Introduction

Medicinal plants have been used for centuries as remedies for human diseases because they contain components of therapeutic value. Free radical role in causing diseases can be compared with role of microorganism in infections disease (1).  Reactive oxygen species are derived from the metabolism of oxygen, this includes superoxide radical (O2-), hydroxyl radical (OH^.-) and hydrogen peroxide (2, 3). Oxidation process is important in producing free radicals in living system (4). In cellular oxidation reactions superoxide transformed into other potentially harmful free radicals causing peroxidation of lipid, protein and DNA damage, (5).Oxidation process can be augmented by antioxidants defence system by scavenging free radicals giving maximum intracellular protection. Due to depletion of natural antioxidants in different maladies, consumption of antioxidants as free radical scavengers has become necessary, inspite of having synthetic antioxidants (6, 7).

Many plant species have been investigated in search for novel antioxidants in the recent time (8,9) but demand still persist .In particular, despite widespread of medicinal plants, the literature supports the importance of medicinal properties of these plants. N. jatamansi (Valerianaceae), the dried rhizomes are known as stimulants, bitter tonic. (10). S.chirayita (Gentianaceae), stem has antipyretic, (11). G. glabra (Leguminosae) roots used to relieve coughs, gastric inflammation (12). Z. officinale (Zingiberacea) roots used as cardiac stimulant, (13). C.carvi (Apiaceae) seeds used in stomach disorders to relieve pain, scabies, (14). T. ammi (Apiaceace) seeds have platelet aggregation inhibitory action .M. indica (Sapotaceae) fruits are used to cure piles, bronchitis. B.aristata (Berberidaceae) roots used  in piles, pain reliever, antituberculosis. F.vulgare (Umbelliferae) seeds are used in bronchitis,. M.fragans (Myristicaceae) seeds anti-platelet aggregation, anti-inflammatory activities.

Materials and Methods

1, 1, Diphenyl-2-picrylhydrazyl, 2, 4, 6-tripyridyl-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 37⁰C, absorbance was taken at 517nm using Systronic UV-visible spectrophotometer The IC⁵⁰ of inhibition was calculated from following equation A₀-Ax100/A_0. Therefore, A₀ is absorbance of control and A is absorbance of sample. IC₅₀ 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 non-enzymatic  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),0.4ml NBT (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 IC₅₀ of inhibition was calculated from following equation. A₀-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 ₂O ₂  (1mM) and 800µl of phosphate buffer so that the final volume is 3ml. After incubation  for 1hr at 37 ⁰C 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 ⁰C 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 IC₅₀ of inhibition was calculated from following equation A₀-Ax100/A_0.

Invitro inhibition of lipid peroxidation

Lipid peroxidation induced by FeSO 4 –ascorbate 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 ⁰C , 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 ⁰C, 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-s-trizine(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 FeSO4 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.

Analysis of bioactive compounds

Presence of major bioactive compounds were analysed by qualitative test for alkaloids (Hagers test, Mayers test, Wagners test), flavonoids (Pew’s,Shinoda test), phenolics (ferric chloride test).

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 disease has shown the involvement of ROS (4). Evidences say that antioxidants may overcome these deleterious consequences of oxidative stress. Free radical scavenging activity was evaluated by DPPH free radical scavenging   method. Ethanol due to high polarity extracts most of the compounds hence exhibit high activity. Increasing concentration of extracts ranging from (1gm, 5gm, 10gm, 15gm, 20gm/100ml) were analysed to find out any proportionality in activity with increase in concentration.  The DPPH assay measured hydrogen atom (or one electron) donating activity and hence provided an evaluation of antioxidant activity due to free radical scavenging. Plant extracts showed decrease in absorbency with a stable DPPH^.-  radicals which indicate a high level of radical scavenging  activity. Table: 1 it infers that B.aristata and M.fragans has shown highest radical scavenging activity in dose dependent order S. chirayita  Z.officinale C. carvi F. vulgares difference in result did not contributed satisfactory activity. Superoxide is biologically important as it can for potent oxidative species, which can damage cellular components . The results are shown in table:-2 gives % of inhibition which is concentration dependent and N.jatamansi, M.fragans showed almost similar pattern highest activity shown by B.aristata BHT (1mg/ml) a synthetic antioxidant used as standard for comparision Hydroxyl radical is very reactive and can be generated in biological cells through Fenton reaction radical scavenging activity .Table: 3 showed 97% Radical scavenging by B.aristata which is significantly high. Initiation of lipid peroxidation by ferrous takes place through hydroxyl radical by Fenton reaction. The degree of inhibition of FeSO4 induced lipid peroxidation in sheep liver homogenate in a function of dose dependent. The inhibition could be due to scavenging hydroxyl radical or superoxide radical or by chelating iron Hydroxyl radicals can interact with membrane lipids to form lipid hydroperoxides (Valentao et al., 2002).This can lead to production of alkoxy and peroxy radicals causing DNA damage (Reimersma et al., 2000) Table:-4 gives data regarding Inhibition of lipid peroxidation which was significant with all plant extracts.

Table 1: DPPH radical scavenging activity with different concentration of ethanolic Extract Comparison of IC₅₀ with standard BHT (1mg/ml).The values are expressed as scavenging %. BHT shown 74.8% inhibition.

Table 2: Superoxide radical scavenging activity of ethanolic plant extract .Comparison of IC₅₀ with standard BHT (1mg/ml), with 69.4% of inhibition 

Table 3: Gives IC₅₀ value which is   concentration dependent and compared with BHT (1mg/ml) standard which exhibited 71% scavenging activity .

Table 4: In vitro lipid peroxidation of ethanolic extract in comparison with standard BHT (1mg/ml) 79.3% of inhibition.

Table 5: Total antioxidants activity by FRAP method with different concentration of ethanolic extract (concentration in mM).

Table 6: Qualitative phytochemical analyses for the presence of alkaloids, Flavonoids and Phenolics.

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.

Acknowledgement

I am thankful to M.V.R.P.G college, Gajuwaka, Visakhapatnam management for their support to provide requirements to carry out the work, Dr. M.Ramarao for his valuable guidance.

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