In vitro free radical scavenging activities of aerial parts’ aqueous extract and extract fractions of Ampelocissus latifolia (Roxb.) Planch. in relation to total phenolics and flavonoid contents

2.4.1 Ferric ion reducing antioxidant power (FRAP) assay

The reductive capability of AAEAL and its fractions were investigated and compared with the standard ascorbic acid by measuring the Fe₃⁺-Fe₂⁺ transformation (Oyaizu, 1986). Phosphate buffer (0.20 M, 1 mL), and K₃Fe(CN)₆ (1%, 1 mL) were added to the sample (0.50 mL) and incubated at 50 °C for 20 min. After that, trichloroacetic acid (10%, 1 mL) was mixed to it and then centrifuged at 3000 rpm for 10 min. 1.50 mL supernatant solution was added to 1.50 mL of distilled water and 0.10 mL of FeCl₃ (0.10%) solution. The absorbance was recorded at 700 nm using a spectrophotometer (UV-1800 Series, Shimadzu, Japan).

2.4.2 DPPH free radical scavenging assay

The antioxidant capacity of AAEAL and its extract fractions were determined by DPPH free radical scavenging assay (Ayoola et al., 2008) and were compared with the standard ascorbic acid. Methanol (3 mL) and DPPH solution (0.002%, 1 mM, 0.50 mL) were mixed with 1 mL of different concentrations (5, 10, 15, 20, 25, 50 and 100 μg/mL) of the sample solution. The whole reaction mixture was kept in dark for 30 min and after that, the absorbance was measured at 517 nm using a spectrophotometer (UV-1800 Series, Shimadzu, Japan).

The decrease of the absorbance indicates an increase in the radical-scavenging activity. The IC₅₀ values (concentration at which 50% scavenging occurs) were calculated from the graph between the inhibition (%) versus the concentrations of the samples. The DPPH free radical-scavenging activity was calculated as $$\%\text{Inhibition}=\left[\left(\left(A_0-A_1\right)/A_0\right)\right]\times 100$$ [A0 = absorbance of the control (without extract), A1 = absorbance in the presence of the extract].

2.4.3 Total antioxidant capacity assay

The phosphomolybdate assay is a widely accepted method for the determination of the total antioxidant capacity of plant extracts. The basic principle is that, here, Mo (VI) is reduced to Mo (V) by the sample analyte and a green colored phosphate Mo (V) complex forms at the acidic range of pH (Umamaheswari and Chatterjee, 2008). The total antioxidant potentials of the different extract fractions of AAEAL were measured and compared with standard ascorbic acid. The sample solution (100 μg/mL) was mixed with 3 times volume of the reagent mixture containing sulfuric acid (0.60 M), ammonium molybdate (4 mM), and sodium phosphate (28 mM) and incubated at 95 °C for 90 min. The absorbance was recorded at 695 nm using a spectrophotometer (UV-1800 Series, Shimadzu, Japan) and the data are represented in terms of µg ascorbic acid equivalents/100 µg dry extract matter.

2.5.1 Total phenolic content (TPC)

The total phenolic content of AAEAL and its extract fractions were estimated with Folin-Ciocalteu’s phenol reagent (FCP) (Makkar et al., 1993; Ray et al., 2013). 10 μL of sample solution of the extract/extract fractions was added to 990 µL distilled water and 0.50 mL FCP reagent (1 N) and were mixed thoroughly. 2.50 mL 20% Na₂CO₃ solution was added, mixed properly and kept for 40 min at room temperature (25 ± 2 °C). The absorbance was recorded at 725 nm using a spectrophotometer (UV-1800 Series, Shimadzu, Japan). The TPC was estimated as tannic acid equivalent (TAE) using the tannic acid (TA) standard curve (2.50–25 μg/mL). Data are represented as Mean ± SEM of nine sets of experiments.

2.5.2 Total flavonoid content (TFC)

The total flavonoid content of the extract/extract fractions was estimated by AlCl₃ method (Chang et al., 2002; Chaudhuri and Ray, 2015a). Distilled water (1 mL) and NaNO₂ (5%, 1.50 mL) were added to the sample solution (0.50 mL) and allowed to settle for 5 min. Later on, AlCl₃ (10%, 0.15 mL) was added followed by the addition of NaOH solution (1 M, 1 mL). Finally, distilled water (0.85 mL) was added. The absorbance reading was taken at 510 nm using a spectrophotometer (UV-1800 Series, Shimadzu, Japan). Here, flavonoid content was estimated as quercetin equivalent (QE) using a standard quercetin calibration curve. The data are represented as Mean ± SEM of nine sets of experiments.

4.1.1 FRAP assay

The data indicate a concentration-dependent (10–100 µg/mL) increase in the FRAP activities of the AAEAL and its extract fractions as shown by their increasing OD values (Table SI). The half maximum effective reducing power concentrations (EC₅₀) for ascorbic acid (84.86 ± 7.22 μg/mL) was found to be lower than that for AAEAL (193.06 ± 9.51 μg/mL) which is a crude extract having a combination of phytochemicals, some with oxidative or some with antioxidant actions (Chaudhuri and Ray, 2014; Dutta and Ray, 2015). The AAEAL was further fractionated sequentially with petroleum ether (AAEALPE), chloroform (AAEALCH), ethyl acetate (AAEALEA), and n-butanol (AAEALNB) to isolate the bioactive fractions (Li et al., 2009). The fractions were checked for FRAP activities and their EC₅₀ values were compared (Table SI, Fig. 1). The EC₅₀ values for AAEALPE, AAEALCH, AAEALEA, AAEALNB, and AAEAL remaining fraction were calculated as 367.02 ± 21.15, 259.67 ± 13.35, 90.8 ± 4.70, 104.80 ± 5.29, and 139.17 ± 6.99 μg/mL respectively.

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Fig. 1

EC₅₀ values (μg/mL) of FRAP activity of ascorbic acid, AAEAL and its fractions. Assays were done in triplicate and the data are expressed as Mean ± SEM. The level of confidence was considered at 95% (p_α = 0.05) (ANOVA) for the determination of differences among the groups, followed by Post Hoc Tukey test [*p < 0.05].

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There was no significant difference in the antioxidant activities among the AAEALEA, AAEALNB, and the remaining fraction. The antioxidant activity of AAEALEA is significantly higher than the AAEAL, AAEALPE, and AAEALCH fractions, but no significant difference was present between the antioxidant activity of ascorbic acid and AAEALEA, indicating the AAEALEA is a potential fraction having ascorbic acid equivalent antioxidant activity (Fig. 1).

4.1.2 DPPH assay

The DPPH assay is used for the determination of the free radical scavenging activity of any agents. In the presence of an antioxidant compound, DPPH, a stable free radical, is reduced and it changes color from purple to yellow due to the formation of 1,1-diphenyl-2-picryl hydrazine from the hydrazyl crystal. The extent of discoloration indicates the free radical scavenging potentials. Results indicate a concentration-dependent increase in DPPH free radical scavenging activity of the extract/extract fractions. Here, the concentration of AAEAL was required nearly 2.40 times (Fig. 2) higher than that of the ascorbic acid (Ascorbic acid, IC₅₀: 18.35 ± 1.15 µg/mL; AAEAL, IC₅₀: 44.85 ± 4.95 µg/mL), which may be due to AAEAL being in a crude form. There are similar reports showing the antioxidant activity of various crude extracts (Ayoola et al., 2008; Deng et al., 2012; Dutta and Ray, 2015, 2020; Fu et al., 2011.).

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Fig. 2

IC₅₀ values (μg/mL) of DPPH free radical scavenging activity of ascorbic acid, AAEAL, and different fractions of AAEAL. Assays were done in triplicate and the data are expressed as Mean ± SEM. The level of confidence was considered at 95% (p_α = 0.05) (ANOVA) for the determination of differences among the groups followed by Post Hoc Tukey test [*p < 0.05].

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Comparison of the DPPH scavenging activities of fractionated extracts revealed that some of the fractions possess higher antioxidant activity than the AAEAL itself in the decreasing order of AAEALEA (IC₅₀ = 19.50 ± 0.50 µg/mL) > AAEALNB (IC₅₀ = 34.95 ± 1.05 µg/mL) > remaining fraction of AAEAL (IC₅₀ = 36.15 ± 2.85 µg/mL) (Fig. 2). The AAEALCH and AAEALPE possess a lower free radical scavenging activity than the AAEAL (Figure S1), indicating a differential free radical scavenging activity of the different extract fractions of AAEAL. Here, AAEALEA showed the maximum free radical scavenging activity. The one way analysis of variance (ANOVA) at the confidence level of 95% (p_α = 0.05), followed by the Post Hoc Tukey test [*p < 0.05] was done to test whether any significant differences were present among the free radical scavenging activities of the different extract fractions (Fig. 2). The plant crude extract contains many types of phytochemicals, while each fraction obtained by the fractionation method yields only a few types of molecules and this is mainly due to the solvent polarity and affinity of the compounds for specific solvents (Umamaheswari and Chatterjee, 2008). Here, the IC₅₀ value of AAEALEA (19.5 ± 0.50 µg/mL) was found to be more than two times lower than that of the crude AAEAL (44.85 ± 4.95 µg/mL), indicating that the effective antioxidants were extracted in ethyl acetate fraction of AAEAL. Like FRAP assay, the data of DPPH assay also indicate that AAEALEA is the best depository of phytochemicals having antioxidant activity. A similar report also exists showing that fractionation of hydromethanolic extract of Coccinia grandis resulted in the differential antioxidant potential of the different extract fractions (Umamaheswari and Chatterjee, 2008).

4.1.3 Total antioxidant capacity assay

Here, the total antioxidant capacity was analyzed through the phosphomolybdate assay, which is a widely used method for the determination of the total antioxidant potential of plant extracts (Umamaheswari and Chatterjee, 2008). Data indicate a decreasing order of total antioxidant capacity of the extract fractions as AAEALEA > AAEALNB > AAEAL remaining fraction > AAEALCH > AAEALPE. The AAEALEA showed a significantly higher (*p < 0.05, analyzed by one way ANOVA followed by Post Hoc Tukey test) antioxidant capacity (37.85 ± 1.55 µg ascorbic acid equivalents/100 µg dry extract) than the other extract fractions (Fig. 3). Previously Umamaheswari and Chatterjee (2008) reported the differential total antioxidant activity of the petroleum ether, chloroform, ethyl acetate, and remaining extract fractions of the methanolic extract of Coccinia grandis. These differences in the total antioxidant property of the extract fractions may be due to the variation in chemical nature and the phenolics content.

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Fig. 3

The total antioxidant activity (phosphomolybdate assay) of the successive organic solvent extracts of AAEAL. Assays were performed in triplicate and the data are expressed as Mean ± SEM. The level of confidence was considered at 95% (p_α = 0.05) by ANOVA for the determination of differences among the groups. The Post hoc Tukey test shows a significant difference (*p < 0.05) between the two fractions.

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