INTRODUCTION:

Oxygen required in many metabolic reactions in the body particularly for the release of energy. In the process oxygen can be transformed under some condition into singlet oxygen hydroxyradical, superoxide anion and hydrogen peroxide. They are highly reactive and cause damage to biomolecules, cells and tissues. These free radicals have been implicated in the progress of several diseases such as cardiovascular disease, cancer, respiratory disease, diabetes and other degenerative disease¹_.Many plants extract and phytochemicals especially the phenolic compounds such as flavonoids and tannins have been shown to have antioxidant free radical scavening properties².

Thespesia populnea soland ex Correa (family Malvaceae) is a large tree found in the tropical regions and coastal forests in India and cultivated in the gardens. All the parts of the plant used in traditional system of medicine. The bark, leaves, flower and fruits are useful in cutaneous infection such as scabies, psoriasis, eczema, ringworm, and guinea worm. The decoction of the bark is commonly used for the treatment of skin and liver diseases. A compound oil of bark and capsules is useful in urethritits and gonorrhea. The bark, root, fruits were used in dysentery, cholera and hemorrhoids³. The fruits of the plant are used in ayurveda for the control of diabetes⁴. An ayurvedic preparation contains Thespesia populnea, namely “panchvalkala” posses’ free radical scavenging activity⁵. The barks and flowers posses astringent, hepatoprotective, antioxidant and anti-inflammatory activities in rats^6-8 and also supposed to improve the memory⁹.

Natural occuring polyphenolic compounds such as tannins, flavinoids are having good antioxidant activity. Several studies have been to access the antioxidant properties of natural products¹⁰. Scientific information on chemical constitutent and antioxidant property of various plants less widely used in the medicine is still rather scare. However relevant experimental work has not been exploited. Therefore our aim in this study was to evaluate the in vitro antioxidant activity of methanolic extract of bark and leaf of Thespesia populnea to determine the relation between its antioxidant properties and its use in traditional medicine.

MATERIALS AND METHODS:

Plant material:

The bark and leaf of the plant were collected from our university Selaiyur campus in Chennai, India. The plant material was taxonomically identified and authenticated by Director, National institute of Herbal Sciences, Chennai. A voucher specimen (PARC/236/07) has been deposited in the herbarium of the same department. The plant materials were powdered. The dried and powdered leaves and stem bark (each 50gm) extracted with 500ml of methanol by maceration process separately for 48 hrs. The methanol was removed under vacuum and a solid mass were obtained.

Estimation of total phenolic content:

The total phenolic content of the extract was estimated according to the method described by Singleton and Rossi¹¹ . The method is as follows; Ten milligrams of standard gallic acid was dissolved in 100 ml distilled water in a volumetric flask (100 mg/ml of stock solution). From the above stock solution 0.5 to 2.5 ml of aliquots were pipetted out into 25 ml volumetric flasks. Ten ml of distilled water and 1.5 ml of Folin Ciocalteu's reagent (diluted according to the label specification) were added to each of the above volumetric flasks. After 5 min, 4 ml of 20% sodium carbonate solution was added and the volume was made up to 25 ml with distilled water and incubated at room temperature for 30 min and the absorbance of the solution was recorded at 765 nm and a standard curve of absorbance verses concentration of gallic acid (50-250 μg) was plotted.

One gram of the powdered drug was extracted with 70% methanol (15 x 3 times), filtered, pooled and the volume was adjusted to 50 ml with 70% methanol in a volumetric flask. From the stock solution, suitable quantity of the extract was taken into a 25 ml volumetric flask and 10 ml of water and 1.5 ml of Folin Ciocalteu reagent were added to it. The mixture was kept for 5 min, and then 4 ml of 20% sodium carbonate solution was added and made up to 25 ml with double distilled water. The mixture was incubated at room temperature for 30 min and the absorbance was recorded at 765 nm in a spectrophotometer. Percentage of total phenolics was calculated from calibration curve of gallic acid (50-250 μg) plotted using the above procedure and total phenolics were expressed as % gallic acid.

Estimation of total tannins¹² :

Two grams of the powdered drug was extracted for 20 h with petroleum ether. The residue was boiled for 2 h with 300 ml of double distilled water. It was cooled, filtered with Whatman No. 1 filter paper and diluted to 500 ml with double distilled water. 25 ml of this infusion was pipetted into 2 liter porcelain dish to which 20 ml indigo solution and 750 ml double distilled water was added. This was titrated with standard KMnO₄ (0.1 N) solution by adding 1 ml at a time, until blue solution changed to green, after which a few drops were added at a time until solution turned golden yellow in colour (A). Similarly, a mixture of 20 ml indigo solution and 750 ml of double distilled water was titrated (B). The percentage of total tannins was calculated using the formula, % Total tannins = [(A-B) x Actual Normality of KMnO₄ solution x 0.004157 x 1000]/Weight of drug sample taken x 0.1. Each ml of 0.1 N KMnO₄ = 0.004157 g of total tannins.

Free radical scavening activity:

Hundred milligram of dried methanolic extract was dissolved in 100ml of methanol to make a stock solution of 1mg/ml. Aliquots from this stock solution were further diluted with methanol as per the concentration required. Free radical scavenging activity of the methanol extract was tested in three in vitro models.

Inhibition of superoxide radicals by riboflavin photo reduction method¹³:

Superoxide scavenging activity of Thespesia populnea was determined by the NBT (Nitroblue tetrazolium) reduction method. The reaction mixture contained EDTA (0.1M) containing 0.0015% NaCN, riboflavin (0.12μM), NBT (1.5μM), various concentration of the extract and phosphate buffer (M/15M pH 7.8) in a final volume of 3ml. The tubes were uniformly illuminated under an incendescent lamp for 15 mins and the optical density was measured at 530nm before and after illumination. The percentage inhibition of superoxide generation was evaluated by comparing the absorbance values of the control and experimental tubes.

Inhibition of lipid peroxide formation by Fe²⁺/ ascorbate system¹⁴:

The reaction mixture contained rat liver homogenate (0.1ml, 25%w/v) in Tris. HCl buffer (20μM, pH 7.0), KCl (150μM), Ferrous ammonium sulphate (0.8μM) and ascorbic acid (0.3μM) in a final volume of 0.5ml and was incubated for 1 hrs at 37⁰C. the lipid peroxide formation was measured. The incubated reaction mixture (0.4ml) was treated with SDS (0.2ml, 8%), thiobarbituric acid (1.5ml, 20%, pH 3.5). The total volume was then made upto 4ml by adding distilled water and kept in a water bath maintained at 100⁰C for 1hrs. After cooling 1ml of distilled water and 5ml of mixture of n-butanol with pyridine (15:1 v/v) were added and shaken vigorously. The absorbance of the organic layer was measured at 560nm after centrifugation. The percentage inhibition of lipid peroxide formation was determined by comparing the results of the drug treated and nontreated samples.

Hydroxyl radical scavenging activity¹⁵:

Hydroxyl radical scavenging was measured by studying the competition between deoxyribose and the extract for hydroxyl radicals generated for the Fe³⁺/ ascorbate/ EDTA/ H₂O₂ system. The hydroxyl radicals attack deoxyribose, which eventually results in TBARS formation. The reaction mixture contained deoxyribose (2-8μM), Fecl₃ (0.1μM), EDTA (0.1μM), ascorbate (0.1μM), potassium hydrogen phosphate-potassium hydroxide buffer (20 μM, pH 7.4) and various concentrations of the drug in a final volume of 1ml. The reaction mixture was incubated for 1hr at 37⁰C. Deoxyribose degradation was measured on TBARS and percentage inhibition was calculated.

RESULT:

Herbal drugs containing free radical scavengers like phenolic, tannins, and flavonoids are known for their therapeutic activity. In the present study the quantitative estimation revealed that the total phenolic content was 10.5 w/w and the total tannin was 3.25 w/w. The bark and leaf extract of Thespesia populnea was found to scavenge the superoxide generated by photoreduction of riboflavin (Figure-1). The concentration of bark and leaf extract needed for 50% scavenging of superoxide was found to be 170μg/ml and 225μg/ml respectively.

Figure-1

The generation of lipid peroxide by Fe²⁺ /ascorbate in rat liver peroxide homogenate was found to be inhibited by the addition of bark and leaf extract of Thespesia populnea (Figure-2). The concentration of extracts needed for 50% inhibition was 200μg/ml and 270μg/ml of bark and leaf extract respectively.

Figure-2

Degradation of deoxyribose mediated by hydroxyl radicals generated by the Fe³⁺ /ascorbate /EDTA /H₂O₂ system was also found to be inhibited by Thespesia populnea extracts (Figure-3). The concentration of extract needed for 50% inhibition was 145μg/ml and 195μg/ml respectively for leaf and bark extracts.

Figure-3

DISCUSSION:

Our finding in this study shows that Thespesia populnea extracts possess potent antioxidant effect. Many plant products are increasingly recognized as having a protective role in coronary heart disease, stroke through several mechanisms including antioxidant¹⁶. Free radicals such as superoxide, nitric oxide and their adducts peroxynitrite, hydrogen peroxide, hydroxyl radicals as well as alkyl peroxy radicals have been implicated in the causation of several diseases¹⁷. Hence, antioxidant activity has important application in therapeutic intervention. Membrane lipids are particularly susceptible to lipid peroxidation¹⁸. Experimental and clinical evidence suggest that aldehyde products of lipid peroxidation can also act as bioactive molecule in physiological and pathological conditions¹⁹. The initial products of peroxidation are conjugated dienes, to which is added oxygen to form lipid peroxide that further breaks down to stable aldehyde and reacts with TBA to form thiobarbituric acid-malanodialdehyde. The present finding strongly suggest that the use of these extracts prevent lipid peroxidation leading to membrane damage. Trace amount of metal ions, primarily ferrous ion, react with H₂O₂ in iron-catalysed ferrous reaction to produce hydroxyl radical. Hydroxyl radical cleave covalent bonds in proteins and carbohydarates, causes lipid peroxidation and destroy cell membranes. Our present study shows that at a concentration of about 145μg/ml and 195μg/ml the extracts showed significant hydroxyl radical scavenging activity.

CONCLUSION:

From the above experiments it is clear that Thespesia populnea and its components showed good free radical scavenging activity which can be attributed in tannins and phenolics along with their compounds.

REFERENCE:

1. Halliwell B, Gutteridge JMC, Cross CE. Free radicals antioxidants and human diseases: Where are we now. J. Lab of Clinical Medicine 1992; 119: 598-620.

2. Larson RA. The antioxidants of higher plants. Phytochemistry 1998; 27:969-978.

3. The Wealth of India. Publication and Information directorate (CSIR), New Delhi. 1995: 223-275.

4. Satyanarayana T, Sarita T, Balaji M, Ramesh A, Boini M.K. Anti-hyperglycemic and hypoglycemic effects of Thespesia populnea fruits in normal and all oxan-induced diabetes in rabbits. Saudi Pharmaceutical Journal 2004; 12: 107-111.

5. Sheetal Anandjiwala, MS Basnl, M Parabia, M Rajani. Evaluation of free radical scavenging activity of an ayurvedic formulation, Panchvalkala. Indian Journal of Pharm. Sciences 2008; 70: 31-35.

6. Illavarasan R, Vasudevan M, Anbazhagan S, Venkataraman S. Antioxidant activity of Thespesia populnea bark extracts against carbon tetrachloride induced liver injury in rats. J. Ethanopharmacology 2003; Aug 87 (2-3): 227-230.

7. Ilavarasan R, Vasudevan M, Anbazhagan S, Venkataraman S, Sridher S.K, Hepatoprotective activity of Thespesia populnea bark extracts against carbon tetrachloride-induced liver injury in rats. Natural Product Sciences 2003; 9: 83-86.

8. Manivasudevan M, Kumar Kishore Gunman, Milind Parle. Antinociceptive and anti-inflammatory effects of Thespesia populnea bark extract. Journal of Ethnopharmacology 2007; 109: 264-270.

9. Vasudevan M, Parle M. Pharmacological action of Thespesia populnea relevant to Alzheimer disease. Phytomedicine. 2006; Nov.13 (9-10): 677-87.

10. Miliauskas G, Venskulonis P.R, Beak T.A. Screening of radical scavenging activity of some medicinal and aromatic plants extract. Food Chemistry. 2004; 85: 237-437.

11. Singleton VL, Rossi JA Jr. Colorimetry of total phenolics with phosphomolybdic acid-phosphotungstic acid reagents. Am J Enol Viticult 1965; 16: 144-58.

12. William H, editor. Official Methods of Analysis. Washington DC: Association of Official Agriculture Chemists 1960: 185.

13. McCord J.M, Fridorich L. Superoxide dismutase, an enzymatic function for erythropoetin. J. Bio. Chem. 1969; 244: 6049-55.

14. Ohkawa, H. Ohishi, N. Yagi, K. Assay for lipid peroxides in animal tissues by thiobarbituric acid reaction. Anal. Biochem. 1979; 95: 351-58.

15. Halliwell B. and Auroma O.I. The deoxyribose method: A simple ‘test-tube’ assay for determination of rate constants for reactions of hydroxyl radicals. Anal. Biochem. 1987; 165: 215-219.

16. Farmica JR. Regelson W. Review of the biology of quercetin and related bioflavonoids. J. Biochem. 1969: 244: 6049-55.

17. Jose N. Janardhanan K.K. Antioxidant and antitumor activity of Plennotus florida. Current Science. 2000; 79: 941-943.

18. Girotti. A.W. Photodynamic lipid peroxidation in biological systems. Photochem and Photobio. 1990; 51: 497-509.

19. Gizzocrea S, Riley DP, Ceputi AP, Salvemini D. Antioxidant therapy-A new pharmacological approach in reperfusion injury. Pharmacol. Rev. 2001; 53, 135-139.

Received on 04.11.2015 Modified on 26.11.2015

Res. J. Pharmacognosy & Phytochem. 8(1): Jan.- Mar. 2016; Page 01-04

DOI: 10.5958/0975-4385.2016.00001.7