Acta agriculturae Slovenica, 116/2, 261–271, Ljubljana 2020 doi:10.14720/aas.2020.116.2.1031 Original research article / izvirni znanstveni članek Antioxidant activity and acute toxicity of two Lagenaria siceraria (Molina) Standl. varieties from Sudan Abdalbasit Adam MARIOD 1, 2, 3 , Makarim Mutasim Mohamed MUSTAFA 4 , Abdelazim Ahmed Mohamed NOUR 5 , Mahmood Ameen ABDALLA 6 , Suzy Munir SALAMA 7 , Nahla Saeed Al W AJEEH 7 Received January 22, 2019; accepted October26,2020. Delo je prispelo 22. januarja 2019, sprejeto 26. oktobra 2020. 1 University of Jeddah, College of Science and Arts, Department of Biology, Alkamil, KSA 2 Ghibaish College of Science & Technology, Indeginous Knowledge and Heritage Centre, Ghibaish, Sudan 3 Corresponding author, e-mail: basitmariod58@gmail.com, aalnadif@uj.edu.sa 4 University of Bahri, Department of Food Science, Khartoum North, Sudan 5 University, Khartoum, Faculty of Agriculture, Department of Food Science & Technology, Sudan 6 University of Malaya, Faculty of Medicine, Department of Molecular Medicine, Kuala Lumpur, Malaysia 7 University Malaya, Faculty of Medicine, Department of Biomedical Science, Kuala Lumpur, Malaysia Antioxidant activity and acute toxicity of two Lagenaria sicer- aria (Molina) Standl. varieties from Sudan Abstract: The present study was conducted to evaluate the antioxidant capacity and acute toxicity of the methanol extract of two Lagenaria siceraria (Molina)  Standl. varieties of dried seeds, Sweet gourd Water Jug (WJ) and bitter gourd Basket Ball (BB). The seed extracts were tested for their total phenolic contents (TPC), total flavonoid contents (TFC), ferric reduc- ing antioxidant power (FRAP) and scavenging activity to the stable free radical 2,2-diphenyl-1-picrylhydrazyl (DPPH). Both dried seed extracts were then tested for acute toxicity at doses 2 and 5 g kg -1 each. Sixty female and male rats were assigned into five groups per sex. Four groups were given low and high doses of each dried seed extract and the fifth group was given 10 % Tween 20 as a control. Kidneys and livers of all rats were assessed for biochemistry and histopathology. The seed extract of WJ revealed higher TPC, FRAP and DPPH activities com- pared to BB seeds, while TFC results were reversed. Toxicologi- cally, no toxicity signs were recorded in animals. Biochemistry results were within the normal ranges with a slight increase in bilirubin and Alanine aminotransferase (ALT), and histology of kidneys and livers showed normal architecture. In conclusion, WJ and BB dried seed extracts exhibited high antioxidant activ- ity suggesting promising therapeutic regimen against oxidative stress. Key words: Lagenaria siceraria; antioxidant; acute toxicity Antioksidacijska aktivnost in akutna toksičnost dveh sort vo- dnjače (Lagenaria siceraria (Molina) Standl.) iz Sudana Izvleček: Raziskava je bila izvedena za ovrednotenje an- tioksidacijske sposobnosti in akutne toksičnosti metanolnih izvlečkov suhih semen dveh sort vodnjače (Lagenaria siceraria (Molina) Standl.)), sladke ‘Water Jug’ (WJ) in grenke vodnjače ‘Basket Ball’ (BB). V izvlečkih semen so bili analizirani celoku- pna vsebnost fenolov (TPC), celukupna vsebnost flavonoidov (TFC), velikost redukcije železa (FRAP) in lovilna aktivnost sta- bilnega prostega radikala 2,2-difenil-1-pikrilhidrazila (DPPH). Oba izvlečka sta bila potem preiskušena na akutno toksičnost z odmerkoma 2 in 5 g kg -1 . 60 podganjih samic in samcev je bilo razdeljeno glede na spol v pet skupin. Vsaka od štirih skupin je dobivala velike in majhne odmerke izvlečka semen, peta sku- pina pa je dobila 10 % Tween 20 kot kontrolo. Ledvice in jetra vseh podgan so bile ocenjene po biokemičnih in histopatolo- ških parametrih. Izvleček semen WJ je pokazal večjo aktivnost TPC, FRAP in DPPH v primerjavi s semeni BB, a rezultati TFC so bili obratni. Na živalih ni bilo opaziti nobenih znakov za- strupitve. Biokemični parametri so bili znotraj normalih meja z rahlim povečanjem vsebnosti bilirubina in aktivnosti alanin aminotransferaze (ALT). Tudi histološki pregled ledvic in jeter je pokazal normalno zgradbo. Zaključimo lahko, da imajo iz- vlečki suhih semen WJ in BB veliko antioksidacijsko aktivnost, kar obeta njihovo uporabo pri blaženju oksidacijskega stresa. Ključne besede: Lagenaria siceraria; antioksidant; akutna toksičnost Acta agriculturae Slovenica, 116/2 – 2020 262 A. A. MARIOD et al. 1 INTRODUCTION Medicinal plants are still considered as cheap and safe, natural resource of drugs with less toxicity all over the world. Sudanese locals and people of other develop- ing countries have relied on traditional herbs to treat their diseases (Elhadi et al., 2013). Therefore, it is use- ful to explore more about medicinal plants (Koko et al., 2008). Family Cucurbitaceae is a very large family com- posed of 118 genera and 825 species. One of the most important genera of this family is Lagenaria (Molina) which is also known as bottle gourd, calabash gourd or white flowered gourd plant (Fader et al., 2013). In ad- dition to Lagenaria siceraria, genus Lagenaria consists of five other wild species, namely L. brevifilora (Benth) Roberty, L. rufa (Gilg) C Jeffrey, L sphaerica E Mey, L. abyssinia (Hook. F.) C Jeffrey and L. guineensis (G Den) C Jeffrey. L. siceraria is an indigenous species native to India (Shah et al., 2010) and became widely spread in Africa and the most cultivable species in Asia and America (Erickson et al., 2005). Shah et al. (2010) re- ported that Lagenaria has two distinguished varieties, one is bitter bottle gourd and sweet variety useful bottle gourd. The bitter variety is wild plant and used for the pharmacological application, while sweet variety is used as a vegetable and medicinal. In Sudan, varieties of L. si- ceraria ,the sorcerer is broadly diffused in different areas of the South and West, while some of them are edible such as snake gourd. Recently, researchers focused their study on L. siceraria varieties exploring more about their chemical composition. Studies reported that they can be used as nutritious source, in cosmetics and medicine (Mariod et al., 2015). Literature review reveals that L. si- ceraria showed a broad spectrum of pharmacological ac- tivities such as antioxidant (Mayakrishnan et al., 2013), antimutagenic (Thakkar, 2013), antiulcer (Srivastava et al., 2011), anti-inflammatory (Ghule et al., 2006), anti- diabetic, hepatoprotective (Deshpande et al., 2008) and cardioprotective (Upaganlawar and Balaraman, 2011). Due to the presence of high percentage of alkaloids, phe- nolic glycosides, carbohydrates, proteins and minerals, L. siceraria can be suggested to replace allopathic medi- cines (Mariod et al., 2015; Sivannarayana et al., 2013). In folk medicine, different parts (leaves, stem, flower, root, and seeds, fresh and dry fruits) of L. siceraria have been used in the ointment for ailment of various diseases (Ku- mar et al., 2015). Traditionally, L. siceraria seeds can be cooked, dried and served as snacks in some countries such as Egypt. Also can be ground and fermented for use as a flavor enhancer in gravies and soups in West Africa (Achigan- Dako et al., 2008). At the phytochemistry level, stud- ies proved the presence of various phytochemicals like cardiac glycosides, terpenoides, carbohydrates, resins, saponins and phytosterols in L. siceraria seeds (Sood et al., 2012). Singh et al. (2012) suggested that the ethanol extract of L. siceraria seeds exhibits cardioprotective po- tential in experimental animals against cardiac toxicity induced by doxorubicin. Another study revealed that the ethanol extract of L. siceraria seeds showed excellent free radical scavenging activity (Gill et al., 2012). As an extension to our previous study on the eight Sudanese Lagenaria varieties (Mariod et al., 2015), the present study was conducted to determine the total phenol and total flavonoid contents, and acute oral toxicity of the methanol extract of sweet and bitter varieties of L. sicer- aria seeds from Sudan. 2 MATERIAL AND METHODS 2.1 IN VITRO ANTIOXIDANT CAPACITY STUDY OF WJ AND BB METHANOL EXTRACTS OF DRIED SEEDS 2.1.1 Plant material Two varieties of Lagenaria siceraria dry fruits {Sweet gourd ‘Water Jug’ (WJ) and bitter gourd ‘Basket Ball’ (BB)} were obtained from the market of Omdur- man, State of Khartoum, Sudan. 2.1.2 Preparation of seeds extracts By the aid of 4.8 mm knife, the seeds were removed from the collected dried fruits. The removed seeds were properly washed and left dried at 50 °C in drying oven (FD 115; Fisher Scientific, Loughborough, Leicester- shire, UK). The dried seeds were then ground to a pow- der using home blender and stored in polyethylene bags in the refrigerator at 4 °C till the onset of the experiment as previously done (Mariod et al., 2015). For seed extrac- tion, the seed powder was firstly defatted using n-hexane at 50–60 °C in a Soxhlet apparatus for six hours follow- ing the AOCS method (Firestone, 2009). The dried seeds (seedcake) were extracted for 72 h in methanol 95 % at room temperature with occasional stirring. The solvent was filtered off using filter paper (Whatman No.1, Fitch- burg, WI, USA) and the filtrate obtained was concen- trated under reduced pressure on a rotary evaporator at 40 °C and finally freeze dried using a freeze-drying machine (LabConco, Kansas City, MO, USA). The dried seed extracts obtained from both water jug seeds (WJ) and basketball seeds (BB) were then stored at 4 °C for further use for various investigations. Acta agriculturae Slovenica, 116/2 – 2020 263 Antioxidant activity and acute toxicity of two Lagenaria siceraria (Molina) Standl. varieties from Sudan 2.1.3 Total phenol content (TPC) determination Total phenolic content was determined using Fo- lin-Ciocalteu reagent following the method of Single- ton and Rossi, (1965) and using gallic acid as a stand- ard. An amount of 10 µl of each WJ and BB dried seed extract solution (1 mg ml -1 ) was added in a test tube followed by the addition of 0.5 ml of 1:10 Folin-Ciocal- teu reagent. The mixture was incubated at room tem- perature for 5 minutes. Following incubation, 0.35 ml of 115 mg ml -1 natrium carbonate (Na 2 CO 3 ) was added and mixed thoroughly. The mixture was then allowed to stand at room temperature for 2 hours. Absorbance reading taken spectrophotometrically at 765 nm and all determinations were done in triplicates. The total phenolic content of WJ and BB were expressed as mil- ligrams of gallic acid equivalent to grams of dried seed extract. 2.1.4 Total flavonoid content (TFC) determination Total flavonoid content of WJ and BB seed ex- tracts were determined by aluminum chloride colori- metric method using quercetin as a standard (Chang et al., 2002). Briefly, 0.5 ml of each seed extract solu- tion (1 mg ml -1 ) was sampled in a test tube and mixed with 1.5 ml ethanol 95 %, 0.1 ml aluminum chloride and 2.8 ml distilled water. The mixtures were left to in- cubate for 30 minutes at room temperature followed by reading the absorbance at 415 nm. The total flavonoid content of WJ and BB were expressed as milligrams of quercetin equivalent to grams of dried plant material. 2.1.5 Ferric reducing antioxidant power (FRAP) de- termination Ferric reducing activity of the WJ and BB seed extracts was estimated using the method developed by Benzie and Strain (1996), while vitamin C (Vit. C) was taken as reference. The principle of the method depends on the chelating power of substances to ferric tripyridyltriazine complex. Briefly, the working FRAP reagent was freshly prepared by mixing 300 mmol l -1 acetate buffer, 10 mmol l -1 TPTZ (2, 4, 6-tripyridyl- s-triazine) in 40 mmol l -1 of HCL and 20 mmol l -1 of FeCl 3 .6H 2 O. The prepared mixture was then incubated at 35 o C in water bath for five minutes and then a blank reading was taken spectrophotometrically at 593 nm. A quantity of 10 µl of each extract/reference was sampled separately and mixed with 290 µl of the working FRAP reagent followed by vortexing and reading the absorb- ance immediately at 593 nm to get the record of zero minutes. Thereafter, the absorbance reading was taken every four minutes for a period of 2 hours. All the re- sults were expressed as mmol ferric reducing activity of the extract per gram of dried weight based on three experiments. 2.1.6 DPPH free radical scavenging activity deter- mination In the DPPH radical scavenging test, the scaveng- ing of DPPH is followed by monitoring the decrease in absorbance at 515 nm that occurs due to reduction by the antioxidant or reaction with a radical species. DPPH is widely used to test for the ability of compounds to act as hydrogen donors or free radical scavengers and to evaluate antioxidant activity of foods (Kadhim et al., 2019). Scavenging activity of the WJ and BB dried seed extracts to the stable 2, 2 diphenyl-1-picryl dyhydrazyl (DPPH) free radical was determined according to the method of Gorinstein et al. (2005) with slight modifica- tion. Briefly, the dried seed extracts and the reference standard (ascorbic acid) were prepared (1 mg ml -1 ) as separate stock solutions and then serial dilution with 8 varying concentrations were prepared (0.37, 0.78, 1.56, 3.125, 6.25, 12.5, 25, 50 µl) from each stock solution. Five microliters of each sample was loaded in 96-well plate followed by the addition of 195 µl of DPPH rea- gent; the mixtures were then incubated in the dark at room temperature for 2 hours. At the end of the incuba- tion period, the absorbance was measured spectropho- tometrically at 515 nm with a Hitachi spectrophotom- eter (Hitachi, LTD.Tokyo, Japan), the DPPH percentage inhibition was calculated using the following formula: DPPH (%) = [(Abs of blank-Abs of sample)/Abs of blank] ×100. The results were expressed as IC 50 value which is the extract concentration required to reduce 50 % of the DPPH free radical. 2.2 IN VIVO ACUTE TOXICITY STUDY OF WJ AND BB METHANOL EXTRACTS OF DRIED SEEDS 2.2.1 Experimental animals Sixty adult and healthy Sprague Dawley (SD) rats (6–8 weeks old) were obtained from the Animal House, Faculty of Medicine, University of Malaya, Kuala Lum- pur (Ethic no.PM/30/05/2012/NSIAW (R). The body Acta agriculturae Slovenica, 116/2 – 2020 264 A. A. MARIOD et al. mass of SD rats was between 195–210 g. The animals were fed standard rat pellets and tap water. 2.2.2 Acute toxicity test The acute toxicity study was performed to deter- mine the safe dosage of the dried seed extracts. Sixty SD rats (30 males and 30 females) were randomly and equally divided into 5 groups per sex. The groups were categorized as control (10 % Tween 20, 5 ml kg -1 ), high dose WJ extract 5 g kg (HD-WJ), low dose WJ extract 2 g kg -1 (LD-WJ), high dose BB extract 5 g kg -1 (HD-BB), and low dose BB extract 2 g kg -1 (LD-BB) (Co-operation and Development, 2002). To prepare the animals for dosing, they were allowed to fast for 14 hours without accessing food but with free access to water. All the rats were weighed recording the body mass on day 0. After dosing, food was prevented for an additional four hours and the rats were observed for 30 min and 2, 4, 8, 24 and 48 hours, post dosing for the appearance of any clinical or toxicological signs such as respiration, salivation, diar- rhoea, tremors, eyes and mucus membrane, skin and fur, sleep pattern and any signs associated with the nervous system. The presence of any morbidity, mortality or be- havioural changes was recorded. At the end of the experi- mental period, the body mass of all the rats was recorded. On the 15th day, the animals were given over-dose of an- aesthesia (xylazine with ketamine) then sacrificed to col- lect their blood for biochemistry study and kidneys and livers for histology study. Serum biochemical parameters were investigated at University of Malaya Medical Cen- tre using a Hitachi Autoanalyzer, Japan, after optimizing the machine as previously performed (Al Batran et al., 2013). Liver function parameters were assayed for total protein, albumin, globulin and conjugated bilirubin. In addition aspartate aminotransferase (AST), alanine ami- notransferase (ALT) and gamma-glutamyl tansferase (GGT) were measured as markers of liver injury (Fujii 1997; Y oung et al., 2008). 2.3 STATISTICAL ANALYSIS Statistical analysis of the data was carried out by us- ing the statistical program for the social sciences (SPSS) version 16 while applying the ANOVA test for compar- ing means, followed by Post-Hoc Tukey test. All the data were expressed as mean ± standard deviation (SD) of triplicates of the in vitro study and n = 6 for the in vivo study. The statistical value p ≤ 0.05 was considered sig- nificant. 3 RESULTS AND DISCUSSION 3.1 IN VITRO ANTIOXIDANT RESULTS 3.1.1 TPC and TFC of WJ and BB methanol extracts of dried seeds Antioxidants are the compounds that when added to food products, especially to lipids and lipid-containing foods, can increase the shelf life retarding the process of lipid peroxidation, which is one of the major reasons for deterioration of food products during processing and storage. Synthetic antioxidants, such as butylatedhy- droxyanisole (BHA) and butylatedhydroxytolune (BHT), have restricted use in foods as they are suspected to be carcinogenic via their binding ability to DNA and cellu- Figure 1: Total phenol content (TPC) of the methanol extracts of sweet gourd ‘Water Jug’ (WJ) and bitter gourd ‘Basket Ball’ (BB) dried seed extract Acta agriculturae Slovenica, 116/2 – 2020 265 Antioxidant activity and acute toxicity of two Lagenaria siceraria (Molina) Standl. varieties from Sudan lar components (Dolatabadi and Kashanian, 2010; Mirza, Asema, and Kasim, 2017). Therefore, the exploitation of natural antioxidants, especially those of plant origin became necessary and attracted the attention of researchers in re- cent years (Moayedi et al., 2017). Aras et al. (2017) defined antioxidants as the substances that are capable of quenching or stabilizing free radicals. In the present study, the seeds of dry fruits of two La- genaria siceraria varieties, (sweet gourd Water Jug (WJ) and bitter gourd Basket Ball (BB) were ground, defatted and ex- tracted in methanol and then tested for their total phenol and total flavonoid contents. The results of total phenol con- tent (TPC) measured from the methanol extract of WJ and BB dried seeds are displayed in Fig.1. Results revealed that WJ seeds measured approximately two and half folds more than BB seeds indicating higher TPC of WJ seeds compared to BB seeds. The results of total flavonoid content (TFC) determined from the methanol extract of WJ and BB dried seeds are illustrated in Fig.2. Presented data recorded that the total flavonoid content of BB seeds was higher compared to that of WJ seeds. 3.1.2 FRAP and DPPH results of WJ and BB methanol extracts of dried seed Free radicals, often called reactive oxygen species (ROS) and they are the core of any biochemical process rep- resenting a vital part in metabolism (Sangeetha and Ven- katalakshmi, 2017). On the other hand, free radicals are highly generated in many pathological conditions and in- volved in the development and progress of common chron- ic degenerative diseases such as cardiovascular and neuro- degenerative diseases, diabetes and cancer (Umeno et al., 2017). Disturbance in the balance between the formation of free radicals in the cells and their antioxidants load leads to oxidative stress that damages cellular components such as proteins, lipids and nucleic acids, and eventually leads to cell death (Shaker and Mnaa, 2017). The FRAP values of WJ and BB seed extracts were displayed in Fig. 3 compared to vitamin C standard. The scavenging abilities of WJ and BB seed extracts to the stable DPPH free radical is diagrammed in Fig. 4 in comparison with ascorbic acid standard (vitamin C). Results recorded that IC 50 of both dried seed extracts were significantly high- er than that of ascorbic acid indicating less DPPH scaveng- ing activity compared to ascorbic acid. However, the WJ seed extract showed higher DPPH scavenging activity as in- dicated by the lower IC 50 value compared to BB seed extract. Studying the chemical composition of natural prod- ucts plays a crucial role in screening their biological ac- tivities. Natural products having a sufficient antioxidant ca- pacity contain mainly phenolic and flavonoid compounds which exhibit a broad range of biological activities such as anticancer, anti-inflammation, anti-aging and anti-athero- sclerosis (Rahman et al., 2013). Antia et al. (2015) studied the antioxidant activity of short-hybrid variety of Lagenaria siceraria seeds reporting that the methanol seed extract of that variety acquires significant antioxidant activity and the seeds can be used as excellent natural antioxidant. Later, another study conducted on the seed oil of short-hybrid variety and revealed significant DPPH scavenging activity (Antia et al. 2016). In the present study, the seeds of dry fruits of two La- genaria siceraria varieties, (sweet gourd ‘Water Jug’ (WJ) and bitter gourd ‘Basket Ball’ (BB) were ground, defatted and extracted in methanol and then tested for their total phenol and total flavonoid contents, their reducing power to Fe (III) and their ability to scavenge the stable DPPH free radical. The methanol extract of both seeds exposed high Figure 2: Total flavonoid content (TFC) of the methanol extracts of sweet gourd ‘Water Jug’ (WJ) and bitter gourd ‘Basket Ball’ (BB) dried seed extract Acta agriculturae Slovenica, 116/2 – 2020 266 A. A. MARIOD et al. antioxidant activity. Although WJ seeds acquired higher phenolic contents, FRAP and DPPH activity more than BB seeds extract, but BB seeds showed higher TFC com- pared to WJ seeds. Our study is consistent with a previ- ous study on the remarkable antioxidant capacity of L. siceraria seeds (Satvir et al., 2012). Based on the results of DPPH of ascorbic acid, plant extracts WJ and BB, the R-squared (R 2 ) values of the standard curve equations generated were 0.9516, 0.9478 and 0.9765, respectively. All values were close to 1.0 indicating acceptable stand- ard curves for all the tested samples ascorbic acid, WJ and BB (Salama et al., 2013). Recent studies on the anti- oxidant power of natural products manifested that they are very well recognized antioxidants, playing vital role in protecting biological cells and tissues from the detri- mental effects of free radicals (Zeb and Mehmood, 2004). Therefore, they can be promising pharmaceutical com- pounds in preventing cancer and degenerative diseases as well (Olszowy, 2019). 3.2 IN VIVO ACUTE TOXICITY RESULTS Acute toxicity studies in animals are usually neces- sary for any pharmaceutical substance intended for hu- man use. The information obtained from these studies is useful in selecting doses for repeat-dose studies, provid- ing preliminary identification of target organs of toxicity, and, occasionally, revealing delayed toxicity. Acute toxic- ity studies may also aid in the selection of starting doses for Phase 1 human studies, and provide information rel- evant to acute overdosing in humans (CDER and FDA, 1996). Past studies on acute toxicity of petroleum ether, ethanol and chloroform extracts of L. siceraria fruits in Figure 3: Ferric reducing antioxidant power (FRAP) of the methanol extracts of sweet gourd ‘Water Jug’ (WJ) and bitter gourd ‘Basket Ball’ (BB) dried seeds Figure 4: DPPH scavenging activities of the methanol extracts of sweet gourd ‘Water Jug’ (WJ) and bitter gourd ‘Basket Ball’ (BB) dried seeds compared to ascorbic acid standard (vitamin C). IC 50 values are indicated on the bars. Acta agriculturae Slovenica, 116/2 – 2020 267 Antioxidant activity and acute toxicity of two Lagenaria siceraria (Molina) Standl. varieties from Sudan rats recommended safe dose up to 2 g kg -1 (Deshpande et al., 2008). Additionally, previous study on the acute toxicity and sub-chronic toxicity of the methanol ex- tract of L. siceraria aerial parts showed that it is quite safe and can be used in the treatment of chronic diseas- es like diabetes (Saha et al., 2011). In the present study, the methanol extracts of WJ and BB dried seeds of L. siceraria revealed safe dose up to 5 g kg -1 . The findings from the acute toxicity study did not indicate any morbidity or mortality from treating male and female SD rats with the methanol extracts of WJ and BB dried seeds at doses 2 g kg -1 and 5 g kg -1 throughout the 14 days study period. Physical obser- vation of the treated rats throughout the study didn’t show any changes on skin, fur, eyes, mucus membrane, behaviour pattern, tremors, salivation or sleep. In addi- tion no diarrhea or coma observed on any of the treated rats from both sexes. Groups Body mass (g) day 0 Body mass (g) day 14 10 % Tween 20, male 202.50±4.4 208.16±3.7 10 % Tween 20, female 194.16±8.5 196.83±8.9 HD-WJ, male 205.16±4.5 207.66±5.3 HD-WJ, female 195.33±7.5 198.00±7.4 LD-WJ, male 205.16±4.8 207.66±5.1 LD-WJ, female 194.16±8.4 196.00±7.0 HD-BB, male 205.83±3.6 207.33±3.6 HD-BB, female 194.50±7.9 196.00±7.5 LD-BB, male 205.83±9.3 208.00±8.1 LD-BB, female 193.16±10.5 196.00±10.0 Table 1: Effects of WJ and BB dried seed extracts on the body mass of the rats Values expressed as mean ± SD, the significant value was set at p ≤ 0.05. Groups Sodium mmol l -1 Potassium mmol l -1 Chloride mmol l -1 Carbon Dioxide mmol l -1 Anion Gap mmol l -1 Urea mmol l -1 Creatinine umol l -1 10 % Tween20 Male 146.5±2.3 5.66±1.20 105.00±1.2 26.06±1.10 23.70±0.89 5.00±1.90 36.30±2.14 10 % Tween20 Female 147.3±0.8 5.66±1.20 102.00±1.5 24.07±1.23 22.00±0.76 5.40±2.16 36.70±2.31 HD-WJ, Male 145.3±1.3 4.95±1.20 103.66±1.0 25.96±0.80 20.83±0.75 4.25±0.20 35.50±2`94 HD-WJ Female 146.3±1.2 4.66±.019 104.16±0.9 23.88±2.00 22.66±1.90 6.10±0.95 37.00±1.78 LD-WJ Male 144.3 ±1.3 5.85±0.72 103.58±1.6 24.25±1.70 23.00±1.30 5.81±0.33 33.33±2.64 LD-WJ Female 144.5±2.0 5.03±0.08 103.83±1.1 23.33±1.80 21.50±1.10 5.80±0.54 37.16±1.66 HD-BB Male 144.50±0.8 4.80±0.90 102.83±1.1 26.00±0.57 20.33±0.50 5.45±0.53 38.33±2.73 HD-BB Female 141.83±1.7 4.68±0.50 100.50±0.8 24.13±1.71 22.00±1.10 5.66±0.71 37.66±7.45 LD-BB Male 144.16±1.6 4.60±0.30 101.16±2.1 25.78±1.58 22.00±1.00 5.70±0.90 37.33±4.90 LD-BB Female 141.66±1.5 4.33±0.50 100.00±1.2 24.15±2.70 21.83±1.80 6.26±0.38 38.16±9.92 Table 2: Effects of WJ and BB dried seed extracts on renal function parameters Values expressed as mean ± SD, the significant value was set at p ≤ 0.05. Acta agriculturae Slovenica, 116/2 – 2020 268 A. A. MARIOD et al. 3.2.1 Effect of WJ and BB dried seed extracts on body mass of rats The body mass measurements of the treated and control rats on day 0 and day 14 are shown in Table 1. Data showed reasonable increase in the body mass of the treated and control rats. In addition, the body mass of the treated rats were insignificantly different as compared to control. 3.2.2 Effect of WJ and BB dried seed extracts on renal function test Renal function parameters of both WJ-treated and BB-treated rats are presented in Table 2. From the results, the concentration of all measured parameters (sodium, potassium, chloride, carbon dioxide, anion gap, urea, and creatinine) in the animals treated with the low dose (2 g kg -1 ) and high dose (5 g kg -1 ) of both seed extracts were insignificant compared to control rats. The concentration of all measured parameters (sodium, potassium, chloride, carbon dioxide, anion gap, urea, and creatinine) is lying within the normal range, however there was slight increase in the con- centration of anion gap and decrease in creatinine level, but they were alike in both control and treated groups indicating safe treatment. This showed that the seed extract at different levels tested did not pro- duce considerable change in the levels of the different parameters studied. 3.2.3 Effect of WJ and BB seed extracts on liver function test Table 3 reorded the effects of the low dose (2 g kg -1 ) and high dose (5 g kg -1 ) of both WJ and BB methanol extracts of dried seeds on liver function parameters. No significant changes were observed in the values of the parameters studied [(total protein, albumin, globulin, conjugated bilirubin, aspartate aminotransferase (AST) and gama glutamyl trans- ferase (GGT),] in comparison with the control ani- mals and the values obtained were within the normal biological and laboratory limit. On the other hand, the total bilirubin recorded slight decrease than nor- mal ranges and showed significant difference in the low and high doses of BB-treated rats of both sexes compared to control. Additionally, alanine ami- notransferase (ALT) revealed slight elevation than normal ranges. Groups Total protein Albumin Globulin Total bilirubin ALT AST GGT g l -1 g l -1 g l -1 mmol l -1 IU l -1 IU l -1 IU l 10% Tween 20 Male 69.33±2.9 40.33±0.80 28.67±1.20 3.50±1.00 46.83±2.00 47.12±9.1 <3 10% Tween 20 Female 72.66±4.3 37.83±0.70 34.83±1.90 3.50±0.54 45.16±2.30 46.66±8.3 <3 HD-WJ Male 68.16±3.3 36.83±0.40 31.33±0.51 3.00±0.00 49.16±300 44.92±5.6 <3 HD-WJ Female 70.60±40 35.00±0.80 35.60±2.00 2.83±0,40 41.50±2.10 48.33±60 <3 LD-WJ Male 68.83±0.88 39.00±0.63 29.83±2.10 2.83±0.46 48.66±2.70 49.45±7.2 <3 LD-WJ Female 66.66±1.4 43.16±1.30 23.50±1.80 3.33±1.80 46.00±1.90 46.66±11 <3 HD-BB Male 63.66±2.2 37.33±0.80 26.33±5.30 2.33±0.50* 41.83±8.70 49.66±10 <3 HD-BB Female 69.16±2.1 35.00±0.90 34.16±7.10 1.50±1.70* 38.33±6.20 45.00±13 <3 LD-BB Male 62.50±1.9 36.00±0.60 26.50±5.50 1.83±1.90* 43.83±5.10 42.83±16 <3 LD-BB Female 68.83±3.9 38.50±2.20 30.33±4.10 1.33±2.50* 40.16±3.40 47.50±9.5 4 Table 3: Effects of WJ and BB dried seed extracts on liver function parameters Values expressed as mean ± SD, the significant value was set at p ≤ 0.05. *p <0.05 compared to normal control Acta agriculturae Slovenica, 116/2 – 2020 269 Antioxidant activity and acute toxicity of two Lagenaria siceraria (Molina) Standl. varieties from Sudan 3.2.4 Effect of WJ and BB dried seed extracts on the histopathology of liver and kidney of rats The qualitative data from the histological sections of kidneys and livers collected from the control and rats treated with WJ and BB seed extracts at the highest dose tested (5 g kg -1 ) are illustrated on Fig. 5. No significant difference was observed between the architecture of liv- ers or kidneys collected from the WJ-treated and BB- treated animals compared to control group. 4 CONCLUSION In conclusion, the current study showed that the methanol extracts of both WJ and BB dried seeds of L. siceraria possesses high antioxidant power. The WJ seeds variety reported higher TPC, FRAP and DPPH values than BB variety, while the TFC results of BB seeds variety recorded higher value than that of WJ. The acute toxicity test of the methanol extract of both seed varieties report- ed safe dose up to 5 g kg -1 in both sexes of rats. 5 ACKNOWLEDGEMENTS The authors would like to express their great thank- ful to the staff of immunology lab, Department of Mo- lecular Medicine, Faculty of Medicine and Lipid lab, Department of Chemistry, Faculty of Science, Univer- sity Malaya for their un limited support and cooperation during the practical period of the study. 6 REFERENCES Achigan-Dako, E. 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