INTRODUCTION:

Herbal medicines are employed in a wide variety of health related applications ranging from treatment of common colds to treatment of cancer [1].

Odina woodier was found to be effective in healing of wounds. Its bark is used as a lotion for ulcers of leprosy and a decoction of the bark is used for toothache. The leaves have been reported to used in the treatment of inflammation and Elephantiasis of the legs. Juice of green branches is used as an emetic in case of coma or insensibility produced by narcotic [2]. Boiled leaves are applied in sprains and bruises and local swellings, to relieve pains. The paste of Odina woodier fruits are applied externally for bone fracture [3, 4, 5]. Anti oxidant play an important role in the alleviation of diabetes and obesity due to oxidation stress. Many plants often contain substantial amounts of anti oxidants including VitaminC and E, Carotenoids, flavanoids and tannins and can be utilized to scavenge the excess free radicals from human body[6].

Antioxidant-based drug formulation is used for the prevention and treatment of complex diseases and cancer [7] In the present study, our investigation aims to find out the constituents by preliminary phytochemical analysis and to investigate the anti oxidant activity of methanol and ethyl acetate extract via, DPPH radical model of odina woodier leaves.

MATERIALS AND METHODS:

Materials:

a. Collection of plant part:

Odina woodier leaves were collected in the S.F.R. College campus, Sivakasi, Virudhunager District in Tamil Nadu. It was cleaned with running tap water to remove adhering elements, shadow dried and powdered in a domestic mixer.

b. Chemicals used:

All chemicals were of highest purity (≥ 99.0%). Ferric chloride, HCl, Dragendroff's reagent, magnesium metal strips, methanol, H₂SO₄, Na₂CO₃, aluminium chloride, potassium acetate, phosphate buffer, 2, 2-diphenyl-1-picrylhydrazyl (DPPH), sodium nitroprusside, hydrogen peroxide, VitaminC, Fehling’s reagent, diethyl ether, and various solvents such as ethanol, methanol, ethyl acetate(SD fine grade) were used. These solvents were distilled above their boiling point for purification and then used.

METHODS USED:

Plant extraction:

The powdered leaves of Odina woodier were extracted by using ethanol, ethyl acetate, and methanol in soxhlet apparatus by using standard procedure[8]. The distillates were collected and distilled separately to yield the concentrated extracts. These extracts on cooling produces powdered mass. Preliminary phytochemical analysis and anti oxidant studies were carried out.

Preliminary phytochemical studies:

Preliminary Phytochemical studies were performed for testing different plant constituent present in ethanol, methanol and ethyl acetate extract of odina woodier leaf.A small portion of the extract were used for the phytochemical tests for compounds which include tannins, flavanoids, alkaloids and steroids with corresponding methods. 1.0 g of plant extract was dissolved in10 ml of distilled water and filtered (using what man No.1 filter paper). A blue colouration resulting from the addition of ferric chloride reagent to the filtrate indicated the presence of tannins in the extract. 0.5 g of the plant extract was dissolved in 5 ml of 1% HCl on steam bath. A millilitre of the filtrate was treated with few drops of Dragendroff's reagent. Turbidity or precipitation indicated the presence of alkaloid.

0.2 g of the extract was dissolved in 2 ml of methanol and heated. A chip of magnesium metal was added to the above mixture followed by the addition of a few drops of concentrated HCl. The occurrence of a red or orange colouration indicates the presence of flavanoids. 0.5 g of the extract was dissolved in 3 ml of chloroform and filtered. Concentrated H₂SO₄was added carefully to the filtrate to form lower layer. A reddish brown ring was formed between filtrate and sulphuric acid indicated the presence of steroid [9-11].

Determination of anti oxidant activity:

Antioxidant studies of methanol and ethyl acetate extract of odina woodier leaf were tested for its free radical scavenging property using DPPH model. These experiments were performed thrice and the results were averaged. The DPPH reacted with methanol or absolute ethanol to yield a purple color DPPH (1, 1-Dipheny -2- picryl - hydracyl) radical. The presence of antioxidants which include flavanoids in the sample will scavenge the formed DPPH radical there by a decreased in the color will be observed which is spectrophotometrically measured at 517nm. 0.1mM solution of DDPH in ethanol was prepared and 1ml of this solution was added to 3ml of plant extract in methanol at different concentration (25, 50, 100, 150, 200 µg/ml).After thirty minutes the absorbance was measured at 517nm. Decrease in the absorbance of the reaction mixture indicates higher free radical scavenging activity [12]. The capability to scavenge the DDPH radical was calculated using the following equation

DDPH scavenging effect = [(A_c – A_t/ A_s)* 100]

A_c = absorbance of negative control

A_t = negative control + extract

A_s = ascorbic acid was (100mM) used as a positive control

The above procedure was also performed for the ethyl acetate extract at different concentration (25µg/ml - 250µg/ml)

RESULTS AND DISCUSSION:

A. Preliminary phytochemical analysis:

Preliminary phytochemical analysis of Odina woodier leaf extracts were listed in Table- I.

Table -1-Phytochemical constituents of Odina woodier leaf extract

S.No	Plant constituent	Ethanol extract	Methanol extract	Ethyl acetate extract
1	Alkaloid	+ +ve	+ +ve	+ve
2	Carbohydrate	-ve	-ve	-ve
3	Steroids	+ +ve	+ +ve	+ve
4	Proteins	-ve	-ve	-ve
5	Tannins	+ +ve	+ +ve	+ve
6	Phenolic compounds	-ve	-ve	-ve
7	Flavonoids	+ +ve	+ +ve	+ve
8	Glycosides	-ve	-ve	-ve
9	Saponins	-ve	-ve	-ve

++ - presence of constituents with more concentration

+ - presence of constituents

- - absences of constituents

Phytochemical constituents present in different extract of Odina woodier leaf was performed by generally accepted laboratory technique for qualitative determination. Alkaloids, flavanoids, tannins, steroids were present more in ethanol and methanol extract and were present in lesser amount in ethyl acetate extract. Other constituents like carbohydrate, phenolic compounds etc., were found to be absent in all the three extracts.

Table-II- Antioxidant activity of methanol extract of Odina woodier leaf

S.No	Samples (concentration)	Absorbance			Mean of absorbance	% of activity
1	DDPH control (50µg/ml)	0.925	0.998	1.007	0.976
2	Standard Vit. C (100µg/ml)	0.138	0.161	0.155	0.151	84%
3	Methanol extract (25µg/ml)	0.225	0.238	0.242	0.235	75%
4	Methanol extract (50µg/ml)	0.172	0.168	0.170	0.170	82%
5	Methanol extract (100µg/ml)	0.125	0.128	0.132	0.128	86%
6	Methanol extract (150µg/ml)	0.072	0.070	0.065	0.069	92%
7	Methanol extract (200µg/ml)	0.036	0.034	0.027	0.032	96%

Table-III-Anti oxidant activity of ethyl acetate extract of Odina woodier leaf

S.No	Samples (concentration)	Absorbance			Mean of absorbance	% of activity
1	DDPH control (50µg/ml)	1.596	1.590	1.582	1.589
2	Standard Vit.C (100µg/ml)	0.138	0.161	0.155	0.151	90%
3	Ethyl acetate extract (25µg/ml)	1.000	0.992	0.995	0.995	37%
4	Ethyl acetate extract (50µg/ml)	0.983	0.980	0.982	0.981	38%
5	Ethyl acetate extract (100µg/ml)	0.955	0.956	0.958	0.956	39%
6	Ethyl acetate extract (150µg/ml)	0.923	0.920	0.921	0.921	42%
7	Ethyl acetate extract (200µg/ml)	0.856	0.850	0.848	0.851	46%

B. Antioxidant activity:

Anti oxidant activity recorded for the Odina woodier leaf extracts were using DPPH control and standard Vitamin C. Anti oxidant activity of the Odina woodier leaf extracts are listed in Table II and III

In-vitro antioxidant study of DPPH radical scavenging at different concentration (25-200µg/ml) in methanol, ethyl acetate extract of Odina woodier leaf was measured. The radical scavenging effect was found to increase with increasing concentrations. The methanol extracts showed the maximum activity of 96% at a concentration of 200µg/ml where as the ethyl acetate extracts showed only 46% of antioxidant activity at same concentration of 200µg/ml.

CONCLUSION:

Preliminary phytochemical studies of Odina woodier indicate the presence of alkaloids, flavanoids, tannins and steroids in more amounts in ethanol and methanol extract and in lesser amount in ethyl acetate extract. Other constituents like carbohydrate, phenolic compounds etc., were found to be absent in methanol, ethanol, and ethyl acetate extracts. Two extracts (methanol, ethyl acetate) exhibited satisfactory scavenging effect in DPPH radical scavenging assay. Among the two extracts methanol extract was found to show more anti oxidant effect (96%) than the ethyl acetate extract (46%).

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Received on 18.09.2012 Accepted on 13.10.2012

Asian J. Pharm. Res. 2(4): Oct. - Dec. 2012; Page 153-155