Comparative Study of Erythrina indica Lam. (Febaceae) Leaves Extracts for Antioxidant Activity

The present study was designed to investigate the antioxidant activity of aqueous and methanol extracts of Erythrina indica Lam leaves by in vitro methods viz. 1, 1-Diphenyl-2-Picrylhydrazyl, nitric oxide radical scavenging activity, and inhibition of lipid peroxidation by thiobarbituric acid reactive substances (TBARS) method on isolated rat liver tissues. Quantitative analysis of antioxidative components like total amount of phenolics, flavonoids, and flavonols were estimated using the spectrophotometric method. Linear regression analysis was used to calculate the IC50 value. Results showed that the aqueous and methanol extracts exhibited significant DPPH radicals scavenging activity with an IC50 value 342.59 ± 19.59, 283.24 ± 12.28 µg/mL respectively. Nitric oxide radicals were significantly scavenged by the aqueous and methanol extracts (IC50 = 250.12 ± 10.66; 328.29 ± 3.74 µg/mL). Lipid peroxidation induced by the Fe2+ was inhibited by the aqueous extract with low IC50 value (97.29 ± 2.05 µg/mL) as compared to methanol extract (IC50 = 283.74 ± 5.70 µg/mL). Both the extracts were exhibited similar quantities of total phenolics. Total flavonoids were found to be in higher quantities than total flavonols in aqueous extract as compared to methanol extract. From the results, it is concluded that the aqueous and methanol extracts of E. indica leaves possesses significant antioxidant activity that may be due to the presence of flavonoids and related polyphenolic compounds.


INTRODUCTION
activities. [9,10] Inhibition of free radical induced damage by supplementation of antioxidants has become an attractive therapeutic strategy for reducing the risk of diseases. [11] Among the activities claimed to be present in most of the nutraceuticals and cosmeceuticals is the antioxidant activity. Several synthetic antioxidants are available, but are quite unsafe and their toxicity is of concern. [12] Natural products with antioxidant activity may be used for human consumption because of their safety. Hence, the present work was undertaken to investigate the antioxidant activity of aqueous and methanol extracts of E. indica Lam leaves using different in-vitro techniques.

Plant material
The fresh leaves of E. indica Lam. were collected from the mature plant in and around the city of Mumbai, Maharashtra, India, during the month of August 2008 and dried under shade. The plant was authenticated by Dr. Ganesh Iyer, Botanist, Ramnarayan Ruia College, Matunga, Mumbai. A voucher specimen (2007/08/07) has been kept in our laboratory for future reference.

Preparation of plant extracts
The dried powdered leaves of E. indica were defatted using petroleum ether (60-80°C) and successively extracted with methanol in the soxhlet extractor. Aqueous extract was prepared by the cold maceration method. Both the extracts were filtered through vacuum filter and the filtrate was concentrated in vacuum evaporator. Dried extracts were used for the further studies.

Assessment of antioxidant activity
1, 1-Diphenyl-2-picrylhydrazyl radical scavenging activity The ability of the extracts to scavenge DPPH . was determined by the method of Gyamfi et al. [14] with minor modifications. A 0.5 mL of aliquot of each extract at different concentrations (50-400 mg/mL) in methanol was mixed with 0.5 mL of 100 mM methanolic solution of DPPH. After 30 min incubation in darkness and at ambient temperature, the resultant absorbance was recorded at 517 nm. The percentage inhibition was calculated using the following formula.
Percentage inhibition 5 (Abs Control 2Abs Sample ) 3 100/Abs Control IC 50 values were calculated as the average of triplicate analyses

Determination of nitric oxide radical scavenging activity
The compound SNP is known to decompose in aqueous solution at physiological pH (7.2) producing nitric oxide radicals (NO . ). Under aerobic conditions, NO . reacts with oxygen to produce stable products (nitrate and nitrite). The quantities of which can be determined using Griess reagent. The scavenging effect of the plant extracts on nitric oxide was measured according to the modified method of Marcocci et al. [15] 1 mL of extracts solution at different concentrations (25-400 mg/mL) were added in the test tubes to 1 mL of SNP solution (100 mM) and the tubes were incubated at 29°C for 2.5 h. An aliquot of 1 mL of the incubation solution was removed and diluted with 1 mL of Griess reagent (1% Sulfanilamide in 2% H 3 PO 4 and 0.1% N-1-Naphthylethylenediamine dihydrochloride). The absorbance of the chromophore that formed during with Naphthylethylenediamine dihydrochloride was immediately read at 540 nm. The percentage inhibition was calculated using the formula mentioned above.

Inhibition of lipid peroxidation by thiobarbituric acid reactive substances method
Lipid peroxide formation was measured (lipid peroxidation assay) by the modified methods of Ohkawa et al. [16] and Masao et al. [17] Male Sprague-Dawley rat (weighing 200-250 g) was sacrificed by dislocation of the neck. The abdomen was opened; the liver was removed and homogenized in phosphate buffer saline (pH 7.0). Then, 1 mL of liver homogenate (10%, w/v) was added to the test extracts of different concentrations (25-400 mg/mL). The lipid peroxidation was initiated by adding 100 (L of 15 mM FeSO 4 solution. After 30 min of incubation at room temperature, 0.1 mL of reaction mixture (liver homogenate 1 test drug) was taken in a tube containing 0.1 mL of SDS (8.1%w/v), 0.75 mL of 20% acetic acid, and 0.75 mL of 0.8% TBA solution. The volume in each tube was made to 2 ml with distilled water and then heated on water bath at 95°C for 60 min. After 60 min, the volume in each tube was made up to 2.5 mL and then 2.5 mL of N-butanol: Pyridine (5:1) was added in each tube. The reaction mixture was vortexed and centrifuged at 4000 rpm for 10 min. The organic layer was removed and absorbance was read at 532 nm in a UV spectrophotometer. The experiment was performed in triplicate. The percentage inhibition was calculated using the formula mentioned above.

Quantitative analysis of antioxidative components
Determination of total phenolics, flavonoids, and flavonols Total phenolics content were determined according to the method of Hammerschmidt et al. [18] Briefly, 0.2 mL of the test solution (10 mg/mL) was mixed with 1 mL of 10% Folin-Ciocalteu solution and 0.8 mL of 7.5% sodium carbonate solution. The mixture was incubated for 1 h at room temperature. The absorbance at 760 nm was measured and converted to phenolic contents according to the calibration curve of gallic acid.
Total flavonoids content was estimated colorimetrically based on the method modified by Zhishen et al. [19] To 0.1 mL of test extract (10 mg/mL) in a 10 mL volumetric flask, distilled water was added to make the volume to 5 mL and 0.3 mL 5% NaNO 2 was added to this. 3 mL of 10% AlCl 3 was added 5 min later. After 6 min, 2 mL of 1 M sodium hydroxide was added and the absorbance was measured at 510 nm. Rutin was used as a standard for constructing a calibration curve.
Total flavonols were estimated as rutin equivalents and expressed as mg of rutin per gram of dry extract by the method of Miliauskas et al. [20] The rutin calibration curve was prepared by mixing rutin solution with 2 mL (20 gm/L) AlCl 3 and 6 mL (50 gm/L) sodium acetate. The absorption at 440 nm was read after 2.5 h at 20 o C. The same procedure was carried out with 2 mL of plant extract (10 gm/L) instead of rutin solution. All determinations were carried out in triplicate and the mean values were used.

Statistical analysis
The results are expressed as the mean 6 SD for three replicates. Linear regression analysis was used to calculate the IC 50 value.

Phytochemical analysis
The phytochemical evaluation and extractive yield of the aqueous and methanol extracts of E. indica leaves are shown in Table 1. The aqueous extract revealed the presence of carbohydrates, proteins, glycosides, saponins, alkaloids, flavonoids, tannins, and phenolic compounds, while methanol extract showed the presence of carbohydrates, proteins, steroids, saponins, alkaloids, flavonoids, tannins, and phenolic compounds. Extractive yield of the aqueous and methanol extracts were found to be 14.26 and 7.89% w/w, respectively.

Assessment of antioxidant activity
1, 1-Diphenyl-2-picrylhydrazyl radical scavenging activity 1, 1-Diphenyl-2-picrylhydrazyl radical scavenging assay is the most widely used method for screening antioxidant activity, since it can accommodate many samples in a short period and detect active ingredients at low concentration. [21,22] The decrease in absorbance of the DPPH caused by antioxidant was due to the scavenging of the radical by hydrogen donation. It is visually noticeable as a color change from purple to yellow. The aqueous and methanol extracts of E. indica showed DPPH radical scavenging activity in a concentration-dependent manner, with an IC 50 value of 342.59 6 19.59 and 283.24 6 12.28 mg/mL, respectively [ Figure 1].

Determination of nitric oxide radical scavenging activity
The effect of E. indica extracts on nitric oxide radical scavenging activity is shown in Figure 2. The compound SNP is known to decompose in aqueous solution at physiological pH (7.2) producing nitric oxide radicals (NO.). Under aerobic conditions, NO. reacts with oxygen to produce stable products (nitrate and nitrite). This leads to reduction of nitrite concentration in the assay media. [15] The aqueous extract of E. indica exhibited potent nitric oxide radical scavenging activity (IC 50 5 250.12 6 10.66 mg/mL) as compared with methanol extract (IC 50 5 328.29 6 3.74 mg/mL).

Inhibition of lipid peroxidation by thiobarbituric acid reactive substances method
Lipid peroxidation is very important process in free radical pathology as it is damaging to cells. The liver of rat was used as a source of polyunsaturated fatty acids for determining the extent of lipid peroxidation. Malondialdehyde, a lipid peroxidation product, is an indicator of reactive oxygen species (ROS) generation in the tissue. [23] The inhibition of lipid peroxide formation by E. indica extracts is shown in Figure 3. The aqueous extract showed the maximum inhibition of peroxide formation with low IC 50 value of 97.29 6 2.05 mg/ mL, whereas methanol extract showed IC 50 value of 283.74 6 5.70 mg/mL.

Determination of total phenolics, flavonoids, and flavonols
The phenolic substances are known to possess the ability to reduce oxidative damage and act as antioxidants. [24] They can trap the free radicals directly or scavenge them through a series of coupled reactions with antioxidant enzymes. [25] In addition, it was reported that phenolic substances were associated with antioxidant activity and played important role in stabilizing lipid peroxidation. [26] This activity is believed to be mainly due to their redox properties, which play an important role in adsorbing and neutralizing free radicals, quenching singlet and triplet oxygen, or decomposing peroxides. [27] The aqueous and methanol extracts of E. indica showed the total phenol contents of 24.91 6 0.00 and 25.62 6 0.00 mg of gallic acid equivalents per gram of dry extract, respectively. Total flavonoids and total flavonols in aqueous extract were found to be 357.55 6 33.38 and 265.14 6 7.30 mg of rutin equivalents, respectively, whereas methanol extract showed total flavonoids 524.22 6 16.17 and flavonols 167.21 6 11.16 mg of rutin equivalents [ Table 2].