1. INTRODUCTION:

Since ancient times, many official herbs have provoked interest as sources of natural products. They have been screened for their potential uses as alternative remedies for the treatment of many diseases from the toxic effects of oxidants¹. A number of compounds with strong antioxidant activity have been identified in these plant extracts². The potential of the antioxidant constituents of plant materials for the maintenance of health and protection from coronary heart disease is also raising interest among scientists³.

The main characteristic of an antioxidant is its ability to trap free radicals. Free radicals which have one or more unpaired electrons are produced during normal and pathological cell metabolism. Reactive oxygen species (ROS) react easily with free radicals to become radicals themselves.

ROS are various forms of activated oxygen, which include free radicals such as superoxide anion radicals (O₂^.) and hydroxyl radicals (OH^.), as well as non-free radical species (H₂O₂) and the singlet oxygen (¹O₂)⁴. Antioxidants are compounds that can delay or inhibit the oxidation of lipids or other molecules by inhibiting the initiation or propagation of oxidative chain reactions⁵. The antioxidative effect is mainly due to components, such as flavonoids⁶, phenolic acids, and phenolic diterpenes⁷. The antioxidant activity of compounds is mainly due to their redox properties, which can play an important role in absorbing and neutralizing free radicals, quenching singlet and triplet oxygen, or decomposing peroxides⁸. Antioxidant compounds scavenge free radicals such as peroxide, hydroperoxide or lipid peroxyl and thus inhibit the oxidative mechanisms that lead to degenerative diseases⁹. Recently, antioxidants have potential role in the treatment of atherosclerosis, heart failure, liver dysfunction, neurodegenerative disorders, cancer, and diabetes mellitus¹⁰.

Ethnomedical literature contains a large number of plants that can be used against diseases, in which reactive oxygen species and free radical play important role. There is a plethora of plants that have been found to possess strong antioxidant activity¹¹. Hemidesmus indicus is also known as Anantmool, Sariva and Indian Sarsaparilla belonging to the family asclepiadaceae. They are found all over the greater part of India. These are slender lactiferous, twining, semi erect shrub. They have woody roots, slender and thick stems, leaves are opposite, short petiolate. Flowers are greenish outside and purplish inside¹². It is widely recognized in folk medicine and as ingredient in Ayurvedic and Unani preparations against disease of biliousness, blood diseases, diarrhea, skin diseases, respiratory diseases, fever, eye diseases, burning sensation, rheumatism and gastric disorders¹³. The roots are used as antipyretic, antidiarrhoeal, astrigent, blood purifier, diuretic, refrigerant and tonic. Stem of Hemidesmus indicus used as diaphoretic, diuretic, laxative, and in treating brain, liver and kidney diseases, syphilis, urinary discharges, uterine complaints, leucoderma, cough and asthma. Main chemical constituents are sarsaponin, smilacin, para-methoxy salicylic aldehyde, beta-sitosterol, sarsapogenin, smilgenin, sitosterol, stigmasterol fatty acids and tannins¹⁴.

The aim of this study was to investigate the antioxidant properties of 70% methanolic extract of roots of Hemidesmus indicus (MEHI) against the free radicals.

2. MATERIALS AND METHODS:

2.1 Plant Material

The roots of Hemidesmus indicus was collected from Dawa bazaar, Maharashtra, India. The plant was identified and authenticated by Dr. Harshad Pandit, Department of Botany, Guru Nanak Khalsa College, Matunga, Mumbai, and Maharashtra, India. A voucher specimen is preserved in our laboratory for further reference at Bharati Vidyapeeth’s College of Pharmacy, Navi Mumbai, and Maharashtra, India.

2.2 Extract preparation

The root was ground to a coarse powder and extracted using maceration method in 70% methanol. The extract was filtered through a cotton plug, followed by whattman filter paper (No.1) and then concentrated by using a rotary evaporator at a low temperature (40-60 ⁰C). The extract was preserved in a dessicator till further use.

2.3 Chemicals

1,1-Diphenyl-2-picrylhydrazyl (DPPH), ascorbic acid, potassium ferricyanide, ferric chloride, sodium bicarbonate, trichloroacetic acid (TCA), hydrogen peroxide, sodium nitroprusside, sulphanilamide, phosphoric acid (H3PO4), napthylethylenediamine dihydrochloride, tribromoacetic acid (TBA) , Ethylene diamine tetraacetate(EDTA), methanol, phosphate buffer, phosphate– buffered saline (PBS) , deoxyribose, sodium hydroxide, butylated hydroxy anisole (BHA ). All chemicals used including solvents were of analytical grade.

2.4 Free radical scavenging activity (DPPH method)

The antioxidant activity of the MEHI and standard were assessed on the basis of the radical scavenging effect of the stable DPPH free radical¹⁵. About 10-100 µl of each extract or standard was added to 2 ml of DPPH (HiMedia Laboratories Pvt. Ltd., Mumbai) in methanol (0.33%) in a test tube. After incubation at 37^oC for 30 minutes the absorbance of each solution was determined at 517 nm using spectrophotometer¹⁶. The corresponding blank reading were also taken and the remaining DPPH was calculated by using the following formula,

DPPH radical scavenging activity (%) =

(Absorbance (control) – Absorbance (standard)

-------------------------------------------------------------- x 100

Absorbance (control)]

Where, Absorbance (control): Absorbance of DPPH radical + methanol

Absorbance (standard): Absorbance of DPPH radical+ extract standard.

IC50 value is the concentration of the sample required to scavenge 50% DPPH free radical.

2.5 Reducing power assay

The reducing power of the MEHI was determined according to the method of Oyaizu¹⁷. Different concentrations of MEHI (10–100 µg/ml) in 1.0 ml of deionised water were mixed with phosphate buffer (2.5 ml, 0.2 M, p^H 6.6) and potassium ferrocyanide (2.5 ml, 1%). The mixture was incubated at 50^oC for 20 min. A portion of trichloroacetic acid (2.5 ml, 10%) was added to the mixture, which was then centrifuged at 3000 rpm for 10 min. The upper layer of the solution (2.5 ml) was mixed with distilled water (2.5 ml) and FeCl₃ (0.5 ml, 0.1%) and the absorbance was measured at 700 nm and compared with standards. Increased absorbance of the reaction mixture indicated increased reducing power.

2.6 Scavenging of Hydrogen peroxide

The ability of the MEHI to scavenge hydrogen peroxide was determined according to the method of Ruch, Cheng and Klaunig¹⁸. A solution of hydrogen peroxide (2 mmol/l) (Fine Chem Industries, Mumbai) was prepared in phosphate buffer (p^H 7.4). Extracts (10– 100 µg /ml) were added to hydrogen peroxide solution (0.6 ml). Absorbance of hydrogen peroxide at 230 nm was determined after 10 min against a blank solution containing phosphate buffer without hydrogen peroxide. For each concentration, a separate blank sample was used for background subtraction. The percentage scavenging activity of hydrogen peroxide by MEHI was calculated using the following formula,

% scavenging activity [H₂O₂] = [Absorbance (control) – Absorbance (standard) / Absorbance (control)] × 100.

Where, Absorbance (control): Absorbance of the control and Absorbance (standard): Absorbance of the extract/standard.

2.7 Nitric oxide radical scavenging activity

Nitric oxide was generated from nitroprusside and measured by the Greiss reaction. Sodium nitroprusside in aqueous solution at physiological pH spontaneously generates nitric oxide^19,
20, which interacts with oxygen to produce nitric oxide which, interacts with oxygen to produce nitric ions that can be estimated by use of Griess reagent. Scavengers of nitric oxide compete with oxygen leading to reduced production of nitric oxide¹⁸. Sodium nitroprusside (5 mM) in phosphate– buffered saline (PBS) was mixed with 3.0 ml of different concentrations (10-100 µg /ml) of the drugs dissolved in the suitable solvent systems and incubated at 25⁰C for 150 min. The samples from the above were reacted with Griess reagent (1% sulphanilamide, 2% H₃PO₄ and 0.1% napthylethylene diamine dihydrochloride). The absorbance of the chromophore formed during the diazotization of nitrite with sulphanilamide and subsequent coupling with napthylethylenediamine was read at 546 nm and referred to the absorbance at standard solutions of potassium nitrite, treated in the same way with Greiss reagent.

NO scavenged (%) =

Absorbance (control) – Absorbance (standard) × 100.

Absorbance (control)

Where, Abs (control): Absorbance of the control reaction and Abs (standard): Absorbance of the extract/standard

2.8 Hydroxyl radical scavenging activity

The scavenging capacity of MEHI for hydroxyl radical was measured according to the modified method of Halliwell²¹. The assay was performed by adding 0.1 ml of 1mM EDTA, 0.01 ml of 10 mM FeCl3, 0.1 ml of 10 mM H₂O₂, 0.36 ml of 10 mM deoxyribose, 1.0 ml of different dilutions of the extract (10 – 100 µg/ml) dissolved in distilled water, 0.33 ml of phosphate buffer (50 mM, pH 7.4) and 0.1 ml of ascorbic acid in sequence. The mixture was then incubated at 37 °C for 1 h. A 1.0 ml portion of the incubated mixture was mixed with 1.0 ml of 10% TCA (S. D. Fine Chem. Ltd., Mumbai.) and 1.0 ml of 0.5% TBA (Central Drug House, New Delhi.) to develop the pink chromogen measured at 532 nm. The hydroxyl radical scavenging activity of the extract is reported as % inhibition of deoxyribose degradation and is calculated as,

OH^- scavenged (%) =

Absorbance (control) – Absorbance (standard) × 100.

Absorbance (control)

Where, Absorbance (control): Absorbance of the control reaction and Absorbance (standard): Absorbance of the extract/standard.

2.9 Statistical analysis

Results are expressed as mean ± S.E.M. of three determinants. Comparisons among the groups were tested by two-way ANOVA using Graph Pad Prism, Version 5.0 (Graph Pad Software, San Diego, CA, USA). P- Values < 0.005 were considered significant.

3. RESULTS AND DISCUSSION:

3.1. Free radical scavenging activity

Figure 1 shows the dose-response curve of DPPH radical scavenging activity of the MEHI, compared with ascorbic acid, as standard. At concentration of 50 µg/ ml, the scavenging activity of MEHI was 48.5%, while at the same concentration, that of the standard ascorbic acid was 68%. The effect of antioxidants on DPPH is thought to be due to their hydrogen donating ability²². Though the DPPH radical scavenging abilities of the extracts were less than those of ascorbic acid at 50 µg/ ml.

Figure 1

3.2 Reducing power assay

Reducing power is associated with antioxidant activity and may serve as a significant reflection of the antioxidant activity²³. Compounds with reducing power indicate that they are electron donors and can reduce the oxidized intermediates of lipid peroxidation processes, so that they can act as primary and secondary antioxidants²⁴.

Figure 2 shows the reductive capabilities of the plant extract compared to ascorbic acid. The reducing power of MEHI was very potent and was found to increasing with increased concentration of MEHI. The MEHI could reduce the most Fe³⁺ ions, which had a lesser reductive activity than the standard of ascorbic acid. Increased absorbance of the reaction indicated increased reducing power.

Figure 2

3.3 Scavenging of hydrogen peroxide

Hydrogen peroxide itself is not very reactive, but can sometimes be toxic to cell because it may give rise to hydroxyl radical in the cells²¹. Scavenging of H₂O₂ by extracts may be attributed to their phenolics, which can donate electrons to H₂O₂, thus neutralizing it to water. The extract was capable of scavenging hydrogen peroxide in a concentration-dependent manner. From figure 3 shows that MEHI shows less scavenging activity (H₂O₂) than that of Ascorbic acid. The IC50 value for scavenging of H₂O₂ for MEHI was 66.5 µg/ ml while IC50 value for ascorbic acid was 71 µg/ ml.

Figure 3

3.4 Nitric oxide scavenging activity

Nitric oxide (NO), being a potent pleiotropic mediator in physiological processes and a diffusible free radical in the pathological conditions, reacts with superoxide anion and form a potentially cytotoxic molecule, the peroxynitrite (ONOO‐). Its protonated form, peroxynitrous acid (ONOOH), is a very strong oxidant²⁵. The main route of damage is the nitration or hydroxylation of aromatic compounds, particularly tyrosine. Under physiological conditions, peroxynitrite also forms an adduct with carbon dioxide dissolved in body fluid and responsible for oxidative damage of proteins^{26, 27} in living systems. From figure 4, MEHI showed moderately good nitric oxide scavenging activity between 10 and 100 µg/ ml. The percentages of inhibitions were increased with increasing concentration of the extracts. IC50 value for scavenging of nitric oxide for MEHI was 59 µg/ ml while IC50 value for ascorbic acid was 66 µg/ ml. In addition to reactive oxygen species, nitric oxide is also implicated in inflammation, cancer and other pathological condition.

Figure 4

3.5. Hydroxyl radical scavenging activity

The highly reactive hydroxyl radicals can cause oxidative damage to DNA, lipids and proteins ²⁸. This assay shows the abilities of the extract and standard mannitol to inhibit hydroxyl radical-mediated deoxyribose degradation in Fe3+-EDTA-ascorbic acid and H₂O₂ reaction mixture. From figure 5 shows that MEHI shows less scavenging activity (H₂O₂) than that of Ascorbic acid. The IC50 values of the extract and standard in this assay were 47 µg/ ml and 56 µg/ ml. The IC50 value of the extract was less than that of the standard.

Figure 5

4. CONCLUSION:

On the basis of the results obtained in the present study, it is concluded that an 70% methanolic extracts of roots of Hemidesmus indicus exhibits high antioxidant and free radical scavenging activities. It also chelates iron and has reducing power. These in vitro assays indicate that this plant extract is a significant source of natural antioxidant, which might be helpful in preventing the progress of various oxidative stresses. However, the components responsible for the antioxidative activity are currently unclear. Therefore, further investigations need to be carried out to isolate and identify the antioxidant compound(s) present in the plant extract. Furthermore, the in vivo antioxidant activity of this extract needs to be assessed prior to clinical use.

5. ACKNOWLEDGMENTS:

Authors are grateful to Dr. Harshad Pandit for plant identification, Department of Botany, Guru Nanak Khalsa College, Matunga, Mumbai, India.

6. REFERENCE:

1. K. Hirasa and M. Takemasa, Spice Science and Technology, Marcel Dekker, New York 1998.

2. N. Nakatani, Antioxidants from Spices and Herbs, in Natural Antioxidants: Chemistry, Health Effects, and Applications (Ed. F. Shahidi), AOCS Press, Champaign, 1997, pp. 64–75.

3. J. Loliger, The Use of Antioxidants in Food, in Free Radicals and Food Additives (Eds. O. I. Aruoma and B. Halliwell), Taylor and Francis, London 1991 pp. 129–150.

4. Adedapol A.A., et al. Jimoh F.O., Afolayan A.J. and Masika P.J., et al. Antioxidant properties of the methanol extracts of the leaves and stems of Celtis Africana, Rec. Nat. Prod., 2009, 3, 23-31.

5. Y. S. Velioglu; et al. G. Mazza, L. Gao and B. D. Oomah, Antioxidant activity and total phenolics in selected fruits, vegetables and grain products, J. Agric. Food Chem. 46 (1998) 4113–4117.

6. P. G. Pietta, Flavonoids in Medicinal Plants, in Flavonoids in Health and Diseases (Eds. C. A. Rice- -Evans and L. Packer), Marcel Dekker, New York, 1998, pp. 61–110.

7. F. Shahidi; et al. P. K. Janitha and P. D. Wanasundara, Phenolic antioxidants, Crit. Rev. Food Sci. Nutr. 31, 1992, 67–103.

8. T. Osawa, Novel Natural Antioxidants for Utilization in Food and Biological Systems, in Postharvest Biochemistry of Plant Food-materials in the Tropics (Eds. I. Uritani, V. V. Garcia and E. M. Mendoza), Japan Scientific Societies Press, Tokyo, 1994, pp. 241–251.

9. Kumaraswamy M.V. and Satish S., Antioxidant and Anti-Lipoxygenase activity of Thespesia lampas Dalz & Gibs, Advan. Biol. Res., 2008, 2(3-4), 5659.

10. Ajitha M. and Rajnarayana K., Role of oxygen free radicals in human diseases, Indian Drugs, 2001, 38, 545-554.

11. Badami S., et al. Gupta M.K. and Suresh B., Antioxidant activity of ethanolic extract of Striga orobanchioides, J. Ethnopharmacol., 2003, 85, 227- 230.

12. Anonymous,1997. the wealth of india. raw materials.vol.3,5 and 10,CSIR, New Delhi,India.

13. Nadkarni, A.N.,1989. Indian Material medica, Vol.1, Popular Book Depot, Bombay,India

14. Austin A. A review on Indian sarsaparilla, Hemidesmus indicus (L.) R. Br. J Bio Sci; 8 (1): 1-12, (2008).

15. Blois M.S. Antioxidant determinations by the use of stable free radical, Nature, 1958, 181, 1199-1200.

16. Hwang B.Y., et al. Kim H.S., Lee J.H., Hong Y.S., Ro J.S. and Lee K.S., Antioxidant benzoylated flavan- 3-ol glycoside from Celastrus orbiculatus, J. Nat. Prod., 2001, 64, 82-84.

17. Gupta M., et al. Mazumdar U.K. and Gomathi P., In vitro antioxidant and free radical scavenging activities of Galenga purpurea root, PHCOG MAG., 2007, 3, 219-225.

18. Ruch R.J., et al. Cheng S.J. and Klaunig J.E., Prevention of cytotoxicity and inhibition of intracellular communication by antioxidant catechins isolated from chinese green tea. Carcinogenesis, 1989, 10, 1003-1008.

19. Green L.C., et al. Wagner D.A., Glogowski J., Skipper P.L., Wishnok J.S. and Tannenbaum S.R., Analysis of nitrate, nitrite and [15N] nitrate in biological fluids, Anal Biochem., 1982, 126, 131-138.

20. Marcocci L., et al. Maguire J.J., Droy-Lefaix M.T. and Packer L., The nitric oxide-scavenging properties of gingko biloba ECB 761, Biochem Biophy Res Commun., 1994, 15, 748-755.

21. Halliwell B., et al. Gutteridge J.M. and Aruoma O.I., The deoxyribose method: a simple “test-tube” assay for determination of rate constant for reactions of hydroxyl radicals, Anal. Biochem., 1987, 165, 215- 219.

22. Shirwaikar A., et al. Prabhu K.S. and Punitha I.S.R., In vitro antioxidant studies of Sphaeranthus indicus (Linn), Indian J Exp Biol., 2006, 44, 993-996.

23. Oktay M, Gulcin I, Kufrevioglu OI. Determination of invitro antioxidant activity of fennel (Foeniculum vulgare) seed extracts. Lebensum.Wiss. U.Technol 2003; 36:263‐271.

24. Chanda S, Dave R. In vitro models for antioxidant activity evaluation and some medicinal plants possessing antioxidant properties: An overview. African Journal of Microbiology Research 2009; 3(13):981‐996.

25. Malinski T. Nitric oxide and nitroxidative stress in Alzheimer's disease. J. Alzheimers Dis 2007; 11: 207‐218.

26. Szabó C, Ischiropoulos H, Radi R. Peroxynitrite: Biochemistry, pathophysiology and development of therapeutics. Drug discovery 2007; 6: 662.

27. Burney S, Niles JC, Dedon PC, Tannenbaum SR. DNA damage in deoxynucleosides and oligonucleotides treated with peroxynitrite.Chem. Res. Toxicol 1999; 12: 513‐520.

28. Spencer J.P., et al. Jenner A., Aruoma O.I., Evans P.J., Kaur H., Dexter D.T., Jenner P., Lees A.J., Marsden D.C. and Haliwell B., Intense oxidative DNA damage promoted by L-dopa and its metabolites: Implications for neurodegenerative disease, FEBS Lett., 1984, 353(3), 246-250.

Received on 11.08.2012 Modified on 25.08.2012

Research J. Pharm. and Tech. 5(9): September 2012; Page 1241-1245