In Vitro Antioxidant Activities in Various Beans Extracts of Five Legumes from West of Java-Indonesia Using DPPH and ABTS Methods

The aim of this research were to determine antioxidant activity from different polarities beans extracts of five legumes beans using two methods of antioxidant testing which were DPPH (2,2-diphenyl-1-picrylhydrazyl) and ABTS (2,2’azino-bis (3-ethylbenzthiazoline-6-sulfonic acid) and correlation of total phenolic, flavonoid and carotenoid content in various extracts of five legumes beans with their IC50 of DPPH and IC50 of ABTS antioxidant activities. Extraction was done by reflux using different polarity solvents. The extracts were evaporated using rotary evaporator. Antioxidant activities using DPPH and ABTS assays, determination of total phenolic, flavonoid and carotenoid content were conducted by UV-visible spectrophotometry and its correlation with IC50 of DPPH and IC50 of ABTS scavenging activities were analyzed by Pearson’s method. All of ethanol extracts from five legumes beans were categorized as very strong antioxidant by DPPH and ABTS methods. Phenolic compounds in red kidney bean extracts were the major contributor in IC50 of DPPH and IC50 of ABTS scavenging activities. DPPH and ABTS assays showed linear results in red kidney bean and bogor peanut sample. keywords: Antioxidant, DPPH, ABTS, legumes beans, phenolic, flavonoid, carotenoid INTRODUCTION Negative effect of free radical can be inhibited by antioxidant. Antioxidant is compound that can inhibit oxidation reaction by scavenging free radical. The excessive of free radical related with oxidative stress condition which can cause many diseases. Many plants have active compounds which have antioxidant properties such as flavonoid and phenolic compounds, which have known to multiple biological effects, included antibacterial and antioxidant activity. Previous study expressed that phenolic and flavonoid content could be correlated to their antioxidant activities. Plants including legumes contained phenolic and flavonoid compounds. DPPH (2,2-diphenyl-1-picrylhydrazyl), FRAP (Ferric Reducing Antioxidant Power) and ABTS (2,2’-azino-bis (3-ethylbenzthiazoline-6-sulfonic acid) can be used to predict antioxidant activity of vegetables, fruits and food. Previous researches revealed that DPPH, FRAP, CUPRAC and ABTS methods could be used to determine antioxidant activity in many plants extracts. The previous studies reported that legumes had antioxidant activities by using ABTS, DPPH, and FRAP assays. The aim of this research were to determine antioxidant activities of three different polarities extracts (n-hexane, ethyl acetate and ethanol) of five legumes beans using DPPH and ABTS assays, and correlations of total phenolic, flavonoid and carotenoid content with their antioxidant activities. MATERIALS AND METHODS Materials DPPH (2,2-diphenyl-1-picrylhydrazyl), ABTS (2,2’azino-bis (3-ethylbenzthiazoline-6-sulfonic acid) diammonium salt), gallic acid, quercetin, beta carotene was purchased from Sigma-Aldrich (MO, USA), legumes beans, ethanol. All other reagents were analytical grades. Preparation of sample Legumes beans which were: soybean (Glycine max) namely as sample GM, green bean (Phaseolus radiatus) as PR, peanut (Arachis hypogaea) as AH, red kidney bean (Phaseolus vulgaris) as PV were collected from Cirebon-West of Java and bogor peanut (Vigna subterranea) as VS was collected from Bogor-West of Java. All of samples were thoroughly washed with tap water, wet sortation, cut, dried and grinded into powder. Extraction Three hundred gram of powdered sample was extracted by reflux using different polarities solvents. Extraction using n-hexane was repeated three times. The remaining residue was then extracted three times by using ethyl acetate. Finally, the remaining residue was extracted three times using ethanol. So totally there were fifteen extracts: five of n-hexane extracts (namely GM 1, PR 1, AH 1, PV 1 and VS 1), five of ethyl acetate extracts (GM 2, PR 2, AH 2, PV 2 and VS 2) and five of ethanolic extracts (GM 3, PR 3, AH 3, PV 3 and VS 3). IC50 of DPPH scavenging activity Irda et al. / In Vitro Antioxidant... IJPPR, Volume 8, Issue 3: March 2016 Page 471 Preparation of DPPH solution was performed using Blois’s method with minor modification. Various concentration of each extract were pipetted into DPPH solution 50 μg/ml (volume 1:1) to initiate the reaction for obtaining a calibration curve. The absorbance was measured after 30 minutes incubation at wavelength 515 nm by using spectrophotometer UV-Vis Hewlett Packard 8435. Methanol was used as a blank. DPPH solution 50 μg/ml was used as control. Ascorbic acid was used as standard. Analysis was done in triplicate for standard and each extract. Antioxidant activity of each extract by DPPH method was determined by calculating percentage of antioxidant activity using reduction of DPPH absorbance. IC50 of DPPH scavenging activity of each extract can be calculated using its calibration curve. IC50 of ABTS scavenging activity Preparation of ABTS solution was conducted using Li method with minor modification. ABTS diammonium salt solution 7.6 mM in aquadest and potassium persulfate solution 2.5 mM in aquadest were prepared. Each solution was left in dark room for 12 hours. Both solutions were mixed with 30 minutes incubation, left the mixture in refrigerator for 24 hours, then diluted in ethanol. Various concentration of each extract were pipetted into ABTS solution 50 μg/ml (volume 1:1) to initiate the reaction for obtaining a calibration curve. The absorbance was read at wavelength 734 nm using UV-Vis spectrophotometer Hewlett Packard 8435. Ethanol (95%) was used as a blank, ABTS solution 50 μg/ml as control and ascorbic acid as standard. Analysis was done in triplicate for standard and each extract. Antioxidant capacity of each extract by ABTS method was determined by calculating percentage of antioxidant activity using reduction of ABTS absorbance. IC50 of ABTS scavenging activity of each extract can be calculated using its calibration curve. Total phenolic content (TPC) Total phenolic content was conducted by using the modified Folin-Ciolcalteu. The absorbance was read at wavelength 765 nm. Analysis was done in triplicate for each extract. Gallic acid standard solution (40-165 g/ml) was used to obtain a calibration curve. Total phenolic content was expressed as percentage of total gallic acid equivalent per 100 g extract (g GAE /100 g). Total flavonoid content (TFC) Total flavonoid content was adapted from Chang et al with minor modification. The absorbance was read at wavelength 415 nm. Analysis was done in triplicate for each extract. Quercetin standard solution (36-120 g/ml) was used to obtain a calibration curve. The total flavonoid content was expressed as percentage of total quercetin equivalent per 100 g extract (g QE/100 g). Total carotenoid content (TCC) Total carotenoid content was measured using modified method which was adapted from Thaipong et al. Each extract was diluted in n-hexane. The absorbance was read at wavelength 470 nm. Analysis was done in triplicate for each extract. Beta carotene standard solution (15-55 g/ml) was used to obtain a calibration curve. The total carotenoid content was expressed as percentage of total beta carotene equivalent per 100 g extract (g BE/100 g). Statistical Analysis Each sample analysis was conducted in triplicate. All results presented are means (± standard deviation) of at least three independent experiments. Statistical analysis using ANOVA with a statistical significance level set at p < 0.05 and post-hoc Tukey procedure was carried out with SPSS 16 for Windows. Correlation between the total phenolic, flavonoid, carotenoid content and antioxidant activities, and correlation between two antioxidant activity methods were performed using the Pearson’s method. RESULTS IC50 of DPPH and IC50 of ABTS scavenging activity The IC50 of DPPH and IC50 of ABTS scavenging activities in different polarities extracts from five legumes beans using DPPH and ABTS assays were shown in Fig 1 and Fig 2. IC50 of DPPH and IC50 of ABTS scavenging activities of each extract were compared to IC50 ascorbic acid as standard. The lowest value of IC50 means had the highest antioxidant activity. Total phenolic content (TPC) TPC among the various extracts were revealed in term of gallic acid equivalent using the standard curve equation y = 0.006x 0.055, R = 0.998. The TPC in fifteen extracts from three different regions of legumes beans exposed different result in the range of 0.19 – 3.04 g GAE/100 g. Ethanolic extract of bogor peanut beans (VS3) revealed that the highest phenolic content (3.04 g GAE/100 g) and the lowest was given by n-hexane extract of soybean (GM1). Total flavonoid content (TFC) TFC among the various extracts were reported in term of quercetin equivalent using the standard curve equation y = 0.006 x 0.0191, R = 0.998. The TFC in fifteen extracts from three different regions of legumes beans showed different result ranged from 0.27 to 8.69 g QE/100 g. Ethyl acetate extract of red kidney beans (PV2) had the highest total flavonoid content (8.69 g QE/100 g). Total carotenoid content (TCC) TCC among the various extracts were expressed in term of beta carotene equivalent using the standard curve equation y = 0.007x 0.027, R = 0.995. The TCC in fifteen extracts from three different regions of legumes beans gave different result in the range of 0.03 – 2.96 g BE/100 g. The highest carotenoid content (2.96 g BE/100 g) was given by ethanol extract of peanut (AH3). Correlations between total phenolic, flavonoid, carotenoid content in various legumes beans extracts and IC50 of DPPH, IC50 of ABTS scavenging activities Pearson’s correlation coefficient between TPC in various extracts of legumes beans and their antioxidant activities exposed that TPC in red kidney bean and bogor peanut extracts (PV and VS) had negative and high significant correlation with IC50 of DPPH scavenging activities (r = 0.888; r = -0.865; p<0.01, respectively) and TPC in green bean, peanut and red kidney bean extracts had negatively Irda et al. / In Vitro Antioxidant... IJPPR, Volume 8, Issue 3: March 2016 Page 472 high correlation with their IC50 of ABTS scavenging activities (r = -0.980, p<0.01; r = -0.721, p<0.05; r = 0.941, p<0.01, respectively). TFC in all of extracts had no significant correlation with their IC50 of DPPH scavenging activities (except peanut and bogor peanut extracts) and IC50 of ABTS scavenging activities, and only TCC in green bean extracts had negative and high correlation with their IC50 of DPPH scavenging activities (r = -0.634, p<0.05). DISCUSSION The previous research reported that legumes had antioxidant capacity. There were no research regarding antioxidant activity of various beans extracts (which were n-hexane, ethyl acetate and ethanol) of five legumes from West JavaIndonesia using DPPH and ABTS assays. ABTS and DPPH free radicals give characteristic absorption at wavelength 734 nm and 516 nm, respectively in ethanol and methanol. Colors of DPPH and ABTS would be changed when the free radicals were scavenged by antioxidant. DPPH would be changed from purple to yellow color, while ABTS changed from turquoise to white color. The IC50 of DPPH scavenging activities and IC50 of ABTS scavenging activities in various legumes beans extracts using DPPH and ABTS assays were shown in Fig 1 and Fig 2. The IC50 of DPPH and IC50 of ABTS scavenging activities in various extracts compared to IC50 of ascorbic acid standard. The lowest value of IC50 means had the highest antioxidant activity. IC50 were used to determine antioxidant activity was compared to standard. Sample which had IC50 lower than 50 g/ml was a very strong antioxidant, 50-100 Table 1: Total phenol, flavonoid, carotenoid content in different polarities extracts from five legumes beans Sample TPC (g GAE/100 g) TFC (g QE/100 g) TCC (g BE/100 g) GM 1 0.19 ± 0.01 0.99 ± 0.07 0.03 ± 0.003 PR 1 1.50 ± 0.01 2.80 ± 0.36 0.03 ± 0.001 AH 1 0.63 ± 0.03 2.16 ± 0.19 0.03 ± 0.002 PV 1 0.33 ± 0.01 2.93 ± 0.20 0.05 ± 0.010 VS 1 0.36 ± 0.02 2.58 ± 0.07 0.06 ± 0.010 GM 2 1.51 ± 0.01 4.56 ± 0.16 0.75 ± 0.010 PR 2 0.76 ± 0.07 4.35 ± 0.25 0.17 ± 0.001 AH 2 1.38 ± 0.07 3.22 ± 0.20 0.09 ± 0.003 PV 2 1.50 ± 0.03 8.32 ± 0.23 0.15 ± 0.010 VS 2 1.08 ± 0.04 8.70 ± 0.27 2.03 ± 0.080 GM 3 0.34 ± 0.01 0.37 ± 0.03 0.53 ± 0.003 PR 3 1.01 ± 0.08 0.66 ± 0.09 0.03 ± 0.001 AH 3 0.80 ± 0.03 0.44 ± 0.03 2.96 ± 0.040 PV 3 0.63 ± 0.01 0.27 ± 0.02 1.19 ± 0.002 VS 3 3.04 ± 0.13 0.49 ± 0.03 0.55 ± 0.010 GM 1 = n-hexane soybean extract, PR 2 = ethyl acetate green bean extract, AH 3 = ethanolic peanut extract, PV = red kidney bean, VS = bogor peanut, TPC = total phenolic content, TFC = total flavonoid content, TCC = total carotenoid content, GAE = gallic acid equivalent, QE = quercetin equivalent, BE = betacaroten equivalent Table 2: Pearson’s correlation coefficient of total phenolic, flavonoid, carotenoid content in various legumes beans extracts with their IC50 of DPPH and IC50 of ABTS scavenging activities Antioxidant activities Coefficient correlation Pearson (r) TPC TFC TCC IC50 ABTS