Study on radical scavenging activity and analysis of bioactive compounds in selected Indian medicinal plants

Antioxidant therapy promises effective role in treatment of diseases caused due to free radicals. The present study is to examine the antioxidant potential of selected medicinal plants namely Nardostachys jatamansi, Swertia chirayita, Glycyrrhiza glabra, Zingiber officinale, Carum carvi, Trachyspermum ammi, Madhucana indica ,Berberries aristata, Fenniculum vulgare, Myristica fragans. Ethanolic extract were used for analysis. B.aristata showed highest radical scavenging effect on the stable DPPH. radical with IC50 (95.9%) followed by M. fragrans with IC50 (91.8%) which is high in comparison with synthetic antioxidant BHT (74.8%). Superoxide radical scavenging activity, hydroxyl radical scavenging activities, Total antioxidant power, invitro lipid peroxidation were evaluated using concentration range of 1gm,5gm,10gm,15gm,20gm/100ml .Antioxidant activity is reported due to presence of bioactive compounds hence analysed presence of phenols, flavonoids, alkaloids.


MATERIAL AND METHODS
1, 1, Diphenyl-2-picrylhydrazyl, 2, 4, 6tripyridyl-s-trizine(TPTZ) purchased from sigma chemicals co.The other chemicals and solvents used in the present study were of analytical grade obtained from local supplier in pure quality .The plant material were purchased from local supplier.

Preparation of plant extract
The plant material were thoroughly cleaned, shade dried and coarse powdered in a mechanical blender.The powder was successively ethanol, by soxhlet extraction method run for 48hrs.Concentration ranging from was prepared (1gm, 5gm, 10gm, 15gm, and 20gm/100ml).

DPPH radical scavenging activity
DPPH scavenging activity was measured by the method of (Cuedet etal, 1997).To 5 ml of a methanolic solution of DPPH (0.004%), 50µl of test extract (1gm, 5gm, 10gm, 15gm, and 20gm/100ml) were added.BHT was used as standard, for control test extract were replaced by ethanol.The reaction mixture were incubated for 30 minutes at 37 0 C, absorbance was taken at 517nm using Systronic UV-visible spectrophotometer The IC 50 of inhibition was calculated from following equation A 0 -Ax100/ A 0. Therefore, A 0 is absorbance of control and A is absorbance of sample.IC 50 value denotes the concentration of sample required to scavenge 50% of free radicals.

Superoxide radical scavenging activity
The superoxide radical scavenging activity was measured by (Beauchamp and Fedovich 1976) method.Superoxide anion were generated in nonenzymatic hydroxylamine (HA)-EDTA system through the reaction of HA, EDTA and oxygen .It was assayed by reduction of nitroblue tetrazolium .The superoxide anion were generated in reaction mixture containing 1.0ml of sodium carbonate (125mM),0.4mlNBT (24µM) and 0.2 ml of EDTA(0.1mM).The reaction was initiated by adding 0.4ml of hydroxylamine(1mM) and 0.5ml of plant extracts of different concentrations , in control test extract were replaced by ethanol.After 5 minutes of incubation at room temperature, the absorbance was measured at 560nm.The IC 50 of inhibition was calculated from following equation.A 0 -Ax100/A 0.

Hydroxyl radical scavenging activity
The ability of sample to inhibit hydroxyl radical mediated peroxidation was measured by (Kunchandy and Rao, 1990) with some adaptations.The reaction mixture contained 100µl of plant extracts 500µl of (0.6mM) of deoxyribose in phosphate buffer (20mM, pH 7.4), 500 µl ferric chloride (0.1mM) 500µl EDTA(0.1mM),500µl of ascorbic acid (0.1mM) and 100µl of H 2 O 2 (1mM) and 800µl of phosphate buffer so that the final volume is 3ml.After incubation for 1hr at 37 0 C add 1.0 ml of TCA(2.8%) and 1.0ml of (thiobarbituric acid) TBA (1%) place the reaction mixture in water bath for 20 minutes at 100 0 C cool and centrifuge if necessary , the absorbance was measured at 532 nm.BHT was used as standard, in control test extract were replaced by ethanol.The IC 50 of inhibition was calculated from following equation A 0 -Ax100/A 0.

Invitro inhibition of lipid peroxidation
Lipid peroxidation induced by FeSO 4ascorbate system in sheep liver homogenate by method of (Bishayee and Balasubramaniyam, 1971) and the formed thiobarbituric acid reactive substance (TBARS) was estimated by (Ohkawa etal1979).The liver was obtained from slaughter house collected and washed number of times with normal saline To 0.1ml of sheep liver homogenate in (25%) in Tris-HCl buffer (40mM, pH 7.0 ; KCl (30mM);0.16mM of ferrous ammonium sulphate , 0.06mM ascorbic acid), add 0.4ml of plant extract (1gm,5gm,10gm,15gm,20gm/100ml) and incubate for 1hr at 37 0 C , remove 0.4ml of the mixture and add 0.2 ml of (8.1%) SDS, 1.5 ml of (20%) acetic acid, 1.5ml of (0.8%) TBA and incubate in water bath for 1hr at 92 0 C, cool and add 1mlof distilled water and 5ml of butanol: pyridine (15:1) mixture.Shake the reaction mixture and centrifuge at 4000 rpm for 15 minutes and the absorbance of organic layer was measured at 532 nm.

Total antioxidant by FRAP method
The total antioxidant power was determined by the modified FRAP (ferric chloride reducing ability of plasma) method by (Benzie and Strain method, 1996) In this assay FRAP reagent was prepared by adding 2,4,6-tripyridyl-strizine(TPTZ) and ferric chloride forming Fe +3 -TPTZ complex is reduced Fe +2 -TPTZ complex which gives an intense blue colour at 595nm .The calibration curve was prepared using FeSO 4 with concentration ranging from100-1000(M to 1.5ml of FRAP reagent (2, 4, 6-tripyridyl-s-triazine and ferric chloride) add 50(l of plant extracts.The absorbance was measured at 593 nm.The results were expressed as Ascorbic acid Equivalent Antioxidant Capacity (AEAC) in terms of mM.

Statistical analysis
The values are expressed as the means + S.D of three determinants.

RESULTS AND DISCUSSION
The etiology of various human diseases as arteriosclerosis, cancer, neurodegenerative  4 gives data regarding Inhibition of lipid peroxidation which was significant with all plant extracts.
It has been recognized that most of the plants considered in the present study showed significant activity when compared with synthetic antioxidant like BHT among these B.aristata ,M.fragans, N.jatamansi were proved to be most promising source of antioxidant and can be used as natural future source of antioxidants .Hence studies has to done to analyze bioactive responsible for radical scavenging activity In long term study on these plant species may be valuable in treatment for free radical induced damage.

Table 6 : Qualitative phytochemical analyses for the presence of alkaloids, Flavonoids and Phenolics Plants species Bioactive compounds
Valentao et al., 2002).This can lead to production of alkoxy and peroxy radicals causing DNA damage(Reimersma et al., 2000)Table