction was refluxed for 3 h and then added into a beaker containing a mixture of ice and water. The precipitated products were collected by filtration and recrystallized from N,N-dimethylformamide. 2-Cyano-N-(3-cyano-4-phenyl-5,6,7,8-tetrahydro-4H- chromen-2-yl)acetamide (6a). Pale brown crystals, yield: 3.18 g (99%), m.p. 250–253 °C. IR (ν, cm–1): 3417–3227 (NH), 3033 (CH-aromatic), 2932–2861 (CH2), 2260, 2209 (2CN), 1647 (C=O), 1600, 1448 (C=C). 1H NMR (δ, ppm): 1.45–1.49 (m, 4H, 2CH2), 2.16–2.20 (m, 4H, 2CH2), 3.80 (s, 2H, CH2), 5.73 (s, 1H, CH-pyran), 7.33–7.43 (m, 5H, C6H5), 10.01 (s, 1H, NH). 13C NMR (δ, ppm): 21.0, 24.8, 27.0, 42.9, 81.5, 112.3, 116.2, 126.9, 128.6, 128.8, 128.9, 129.3, 132.4, 134.6, 143.6. MS m/z (%): 317 [M+ – 2] (0.31), 300 (100.00), 77 [C6H5]+ (2.82). Anal. Calcd for C19H17N3O2 (319.36): C, 71.46; H, 5.37; N, 13.16. Found: C, 71.83; H, 5.38; N, 13.19. 2-Cyano-N-(3-cyano-4-(4-methoxyphenyl)-5,6,7,8-tet- rahydro-4H-chromen-2-yl)acetamide (6b). Reddish brown crystals, yield: 1.74 g (50%), m.p. 122–125 °C. IR (ν, cm–1): 3426–3242 (NH), 3100 (CH-aromatic), 2936 (CH2, CH3), 2260, 2208 (2CN), 1714 (C=O), 1593, 1439 (C=C). 1H NMR (δ, ppm): 1.66–1.71 (m, 4H, 2CH2), 2.10–2.20 (m, 4H, 2CH2), 3.73 (s, 3H, OCH3), 4.34 (s, 2H, CH2), 5.70 (s, 1H, CH-pyran), 6.91–7.90 (m, 4H, C6H4), 8.31 (s, 1H, 265Acta Chim. Slov. 2023, 70, 261–273 Abdallah et al.: Novel 5,6,7,8-tetrahydrobenzo[b]pyran Derivatives: ... NH). MS m/z (%): 347 [M+ – 2] (15.34), 330 (100.00). Anal.Calcd for C20H19N3O3 (349.38): C, 68.75; H, 5.48; N, 12.03. Found: C, 69.03; H, 5.49; N, 12.27 2. 1. 7. General Method for the Preparation of 3, 5-Diamino-4-cyano-N-(3-cyano-4-phenyl- 5,6,7,8-tetrahydro-4H-chromen-2-yl) thiophene-2-carboxamide derivatives 7a,b To an equimolar amounts of 6a (3.19 g, 0.01 mol) in absolute ethanol (25 mL) and triethylamine (0.50 mL), and either malononitrile (0.66 g, 0.01 mol) or ethyl cyano- acetate (1.13 g, 0.01 mol) was added with elemental sulfur (0.32 g, 0.01 mol). The reaction was refluxed for 3 h and then added onto the mixture of ice, water and HCl (a few drops). The precipitated products were collected by filtra- tion and then recrystallized from ethanol. 3,5-Diamino-4-cyano-N-(3-cyano-4-phenyl-5,6,7,8-tet- rahydro-4H-chromen-2-yl)thiophene-2-carboxamide (7a). Reddish brown crystals, yield: 2.97 g (71%), m.p. 201–204 °C. IR (ν, cm–1): 3417, 3340 (2NH2), 3244 (NH), 3034 (CH-aromatic), 2932, 2864 (CH2), 2260, 2209 (2CN), 1644 (C=O), 1601, 1448 (C=C). 1H NMR (δ, ppm): 1.45– 1.71 (m, 4H, 2CH2), 2.01–2.24 (m, 4H, 2CH2), 5.73 (s, 1H, CH-pyran), 7.28–7.62 (m, 9H, C6H5, 2NH2), 8.50 (s, 1H, NH). 13C NMR (δ, ppm): 21.0, 22.0, 24.8, 26.5, 42.8, 81.5, 112.3, 113.7, 116.2, 124.2, 128.2, 128.6, 128.7, 128.8, 132.3, 133.4, 134.6, 143.5. MS m/z (%): 418 [M+ + 1] (4.38), 417 [M+] (6.65), 300 (100.00). Anal. Calcd for C22H19N5O2S (417.48): C, 63.29; H, 4.59; N, 16.78; S, 7.68. Found: C, 63.30; H, 4.70; N, 16.98; S, 7.86. Ethyl 2,4-diamino-5-((3-cyano-4-phenyl--5,6,7,8-tet- rahydro-4H-chromen-2-yl)carbamyle)thiophene-3-car- boxylate (7b). Brown crystals, yield: 2.78 g (60%), m.p. 232–235 °C. IR (ν, cm–1): 3418, 3340 (2NH2), 3251 (NH), 3035 (CH-aromatic), 2933–2831 (CH2, CH3), 2209 (CN), 1709 (C=O), 1645, 1450 (C=C). 1H NMR (δ, ppm): 1.29– 1.31 (t, 3H, CH3), 1.66–1.71 (m, 4H, 2CH2), 2.15–2.20 (m, 4H, 2CH2), 4.31–4.34 (q, 2H, CH2), 5.73 (s, 1H, CH- pyran), 7.03–7.63 (m, 9H, C6H5, 2NH2), 8.41 (s, 1H, NH). Anal. Calcd for C24H24N4O4S (464.54): C, 62.05; H, 5.21; N, 12.06; S, 6.90. Found: C, 62.16; H, 5.23; N, 12.25; S, 7.11. 2. 1. 8. Synthesis of N-(3-Cyano-4-phenyl-5,6,7,8- tetrahydro-4H-chromen-2-yl)-2-oxo-2H- chromene-3-carboxamide (8) A solution of compound 6a (3.19 g, 0.01 mol) is made by adding absolute ethanol (25 mL) and piperidine (0.50 mL) with salicylaldehyde (1.22 g, 0.01 mol). The chemical reaction was refluxed for 3 h and then added into a beaker containing a mixture of ice and water. The precip- itated product was filtered and then recrystallized from ethanol. Brown crystals, yield: 2.13 g (50%), m.p. 91–94 °C. IR (ν, cm–1): 3434, 3245 (NH), 3054 (CH-aromatic), 2932, 2855 (CH2), 2215 (CN), 1738, 1696 (2C=O), 1598, 1441 (C=C). 1H NMR (δ, ppm): 1.69–1.71 (m, 4H, 2CH2), 2.10– 2.20 (m, 4H, 2CH2), 6.30 (s, 1H, CH-pyran), 6.89 (s, 1H, CH-coumarin), 6.92–7.37 (m, 9H, C6H4, C6H5), 8.25 (s, 1H, NH). 13C NMR (δ, ppm): 22.1, 22.3, 23.6, 25.6, 43.8, 87.9, 113.6, 114.6, 116.4, 118.2, 119.2, 125.2, 125.9, 127.2, 127.7, 128.1, 128.3, 128.6, 128.7, 129.1, 146.9, 148.3, 150.8, 153.2, 158.7, 163.2. MS m/z (%): 426 [M+ + 2] (0.45), 425 [M+ + 1] (1.54), 424 [M+] (4.77), 423 [M+ – 1] (17.73), 422 [M+ – 2] (31.09), 77 [C6H5]+ (1.97), 76 [C6H4]+ (0.83). Anal. Calcd for C26H20N2O4 (424.45): C, 73.57; H, 4.75; N, 6.60. Found: C, 73.60; H, 4.89; N, 6.98. 2. 1. 9. General Method for the Preparation of 1-(3-Cyano-4-phenyl-5,6,7,8-tetrahydro- 4H-chromen-2-yl)-4,6-dimethyl-2-oxo-1,2- dihydropyridine -3-carbonitrile derivatives 9a,b To a solution of compound 6a (3.19 g, 0.01 mol) in absolute ethanol (25 mL) and piperidine (0.50 mL), either acetylacetone (1.00 g, 0.01 mol) or ethyl acetoacetate (1.30 g, 0.01 mol) was added. The reaction was carried out for 3 h. Thereafter, the reaction mixture was poured onto the mixture of ice/water with a few drops of HC1 added. The precipitated products were collected by filtration and then recrystallized from ethanol. 1-(3-Cyano-4-phenyl-5,6,7,8-tetrahydro-4H-chromen- 2-yl)-4,6-dimethyl-2-oxo-1,2-dihydropyridine-3-car- bonitrile (9a). Reddish brown crystals, yield: 1.91 g (50 %), m.p. 71–74 °C. IR (ν, cm–1): 3034 (CH-aromatic), 2943, 2866 (CH2, CH3), 2258, 2218 (2CN), 1751 (C=O), 1636, 1447 (C=C). 1H NMR (δ, ppm): 1.02–1.06 (s, 3H, CH3), 1.08–1.29 (s, 3H, CH3), 1.60–1.70 (m, 4H, 2CH2), 2.10–2.20 (m, 4H, 2CH2), 5.70 (s, 1H, CH-pyran), 6.30 (s, 1H, CH-pyridine), 6.96–7.53 (m, 5H, C6H5). 13C NMR (δ, ppm): 21.5, 22.1, 22.3, 26.9, 27.1, 28.2, 40.5, 53.3, 113.6, 115.1, 115.5, 115.8, 120.6, 125.9, 127.0, 127.7, 128.6, 128.7, 143.5, 146.9, 150.1, 150.7, 158.7, 166.0. Anal. Calcd for C24H21N3O2 (383.44): C, 75.18; H, 5.52; N, 10.96. Found: C, 75.19; H, 5.81; N, 11.15. 1-(3-Cyano-4-phenyl-5,6,7,8-tetrahydro-4H-chromen- 2-yl)-6-hydroxy-4-methyl-2-oxo-1,2-dihydropyridine- 3-carbonitrile (9b). Brown crystals, yield: 2.76 g (72%), m.p. 56–58 °C. IR (ν, cm–1): 3442–3245 (OH), 3100 (CH-aromatic), 2940, 2869 (CH2), 2260, 2219 (2CN), 1745 (C=O), 1635, 1447 (C=C). 1H NMR (δ, ppm): 1.24–1.29 (s, 3H, CH3), 1.60–1.70 (m, 4H, 2CH2), 2.10–2.27 (m, 4H, 2CH2), 5.71 (s, 1H, CH-pyran), 6.26 (s, 1H, CH-pyridine), 7.26–7.53 (m, 5H, C6H5), 8.40 (s, 1H, OH). MS m/z (%): 387 [M+ + 2] (32.13), 386 [M+ + 1] (26.38), 385 [M+] (15.59), 384 [M+ – 1] (13.19), 383 [M+ – 2] (19.66), 346 266 Acta Chim. Slov. 2023, 70, 261–273 Abdallah et al.: Novel 5,6,7,8-tetrahydrobenzo[b]pyran Derivatives: ... (100.00). Anal. Calcd for C23H19N3O3 (385.42): C, 71.67; H, 4.97; N, 10.90. Found: C, 71.76; H, 5.24; N, 11.02. 2. 1. 10. Synthesis of 2-Cyano-N-(3-cyano-4- phenyl-5,6,7,8-tetrahydro-4H-chromen-2- yl)-3-ethoxyacrylamide (10) To a mixture of equimolar amounts of 6a (3.19 g. 0.01 mol) in acetic acid (25 mL), triethyl orthoformate (1.45 g, 0.01 mol) was added. The chemical reaction was refluxed for 1 h and then added into a beaker containing a mixture of ice and water. The resultant product was filtered and then recrystallized from acetic acid. Yellow powder, yield: 2.25 g (60%), m.p. 172–175 °C. IR (ν, cm–1): 3427–3245 (NH), 3100 (CH-aromatic), 2934, 2864 (CH2, CH3), 2260–2199 (2CN), 1701 (C=O), 1638– 1490 (C=C). 1H NMR (δ, ppm): 1.05–1.10 (t, 3H, CH3), 1.50–1.99 (m, 4H, 2CH2), 2.06–2.27 (m, 4H, 2CH2), 4.22– 4.24 (q, 2H, CH2), 5.80 (s, 1H, CH-pyran), 6.80–7.53 (m, 6H, C6H5, CH), 11.10 (s, 1H, NH). MS m/z (%): 377 [M+ + 2] (10.63), 376 [M+ + 1] (7.20), 375 [M+] (12.60), 374 [M+ – 1] (6.39), 373 [M+ – 2] (7.67), 329 (100.00), 77 [C6H5]+ (21.72). Anal. Calcd for C22H21N3O3 (375.42): C, 70.38; H, 5.64; N, 12.79. Found: C, 70.62; H, 5.82; N, 12.96. 2. 1. 11. Synthesis of 2-Cyano-N-(3-cyano-phenyl- 5,6,7,8-tetrahydro-4H-chromen-2-yl)-3- (phenyl amino) acrylamide (11) To a solution of compound 10 (3.75 g. 0.01 mol) in absolute ethanol (25 mL), aniline (0.93 g, 0.01 mol) was add- ed. The reaction was refluxed for 3 h and then poured onto an ice/water mixture with a few drops of HC1 added. The ob- tained product was filtered and recrystallized from ethanol. Yellow crystals, yield: 2.78 g (65%), m.p. 210–213 °C. IR (ν, cm–1): 3422–3244 (2NH), 3061 (CH-aromatic), 2934–2864 (CH, CH2), 2260, 2211 (2CN), 1696 (C=O), 1638, 1490 (C=C). 1H NMR (δ, ppm): 1.56–1.74 (m, 4H, 2CH2), 2.13–2.17 (m, 4H, 2CH2), 5.80 (s, 1H, CH-pyran), 6.96–7.50 (m, 11H, 2C6H5, CH), 8.50, 10.10 (2s, 2H, 2NH). MS m/z (%): 424 [M+ + 2] (33.57), 423 [M+ + 1] (20.37), 422 [M+] (14.63), 273 (100.00), 77 [C6H5]+ (42.32). Anal. Calcd for C26H22N4O2 (422.48): C, 73.92; H, 5.25; N, 13.26. Found: C, 74.09; H, 5.29; N, 13.28. 2. 1. 12. General Method for the Preparation of 6-Amino-1-(3-cyano-4-phenyl-5,6,7,8- tetrahydro-4H-chromen-2-yl)-2-oxo- 1,2-dihydropyridine-3,5-dicarbonitrile derivatives 12a,b To a solution of 10 (3.75 g, 0.01 mol) in absolute eth- anol (25 mL), either malononitrile (0.66 g, 0.01 mol) or ethyl cyanoacetate (1.13 g, 0.01 mol) was added. On the reflux system, the reaction was heated for 3 h. The resultant product was poured onto the ice/water mixture with a few drops of HCl added. The precipitated products were col- lected by filtration and then recrystallized from ethanol. 6-Amino-1-(3-cyano-4-phenyl-5,6,7,8-tetrahydro-4H- chromen-2-yl)-2-oxo-1,2-dihydropyridine-3,5-dicarbo- nitrile (12a). Brown crystals, yield: 3.00 g (76%), m.p. 112–115 °C. IR (ν, cm–1): 3442, 3351 (NH2), 3242 (NH), 3100 (CH-aromatic), 2935, 2865 (CH2), 2260, 2220, 2199 (3CN), 1699 (C=O), 1638, 1449 (C=C). 1H NMR (δ, ppm): 1.54–1.74 (m, 4H, 2CH2), 2.13–2.17 (m, 4H, 2CH2), 5.80 (s, 1H, CH-pyran), 6.37 (s, 1H, CH-pyridine), 6.95–7.53 (m, 7H, C6H5, NH2). 13C NMR (δ, ppm): 21.4, 22.0, 22.4, 26.9, 42.9, 95.7, 115.4, 115.8, 124.2, 127.6, 127.9, 128.5, 128.7, 137.3, 146.9, 150.1, 150.7. MS m/z (%): 397 [M+ + 2] (2.57), 396 [M+ + 1] (2.71), 395 [M+] (3.46), 394 [M+ – 1] (4.29), 393 [M+ – 2] (10.61), 391 (100.00), 77 [C6H5]+ (5.32). Anal. Calcd for C23H17N5O2 (395.41): C, 69.86; H, 4.33; N, 17.71. Found: C, 70.01; H, 4.49; N, 17.98. 6-Amino-1-(3-cyano-4-phenyl-5,6,7,8-tetrahydro-4H- chromen-2-yl)-2-oxo-1,2-dihydropyridine-3,5-dicarbo- nitrile (12b). Yellowish brown crystals, yield: 3.75 g (95%), m.p. 87–90 °C. IR (ν, cm–1): 3419–3240 (OH), 3100 (CH-aromatic), 2935, 2864 (CH2), 2260, 2240, 2209 (3CN), 1696 (C=O), 1640, 1448 (C=C). 1H NMR (δ, ppm): 1.54–1.73 (m, 4H, 2CH2), 2.13–2.17 (m, 4H, 2CH2), 5.70 (s, 1H, CH-pyran), 6.36 (s, 1H, CH-pyridine), 7.20–7.52 (m, 5H, C6H5), 8.40 (s, 1H, OH). MS m/z (%): 397 [M+ + 1] (4.82), 396 [M+] (8.26), 300 (100.00), 77 [C6H5]+ (13.12). Anal. Calcd for C23H16N4O3 (396.40): C, 69.69; H, 4.07; N, 14.13. Found: C, 69.93; H, 4.29; N, 14.39. 2. 2. Biological Evaluations 2. 2. 1. Materials and Methods – Gibco Invitrogen Company (Scotland, UK): Provide fe- tal bovine serum (FBS) and L-glutamine. – Cambrex (New Jersey, USA): Provide RPMI-1640 medi- um – Sigma Chemical Company. (Saint Louis, MO, USA): Provide dimethyl sulfoxide (DMSO), foretinib, penicil- lin, streptomycin, and sulforhodamine B (SRB). 2. 2. 2. Samples Tumor Cell Proliferation Assay: The effects of 1a,b to 12a,b on the in vitro proliferation of human cancer cell lines were tested. The method were obtained from the Na- tional Cancer Institute (NCI, USA) in the In vitro Antican- cer Drug Discovery Screen using the protein-binding dye sulforhodamine B to assess cell proliferation. 2. 2. 3. Cell Cultures The three human cancer cell lines were A-549 (lung carcinoma), HC-29 (colorectal adenocarcinoma), 267Acta Chim. Slov. 2023, 70, 261–273 Abdallah et al.: Novel 5,6,7,8-tetrahydrobenzo[b]pyran Derivatives: ... and MKN-45 (gastric cancer). The later cells were ob- tained from the National Cancer Institute (NCI), Cairo, Egypt. The cell cultures were prepared as the following: They were grown as monolayers and plated in RPMI 1640 medium supplemented with 5% heat-inactivated FBS, 2 mM glutamine and antibiotics (penicillin 100 U/mL and streptomycin 100 μg/mL) in a humidified atmosphere at 37 °C. Permanently maintained at 5% CO2, exponentially growing cells were plated at 0.75 × 104 cells/mL followed by 1.5 × 105 cells/mL for MCF-7 and SF-268 and 0.75 × 104 cells/mL for three-cell line and maintained the incuba- tion for 48 h. The effect of carrier solvent (DMSO) on the growth of these cell lines was examined in all experiments by exposing untreated control cells to the highest concen- tration of DMSO used in each assay (0.5%). Scheme 1. Synthesis of tetrahydrobenzo[b]pyran derivatives 1a,b, 2a,b and 3a–d. 268 Acta Chim. Slov. 2023, 70, 261–273 Abdallah et al.: Novel 5,6,7,8-tetrahydrobenzo[b]pyran Derivatives: ... 3. Results and Discussion 3. 1. Chemistry The reaction of cyclohexanone with malononitrile and either of benzaldehyde or 4-methoxybenzaldehyde gave the pyran derivatives 1a and 1b,31 respectively (Scheme 1). According to the data obtained from the spec- troscopic analysis methods, the structures of the resultant products were indicated. The obtained structures were confirmed by 1H NMR and IR spectroscopy. Thus, for 1H NMR spectrum of the compound 1a, a multiplet at δ 1.66– 1.71 ppm for 2CH2 cyclohexene, a multiplet at 2.16–2.81 ppm for the other 2CH2 of cyclohexene ring and a singlet at 5.73 ppm for CH pyran were observed. Moreover, the presence of a multiplet at δ 7.14–7.89 ppm for phenyl moi- ety and NH2 group and cyano group in the IR spectrum in the range of 2209 cm–1 supported the proposed structure. Besides, for compound 1b, the presence of the methoxy group in the 1H NMR in the range of 3.87 ppm confirmed its structure. The reaction of compound 1a or 1b with triethylo- rthoformate in acetic acid gave the 2-N-ethoxymethino derivatives 2a and 2b, respectively (Scheme 1). The disap- pearance of the NH2 group signal in the 1H NMR and IR spectrum of the compounds 2a and 2b, confirmed the structures. The appearance of the ethoxy group in the range between 1.10–1.20 ppm for the CH3 group and 4.25–4.30 ppm for the CH2 confirmed the structures. Previously obtained products 2a or 2b were reacted with either of hydrazine hydrate or phenylhydrazine to give hydrazino derivatives 3a–d, respectively (Scheme 1). The 1H NMR spectrum of 3a indicated a multiplet at δ Scheme 2. Synthesis of fused pyran systems 4, 5a–d and 6a–b. 269Acta Chim. Slov. 2023, 70, 261–273 Abdallah et al.: Novel 5,6,7,8-tetrahydrobenzo[b]pyran Derivatives: ... 1.58–1.74 ppm for 2CH2 cyclohexene moiety, a multiplet at δ 2.13–2.15 ppm for the other 2CH2, a singlet at δ 6.40 ppm for CH-pyran ring, a singlet at δ 6.96 ppm for CH group, a multiplet at δ 7.20–7.54 ppm for NH2 group and phenyl ring. Moreover, the appearance of the singlet at δ 10.80 ppm for NH group elucidated the chemical structure of compound 3a. Compound 2a upon reaction with aniline in ethanol gave the aniline derivative 4 (Scheme 2). The reaction of compound 2a or 2b with either of malononitrile or ethyl cyanoacetate gave 2-N-alkyl products 5a–d, respectively (Scheme 2). The structures of these products were con- firmed by the presence of the ethoxy groups in the 1H NMR spectra for compounds 5b and 5d in the range at δ 1.06–1.21 ppm for CH3 group and 4.19–4.24 ppm for CH2 group. On the other hand, the appearance in the IR spectra of compounds 5a and 5c of the three cyano moieties in the range at ν 2190–2260 cm–1 elucidated the proposed struc- tures. Compounds 1a and 1b showed interesting reactivity towards amide formation. Thus, the reaction of either of compound 1a or 1b with ethyl cyanoacetate gave the cy- anoacetamide derivatives 6a and 6b, respectively (Scheme 2). The analytical and spectral data of 6a and 6b elucidated their structures. Thus, the 1H NMR of 6a contains a multi- plet at δ 1.45–1.49 ppm for 2CH2 cyclohexene ring, a mul- tiplet at δ 2.16–2.20 ppm for the second 2CH2 cyclohexene moiety, a singlet at δ 5.73 ppm CH-pyran ring, a multiplet at δ 7.33–7.43 ppm for phenyl ring and a singlet at δ 10.01 ppm for NH group; also spectrum of 6b revealed a multi- Scheme 3. Synthesis of thiophene 7a–b, coumarin 8 and pyridine derivatives 9a–b. 270 Acta Chim. Slov. 2023, 70, 261–273 Abdallah et al.: Novel 5,6,7,8-tetrahydrobenzo[b]pyran Derivatives: ... plet at δ 1.66–1.71 ppm for 2CH2 groups, a multiplet at δ 2.10–2.20 ppm for the other 2CH2 cyclohexene moiety, a singlet at δ 3.73 ppm for OCH3 group, a singlet at δ 4.34 ppm for CH2 group, a singlet at δ 5.70 ppm for CH-pyran ring, a multiplet at δ 6.91–7.90 ppm for C6H4 moiety and a singlet at δ 8.31 ppm for NH group. Compound 6a underwent the Gewald’s thiophene synthesis32–34 by the reaction of either of malononitrile or ethyl cyanoacetate with elemental sulfur in ethanol and tri- ethylamine to give the thiophene derivatives 7a and 7b, re- spectively (Scheme 3). The formation of the latter products was confirmed by the 1H NMR spectrum via the presence of the two NH2 moieties in the range between δ 7.19–7.63 ppm with the phenyl groups. In addition, the IR spectrum of compounds 7a and 7b showed two bands in the range between ν 3417–3340 cm–1 due to the presence of the two NH2 groups. Moreover, compound 6a upon the reaction with salicylaldehyde in ethanol and piperidine gave the coumarin derivative 8 (Scheme 3). Mass spectrum of 8 ex- hibited molecular ion at m/z 424 corresponding to the mo- lecular formula C26H20N2O4, which confirmed the assign- ment for coumarin structure 8. The other resulting peaks which confirmed the molecular ion peak were observed at m/z 426, 425, 423, 422, 77 and 76 which correspond to [M+ + 2], [M+ + 1], [M+ – 1], [M+ – 2], [C6H5]+ and [C6H4]+, respectively. In addition, the structure of 8 was elucidated via the 13C NMR which confirmed the presence of two car- bonyl groups at δ 158.7 and 163.2 ppm. The reactivity of compound 6a towards 1,3-dicarbo- nyl compounds was studied to give bioactive pyridine de- rivatives. Compound 6a reacted with either of acetylace- tone or ethyl acetoacetate to afford the pyridine derivatives 9a and 9b, respectively (Scheme 3). The structures of the latter products were confirmed according to the results of the spectral data. Thus, the 13C NMR spectrum of 9a showed the carbonyl carbon signal at δ 166.00 ppm. More- over, the mass spectrum of 9b exhibited a molecular ion peak [M+] at m/z 385 corresponding to the molecular for- mula C23H19N3O3. Many other peaks were observed to confirm the final chemical structure of 9b, such as the peak at m/z 387, 386, 384 and 383 which corresponds to [M+ + 2], [M+ + 1], [M+ – 1] and [M+ – 2], respectively. Scheme 4. Synthesis of tetrahydrobenzo[b]pyran derivatives 10, 11 and pyridines 12a,b. 271Acta Chim. Slov. 2023, 70, 261–273 Abdallah et al.: Novel 5,6,7,8-tetrahydrobenzo[b]pyran Derivatives: ... Compound 6a upon the reaction with ethyl ortho- formate gave ethoxyvinyl product 10 which reacted with aniline to give the aniline derivative 11 (Scheme 4). The structure of 10 was confirmed based on analytical and spectral data. Thus, the IR spectrum revealed absorption bands at ν 1701 cm–1 corresponding to C=O. 1H NMR showed a triplet in the range at δ 1.05–1.10 ppm for the CH3 group and quartet in the range at δ 4.22–4.24 ppm for the CH2 moiety which confirmed the presence of the ethyl group in compound 10. Mass spectra of compounds 10 and 11 revealed molecular ion peaks at m/z 375 and 422, respectively, corresponding to the respective molecular formulas C22H21N3O3 and C26H22N4O2. Compound 10 was reacted with either of malononitrile or ethyl cyanoac- etate in ethanol under reflux to give pyridine derivatives 12a and 12b, respectively (Scheme 4). The latter products were formed through the intermediate acyclic products C and D, respectively. Compounds 12a,b were confirmed; thus, the 1H NMR spectrum of 12a showed a multiplet at δ 1.54–1.74 ppm for 2CH2, a multiplet at δ 2.13–2.17 ppm for the other 2CH2 groups, a singlet at δ 6.37 ppm for CH-pyridine ring, a singlet at δ 5.80 ppm for CH-pyran moiety and a multiplet at δ 6.95–7.53 ppm for NH2 group and C6H5 moiety. In addition, the mass spectrum of 14b showed molecular ion peak [M+] 396 corresponding to its molecular formula C23H16N4O3. 3. 2. Biological Activity Evaluations 3. 2. 1. Structure Activity Relationship Table 1 demonstrates the cytotoxicity of the prepared products on the three cancer cell lines comparing com- pounds 1a and 1b, where compound 1b has more potency than compound 1a due to the 4-methoxyphenyl group present in compound 1b. The same also appears in com- pound 2b which has higher cytotoxicity than 2a. By com- paring compound 3a–d, it can be noticed that compound 3b has the highest cytotoxic effect among the other com- pounds 3. Moreover, in the case of the pyran compounds 5a–d, compound 5b with the ethoxy carbonyl group has the highest potency within the four compounds; reaction of compounds 1a and 1b with ethyl cyanoacetate gave compounds 6a and 6b. Compound 6b with the 4-meth- oxyaryl group demonstrated higher potency than com- pound 6a. Reaction of compound 6a with ethyl orthofor- mate gave the ethoxy metheno derivative 10 possessing moderate cytotoxicity. Besides, compound 11 obtained from the reaction of 10 with aniline has shown the same moderate cytotoxicity. Comparing compounds 7a and 7b explains that compound 7a with the electronegative cyano group exhib- ited higher potency than 7b with the ester group. The cou- marine derivative 8 shows a high potency. The pyridine derivatives 9a and 9b showed similar cytotoxicity. The cy- totoxic effect for the compounds 12a and 12b represents moderate activity, but compound 12b showed a higher ef- fect than 12a especially for the A-549 and MKN-45 cell lines. The latter activity is attributed to the presence of the hydroxyl group in compound 12b. Finally, the presence of the two phenyl rings, meth- oxy group and coumarin moiety in the compounds 3b, 6b and 8, respectively, were responsible for the highest effect of these compounds among all the other tested com- pounds. Table 1. The cytotoxic effect of the prepared products against three cancer cell lines Compd. [GI50(mM)] Number A-549 HC-29 MKN-45 1a 29.48±5.43 40.69±4.61 48.90±12.53 1b 18.48±1.84 19.54±2.80 11.85±4.75 2a 49.11±10.42 52.2±10.32 36.59±4.80 2b 0.26±0.08 1.69±0.59 0.86±0.04 3a 45.24±6.55 70.2±10.50 64.21±10.33 3b 0.08±0.002 0.09±0.09 0.1±0.01 3c 48.29±6.81 73.2±12.53 69.31±12.59 3d 14.23±1.80 15.80±2.79 12.64±2.55 4 14.70±1.83 18.11±2.82 20.12±4.15 5a 40.63±8.62 45.60 ± 3.51 37.39± 4.21 5b 4.70±1.93 0.1±0.02 0.02±0.005 5c 28.19±6.73 19.26±2.60 22.80±4.76 5d 38.41±6.80 22.59±6.90 29.30±5.70 6a 4.73±1.69 5.80±0.98 2.66±0.39 6b 0.03±0.002 0.06±0.09 0.2±0.01 7a 8.09±2.70 10.39±4.62 8.39±3.77 7b 12.37±2.75 6.19±1.65 8.62±2.63 8 0.08±0.003 1.20±0.22 0.07±0.01 9a 10.69±2.73 12.70±2.84 12.61±3.74 9b 12.69±2.59 14.72±2.80 8.91±3.76 10 8.33±1.75 6.29±1.39 4.28±1.30 11 10.50±2.65 6.08±1.27 14.59±1.19 12a 4.82±0.27 3.79±0.92 10.55±1.76 12b 4.73±1.69 5.80±0.98 2.66±0.39 Foretinib 0.18±0.09 0.24±0.023 0.021±0.0016 (mM) 4. Conclusions The current research describes a practical synthesis method for 24 novel pyran derivatives. The variety of the final products prepared can be attributed to the various ways of possible attacks of chosen reagents on the reactive points in the pyran system. Moreover, anti-cancer activi- ties of all the compounds were examined on three human cancer cell lines. Some of the tested products were shown to be favorable as anti-proliferative agents. The most promising compounds were 3b, 6b, and 8 against the three tumor cell lines such A-549 (lung carcinoma), HC-29 (colorectal adenocarcinoma), and MKN-45 (gastric can- cer). 272 Acta Chim. Slov. 2023, 70, 261–273 Abdallah et al.: Novel 5,6,7,8-tetrahydrobenzo[b]pyran Derivatives: ... Conflict of Interests The authors do not report any conflicts of interest in this work. Compliance with Ethical Standards Any of the author’s experiments involving animals or human subjects are not included in this article. 5. References 1. E. Middleton, C. Kandaswami, T. C. Theoharides, Pharmacol Rev. 2000, 52, 673–751. 2. H. F. Roaiah, S. S. El-Nakkady, W. S. El-Serwy, M. A. A. Ali, A. H. Abd El-Rahman, Z. El-Bazzad, Nat Sci. 2010, 8, 20–29. DOI:10.7537/marsnsj080710.04 3. V. R. Rao, P. V. Kumar, V. R. Reddy, K. M. Reddy, Heterocycl. Commun. 2005, 11, 273–284. DOI:10.1515/HC.2005.11.3-4.273 4. M. Grażul, A. Kufelnicki, M. Wozniczka, I.-P. Lorenz, P. May- er, A. Jóźwiak, M. Czyz, E. Budzisz, Polyhedron. 2012, 31, 150–158. DOI:10.1016/j.poly.2011.09.003 5. D. A. Vasselin, A. D. Westwell, C. S. Matthews, T. D. Brad- shaw, M. F. Stevens, J. Med. Chem. 2006, 49, 3973–3981. DOI:10.1021/jm060359j 6. M. M. F. Ismail, H. S. Rateb, M. M. M. Hussein, Eur. J. Med. Chem. 2010, 45, 3950–3959. DOI:10.1016/j.ejmech.2010.05.050 7. Z.-F. Wang, X.-L. Nai, Y. Xu, F.-H. Pan, F.-S. Tang, Q.-P. Qin, L. Yang, S.-H. Zhang, Dalton Trans. 2022, 51, 12866–12875. DOI:10.1039/D2DT01929A 8. D. Ashok, K. Pallavi, Heterocycl. Commun. 2006, 12, 103–106. DOI:10.1111/j.1355-0691.2006.01241_1.x 9. N. Artizzu, L. Bonsignore, G. Loy, A. Calignano, Farmaco 1995, 50, 853–856. 10. L. Bonsignore, G. Loy, D. Secci, A. Calignano, Eur. J. Med. Chem. 1993, 28, 517–520. DOI:10.1016/0223-5234(93)90020-F 11. A. Dutta, N. Rahman, J. E. Kumar, J. Rabha, T. Phukan, R. Nongkhlaw, Synth. Commun. 2021, 51, 263–278. DOI:10.1080/00397911.2020.1825741 12. M. R. P. Heravi, P. Aghamohammadi, E. Vessally, J. Mol. Struct. 2022, 1249, 131534. DOI:10.1016/j.molstruc.2021.131534 13. J. Li, C.-W. Lv, X.-J. Li; D. Qu, Z. Hou, M. Jia, X.-X. Luo, X. Li, M.-K. Li, Molecules 2015, 20, 17469–17482. DOI:10.3390/molecules200917469 14. I. E. Bylov, M. V. Vasylyev, Y. V. Bilokin, Eur. J. Med. Chem. 1999, 34, 997–1001. DOI:10.1016/S0223-5234(99)00119-1 15. A. Nohara, T. Umetani, Y. Sanno, Tetrahedron 1974, 30, 3553– 3561. DOI:10.1016/S0040-4020(01)97034-6 16. M. Biglari, F. Shirini, N. O. Mahmoodi, M. Zabihzadeh, M. Mashhadinezhad, J. Mol. Struct. 2020, 1205, 127652. DOI:10.1016/j.molstruc.2019.127652 17. H. R. Saadati-Moshtaghin, F. M. Zonoz, Mater Chem. Phys. 2017, 199, 159–165. DOI:10.1016/j.matchemphys.2017.06.066 18. A. Thongni, P. T. Phanrang, A. Dutta, R. Nongkhlaw, Synth. Commun. 2022, 52, 43–62. DOI:10.1080/00397911.2021.1998535 19. J. Yang, S. Liu, H. Hu, S. Ren, A. Ying, Chin. J. Chem. Eng. 2015, 23, 1416–1420. DOI:10.1016/j.cjche.2015.04.020 20. F. Sepehr, S. Allameh, A. Davoodnia, Ann. Romanian Soc. Cell Biol. 2021, 25, 19067–19072. 21. F. Mohamadpour, J. Taiwan Inst. Chem. Eng. 2021, 129, 52– 63. DOI:10.1016/j.jtice.2021.09.017 22. F. Mohamadpour, Polycycl. Aromat. Compd. 2021, 42, 7607– 7615. DOI:10.1080/10406638.2021.2006244 23. I. Devi, P. J. Bhuyan, Tetrahedron Lett. 2004, 45, 8625–8627. DOI:10.1016/j.tetlet.2004.09.158 24. J.-T. Li, W.-Z. Xu, L.-C. Yang, T.-S. Li, Synth. Commun. 2004, 34, 4565–4571. DOI:10.1081/SCC-200043233 25. D. Elhamifar, Z. Ramazani, M. Norouzi, R. Mirbagheri, J. Col- loid Interface Sci. 2018, 511, 392–401. DOI:10.1016/j.jcis.2017.10.013 26. A. E. M. Abdallah, R. M. Mohareb. Pigm. Resin Technol. 2019, 48, 89–103. DOI:10.1108/PRT-11-2017-0085 27. A. E. M. Abdallah, R. M. Mohareb, E. A. Ahmed, J. Heterocycl. Chem. 2019, 56, 3017–3029. DOI:10.1002/jhet.3697 28. R. M. Mohareb, E. M. Khalil, A. E. Mayhoub, A. E. M. Abdal- lah, J. Heterocycl. Chem. 2020, 57, 1330–1343. DOI:10.1002/jhet.3870 29. A. E. M. Abdallah, R. M. Mohareb, M. H. E. Helal, G. J. Mo- feed. Acta Chim. Slov. 2021, 68, 604–616. DOI:10.17344/acsi.2020.6446 30. R. M. Mohareb, M. H. E. Helal, S. S. Mohamed, A. E. M. Ab- dallah, Anti-Cancer Agents in Med. Chem. 2022, 22, 2327– 2339. DOI:10.2174/1871520622666211224102301 31. F. Javadi, R. Tayebee, Micropor. Mesopor. Mat. 2016, 231, 100– 109. DOI:10.1016/j.micromeso.2016.05.025 32. D. Wang, J. Wu, Q. Cui, Chin. Chem. Lett. 2014, 25, 1591– 1594. DOI:10.1016/j.cclet.2014.07.007 33. Y. Han, W.-Q. Tang, C.-G. Yan, Tetrahedron Lett. 2014, 55, 1441–1443. DOI:10.1016/j.tetlet.2014.01.043 34. V. D. Dyachenko, A. N. Chernega, Russ. J. Gen. Chem. 2005, 75, 952–960. DOI:10.1007/s11176-005-0351-6 273Acta Chim. Slov. 2023, 70, 261–273 Abdallah et al.: Novel 5,6,7,8-tetrahydrobenzo[b]pyran Derivatives: ... Povzetek Doslej so bili načrtovani in pripravljeni že mnogi novi ciklični piranski sistemi s potencialnimi delovanjem proti raku. Poleg tega piranski sistemi kažejo tudi visoko reaktivnost do mnogih kemijskih reagentov. Pripravili smo 24 produktov in jih preizkusili kot morebitne protirakave učinkovine (v mM območju). Rezultati kažejo, da so spojine 3b, 6b in 8 široko učinkovite proti trem rakavim celičnim linijam in sicer A-549 (pljučni karcinom), HC-29 (kolorektalni adenokarcinom) in MKN-45 (rak želodca) in kažejo primerljivo aktivnost glede na standarndo referenčno kontrolno spojino foretinib. Except when otherwise noted, articles in this journal are published under the terms and conditions of the  Creative Commons Attribution 4.0 International License 274 Acta Chim. Slov. 2023, 70, 274–280 Pavlin et al.: Direct Determination of Kynurenic Acid ... DOI: 10.17344/acsi.2023.8079 Scientific paper Direct Determination of Kynurenic Acid with HPLC-MS/MS Method in Honey Anže Pavlin, Matevž Pompe and Drago Kočar* Department of Chemistry and Biochemistry, Faculty of Chemistry and Chemical Technology, University of Ljubljana, Večna pot 113, SI-1000 Ljubljana, Slovenia Faculty of Chemistry and Chemical Technology, University of Ljubljana, Slovenia * Corresponding author: E-mail: drago.kocar@fkkt.uni-lj.si Received: 02-16-2023 Abstract Kynurenic acid (KYNA) has been attributed many beneficial properties, such as antioxidant, antiproliferative, anti-in- flammatory, and anti-obesogenic, as it is believed to affect metabolism and weight gain. A rapid and simple HPLC-MS/ MS method for the determination of kynurenic acid (KYNA) in honey has been developed. HPLC-MS/MS system al- lowed us to perform the analyzes without any special extraction or treatment of the samples. The study was carried out on different honeys: Chestnut (C), Linden (L), Acacia (A), Spruce (S), Silver Fir (SF), Forest (Fo) and Flower (F). The highest mean concentration, 682 μg/g, was determined for chestnut honey, making it one of the foods with the highest KYNA content. Keywords: Kynurenic acid, honey, HPLC-MS/MS, SRM 1. Introduction 4-Hydroxyquinoline-2-carboxylic acid (structural formula shown in Figure 1), also known as kynurenic acid (KYNA), is a tryptophan metabolite, a byproduct of the kynurenine metabolic pathway, and was discovered by Liebig in 1853.1 The kynurenine metabolic pathway is a process of dietary tryptophan metabolism and production of the cofactor nicotinamide adenine dinucleotide (NAD +). It is formed directly from kynurenine in a reaction catalyzed by kynurenine aminotransferases.2 Studies and analyzes of KYNA have been performed on various sam- ples. It has been detected in samples ranging from honey- bee products, various plants, herbs and spices to cells and human and animal tissues and excretions showing anti- convulsant and neuroprotective activity.3–15 In this study, the presence of KYNA in food and honeybee products was investigated. KYNA was found in all 37 tested samples of food and honeybee products. The highest concentration of KYNA was obtained from honeybee products’ samples, propolis (9.6 nmol/g). Many properties were attributed to it, such as anti-ulcer, anti-inflammatory and anti-prolifera- tion.12,16–18 High antioxidant capacity and regulation of bacterial growth have also been observed along with prop- erties of reducing hypermotility and antagonizing iono- tropic glutamate receptors.15,18–21 Figure 1: Structural formula of kynurenic acid (KYNA). It has also been observed that KYNA concentration deviates from normal value if subject suffers from irrita- ble bowel syndrome, Parkinson’s disease, Huntington’s disease, or multiple sclerosis, resulting in a decrease in concentration, while the opposite phenomenon has been observed in colon lesions such as adenomas or adenocar- cinomas and inflammatory bowel disease, in which the concentration of KYNA is increased.22–28 There has also been a suspected association be- tween lower KYNA levels and various types of mood dis- orders, a phenomenon which occurs primarily in wom- en.29 Consequently tryptophan and its metabolites such as kynurenine and kynurenic acid were investigated in human plasma.30 KYNA plays an important role as antagonist of iono- tropic glutamate receptor and an agonist for the orphan G 275Acta Chim. Slov. 2023, 70, 274–280 Pavlin et al.: Direct Determination of Kynurenic Acid ... protein-coupled receptor GPR35, which is found in the gastrointestinal tract and immune cells.13,21 Mainly the positive properties in gastrointestinal tract and capability of decreasing hypermotility call for broader investigation of KYNA intake from food.18,20 KYNA is found in many herbs, spices, and other remedies used to relieve digestive system problems.10,11 For example common nettle or St. John’s wort, both KYNA rich substances, are often used as remedies for reducing the symptoms of digestive system diseases.10 This means that KYNA, among many other beneficial properties, may play an important role in diges- tion and also weight gain. KYNA has been suggested to be an anti-obesogenic compound which can influence weight gain. The concentration of KYNA has been studied in hu- man breast milk and in baby formula, suggesting that a deficiency of KYNA in baby formula may lead to obesity of infants and children. This was further tested on rats, re- sulting in lower weight gain in rats postnatally fed with KYNA supplements compared to rats without it.7 In general, the methods developed so far require complex preparation of samples. This means mainly ho- mogenization, centrifugation, and various extraction methods (such as Solid Phase Extraction – SPE) which potentially eliminate possible interferences. The method for determination of KYNA in potatoes and flour consist- ed of homogenization of samples and further centrifuga- tion. KYNA was later extracted from the supernatant by SPE method using a cation exchange resin.8 The same SPE method was used to determine KYNA in honey.9 Howev- er, a RP-SPE cartridge filled with solid phase, was used in NMR and MS study of KYNA in plants.4,31 Our goal was to simplify sample preparation. Selectivity can also be improved in other ways, for example by using a liquid chromatography system coupled to a triple quadrupole mass spectrometry system in Select- ed Reaction Monitoring (SRM) mode to observe only the molecule of interest.30,32,33 All mentioned positive properties of KYNA and the need to optimize the preparation methods make honey of different botanical sources an interesting target for the study of KYNA content. Our main goal was devel- oping a method avoiding all mentioned complex and not necessary preparations steps which resulted in cheaper and less time-consuming method for analysis of KYNA in honey matrix. Method was developed for HPLC-MS/ MS system in SRM mode. An example of the method LC-MS/MS in SRM mode is shown in Figure 2. Newly developed method does not require any centrifugation or special extraction of the analyte and its selectivity does not depend on compounds fluorescence or UV light ab- sorption so no other detector is necessary.30,32,34 Analyz- es were performed on Chestnut (C), Linden (L), Acacia (A), Spruce (S), Silver Fir (SF), Forest (Fo) and Flower (F) honey types. 2. Experimental Honey Different honey samples, in total 129, including chestnut (6), linden (14), spruce (2), acacia (20), silver fir (2), flower (59) and forest (26) honey, were obtained from local bee keepers and Medex (Medex d.o.o., Slovenia). Sample preparation KYNA (Sigma, USA) standard for calibration curve was dissolved in 0.1% NH3 (Gram-Mol d.o.o., Republic of Croatia), as were the honey samples. 0.1 g of chestnut hon- ey, 1 g of flower, forest, acacia and linden honey and 3 g of spruce and silver fir honey were separately dissolved in 0.1 % NH3, mixed thoroughly, and filled to mark in 50 mL volumetric flask. The solution was filtered through nylon filter (pore size 0.45 μm) and transferred to vial. (a) (b) Figure 2: (a) MS spectrum of KYNA precursor ion; (b) MS spectrum of KYNA product ions. 276 Acta Chim. Slov. 2023, 70, 274–280 Pavlin et al.: Direct Determination of Kynurenic Acid ... For purposes of recovery determination L 131 and A 15 were prepared the same way and additionally spiked with KYNA standard, 105.5 mg and 9.1 mg, respectively. HPLC-MS/MS Analysis Analysis was developed for UHPLC system (Van- quish™ Flex UHPLC, Thermo Scientific™, USA) coupled with H-ESI-MS system (TSQ Quantis™ Triple Quadrupole Mass Spectrometer, Thermo Scientific™, USA). Chromatographic conditions Sampler compartment was thermostated at tempera- ture 20 °C. Injection volume was 10 μL. Column (Kinetex® 2.6 µm C18 100 Å, LC Column 150 × 4.6 mm, Phenomen- ex Inc., USA) was thermostated at temperature 25 °C. Mo- bile phase A was 0.1% formic acid (Honeywell, USA) in ultrapure MilliQ water (Millipore, USA), mobile phase B was acetonitrile (Fisher Chemical, UK). Gradient method was developed at flow 0.7 mL/min. Elution was used as fol- lows: time 1 min, 10% B; time 8 min, 80% B; time 11 min, 80% B; time 13 min, 10% B; time 18 min, 10% B. Mass spectrometric conditions A SRM method for H-ESI-MS was developed. H-ESI-MS was used in positive mode with spray voltage +190 V. Gases were optimized at following values: Sheath gas 8.55 L/min; Auxiliary gas 14.29 L/min; Sweep gas 1.5 L/min. Ion Transfer Tube Temperature was held at 350 °C and Vaporizer Temperature was held at 400 °C. SRM parameters for KYNA were: Precursor ion (m/z) 190.08; Product ion (m/z) 144.02; Collision energy 18 V.30,32 Determination of KYNA The concentration and content of KYNA in honey was determined using method of calibration curve in con- centration range from 0.01 mg/L to 20 mg/L. 3. Results and Discussion This research was aimed at developing a HPLC-MS/ MS method for determination of KYNA in honey matrix with application advantages such as avoiding the use of any special extraction method or other sample pretreat- ment consistently used laboratory practice so far. 129 honey samples were analyzed. Our study confirmed that KYNA is poorly soluble in acetonitrile and methanol.34 The best MS-compatible solvent was determined to be 0.1 % ammonia; alkaline solvent improves solubility as well as stability of kynurenic acid. During optimization of MS ion source conditions, we tested different ionization voltages. +190 V turned out to give the same, if not better, results as some higher volt- ages, used in experiments described in literature.30,32 Easy ionization may be due to free electron pair on nitrogen while formic acid from mobile phase acts like an excellent proton donor. Calibration curve, LOD, LOQ Concentrations were determined by a calibration curve. 3 calibration curves, covering the entire linearity range from 0.01 mg/L to 20 mg/L, were used. Equation for calibration curve in range (0.01 – 0.1) mg/L was y = 2*107x + 14246 with coefficient of determination R2 = 0.9987. Equation for calibration curve in range (0.1–1) mg/L was y = 2*107x + 282943 with coefficient of deter- mination R2 = 0.9994. Equation for calibration curve in range (1–20) mg/L was y = 2*107 + 8*106 with coefficient of determination R2 = 0.9957. LOQ and LOD were de- termined experimentally analyzing low concentration standard solutions. LOQ was determined to be 0.01 mg/L (S/N = 10), while LOD was determined to be 0.001 mg/L (S/N = 3), which would give a KYNA content of 0.5 μg/g and 0.05 μg/g, respectively, given the weight of the honey sample was 1 g. Repeatability, stability and recovery Repeatability, shown in Table 1, was investigated for each honey type. Three parallel samples of each represent- ative honey type were prepared. Relative standard devia- tion (RSD) of the peak area was calculated. Table 1: Repeatability of each honey type. SAMPLE RSD [%] Chestnut 2.9 Spruce 2.2 Silver Fir 2.8 Linden 1.2 Acacia 2.8 Forest 4.0 Flower 1.0 The stability of three KYNA standard solutions at concentrations of 0.05 mg/L, 0.5 mg/L, and 10 mg/L at 20 °C was studied over a 21-day period. They were all found to be stable over this period with relative standard devia- tions of instrument response over time of 11.0%, 9.6% and 8.7%, respectively. The stability of the standard solution with a concentration of 0.5 mg/L over time is presented in Figure 3. For purposes of recovery determination L 131 and A 15 were spiked with KYNA standard. The recoveries for L 131 and A 15 were 106% and 113%, respective- 277Acta Chim. Slov. 2023, 70, 274–280 Pavlin et al.: Direct Determination of Kynurenic Acid ... ly. These specific samples were selected based on their KYNA content, which was moderate in L 131 (106.2 μg/g) and low in A 15 (0.7 μg/g), making them perfect for the recovery study. L 131 was spiked with 105.5 μg and A 15 was spiked with 9.1 μg of KYNA. Chromatogram of L 131 sample and L 131 spiked with KYNA standard can be seen in Figure 4. Concentration in honey As shown in Figure 5 concentration of KYNA is the highest in chestnut honey in range (327.8–1015.7) μg/g fol- lowed by linden honey in general range (24.6–188.7) μg/g. Next, in general order is spruce honey in range (8.0–8.9) μg/g followed by acacia honey in general range (0.7–5.3) μg/g. Somewhere in between acacia honey range is silver fir honey with concentration range (1.4–2.2) μg/g. Results of spruce, acacia and silver fir honey are presented in Figure 6. As expected the range of mixed honey samples, such as flower honey and forest honey, varies from low to high concen- trations with no observable order. Therefore, the concentration of KYNA in forest honey and flower honey samples ranged from (0.8–397.7) μg/g and (1.4–194.2) μg/g, respectively. The content of KYNA is the highest in chestnut honey then followed by linden honey and others. It can Figure 3: Stability of standard solution with concentration 0.5 mg/L of KYNA. Standard deviations were determined with three consec- utive parallel determinations. Figure 4: SRM chromatogram of Linden 131 sample and Linden 131 spiked with KYNA standard. Figure 5: KYNA concentration in all chestnut (C) honeys (samples C 1–6) and three samples of linden (L) honey with maximal con- centration (sequence label L 99.2, L 99.1 and L 88) and three sam- ples with minimal concentration (sequence label L 77, L 66 and L 22) of KYNA. Standard deviations were determined on three con- secutive parallel determinations. Figure 6: KYNA concentration in both samples of spruce (sequence label S 143.2 and S 143.1) and silver fir (sequence label S 118.1 and S 118.2) honeys and three samples of acacia (A) honey with maxi- mal concentration (sequence label A 122, A 42 and A 46) and three samples with minimal concentration (sequence label A 105, A 43 and A 15) of KYNA. Standard deviations were determined on three consecutive parallel determinations. 278 Acta Chim. Slov. 2023, 70, 274–280 Pavlin et al.: Direct Determination of Kynurenic Acid ... be noticed that concentrations of KYNA in some acacia honey samples are higher than its general range, as the concentration of KYNA in some linden honey samples are lower than its general range which could be explained as presence of some other type of honey which increases or decreases KYNA concentration. Reason for this could be mixing honey with honey of different type, intention- al dilution of honey or heterogeneity of honey bee apiary area.35,36 As for flower and forest honey it was expected to have wide concentration range of KYNA. Besides the men- tioned reasons, for out of general range concentrations, explanation of this phenomenon, could be wide range of botanical sources of honey as this is not honey from one specific plant. High concentration of KYNA in some forest and flower honey may suggest a greater share of chestnut honey or even linden honey. Comparison with other KYNA rich substances Similar study was also conducted on honeys from different countries where it was also evident that chest- nut honey has the highest KYNA content. Concentration range was determined to be (103–141) μg/g or in another study (129–601) μg/g for chestnut honey, (0.177–0.391) μg/g for linden honey and (0.093–0.124) μg/g for flower honey.9,33 These results somehow overlap with our re- sults for chestnut honey, but are significantly lower in comparison with our results for linden and flower hon- ey. From another set of results containing flower honey it is evident that the determined concentration of KYNA is 0.878 μg/g.3 This may suggest that concentration of KYNA may also be dependent on soil, environment or fertilizer.10 Concentration of KYNA in honey samples is also high in comparison with other food. In other studies potato was suggested as food with high KYNA content where concentration varies (0.239–3.240) μg/g dry weight which quite coincides with concentration range of KYNA in acacia honeys.8 There are also some herbs and spices with high amount of KYNA with basil as one of the most prominent representatives with concentration 14.08 μg/g which positions basil in between linden and spruce hon- ey, but not even close the amount of KYNA in chestnut honey.11 Based on our results we can say that chestnut and linden honey are KYNA rich substances. Since the con- centration of KYNA is considerably high in chestnut honey the source of it must be a part of chestnut tree. Research on chestnut tree parts (flower, peeled chestnut, nectar, pollen, …) suggest that the source of KYNA is nectar of male flowers, since female flowers do not pro- duce nectar.9,37 KYNA in chestnut nectar is also observed in another study where the content of honey bee’s stom- ach of honey bee collecting in chestnut wood was inves- tigated.38 4. Conclusions Our main objective was to develop an optimal meth- od for preparation and analysis of KYNA in honey samples resulting in a fast and simple method with very few steps, avoiding any kind of extraction or other special pretreat- ment of samples that have been used by most so far. HPLC- MS method, where MS detector was used in SRM mode, was developed. Main focus was the optimization of SRM parameters allowing us to perform a selective analysis of KYNA within the untreated samples (except dilution and filtration). Mainly the spray voltage and collision energy needed to be optimized for better selectivity and flows of sheath, auxiliary and sweep gasses for better limit of detec- tion. Interestingly the spray voltage in positive was deter- mined to be only 190 V, which can be attributed to using the correct mobile phase and other MS parameters. The fragmentation of precursor ion was also investigated, since there is many product ions, and it was determined that the fragmentation to the product ion (m/z) 144.02 is the most suitable for selective determination; collision energy for that reaction was optimized and determined at 18 V. Hon- eys of chestnut and linden botanical species were found to be rich in KYNA, with average contents of 682 mg/g and 85 mg/g, respectively, followed by spruce (8.5 mg/g), acacia (2.2 mg/g), and silver fir (1.8 mg/g) honeys. For- est honey (0.8–397.7 mg/g) and flower honey (1.4–194.2 mg/g) show a very wide range of concentrations, which could be attributed to them being honey of various floral sources or heterogeneity of apiary area; a higher KYNA content could also suggest presence of chestnut or even linden honey. These results could lead chestnut or even linden honey under consideration as food supplement for relieving of digestive problems or influencing digestion and body weight. In addition, the results could as well set values for KYNA content to detect altered honeys. Acknowledgements The authors thank Medex d.o.o. for providing honey samples and Mikro+Polo d.o.o., for providing laboratory material. This work was supported by the Slovenian re- search agency ARRS, grant number P1-153. 5. References 1. J. Liebig, Justus Liebigs Ann. Chem. 1853, 86, 125–126. DOI:10.1002/jlac.18530860115 2. M. Dehhaghi, H. Kazemi Shariat Panahi, G. J. Guillemin, Int. J. Tryptophan Res. 2019, 12, 1–10. DOI:10.1177/1178646919852996 3. M. P. Turski, M. Turska, W. Zgrajka, D. Kuc, W. A. Turski, Amino Acids, 2009, 36, 75–80. DOI:10.1007/s00726-008-0031-z 4. G. Beretta, E. Caneva, R. M. Facino, Planta Med. 2007, 73, 279Acta Chim. Slov. 2023, 70, 274–280 Pavlin et al.: Direct Determination of Kynurenic Acid ... 1592–1595. DOI:10.1055/s-2007-993740 5. M. Kaihara, J. .M Price, H. Takahashi, J. Biol. Chem. 1956, 223, 705–708. DOI:10.1016/S0021-9258(18)65070-7 6. M. Kaihara, J. M. Price, J. Biol. Chem. 1962, 237, 1727–1729. DOI:10.1016/S0021-9258(19)83769-9 7. P. Milart, P. Paluszkiewicz, P. Dobrowolski, E. Tomaszewska, K. Smolinska, I. Debinska, K. Gawel, K. Walczak, J. Bednar- ski, M. Turska, M. Raban, T. Kocki, W. A. Turski, Sci. Rep. 2019, 9, 6108–6115. DOI:10.1038/s41598-019-42646-4 8. M. P. Turski, P. Kamiński, W. Zgrajka, M. Turska, W. A. Tur- ski, W, Plant Foods Hum. Nutr. 2012, 67, 17–23. DOI:10.1007/s11130-012-0283-3 9. M. P. Turski, S. Chwil, M. Turska, M. Chwil, T. Kocki, G. Ra- jtar, J. Palada-Turska, J. Food Compos. Anal. 2016, 48, 67–72. DOI:10.1016/j.jfca.2016.02.003 10. M. P. Turski, M. Turska, W. Zgrajka, M. Bartnik, T. Kocki, W. A. Turski, Planta Med. 2010, 77, 858–864. DOI:10.1055/s-0030-1250604 11. M. P. Turski, M. Turska, T. Kocki, W. A. Turski, P. Paluszkie- wicz, J. Chem. 2015, 1, 1– 6. DOI:10.1155/2015/617571 12. K. Walczak, W. A. Turski, G. Rajtar, Amino Acids, 2014, 46, 2393–2401. DOI:10.1007/s00726-014-1790-3 13. J. Wang, N. Simonavicius, X. Wu, G. Swaminath, J. Reagan, H. Tian, L. Ling, J. Biol. Chem. 2006, 281, 22021–22028. DOI:10.1074/jbc.M603503200 14. W. A. Turski, N. Nakamura, W. P. Todd, B. K. Carpenter, W. O. Whetsell Jr., R. Schwarcz, Brain Res. 1988, 454, 164–169. DOI:10.1016/0006-8993(88)90815-3 15. R. Lugo-Huitrón, T. Blanco-Ayala, P. Ugalde-Muñiz, P. Carri- lo-Mora, J Pedraza-Chaverrí, D. Silva-Adaya, P. D. Maldona- do, I. Torres, E. Pinzón, E. Ortiz-Islas, T. López, E. Garcia, B. Pineda, M. Torres-Ramos, A. Santamaría, V. Pérez-De La Cruz, Neurotoxicol. Teratol. 2011, 33, 538–547. DOI:10.1016/j.ntt.2011.07.002 16. G. B. Glavin, C. Pinsky, Res. Commun. Chem. Pathol. Phar- macol. 1989, 64, 111–119. 17. G. B. Glavin, R. Bose, C. Pinsky, Prog. Neuro-Psychopharma- cology Biol. Psychiatry, 1989, 13, 569–572. DOI:10.1016/0278-5846(89)90148-6 18. G. Varga, D. Erces, B. Fazekas, M. Fülöp, T. Kovács, J. Kaszaki, F. Fülöp, L. Vëcsei, M. Boros, Neurogastroenterol. Motil. 2010, 22, 7–9. 19. J. Dolecka, T. Urbanik-Sypniewska, B. Skrzydło-Radomańs- ka, J. Parada-Turska, Pharmacol. Reports, 2011, 63, 548–551. DOI:10.1016/S1734-1140(11)70522-9 20. J. Kaszaki, Z. Palásthy, D. Erczes, A. Rácz, C. Torday, G. Var- ga, L. Vécsei, M. Boros, Neurogastroenterol. Motil. 2008, 20, 53–62. 21. M. H. S. Mok, A. C. Fricker, A. Weil, J. N. C. Kew, Neurophar- macology, 2009, 57, 242–249. DOI:10.1016/j.neuropharm.2009.06.003 22. G. Clarke, P. Fitzgerald, J. F. Cryan, E. M. Cassidy, E. M. Quig- ley, T. G. Dinan, BMC Gastroenterol. 2009, 9, 6–12. DOI:10.1186/1471-230X-9-6 23. T. Ogawa, W. R. Matson, M. F. Beal, R. H. Myers, E. D. Bird, P. Milbury, S. Saso, Neurology, 1992, 42, 1702 LP – 1706. DOI:10.1212/WNL.42.9.1702 24. M. F. Beal, W. R. Matson, K. J. Swartz, P. H. Gamache, E. D. Bird, K, J. Neurochem. 1990, 55, 1327–1339. DOI:10.1111/j.1471-4159.1990.tb03143.x 25. M. F. Beal, W. R. Matson, E. Storey, P. Milbury, E. A. Ryan, T. Ogawa, E. D. Bird, J. Neurol. Sci. 1992, 108, 80–87. DOI:10.1016/0022-510X(92)90191-M 26. K. Rejdak, H. Bartosik-Psujek,  B. Dobosz,  T. Kocki,  P. Grieb, G. Giovannoni, W. A. Turski, Z. Stelmasiak, Neurosci. Lett. 2002, 331, 63–65. DOI:10.1016/S0304-3940(02)00710-3 27. K. Walczak, W. Dąbrowski, E.Langner, W. Zgrajka, J. Piłat, T. Kocki, W. Rzeski, W. A. Turski, Scand. J. Gastroenterol. 2011, 46, 903–912. DOI:10.3109/00365521.2011.579159 28. C. M. Forrest, S. R. Gould, L. G. Darlington, T. W. Stone, Adv. Exp. Med. Biol. 2003, 527, 395–400. DOI:10.1007/978-1-4615-0135-0_46 29. T. B. Meier, W. C. Drevets, T. Kent Teague, B. E. Wurfel, S. C. Mueller,  J. Bodurka, R. Dantzer,  J. Savitz, Brain. Behav. Im- mun. 2018, 67, 59–64. DOI:10.1016/j.bbi.2017.08.024 30. L. J. Hu, X. F. Li, J. Q. Hu, X. J. Ni, H. Y. Lu, J. J. Wang , X. N. Huang, C. X. Lin, D. W. Shang, Y. G. Wen, J. Anal. Toxicol. 2017, 41, 37–44. DOI:10.1093/jat/bkw071 31. G. Beretta, R. Artali, E. Caneva, S. Orlandini, M. Centini, R. M. Facino, J. Pharm. Biomed. Anal. 2009, 50, 432–439. DOI:10.1016/j.jpba.2009.05.029 32. Q. Tong, J.Song, G. Yang, L. FaN, W. Xiong, J. Fang, Biomed. Chromatogr. 2018, 32. DOI:10.1002/bmc.4156 33. M. E. Soto, A. M. Ares, J. Bernal, M. J. Nozal, J. L. Bernal, J. Chromatogr. A, 2011, 1218, 7592–7600. DOI:10.1016/j.chroma.2011.06.105 34. T. Du, T. Cui, H. M. Qiu, N. R. Wang, D. Huang, X. H. Jiang, J. Pharm. Biomed. Anal. 2018, 158, 8–14. DOI:10.1016/j.jpba.2018.05.032 35. R. Fakhlaei, J. Selamat, A. Khatib, A. F. A. Razi, R. Sukor, S. Ahmad, A. A. Babadi, Foods. 2020, 9, 1538–1558. DOI:10.3390/foods9111538 36. T. Ždiniaková, C. Lörchner, O. De Rudder, T. Dimitrova, G. Kaklamanos, A. Breidbach, A. Respaldiza, I. Vaz Silva, V. Paiano, F. Ulberth, A. Maquet, Publications Office of the Eu- ropean Union, https://op.europa.eu/en/publication-detail/-/ publication/330ee93f-c9fc-11ed-a05c-01aa75ed71a1/lan- guage-en, (accessed: April 16, 2023). 37. Á. Farkas, E. Zajácz, Eur. J. Plant Sci. Biotechnol. 2007, 1, 125– 151. 38. P. Truchado, I. Martos, L. Bortolotti, A. G. Sabatini, F. Fer- reres, F. A. Tomas-Barberan, J. Agric. Food Chem. 2009, 57, 5680–5686. DOI:10.1021/jf900766v 280 Acta Chim. Slov. 2023, 70, 274–280 Pavlin et al.: Direct Determination of Kynurenic Acid ... Except when otherwise noted, articles in this journal are published under the terms and conditions of the  Creative Commons Attribution 4.0 International License Povzetek Kinurenski kislini (KYNA) pripisujejo številne koristne lastnosti, kot so antioksidativne, antiproliferativne, protivnetne in anti-obesogene značilnosti. S svojim delovanjem naj bi vplivala na metabolizem in s tem na uravnavanje telesne mase. Razvili smo hitro, enostavno in zanesljivo HPLC-MS/MS metodo za določanje kinurenske kisline (KYNA) v medu. Sistem HPLC-MS/MS nam je omogočil izvedbo analiz brez posebne ekstrakcije ali obdelave vzorcev. V raziskavi smo analizirali med različnih botaničnih vrst, in sicer kostanjev (C), lipov (L), akacijev (A), smrekov (S), hojev (SF), gozdni (Fo) in cvetlični (F) med. Najvišjo povprečno koncentracijo kinurenske kisline (682 μg/g) smo določili v kostanjevem medu, kar ga uvršča med živila z najvišjo vsebnostjo KYNA. 281Acta Chim. Slov. 2023, 70, 281–293 Tahir et al.: Synthesis, SC XRD Based Structure Elucidation, ... DOI: 10.17344/acsi.2023.8108 Scientific paper Synthesis, SC XRD Based Structure Elucidation, Supramolecular Assembly Exploration Via Hirshfeld Surface Analysis, Computational and QTAIM Study of Functionalized Anilide Muhammad Nawaz Tahir,1 Muhammad Ashfaq,1* Akbar Ali,2* Chin Hung Lai,3,4 Bojja Rajeshwar Rao,5 Khurram Shahzad Munawar6,7 and Irshad Ali Shahid1 1 Department of Physics, University of Sargodha, Sargodha 40100, Pakistan 2 Department of Chemistry, Government College University, Faisalabad, Pakistan 3 Department of Medical Applied Chemistry, Chung Shan Medical University, Taichung 40241, Taiwan 4 Department of Medical Education, Chung Shan Medical University Hospital, Taichung 402, Taiwan 5 Senior Chemist (Retired), Chemical Division, Kakatiya Thermal Power Project (O&M) Chelpur-506 170, Telangana State, India 6 Department of Chemistry, University of Mianwali, 42200, Pakistan 7 Department of Chemistry, University of Sargodha, 40100, Pakistan * Corresponding author: E-mail: ashfaq.muhammad@uos.edu.pk Received: 03-03-2023 Abstract The anilide compound named (Z)-4-(2-methoxy-4-nitrophenyl)amino)-4-oxobut-2-enoic acid (MAOA) has been synthesized by the chemical reaction of 2-methoxy-4-nitroaniline and maleic anhydride in ethyl acetate. The synthesized compound was characterized by elemental analysis, FT-IR and UV-Vis spectroscopy, and TGA/DSC technique. Further- more, the crystal structure was analyzed by the single crystal X-ray diffraction (SC XRD) technique. The supramolecular assembly of MAOA in terms of non-covalent interactions was explored by Hirshfeld surface analysis. Void analysis inferred that MAOA is expected to have good mechanical properties. The crystal packing environment was further in- vestigated by interaction energy between molecular pairs and energy frameworks. Moreover, the result of the gas-phase DFT study showed that there is an intramolecular N–H···O and O–H···O hydrogen bond in MAOA due to the distance between D and A being smaller than the sum of their van der Waals radii. The result of the QTAIM study showed that there should also be an intramolecular CH···O hydrogen bond with a strength of 3.40 kcal/mol in MAOA. Keywords: Anilide; Crystal structure; Supramolecular assembly; Non-covalent interactions; Gas-phase DFT 1. Introduction Substituted anilines are very important chemical species that could be used as a starting material for the synthesis of valuable triazole-based medicines like flu- conazole, itraconazole, voriconazole, and posaconazole.1 Another important method of aniline modification is the N-alkylation followed by photochemical radical cycliza- tion reaction for the synthesis of indoles,2 as a precursor for the synthesis of the acetaminophen (paracetamol) that is widely used as a medication to treat the pain and fever,3 and for the photochemical cyclization to accomplish the highly substituted indulines.4 Aniline could also be used as a precursor for the synthesis of quinoline which is an im- portant heterocyclic aromatic compound with medicinal and chemical significance.5 Acid anhydrides are also valu- able chemical building blocks with the speciality of high reactivity that can be used for the synthesis of new chemi- cal architectures that might be used as intermediates or the 282 Acta Chim. Slov. 2023, 70, 281–293 Tahir et al.: Synthesis, SC XRD Based Structure Elucidation, ... final products for utilization in the field of chemical mod- ification specially anilides. Anilides, mostly produced by the reaction of substi- tuted anilines and anhydrides are well-recognized chemi- cal building blocks in the field of medicinal chemistry be- cause of their broad bioactive spectrum. Anilides such as MAOA having the nitro group and carboxylic acid func- tionality could be exciting chemical compounds as the hetero atoms might be responsible for non-covalent inter- actions and could also be used as potential ligands in coor- dination chemistry. Nitro group containing anilides have shown several biological activities like antidepressants and anticancer,6,7 analgesic and antimicrobial,7 caspases acti- vators and apoptosis inducers,8 and anti-HIV-I agents9 as shown in Figure 1. Figure 1. Anilide functionality embedded molecules with their bio- logical potential. Currently, the synthesis of crystalline organic com- pounds and their single crystal analysis together with the computational investigation are gaining enormous atten- tion in order to predict various electronic features such as non-linear optical properties,10–13 frontier molecular or- bitals14 and non-covalent interactions etc.15–17 Maleic an- hydride could also be utilized for the N-alkylation of pri- mary amines to produce functionalized anilides. Herein, we are presenting our findings concerning the synthesis, single crystal analysis-based structural investigation and computational exploration of the MAOA. 2. Experimental 2. 1. Materials and methods The 2-methoxy-4-nitroaniline, maleic anhydride, and other reagents used in the current work were of ana- lytical grade and purchased from Sigma-Aldrich, Merck, Daejung, and Alfa Aesar. The combustion analysis for the estimation of C, H, and N was carried out using a Vario EL elemental analyzer. The melting point of the synthesized compound was measured in an open capillary using the Gallen Kamp electrochemical melting point device. Func- tional groups present in the sample were analyzed by using Fourier transform infrared spectroscopy from 400 to 4000 cm–1 using IRSpirit-T equipped with diamond ATR (Shi- madzu). The thermal data (TGA/DSC) was collected using Discovery 650 SDT simultaneous thermal analyzer (TA Instruments)  with a temperature range from ambient to 400 °C. The heating rate was 10 °C/minute under a 99.999% nitrogen atmosphere with a flow rate of 50 mL/min. The absorption spectra were measured on a CE 7200 dou- ble-beam UV-Visible spectrophotometer using DMSO as a solvent. For the sake of thin-layer chromatography, the pre-coated silica was employed to monitor the progress of the chemical reaction and to ensure the purity of the prod- uct formed. 2. 2. Synthesis of (Z)-4-(2-Methoxy-4- nitroanilino)-4-oxobut-2-enoic acid (MAOA) Equimolar amounts of 2-methoxy-4-nitroaniline (1.0 mmol, 0.168 g) and maleic anhydride (1.0 mmol, 0.098 g) were dissolved separatelyin 10 mL of ethyl acetate in 50 mL of beakers. Both solutions were then mixed drop by drop with continuous stirring. After complete addition, the mixture was further stirred for 5 hours. The progress and completion of the reaction and purity of the product were continuously monitored with the help of thin-layer chromatography. The yellow solid product was then ob- tained by evaporation of solvent via a rotary evaporator. Recrystallization of the obtained product was done in methanol to get light yellow crystals (0.213 g) of good quality (Scheme 1). MAOA: Yield: 80 %; M.P: 171 °C; Color: Light Yel- low; Anal. Calc. for C11H10N2O6: C, 49.63; H, 3.79; N, 10.52; Found: C, 49.52; H, 3.71; N, 10.55 %; FT-IR (cm–1); 3328 (νN–H), 3055 (νCH aromatic), 3001 (νC–H alkene), ~2970 (νC–H aliphatic), 1713 (νCOO), 1615 (νC=O), 1582 (νC=C), 1552 and 1349 (νNO2), 1017 (νC–N) [Figure S1]; UV-Vis (DMSO); λmax= 360 (π–π*) [Figure S2]; TGA/ DSC; 71% weight loss from 130 to 260 oC, Enthalpy (nor- malized); 1361 J/g, Phase change at 171 °C with heat flow –0.671 W/g, Residue 12.97% [Figure S3]. 2. 3. X-ray Crystallography Details Raw data of single crystal by X-ray was collected on Bruker Kappa Apex-II CCD diffractometer with a target made of molybdenum and λ = 0.71073 Å. APEX-II soft- ware was employed for data collection. The structure was solved and refined on SHELXT201418 and SHELXL-2019/2,19 respectively. Refinement of all atoms other than H-atoms was performed by employing aniso- 283Acta Chim. Slov. 2023, 70, 281–293 Tahir et al.: Synthesis, SC XRD Based Structure Elucidation, ... tropic displacement parameters of atoms whereas refine- ment of H-atoms was performed with relative isotropic displacement parameters by using the riding model. OR- TEP-3,20 PLATON21 and Mercury 4.022 software were em- ployed for the graphical representation of results. Table 1. Crystal data and refinement parameter for MAOA. Chemical formula C11H10N2O6 Molecular weight 266.21 Temperature 296(2) K Crystal system Monoclinic Space group P21/c a (Å) 3.8664(6) b (Å) 23.282(4) c (Å) 11.2114(17) α (°) 90 β (°) 96.461(9) γ (°) 90 V (Å3) 1132.0(3) Z 4 µ (mm–1) 0.130 F(000) 552 Reflections collected 8207 Unique reflections 2168 Observed reflections [I > 2σ(I)] 1388 Data/restraints/parameters 2168/0/174 Rint 0.078 S 1.071 R1, wR2 [I ≥ 2σ(I)] 0.0811, 0.1770 R1, wR2 (all data) 0.1191, 0.1988 2. 4. Procedure of Hirshfeld Surface Analysis and Interaction Energy Between Molecular Pairs Hirshfeld surface analysis is a unique way for the ex- ploration of strong as well as weak intermolecular interac- tions in single crystals. The analysis is done on Crystal Ex- plorer version 21.5.23 Hirshfeld surfaces providing information about the intermolecular interactions by color coding.24 We further explored the crystal packing environ- ment by finding the interaction energy between molecules. Crystal Explorer version 21.5 is used for interaction ener- gy calculations along with B3LYP/6-31G(d,p) electron density model. The interaction energy is the sum of four kinds of energies named as electrostatic (E_ele), polariza- tion (E_pol), dispersion (E_dis) and exchange repulsion (E_rep).25 Electrostatic energy can be attractive or repul- sive whereas polarization and dispersion energy are always attractive. 2. 5. Computational Details 2. 5. 1. DFT and NBO Studies The DFT study is utilized to examine the title com- pound’s gas-phase structures. The DFT calculations were done using the hybrid B3LYP approach, which is a combi- nation of the exact exchange (HF) and Becke functionals, as well as the LYP correlation functional, and is based on Becke’s notion.26–28 A B3LYP calculation was performed with the basis set 6-311++G**.29 After obtaining the con- verged geometry, the vibrational harmonic frequencies are calculated at the same theoretical level to ensure that the imaginary frequency number is zero for the saddle point. For the study of the intrinsic electronic properties of the studied compound, the NBO analysis on the studied com- pound is performed at the same theoretical level. All men- tioned calculations are performed by Gaussian 16.30 More- over, the molecular conformational is acquired by Austin Model method and compared with the molecular confor- mation by SC XRD. The details and findings of Austin Model method are given in the supplementary informa- tion file. 2. 5. 2. QTAIM Study The quantum theory of atoms in molecules (QTAIM) also called atoms in molecules (AIM) is a model of mole- cules and condensed matters. In this model, the major ob- jects of molecules and condensed matters, i.e., atoms and bonds are naturally expressed by the distribution function of the observable electronic density of a molecule. The electron density distribution of a molecule is a probability distribution and describes the average distribution of the electronic charge in the field of attraction exerted by the nuclei. According to QTAIM, the molecular structure is revealed by the stationary points and the gradient paths of electron density. The gradient paths of a molecule’s elec- tron density are originated and terminated from the sta- tionary points. In this study, the QTAIM analysis is per- formed using the multiwfn program.31 Scheme 1. Synthetic scheme for the (Z)-4-(2-methoxy-4-nitroanilino)-4-oxobut-2-enoic acid (MAOA). 284 Acta Chim. Slov. 2023, 70, 281–293 Tahir et al.: Synthesis, SC XRD Based Structure Elucidation, ... 3. Results and Discussion 3. 1. Synthesis and Analysis A new functionalized anilide (MAOA) was synthe- sized by reacting 2-methoxy-4-nitroaniline with maleic anhydride in ethyl acetate under stirring conditions. The obtained yellow solid product was recrystallized in metha- nol to get pure crystals suitable for X-ray diffraction anal- ysis and spectroscopic characterizations (Scheme 1). 3. 2. Single Crystal X-ray Diffraction Analysis of MAOA The Cambridge structure database search confirmed that the crystal structure of MAOA is novel. The search provides a lot of crystal structures that have some similar- ities with the crystal structure of MAOA. The crystal structure of MAOA is compared with the closely related crystal structures. The molecular configuration of MAOA (Figure 2, Table 2) is stabilized by intramolecular N–H···O and O–H···O bonding. S(5) and S(7) H-bonded loops are formed by the intramolecular N–H···O and O–H···O bonding, respectively.32 The (Z)-4-oxobut-2-enoic acid group A (C1–C4/O1–O3) and 2-methoxyanilinic group B (C5–C11/N1/O4) are roughly planar with root mean square (r.m.s.) deviations of 0.0694 and 0.0159 Å, respec- tively. The corresponding dihedral angle A/B is 6.73(14)°. The nitro group C (N2/O5/O6) is twisted at the dihedral angle of 10.3(4)° with respect to group B. The substitutions on the phenyl ring make the molecule non-planar. The molecules are connected in the form of dimers through N–H···O and C–H···O bonding to form R12(6) loop (Fig- ure 3, Table 2). In both H-bonding, the acceptor O-atom is from the carbonyl O-atom (O2) of the carboxylate group (C1/O1/O2). In C–H···O bonding, the H-bond donor is from group A. The phenyl ring, carbonyl O-atoms (O3/ O4) and nitro group are not involved in any intermolecu- lar H-bonding. Due to the intermolecular bonding, a monoperiodic infinite chain of molecules is formed with a base vector [2 0 1]. Moreover, solid-state packing is further stabilized by π···π stacking. The phenyl rings of the mole- cule present in the asymmetric unit are involved in off-set π···π stacking interactions with the phenyl rings of the symmetry-related molecules (1 – x, y, z and 1 + x, y, z). Inter-centroid separation of this interaction is 3.866 Å and the ring off-set range is from 1.527 to 3.866 Å as displayed Figure 3. Packing diagram of MAOA showing dimerization of molecules. Figure 2. ORTEP diagram of MAOA that is drawn at the probabil- ity level of 50% with H-atoms are displayed by small circles of arbi- trary radii. 285Acta Chim. Slov. 2023, 70, 281–293 Tahir et al.: Synthesis, SC XRD Based Structure Elucidation, ... in Figure 4. The other weak interactions such as C–H···π, N–O···π and C–O···π are not found in the crystal packing of MAOA. The Cambridge structure database search pro- vides more than 50 crystal structures that have some simi- larity with the crystal structure of MAOA. The close in- spection of these structures inferred that 5 out of 50 have close similarity with the crystal structure of MAOA. Two structures with reference code JAYGEW33 and MNP- MAL0134 have nitro-substituted phenyl rings whereas, the other two structures with reference code LAQJEU35 and SAGFIR36 have disubstituted phenyl rings (nitro and chloro). The molecular configuration of JAYGEW and MNPMAL01 is stabilized by intramolecular O–H···O and C–H···O bonding along with intermolecular N–H···O and C–H···O bonding. O–H···O intramolecular H-bonding is found in LAQJEU and SAGFIR along with N–H···Cl bond- ing which is present in SAGFIR. π···π stacking interaction is found in these four selected literature crystal structures. Table 2. Hydrogen-bond geometry (Å, º) for MAOA. D—H···A D—H H···A D···A <(D—H···A)° N1–H1A···O4 0.86 2.22 2.621 (3) 108 O1–H1···O3 0.82 1.74 2.545 (3) 167 N1–H1A···O2i 0.86 2.39 3.198 (4) 156 C3–H3···O2i 0.93 2.31 3.176 (4) 154 Symmetry codes: (i) x − 1, −y + ½, z − ½. 3. 3. Hirshfeld Surface Analysis Hirshfeld surface (HS) mapped over dnorm is dis- played in Figure 5a. The surface uses three colors, red, white and blue to classify interatomic contacts by their strength. Red and blue spots stand for short and long con- tacts, respectively. The contacts for which the distance be- tween the interacting atoms is equal to the sum of the van der Waal radii are shown by white spots on the surface. The most dominant interactions in the crystal packing are indicated by red spots on the HS whereas nearly negligible and intermediate intermolecular interactions are indicated by blue and white color, respectively. The red spot around the NH group, one of the CH of group A and the carbonyl O-atom of the carboxylate group indicate that these atoms are involved in short contacts or H-bonding. The H-bond- ing is represented by the green dotted line in Figure 5a. π···π stacking interactions can be visualized by plotting HS over the shape index. The presence of consecutive red and Figure 5. Hirshfeld surface plotted over (a) dnorm, (b) shape index. Figure 4. Graphical representation of chain along a-axis that is formed by off-set π···π stacking interactions. H-atoms are not shown for clarity. Distances are measured in Å. 286 Acta Chim. Slov. 2023, 70, 281–293 Tahir et al.: Synthesis, SC XRD Based Structure Elucidation, ... blue triangular-shaped regions around phenyl rings in Fig- ure 5b indicates that stacking interactions are present in MAOA. The intermolecular interactions can be further ex- plored with the utilization of two-dimensional finger- print plots which is a key analysis to separately identify and quantify the interatomic contacts.37–40 Figure 6a is the 2D fingerprint plot for overall interactions on which short interactions contacts are shown by large spikes. In most crystal structures of organic compounds, H···H contacts are the most significant contributors in the crys- tal packing but in our case, the O···H contacts are the most significant contributors in the crystal packing with a percentage contribution of 39.8% (Figure 6b). The oth- er significant interatomic contributors responsible for the overall packing of molecules are H···H, C···H, O···O, C···C and O···C with percentage contributions of 22.6%, 12.7%, 7.2%, 6.2%, and 5.9% (Figure 6c–g), respectively. The enrichment ratio provides the tendency of the pair of chemical species in the single crystal to form crys- tal-packing interactions. Each pair of chemical species has a unique ability to be involved in the crystal packing. Some pairs have a higher tendency to be involved in the crystal packing interactions than others. The enrichment ratio for a pair (X, Y) is acquired by dividing the propor- tion of the actual contact by the proportion of the ran- Figure 6. Two-dimensional fingerprint plots in MAOA for (a) overall interactions and (b-i) individual interatomic contacts. 287Acta Chim. Slov. 2023, 70, 281–293 Tahir et al.: Synthesis, SC XRD Based Structure Elucidation, ... dom contact calculated theoretically.41 The results of this study are summarized in Table S1. Although the O···H contacts are the most significant contributors to the crys- tal packing but the contact which has the highest tenden- cy to form crystal packing is C···C with an enrichment ratio of 2.58. The other higher tendency contacts are O···N and O···H with enrichment ratios of 2.15 and 1.26, respectively. The H···H contacts are not favorable as the enrichment ratio for this contact is less than 1. For the sake of further exploration of the crystal packing in MAOA, the interaction of an atom of a mole- cule with all other atoms present in its surrounding is calculated.42 Figure 7a gives a quantitative description of the interaction of an atom present inside the HS to the atoms present in the surrounding HS. The H-atoms pres- ent inside the HS interact strongly with atoms present in the surrounding HS with a percentage contribution of 46.6%. The quantitative contribution of other such inter- actions O-ALL, C-ALL and N-ALL is 33.2%, 17% and 3.2%, respectively. Figure 7b gives a quantitative descrip- tion of the interaction of an atom present outside the HS with all the atoms present inside the HS. The H-atoms present outside the HS interact strongly with atoms pres- ent inside the HS with a percentage contribution of 53%. The quantitative contribution of other such interactions ALL-O, ALL-C and ALL-N is 30.6%, 14.1%, and 2.3%, respectively. 3. 4. Interaction Energy and Energy Frames Analysis The molecule present in the asymmetric unit is taken as a reference molecule and molecules present in the vicini- ty of the reference molecule (3.8 Å) are taken in calcula- tions. The results of interaction energy calculations are giv- en in Table 3. The total energy is maximum for the molecular pairs with the center-to-center separation of 11.07 Å that are related to each other by inversion symmetry and for this pair, the hydrogen-bonded contacts are the major controller of the interaction energy as compared to the π···π stacking interaction. The net attractive energy is maximum for the molecular pairs with the center-to-center separation of 9.91 Å that is related to each other by symmetry (–x, –y + ½, z + ½). The electrostatic energy is repulsive for two molecular pairs with the center-to-center separation of 3.87 and 6.07 Å. For the molecular pair with an intermolecular distance of 3.87 Å, hydrogen-bonded contacts and π···π stacking interaction both are the significant controller of the interaction energy. For all other pairs, the hydrogen-bonded contacts are the significant controller of the interaction en- ergy. The strength of a particular type of interaction energy can be visualized by energy frameworks that contained cyl- inders whose width is directly proportional to the strength of the interaction.43 Figure 8 represents energy frames of the electrostatic and dispersion energy, respectively. Energy Table 3. Intermolecular interaction energies in kJ mol–1 calculated at B3LYP/6-31G(d,p) electron density model for MAOA. %E_attract contribution Colour Symmetry N R E_ele E_pol E_dis E_rep E_tot %E_attract %E_ele %E_pol %E_dis codes   (i) 1 6.07 1.9 –1.6 –23.1 10 –12.8 –24.7 0 6.48 93.5   (ii) 2 3.87 15.8 –6 –62 28 –24.2 –68 0 8.82 91.2   (i) 1 11.07 –26.9 –5.1 –14.6 18.4 –28.2 –46.6 57.7 10.9 31.3   (ii) 2 11.44 –14.8 –2.8 –6.7 5 –19.3 –24.3 60.9 11.5 27.6   (iii) 2 9.91 –30.2 –9.1 –15.9 30.7 –24.5 –55.2 54.7 16.5 28.8   (i) 1 10.57 –21 –3.9 –17.2 11.3 –30.8 –42.1 49.9 9.26 40.9   (iii) 2 9.39 –0.8 –5.6 –18.4 13.4 –11.4 –24.8 3.23 22.6 74.2   (i) 1 6.80 13.8 –2.5 –17.4 7.9 4.2 –19.9 0 12.6 87.4 Symmetry codes: (i) –x, –y, –z; (ii) x, y, z; (iii) –x, –y + ½, z + ½. Figure 7. Graphical summary of the (a) Interaction of an atom present inside the HS to atoms present in the surrounding of HS, (b) Interaction of an atom present outside the HS to atoms present in the HS. 288 Acta Chim. Slov. 2023, 70, 281–293 Tahir et al.: Synthesis, SC XRD Based Structure Elucidation, ... frames show that the contribution of the dispersion energy in defining the total interaction energy is greater than the contribution of the electrostatic energy. 3. 5. Void Analysis The percentage of void volume in the unit cell of a compound is the demonstration of how strongly the mole- cules are packed with each other. Figure S4 is a graphical view of voids that is obtained by summation of electron den- sities of spherically symmetric atoms at the pertinent nuclear positions.44–47 The computations of the crystal void infer that the void volume is of the order of 80.53 Å3. It is found that the calculated void volume of the entitled compound is near- ly equal to 7% indicating that the crystal has a high packing factor without a large cavity in the crystal packing. 3. 6. DFT Exploration A gas-phase DFT study was performed utilizing the B3LYP functional to rationalize the relationship between the intrinsic electronic properties, the chemical reactivity, and the biological activities of the title compound. The B3LYP-optimized geometry of the title compound is de- picted in Figure 9. Moreover, the detailed comparison be- tween the optimized geometry and the crystallographical one could be seen in the Supporting information (Table S2). Accordingly, the B3LYP/6-311+G** theoretical level which was utilized in this study is proved to be a suitable one to investigate anilide derivatives. According to the frontier molecular orbital theory, one can determine a molecule’s nucleophilicity or elec- trophilicity by focusing on the highest occupied and lowest unoccupied molecular orbitals (HOMO and LU- MO).48 Instead of considering the total electron density as a nucleophile, evaluate the localization of the HOMO orbital since electrons from this orbital have the best probability of participating in the nucleophilic attack, whereas a site with the lowest empty orbital is a suitable electrophilic site. The title compound’s frontier molecu- lar orbital is thus studied further in this work. As depict- ed in Figure 10, in the studied chemical, the orbital Figure 8. Energy frames for (a) electrostatic energy, (b) dispersion energy. Figure 9. The B3LYP-optimized geometry of the title compound. 289Acta Chim. Slov. 2023, 70, 281–293 Tahir et al.: Synthesis, SC XRD Based Structure Elucidation, ... transfer from HOMO to LUMO belongs to a π-π* transi- tion. Molecular electrostatic potentials (MEPs) are essen- tial assessments of the strength of interactions between neighboring charges, nuclei, and electrons at a specific point, allowing us to examine charge distribution and charge-related features of molecules. A graphic depiction with different colors is utilized to make the electrostatic potential data easier to understand. Electrophiles may be attracted to reading since it reflects the lowest electrostatic potential value. Blue, on the other hand, has the largest electrical potential and may be attractive to nucleophiles. The entire density of the title compound is calculated us- ing the whole density matrix, and the resulting MEP is mapped on its surface. As depicted in Figure 11, the oxy- gen atoms are the most sensitive site for the attack by an electrophile among the title compound. 3. 6. 1. NBO Analysis The second-order perturbation theory is used to analyze the relative strength of the intramolecular hydro- gen bonds in the tested molecule in this study. When a hy- drogen bond occurs, there should be an orbital interaction between the nonbonding orbital of the hydrogen-bonded acceptor (nA) and the antibonding orbital of the H-donor bond (σH-D*). As a result of this orbital interaction, the H-D bond’s bond strength and bond order should be re- duced and decreased, respectively. Therefore, the interac- tion between a lone pair and the X–H antibonding orbital is summarized in Table 4. It is noteworthy that such orbital interactions with interaction energies larger than 1 kcal/ mol in the whole studied chemical are only listed in Table 4. As expected, the oxygen atom has two lone pairs (LPs) whereas the nitrogen atom has only one LP. The two LPs on the oxygen atom are represented as LP, and LP’, respec- tively. The LPs on the oxygen atom of the methoxy group in the title chemical have three orbital interactions by in- teraction energies larger than 1 kcal/mol with respect to the antibonding orbitals of the C-H bond in the methyl group. However, the LPs on the oxygen atom of the meth- oxy group in the title chemical don’t form orbital interac- tions by interaction energies larger than 1 kcal/mol (about 0.8 kcal/mol) with respect to the antibonding orbital of the Figure 11. The MEP of the studied compound (the isovalue = 0.0004 a.u). Figure 10. The HOMOs and LUMOs of the studied compound (the isovalue = 0.02 a.u.). 290 Acta Chim. Slov. 2023, 70, 281–293 Tahir et al.: Synthesis, SC XRD Based Structure Elucidation, ... neighboring N–H bond. Moreover, the lone pair on the nitrogen atom in the title compound doesn’t show orbital interactions by interaction energies larger than 1 kcal/mol with respect to any antibonding orbitals. 3. 7. QTAIM Study The aforementioned results of the NBO analysis which show that the LPs of the oxygen atom in the meth- oxy group do not form orbital interactions by more than l kcal/mol with the antibonding orbital of the neighboring N-H bond motivated us to investigate whether there is no intramolecular N-H···O hydrogen bond in the studied compound. The QTAIM study is thus performed using the Multiwfn program. Under the condition that the Poin- care-Hopf relationship is satisfied, the calculated critical points (CPs) have a total of 65 (in Figure 13). There are 29, 32, 4, and 0 for the (3,–3), (3,–1), (3,+1) and (3,+3) CP, respectively. As depicted in Figure S5, the (3,–1) CP desig- nated as points 32, 34, 61 may indicate that there is an in- tramolecular O1–H2···O4, C24–H25···O4, and N8– H9···O5 hydrogen bond, respectively. The QTAIM study has already been used as a powerful tool to investigate in- tra- or intermolecular hydrogen bonding of several sys- Table 4. The NBO results of the title compound. The type of nA The electron The orbital The interaction The occupancy The bond order configuration of nA interaction energy (in kcal/mol)b of σH-D* of σH-Dc LP(O4)a s(56.05%) LP(O4)···σ*(O1-H2)a 7.44 0.06057 0.5873 p(43.92%) d(0.02%) LP’(O4)a s(3.32%) LP’(O4)···σ*(O1-H2)a 25.67 0.06057 0.5873 p(96.62%) d(0.06%) LP(O5)a s(36.50%) LP(O5)···σ*(C26-H27)a 1.06 0.01534 0.7942 p(63.47%) d(0.03%) LP(O5)a s(36.50%) LP(O5)···σ*(C26-H28)a 3.23 0.00770 0.7942 p(63.47%) d(0.03%) LP’(O5)a s(0.00%) LP’(O5)···σ*(C26-H27)a 4.11 0.01534 0.7942 p(99.96%) d(0.04%) a) Please see the atomic designations in Figure 9. b) The interaction energy was calculated based on the second-order perturbation theory. c) The listed values were the atom-atom overlap-weighted NAO bond order. Figure 12. All the critical points of MAOA. 291Acta Chim. Slov. 2023, 70, 281–293 Tahir et al.: Synthesis, SC XRD Based Structure Elucidation, ... tems.49–56 According to QTAIM, if D-H forms a hydrogen bond with A, there should be a CP between H and A. In addition, criteria about the electron density (ρb) and the Laplacian of electron density (∇2ρb) at BCPs have been es- tablished by Koch and Popelier to distinguish hydrogen bonding from van der Waals interactions. Moreover, Liu and coworkers have established a relationship between the hydrogen bonding strength (BE) and the electron density (ρ) at the CP corresponding to the hydrogen bond. The relationship could be described as Eq. (1) shows BE (in kcal/mol) ≈ –223.08 × ρ + 0.7423 (1) Accordingly, the strength of the aforementioned in- tramolecular O1–H2···O4, C24–H25···O4, and N8– H9···O5 hydrogen bonds is calculated as –11.43, –3.40, and –3.99 kcal/mol, respectively. 4. Conclusion The (Z)-4-(2-methoxy-4-nitrophenyl)amino)-4-ox- obut-2-enoic acid is synthesized and characterized by sin- gle crystal X-ray diffraction (SC XRD), UV-Vis, FT-IR, TGA/DSC techniques. SC-XRD analysis inferred that the strong intermolecular H-bonding of type O–H···O, N–H···O and comparatively weak C–H···O bonding and π···π stacking interactions are responsible for crystal packing. UV-Vis spectrum showed λmax at 360 nm due to π-π* transitions. FT-IR result confirms the formation of the compound by showing characteristics carboxylic acid peak at 1713 cm–1. TGA/DSC results represent the major weight loss (71%) in a single step from 130 to 260 °C with the loss of main fragments leaving behind residue com- prised of carbon in the form of coke. It is evident from heat flow that the sample changed its phase from solid to liquid around 171 °C. Hirshfeld surface analysis shows that O–H/H–O inter-atomic contact is the most significant contributor to the overall strengthening of packing of mol- ecules with a percentage contribution of 39.8%. The void analysis predicted that MAOA will have good mechanical properties. The interaction energy between molecular pairs and energy framework analysis showed that for the stabilization of the supramolecular assembly in MAOA, the dispersion energy is the dominant energy as compared to other types of energies. According to the results of the DFT and QTAIM studies, MAOA could be stabilized by the intramolecular O–H···O, N–H···O, and C–H··· hydro- gen bonds in the gas phase. Supplementary data CCDC 2009600 contains the supplementary crystal- lographic data for (MAOA). The data can be obtained free of charge via http://www.ccdc.cam.ac.uk/conts/retrieving. html, or from the Cambridge Crystallographic Data Cen- tre, 12 Union Road, Cambridge CB2 1EZ, UK; fax: (+44) 1223-336-033; or e-mail: deposit@ccdc.cam.ac.uk. Acknowledgement Akbar Ali greatly acknowledges the support of HEC Pakistan and Khurram Shahzad Munawar is highly thank- ful to the University of Mianwali for characterization. Conflict of interest: The authors declare that they have no conflict of interest. 5. References 1. A. Ali, A. G. Corrêa, D. Alves, J. Zukerman-Schpector, B. Westermann, M. A. Ferreira, M. W. Paixão, ChemComm. 2014, 50, 11926–11929. DOI:10.1039/C4CC04678A 2. G. P. da Silva, A. Ali, R. C. da Silva, H. Jiang, M. W. Paixao, ChemComm. 2015, 51, 15110–15113. DOI:10.1039/C5CC06329A 3. W. M. Lee, J. Hepatol. 2017, 67, 1324–1331. DOI:10.1016/j.jhep.2017.07.005 4. C. A. Caiuby, A. Ali, V. T. Santana, F. W. d. S. Lucas, M. S. San- tos, A. G. Corrêa, O. R. Nascimento, H. Jiang, M. W. Paixão, RSC Adv. 2018, 8, 12879–12886. DOI:10.1039/C8RA01787E 5. S. M. Prajapati, K. D. Patel, R. H. Vekariya, S. N. Panchal, H. D. Patel, RSC Adv. 2014, 4, 24463–24476. DOI:10.1039/C4RA01814A 6. P. Theivendren, A. Subramanian, I. Murugan, S. D. Joshi, U. A. More, Chem. Biol. Drug Des. 2017, 89, 714–722. DOI:10.1111/cbdd.12894 7. T. Panneerselvam, S. Arumugam, M. A. Ali, K. Selvaraj, M. Indhumathy, A. Sivakumar, S. D. Joshi, ChemistrySelect 2017, 2, 2341–2347. DOI:10.1002/slct.201601763 8. W. Kemnitzer, S. Cai, J. Drewe, N. Sirisoma, Substituted N-Aryl-9-Oxo-9H-Fluorene-1-Carboxamides and Analogs as Activators of Caspases and Inducers of Apoptosis, Patent Number WO2006039356A3, date of patent April 13, 2006. 9. H. J. Breslin, M. J. Kukla, D. W. Ludovici, R. Mohrbacher, W. Ho, M. Miranda, J. D. Rodgers, T. K. Hitchens, G. Leo, J. Med. Chem. 1995, 38, 771–793. DOI:10.1021/jm00005a005 10. A. Ali, M. Khalid, M. F. u. Rehman, S. Haq, A. Ali, M. N. Ta- hir, M. Ashfaq, F. Rasool, A. A. C. Braga, ACS Omega 2020, 5, 15115–15128. DOI:10.1021/acsomega.0c00975 11. B. Khan, M. Khalid, M. R. Shah, M. N. Tahir, M. U. Khan, A. Ali, S. Muhammad, ChemistrySelect 2019, 4, 9274–9284. DOI:10.1002/slct.201901422 12. M. Khalid, A. Ali, M. Adeel, Z. U. Din, M. N. Tahir, E. Rod- rigues-Filho, J. Iqbal, M. U. Khan, J. Mol. Struct. 2020, 1206, 127755. DOI:10.1016/j.molstruc.2020.127755 13. M. Khalid, A. Ali, J. Tariq, M. N. Tahir, H. A. R. Aliabad, I. Hussain, M. Ashfaq, M. U. Khan, ChemistrySelect 2020, 5, 10618–10631. DOI:10.1002/slct.202002653 14. A. Ali, Z. U. Din, M. Khalid, M. N. Tahir, E. Rodrigues‐Fil- 292 Acta Chim. Slov. 2023, 70, 281–293 Tahir et al.: Synthesis, SC XRD Based Structure Elucidation, ... ho, B. Ali, S. Asim, S. Muhammad, ChemistrySelect 2020, 5, 3735–3745. DOI:10.1002/slct.201904757 15. A. Ali, M. Khalid, S. Abid, M. N. Tahir, J. Iqbal, M. Ashfaq, F. Kanwal, C. Lu, Crystals 2020, 10, 778. DOI:10.3390/cryst10090778 16. M. Khalid, A. Ali, S. Asim, M. N. Tahir, M. U. Khan, L. C. Curcino Vieira, A. F. de la Torre, M. Usman, J. Phys. Chem. Solids 2021, 148, 109679. DOI:10.1016/j.jpcs.2020.109679. 17. M. Khalid, A. Ali, S. Haq, M. N. Tahir, J. Iqbal, A. A. C. Braga, M. Ashfaq, S. U. H. Akhtar, J. Mol. Struct. 2021, 1224, 129308. DOI:10.1016/j.molstruc.2020.129308 18. G. M. Sheldrick, Acta Crystallogr., Sect. A: Found. Adv. 2015, 71, 3–8. DOI:10.1107/S2053273314026370 19. G. M. Sheldrick, Acta Crystallogr. C Struct. Chem. 2015, 71, 3–8. DOI:10.1107/S2053229614024218 20. L. J. Farrugia, J. Appl. Crystallogr. 2012, 45, 849–854. DOI:10.1107/S0021889812029111 21. A. L. Spek, Acta Crystallogr. D Biol. Crystallogr. 2009, 65, 148– 155. DOI:10.1107/S090744490804362X 22. C. F. Macrae, I. Sovago, S. J. Cottrell, P. T. Galek, P. McCabe, E. Pidcock, M. Platings, G. P. Shields, J. S. Stevens, M. Towler, J. Appl. Crystallogr. 2020, 53, 226–235. DOI:10.1107/S1600576719014092 23. P. R. Spackman, M. J. Turner, J. J. McKinnon, S. K. Wolff, D. J. Grimwood, D. Jayatilaka, M. A. Spackman, J. Appl. Crystallogr. 2021, 54, 1006–1011. DOI:10.1107/S1600576721002910 24. M. A. Spackman, D. Jayatilaka, CrystEngComm 2009, 11, 19–32. DOI:10.1039/B818330A 25. M. J. Turner, S. Grabowsky, D. Jayatilaka, M. A. Spackman, J. Phys. Chem. Lett. 2014, 5, 4249–4255. DOI:10.1021/jz502271c 26. A. D. Becke, Phys. Rev. A 1988, 38, 3098–3100. DOI:10.1103/PhysRevA.38.3098 27. B. Miehlich, A. Savin, H. Stoll, H. Preuss, Chem. Phys. Lett. 1989, 157, 200–206. DOI:10.1016/0009-2614(89)87234-3 28. C. Lee, W. Yang, R. G. Parr, Phys. Rev. B 1988, 37, 785–789. DOI:10.1103/PhysRevB.37.785 29. A. McLean, G. Chandler, J. Chem. Phys. 1980, 72, 5639–5648. DOI:10.1063/1.438980 30. M. J. Frisch, G. Trucks, H. Schlegel, G. Scuseria, M. Robb, J. Cheeseman, G. Scalmani, V. Barone, G. Petersson, H. Nakat- suji, Gaussian 16, Gaussian, Inc. Wallingford, CT, 2016. 31. T. Lu, F. Chen, J. Comput. Chem. 2012, 33, 580–592. DOI:10.1002/jcc.22885 32. J. Bernstein, R. E. Davis, L. Shimoni, N. L. Chang, Angew Chem. Int. Ed. Engl. 1995, 34, 1555–1573. DOI:10.1002/anie.199515551 33. J. L. Wardell, J. M. Skakle, J. N. Low, C. Glidewell, Acta Crys- tallogr., Sect. E: Struct. Rep. Online 2005, 61, o3849–o3851. DOI:10.1107/S160053680503374X 34. B. T. Gowda, M. Tokarčík, K. Shakuntala, J. Kožíšek, H. Fuess, Acta Crystallogr., Sect. E: Struct. Rep. Online 2010, 66, o1671– o1672. DOI:10.1107/S1600536810022245 35. U. Chaithanya, S. Foro, B. T. Gowda, Acta Crystallogr., Sect. E: Struct. Rep. Online 2012, 68, o873–o873. DOI:10.1107/S1600536812008021 36. K. Shakuntala, M. Fronc, B. T. Gowda, J. Kožíšek, Acta Crys- tallogr., Sect. E: Struct. Rep. Online 2012, 68, o99–o100. DOI:10.1107/S1600536811052573 37. J. J. McKinnon, D. Jayatilaka, M. A. Spackman, ChemComm. 2007, 3814–3816. DOI:10.1039/b704980c 38. N. Kitanovski, M. Počkaj, Acta Chim. Slov. 2021, 68, 475–482. DOI:10.17344/acsi.2020.6634 39. T. Topal, Acta Chim. Slov. 2021, 68, 88–101. DOI:10.17344/acsi.2020.6183 40. J.-J. Wang, L.-N. Dun, B.-S. Zhang, Z.-H. Wang, H. Wang, C.- B. Li, W. Liang, Acta Chim. Slov. 2021, 68, 239–246. DOI:10.17344/acsi.2020.6438 41. C. Jelsch, K. Ejsmont, L. Huder, IUCrJ 2014, 1, 119–128. DOI:10.1107/S2052252514003327 42. H. Kargar, M. Fallah-Mehrjardi, R. Behjatmanesh-Ardakani, K. S. Munawar, M. Ashfaq, M. N. Tahir, Inorg. Chim. Acta. 2021, 526, 120535. DOI:10.1016/j.ica.2021.120535 43. C. F. Mackenzie, P. R. Spackman, D. Jayatilaka, M. A. Spack- man, IUCrJ 2017, 4, 575–587. DOI:10.1107/S205225251700848X 44. M. J. Turner, J. J. McKinnon, D. Jayatilaka, M. A. Spackman, CrystEngComm 2011, 13, 1804–1813. DOI:10.1039/C0CE00683A 45. M. Ashfaq, M. Khalid, M. N. Tahir, A. Ali, M. N. Arshad, A. M. Asiri, ACS Omega 2022, 7, 9867–9878. DOI:10.1021/acsomega.2c00288 46. H. Kargar, M. Ashfaq, M. Fallah-Mehrjardi, R. Behjat- manesh-Ardakani, K. S. Munawar, M. N. Tahir, J. Mol. Struct. 2022, 1253, 132264. DOI:10.1016/j.molstruc.2021.132264 47. M. Ashfaq, M. N. Tahir, S. Muhammad, K. S. Munawar, A. Ali, G. Bogdanov, S. S. Alarfaji, ACS Omega 2021, 6, 31211– 31225. DOI:10.1021/acsomega.1c04884 48. K. O. Ali, H. A. Mohamad, T. Gerber, E. Hosten, Acta Chim. Slov. 2022, 69, 905–912. DOI:10.17344/acsi.2022.7682 49. U. Koch, P. L. Popelier, J. Phys. Chem. 1995, 99, 9747–9754. DOI:10.1021/j100024a016 50. A. Saeed, M. Bolte, M. F. Erben, H. Pérez, CrystEngComm 2015, 17, 7551–7563. DOI:10.1039/C5CE01373A 51. M. Xu, B. Zhang, Q. Wang, Y. Yuan, L. Sun, Z. Huang, J. Chil. Chem. Soc. 2018, 63, 3788–3794. DOI:10.4067/s0717-97072018000103788 52. M. J. Javan, Comput. Theor. Chem. 2021, 1205, 113440. DOI:10.1016/j.comptc.2021.113440 53. M. K. Chaudhary, T. Karthick, B. D. Joshi, P. Prajapati, M. S. A. de Santana, A. P. Ayala, V. J. Reeda, P. Tandon, Spectrochim. Acta A Mol. Biomol. Spectrosc. 2021, 246, 118976. DOI:10.1016/j.saa.2020.118976 54. I. N. Kolesnikova, N. A. Chegodaev, P. Y. Sharanov, I. F. Shish- kov, Chem. Phys. Lett. 2022, 793, 139447. DOI:10.1016/j.cplett.2022.139447 55. A. Jezierska, J. J. Panek, K. Błaziak, K. Raczyński, A. Koll, Mol- ecules 2022, 27, 792–810. DOI:10.3390/molecules27030792 56. S. Emamian, T. Lu, H. Kruse, H. Emamian, J. Comput. Chem. 2019, 40, 2868–2881. DOI:10.1002/jcc.26068 293Acta Chim. Slov. 2023, 70, 281–293 Tahir et al.: Synthesis, SC XRD Based Structure Elucidation, ... Except when otherwise noted, articles in this journal are published under the terms and conditions of the  Creative Commons Attribution 4.0 International License Povzetek (Z)-4-(2-metoksi-4-nitrofenil)amino)-4-oksobut-2-enojsko kislino (MAOA) smo sintetizirali z reakcijo 2-metok- si-4-nitroanilina in maleinskega anhidrida v etil acetatu. Sintetizirano spojino smo okarakterizirali z elementarno anali- zo, FT-IR in UV-Vis spektroskopijo in TGA/DSC analizo. Kristalno strukturo smo določili z monokristalno rentgensko difrakcijo (SC XRD). Supramolekularno strukturo MAOA glede na nekovalentne interakcije smo raziskali z analizo Hirshfeldove površine. Analiza praznin je pokazala, da naj bi imela MAOA dobre mehanske lastnosti. Okolje kristalnega pakiranja smo nadalje raziskali z interakcijsko energijo med molekularnimi pari in energijskimi mrežami. Poleg tega je rezultat DFT študije v plinski fazi pokazal, da v MAOA obstajata N–H···O in O–H···O intramolekularni vodikovi vezi, ker je razdalja med D in A manjša od vsote njunih van der Waalsovih radijev. Rezultat študije QTAIM je pokazal, da bi morala v MAOA obstajati tudi intramolekularna vodikova vez CH···O z močjo 3,40 kcal/mol. S39Acta Chim. Slov. 2023, 70, (2), Supplement Društvene vesti in druge aktivnosti DRUŠTVENE VESTI IN DRUGE AKTIVNOSTI SOCIETY NEWS, ANNOUNCEMENTS, ACTIVITIES Vsebina Poročilo o delu v letu 2022 ..................................................................................................... S41 Koledar važnejših znanstvenih srečanj s področja kemije in kemijske tehnologije ......... S48 Navodila za avtorje .................................................................................................................. S50 Contents Report for 2022 ........................................................................................................................ S41 Scientific meetings – Chemistry and chemical engineering ................................................ S48 Instructions for authors .......................................................................................................... S50 S40 Acta Chim. Slov. 2023, 70, (2), Supplement Društvene vesti in druge aktivnosti S41Acta Chim. Slov. 2023, 70, (2), Supplement Društvene vesti in druge aktivnosti Tudi v letu 2022 je bilo društvo aktivno na številnih pod- ročjih. Izvajali smo redne letne aktivnosti, pri katerih je bil glavni poudarek na rednem izdajanju društvene revije Acta Chimica Slovenica (ACSi) ter organizaciji največjega letnega dogodka društva, konference »Slovenski kemijski dnevi 2022«. V letu 2021 je društvo praznovalo 70- letnico. Slovesnost ob jubileju je bila zaradi Covida 19 prestavljena v leto 2022 in je potekala v sklopu konference Slovenski kemijski dnevi v Grand Hotelu Bernardin v Portorožu. Dogodka se je udeležilo več kot 310 povabljenih gostov iz 13 držav, med drugim tudi predsedni- ka hrvaškega in slovaškega kemijskega društva ter predstavniki ECTN in EuChemS. Slavnostni govorci so bili prof. dr. Tamara Lah Turnšek, akad. prof. dr. Branko Stanovnik in prof. dr. Ven- česlav Kaučič, Slovensko kemijsko društvo pa je podelilo prizna- nja 8 častnim članom, 9 zaslužnim članom, 11 zaslužnim inštitu- cijam; priznanja za sodelovanje pri uredništvu znanstvene revije Acta Chimica Slovenica je prejelo 30 sodelavcev. Zahvala gre tudi sponzorjem Slavnostne akademije, ki so omogočili proslavo ob tako pomembni obletnici- hvala torej podjetjem Cinkarna Celje, Salonit Anhovo, Knauf Insulation, Melamin, Aquafil, Novartis, Belinka Perkemija, Krka, Kemomed, Mettler Toledo, Belinka Pr- kemija in Primalab za podporo in sodelovanje. 21. septembra smo prav tako v sklopu omenjene konferen- ce izvedli redni občni zbor društva, kjer smo uradno ustanovili Sekcijo za okolje ter sklenili, da elektorje za volitve v Državni svet izbere Glavni odbor med svojimi člani. Slovenski kemijski dnevi 2022 so bili organizirani v Por- torožu, v Kongresnem centru Grand hotela Bernardin, in sicer v dneh od 21. do 23. septembra 2022. Programskemu in organi- zacijskemu odboru je predsedoval znan. svet. dr. Albin Pintar, skupaj s člani odbora v zasedbi prof. dr. Romana Cerc-Korošec, prof. dr. Zorka Novak Pintarič, prof. dr. Darja Lisjak, doc. dr. Matic Lozinšek, prof. dr. Matjaž Valant, dr. Silvo Zupančič in Marjana Gantar Albreht ter Eva Mihalinec, ki se je društvu pri- ključila v drugi polovici leta. Na konferenci je bilo predstavljenih preko 180 prispev- kov v obliki predavanj in posterjev. Delo je potekalo plenarno in v treh vzporednih sekcijah. Udeleženci konference, bilo jih je 279 iz Slovenije in dvanajstih drugih držav, so bili zelo zadovolj- ni s kakovostjo znanstvenih in strokovnih prispevkov ter dru- žabnim programom srečanja. Na konferenci je sodelovalo tudi 21 razstavljalcev laboratorijske in procesne opreme. Sponzorji dogodka so bili Mikro+Polo, Analysis Adria, Chemass, Donau Lab, Hiden Analytical, Kemomed, Labtim, Merck, Mettler To- ledo, Optik Instruments, Primalab in Vigor ter MDPI. Objavili smo zbornik povzetkov konference, ki je dostopen na USB klju- ču ter na voljo v NUK-u in strokovnih knjižnicah po Sloveniji. Plenarni predavatelji na konferenci so bili prof. dr. Goran Dražić (Kemijski inštitut, Ljubljana), prof. dr. Doris Vollmer (Max Planck Institute for Polymer Research, Mainz, Nemčija) in prof. dr. Ioannis Katsoyiannis (Aristotle University of Thessalo- POROČILO PREDSEDNIKA SLOVENSKEGA KEMIJSKEGA DRUŠTVA O DELU DRUŠTVA V LETU 2022 niki, Grčija). Poleg treh plenarnih predavanj so udeleženci pos- lušali šest »keynote« vabljenih predavanj, ki so jih izvedli dr. Slavko Kralj (Institut Jožef Stefan in Fakulteta za farmacijo UL), prof. dr. Nataša Novak Tušar (Kemijski inštitut in Univerza v Novi Gorici), prof. dr. Layla Martin-Samos (Italian National Re- search Council (CNR-IOM Democritos), Trst, Italija), dr. Dinesh Shetty (Khalifa University, Abu Dhabi, UAE), dr. Nataša Kovače- vić (Kolektor Mobility) in prof. dr. Robin A. Hutchinson (Queen’s University, Kingston (ON), Kanada). Podelili smo tudi nagrade za najboljša študijska dela s področja trajnostne kemije. Strokovno komisijo za izbor naj- boljših del so sestavljali dr. Vid Margon, dr. Ema Žagar, izr. prof. dr. Romana Cerc Korošec in prof. dr. Marjan Veber, ki so se odločili, da nagrado za najboljše diplomsko delo prejme Martin Ciringer, za najboljše magistrsko delo Ana Rebeka Kamšek, za najboljše doktorsko delo pa dr. Maja Čolnik. Sponzor nagrad je bilo podjetje AquafilSLO. Ob zaključku konference smo že tradicionalno podelili nagrade doktorskim študentom za najboljša predavanja in pos- terske predstavitve. V letu 2022 smo v reviji Acta Chimica Slovenica (ACSi) izdali 4 številke revije, v katerih je bilo skupaj objavljenih 92 ori- ginalnih znanstvenih člankov na skupno 943 straneh z dvoko- lonskim tiskom. Članki pokrivajo vsa področja kemije, kemije materialov in kemijskega in biokemijskega inženirstva. Vseh člankov, ki so bili leta 2022 oddani v uredniški sistem, je bilo 469, kar pomeni, da jih je bilo na koncu sprejetih okoli 19 %. Vsi član- ki so objavljeni na spletu in so prosto dostopni. Poleg tega so ob- javljeni tudi v več podatkovnih bazah. Faktor vpliva revije za leto 2021 je bil 1,524.Od objavljenih člankov sta bila dva tako imeno- vana »Feature Articles« (FA). Med FA je bil tudi prispevek razi- skovalne skupine iz tujine, ki raziskuje na področju polimerne kemije pod vodstvom Prof. Heikkija Tenhuja z Univerze v Helsinkih. Z letom 2022 delo odgovorne urednice ACSi in področne urednice za področje Physical Chemistry zaključuje Ksenija Ko- gej. V društvenih vesteh smo objavili seznam diplomskih, ma- gistrskih in doktorskih del FKKT UL, FKKT UM, podiplomske- ga študijskega programa »Znanosti o okolju« in Fakultete za zna- nosti okolju, UNG v letu 2020. Objavili smo tudi letna poročila sekcij. V letu 2022 so društvene vesti obsegale 120 strani. Na društvenih straneh je bila poleg ostalih novic objavljena tudi no- vica o Slavnostni akademiji ob 70. letnici društva. Zahvaljujem se tudi vsem inštitucijam, ki so v letu 2022 finančno podprle izdajanje revije Acta Chimica Slovenica. Te so Fakulteta za kemijo in kemijsko tehnologijo Univerze v Ljubljani, Fakulteta za kemijo in kemijsko tehnologijo Univerze v Maribo- ru, Univerza v Novi Gorici, Kemijski inštitut, Inštitut »Jožef Ste- fan« in Belinka Perkemija. Sponzorji revije so bili z objavo oglasa Krka d.d., Novo mesto, Donau Lab d.o.o. Ljubljana in Helios Domžale, d.o.o. S42 Acta Chim. Slov. 2023, 70, (2), Supplement Društvene vesti in druge aktivnosti V letu 2022 smo nadaljevali z aktivnostmi za pridobivanje novih članov. Medse smo jih privabili 48, od tega 30 študentov. Za komunikacijo s člani smo pogosteje uporabljali Facebook , Twitter in LinkedIn ter jih obveščali o dogodkih po elektronski pošti. V društvu smo prvič objavili razpis za povezovanje dijakov s strokovnjaki kemijske stroke, pripravili pa smo 5 tem razisko- valnih nalog na različnih institucijah. Kot mentorji dijakom so se predstavili prof. dr. Darja Lisjak (Institut Jožef Stefan), asist. Mi- ha Slapničar in Tim Prezelj (Univerza v Ljubljani, Pedagoška fa- kulteta), red. prof. Urban Bren in Veronika Furlan (Univerza v Mariboru in Fakulteta za kemijo in kemijsko tehnologijo), dr. Annamaria Vujanovic in izr. Prof. Lidija Čuček (Univerza v Ma- riboru in Fakulteta za kemijo in kemijsko tehnologijo) in dr. Jan Bitenc (Kemijski inštitut). Člani Slovenskega kemijskega društva so bili aktivni tudi na področju mednarodnega sodelovanja. Predvsem je potrebno omeniti članstvo društva v mednarodnih združenjih IUPAC, ECTN, IUCr, EURACHEM, EuChemS, EFCE, EPF, ECA in EF- CATS. Društvo se je v letu 2022 prijavilo na Javni razpis ARRS za sofinanciranje delovanja v mednarodnih znanstvenih združenjih v letu 2022, kjer smo bili uspešni pri vseh oddanih vlogah. Konec leta smo oddali tudi prijavo na Javni razpis ARRS za za sofinan- ciranje izdajanja domačih periodičnih znanstvenih publikacij v letih 2023 in 2024, rezultati pa bodo znani v 2023. dr. Peter Venturini, predsednik društva dr. Albin Pintar, predsednik organizacijskega odbora konference Slovenski kemijski dnevi prof. dr. Ksenija Kogej, glavna urednica ACSi S43Acta Chim. Slov. 2023, 70, (2), Supplement Društvene vesti in druge aktivnosti Mariborska podružnica se je v letu 2022 usmerila v izpolnitev ciljev, ki si jih je zastavila v preteklem letu. Kot vsako leto smo se tudi letos v Portorožu udeleži- li konference Slovenski kemijski dnevi, kjer smo predsedo- vali različnim sekcijam, sodelovali kot predavatelji in kot predstavniki prispevkov na posterjih ter v komisiji za oce- njevanje študentskih del. Skrb Mariborske podružnice je tudi stalno izobraževanje članov. V ta namen smo organi- zirali strokovna predavanja in razne seminarje, na katerih so predavali priznani tuji in domači strokovnjaki. Predava- nja so pokrivala pomembna področja teoretične in upo- rabne kemije, kemijske in procesne tehnike ter kemijskega izobraževanja. V letu 2022 smo gostili predstavnike številnih podje- tij (Kansai Helios, Lek, Etol, Krka, Microinnova …) z na- menom sodelovanja fakultete z industrijskimi partnerji. Izvedli smo promocijo kemijskih znanosti na 14. srednjih šolah po Sloveniji. Organizirali smo razpravo s srednješol- skimi učitelji na FKKT UM o načinu poučevanja. Z dijaki smo izvedli delavnici z naslovom angl. Belle 2 master clas- Poročilo o delovanju in aktivnostih Mariborske podružnice za leto 2022 ses. Dvakrat smo se udeležili tudi kariernega sejma. Pojavljamo se v medijih kot so RTV Slovenija, Delo, Večer, dijaški.net, FAX VPISNIK, kjer predstavljamo ob- novo naše fakultete, novo raziskovalno opremo in dosežke najuspešnejših raziskovalcev (npr. uvrstitev sodelavcev na Stanfordovo lestvico najuspešnejših raziskovalcev na sve- tu). Prvič do sedaj smo v avgustu 2022 organizirali pole- tno šolo kemije in kemijskega inženirstva za dijake sre- dnjih šol. Aktivno smo sodelovali tudi pri mednarodnih poletnih šolah. Na naši fakulteti je v sodelovanju s fakulteto iz Grad- ca potekala mednarodna poletna šola na temo visokotlač- nih tehnologij: ‘ESS-HPT 2022’ The Eurpean Summer school in High Pressure Technology, ki jo je organiziral Laboratorij za separacijske procese in produktno tehniko v sodelovanju s Tehnološko fakulteto v Gradcu. izr. prof. dr. Matjaž Finšgar Komisija za slovensko kemijsko terminologijo in no- menklaturo je tudi v preteklem letu sodelovala pri delu Tehniške komisije Sekcije za terminološke slovarje pri In- stitutu za slovenski jezik ZRC SAZU. Člana tehniške komi- sije za področje kemije in kemijske tehnologije sta Andrej Šmalc iz ljubljanske ter Peter Glavič iz mariborske podru- žnice Slovenskega kemijskega društva. V letu 2022 je potekala končna redakcija gradiva s področja kemije, kemijske tehnologije in kemijske tehnike za novo izdajo splošnega tehniškega slovarja, ki bo predvi- doma končana v letu 2023 Obenem s tem je potekalo nadaljnje zbiranje gradiva za novi Kemijski slovar, ki je že nekaj let na spletu in pred- stavlja pomembno dopolnitev bodočega Splošnega tehni- Poročilo Komisije za slovensko kemijsko terminologijo in nomenklaturo za leto 2022 škega slovarja. Prednost spletne oblike slovarja je prav v tem, da ga je mogoče ves čas dopolnjevati in po potrebi tudi popravljati. V letu 2022 je bilo obdelanih 688 gesel, ki bodo v naslednjem letu vnesena v slovar. Za leto 2023 je predvideno nadaljnje sodelovanje pri končni redakciji Slovenskega tehniškega slovarja, kot tudi nadaljnje zbiranje gradiva za nova gesla ter njihova obde- lava in vnašanje v Kemijski slovar. Poleg dela v zvez s Kemijskim slovarjem bomo še na- dalje sodelovali pri prevodu mednarodnega standarda ISO 80000: Veličine in enote s strokovnim pregledom novih pre- vodov, ki bo nadomestil sedanji standard SIST ISO 31. prof. dr. Andrej Šmalc S44 Acta Chim. Slov. 2023, 70, (2), Supplement Društvene vesti in druge aktivnosti Sekcija za polimere je članica Evropske polimerne federacije (EPF), kateri je v letu 2022 predsedoval prof. dr. Jiri Kotek (Inštitut za makromolekularno kemijo, Češka). Zaradi COVID-19 epidemije je prof. dr. Jiri Kotek z eno letno zamudo lani organiziral največji evropski polimerni kongres, EPF 2022, pri katerem predstavniki nacionalnih polimernih sekcij in društev iz 28 držav delujemo kot mednarodni svetovalni odbor. Na generalni skupščini EPF, ki smo jo imeli Februarja v Pragi, Češka, smo izvolili novo predsednico EPF prof. dr. Katjo Loos (Univerza v Gronin- genu, Nizozemska), ki bo predsedovala do leta 2025, ko bo organizirala naslednji EPF kongres v Groningenu, Nizo- Sekcija mladih je v letu 2022 izvedla sledeče dogodke in projekte: Razpis raziskovalnih nalog za dijake V februarju 2022 smo pričeli z razpisom za povezo- vanje dijakov s strokovnjaki kemijske stroke, ki bi jim bili mentorji pri raziskovalnih nalogah. Razpis skupaj s seznamom in opisom tem (4 teme) smo poslali po slovenskih srednjih šolah. Dijaki so imeli čas za prijavo do sredine aprila, strokovna komisija SKD je pregledala prijave in povezala dijake z izbranimi mentorji. Ekskurzija v KRKO (v sodelovanju s Študentsko or- ganizacijo FKKT) V aprilu 2022 smo organizirali v sodelovanju s štu- dentsko organizacijo UL FKKT strokovno ekskurzijo v Članice in člani sekcije za katalizo smo bili angažira- ni pri organizaciji mednarodnega znanstvenega srečanja, tj. 6th International Conference on New Photocatalytic Materials for Environment, Energy and Sustainability (NPM-6) & 7th International Conference on Photoca- talytic and Advanced Oxidation Technologies for the Tre- atment of Water, Air, Soil and Surface (PAOT-7), ki je po- tekal na Kemijskem inštitutu v Ljubljani od 4. do 6. aprila 2022. Prav tako smo leta 2022 pridobili organizacijo med- narodne znanstvene konference z naslovom “4th Interna- tional Conference on Fundamentals and Applications of Cerium Dioxide in Catalysis”, ki bo potekala od 17. do 20. septembra 2024 v kongresnem centru Grand hotela Ber- nardin v Portorožu. Več informacij o dogodku je na voljo na konferenčni spletni strani: https://ceria2024.chem-soc. si/. Poročilo Sekcije za polimere za leto 2022 Poročilo Sekcije mladih SKD za leto 2022 Poročilo Sekcije za katalizo za leto 2022 zemska. Na sestanku smo med drugim potrdili drugega prejemnika EPF nagrade, ki jo je prejel prof. dr. Chris- topher Barner-Kowollik (Centre for Materials Science, Av- stralija) za pionirsko delo na področju makromolekularne fotokemije. Člani sekcije so bili v juniju povabljeni na predavanje prof. dr. Michael S. Silversteina, ki je v sklopu Preglovih predavateljev na Kemijskem inštitutu imel predavanje z naslovom: „Accessing Innovative Polymers through Emu- lsion Templating“. dr. David Pahovnik farmacevtsko podjetje Krka. Ekskurzijo smo kombinirali z ogledom vinske kleti. V sklopu ekskurzije so se lahko štu- denti včlanili v društvo, za člane je bila cena prevoza nižja. Na tak način smo uspeli privabiti nove člane. Javni razpis: nagrade za najboljša študijska dela s področja trajnostne kemije Poleti smo v sodelovanju s podjetjem Aquafil objavi- li razpis za nagrade za najboljša diplomsko, magistrsko in doktorsko delo iz trajnostne kemije. Strokovna komisija SKD je pregledala prijave in ocenila dela. Nagrada je obse- gala plaketo in denadno nagrado. Nagrade smo podelili na konferenci Slovenski Kemijski dnevi 2022. dr. Tina Paljk Izvajamo tudi aktivnosti za pridobitev organizacije mednarodne znanstvene konference “13th European Con- ference on Solar Chemistry and Photocatalysis: Envi- ronmental Applications (SPEA)”, ki jo bomo v primeru potrditve kandidature organizirali leta 2026. Nataša Novak Tušar in Albin Pintar sta kot nacional- na predstavnika sodelovala pri izvajanju aktivnosti pri Evropski federaciji katalitskih združenj (EFCATS). V sekciji za katalizo smo v lanskem letu sodelovali pri organizaciji predavanj vabljenih tujih raziskovalcev, ki smo jih pripravili v sodelovanju z raziskovalnimi in aka- demskimi inštitucijami, zelo angažirani pa smo bili pri or- ganizaciji konference “Slovenski kemijski dnevi 2022”, ka- kor tudi pri sodelovanju na dogodku s predstavitvami velikega števila prispevkov. dr. Albin Pintar S45Acta Chim. Slov. 2023, 70, (2), Supplement Društvene vesti in druge aktivnosti Sekcija za okolje SKD je bila ustanovljena v letu 2022 na pobudo Darje Lisjak. 18.1.2022 smo organizirali uvo- dni sestanek sekcije, na katerem smo izbrali predsednika sekcije (Marko Štrok) ter podpredsednico in tajnico sekci- je (Janja Vidmar). Po sestanku je Janja Vidmar vzpostavila oblak, na katerem se zbirajo dokumenti in zapisniki sestankov, vezani na delo sekcije (https://1drv.ms/u/ s!AgGv2g6_iqYaxFGqYul2Qt2xwvMK?e=OKiqEx). Prav tako je bila pripravljena novica o ustanavljanju sekcije in objavljena na različnih internetnih straneh in socialnih medijih z namenom seznaniti zainteresirane o ustanavlja- nju sekcije in pritegniti čim večje število članov SKD k članstvu v sekciji. Na podlagi sestanka smo pripravili tudi vizijo, pos- lanstvo in program dela sekcije. Poslanstvo sekcije je zdru- ževanje članov SKD, ki jih zanimajo tematike s področja kemije okolja z namenom izboljšati razumevanje in per- cepcijo kemije okolja med različnimi deležniki, ter tako vplivati na kvaliteto življenja. Vizija sekcije je postati eno izmed vodilnih združenj strokovnjakov v Sloveniji na po- V letu 2022 smo člani kristalografske sekcije aktivno sodelovali pri organizaciji dveh mednarodnih konferenc. Obe bi morali biti izvedeni že v predhodnih letih, pa sta bili zaradi pandemije COVID-19 preloženi na primernej- ši čas. Prvi od teh dveh dogodkov je bila Evropska konfe- renca o praškovni difrakciji (EPDIC17), ki je bila izvedena v Šibeniku od 31. maja do 3. junija 2022. Kot svetovalec organizacijskega odbora je bil v delo pri organizaciji zaradi izkušenj večletnega članstva v Evropskem odboru za pra- škovno difrakcijo vključen tudi vodja sekcije za kristalo- grafijo pri SKD Anton Meden. Konferenca je bila zelo uspešna in na njej je aktivno sodelovala tudi osmerica slo- venskih udeležencev. Program je obsegal pet plenarnih predavanj in 12 mikrosimpozijev, udeležilo se ga je več kot 20 sponzorjev/razstavljavcev. Dobro sta bili obiskani tudi dve delavnici pred začetkom konference (za delo s progra- mom Topas za Rietveldovo analizo in za uporabo zbirke podatkov PDF). Drugi dogodek je bilo tradicionalno 28. Hrvaško- -slovensko kristalografsko srečanje od 8. do 11. septembra v Poreču. V organizacijskem odboru tega srečanja je sode- lovalo pet članov iz Slovenije, pet iz Hrvaške in eden iz Poročilo o delu Sekcije za okolje SKD za leto 2022 Poročilo Sekcije za kristalografijo pri Slovenskem kemijskem društvu za leto 2022 dročju tematik, povezanih s kemijo okolja z namenom ozaveščanja, razširjanja, izobraževanja in svetovanja raz- ličnim deležnikom na tem področju. Cilji sekcije pa so sle- deči: • Povezovanje članov Slovenskega kemijskega društva, ki delujejo na področju kemije okolja • Postati stičišče različnih deležnikov na področju kemije okolja z namenom povezovanja in sku- pnega sodelovanja na področjih aktualnih za kemijo okolja • Izboljšati ozaveščenost splošne in strokovne jav- nosti o tematikah povezanih s kemijo okolja Sekcija pa bo delovala predvsem na naslednjih pod- ročjih: • Ozaveščanje in razširjanje spoznanj, povezanih s kemijo okolja • Izobraževanje • Mednarodno sodelovanje Program sekcije je bil predstavljen in sprejet na 3. se- ji glavnega odbora društva 6.6.2022. Poljske. Tudi tokrat je uspelo pridobiti dovolj sponzorskih sredstev, da kotizacija ni bila potrebna. Lahko smo celo podelili nekaj nagrad za najbolje predstavitve v različnih kategorijah raziskovalcev. Tako brezplačna udeležba kot kandidiranje za nagrade je za udeležbo mladih raziskoval- cev dobra spodbuda in odlična priložnost za pridobivanje kompetenc, saj so vsi prispevki v obliki kratkih predavanj. Udeležba je bila zelo dobra, program pa kakovosten. Plenarna predavanja so bila štiri, kratkih predavanj 66, od tega 21 iz Slovenije. Plenarna predavanja so bila na različ- ne teme: materiali za sproščanje tekočih sestavin (Alessina Bacchi, Parma, Italija), strukturna analiza nanomaterialov z elektronsko difrakcijo (Mariana Klementova, Praga, Če- ška), strukturni vpogled v pripravo in molekularno delo- vanje bioaktivnih kovinskih spojin (Jakob Kljun Ljubljana, Slovenija) in kristalografija kot zmogljiva metoda v znano- sti o materialih (Anna Moliterni, Bari, Italija). Že v letu 2022 so stekle tudi priprave na 29. sloven- sko-hrvaško kristalografsko srečanje, ki bo od 14. do 18. junija 2023 v Topolšici (https://sccm2023.fkkt.uni-lj.si/). Pridobili smo že 10 sponzorjev in 4 vabljene predavatelje, kar obeta, da bo tudi ta znanstveni dogodek uspešen. prof. dr. Anton Meden S46 Acta Chim. Slov. 2023, 70, (2), Supplement Društvene vesti in druge aktivnosti Z namenom, da bi pripravili kratkoročni program dela sekcije za leti 2022-2023, smo 27.5.2022 organizirali drugi sestanek članov sekcije. Na podlagi tega programa smo v letu 2022 na podro- čju ozaveščanja in razširjanja spoznanj, povezanih s kemi- jo okolja in izobraževanja, na pobudo Andree Oarga-Mu- lec in Vesne Mislej začeli s projektom snemanja izobraže- valnih / promocijskih videov na tematiko ponovne uporabe blata iz čistilnih naprav. Projekt je večplasten in vključuje študente Akademije umetnosti UNG, ki se pod mentorstvom Jasne Hribernik učijo snemati promocijske filme, strokovnjake na področju čiščenja odpadnih vod, kot sta Vesna Mislej iz CČN Ljubljana in Mojca Müller iz CČN Škofja Loka, ter učitelje in učence osnovnih in sre- dnjih šol. Pri izvedbi projekta pa aktivno sodelujejo s svojo podporo tudi Andreea Oarga-Mulec, Janja Vidmar in Marko Štrok. Ideja projekta je, da bi študenti Akademije umetnosti v sklopu svojega izobraževanja posneli izobra- ževalne / promocijske filme na tematiko blata iz čistilnih naprav in pomembnosti čistilnih naprav za okolje. Ti videi pa bodo podlaga za pripravo učnega paketa za osnovne in srednje šole, ki bi ga bodo učitelji uporabili pri poučevanju v šolah. V letu 2022 nam je Vesna Mislej pomagala pri is- kanju lokacije za snemanje. Na koncu smo dobili zeleno luč za snemanje CČN Škofje Loke, ki jo strokovno vodi Mojca Müller. 20.9.2022 smo si tako ogledali lokacijo sne- manja, 12.11.2022 pa je Jasna Hribernik s svojimi študenti izvedla snemalni dan na CČN Škofja Loka. Material, ki so ga študentje posneli, je sedaj v post produkciji, tako da se izvedba projekta nadaljuje v letu 2023. V letu 2022 smo s pomočjo Iztoka Devetaka poslali vabilo učiteljem osnov- nih in srednjih šol, ki bi se želeli priključiti projektu. in na podlagi vabila so pripravljenost izrazile štiri osnovnošol- ske in ena srednješolska učiteljica. 25.8.2022 smo se člani sekcije (Andreea Oarga-Mu- lec, Janja Vidmar in Marko Štrok) na pobudo Andree Oar- ga-Mulec udeležili sestanka z Izobraževalnim centrom za zaščito in reševanje Ig. Jože Pogačar in njegovi sodelavci so nam predstavili delovanje centra, pogovarjali smo se tudi o morebitnem sodelovanju med sekcijo in centrom. Tako smo se dogovorili, da bi lahko v letu 2023 organizirali ogled centra in njegovih aktivnosti oz. študijo primera nesreče Kemis za člane sekcije oz. društva. Sekcija je tudi sodelovala pri pripravi javnega razpisa za nagrade za najboljša študijska dela s področja trajno- stne kemije, kjer je Marko Štrok podal pripombe na osnu- tek razpisa in predlagal članico komisije za izbor. V letu 2022 so se člani sekcije aktivno udeležili tudi Slovenskih kemijskih dnevov v Portorožu med 21.- 23.9.2022, kjer je na podlagi našega predloga imel tudi predavanje plenarni predavatelj prof. dr. Ioannis Katsoyi- annis, ki je predsednik EuChemS Division of Chemistry and the Environment. Na Slovenskih kemijskih dnevih je potekal tudi izredni občni zbor SKD, kjer je bila Sekcija za kemijo okolja tudi formalno ustanovljena. Po podatkih tajništva SKD znaša število članov sek- cije 27 na dan 23.2.2023, od tega se jih je približno tretjina aktivno udeleževala in prispevala k aktivnostim sekcije v letu 2022. dr. Marko Štrok Osnovna dejavnost sekcije za Analizno kemijo v okviru Slovenskega kemijskega društva je organiziranje mednarodnih in domačih znanstvenih ter strokovnih sre- čanj, predavanj domačih in tujih strokovnjakov ter izvedba različnih delavnic, seminarjev in simpozijev. Člani sekcije so aktivni tudi znotraj delovnih skupin Eurachem in dru- gih združenj v evropskem prostoru (DAC, FECS) in tako dodatno prispevajo k prepoznavnosti Slovenskega kemij- skega društva. Po letih 2020 in 2021, na kateri je ključno vplivala epidemija Sars-Covid 19 in so bile planirane aktivnosti sekcije praviloma odpovedane ali prestavljene, je leto 2022 potekalo brez tovrstnih omejitev. Med 4. in 7. 7. 2022 smo izvedli tradicionalno 27. mednarodno srečanje podiplom- skih študentov in njihovih mentorjev YISAC (Young Inve- stigators Seminar on Analytical Chemistry) na Univerzi v Poročilo Analizne sekcije za leto 2022 Lodzu na Poljskem. Med aktivnostmi sekcije v Sloveniji velja izpostaviti 28. jubilejno konferenco »Slovenski kemij- ski dnevi 2022«, ki se je odvijala septembra 2022 v Porto- rožu. Področje analizne kemije je bilo dobro zastopano, konference se je udeležilo veliko kolegov iz različnih insti- tucij, ki so predstavili številne zanimive raziskave s podro- čij spektroskopije, kromatografije, elektrokemije, materia- lov in okolja. V prihodnje želimo v sekcijo aktivno vključiti mlajše kolege in nadaljevati z organizacijo domačih ter tujih sre- čanj, predavanj in konferenc. Posebej želimo okrepiti po- vezovanje in prenos znanja iz univerzitetnih in raziskoval- nih laboratorijev v industrijo. prof. dr. Mitja Kolar S47Acta Chim. Slov. 2023, 70, (2), Supplement Društvene vesti in druge aktivnosti Člani Sekcije za živilsko kemijo (SŽK) smo se aktiv- no udeležili različnih znanstvenih in strokovnih srečanj, kjer so predstavili svoje raziskovalne dosežke. Aktivni pa so bili tudi na delavnicah in na področju izobraževanja in popularizacije kemije in še posebej živilske kemije. Člani SŽK so sodelovali pri organizacijah mednarodnih znan- stvenih srečanj, katerih organizacijo je podprlo tudi Slo- vensko kemijsko društvo: • 26th International Symposium on Separation Sciences (ISSS 2022, www.isss2020.si), • 25th International Symposium for High-Perfor- mance Thin-Layer Chromatography (HPTLC 2022, www.hptlc2020.si), • 11th Central European Congress on Food and Nutrition: “Food, Technology and Nutrition for Healthy People in a Healthy Environment“ (CE- Food 2022, www.cefood2022.si). Aktivno smo sodelovali tudi pri različnih dejavno- stih Sekcije za živilsko kemijo (Food Chemistry Division, FCD) Evropskega kemijskega združenja (European Che- mical Society - EuChemS), kjer predstavljamo Slovensko kemijsko društvo. Prvič smo organizirali letni sestanek FCD v Sloveniji (30. 9. 2022). V okviru FCD smo sodelo- vali pri izboru tematik mednarodnega kongresa »XXII Eu- roFoodChem Congress«, ki bo leta 2023. Med te aktualne tematike spadajo: kakovost in varnost hrane; hrana in traj- nostni razvoj (vključno z valorizacijo stranskih proizvo- dov); nova živila; hrana in zdravje, funkcionalna živila in sestavine s funkcionalnimi lastnostmi; kemijske reakcije in interakcije med sestavinami v živilih; kemijske spremembe v hrani med predelavo in skladiščenjem; ponarejanje živil, V Sloveniji je do sedaj večina preskusnih laboratori- jev akreditiranih v skladu z ISO 17025, tako da večjih po- treb po izobraževanjih za preskusne laboratorije s področ- ja, ki ga pokriva Eurachem, ni več. Izjema so medicinski laboratoriji, za katere velja ISO 15189. ki se vsebinsko ne razlikuje bistveno od 17025. Za tiste, ki želijo pridobiti znanja s področja kakovo- sti merjenj v kemijskih/biokemiojskih laboratorijh, je tudi v letu 2022 organiziral SIQ tako Šolo kakovosti za analitske laboratorije, kot tudi celodnevna izobraževanja za posa- mezne vsebine (meroslovna sledljivost,, validacija meril- Poročilo Sekcije za živilsko kemijo za leto 2022 Poročilo sekcije EURACHEM Sloveniija za leto 2022 avtentičnost in sledljivost; nove metode analize živil; kon- taminanti v hrani. V okviru Skupine za organizacijo in iz- vedbo webinarjev, ki od leta 2021 deluje v okviru FCD, smo tudi leta 2022 sodelovali tudi pri izboru tematik in organizaciji webinarjev: • »Fighting global food crime with analytical che- mistry«, prof. dr. Chris Elliott z Institute for Global Food Security, Queen’s University, Bel- fast; • »Interpreting the gut microbiota function by employing LC/GC-MS metabolomic approa- ches« by Dr. Josep Rubert z Wageningen Uni- versity, Wageningen. V okviru FCD je potekalo tudi sodelovanje pri prip- ravi razpisa za mednarodno nagrado »The EuChemS Food Chemistry Division Young Researcher Award 2023«, ki bo podeljena mladi raziskovalki ali mlademu raziskovalcu za raziskave izvedene v okviru doktorske disertacije na po- dročju živilske kemije in sorodnih področij. (https://www. euchems.eu/divisions/food-chemistry-2/news/). V planu za leto 2023 smo predvideli aktivno udelež- bo na mednarodnih simpozijih. Načrtujemo večjo udelež- bo na »XXII EuroFoodChem Congress«, ki bo od 14. do 16. 6. 2023 v Beogradu, saj gre za dogodek iz najpomemb- nejše serije kongresov in simpozijev, ki jih organizira FCD. Udeležili se bomo tudi sestanka FCD, kjer bomo prestavili naše delo in z ostalimi člani FCD naredili načrt dela za prihodnje leto. dr. Irena Vovk nih postopkov, ovrednotenje merilne negotovosti). Preda- vateljici sva dr. Monika Inkret in jaz. V lanskem letu je bilo vzpodbudno predvsem to, da so se za udeležbo na SIQ iz- obraževanju odločili tudi raziskovalni laboratoriji, ki se nimajo namena akreditirati. To bi vsekakor veljalo vzpod- bujati in razširiti na druge raziskovalne laboratorije. Tudi v okviru Eurachem-a ni bilo večjih novosti, kar je pričakovano, saj so koncepti, ki so se intenzivno obliko- vali v začetku tega tisočletja, do sedaj definirani in so v pretežni meri ‚zgolj‘ stvar implementacije, poleg seveda širitve na biopodročja. dr. Nina Hrastelj S48 Acta Chim. Slov. 2023, 70, (2), Supplement Društvene vesti in druge aktivnosti 2023 June 2023 14 – 16 XXII EUROFOODCHEM CONGRESS Belgrade, Serbia Information: https://xxiieurofoodchem.com/ 18 – 21 33RD EUROPEAN SYMPOSIUM ON COMPUTER-AIDED PROCESS ENGINEERING (ESCAPE-33) Athens, Greece Information: https://escape33-ath.gr/ 28 – 30 MASS SPECTROMETRY CONGRESS IN ITALY – MASSA 2023 Torino, italy Information: https://torino2023.spettrometriadimassa.it/ July 2023 2 – 5 17TH EUROPEAN CONFERENCE ON MIXING Porto, Portugal Information: http://mixing17.eu/ 2 – 6 FEZA 2023 – 9TH CONFERENCE OF THE FEDERATION OF THE EUROPEAN ZEOLITE ASSOCIATIONS Portorož-Portorose, Slovenia Information: https://feza2023.org/en/ 2 – 7 XV POSTGRADUATE SUMMER SCHOOL ON GREEN CHEMISTRY Venice, Italy Information: https://www.greenchemistry.school/ 3 – 7 SCIENCE, TECHNOLOGY, SOCIETY AND WIKIPEDIA Milano, Italy Information: https://iupac.org/project/2018-038-1-400/ 4 – 7 EURODRYING 2023 Lodz, Poland Information: https://www.eurodrying2023.p.lodz.pl/ 7 – 11 9TH EUCHEMS CHEMISTRY CONGRESS (ECC9) Dublin, Ireland 9 – 14 38TH INTERNATIONAL CONFERENCE ON SOLUTION CHEMISTRY Belgrade, Serbia Information: https://icsc2023.pmf.uns.ac.rs/ KOLEDAR VAŽNEJŠIH ZNANSTVENIH SREČANJ S PODROČJA KEMIJE IN KEMIJSKE TEHNOLOGIJE SCIENTIFIC MEETINGS – CHEMISTRY AND CHEMICAL ENGINEERING S49Acta Chim. Slov. 2023, 70, (2), Supplement Društvene vesti in druge aktivnosti August 2023 7 – 11 CHEMISTRY AND INTERDISCIPLINARY RESEARCH TOWARDS SDGS Online/ Virtual Information: https://sites.google.com/uom.ac.mu/vcca-2023 18 – 25 52ND IUPAC GENERAL ASSEMBLY The Hague, Netherlands Information: https://iupac.org/event/ga2023/ 20 – 27 IUPAC WORLD CHEMISTRY CONGRESS 2023 The Hague, Netherlands Information: https://iupac2023.org/ 27 – 31 EUROANALYSIS 2023 Geneva, Switzerland Information: https://www.euroanalysis2023.ch/ 30 – Sept 1 28TH INTERNATIONAL WORKSHOP ON INDUSTRIAL CRYSTALLIZATION - BIWIC 2023 Stockholm, Sweden Information: https://www.biwic2023.se/ September 2023 4 – 7 POLYMER MEETING 15 IN BRATISLAVA (PM15) Bratislava, Slovakia Information: https://pm15.sav.sk/ 5 – 8 22ND INTERNATIONAL SYMPOSIUM ON INDUSTRIAL CRYSTALLIZATION - ISIC 2023 Glasgow, Scotland Information: https://www.isic2023.com/ 5 – 8 12TH INTERNATIONAL WORKSHOP ON POLYMER REACTION ENGINEERING (PRE) Potsdam, Germany Information: https://dechema.de/en/PRE2023.html 12 – 14 8TH INTERNATIONAL FAPS POLYMER CONGRESS Istanbul, Turkey Information: https://www.faps2023.com/home 13 – 15 SCS ANNUAL MEETING 2023 Portorose, Slovenia Information: https://skd2023.chem-soc.si/en/ 17 – 21 14TH EUROPEAN CONGRESS OF CHEMICAL ENGINEERING AND 7TH EUROPEAN CONGRESS OF APPLIED BIOTECHNOLOGY Berlin, Germany Information: https://ecce-ecab2023.eu/ 17 – 22 6TH INTERNATIONAL MASS SPECTROMETRY SCHOOL Cagliari, Italy Information: https://www.spettrometriadimassa.it/imss2023/ October 2023 15 – 19 31ST INTERNATIONAL SYMPOSIUM ON THE CHEMISTRY OF NATURAL PRODUCTS AND 11TH INTERNATIONAL CONGRESS ON BIODIVERSITY Naples, Italy Information: https://www.iscnp31-icob11.org/ 22 – 25 2ND INTERNATIONAL CONFERENCE ON ENERGY, ENVIRONMENT & DIGITAL TRANSITION – E2DT 2023 Palermo, Italy Information: https://www.aidic.it/e2dt2023/ S50 Acta Chim. 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Additional information • Citing spectral and analytical data • Depositing X-ray data Sub mis sion ma te rial Typi cal sub mis sion con sists of: • full manuscript (PDF file, with title, authors, ab- stract, keywords, figures and tables embedded, and references) • supplementary files – Full manuscript (original Word file) – Statement of novelty (Word file) – List of suggested reviewers (Word file) – ZIP file containing graphics (figures, illustra- tions, images, photographs) – Graphical abstract (single graphics file) – Proposed cover picture (optional, single graphics file) – Appendices (optional, Word files, graphics files) Incomplete or not properly prepared submissions will be rejected. Sub mis sion pro cess Before submission, authors should go through the checklist at the bottom of the page and prepare for submission. Submission process consists of 5 steps. Step 1: Star ting the sub mis sion • Choo se one of the jour nal sections. • Con firm all the re qui re ments of the chec klist. • Ad di tio nal plain text com ments for the edi tor can be pro vi ded in the re le vant text field. Step 2: Up load sub mis sion • Up load full ma nus cript in the form of a Word fi­ le (with tit le, aut hors, ab stract, key words, fi gu res and tab les em bed ded, and re fe ren ces). Step 3: En ter me ta da ta • First na me, last na me, con tact email and af lia tion for all aut hors, in re le vant or der, must be pro vi ded. Cor res pon ding aut hor has to be se lec ted. Full po- stal ad dress and pho ne num ber of the cor res pon- ding aut hor has to be pro vi ded. • Tit le and ab stract must be pro vi ded in plain text. • Key words must be pro vi ded (max. 6, se pa ra ted by se mi co lons). • Data about con tri bu tors and sup por ting agen cies may be en te red. • Re fe ren ces in plain text must be pro vi ded in the re le vant text fi led. Step 4: Up load sup ple men tary fi les • Original Word file (original of the PDF uploaded in the step 2) • List of suggested reviewers with at least five re- viewers with two recent references from the field of submitted manuscript must be uploaded as a Word file. At the same time, authors should declare (i) that they have no conflict of interest with suggest- ed reviewers and (ii) that suggested reviewers are experts in the field of the submitted manuscript. • All grap hics ha ve to be up loa ded in a sin gle ZIP fi le. Grap hics should be na med Fi gu re 1.jpg, Fi gu re 2.eps, etc. • Grap hi cal ab stract ima ge must be uploaded separately • Pro po sed co ver pic tu re (op tio nal) should be up- loa ded se pa ra tely. • Any ad di tio nal ap pen di ces (optional) to the paper may be uploaded. Appendices may be published as a supplementary material to the paper, if accepted. • For each uploaded file the author is asked for addi- tional metadata which may be provided. Depending of the type of the file please provide the relevant title (Statement of novelty, List of suggested re- viewers, Figures, Graphical abstract, Proposed cov- er picture, Appendix). Step 5: Con fir ma tion • Fi nal con fir ma tion is re qui red. Ar tic le Types Feature Articles are contributions that are written on Editor’s invitation. They should be clear and concise summaries of the author’s most recent work written with the broad scope of ACSi in mind. They are intend- ed to be general overviews of the authors’ subfield of research but should be written in a way that engages and informs scientists in other areas. They should con- tain the following (see also general guidelines for arti- cle structure below): (1) an introduction that acquaints readers with the authors’ research field and outlines the important questions for which answers are being sought; (2) interesting, novel, and recent contributions of the author(s) to the field; and (3) a summary that presents possible future directions. Manuscripts should normally not exceed 40 pages of one column format (font size 12, 33 lines per page). Generally, experts who have made an important contribution to a specific field in recent years will be invited by the Editor to contrib- ute a Feature Article. Individuals may, however, send a proposal (of no more than one page) for a Feature Article to the Editor-in-Chief for consideration. Acta Chimica Slovenica Author Guidelines S51Acta Chim. Slov. 2023, 70, (2), Supplement Društvene vesti in druge aktivnosti Scien ti fic ar tic les should report significant and inno- vative achievements in chemistry and related scienc- es and should exhibit a high level of originality. They should have the following structure: 1. Tit le (max. 150 cha rac ters), 2. Aut hors and af lia tions, 3. Ab stract (max. 1000 cha rac ters), 4. Key words (max. 6), 5. Intro duc tion, 6. Experimental, 7. Re sults and Dis cus sion, 8. Conc lu sions, 9. Acknowledgements, 10. Re fe ren ces. The sections should be arranged in the sequence gen- erally accepted for publications in the respective fields and should be successively numbered. Short com mu ni ca tions generally follow the same order of sections as Scientific articles, but should be short (max. 2500 words) and report a significant as- pect of research work meriting separate publication. Editors may decide that a Scientific paper is catego- rized as a Short Communication if its length is short. Tech ni cal ar tic les report applications of an already described innovation. Typically, technical articles are not based on new experiments. Pre pa ra tion of Sub mis sions Text of the submitted articles must be prepared with Microsoft Word. Normal style set to single column, 1.5 line spacing, and 12 pt Times New Roman font is recommended. Line numbering (continuous, for the whole document) must be enabled to simplify the re- viewing process. For any other format, please consult the editor. Articles should be written in English. Correct spelling and grammar are the sole responsibility of the author(s). Papers should be written in a concise and succinct manner. The authors shall respect the ISO 80000 standard [1], and IUPAC Green Book [2] rules on the names and symbols of quantities and units. The Système International d’Unités (SI) must be used for all dimensional quantities. Grap hics (figures, graphs, illustrations, digital imag- es, photographs) should be inserted in the text where appropriate. The captions should be self-explanatory. Lettering should be readable (suggested 8 point Arial font) with equal size in all figures. Use common pro- grams such as MS Excel or similar to prepare figures (graphs) and ChemDraw to prepare structures in their final size. Width of graphs in the manuscript should be 8 cm. Only in special cases (in case of numerous data, visibility issues) graphs can be 17 cm wide. All graphs in the manuscript should be inserted in relevant places and aligned left. The same graphs should be provid- ed separately as images of appropriate resolution (see below) and submitted together in a ZIP file (Graphics ZIP). Please do not submit figures as a Word file. In graphs, only the graph area determined by both axes should be in the frame, while a frame around the whole graph should be omitted. The graph area should be white. The legend should be inside the graph area. The style of all graphs should be the same. Figures and illustrations should be of sufcient quality for the printed version, i.e. 300 dpi minimum. Digital images and photographs should be of high quality (minimum 250 dpi resolution). On submission, figures should be of good enough resolution to be assessed by the refer- ees, ideally as JPEGs. High­resolution figures (in JPEG, TIFF, or EPS format) might be required if the paper is accepted for publication. Tab les should be prepared in the Word file of the pa- per as usual Word tables. The captions should appear above the table and should be self-explanatory. Re fe ren ces should be numbered and ordered se- quentially as they appear in the text, likewise meth- ods, tables, figure captions. When cited in the text, reference numbers should be superscripted, follow- ing punctuation marks. It is the sole responsibility of authors to cite articles that have been submitted to a journal or were in print at the time of submission to ACSi. Formatting of references to published work should follow the journal style; please also consult a recent issue: 1. J. W. Smith, A. G. Whi te, Ac ta Chim. Slov. 2008, 55, 1055–1059. 2. M. F. Kem me re, T. F. Keu rent jes, in: S. P. Nu nes, K. V. Pei ne mann (Ed.): Mem bra ne Tech no logy in the Che mi cal In du stry, Wi ley­VCH, Wein heim, Ger­ many, 2008, pp. 229–255. 3. J. Le vec, Ar ran ge ment and pro cess for oxi di zing an aqu e ous me dium, US Pa tent Num ber 5,928,521, da te of pa tent July 27, 1999. 4. L. A. Bur sill, J. M. Tho mas, in: R. Ser sa le, C. Col le la, R. Aiel lo (Eds.), Re cent Pro gress Re port and Dis cus­ sions: 5th In ter na tional Zeo li te Con fe ren ce, Na ples, Italy, 1980, Gia ni ni, Na ples, 1981, pp. 25–30. 5. J. Sze gez di, F. Csiz ma dia, Pre dic tion of dis so cia tion con stant using mi cro con stants, http://www. che­ ma xon.com/conf/Pre dic tion_of_dis so cia tion _con­ stant_using_mi cro co nstants.pdf, (as ses sed: March 31, 2008) Titles of journals should be abbreviated according to Chemical Abstracts Service Source Index (CASSI). Spe cial No tes • Com ple te cha rac te ri za tion, inc lu ding cry stal struc tu re, should be gi ven when the synthe sis of new com pounds in cry stal form is re por ted. • Nu me ri cal da ta should be re por ted with the num ber of sig ni fi cant di gits cor res pon ding to the mag ni tu de of ex pe ri men tal un cer tainty. • The SI system of units and IUPAC re com men­ da tions for nomenclature, symbols and abbrevia- tions should be followed closely. Additionally, the authors should follow the general guidelines when citing spectral and analytical data, and depositing crystallographic data. • Cha rac ters should be correctly represented throughout the manuscript: for example, 1 (one) and l (ell), 0 (zero) and O (oh), x (ex), D7 (times sign), B0 (degree sign). Use Symbol font for all Greek letters and mathematical symbols. • The ru les and re com men da tions of the IUBMB and the In ter na tio nal Union of Pure and Ap plied Che mi stry (IUPAC) should be used for abbreviation of chemical names, nomenclature of chemical com- pounds, enzyme nomenclature, isotopic compounds, optically active isomers, and spectroscopic data. • A conf ict of in te rest occurs when an individual (author, reviewer, editor) or its organization is in- S52 Acta Chim. Slov. 2023, 70, (2), Supplement Društvene vesti in druge aktivnosti volved in multiple interests, one of which could pos- sibly corrupt the motivation for an act in the other. Financial relationships are the most easily identifi- able conflicts of interest, while conflicts can occur also as personal relationships, academic competi- tion, etc. The Edi tors will make effort to ensure that conflicts of interest will not compromise the evaluation process; potential editors and reviewers will be asked to exempt themselves from review process when such conflict of interest exists. When the manuscript is submitted for publication, the aut hors are expected to disclose any relationships that might pose potential conflict of interest with respect to results reported in that manuscript. In the Acknowledgement section the source of fund- ing support should be mentioned. The statement of disclosure must be provided as Comments to Editor during the submission process. • Pub lis hed sta te ment of In for med Con sent. Research described in papers submitted to ACSi must adhere to the principles of the Declaration of Helsinki (http://www.wma.net/e/po licy/ b3.htm). These studies must be approved by an appropriate institutional review board or commit- tee, and informed consent must be obtained from subjects. The Methods section of the paper must include: 1) a statement of protocol approval from an institutional review board or committee and 2), a statement that informed consent was obtained from the human subjects or their representatives. • Pub lis hed Sta te ment of Hu man and Ani mal Rights.When reporting experiments on human subjects, authors should indicate whether the procedures followed were in accordance with the ethical standards of the responsible committee on human experimentation (institutional and na- tional) and with the Helsinki Declaration of 1975, as revised in 2008. If doubt exists whether the research was conducted in accordance with the Helsinki Declaration, the authors must explain the rationale for their approach and demonstrate that the institutional review body explicitly ap- proved the doubtful aspects of the study. When reporting experiments on animals, authors should indicate whether the institutional and national guide for the care and use of laboratory animals was followed. • To avoid conflict of interest between authors and referees we expect that not more than one referee is from the same country as the corresponding au- thor(s), however, not from the same institution. • Con tri bu tions aut ho red by Slo ve nian scien tists are evaluated by non-Slovenian referees. • Pa pers des cri bing mi cro wa ve­as si sted reac­ tions performed in domestic microwave ovens are not considered for publication in Acta Chimica Slovenica. • Ma nus cripts that are not pre pa red and sub mit­ ted in ac cord with the in struc tions for aut hors are not con si de red for pub li ca tion. Ap pen di ces Authors are encouraged to make use of supporting in- formation for publication, which is supplementary ma- terial (appendices) that is submitted at the same time as the manuscript. It is made available on the Journal’s web site and is linked to the article in the Journal’s Web edition. The use of supporting information is particular- ly appropriate for presenting additional graphs, spectra, tables and discussion and is more likely to be of interest to specialists than to general readers. When preparing supporting information, authors should keep in mind that the supporting information files will not be edited by the editorial staff. In addition, the files should be not too large (upper limit 10 MB) and should be provided in common widely known file formats to be accessible to readers without difculty. All files of supplementary materials are loaded separately during the submission process as supplementary files. Pro po sed Co ver Pic tu re and Grap hi cal Ab stract Image Grap hi cal con tent: an ideally full-colour illustration of resolution 300 dpi from the manuscript must be proposed with the submission. Graphical abstract pic- tures are printed in size 6.5 x 4 cm (hence minimal resolution of 770 x 470 pixels). Cover picture is print- ed in size 11 x 9.5 cm (hence minimal resolution of 1300 x 1130 pixels) Authors are encouraged to submit illustrations as can- didates for the journal Cover Picture*. The illustration must be related to the subject matter of the paper. Usually both proposed cover picture and graphical ab- stract are the same, but authors may provide different pictures as well. * The authors will be asked to contribute to the costs of the cover picture production. Sta te ment of no velty Statement of novelty is provided in a Word file and submitted as a supplementary file in step 4 of sub- mission process. Authors should in no more than 100 words emphasize the scientific novelty of the present- ed research. Do not repeat for this purpose the con- tent of your abstract. List of sug ge sted re vie wers List of suggested reviewers is a Word file submitted as a supplementary file in step 4 of submission pro- cess. Authors should propose the names, full afliation (department, institution, city and country) and e­mail addresses of five potential referees. Field of expertise and at least two references relevant to the scientif- ic field of the submitted manuscript must be provid- ed for each of the suggested reviewers. The referees should be knowledgeable about the subject but have no close connection with any of the authors. In addi- tion, referees should be from institutions other than (and countries other than) those of any of the authors. Authors declare no conflict of interest with suggested reviewers. Authors declare that suggested reviewers are experts in the field of submitted manuscript. How to Sub mit Users registered in the role of author can start sub- mission by choosing USER HOME link on the top of the page, then choosing the role of the Author and follow the relevant link for starting the submission process. Prior to submission we strongly recommend that you familiarize yourself with the ACSi style by browsing the journal, particularly if you have not submitted to the ACSi before or recently. S53Acta Chim. Slov. 2023, 70, (2), Supplement Društvene vesti in druge aktivnosti Cor res pon den ce All correspondence with the ACSi editor regarding the paper goes through this web site and emails. Emails are sent and recorded in the web site database. In the correspondence with the editorial ofce please provide ID number of your manuscript. All emails you receive from the system contain relevant links. Please do not answer the emails directly but use the embed­ ded links in the emails for carrying out relevant actions. Alternatively, you can carry out all the ac- tions and correspondence through the online system by logging in and selecting relevant options. Proofs Proofs will be dispatched via e-mail and corrections should be returned to the editor by e­mail as quick- ly as possible, normally within 48 hours of receipt. Typing errors should be corrected; other changes of contents will be treated as new submissions. Sub mis sion Pre pa ra tion Chec klist As part of the submission process, authors are required to check off their submission’s compliance with all of the following items, and submissions may be returned to authors that do not adhere to these guidelines. 1. The submission has not been previously published, nor is it under consideration for publication in any other journal (or an explanation has been provid- ed in Comments to the Editor). 2. All the listed authors have agreed on the content and the corresponding (submitting) author is re- sponsible for having ensured that this agreement has been reached. 3. The submission files are in the correct format: manuscript is created in MS Word but will be sub­ mitted in PDF (for reviewers) as well as in orig- inal MS Word format (as a supplementary file for technical editing); diagrams and graphs are cre- ated in Excel and saved in one of the file formats: TIFF, EPS or JPG; illustrations are also saved in one of these formats. The preferred position of graphic files in a document is to embed them close to the place where they are mentioned in the text (See Author guidelines for details). 4. The ma nus cript has been exa mi ned for spel ling and gram mar (spell chec ked). 5. The tit le (ma xi mum 150 cha rac ters) briefly ex­ plains the con tents of the ma nus cript. 6. Full names (first and last) of all authors together with the afliation address are provided. Name of author(s) denoted as the corresponding author(s), together with their e­mail address, full postal ad- dress and telephone/fax numbers are given. 7. The ab stract sta tes the ob jec ti ve and conc lu­ sions of the re search con ci sely in no mo re than 150 words. 8. Keywords (minimum three, maximum six) are provided. 9. Sta te ment of no velty (maximum 100 words) clearly explaining new findings reported in the manuscript should be prepared as a separate Word file. 10. The text adheres to the stylistic and bibliographic requirements outlined in the Aut hor gui de li nes. 11. Text in normal style is set to single column, 1.5 line spacing, and 12 pt. Times New Roman font is recommended. All tables, figures and illustrations have appropriate captions and are placed within the text at the appropriate points. 12. Mathematical and chemical equations are provided in separate lines and numbered (Arabic numbers) consecutively in parenthesis at the end of the line. All equation numbers are (if necessary) appropri- ately included in the text. Corresponding numbers are checked. 13. Tables, Figures, illustrations, are prepared in cor- rect format and resolution (see Aut hor gui de li­ nes). 14. The let te ring used in the fi gu res and graphs do not vary greatly in si ze. The re com men ded let te ring si ze is 8 point Arial. 15. Separate files for each figure and illustration are prepared. The names (numbers) of the separate files are the same as they appear in the text. All the figure files are packed for uploading in a single ZIP file. 16. Aut hors ha ve read spe cial no tes and ha ve ac cor- dingly pre pa red their ma nus cript (if ne ces sary). 17. Re fe ren ces in the text and in the Re fe ren ces are cor rectly ci ted. (see Aut hor gui de li nes). All ref- erences mentioned in the Reference list are cited in the text, and vice versa. 18. Permission has been obtained for use of copy- righted material from other sources (including the Web). 19. The names, full afliation (department, institution, city and country), e­mail addresses and referenc- es of five potential referees from institutions other than (and countries other than) those of any of the authors are prepared in the word file. At least two relevant references (important recent papers with high impact factor, head positions of departments, labs, research groups, etc.) for each suggested re- viewer must be provided. Authors declare no con- flict of interest with suggested reviewers. Authors declare that suggested reviewers are experts in the field of submitted manuscript. 20. Full-colour illustration or graph from the manu- script is proposed for graphical abstract. 21. Ap pen di ces (if appropriate) as supplementary material are prepared and will be submitted at the same time as the manuscript. Pri vacy Sta te ment The na mes and email ad dres ses en te red in this journal si te will be used exc lu si vely for the sta ted pur po ses of this jour nal and will not be ma de avai lab le for any ot­ her pur po se or to any ot her party. ISSN: 1580­3155 S54 Acta Chim. Slov. 2023, 70, (2), Supplement Društvene vesti in druge aktivnosti Slovensko kemijsko društvo www.chem-soc.si e-mail: chem.soc@ki.si Wessex Institute of Technology www.wessex.ac.uk SETAC www.setac.org European Water Association http://www.ewa-online.eu/ European Science Foundation www.esf.org European Federation of Chemical Engineering https://efce.info/ International Union of Pure and Applied Chemistry https://iupac.org/ Brussels News Updates http://www.euchems.eu/newsletters/ Novice europske zveze kemijskih društev EuChemS najdete na: Koristni naslovi Komore za testiranje baterij Vakuumski sušilniki Klimatske komore Donau Lab d.o.o., Ljubljana Tbilisijska 85 SI-1000 Ljubljana www.donaulab.si office-si@donaulab.com BINDER Acta Chimica oglas.indd 1 21. 11. 2021 20:29:54 Belinka Perkemija, d. o. o., Zasavska cesta 95, Ljubljana perkemija@belinka.si, www.belinka-perkemija.com Contact person: mag. Ivan Grčar: igc@belinka.si Member of KANSAI HELIOS. Part of KANSAI PAINT. Razvojna usmerjenost • Nove formulacije na bazi vodikovega peroksida • Širjenje uporab vodikovega peroksida na različnih področjih Področja uporabe V svoje vrste vabimo mlade raziskovalce, diplomante in post diplomante, ki bodo skupaj z nami reševali nove izzive. Hydrogen Peroxide H202 CosmeticsDetergents Pharmacy Medicine Pulp & Paper Veterinary AgricultureFood & Beverage Ecology www.helios-group.eu Znanje, kreativnost zaposlenih in inovacije so ključnega pomena v okolju, kjer nastajajo pametni premazi skupine KANSAI HELIOS. Z rešitvami, ki zadostijo široki paleti potreb, kontinuiranim razvojem ter s kakovostnimi izdelki, Helios predstavlja evropski center za inovacije in poslovni razvoj skupine Kansai Paint. Razvoj in inovacije za globalno uspešnost Odpihnite alergijo! www.dasselta.si Da ss el ta co nt ro l v se bu je d es lo ra ta di n. Pred uporabo natančno preberite navodilo! O tveganju in neželenih učinkih se posvetujte z zdravnikom ali s farmacevtom. Lajša simptome alergijskega rinitisa oz. senenega nahoda. Deluje 24 ur. Ne povzroča zaspanosti. NOVO 359702-2023 Dasselta control Ad A4 SI.indd 1 27. 01. 2023 12:00:09 4 n Year 2023, Vol. 70, No. 2 ActaChimicaSlovenica ActaChimicaSlovenica ActaChimicaSlovenica ActaChimicaSlovenica SlovenicaActaChim A cta C him ica Slovenica 70/2023 Pages 173–294 Pages 173–294 n Year 2023, Vol. 70, No. 2 http://acta.chem-soc.si 2 70/2023 2 ISSN 1580-3155 Kynurenic acid is metabolite of tryptophane that can be found in different foods, also in honey. The content of kynurenic acid was determined in more than 100 samples of different botanical sources. Results have shown that chestnut honey has much higher concentration of kynurenic acid than other honeys.