INVITRO AND INVIVO ANTIOXIDANT ACTIVITY OF PSEUDARTHRIA VISCIDA Majority Of the diseases/disorders are mainly linked to oxidative stress

due to free radicals(Benzie .,2003). Free radicals are fundamental to any biochemical process and represent an essential part of aerobic life and metabolism(Fang etal.,2002,manavalan .,2002). The most common reactive oxygen spices (ROS) include super oxide anion (O2.), hydroxyl radical (OH.), hydrogen peroxide (H2O2) peroxyl radical radicals (ROO.). The nitrogen derived free radicals are nitric oxide (NO.) and peroxynitrite anion (ONOO.)( Nagendrappa.,2005). ROS have been implicated in over a hundreds of diseases states which range from arthritis and connective tissue disorders to carcinogenesis, aging, physical injury, infection and cardio vascular malfunction(Nordberg J.,2001, Ray.,2001). In treatments of these diseases, antioxidant therapy has gained an immense importance. Current research is now directed towards finding naturally occurring antioxidants of plant origin. Antioxidants have been reported to prevent oxidative damage by free radical and ROS; any may prevent the occurrence of disease, cancer and aging. It can interfere with the oxidation process by reacting with free radicals, chelating, catalytic metals, and also by acting as oxygen scavengers(Halliwell.,1999, Daniel et al.,1998). Plant and plant products are being used as a source of medicine since long. The medicinal properties of plants have been investigated in the recent scientific developments throughout the world, due to their potent antioxidant activities, no side effects and economic viability(Agarwal.,2005, Chaurasia et al.,1995). Chronic diseases is the involvement of oxidative stress, related to the production of reactive oxygen species (ROS). Due to their high reactivity and low stability, ROS (hydroxyl radical, super oxide anion radical, hydrogen peroxide, singlet oxygen, nitric oxide radical, hypochlorite radical, and various lipid peroxides) enter reactions to lipids, proteins and deoxyribonucleic acid (DNA), generating oxydized metabolites and DNA adducts(Kimura et al .,2005). The ubiquitos nature of ROS targets explains the large array of damage that these reactive molecules bring about in any living organism, causing a progressive functional deterioration of cells, tissues and organ systems(Valko et al ., 2007). The unifying theory of oxidative damage provides a plausible and currently accepted global mechanism underlying a diversity of diseases, ranging from atherosclerosis, inflammations, degenerative disorders of the locomotor and nervous systems, up to cancer. Aging as well is characterized by an imbalance between damage inflicted by reactive oxygen species (ROS), and the antioxidative defenses of the organism(Afanasev.,2005). As oxidative damage has been pointed out as a major factor in the molecular mechanisms of several conditions and aging, antioxidative capacity is likely to represent an important feature of modern medications which are able to act on multiple levels of the disease processes. Commercially available antioxidants obtained through chemical synthesis usually possess very strong and unspecific antioxidative effects, blocking the signaling pathways using ROS(Hancock., 2001).Pseudarthria Viscida is a pennial viscid pubescent semi-erect diffuse under shrub, 60-120cm. . It alleviates all the three doshas. It possesses heavy and oily attributes. It has antipyretic, aphrodisiac and rejuvenative properties and is used in the diseases like fever, bronchial asthma, hemorrhoids, edema, diabetes mellitus, diarrhea and tuberculosis(Nadkarni.,1982).

MATERIALS AND METHODS

Plant collection and Authentification: The roots of Pseudarthria Viscida were collected from the Nellore district and the sample was authenticated for their correct botanical identity by Professor Jayaraman, National institute of herbal science, Chennai. Processing of plant material Plant material was washed thoroughly with distilled water, to remove soil particles and other impurities and shade dried. The shade dried plant was powdered by the mixer. Plant extraction by maceration: The dried powdered roots (500gm) were defatted with petroleum ether and the material was extracted with ethanol (2L) for five days and filtered with muslin cloth and the marc was pressed. The ethanolic extract was evaporated until it was dried. The yield of Pseudarthria viscida ethanolic extract was 0.86% respectively. The ethanolic extract was stored in refrigeration condition until it was used for further tests. INVITROANTIOXIDANT STUDIES Hydroxyl Radical Scavenging activity The hydroxyl radical scavenging activity was measured by studying the competition between deoxyribose and the extract for hydroxyl radicals generated from the Fe 3+/ ascorbate / EDTA / H2O2 system. The reaction mixture contained deoxyribose (2.8mM), Fecl 3 (0.1mM), EDTA (0.1mM, H2O2 (1mM), ascorbate (0.1mM), KH 2PO4-KOH buffer (20mM, pH 7.4) and various concentrations (100-300 g/ml) in a final volume of 1 ml. The reaction mixture was incubated for 1 hr at 37 oC. Deoxyribose degradation was measured at 532 nm(Mary et al ., 2002) Hydrogen peroxide scavenging activity Hydrogen peroxide solution(2mM) was prepared with standard phosphate buffer (pH,7.4). Extarct samples (10-160 g/ml) in distilled water added to hydrogen peroxide solution(0.6ml). Absorbance of hydrogen peroxide at 230nm ws determined after 10min against a blank solution containing phosphate buffer without hydrogen peroxide. The percentage scavenging of hydrogen peroxide of plant extract was determined (Mary et al ., 2002) . Determination of reducing power 10mg of ethanolic extract of Pseudarthria Viscida in 1ml of distilled water was mixed with phosphate buffer (2.5 ml, 0.2 M, pH 6.6) and potassium ferricyanide [K 3 Fe(CN)6] (2.5 ml, 1%). The mixture was incubated at 50C for 20 min. A portion (2.5 ml) of trichloroacetic acid (15%) 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 ferric chloride, (0.5 ml, 0.1%), and the absorbance was measured at 700 nm. Increased absorbance of the reaction mixture indicates increased reducing power. (Nagulendran et al.,2007). Nitric Oxide Scavenging: Nitric oxide scavenging activity was measured by using a spectrophotometer. Sodium nitroprusside (5mM) in phosphate buffered saline was mixed with different concentrations of the extract (10-160 g/ml) dissolved in methanol and incubated at 25C for 30 min. A control without test compound but with equivalent amount of methanol was taken. After 30 min 1.5 ml of the incubation solution were removed and diluted with 1.5 ml of Griess reagent (1% sulphanilamide, 2% phosphoric acid, and 0.1% napthyl ethylene diamine dihyrochloride). The absorbance of the chromophore formed during diazotization of the nitrite with sulphanilamide and subsequent coupling with napthylethylene diamine was measured at 546 nm(Balakrishnan et al.,2002).. INVIVO ANTIOXIDANT STUDIES Rats were divided into three groups consisting of 6 per group.Group 1 received Vehicle 2%v/v Tween 80, Group 2 received ethanolic extract of Pseudarthria Viscida (100 mg/kg, p.o) and Group 3 received ethanolic extract of Pseudarthria Viscida (200 mg/kg, p.o) respectively for 14 days .On 15th day animals were decapitated under ether anesthesia and the brains quickly removed, cleaned with ice-cold saline . Brain tissue samples were homogenized with 10 times (w/v) by homogenizer in ice-cold 0.1 M phosphate buffer (pH 7.4). Homogenates were centrifuged and supernatant was then used to determine protein, lipid peroxidation and glutathione. Catalase activity was determined immediately after sample preparation and SOD was determined with in 24 h. Protein concentration was determined according to (Lowry et al. 1951) Estimation of MDA Malondialdehyde (MDA) a measure of lipid peroxidation was measured as described by (Jainkang et al.,1990). Reagents acetic acid 1.5 ml (20%) pH 3.5, 1.5 ml thiobarbituric acid (0.8%) and 0.2 ml sodium dodecylsulphate (8.1%) were added to 0.1 ml of processed tissue samples, then heated at 100 C for 60 min. Mixture was cooled under tap water and 5 ml of nbutanolpyridine (15:1), 1 ml of distilled water was added and vortexed vigorously. After centrifugation at 4000 rpm for 10 min, the organic layer was separatedand absorbance was measured at 532 nm using a spectrophotometer and concentration of MDA was expressed as nmol/g tissue.

Estimation of glutathione Glutathione was measured according to the method of Ellman (1959). The equal quantity of homogenate was mixed with 10% trichloroacetic acid and centrifuged to separate the proteins. To 0.01 ml of this supernatant, 2 ml of phosphate buffer (pH 8.4), 0.5 mlof 5_5-dithiobis(2nitrobenzoic acid), and 0.4 ml of double distilled water was added. Mixture was vortexed and the absorbance read at 412 nm within 15 min. The concentration of glutathione was expressed as g/gtissue.

Estimation of catalase Catalase activity was measured by the method of Aebi (1974). 0.1 ml of supernatant was added to cuvette containing 1.9 ml of 50 mM phosphate buffer (pH 7.0).Reaction was started by the addition of 1.0 ml of freshly prepared 30 mM H2O2. The rate of decomposition of H2O2 was measured spectrophotometrically from changes in absorbance at 240 nm. Activity of catalase was expressed as units/mg protein. Estimation of Superoxide dismutase (SOD) To 50 l of the suspension, 75 mM of Tris-HCl buffer (pH 8.2), 30 mM EDTA and 2 mM of pyrogallol were added. An increase in absorbance was recorded at 420 nm for 3 min by spectrophotometer. One unit of enzyme activity is 50% inhibition of the rate of autooxidation of pyrogallol as determined by change in absorbance/min at 420 nm. The activity of SOD is expressed as units/mg protein. (McCord &Fridovich.,1969). RESULTS

PRELIMINARY PHYTOCHEMICAL TESTS SL.NO PHYTOCHEMICAL TESTS ETHANOLIC EXTARCT 1 2 3 4 5 6 7 8 Test for Alkaloids Test for Steroids Test for Phytosterols Test for Tannins Test for Flavanoids Test for Saponins Test for Gums and mucilages Test for Carbohydrates -ve -ve -ve +ve +ve +ve -ve +ve

TLC profile of ethanolic extract of Pseudarthria viscida root

The Rf value of the TLC profile of Pseudarthria Viscida is 0.8

INVITROANTIOXIDANT STUDIES

Table -1 Hydroxyl radical scavenging activity of ethanolic extract of Pseudarthria Viscida Sl. No. 1 2 3 4 5 Concentration (g/ml) 10 20 40 80 160 Inhibition (%) 37.80 0.24 48.95 0.46 57.96 0.31 66.81 0.45 93.20 0.15 Ic50 Value((g/ml)

23 0.42

Mannitol 100

63.17 0.92

Values are Mean SEM of 3 replicates.

Hydroxyl radical

100 90 80 70 60 50 40 30 20 10 0 10 20 40
concentration

inhibition %

80

160

Table -2Hydrogen Peroxide Viscida Sl. No. 1 2 3 4 5 Concentration (g/ml) 10 20 40 80 160

Scavenging activity of ethanolic extract of Pseudarthria Ic50 Value((g/ml)

Inhibition (%) 32.92 0.83 63.14 0.62 74.44 0.73 87.64 0.29 91.27 0.28

16 0.72

Values are Mean SEM of 3 replicates.

hydrogen peroxide

100 90 80 70 60 50 40 30 20 10 0 10 20 40
concentration

inhibition %

80

160

Table -3 Determination of reducing power of ethanolic extract of Pseudarthria Viscida Sl. No. 1 2. 3. 4. 5. Concentration (g/ml) 10 20 40 80 160 Absorbance 0.022 0.001 0.078 0.002 0.092 0.001 0.233 0.012 0.241 0.002

Values are Mean SEM of 3parallel measurements.

Table -4 Nitric oxide Scavenging activity of ethanolic extract of Pseudarthria Viscida Sl. No. 1 2 3 4 5 Concentration (g/ml) Inhibition (%) 10 20 40 80 160 46.81 0.65 55.23 1.50 61.87 0.33 70.34 1.13 82.50 1.08 15 0.92 Ic50 Value((g/ml)

Values are Mean SEM of 3 replicates.

Nitric oxide

90 80 70
inhibition %

60 50 40 30 20 10 0 10 20 40
concentration

80

160

INVIVO ANTIOXIDANT STUDIES Table -5 Effect of ethanolic extract of Pseudarthria Viscida 100mg/kg,200mg/kg on MDA

TREATMENT

MDA(nmol/g tissue.) 220.518.4

Vehicle Ethanolic extract of pseudarthria viscida(100mg/kg) 180.36.3* Ethanolic extract of pseudarthria viscida(200mg/kg) 142.7.7.5*

Statistical significance test was done by ANOVA followed by Dunnets t test (n=6) Values are mean SEM of 6 animals per group *P < 0.01 Vs Control

MDA(nmol/g tissue.)
300 250

MDA(nmol/g tissue.)

200 150 100 50 0 vehicle EEPV(100mg/kg) Treatment EEPV(200mg/kg)

Table -6 Effect of ethanolic extract of Pseudarthria Viscida 100mg/kg,200mg/kg on,glutathione

TREATMENT

GLUTATHIONE(g/gtissue)

Vehicle

87.36.4

Ethanolic extract of pseudarthria 120.25.7* viscida(100mg/kg) Ethanolic extract of pseudarthria 137.24.5* viscida(200mg/kg) Statistical significance test was done by ANOVA followed by Dunnets t test (n=6) Values are mean SEM of 6 animals per group * P < 0.01 Vs Control

GLUT AT HIONE(g/gtissue)
160 140

GLUTATHIONE(g/gtissue)

120 100 80 60 40 20 0 vehicle EEPV(100mg/kg) Treatment EEPV(200mg/kg)

Table -7 Effect of ethanolic extract of Pseudarthria Viscida 100mg/kg,200mg/kg on catalase

TREATMENT

CATALASE (units/mg protein)

Vehicle 15.32.5 Ethanolic extract of pseudarthria viscida(100mg/kg) 23.73.4* Ethanolic extract of pseudarthria viscida(200mg/kg) 59.36.5* Statistical significance test was done by ANOVA followed by Dunnets t test (n=6) Values are mean SEM of 6 animals per group P < 0.01 Vs Control

CATALASE (units/mg protein)

CATALASE (units/mg protein)
70 60 50 40 30 20 10 0 vehicle EEPV(100/mg/kg) EEPV(200/mg/kg) Treatment

Table -8 Effect of ethanolic extract of Pseudarthria Viscida 100mg/kg,200mg/kg on SOD

TREATMENT

SOD(units/mg protein.)

Vehicle Ethanolic extract viscida(100mg/kg) Ethanolic extract viscida(200mg/kg)

3.630.3 of of pseudarthria 3.890.2 pseudarthria 3.950.3

Statistical significance test was done by ANOVA followed by Dunnets t test (n=6) Values are mean SEM of 6 animals per group

SOD(units/mg protein.)
5 4.5 4 3.5 3 2.5 2 1.5 1 0.5 0 vehicle EEPV(100mg/kg) EEPV(200mg/kg) Treatment

DISCUSSION The preliminary phytochemical analysis revealed the presence of phenolic substance ,tannins ,flavanoids, terpenes and carbohydrate . Hydroxyl radical is a principle contributor for tissue damage. The formation of hydroxyl radical from fenton reaction was quantified using 2,deoxy -D-ribose degradation. Studies with ethanolic extract of Pseudarthria Viscida have revealed significant hydroxyl scavenging activity in invitro. Ethanolic extract of pseudarthria viscida was capable of scavenging hydroxyl radical in a dose dependent manner. Hydrogen peroxide itself is not very reactive, but it can sometimes be toxic to cell because it can give rise to hydroxyl radical in the cells. Thus the removal of H2O2 is very important for antioxidant defence in cell or food systems. H2O2 can cross membranes and may oxidize a number of compounds. Ethanolic extract of pseudarthria viscida was capable of scavenging Hydrogen peroxide in a amount dependent manner Reducing power of ethanolic extract of Pseudarthria Viscida increased with increased concentration of test compound. The reducing capacity of a compound may serve as a significant indicator of its potential antioxidant activity. The reducing ability is generally associated with the presence of reductones, which breaks the free radical chain by donating a hydrogen atom. The extract had reductive ability which increased with increasing concentrations of the extract. Nitric oxide is a potent pleiotropic mediator of physiological processes such as smooth muscle relaxation, neuronal signaling, inhibition of platelet aggregation and regulation of cell mediated toxicity. It is a diffusible free radical which plays many roles as an effector molecules

SOD(units/mg protein.)

in diverse biological systems including neuronal messenger, vasodilation, antimicrobial and antitumour activities. Scavengers of nitricoxide compete with oxygen leading to the reduced production of nitricoxide. Our finding suggests that the phenolic compounds present in the extract might be responsible for nitricoxide scavenging effect. Ethanolic extract of Pseudarthria Viscida the dose of 100 and 200 mg/kg showed a significant decrease in the brain levels of MDA, which is the end product of lipid peroxidation and a measure of free radical generation. Also there was a simultaneous significant increase in levels of glutathione, a tripeptide found in all cells and reacts with the free radicals to protect cells from superoxide radical, hydroxyl radical and singlet oxygen (Schulz et al., 2000). In our study there was an insignificant increase in levels of SOD at 200 mg/kg, which is the only enzyme uses the superoxide anions as a substrate and produces the hydrogen peroxide as a metabolite, which is more toxic than O2 radical and has to be removed by catalase (Harman, 1991). In the present study there was a significant increase in levels of catalase at 100 and 200 mg/kg as compared to vehicle treated group, indicating the ethanolic extract of Pseudarthria Viscida scavenges the hydrogen peroxide, which is generated by SOD. The present study therefore demonstrate that the ethanolic extract of Pseudarthria Viscida has the antioxidant property by decreasing the lipid peroxidation and augmenting the endogenous antioxidant enzymes in brain .The preliminary phytochemical analysis of our present investigation revealed the presence of flavonoids,tannins,terpenes and carbohydrate . Active constituent of plant extract such as flavonoids, tannins are reported to posses antioxidant potential. The results of in vitro and in vivo model using brain homogenate in experimental rats suggest that use in disease condition like hysteria and nervine tonic reported for Pseudarthria Viscida use in Indian system of medicine may be due to their antioxidant and free radical scavenging activity. SUMMARY Currently there has been an increased interest globally to identify antioxidant compounds from plant sources which are pharmacologically potent and have low or no side effects for use in protective medicine and the food industry. Modern civilization, use of different chemicals, pesticides, pollutant, smoking and alcohol intake and even some of synthetic medicine increases the chance of disease due to free radicals. Plants produces large amount of antioxidants to prevent the oxidative stress, they represent a potential source of new compounds with antioxidant activity. More or less the free radicals plays a role in health of modern era and the diseases caused from free radical are becoming a part of normal life. Based on the results of the present study, we conclude that the plant Pseudarthria Viscida root extract possesses antioxidant potential. However, further studies are necessary to examine underlying mechanisms of antioxidant effects and to isolate the active compound (s) responsible for these pharmacological activities. Therefore it is time for us, to explore and identify our traditional therapeutic knowledge and plant sources and interpret it according to the recent advancements to fight against oxidative stress, in order to give it a deserving place. REFERENCE 1. Aebi, H., 1974. Catalase. In: Bergmeyer, H.V. (Ed.), Methods in Enzymatic Analysis, vol. 2. academic Press, New York, Chemie, Weinheim, FRG, pp. 674684.

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