Antioxidant Activities of Ethanolic and Aqueous Extracts of Asparagus racemosus Roots

INTRODUCTION Asparagus racemosus (AR) wild., (Family: Liliaceae), commonly known as shatavari, satawar or satmuli, is found in all over India, and is commonly known in Thailand as sam-sib or rak-sam-sib. In Ayurveda, the dried root of AR is used as a tonic, galactogogue, aphrodisiac, rejuvenator, antispasmodic, antiulcerous and anti-inflammatory agent. Steroidal saponins and sapogeninsare the pharmacological value of the AR root.1-2 Antioxytocic activities, antihepatotoxic, hepatopathy, dyspepsia and dysentery are the uses of the root of AR for treatment.1 Recent reports on AR indicate that the root extracts show antioxidant and antidiarrheal activities in animal models.3-4 Phytoanalysis of the AR root composition by Visavadiya et al.5 found phytosterols 0.79%, saponins 8.83%, polyphenols 1.69%, flavonoids 0.47% and total ascorbic acid 0.76%. AR contains alkaloids, flavonoids, tannins, saponins, phenols, terpenes, polysaccharides and steroids.5 AR root extract was found to contain flavonoids, polyphenols and vitamin C, which were found to exhibit the greatest antioxidant activity. A phytoestrogenic effect of AR root extracts has recently been found as well as antidiarrhoeal, antidyspepsia, adaptogenic, cardioprotective, antibacterial, immune adjuvant and antitussive effects. The methanolic and aqueous extracts of AR roots have been produced in Antioxidant Activities of Ethanolic and Aqueous Extracts of Asparagus racemosus Roots

100ºC for 10 min.The extracts were concentrated to dryness under a reduce pressure and controlled temperature using an evaporator and a freeze-dryer, respectively.

DPPH radical scavenging assay
8] An ascorbic acid solution was used as a reference standard.A varied dose of each extract (10-1000 ug/mL, dissolved in 10% DMSO) was added to a volume of 750 µL DPPH in absolute ethanol, incubated at room temperature in the dark for 20 min and measured for absorbance at 517 nm using a 96-well plate with microtiter plate reader (BMG LABTECH GmbH, Ortenberg, Germany).The inhibition percentages of the radical scavenging activity of the samples were expressed as follows: %

inhibition = [(A control-A sample)/A control] x 100
The absorbance values of the DPPH solutions without or with a sample added were the control and samples, respectively.The inhibitory concentration of the sample requires a scavenging activity for the DPPH radical of 50% (IC 50 value) as obtaining from a linear regression analysis of a calibration curve.

ABTS radical scavenging activity
A decolorization test was performed using the spectrophotometric method of previous studies with a slight modification. 9-10A stock solution of ABTS was mixed, 7 mM of ABTS stock solution (pH 7.4) with 140 mM potassium persulfate (K 2 S 2 O 8 ).Ethanol was used to dilute the ABTS for the working solution.The test concentrations of the standard ascorbic acid and extracts were as previously described for the DPPH assay.The ABTS working solution (100 µL) with an equal volume of ethanol served as a control and was incubated for 30 min at room temperature.The absorbance of each solution was determined at 743 nm usinga 96-well plate with microtiter plate reader (BMG LABTECH GmbH, Ortenberg, Germany).The percentage of inhibition was expressed as follows:

% inhibition = [(A control-A sample)/A control] x 100
The control was the absorbance of the ABTS solution without a sample.An effective concentration for the sample requires scavenging of the ABTS radical at 50% (EC 50 value) and this was obtained by linear regression analysis of a calibration curve plotting % inhibition versus concentration.

FRAP assay (Ferric reducing antioxidant power)
The FRAP assay was undertaken with some modification to the method of Benzie and Strain. 11The ratio of the 300 mM acetate buffer (pH 3.6), 10 mM TPTZ in 40 mM HCl and 20 mM ferric chloride was 10:1:1 (v/v/v), respectively.A total of 30 µL of the sample extract was added to 270 µL of the FRAP reagent and mixed well.After 30 min incubation at room temperature, the absorbance values of the samples were subsequently measured at 593 nm in three replicates using a microplate reader (BMG LABTECH GmbH, Ortenberg, Germany).The antioxidant capacity of the extract was compared with a trolox calibration curve and expressed as µmol Trolox equivalent (TE) per gram of extract.

Determination of total flavonoid contents
Flavonoid contents were measured by the aluminium chloride colorimetric methodas described by Kim et al. with slight modification. 12Fifty milligrams of each AR extract was suspended in 10 mL of 80% methanol, sonicated for 15 min and filtered through Whatman filter paper No.42 (125 mm).Then mixed 0.3 mL of filtrate, 3.4 mL of 30% methanol, 0.15 mL of 0.5 M NaNO 2 and 0.15 mL of 0.3 M AlCl 3 -6H 2 O in a test tube.Add 1 mL of 1M NaOH for 5 min, and then the absorbance was measured at 510 nm.A standard calibration curve was constructed using known quercetin concentrations against absorbance.Total flavonoid contents were determined on the basis of absorbance and calculated as mg quercetin equivalent per gram of extract (mg QE/g powder).

Determination of total phenolic compound
Total soluble phenolic content in the extracts was determined using the Folin-Ciocalteu method as previously described by Thaipong et al. with a slight modification. 1310% Folin-Ciocalteu solution (400 µl) was mixed with 200 µl of the sample solution (1.0 mg/mL) in a volumetric flask and incubated at room temperature for 10 min before being mixed with 0.2 mL of 7% Na 2 CO 3 solution.Finally the mixture was diluted with deionized distilled water and made up to 10 mL in a volumetric flask.The mixture was kept at room temperature for 2 hours and then had the absorbance measured at 725 nm.The total phenolic compounds in the extract were presented as grams of gallic acid equivalent (GAE) using an equation obtained from the regression line of a standard gallic acid graph: y = 0.2177x+0.0005,r 2 =0.999.Where, y was the absorbance and x was the concentration.Total phenolics were then calculated as mg gallic acid equivalent per gram of extract (mg GAE/g extract).

Statistical analysis
Results were expressed as means ± SEM.Statistical analysis was determined using a one-way ANOVA followed by Student's t-tests.P-values <0.05 were considered to be statistically significant.

RESULTS
The percentage yields of the aqueous and the ethanolic extracts of AR were 27.41 and 20.88, respectively.

DPPH radical scavenging assay
The antioxidant activity of ascorbic acid by DPPH method was found to be greater than those of the ethanolic and the aqueous AR root extracts (Table 1).There was a significant decrease in the concentration of the DPPH radical due to the scavenging ability of both extracts.The ethanolic extract had a scavenging activity that was better than that of the aqueous extract.

ABTS RADICAL SCAVENGING ACTIVITY
There was a decrease in the concentration of the ABTS radical due to the scavenging abilities of both extracts (Table 1).The ethanolic extract of the AR roots had a scavenging activity that was better than that of the aqueous extract.

FRAP assay (Ferric reducing antioxidant power)
The trolox equivalents in the ethanolic and aqueous extracts of AR roots are shown in Table 1.The trolox equivalent scavenging ability of the ethanolic extract of the AR roots was significantly greater than that of the aqueous extract of the AR roots.

Determination of total phenolic compounds
The total phenolic contents in the ethanolic and the aqueous extracts of AR roots are shown in Table 2.The ethanolic extract had significantly higher total phenolic contents than the aqueous extract.

Determination of total flavonoid contents
The total flavonoids in the ethanolic and the aqueous extracts of AR roots are shown in Table 2.The ethanolic extract had significantly higher total flavonoids than the aqueous extract of AR roots.

DISCUSSION
The results of this present study indicated that the plant extracts were moderately free radical scavengers, which could reduce or reverse damage caused by free radicals in the human body.The antioxidant activity of the ethanolic extract of AR roots was better than that from the aqueous extract.The total phenolic contents and total flavonoids in the ethanolic extract were also higher than those in the aqueous extract.This study showed that the scavenging ability, total phenolic compounds and total flavonoid contents of the ethanolic extract were higher than those from the aqueous extract but were four times lower than those reported from a methanolic extract. 2 The scavenging ability of the aqueous extract in this study was better than the study of Kongkaneramit et al. 1 The previous study reported that the main components were saponins such as shatvarin, sarsapogenin and kaempferol.The presence of phenolic compounds is shown mostly via the antioxidant activities of plant sources.Due to their hydroxyl groups, phenols are very important plant constituents with a good scavenging ability.The presence of large quantities of phenolic compounds does not necessarily always correlate with antioxidant effects. 6AR has been reported to have antioxidant activity.-15 In addition, AR has recently been shown to contain saponins, steroidal 16 and racemofuran. 17The aqueous extract has also been shown to exhibit anantioxidant effect in rat liver and inactivation of superoxide dismutase 18 and the amelioration of oxidative stress. 19The antioxidant activity in terms of mean inhibitory concentrations (IC 50 ) of ascorbic acid, ethanolic and aqueous extracts were found to be 3.37, 950.34 and 1121.24µg /mL, respectively, when using the DPPH method.The effective concentration (EC 50 ) of a crude ethanol extract of AR was 381.91 µg/ mL, 20 600 µg /mL. 1 The methanolic extract of the root of AR was 4158.8 µg/mL when using butylated hydroxyl toluene as a standard. 2The present study showed that both the ethanolic and the aqueous extracts of the roots have moderate antioxidant activities.
Although AR extracts exhibited far less antioxidant activity than ascorbic acid when tested in vitro using the DPPH method, 15 -16 when considering the antioxidant enzyme: superoxide dismutase and ascorbic acid were increased by the effects of AR aqueous and methanolic extracts. 1R extracts contained saponins as the main constituents (8.83%), followed by polyphenols (1.69%), phytosterols (0.79%), ascorbic acid (0.76%) and flavonoids (0.47%), respectively. 5These compounds could be responsible for decreased cholesterol levels and increased fecal sterol excretion in hypercholesteremic rats.2] Saponins reduced the plasma cholesterol level by interfere with the enterohepatic circulation of bile acids and so making it unavailable for intestinal absorption of cholesterol. 23olyphenolic compounds and phenolic acids, found in plants, have multiple biological effects, including antioxidant activity. 24Ascorbic acid shows very strong antioxidative activities. 25ur in vitro studies showed that AR has moderate free radical scavenging action.The antioxidant properties of both the ethanolic and the aqueous extracts of AR can be attributed to the presence of ascorbic acid, flavonoids and polyphenols, which in turn may be responsible for its antioxidative effects. 2

CONCLUSION
These results have shown high phenolic and flavonoid contents.The ethanolic extract of AR root possessed higher amounts of phenolic and flavonoid contents than the aqueous extract.In conclusion, the antioxidant capacity of the ethanolic extract was higher than the aqueous extract.

Table 2 : Phenolic and flavonoid contents of AR root extractions. Herbal extracts Total phenolic contents (mg gallic acid/100 g extract) Total flavonoids (mg Quercetin/100 g extract)
*in the same column showed significant differences (p<0.05)