INTRODUCTION:

Coldenia procumbens Linn (Boraginaceae) is widely distributed in India, Srilanka and other tropical countries. It is used for medicinal purposes in the codified Indian systems of medicine namely Ayurveda and Siddha. But it has not been explored properly and remains a silent drug in herbal medicine.¹

Free radicals are highly reactive molecules in the body that can damage by destroying the enzymes, protein molecules and entire cells. The oxidative damage caused by free radicals is a pivotal mechanism implicated in the progression of a large number of human diseases.²

There is a natural balance between the amount of free radicals generated in the body and antioxidants to quench and protect the body against their deleterious effects.³ Some of the diseases that are mediated through free radical process are cancer, aging, atherosclerosis, cardiovascular diseases, brain metabolism, lung diseases, kidney damage and tissue injuries.

This investigation involves an in vitro analysis of the antioxidant activity of ethanolic extract of the plant by diphenyl-1-picryl hydrazyl method (DPPH) and nitric oxide radical scavenging inhibition activity method. ⁴

EXPERIMENTAL:

Plant source:

Leaves of Coldenia procumbens Linn were collected from west Tambaram, Chennai, India and were authenticated by Prof. P. Jayaraman of Plant Anatomy Research Centre (PARC), Chennai. These were freed from earthy material, washed, shade dried and powdered.

EXTRACTION:

One kg of powdered ariel parts of Coldenia procumbens Linn was taken and 2500 ml of 95% methanol was added. It was refluxed for 2 hours and filtered through muslin cloth while hot. Filtrate was concentrated, evaporated and standardized

Diphenyl-1-picryl hydrazyl method (DPPH)^{5, 6}

Preparation of solutions:

Test solution:

105 mg of the plant extract was dissolved in 5 ml of DMSO to get 21 mg/ml solution. This solution was serially diluted with DMSO to obtain lower dilutions.

Standard solution:

105 mg of ascorbic acid was dissolved in 5 ml of DMSO to get 21 mg/ml solution. This solution was serially diluted with DMSO to obtain lower dilutions.

Method:

The assay was carried out in a 96 well microtiter plate. To 200 µl of DPPH solution, 10µl of each of the test sample or standard solutions was added separately in microtiter plates. The final concentration of the test and standard solution used were 400, 200, 100, 50, 25, 12.5, 6.25, 3.125 µg/ml. The plates were incubated at 37ºC for 30 min and absorbance of each solution was measured at 490 nm using microtiter plate reader(ELISA, Biorad Laboratories INC., California, USA, Model 550) against the corresponding test and standard blanks. The remaining DPPH was calculated. IC₅₀ is the concentration of the sample required to scavenge 50% DPPH free radicals. IC₅₀ value was calculated for the test solutions and also compared with the IC₅₀ value of standard.

Nitric oxide radical scavenging inhibition activity:

Preparation of solutions:

Test solution:

105 mg of Coldenia procumbens Linn was dissolved in 5 ml of DMSO to get 21 mg/ml solution. This solution was serially diluted with DMSO to obtain lower dilutions.

Standard solution:

105 mg of rutin was dissolved in 5 ml of DMSO to get 21 mg/ml solution. This solution was serially diluted with DMSO to obtain lower dilutions.

Method:

The reaction mixture (6ml) containing sodium nitro prusside (4 ml), phosphate buffer saline (PBS, 1 ml) and extract (1ml) in DMSO was incubated at 25ºC for 15 minutes. After incubation, 0.5 ml of the reaction mixture containing nitrite was removed, 1 ml of sulphanilic acid reagent was added, mixed well and allowed to stand for 5 min for completion of diazotization and then 1 ml of naphthyl ethylene diamine dihydrochloride was added, mixed and allowed to stand for 30 min in diffused light. A pink coloured chromophore was formed. The absorbances of these solutions were measured at 540 nm against corresponding blank solution in microtiter plates using ELISA reader. IC₅₀ value was calculated. IC₅₀ value is the concentration of sample required to inhibit 50 % of nitric oxide radical generation.

RESULTS AND DISCUSSION:

Anti oxidant activity of Coldenia procumbens Linn by DPPH method

In DPPH method, the free radical scavenging activity was assessed by adding a methanolic solution of DPPH to the test materials dissolved in methanol at various concentrations. Anti oxidants present in the test materials react with DPPH which is stable free radical and convert into, diphenyl1, 2 –picryl hydrazine. Results were expressed as IC₅₀ in Table 1.

Table 1: Anti oxidant activity of Coldenia procumbens Linn by DPPH method

S. No	Extract	IC₅₀ values (µg/ml)
1	Coldenia procumbens Linn	121.30 + 8.04
2	Ascorbic acid	18.5 + 1.48

Anti oxidant activity of Coldenia procumbens Linn by nitric oxide radical scavenging inhibition method:

In this method, nitric oxide generated from sodium nitro prusside in aqueous solution at physiological pH interacts with oxygen to produce nitrite ions which were measured at 540 nm. Results were expressed as IC₅₀ in Table 2.

Table 2: Anti oxidant activity of Coldenia procumbens Linn by nitric oxide radical scavenging inhibition method

S. No	Extract	IC₅₀ values (µg/ml)
1	Coldenia procumbens Linn	313.5 + 5.8
2	Rutin	91.5 + 3.3

CONCLUSION:

The ethanolic extract of the plant showed an IC₅₀ value of 121.30 + 8.04µg/ml in DPPH method and 313.5 + 5.8 µg/ml in nitric oxide radical scavenging inhibition method.

These results shows the anti oxidant property of the plant which is promising for further in vivo studies.

REFERENCES:

1) Senthamarai R., Uvarani M., Jayakar. Pharmacognostical studies on leaf of Coldenia procumbens Linn. Ancient science of life, 2002, Vol. XXII, 1, 67.

2) Bandyopadhyay U., Das.D., Banerjee.R.K. Reactive oxygen species: oxidative damage and pathogenesis. Current science, 1999, 77, 658.

3) Joe B., Lokesh B.R. Role of capsaicin, curcumin and dietary n-3 fatty acids in lowering the generation of reactive oxygen spieces in rat peritoneal macrophages, Biochemica et Biophysica Acta, 1994, 1224, 255

4) Ashok G., Raghu C., Dhanraj S.A., Suresh B., Vijayan P. Free radical scavenging activity of selected essential oils, Indian drugs. 2003, 41.(4), 244

5) Gopinath N., Srinivasan K.K., Mathew J.E. Free radical scavenging properties of the ethanol extract of Saccharum spontaneum. Indian drugs, 2003, 41, (10), 633.

6) Patil S., Jolly C.I., Narayanan S. Free radical scavenging activity of Acacia catechu and Rotula aquatica : implications in cancer therapy. Indian Drugs, 2003, 40(6), 328

Received on 27.05.2009 Modified on 30.07.2010

Asian J. Research Chem. 4(3): March 2011; Page 450-451