408 Acta Chim. Slov. 2008, 55, 408–418 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 Scientific paper Modified 2,2’-Azinobis(3-Ethylbenzo Thiazoline)-6-Sulphonic Acid Radical Cation Decolorization Assay for Antioxidant Activity of Human Plasma and Extracts of Traditional Medicinal Plants Muhammad Nadeem Asghar, Islam Ullah Khan,* Iram Zia, Mukhtar Ahmad# and Fahim Ahmad Qureshi Department of Chemistry, Government College University, Lahore-54000, Pakistan #Department of Biochemistry, Allama Iqbal Medical College, Canal Road, Lahore, Pakistan * Corresponding author: E-mail: iukhan@gcu.edu.pk Office Tel: 92-042-111-000-010 Received: 17-02-2008 Abstract 2,2’-Azinobis(3-ethylbenzothiazoline)-6-sulfonic acid (ABTS) radical cation decolorization assay has been applied under different pH conditions for the determination of trolox equivalent antioxidant capacity (TEAC) of biological and food samples. Due to innate nature of ABTS radical cation, it is unstable at physiological pH. Further, its sensitivity is also lost at acidic pH. No single method has yet claimed to cover both drawbacks of the ABTS procedure. The present investigation was aimed at developing an improved ABTS decolorization method for the measurement of antioxidant activity of human plasma and herbal extracts with greater stability and sufficient sensitivity. A relatively stable ABTS radical cation was generated through a reaction between ABTS and potassium persulfate in the presence of sodium acetate buffer at pH 6.5 (20 mM). The electron or hydrogen atom transfer from antioxidative components of plasma or herbal extracts reduced the radical cation. Standard antioxidants like trolox, butylated hydroxyanisol (BHA), butylated hydroxytoluene (BHT) and ascorbic acid demonstrated stable end-point readings showing a promising aspect of determination of total antioxidant activity (TAA) of all the other samples with reference to these indicators. The modified assay proved to be highly reproducible. TEAC values for plasma samples ranged between 1.410–2.025 mM with mean percent relative standard deviation (% RSD) 1.64 and between 1.72–1.98 with mean % RSD 1.27 for older and modified ABTS assays, respectively. The efficacy of the assay was ascertained further by determining antioxidant activity of organic and aqueous extracts of Cuscuta reflexa and Terminalia arjuna. Keywords: ABTS radical cation, antioxidant activity, TEAC, plasma, potassium persulfate. 1. Introduction A number of decolorization assays are being employed for the measurement of total antioxidant activity of body fluids, food, herbal extracts, and pure compounds.1–17 Amongst these assays, ABTS (2,2’-azinobis(3-ethylbenzot-hiazoline)-6-sulfonic acid; (2Z,2’Z)-2,2’-(hydrazine-1,2-diylidene)bis(3-ethyl-2,3-dihydrobenzo[d]thiazole-6-sulfo-nic acid)) radical cation decolorization assay as developed by Miller et al.1 have gained popularity for its simplicity. ABTS, a phenothiazine drug, when reacting with a suitable oxidizing agent, like potassium persulfate, forms ABTS ra- dical monocation that has been reported to be stable in pH 6.5–3.5 range. On either side of this pH there is an accelerated degradation of ABTS into its metabolites. At physiological pH of 7.4, Cano et al.18 and Erel19 have expressed their reservations about the stability of ABTS radical cation. In order to determine the antioxidant activity of both plasma antioxidants and citrus fruits at pH 7.4, Re et al. have developed an improved ABTS assay.20 Since the stability of ABTS radical cation and the antioxidant activity depend partially upon pH of the radical cation medium, the repro-ducibility and accuracy may not be expected for biological and abiological samples when using ABTS assay at pH 7.4. Recently, Ozgen et al. reported that ABTS radical cation 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 Asghar et al.: Modified 2,2’-Azinobis(3-Ethylbenzo Thiazoline)-6-Sulphonic Acid Radical ... Acta Chim. Slov. I was more stable at pH 4.5 and could be used for the measu-2 rement of antioxidant activity of small fruits that have lower 3 pH values.21 In case of plasma samples, the sensitivity of 4 ABTS assay at pH 4.5, however, was markedly affected. In 5 the present study, the assay was modified to obtain a more 6 stable radical cation with comparable sensitivity for total 7 antioxidant capacity (TAC), for both herbs and plasma sam-8 ples. The results obtained were subsequently compared 9 with ABTS assay performed at pH 7.4. 10 Oxygen, although essential for existence of life, is II paradoxically involved in the generation of reactive oxy-12 gen species (ROS) in living organisms. The over-produc-13 tion of ROS has reportedly been implicated in more than 14 100 degenerative diseases including cardiovascular, dia-15 betes and cancer.22–25 Since phytochemicals like flavo-16 noids, terpenes and other related compounds are thought 17 to be active against ROS generation, exploration of new 18 natural sources of such compounds may be of great inte-19 rest for the scientists. In the sub-continental region, tradi-20 tional medicinal plants have extensively been used for the 21 control and cure of various degenerative diseases. These 22 herbs are either used as dietary supplement or directly as a 23 treatment of several diseases. Cuscuta reflexa and diffe-24 rent parts of Terminalia arjuna have been used as folk me-25 dicine for various diseases. The bark, stem and leaves of 26 T. arjuna have been used for the treatment of various car-27 diovascular diseases in the sub-continental region. In vivo 28 and in vitro studies have shown that T. arjuna is cardio-29 protective,26–29 gastroprotective,30–31 antigenotoxic32 and 30 antimutagenic herb.33–34 It has been reported that tannins, 31 triterpenoid, saponins (arjunic acid, arjunolic acid, arjun-32 genin, arjunglycosides), flavonoids (arjunone, arjunolone, 33 luteolin), gallic acid, ellagic acid, oligomeric proanthoc-34 yanidins (OPCs), phytosterols, calcium, magnesium, zinc, 35 and copper are the major constituents of T. arjuna.35–36 36 Cuscuta reflexa is used as alterative, anthelmintic, 37 carminative and purgative agent by the traditional medici-38 ne practitioners. The plant is employed in treatment of uri-39 nating problems, jaundice, muscle pain and chronic 40 coughs. Antibacterial, antiviral, antisteroidogenic and 41 psychopharmacological activity of C. reflexa have also 42 been reported.37–38 43 Until now, no comprehensive study has been under-44 taken for evaluation of in vitro antioxidant potential of T. 45 arjuna and C. reflexa. Efforts have been undertaken in the 46 present study to evaluate antioxidant activity of these 47 herbs though an improved ABTS assay. The antioxidant 48 activity observed through our study was compared to 49 those determined by other contemporary assays, in order 50 to correlate it with their anti-disease activity. 51 52 53 2. Materials and Methods 54 55 All the chemicals used were of the highest purity 56 grade available. Trolox (Hoffman-La Roche) (6-hydroxy- 8, 55, 408–418 409 2,5,7,8-tetramethylchroman-2-carboxylic acid; Aldrich 1 Chemical Co., Gillingham, Dorset, UK) was used as an 2 antioxidant standard and its solution was prepared in 3 phosphate buffered saline at pH 7.4. Fresh working stan- 4 dards were prepared daily on dilution with either metha- 5 nol or double distilled water. ABTS (2,2’-azinobis(3- 6 ethylbenzothiazoline-6-sulfonic acid)) diammonium salt, 7 potassium persulfate (dipotassium peroxodisulfate, 8 K2S2O8), ascorbic acid, reduced glutathione, BHT (buty- 9 lated hydroxytoluene; 2,6-di-tert-butyl-4-methylphenol), 10 BHA (butylated hydroxyaniline; 2-tert-butyl-4-met- 11 hoxyphenol), gallic acid (3,4,5-trihydroxybenzoic acid), 12 uric acid (2,6,8-trihydroxypurine), 2,2’-methylenebis(6- 13 tert-butyl-4-methylphenol) and kaempferol (3,5,7-trihy- 14 droxy-2-(4-hydroxyphenyl)-4H-chromen-4-one) were ob- 15 tained from Fluka (UK) and HPLC grade ethanol from 16 Rathburn Chemicals Ltd. (Walkerburn, Peebleshire, Scot- 17 land). Experiments were performed on UV-1700 Pharma- 18 Spec UV-Visible Spectrophotometer, Shimadzu, Japan 19 equipped with CPS controller. All the experiments were 20 performed three times and results obtained were averaged. 21 Data obtained, except where specifically mentioned, is 22 mean ±SD (n = 3). 23 24 2. 1. Sample Preparations 25 26 Solutions of standard antioxidants were prepared in 27 either methanol or double distilled water, which depended 28 upon the solubility of the antioxidant. 29 The blood samples were taken from five apparently 30 healthy volunteer men (average age 30 years), with infor- 31 med consent. For plasma, the blood samples, pre-mixed 32 with sodium citrate in clinical concentrations, were cen- 33 trifuged at 5000 rpm and 4 °C for 30 min. Plasma was se- 34 parated and stored in refrigerator at 4 °C. All samples we- 35 re analyzed within three days. To check and compare re- 36 producibility of the modified method with the older 37 ABTS versions, % RSD and mean % RSD values were 38 calculated. Stem (TAS), bark (TAB) and leaves (TAL) 39 samples of T. arjuna and C. reflexa (CR) were obtained in 40 dried form from a local market in Lahore, Pakistan. A fi- 41 nely ground amount (TAS 50 g, TAB 35 g, TAL 10 g and 42 CR 50 g) of these medicinal herbs was soaked in metha- 43 nol in 1:20 (w/v) in cork-fitted flasks separately for 24 h 44 at 30 °C and 240 rpm. The following day, extract obtai- 45 ned was filtered and stored at 4 °C, while the residue was 46 re-soaked in methanol in the same proportion at 240 rpm 47 and 30 °C for 24 h. The filtrate obtained on the second 48 day was mixed with the filtrate from the first day. The 49 methanolic filtrate was concentrated at 30 °C using rotary 50 evaporator. The solid concentrate obtained was weighed 51 and suspended in appropriate volume of distilled water 52 (Table 1). Subsequently, fractions of aqueous extract we- 53 re further extracted in equal volumes of organic solvents 54 (25 × 3 mL) of varying polarity according to the scheme 55 shown in figure 1. 56 Asghar et al.: Modified 2,2’-Azinobis(3-Ethylbenzo Thiazoline)-6-Sulphonic Acid Radical ... 410 Acta Chim. Slov. 2008, 55, 408–418 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 Figure 1. Flow sheet diagram showing extraction of antioxidant components of T. arjuna and C. reflexa in different solvents Table 1. Mass of residue obtained and stock solutions formed for different fractions of T. arjuna and C. reflexa Plant/herb Sample Fraction TAS Stock TAL Stock TAB Stock CR Stock (g) Solution (mg/ml) (g) Solution (mg/ml) (g) Solution (mg/ml) (g) Solution (mg/ml) Methanol 0.100 20.00 0.038 7.74 0.055 11.18 0.100 20.00 n-Hexane 0.190 19.00 0.050 3.33 0.030 3.00 0.580 58.00 Chloroform 0.110 11.00 0.250 25.00 0.940 94.00 1.420 94.66 EtOAc 0.100 9.09 0.030 3.00 0.190 7.60 0.950 95.00 1-Butanol 1.170 39.00 0.810 67.50 0.280 23.33 1.670 167.00 Aq. (AP) 4.980 14.20 1.040 2.45 5.430 27.15 2.840 10.88 Aq. (BP) 8.080 23.00 1.871 4.40 8.614 43.07 10.881 26.26 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 Asghar et al.: Modified 2,2’-Azinobis(3-Ethylbenzo Thiazoline)-6-Sulphonic Acid Radical ... Acta Chim. Slov. 2008, 55, 408–418 411 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 The residue obtained at each extraction step was weighed and dissolved in appropriate volume of respective solvent to obtain stock solution to be used in antioxi-dant assays (Table 1). 2. 2. ABTS•+ Decolorization Assay Protocol ABTS•+ (ABTS radical cation) assay protocol, as developed by Re et al.,20 was followed for the analysis described in this article. ABTS was dissolved in double distilled water to a 7 mM concentration. ABTS+ was produced by reacting ABTS stock solution with 2.45 mM potassium persulfate (final concentration) and allowing the mixture to stand, in the dark, at room temperature for 12–16 h before use. For study of antioxidant activity of standard antioxidant and plasma samples, the ABTS stock solution was diluted with PBS buffer (pH 7.4) to an absor-bance of 0.70 (±0.02) at 745 nm respectively, and equilibrated at 30 °C. For herbal extracts, dilutions were made in the respective organic solvents with which they had been extracted from aqueous solutions. After addition of 10 µL of neat or diluted stock solution (as necessary) to 2.99 mL of diluted ABTS+ solution (A = 0.700 ± 0.020), the absorbance was measured at 30 °C, within exactly 1 min intervals for 8 min. Solvent blanks were run in each assay for accurate readings. All determinations were carried out at least three times in succession and in triplicate at each separate concentration level of the standards. The percentage inhibition of absorbance at 734 nm (I734) was calculated by the following formula. (1) Where Ao is the absorbance of radical cation solution before addition of sample/standard antioxidants and Af is the absorbance after addition of the sample/standard antioxidants. The resultant data was plotted between concentration of antioxidants and that of trolox for the standard reference curve. 2. 3. Modified ABTS•+ Decolorization Assay Protocol ABTS was dissolved in double distilled water to a 7 mM concentration. ABTS•+ was produced by reacting ABTS stock solution (in 20 mM sodium acetate buffer, pH 6.5) with 2.45 mM potassium persulfate (final concentration) and allowing the mixture to stand in the dark at room temperature for 12–16 h before use. For the study of antioxidant activity of standard antioxidants and plasma samples, the ABTS stock solution was diluted with 20 mM sodium acetate buffer (pH 6.5) to absorption value of 0.70 ± 0.02 at 734 nm and equilibrated at 30 °C. After addition of 10 µL of neat or diluted stock solution to 2.99 mL of diluted ABTS•+ solution (A734nm = 0.700 ± 0.020), the absorbance reading was taken after intervals of exact- ly 1 min, for 8 min. Appropriate solvent blanks were run in each assay for accurate readings. In case of plasma samples, the radical cation solution, after mixing with plasma samples were incubated at room temperature for 30 min. All determinations were carried out at least three times, at each separate concentration of the standards and subsequent samples. The percentage inhibition of absor-bance at 734 nm (I734) was calculated as follows. (2) Where Ao is the absorbance of radical cation solution before addition of sample/standard antioxidants while Af is the absorbance after addition of the sample/standard antioxidants. 2. 4. 2,2’-Diphenyl-1-picrylhydrazyl Radical Scavenging Capacity Assay (DPPH Assay) DPPH solution (3 mL, 25 mg/L) in methanol was mixed with appropriate volumes of neat or diluted sample solutions. The reaction progress of the mixture was monitored at 515 nm over a time period until TEC50 was obtained. Upon appropriate reduction, the color of the solution faded. The percentage of the DPPH remaining was calculated as (3) Where [DPPH]T=0 is the concentration of DPPH radical before reaction with antioxidant samples and [DPPH]T=t is the concentration of DPPH radical after reaction with antioxidant sample at time t. A kinetic curve showing the scavenging of DPPH radical in terms of decrease in absorbance at 593 nm as a function of time (min) was plotted for each fraction of the samples. The concentration that causes a decrease in the initial DPPH concentration by 50% (EC50), and the time needed to reach the steady state with EC50 concentration (TEC50) were measured from the equation 3. 2. 5. Total Phenolic Content Assay Stock solution of gallic acid was made by dissolving 0.5 g gallic acid in 10 mL of ethanol in a 100 mL volumetric flask and diluting to volume with doubly distilled water. Sodium carbonate solution was prepared by dissolving 200 g of anhydrous sodium carbonate in 800 mL of double distilled water. After boiling and subsequent cooling of the solution, a few crystals of sodium carbonate were added. The solution was let to stand for 24 h, filtered and volume was increased to 1 L with double distilled wa- 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 Asghar et al.: Modified 2,2’-Azinobis(3-Ethylbenzo Thiazoline)-6-Sulphonic Acid Radical ... 412 Acta Chim. Slov. 2008, 55, 408–418 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 ter. To prepare a calibration curve, 0, 1, 2, 3, 5 and 10 mL of phenol stock solution were added into 100 mL volumetric flask separately and then diluted to volume with double distilled water. The resultant solutions contained concentrations of 0, 50, 100, 150, 250 and 500 mg/L gallic acid. From each calibration solution and sample or blank, 40 µL were pipetted into separate cuvettes, and to each 3.16 mL of double distilled water was added. Folin–Cio-calteu reagent (200 µL) was added and mixed well. After 8 min, 600 µL of sodium carbonate solution was mixed thoroughly in the solution. The solution was allowed to stand at 40 °C for 30 min and absorbance of each solution was determined at 765 nm against the blank (without phenolic solution). A concentration versus absorbance linear plot was thus obtained. The concentration of total phenolic compounds of each fraction of TAS, TAL, TAB and CR (as milligram of gallic acid equivalent (GAE)), as determined by using the following equation was obtained from the standard gallic acid plot. Absorbance = 0.0557 × [gallic acid (mg/L)] 2. 6. Total Antioxidant Activity Total antioxidant activity of aqueous and organic extracts of both plants was determined according to the method employed by Mitsuda et al.39 The solution, which contained 100 L each of neat or diluted plant extract of both plants in 2.5 mL of potassium phosphate buffer (0.04 M, pH 7.0) was added to 2.5 mL of linoleic acid emulsion in potassium phosphate buffer (0.04 M, pH 7.0). Each solution was then incubated at 37 °C in sealed bottles in dark. The solution without added extract was used as a blank, while the solutions containing 100 µL (50 µg/20 µL) of trolox were used as positive control. At intervals of 24 h during incubation, 0.1 mL of each solution was transferred to a beaker containing 3.7 mL of ethanol. After addition of 0.1 mL each of FeCl2 (20 mM in 3.5% HCl) and thiocyanate solution (30%) to ethanolic sample, the solution was stirred for 1 min. The absorbance values of the solutions measured at 500 nm were taken as lipid peroxidation values. 3. Results and Discussion 3. 1. Modification of ABTS•+ Decolorization Assay The basic principle underlying the ABTS•+ decolori-zation assay is that ABTS•+, on reaction with K2S2O8, forms a greenish blue radical cation. Standard and sample antioxidants that are able to transfer an electron to ABTS radical cation scavenge the color of the solution proportionate to their amount. The extent of scavenging depends both upon the concentration of antioxidant and time dura- tion for the reaction under analysis. Standard ABTS assay (pH 7.4) used 734 nm as the absorption maximum. First derivative UV-Visible spectrum of the ABTS radical cation in sodium acetate buffer (20 mM, pH 6.5) is shown in Figure 2. It is quite clear from the figure that the change in pH of the medium did not disturb the position of absorption maximum as indicated by zero crossing at 734 nm. Figure 2. First derivative spectrum of ABTS radical cation in sodium acetate buffer (20 mM, pH 6.5) ABTS decolorization assay has most widely been used at pH 7.4 (PBS, 5 mM). The effectiveness of the assay at pH 7.4 has been questioned due to the vulnerability of ABTS radical cation to degradation at higher pH va-lues.18–19 Ozgen et al.21 has recommended that the antioxi-dant activity of the fruits, which occur naturally at low pH, should have been evaluated by ABTS radical cation stabilized at pH 4.5. Using pH 4.5, stability of ABTS radical cation was appreciably improved. However, its sensitivity to biological samples was lost. The need was, therefore, felt to modify the assay for determination of antioxi-dant activity of both biological and abiological samples at a single pH with greater accuracy. The stability and sensitivity of ABTS radical cation was checked at different pH values using standard antioxi-dants, herbal and plasma samples. Figure 3 shows stability of ABTS radical cation at different pH. Figure 3. Absorbance over time of ABTS radical cation standardized to an initial A734 of 0.7 at pH 4.5, 6.5 and 7.4 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 Asghar et al.: Modified 2,2’-Azinobis(3-Ethylbenzo Thiazoline)-6-Sulphonic Acid Radical ... Acta Chim. Slov. 2008, 55, 408–418 413 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 It is apparent from the figures that increase in the pH of radical cation solution resulted in decrease of stability of the cation, with the lowest stability being observed at pH 7.4. Although maximum stability of ABTS radical cation was observed at pH 4.5, the sensitivity of assay with regards to plasma samples was greatly affected. The A B C D E I—?— pH65-«— pH7.4--------Linear(pH6.5) — Linear (pH7.4) 100 , 60 y = 12.26* + 15.66 R= = 0.9921 *0^^ š 60-1 40- l^^5*^ y = 15.01k + 5.68 20 - **^ f¥ = 0.998 o\ 3 6 9 12 1S Concentration (micromolar) —?— pH6.S -¦— pH7.4--------Linear (pH6.5) Linear (pH7.4) 80 o 60 -iS 40 $ 20 y = 10.111ï+5.735 ^^JÇ_ R* - 0.9871 ^J^'"ÌS^***"*^ fe^5**^ y = 1137x + 0.97 0 R? = 0.9952 3 6 9 12 15 Co n co nt ration ( m Icro m olar) G H Figure 4. Effect of concentration of antioxidants on the inhibition of the ABTS+ in sodium acetate buffer (20 mM, pH 6.5) and PBS (5 mM, pH 7.4) (A) ascorbic acid, (B) BHT, (C) trolox, (D) reduced glutathione, (E) uric acid, (F) BHA, (G) gallic acid, (H) kaempfe-rol, (I) 2,2’-methylenebis(6-tert-butyl-4-methylphenol) 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 F I Asghar et al.: Modified 2,2’-Azinobis(3-Ethylbenzo Thiazoline)-6-Sulphonic Acid Radical ... 414 Acta Chim. Slov. 2008, 55, 408–418 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 maximum stability and sensitivity of ABTS radical cation, with respect to both plant and plasma samples, were observed at pH 6.5 (20 mM sodium acetate buffer). 3. 2. Trolox Equivalent Antioxidant Capacity (TEAC) Assay The method was used to measure the antioxidant activity of both lipophilic and hydrophilic standard antioxi-dants and plasma samples by measuring the extent of scavenging of ABTS radical cation at 734 nm on addition of the sample solution for 8 min for standard antioxidants and plant extracts, and 30 min for plasma samples. Dose-response curves were obtained by plotting the percent inhibition of the original absorbance value (fixed at 0.7 ± 0.02 absorbance units) for a range of concentrations of different standard antioxidant compounds. The kinetics of reaction showed that the reaction with standard antioxi-dant was completed in 0.5–2 min range, by reaching a stable end-point. TEAC values of the sample solutions were obtained by comparing the percent inhibition with standard curve of trolox. A comparison of the dose-response curves of different standard antioxidants by the modified and the old version of ABTS•+ decolorization assays is shown in Figure 4. The values of coefficient of determination (R2) obtained for both assays demonstrate that there was a high degree of correlation between the concentrations of a given antio-xidant compound and % inhibition of the radical cation. A general trend of relative decrease in the antioxidant activity (in terms of % inhibition of the radical cation) of the standard phenols using the modified assay may be attributed to the greater stability of the radical at pH 6.5 value. This also, in turn, minimized the effect of pH, which was reported to cause about 5–20% increase in the observed antioxidant value of standard phenols due to instability of ABTS radical cation at higher pH values (pH 7.4).21 The TEAC assay which was originally described by Miller et al.1 and later modified by Re et al.20 is based upon scavenging of ABTS radical cation on addition of sample antioxidants. The extent of scavenging of radical anion, which is detected spectrophotometrically at 734 nm, can therefore be related to the amount of antioxidants pre- sent in the sample. TEAC value can be determined for all the compounds/samples, which are able to scavenge ABTS•+ radical cation by comparing their scavenging activity with that of trolox, a water-soluble analogue of vitamin E. The modified assay was found to be highly reproducible. TEAC values for plasma sample ranged between 1.410–2.025 mM and 1.72–1.98 mM for older version of ABTS assay and the modified ABTS assay, respectively (Table 2). The mean % RSD was found to be 1.64 and 1.27 for older and modified versions of ABTS•+ assay, respectively. TEAC values for different fractions of C. reflexa and stem, bark and leaves of T. arjuna were also determined using both assays. Comparative analyses of TEAC values of different fractions of the two plants employing two assays are shown in Figure 5. TEAC values obtained by using the modified ABTS•+ assay were comparable with those found by the older version of ABTS assays. A general trend of slightly decreased antioxidant activity with the modified assay may be attributed to the increased stability of ABTS radical cation at pH 6.5. The results showed greater precision with the modified assay showing its superiority over the older versions. 3. 3. Total Phenolic Content (TPC) Assay Phenolic compounds have been reported to be very powerful antioxidants due to the presence of hydroxy groups in their structures.40 For TPC, a previously reported assay was adopted.41 Amongst the three parts of T. ar-juna, high phenolic contents were found in butanol, methanol, aq. (AP) and aq. (BP) fractions of TAB and bu-tanol, methanol, aq. (AP) and ethyl acetate fractions of TAS. Chloroform, ethyl acetate and butanol fractions of CR also showed high content of phenolic compounds (Figure 6). High TPC values may be attributed to the previously reported tannins, triterpenoid, saponins, flavonoids, gallic acid, ellagic acid, OPCs, and phytosterols that are found in T. arjuna.35–36 These results are also depictive of a correlation between the polyphenolic content and the antioxidant activity of the respective fraction, which is in agreement with the already reported results regarding other plants.42–43 Table 2. TEAC values (mM) of human plasma using old version of ABTS assay and the modified ABTS assay TEAC using Old TEAC using version of Modified Sr. Plasma ABTS/K S O %RSD Mean ABTS/K S O %RSD Mean No. Assay (pH 7.4) (mM)* %RSD Assay (pH 6.5) (mM)* %RSD 1. Subject 1 2.025 1.72 1.901 1.41 2. Subject 2 1.740 1.54 1.902 1.24 3. Subject 3 1.499 1.21 1.64 1.750 1.25 1.27 4. Subject 4 1.990 1.81 1.980 1.12 5. Subject 5 1.410 1.91 1.720 1.35 * The data shown is mean ±SD (n = 5) 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 Asghar et al.: Modified 2,2’-Azinobis(3-Ethylbenzo Thiazoline)-6-Sulphonic Acid Radical ... Acta Chim. Slov. 2008, 55, 408–418 415 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 A C D B Figure 5: A comparative analysis of TEAC values (mM) of different fractions of (A) TAS (B) TAL (C) TAB and (D) CR, using ABTS assay (pH 7.4) and the modified assay. n-hexane, chloroform (TAS & TAL) and n-hexane and methanol (CR) fractions were used neat; chloroform, aq. (BP) (CR), EtOAc, 1-butanol, methanol, aq. (BP), aq. (AP) (TAL) and EtOAc, aq. (BP) (TAS) fractions were 10 times diluted while all the other fractions were 20 times diluted for both the ABTS assay (pH 7.4) and the modified ABTS assay. In the case of TAB n-butanol, methanol, aq. (AP), aq. (BP) fractions were 20 times diluted; EtOAc was 10 times diluted and n-hexane fractions were used neat for both assays. Chloroform fraction of TAB was used as neat for the modified assay while it was 10 times diluted for ABTS assay (pH 7.4). Figure 6. Comparison of TPC analysis of different fractions of stem, leaves and bark of T. arjuna and C. Reflexa (butanol fraction of TAS, butanol, aq. (BP), aq. (AP) and methanol fractions of TAB; EtOAc and butanol of CR; and chloroform fraction of CR are 100, 20 and 10 times diluted, respectively) 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 Asghar et al.: Modified 2,2’-Azinobis(3-Ethylbenzo Thiazoline)-6-Sulphonic Acid Radical ... 416 Acta Chim. Slov. 2008, 55, 408–418 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 Table 3. EC50 and TEC50 values of different fractions of C. reflexa and stem, bark and leaves of T. arjuna Plant/herb Sample TA S TAB TAL CR Fraction EC50 µg of dried mass/mL of DPPH EC50 (min) EC50 µg of dried mass/mL of DPPH EC50 (min) EC50 µg of dried mass/mL of DPPH EC50 (min) EC50 µg of dried mass/mL of DPPH EC50 (min) Methanol 5.5 3 3.1 5 8.6 40 665.9 12 n-Hexane Nil* Nil* 416.6 46 18.156 11 966.6 75 Chloroform 61.1 20 1044.4 38 416.6 20 1577.5 3 EtOAc 2.5 20 12.7 25 50.0 65 158.3 4 1-Butanol 3.6 55 8.6 42 11.25 45 278.3 8 Aq. (AP) 5.9 10 6.7 34 2.0 75 362.7 30 Aq. (BP) 19.7 45 18.4 15 7.3 37 739.4 12 * n-hexane fraction of TAS did not show any radical scavenging activity 3. 4. DPPH Free Radical Scavenging Activity DPPH is a commercially available stable free radical, which has been widely used for estimating scavenging activity of pure antioxidants and extracts of herbs.44–45 Violet-colored DPPH radical, after accepting an electron or hydrogen atom from the antioxidant compounds, is converted into a colorless or somewhat yellow diamagnetic DPPH molecule.46 The free radical scavenging activity of aqueous and organic extracts of TAS, TAB, TAL and CR were investigated. A plot between % DPPH remaining and the time after addition of neat or diluted stock solution of TAB is shown in Figure 7. The graph shows a sharp fall in the absorbance of DPPH in the first 5 min after addition of extract from bark of T. arjuna, and then becomes more moderate and gradual for the next 35 min. This behavior indicated the presence of both slow-reacting and fast-reacting antioxidants in the samples. The radical scavenging activity was found to be in the decreasing order of methanol > 1-butanol > aq. (AP) > EtOAc > aq. (BP) > n-hexane > chloroform for TAB. Kinetic graphs for the rest of the fractions of T. arjuna and C. reflexa were obtained and EC50 and TEC50 values for each extract were determined (Table 3). The data showed methanol, 1-butanol, EtOAc, aq. (AP) and aq. (BP) as the Figure 7. DPPH free radical scavenging activity of bark of T. arju-na. Data are mean ±SD (n = 3) best extracting solvents for all three parts of T. arjuna. The radical scavenging activity of C. reflexa was found to be comparatively low. Ethyl acetate, 1-butanol and aq. (AP) showed relatively good radical scavenging activity. 3. 5. Total Antioxidant Capacity Assay Thiocyanate method was employed to determine total antioxidant activity of the extracts of C. reflexa and T. arjuna. During linoleic acid oxidation, peroxides are formed with the ability to oxidize Fe2+ to Fe3+. Iron(III) ion on reaction with thiocyanate ions (SCN–) forms a complex which can be measured spectrophotometrically at 500 nm. The added standard or sample solutions compete to inhibit or slow down oxidation of linoleic acid and thus formation of peroxide values. The low peroxidation values indicate high antioxidant power of the sample and vice versa. Antioxidant activity of aqueous and organic fractions of both plants was determined as a function of time, with standard antioxidant trolox being utilized as a positive control. The results showed that all the fractions of both plants have strong antioxidant activity (Figure 8). The an-tioxidant components of the samples were potent to the extent that even after incubation of 192 h, there was observed a slight increase in the peroxidation value. 4. Acknowledgement We acknowledge financial support of Higher Education Commission of Pakistan, Islamabad for the research work under Research Scholar Grant No. 042-121235-Ps2-286. 5. 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S sedanjo raziskavo smo `eleli razviti izbolj{ano ABTS metodo z razbarvanjem za meritev antioksidativne aktivnosti ~lov{ke plazme in rastlinskih ekstraktov, ki bi bila manj ob~utljiva na pH in hkrati dovolj ob~utljiva. Z reakcijo med ABTS in kalijevim persulfatom v prisotnosti natrijevega acetatnega pufra pri pH 6.5 (20 mM) je nastal relativno stabilen ABTS radikal kation. Prehod elektrona ali vodikovega atoma z antioksidativne komponente v plazmi ali rastlinskem ekstraktu reducira radikal kation. Obi~ajni antioksidanti, kot so troloks, butil hidroksianizol (BHA), butil hidroksitoluen (BHT) in as-korbinska kislina ka`ejo stabilne kon~ne vrednosti pri dolo~anju celotne antioksidativne aktivnosti (TTA) za vse vzorce glede na referen~ne indikatorje. Modificirana metoda se je izkazala za visoko ponovljivo. TEAC vrednosti za vzorce plazme so bile med 1.410–2.025 mM s srednjim odstotnim relativnim standardnim odklonom (% RSD) 1.64 za prvotno ABTS metodo in 1.72–1.98 s srednjim % RSD 1.27 za modificirano ABTS metodo. U~inkovitost metode smo dodatno dokazali z dolo~itvijo antioksidativne aktivnosti organskih in vodnih ekstraktov Cuscuta reflexa in Terminalia arjuna. 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 Asghar et al.: Modified 2,2’-Azinobis(3-Ethylbenzo Thiazoline)-6-Sulphonic Acid Radical ...