Comparative study on some selected species of Ocimum genus on free radical scavenging activity and hepatoprotective activity against CCl 4 induced intoxication in rats

In this study, we compared the in vitro antioxidant property among the selected Ocimum species (O. sanctum, O. americanum, O. basilicum and O. gratissimum) and hepatoprotective activities of their extracts against CCl4 induced intoxication. The results suggested that the four species of Ocimum genus showed variability in Phenolic content and in vitro antioxidant activity against DPPH, superoxide and hydroxyl radicals in the following manner: O. sanctum > O. americanum > O. basilicum > O. gratissimum respectively. Based on serum AST, ALT, ALP and T. Bil levels, the alcoholic extracts of Ocimum species showed a significant dose dependent (250 mg and 500 mg and 750 mg/kg p.o.) and a protective effect against CCl4 induced hepatotoxicity in albino rats. The results further revealed that the potential hepatoprotective activity of Ocimum sanctum among the Ocimum species.


INTRODUCTION
The liver is the most important organ concerned with the biochemical activities in the human body.It has great capacity to detoxicate substances and synthesize useful principles [1].A major cause of these disorders is due to exposure to different environmental pollutants, viruses, and xenobiotics e.g., paracetamol, carbon tetrachloride, thioacetamide, alcohol, etc. Reactive oxygen species are implicated in liver damage [2] through covalent binding and lipid peroxidation and have been shown to augment fibrosis as seen from increased collagen synthesis [3].Scavenging of free radicals by antioxidants could reduce the fibrosis process in the tissues [4].
Ocimum sanctum (Family: Lamiaceae) is globally known for its immense medicinal properties, especially in India, and is popularly known as Tulsi.It is a holy plant common to most Indian households.Ocimum sanctum is known to possess anti-inflammatory, antimicrobial, antidiabetic, hepatoprotective, cardioprotective, antioxidant, adaptogenic, anticancer and radioprotective activities.There are other species in the same genus (Ocimum) and its influence on hepatoprotective activity is not established scientifically.Hence, the selected four species O. sanctum, O. americanum, O. basilicum and O. gratissimum were subjected to the estimation of total phenolic content, antioxidant activity and evaluated for hepatoprotective activity.

Animals
Adult Wistar rats (National Institute of Nutrition, Hyderabad, India) of either sex weighing 200 -250 gms were used in this study.The animals were maintained under standard laboratory conditions at an ambient temperature of 25˚C ± 2˚C having 50% ± 5% relative humidity with 12 h light and dark cycle.The rats were acclimatized to the laboratory conditions for one week prior to experimentation and provided with standard rat pellet diet (Rayans biotech Pvt Ltd., Hyderabad, India) and water ad libitum.The use and care of animals in the experimental protocol has been approved by the local Institutional Animal Ethics Committee (Regd.No. 516/01 /A/CPCSEA) following the guidelines of the committee for the Purpose of Control and Supervision of Experiments on animals (CPCSEA), Government of India.

Total Phenolic Content
Total phenolic content of the extracts was determined by using Folin-Ciocalteau reagent method [5].Folin-Ciocalteau colorimetry is based on a chemical reduction of the reagent with a mixture of tungsten and molybdenum oxides.The products of the metal oxide reduction have a blue colour with absorption maximum at 765 nm.By using standard Gallic acid calibration curve, measured the concentration of phenolic content in Gallic acid total equivalents using units mg/gm.

Superoxide Radical Scavenging Activity
Superoxide scavenging activity of the plant extract was determined by McCord and Fridovich method, 1969 [6] which depends on light induced superoxide generation by riboflavin and the corresponding reduction of Nitroblue tetrazolium was measured spectrophotometrically at 560 nm.

Hydroxyl Radical Scavenging Activity
Hydroxyl radical scavenging activity was measured by studying the competition between deoxyribose and the extracts for hydroxyl radicals generated from the Fe 2+ / EDTA/H 2 O 2 system (fenton reaction).The hydroxyl radical attacks deoxyribose, which eventually results in the formation of thiobarbituric acid reacting substances (TBARS) [7].

DPPH Radical Scavenging Activity
The scavenging activity for DPPH free radicals was measured according to the procedure described by Braca et al., 2003 [8].In DPPH assay, method is based on the reduction of alcoholic DPPH solution (dark blue in colour) in the presence of a hydrogen donating antioxidant converted to the non radical form of yellow colored diphenyl-picrylhydrazine.

CCl 4 Induced Intoxicated Rat Model
The animals were divided into 15 groups, each consisting of 6 animals.The standard and test group animals were treated with 100 mg/kg dose of Silymarin and 250, 500, 750 mg/kg dose of alcoholic extracts of selected plant drugs for 5 days.On the 6th day, 1 h after treatment with standard and test doses, the animals were intoxicated with CCl 4 in liquid paraffin (1:1 v/v, 1 ml of CCl 4 /kg, p.o.).Serum was separated by centrifugation at 37˚C and used for estimation of various biochemical parameters.Biochemical parameters like serum enzymes, Aspartate transaminase (AST) and Alanine transaminase (ALT) were estimated by Reitman and Frankel, 1957 method [9], Serum alkaline phosphatase (ALP) by King and Armstrong, 1980 method [10], Serum Total bilirubin (T.Bil) by Jendrassik and Grof, 1938 method [11] by using commercial reagent kits in Autoanalyzer (RM4000, Biochemical systems International, Italy).

Statistical Analysis
Results of the biochemical estimations are reported as Mean ± S.E.M. Significance was tested by One-Way analysis of variance (ANOVA) followed by Dunnett's post-hoc test using Graphpad Prism-5 software.
Similarly the IC 50 values of superoxide radical and  3).

DISCUSSION
The genus Ocimum consists of more than 30 species and the plants belonging to the genus contains numerous phytochemicals including phenolics.The selected four plant extracts O. sanctum, O. americanum, O. basilicum and O. gratissimum contains phenolics ranging from 38.00 to 70.00 mg/gm gallic acid equivalents (GAE).Mostly the phenolics are possessing antioxidant activity and the plant extracts also showed good antioxidant activity.Among the four extracts O. sanctum showed better antioxidant activity.
Hepatotoxicity produced by CCl 4 seems to be mediated by reactive metabolite trichloro methyl free radical (.CCl 3 ) formed by the hemolytic cleavage of CCl 4 or even by more reactive species trichloro methyl peroxy free radical (Cl 3 COO.)formed by the reaction of .CCl 3 with O 2 caused membrane damage of hepatocytes and resulted in centrilobular necrosis.The four plant extracts showed dose dependent hepatoprotective activity against CCl 4 induced hepatotoxicity and maximum percent protection was observed at a dose of 750 mg/kg.Among the four extracts Ocimum sanctum showed better activity and the results are comparable with that of the standard drug Silymarin.The mechanism of action may be due to its free radical scavenging (antioxidant) activity.Further the study revealed that it is not the quantity but the quality of phenolic compounds is important for showing good hepatoprotective activity and the same was proved by the good antioxidant activity of Ocimum sanctum.

CONCLUSIONS
The selected four species showed variability in phenolic content ranging from 38.00 to 70.00 mg/gm GAE.The selected plant extracts showed free radical scavenging activity in a dose dependent manner.Among the selected plants, Ocimum sanctum showed better free radical scavenging activity and the results are comparable with that of the standard drug Ascorbic acid.The four plant extracts showed hepatoprotective activity against CCl 4 induced intoxication.The study revealed the hepatoprotective activity of O. americanum for the first time and the results are comparable with that of Ocimum sanctum.
Based on ALT levels among the four extracts, Ocimum sanctum showed better activity (83.78 percent production) and the results are comparable with that of the standard drug Silymarin (87.04 percent protection).As per the superoxide and hydroxyl radical scavenging activity, the selected extracts showed good correlation between IC 50 value and percentage of hepatoprotection.Overall the study revealed the selected four plants possessing hepatoprotective activity and showed scopes for biomass utility.

ACKNOWLEDGEMENTS
The present work was carried out at Andhra University, Visakhapatnam,  authors are also thankful to National Institute of Health (NIH), Maryland, USA for their financial assistance.
The palnts O. sanctum, O. americanum, O. basilicum and O. gratissimum were collected in Visakhapatnam region, A.P, India and authenticity of the plants were confirmed by Taxonomist Prof. M. Venkaiah, Department of Botany, Andhra University, Visakhapatnam.Freshly collected whole plants were dried under shade and were made into coarse powder.400 gms of O. sanctum, O. americanum, O. basilicum and O. gratissimum were macerated in 2.5 lts of 95% v/v ethanol.The alcoholic extracts thus obtained were dried under reduced pressure at a room tem-perature not exceeding 40˚C to get the crude extracts.
India in Minority International Research Training (MHIRT) programme-2010 awarded (NIH/NCMHD/MHIRT# 9T37 MD 001532) to lcorn State University for their support and encouragement.The A

Table 1 .
Total Phenolic content of alcoholic extracts of O. sanctum, O. americanum, O. basilicum and O. gratissimum.

Table 3 .
Effect of alcoholic extracts of Ocimum species on Serum parameters in rats intoxicated with CCl 4 .< 0.0001.All groups were compared with CCl 4 group.Values are mean ± S.E.M., n = 6 animals per group.Values in the parenthesis indicate percent protection in individual biochemical parameters from their elevated values caused by the hepatoprotection.The percentage of the protection is calculated as 100 × (values of CCl 4 control-values of sample)/(values of CCl 4 control-values of control).