INTRODUCTION:

Oxygen is essential for survival however, its univalent reduction generates several harmful reactive oxygen species (ROS), inevitable to living cells and highly associated with the wide range of pathogenesis such as diabetes, liver damage, inflammation, aging, neurological disorders and cancer. In spite of comprehensive network of cellular defensive antioxidants, many ROS still escape this surveillance inflicting serious anomalies favoring such diseases states1 (Ester Bauer H, 1996; Halliwell B et al 1993; Sies H 1997). Though synthetic antioxidants, BHT, BHA and radio protector, War far in are being used widely, however, due to their potential health hazards, they are under strict regulation (Satio M et al 2003; . Rades D et al 2004) .Antioxidant principles from natural resources are multifaceted in their multitude/magnitude of activities and provide enormous scope in correcting the imbalance through regular intake of proper diet. Therefore, in the recent years, the interest is centered on antioxidants derived from herbal medicine in view of their medicinal benefits (Kamat JP 2007; Kamat JP et al 2004; Umadevi P et al 1995) .Phyto antioxidants, commonly available, less toxic, serving food and medicinal components have been suggested to reduce threat of wide range of ROS (Winston JC 1999; . Arora S et al 2003)

Mimosa pudica (Mimosaceae) known as chue Mue, is a stout stragling prostrate shrubby plant with the compound leaves which gets sensitive on touching, pinousstipules and globose pinkish flower heads, grows as weed in almost all parts of the country (Ghani, 2003). Leaves and stems of the plant have been reported to contain an alkaloid mimosine, leaves also contain mucilage and root contains tannins (Ghani, 2003). Mimosa pudica is used for its anti diarrhoeal (Balakrishnan, et al., 2006), anti-convulsant (Bum, et al., 2004) and cytotoxic properties (Sadia Afreen Chowdhury, et al., 2008). The plant also contains turgorins, leaves and roots are used in treatment of piles and fistula. Paste of leaves is applied to hydrocele. Cotton impregnated with juice of leaves is used for dressing sinus. Plant is also used in the treatment of sore gum and is used as a blood purifier (Ghani, 2003)

MATERIALS AND METHODS:

Reagents:

DPPH (Sigma – Aldrich, USA), Methanol (HPLC grade, Merck, India),Ascorbic acid (Analytical grade, Merck, India), Rutin, Potassiumpersulphate, ABTS (2, 2’–azino-bis-(3-ethylbenzothiazoline-6-sulfonic acid), Phosphate buffered saline, Dimethyl sulfoxide (Merck,India), Hydrogen peroxide, sodium nitroprusside, Sulphanilic cid(0.3% w/v), Naphthyl ethylene diamine dihydrochloride (NEDD,0.1%).

Preparation of extract: The leaves of Mimosa pudica were procured from the Pattukkottai in the month of Febuary-2010. The coarsely powdered leaves (300g) of Mimosa pudica was extracted to exhaustion in a sock let apparatus at 50oC with 500ml of methanol. The extract was filtered through a cotton plug, followed by what man filter paper (no.1) and then concentrated by using a rotary evaporator at a low temperature (40-60oC) and reduced pressure to provide methanolic extractive of 8.20g.

In vitro antioxidant activity:

DPPH Assay (Badami S et al, 2005)

The assay was carried out in a 96 well microtitre plate. To 200 μl of DPPH solution, 10 μl of various concentrations of the extract or the standard solution was added separately in wells of the microtitre plate. The plates were incubated at 37 ºC for 30 min. Absorbance was measured at 490 nm using ELISA reader. IC50 value is the concentration of the sample required to scavenge, 50 % DPPH free radical.

ABTS radical cation decolourisation assay (Re R, Pellegrini N et al, 1999):

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 ofthe extracts or standards, 1.0 ml of distilled DMSO and 0.16 ml ofABTS 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.

Scavenging of hydrogen peroxide (Jayaprakah GK et al, 2004)

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.

Scavenging of Nitric Oxide radical (Marcooci L et al 1994)

In the present investigation, Griess Ilosvay reagent is modified by using Naphthyl ethylene diamine dihydrochloride (0.1% w/v)in stead of 1‐napthylamine (5%). Nitrite ions react with Griess reagent, which forms a purple azo dye. In presence of test components, likely to be scavengers, the amount of nitrites will decrease. The degree of decrease in the formation of purple azo dye will reflect the extent of scavenging. The reaction mixture (6 ml) containing sodium nitroprusside (10 mM, 4 ml), phosphate buffer saline (PBS, pH 7.4, 1 ml) and extract in DMSO at various concentrations or standard was incubated at 25o C for 150 min. After incubation, 0.5 ml of the reaction mixture containing nitrite ion was removed, 1 ml of sulphanilic acid reagent was added, mixed well and allowed to stand for 5 min for completion of diazotisation. Then, 1ml of NEDD was added, mixed and allowed to stand for 30 min in diffused light. A pink coloured chromophore was formed. The absorbance of these solutions was measured at 540 nm.

RESULTS AND DISCUSSION:

In the present study, ethanolic extract of Mimosa pudica (Mimosaceae) were studied for in vitro antioxidant activity by four radical scavenging methods which is summarized in Table 1. The extract showed potent scavenging activity with IC50 values of 78.1±1.75 and 35.00±1.15μg/ml, respectively in the cases of nitric oxide and DPPH free radicals. The other methods shown moderate activity which is all compared with standards. The variations in activity may be due to the fact that diversity in the basic chemical structure of phytoconstituents possesses different degree of antioxidant activity against different free radicals. The preliminary phytochemical investigation revealed the presence of phenolic compounds in the ethanol extract of the plant. Plant phenolics are known to exhibit potent antioxidant activity (Velioglu YS et al 1998). Hence, the observed antioxidant activity of the extracts of Mimosa pudica (Mimosaceae) may be due to the presence of these constituents. DPPH has been used to evaluate the free radical- scavenging activity of natural antioxidants. DPPH which is a radical itself with a purple color, changes into a stable compound with a yellow color by reacting with an antioxidant and the extent of the reaction depends on the hydrogen donating ability of the antioxidant. (Chen CW et al 1995). The ability of ethanolic extract of Mimosa pudica (Mimosaceae) scavenge DPPH radicals suggests that it is an electron donor and can react with free radicals to convert them to more stable products and terminate radical chain reactions. The technique in ABTS assay involves there action between ABTS and potassium persulphate to produce the ABTS radical cation, a blue green chromogen. In the presence of the antioxidant reductant, the colored radical cation is converted back to colorless. It is applicable for the study of both water‐soluble and lipid‐soluble antioxidants.

Hydrogen peroxide itself not very reactive, but it can sometimes be toxic to cell because of it may give rise to hydroxyl radical in the cells (Halliwell B, 1991). Thus removing of H₂O₂ is very important for antioxidant defense in cell or food systems.

Nitric oxide is produced by several different types of cells, including endothelial cells and macrophages. The early release of nitric oxide through the activity of constitutive nitric-oxide synthase is important in maintaining the dilation of blood vessels the much higher concentrations of nitric oxide produced by inducible nitricoxide synthase in macrophages can result in oxidative damage. Nitric oxide reacts with free radicals, thereby producing the highly damaging peroxy nitrite. Nitric oxide injury takes place for the most part through the peroxynitrite route because peroxy nitrite can directly oxidize LDLs, resulting in irreversible damage to the cell membrane

Table 1: In vitro antioxidant activity of ethanol extract of Mimosa pudica leaves

Extracts /

Standards

IC50 values ± SE μg/ml* by methods

DPPH

ABTS

H202

Nitric oxide

Concent ration

μg/ml

Inhibition

Concent ration

μg/ml

Inhibition

Concent ration

μg/ml

Inhibition

Concent ration

μg/ml

Inhibition

Ethanol

extract of

Mimosa pudica

125

62.5

31.25

15.6

89.50±0.75

82.80±1.65

45.90±0.50

15.70±1.00

500

250

125

62.5

79.50±1.15

71.36±0.80

62.18±0.50

44.50±0.35

500

250

125

62.5

52.65±0.25

41.50±0.27

28.65±0.80

16.90±0.28

500

250

125

62.5

72.36±0.80

61.90±1.50

56.25±0.50

47.80±0.65

IC50 μg/ml

35.00±1.15

81.00±3.85

449.60±1.30

78.10±1.75

Standards

Ascorbic

Rutin

2.69 ± 0.05

11.25 ± 0.49

187.33 ± 1.93

---------

-------

0.51± 0.01

36.66 ± 0.22

65.44±2.56

*Average of four determinations, values were mean ± S.E.M *P<0.001(Turkey‐Kramer equation)

CONCLUSION:

Based on the results of the present study, we conclude that the plant extract possesses antioxidant potential. However, further studies are necessary to examine underlying mechanisms of antioxidant effect sand to isolate the active compound (s) responsible for these pharmacological activities.

REFERENCES:

1. Arora S, Kaur K, Kaur S. Indian medicinal plants as reservoir of protective phytochemicals. Teratog Carcinog Mutagen. Suppl1:295-300. (2003)

2. Balakrishnan N, Suresh D,Pandian GS ,Edwin E,Sheeja E,Anti –diarrhoeal potential of mimosa pudica root extracts .Indian J Nat Prod ., 22(2):21-23. (2006)

3. Badami S, Om Prakash, SH, Dongre, Suresh B. Invitro antioxidant properties of Solanum pseudocapsicum leaf extracts, Indian JPharmacol., 37:251-2(2005)

4. Bum EN, Dawack DL, Schmutz M, Rakotonirina A, Herding P, et al. Anticonvulsant activity of Mimosa pudia decoction. Fitoterapia., 75(3-4):309-314(2004)

5. Chen CW, Ho CT. Antioxidant properties of polyphenols extracted from green and black teas. J Food lipids 2: 35-46.( 1995)

6. Esterbauer H. Estimation of peroxidative damage: A critical review. Pathologies Biol (Paris)., 44: 25-28. (1996)

7. Ghani A. Medicinal plants of Bangladesh, the Asiatic society of Bangladesh, 2nd ed. Dhaka.,. p. 302-3. (2003)

8. Halliwell B, Gutteridge JMC. Free Radicals in Biology and Medicine, 2nd edition. London: Clarendon Oxford Press; (1993 ).

9. Halliwell B, Reactive oxygen species in living systems: Source biochemistry and role in human disease. Am J Med; 91 Suppl., 14–22. (1991)

10. Jayaprakah GK, Lingamallu JR, Kunnumpurath KS. Antioxidant activities of flavibin in different invitro model systems, Bioorg MedChem., 12: 5141-6. (2004)

11. Kamat JP. Phytochemicals and radioprotection. In: Chiranjib Chakraborty, editor. Advances in biochemistry and biotechnology, New Delhi: Daya Publishing House;, In press. (2007)

12. Kamat JP, Venkatachalam SR. Asparagus racemosus and radioprotection, In: K G Ramawat, editor. Biotechnology of medicinal plants vitalizer and therapeutic, New Delhi:Sci.Publ.Inc./Oxford/IBH Pub. Co. p.78-84.( 2004)

13. Marcooci L, Packer L, Sckaki A, Albert, GM, Antioxidant action of Ginkgo biloba extract EGb 766, Methods Enzymol., 234: 462-75(1994)

14. Rades D, Fehlauer F, Bajrovic A, Mahlmann B, Richter E,Alberti W.Serious adverse effects of amifostine during radiotherapy in head and neck cancer patients. Radiotherapy Oncol ., 70: 261-4 (2004)

15. Re R, Pellegrini N., Proteggente, A., Pannala, A., Yang, M, Rice‐Evans, C. Antioxidant activity applying an improved ABTS radicalcation decolorization assay, Free Radic Biol Med ., 26: 1231-7(1999)

16. Satio M, Sakagami H, Fujisawa S. Cytotoxicity and apoptosis induction by butylated hydroxyanisole (BHA) and butylatedhydroxytoluene (BHT). Anticancer Res., 23: 4693-701(2003)

17. Sies H, editor. Antioxidants in disease Mechanisms and therapy, New York: Academic Press; 1997.

18. Velioglu YS, Mazza G, Gao L, Oomah BD, Antioxidant activity and total phenolics in selected fruits, vegetables, and grain products,J Agrig Food Chem.,46:4113–7(1998).

19. Umadevi P, Ganasoundari A. Radioprotective effect of leaf extract of Indian medicinal plant Ocimum Santum. Indian J Exp Biol., 33: 205-8(1995).

20. Winston JC. Health-promoting properties of common herbs. AmerJ Clin Nutr., 70 Suppl: 491-99 (1999).

Received on 06.05.2011 Accepted on 14.06.2011

Asian J. Pharm. Res. 1(2): April-June 2011; Page 44-46