Comparative in vitro Antidiabetic and Antioxidant Activity of Various Extracts of Ficus Species

Introduction: Ficus religiosa, Ficus benghalensis and Ficus glomerata are plants from Ficus species used traditionally for the treatment of various ailments. This study aimed to investigate in vitro antidiabetic and antioxidant activity of three plants from Ficus species and effect of extracting solvents, total flavonoids and phenolics content on its in vitro activity. Methods: Dried leaf powder was extracted successively by using solvents with increasing order of polarity index (PI). In vitro antioxidant (RP: reducing power assay, DPPH: 2,2-diphenyl-1-picrylhydrazyl assay and HP: Hydrogen peroxide assay) and antidiabetic (αA: α-amylase assay and αG: α-glucosidase assay) activities as well as total flavonoid (TF) and total phenolic (TP) contents of extracts were evaluated. The correlation between in vitro activities and solvent polarity index, total flavonoid and phenolic content were established by using pearson’s correlation coefficient (R). Results: Strong positive correlation was observed with PI of extracting solvents and TP content of Ficus religiosa (PI/ TP, R=0.8159) and Ficus glomerata (PI/ TP, R=0.9172). Comparatively benzene and water extracts of Ficus glomerata were found to have significantly (P<0.05) highest in vitro antidiabetic and antioxidant activity respectively. Strong positive correlation was observed between TF and αG inhibitory (TF/ αG, R=0.793) effects of Ficus benghalensis. In addition, strong positive correlation observed between TP and antioxidant activity (TP/DPPH, R=0.9744; TP/RP, R=0.9514 and TP/HP, R=0.8108) of Ficus glomerata. Conclusions: Finding of our research will help in selection of solvents for extracting antidiabetic and antioxidant rich phytoconstituents from Ficus species.

authenticated by Dr. A. S. Upadhye of Agharkar Research Institute, Pune, India.

Preparation of Extracts
The leaves were dried in shade and powdered using mixture grinder.The powder leaf material was successively extracted with the solvents of increasing order polarity such as petroleum ether, benzene, chloroform, ethanol and distilled water.All the extracts obtained are preserved in desiccators for future use.

Determination of total flavonoids content
Total flavonoids content was determined using the method of Ordon et al. 6 A volume of 0.5 ml of 2% AlCl 3 ethanol solution was added to 0.5 ml of sample solution.After one h at room temperature, the absorbance was measured at 420 nm using Jasco UVVIS spectrophotometer.Total flavonoids content was calculated as mg/g QE.

Determination of total phenolics content
Total phenolics content in the extracts were determined by the modified FolinCiocalteu method as described by Wolfe et al. 7 Extracts (100 μl ) was mixed with 5 ml FolinCiocalteu reagent and 4 ml (75 g/l) of sodium carbonate.The tubes were vortexed for 15 sec and allowed to stand for 30 min at 40°C for colour development.Absorbance was recorded against reagent blank at 765 nm using Jasco UVVIS spectrophotometer.Total phenolics content were expressed as mg/g GAE.

DPPH Free radical scavenging assay
The capability of the extracts to scavenge DPPH free radical was evaluated according to the method of Arabshahi and Urooj. 81 ml of sample (0.1 mg/ml) dissolved in methanol was added to 1 ml of a 1 mM methanolic solution of DPPH.Samples were vortex shaken and left in dark for 30 min.The absorbance of the samples was recorded at 517 nm and percentage scavenging activities of extracts were calculated by using following equation:

Reducing power assay
The reducing power was determined according to the method of Oyaizu. 92.5 ml of extracts (0.1 mg/ml) dissolved in methanol were mixed with 2.5 ml of 200 mmol/l sodium phosphate buffer (pH 6.6) and 2.5 ml of 1% potassium ferricyanide.The mixture was incubated at 50 O C for 20 min.After 2.5 ml of 10% trichloroacetic acid (w/v) were added, the mixture was centrifuged at 650 rpm for 10 min.5 ml of upper layer was mixed with 5 ml of water (deionised) and 1 ml (0.1%) ferric chloride, and the absorbance was measured at 700 nm.The higher absorbance indicates higher reducing power.

Hydrogen peroxide radical scavenging assay
Hydrogen peroxide radical scavenging activity of extract was determined according to the method of Jayaprakasha et al 10 with slight modification.1 ml of each extract solution (0.01 mg/ml) prepared in methanol was incubated with 1.5 ml of 20 mM H 2 O 2 solution prepared in PBS pH 7.4 for 10 min.The absorbance of the solution was measured at 230 nm.For each extract separate blank was prepared and their absorbance is subtracted from the absorbance of sample.Percentage hydrogen peroxide radical scavenging activity of extracts was calculated by using following equation:

α-amylase inhibitory assay
The αamylase inhibitory assay was conducted according to the method described by Okutan et al. 11 In brief, 500 μl of 0.02 mM phosphate buffer (pH 6.9, 0.06Mm Nacl) containing αamylase enzyme (0.05 U/ml) and 500 μl extract dissolved in DMSO were incubated at 37 o C for 5 min.Add 500 μl of starch and incubate for exactly 3 min.The reaction was stopped by adding 500 μl of DNS reagent and keep it for heating in water bath maintain at 100 o C for 5 min, removed the test tube from water bath, cooled and add 5 ml of distilled water and measure the absorbance at 540 nm.Percentage αamylase inhibitory activity of extracts was calculated by using following equation:

α-glucosidase inhibitory assay
The effect of the plant extracts on αglucosidase activity was determined according to the method described by Kim et al. 12 The substrate solution of pnitropheynylα-glucopyranoside (pNPG) (3.0 mM) was prepared in phosphate buffer (20 mM), pH 6.9. 100 μL of αglucosidase (1.0 U/ml) was preincubated with 50 μL of the extracts for 10 min.Then 50 μL of 3.0 mM (pNPG) as a substrate dissolved in 20 mM phosphate buffer (pH 6.9) was added to start the reaction.The reaction mixture was incu bated at 37 o C for 20 min and stopped by adding 2 ml of 0.1 M Na 2 CO 3 .The αglucosidase activity was determined by measuring the yellow colored paranitrophenol released from pNPG at 405 nm and percentage αglucosidase inhibitory activity of extracts were calculated by using following equation:

Statistical analysis
All the determinations were performed in triplicate.Statistical analysis was performed using one way ANOVA followed by posthoc Tukey's HSD Test at the significance level P < 0.05 and P < 0.01.Data were also evaluated using Pearson's correlation coefficients to identify relationships between total flavonoids, total phenolics contents and in vitro antioxidant and antidiabetic activities of leaves of Ficus species.

RESULTS AND DISCUSSION
Phytochemicals such as phenolics and flavonoidal compounds present in the various herbs are well known for its antioxidant and antidiabetic activity. 9For this reason there are interests in using phenolics and flavo noids rich extracts in the treatment of diabetes and its complications.In present study five different solvents including petroleum ether, benzene, chloroform, ethanol, water with increase in order of their polarity index were used.Polarity of extracting solvents have significantly (P < 0.05) affected both measured total flavonoids and phenolics content.Total flavonoids content is varied from 361.45 to 93.96 mg QE/g, whereas total phenolics content is varied from 144.04 to 4.50 mg GAE/g of extract.When total flavo noid content of each extract was compared, chloroform extract of Ficus religiosa and Ficus glomerata extracts were found to have significantly (p<0.05)higher content of total flavonoids (Figure 1A).However, when values of αamylase inhibition were observed with benzene and chloro form extracts of Ficus religiosa and ethanol extract of Ficus benghalensis (Figure 2A).Also there is one negative value of αglucosidase inhibition was observed for ethanol extract of Ficus benghalensis (Figure 2A).This indicates activation of these enzymes by the respective extracts rather being inhibited.This type of negative value of enzyme inhibitions was also reported by Bahman N et al. 13 Overall in both the assay benzene extract of Ficus glomerata showed highest inhibition of both the enzymes.Moderate to strong positive correlations were found between total flavonoids content and αamylase and αglucosidase inhibitory activities of Ficus benghalensis and Ficus glomerata (Table 2) which indicate that the presence of flavonoids in the extract might be responsible for their activity.αamylase and αglucosidase are the main enzymes responsible for the conversion of starch to more simple sugars.Thus, the inhibitors of these enzymes delay carbohydrate digestion and reduce the rate of absorption of glucose.As a result, this type of drugs can control post prandial rise in blood glucose level.The antioxidant activity of each extract was monitored by using the DPPH radical scavenging assay, reducing powers assay and hydrogen peroxide radical scavenging assay.All the tested plant extracts of Ficus species exhibited significantly different (P < 0.05) antioxidant activities (Figure 3A, 3B and 3C).Percentage DPPH and hydrogen peroxide radical scavenging activity of extracts were found to be in the range from 99.02 to 47.51 and 49.43 to 38.98 respectively.Whereas reducing power of extracts expressed as absorbance at 700 nm were found to be  the phenolics content of each extract was compared with the others, water extract of Ficus glomerata and ethanol extract of Ficus religiosa were found to have significantly (p<0.05)higher phenolics content (Figure 1B).Strong positive correlation was found between polarity index of extracting solvents and phenolics content of Ficus religiosa and Ficus glomerata (Table 1) which indicate the role of more polar solvent in extraction of phenolics compound.However, weak negative correlation was found between polarity index of extracting solvents and phenolics content of Ficus benghalensis.This different trend indicates that the phenolics content may change from species to species.Moderate to strong negative correlation was found between polarity index of extracting solvents and flavonoids content in all three Ficus species which indicate the ability of less to moderate polar solvents in extracting flavonoidal compounds (Table 1).
In vitro antidiabetic activity of each extract was estimated by αamylase and αglucosidase inhibitory assay.The αamylase and αglucosidase inhibitory activities of extracts were found to be significantly (P < 0.05) differ among the tested plants.Significantly high αamylase inhibitory and αglucosidase inhibitory activity was found with benzene and chloroform extract of Ficus gloemrata (Figure 2A and 2B).Three negative  in the range from 0.4294 to 0.1422.When DPPH and hydrogen peroxide radical scavenging activity of extracts was compared, water, ethanol extract of Ficus glomerata and water extract of Ficus religiosa respectively showed significantly (p<0.05)high antioxidant activity (Figure 3A and 3C).
When reducing power of extracts were compared, water extract of Ficus glomerata and ethanol extract of Ficus bengalesnsis showed significantly (p<0.05)high reducing power (Figure 3B).Overall in all antioxidant activity assays water extracts of Ficus glomerata showed highest anti oxidant activity.Moderate to weak correlations were observed between phenolics content and antioxidant activity of Ficus religiosa whereas strong positive correlation was found between phenolics content and antioxidant activity of Ficus glomerata (Table 3) which indicate that the presence of phenolics content in the extract might be responsible for its antioxidant activity.
Other researchers have also reported such type of favourable positive correlation between antioxidant activity and phenolics content. 14,15,16It was also found that most polar extracting solvents showed more anti oxidant activity and total phenolic content as compared to less polar solvents.It indicate that the polar solvents are important in extracting phytoconstituents with more antioxidant activity and phenolics content which is in agreement with earlier study. 17   positive correlation was observed between phenolics content and anti oxidant activities of Ficus glomerata.The present findings not only showed comparative in vitro antidiabetic and antioxidant activity of three plants from Ficus species but also give clear idea about the effect of extracting solvents, total flavonoids and phenolic content on in vitro antidiabetic and antioxidant activities of these plants.Among all analyzed leaf extracts, Ficus glomerata leaf extracts showed comparatively high in vitro antidiabetic and antioxi dant activity.However, further in vivo studies are needed to confirm the potential of these plants in the treatment of diabetes and oxidative stress related disorders.

CONCLUSION
Total flavonoids and phenolics content of leaves of three Ficus species were significantly different depending on the types of plant and polarity of solvents used for the extraction.More polar solvent like water seems to be more effective in extracting phenolics content from the leaves.In addition, it should be also noted that statistically significant strong

Figure 1 (
Figure 1 (A): Total flavonoids content of leaves of Ficus species.

Figure 2 (
Figure 2 (a): In vitro α-amylase inhibitory activity of leaves of Ficus species.

Figure 1 :
Figure 1: Total flavonoids content (A) and total phenolics con tent (B) of leaves of Ficus species.Data expresses as mean ± SEM (n=3).Lower case letters indicate significant differences of P < 0.05 and capital letters indicate significant difference of P <0.01 as compared to different solvent extracts.PI: Polarity index, TFC: Total flavonoids content, TPC: Total phenolics content, QE: Quercetin equivalent, GAE: Gallic acid equivalent.

Figure 3 (
Figure 3 (b): In vitro reducing power assay of leaves of Ficus species.

Figure 3 (
Figure 3 (c): Figure 3 (C): In vitro hydrogen peroxide radical scavenging activity of leaves of Ficus species.

Figure 3 :
Figure 3: In vitro DPPH radical scavenging activity (A), reducing power (B) and hydrogen peroxide radical scavenging activity (C) of leaves of Ficus species.Data expresses as mean ± SEM (n=3).Lower case letters indicate significant differences of P < 0.05 and capital letters indicate significant difference of P <0.01 as compared to different solvent extracts.PI: Polarity index, DPPH-RSA: 2, 2-diphenyl-1-picrylhydrazyl -radical scavenging activity, RP: Reducing power, HP-RSA: Hydrogen peroxide -radical scavenging activity