Azima tetracantha is used to treat cough, phthisis, asthma, small pox and diarrhea. The decoction of the stem bark is considered astringent, expectorant and antiperiodic (Reddy et a 1991). Hence in the present study the ethanolic extract of Azima tetracantha (Salvadoraceae) were examined for its antioxidant activity.

MATERIALS AND METHODS:

Preparation of the plant extract

The leaves of Azima tetracantha was collected from Thanjavur district, Tamil Nadu, India in the month of april 2012. 500 grams of dried leaves of Azima tetracantha was powdered and then soaked in 1500 ml of ethanol overnight. After filtration, the residue obtained was again resuspended in equal volume of ethanol for 48 hand filtered again. The above two filtrates were mixed and the solvent was evaporated in a rotovac evaporator at 40°- 50°C under reduced pressure. A 16% semisolid dark material obtained was stored at 0-4°C until used.

Chemicals

1, 1 - diphenyl-2-picrylhydrazyl (DPPH) and 2,2’-azino-bis (3-ethylbenzthiazoline-6-sulfonic acid) (ABTS) were obtained from mumbai. Ascorbic acid and rutin were obtained from SD Fine Chemicals Ltd., Mumbai, India. Methanol and dimethyl sulphoxide were obtained from Ranbaxy Laboratories Ltd., Punjab, India. Hydrogen peroxide (30%) was obtained from Qualigen Fine Chemicals, Mumbai, India. All chemicals used were of analytical grade.

Phytochemical studies

Freshly prepared Azima tetracantha leaves extract was subjected to phytochemical screening tests for the detection of various constituents using conventional protocol.

DPPH radical scavenging activity

Free radical scavenging activity of extracts of pericarp of C.maxima were tested by its ability to bleach the stable 1,1-diphenyl 2-picryl-hydrazyl (DPPH) radical. A stock solution of DPPH (0.3mM in methanol) was prepared such that 1ml of it in 3ml methanol gave an initial absorbance of 0.9.Decrease in absorbance in the presence of PECM, CECM and MECM at different concentration(50-500 mg/ml) were noted after 15 min. scavenging activity was expressed as the %inhibition (Ionita et al 2005).

Formula :

% Inhibition =

Absorbance of the control - Absorbance of the test

______________________________________________ x 100

Absorbance of the control

ABTS radical cation decolourisation assay

ABTS (54.8 mg) was dissolved in 50 ml of distilled water to 2 mM concentration and potassium persulphate (17 mM, 0.3 ml) was added. The reaction mixture was left to stand at room temperature overnight in dark before use. To 0.2 ml of various concentrations of the extracts or standards, 1.0 ml of distilled DMSO and 0.16 ml of ABTS solution was added to make a final volume of 1.36 ml. Absorbance was measured spectrophotometrically, after 20 min at 734 nm. The assay was performed in triplicate (Jayaprakasha et al 2004).

Scavenging of hydrogen peroxide

It can be generated through a dismutation reaction from superoxide anion by superoxide dismutase. It can generate the hydroxyl radical in the presence of metal ions and superoxide anion.

O₂ + H₂O₂ OH^- + OH⁺ + O₂

A solution of hydrogen peroxide (20mM) was prepared in phosphate buffered saline (PBS, pH 7.4). Various concentrations of 1ml of the extracts or standards in methanol were added to 2 ml of hydrogen peroxide solutions in PBS. The absorbance was measured at 230 nm, after 10 min against a blank solution that contained extracts in PBS without hydrogen peroxide (Mruthunjaya et al 2008).

RESULTS:

Phytochemical test were carried out to detect the presence of phytoconstituents, viz. alkaloids, flavonoids, triterpenoids, saponins, etc. (Kokate et al 1990)

The antioxidant activity of Azima tetracantha leaves was evaluated by DPPH, ABTS and Hydrogen peroxide radical scavenging methods. Azima tetracantha leaves showed a dose dependent scavenging activity and free radical inhibition of DPPH, ABTS and H₂O₂ comparable to free radical scavenging activity of ascorbic acid. (Table 1, 2 and 3)

DISCUSSION:

Reactive oxygen species (ROS) are consisting of free radicals (O₂, HO) and non free radicals (H₂O₂). Freeradicals produced from oxidation reaction start the chain reaction that damage the cell get involved in immune suppression, cell membrane disintegration, membrane protein damage and DNA mutation, which can further initiate the development of many diseases like cancer, liver injury, cardiovascular diseases, inflammation, diabetes, atherosclerosis etc. The most reactive free radical is the hydroxyl radical which is known to initiate lipid peroxidation and cause fragmentation of DNA leading to mutations. Exogenous antioxidant supplement is helpful to overcome this severe problem of free radicals, which may scavenge these free radicals.

T he free radical scavenging activity of natural compounds can be evaluated through their ability to quench the synthetic free radicals, in which the absorbance of the reaction mixture is taken in visible range to know whether the compound is having antioxidant activity.

DPPH assay is based on the measurement of the scavenging ability of antioxidant towards the stable DPPH radical. DPPH is relatively stable nitrogen centered free radical that can accept an electron or hydrogen radical to become a stable diamagnetic molecule. DPPH radicals react with suitable reducing agent as a result of which electron become paired off forming the corresponding hydrazine. The solution therefore looses color stoichometrically depending on the number of electrons consumed which is measured spectrometrically at 517 nm (Halliwell 1991). From the results it may be postulated that the leaves extract of Azima tetracantha have hydrogen donors, thus scavenge the free radical DPPH.

ABTS assay is relatively recent one, which involves a more drastic radical, chemically produced and, is often used for screening complex antioxidant mixture such as plant extracts, beverages and biological fluids. The solubility in both the organic and aqueous media and the stability in a wide pH range raised the interest in the use of ABTS radical for the estimation of the antioxidant activity( Min et al 2002). The principle behind the technique involves the reaction between ABTS and potassium persulphate to produce the ABTS radical cation (ABTS+) a blue green chromogen. In the presence of antioxidant reductant, the colored radical is converted back to colorless ABTS, the absorbance of which is measured at 734 nm. The leaves extract of Azima tetracantha possessed an antioxidant activity with IC50 value being 211 µg/ml , suggest the free radical scavenging activity of Azima tetracantha leaves extract.

Hydrogen peroxide is a weak oxidizing agent and can inactivate a few enzymes directly, usually by oxidation of essential thiol (-SH) groups. Hydrogen peroxide can cross cell membrane rapidly. Once inside the cell, H₂O₂ can probably react with Fe²⁺and possibly Cu²⁺to form hydroxyl radical and this may be the origin of many of its toxic effects. It is therefore biologically advantageous for cells to control the amount of hydrogen peroxide that is allowed to accumulate (Senthilkumar et al 2008). The decomposition of H₂O₂ by leaves extract of Azima tetracantha may result from its antioxidant and free radical scavenging activity.

CONCLUSION:

The results obtained in the present study indicate that Azima tetracantha leaves extract exhibits free radical scavenging activity. The overall antioxidant activity of Azima tetracantha leaves extract might be attributed to its polyphenolic content and other phytochemical constituents. The findings of the present study suggest that Azima tetracantha leaves could be a potential source of natural antioxidant that could have greater importance as therapeutic agent in preventing or slowing oxidative stress related degenerative diseases

Table -1: DPPH free radical Scavenging activity of Azima tetracantha leaves

Ethanol Extract of Azima tetracantha leaves
	Conc.(mg/ml)	Absorbance ±SD (n=3)	% Inhibition
1	Control	0.948±0.007	---
2	10	0.633±0.039	33.2
3	50	0.536±0.002	43.25
4	100	0.466±0.001	50.84
5	150	0.400±0.001	57.80
6	200	0.293±0.010	69.09
7	300	0.257±0.003	72.89
8	500	0.714±0.002	81.64
9	IC 50	155µg/ml
10	IC 50 (Std.) Ascorbic acid	16μg/ml

Table -2: ABTS radical cation decolourisation assay of Azima tetracantha leaves

Ethanol Extract of Azima tetracantha leaves
	Conc.(mg/ml)	Absorbance ±SD (n=3)	% Inhibition
1	Control	0.916±0.036	---
2	10	0.868±0.038	5.24
3	50	0.730±0.010	20.3
4	100	0.591±0.053	43.34
5	150	0.433±0.056	52.72
6	200	0.417±0.064	54.47
7	300	0.289±0.007	68.4
8	500	0.259±0.005	81.20
9	IC 50	211µg/ml
10	IC 50 (Std.) Ascorbic acid	258μg/ml

Table-3: Hydrogen peroxide scavenging activity of Azima tetracantha leaves

Ethanol Extract of Azima tetracantha leaves
	Conc.(mg/ml)	Absorbance ±SD (n=3)	% Inhibition
1	Control	0.956±0.016
2	100	0.776±0.052	18.82
3	200	0.638±0.127	33.26
4	300	0.515±0.157	46.12
5	500	0.399±0.173	58.26
6	1000	0.263±0.077	72.48
7	1500	0.151±0.043	84.20
8	IC 50	619mg/ml
9	IC 50 (Std.) Ascorbic acid	405μg/ml

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Received on 20.06.2012

Modified on 22.07.2012

Accepted on 26.07.2012

Research J. Science and Tech. 4(4): July-August. 2012: 148-151