Hepatoprotective Activity of Vitex quinata Roots against Paracetamol-Induced Hepatic Injury in Rats.

V. Sreedhar¹*, L.K. Ravindra Nath², N. Madana Gopal¹,K. Venu Gopal³, D.Raju1 and K.K. Rajasekhar⁴

¹Department of Pharmacy, Prabhath Institute of Pharmacy, Nandayal, A.P, INDIA

²Department of Chemistry, Srikrishna Devaraya University, Anantapur, A.P, INDIA

³Dept of Pharmacy, Nirmala College of Pharmacy, Kadapa, A.P, INDIA

⁴Departement Of Pharmaceutical Chemistry, Sri Padmavathi School of Pharmacy, Tiruchanoor, Tirupathi-517503, A.P,INDIA

ABSTRACT:

Methanolic extracts of Vitex quinata roots at 200, 400 and 800 mg/kg and silymarin was prepared and tested for its hepatoprotective effect against paracetamol induced hepatitis in rats. Alteration in the level of biochemical markers of hepatic damage like SGOT, SGPT, ALKP, TPL, CHL, TPTN and ALP were tested in both treated and untreated groups. The methanolic extracts of Vitex quinata roots at 200,400 and 800 mg/kg and silymarin showed a significant (P<0.05) decrease in all the elevated SGOT, SGPT, ALKP, TBL, CHL and significant increase (P<0.05) in TPTN and ALB levels produced by the toxicant Paracetamol.

KEYWORDS: Vitex quinata, Antioxidant activity, Ascorbic acid.

INTRODUCTION:

The liver is the vital organ of paramount importance involved in the maintenance of metabolic function and detoxification from the exogenous and endogenous challenges, like xenobiotic, drugs, viral infection and chronic alcoholism. If during all such exposures to the above mentioned challenges the natural protective mechanisms of the liver are overpowered, the result is hepatic injury. Liver damage is always associated with cellular necrosis, increase in tissue liquid peroxidation and depletion in the tissue GSH levels. In addition serum levels of many biochemical markers like SGOT, SGPT, ALP and billirubin are elevated^{1, 2}. Liver regulates many important metabolic functions, and any injury causes distortion of these metabolic functions. As per an estimate, about 20,000 deaths occur every year due to liver disorders. Hepatocellular carcinoma is 1 of the 10 most common tumors in the world, with over 250,000 new cases registered each year³. It has been reported that 160 phytoconstituents from 101 plants possess hepatoprotective activity⁴. Liver-protective herbal drugs contain a variety of chemical constituents like phenols, coumarins, lignans, essential oil, monoterpenes, carotinoids, glycosides, flavanoids, organic acids, lipids, alkaloids and xanthones derivatives. Extracts of about 25 different plants have been reported to cure liver disorders⁵. In spite of tremendous efforts made in the field of modern medicine, there are hardly any drugs yet designated that stimulate liver function, offer protection to the liver from damage or help regeneration of hepatic cell. ⁶

Many Indian ethno botanic traditions propose a rich repertory of medicinal plants used by the population for treatment of liver diseases. However, there were not enough scientific investigations on the hepatoprotective activities conferred to these plants⁷. In India, about 40 polyherbal commercial formulations are available and prescribed by physicians to treat hepatic disorders, but search for simple and precise herbal drug still poses an intriguing problem. Some of these plant drugs have also been reported to possess strong antioxidant activity^{8, 9, 10}. Vitex quinata (Verbenaceae

) Trees 4-12 m tall, evergreen; bark brown. Branchlets pubescent and glandular when young, glabrescent. Leaves 3-5-foliolate; petiole 2.5-6 cm; petiolules 0.5-2 cm; leaflets obovate-elliptic to obovate or oblong to elliptic, thickly papery, both surfaces shiny. It is found from chintapalli, anantagiri forest, vizag to nallamadala forests in Andhra Pradesh. Exhaustive and up to date review of literature for hepatoprotective activity and their methods of screening and pharmacological review of the selected plant was conducted. Dried powdered roots of Vitex quinata were separately extracted in a Soxhlet apparatus for 6 h successively with methanol. The concentrate is dried under vacuum in a rotary evaporator^11,12.

MATERIALS AND METHODS:

All the chemicals were used analytical grade obtained from S.D. Fine Chemicals Pvt. Ltd., Mumbai, Sigma chemical company, U.S.A. and Loba chemicals, Mumbai.

Plant material:

The roots of Vitex quinata were collected from Ananthagiri forest region, Visakhapatnam District, Andhra Pradesh, India in the months of March and May, 2008. These plant species were authenticated by Dr. M.Venkaiah, Taxonomist, Department of Botany, Andhra University, Visakhapatnam, and Andhra Pradesh, India. The Voucher specimens (VTR-14-03-2008) were deposited in the institutional museum, College of Pharmaceutical Sciences, Andhra University, Visakhapatnam. The collected plants were washed and air-dried under the shade, cut into small pieces, powdered by a mechanical grinder and passed through 40-mesh sieve and stored in a closed vessel for future use.

Preparation of extract:

Shade dried root powder of Vitex quinata, was separately extracted in a Soxhlet apparatus for 6 hrs successively with methanolic concentrated to dryness under reduced pressure. Later, the quantity of root powder taken for extraction. These extract used to test the biochemical markers of hepatic damage like SGOT, SGPT, ALKP, TPL, CHL, TPTN andALP were tested in both treated and untreated groups.

In-vivo hepatoprotective activity:

Hepatoprotective Activity of V. quinata roots against Paracetamol-Induced Hepatic Injury in rats using different in vivo models.

Paracetamol induced hepatototxicity:

The set of experiment was divided into groups consisting of control, toxicant, standard, and test. The protocol followed for paracetamol induced hepatotoxicity ¹³ is given Table 1.

The rats of control group received a single daily dose of 1% Sodium CMC (1 ml/kg p.o.) The rats of Paracetamol group received a single daily dose of vehicle for three days and a single dose of Paracetamol (3 g/kg) 30 min after the administration of the vehicle, on the third day of experiment. The animals in silymarin group received daily dose of silymarin (25 mg/kg p.o.) for three days. Paracetamol (3 g/kg p.o.) was administered 30 min after the third dose of silymarin while test groups were given orally a single daily dose of extracts in sodium CMC for three days and a single dose of parcetamol (3 g/kg p.o.) on the third day 30 min after the administration of respective test suspensions¹³. After 48 h of paracetamol intoxication blood was collected and serum was an analyzed for the biochemical parameters

Assessment of liver function:

Blood was collected from all the groups by puncturing the retro-orbital plexus and was allowed to clot at room temperature, and serum was separated by centrifuging at 2500 rpm for 10 minutes. The serum was used for estimation of biochemical parameters to determine the functional state of the liver. Serum glutamic oxaloacetic transaminase (SGOT) and serum glutamic pyruvic transaminase (SGPT) were estimated by a UV kinetic method based on the reference method of International Federation of Clinical Chemistry. ¹⁴ Alkaline phosphatase (ALKP) was estimated by the method described by Mac Comb and Bowers. ¹⁵Total bilirubin (TBL) was estimated by Jendrassik and Grof method. ¹⁶ Total cholesterol (CHL) was determined by CHOD-PAP method of Richmond. ¹⁷ Total protein (TPTN) was estimated by Biuret method_, ¹⁸ while albumin (ALB) was estimated by BCG.¹⁹ All the estimations were carried out using standard kits on auto-analyzer of Merck make (300 TX, E. Merck-Micro Labs, mumbai).

Histopathological Study:

Histopathological examination of hepatocytes:²⁰

Each rat was laprotomized to obtain the liver immediately after collecting blood under ether anaesthesia. Small fragments of the rat liver were fixed in 10% formalin solution, dehydrated with ethanol solution from 50% to 100%, embedded in paraffin and cut into 5 μm thick sections which were stained using haemotoxylineosin dye for photomicroscopic observation including necrosis, steatosis and fatty change of hepatic cells. (Figure1 and table 2)

Statistical analysis:

The mean values ± SEM were calculated for each parameter. For determining the significant inter-group differences, each parameter was analyzed separately, and one-way analysis of variance ²¹ was carried out. Individual comparisons of the group mean values were done using Dunnet's test. ²² P value less than 0.05 was considered to be a significant difference.

Table-1: The protocol for paracetamol – induced hepatotoxicity

Group	Day 1	Day 2	Day 3	Day 4	Day5
control	Vehicle	Vehicle	Vehicle	vehicle	With drawl of blood
PCML	Vehicle	Vehicle	Vehicle+pcmL	Vehicle
Standard	Silymarin	Silymarin	Silymarin+pcmL	Vehicle
Test	extract	extract	Extract+pcmL	vehicle

Vehicle: 1% Sodium CMC, test: Extracts prepared in 1% Sodium CMC. PCML: Paracetamol

Table 2: Histopathological Study of liver of albino rats.

Control (A) 1 ml/kg	Observed under 100 x H.E of magnification, showed liver tissue with typical lobular arrangement, Individual lobules consist of hepatocytes arranged as cords radiating around centrally placed terminal hepatic veins. Hepatocytes seen are uniform in size, polyherbal in shap, with centrally located large nuclei. The cytoplasm is strongly eosinophilic with a fine basophilic granularity. Portal tracts containing terminal branches of the hepatic portal vein and hepatic artery at the periphery in fibrous stroma are also seen. Impression : Normal liver tissue
Paracetamol Treated(B) 3 g/kg	Observed under 100 x H.E of magnification showed liver tissue with disturbances in the lobular arrangement. Degenerative and early necrotic changes extending across lobules. Hepatocytes show ballooning degeneration and steatotic changes. Some amount of fibrosis seen in portal tracts. Impression: Liver with cytotoxic injury showing mild necrosis and fibrotic changes.
Silymarin treated(C) 25 mg/kg	Observed under 400 x H.E of magnification showed liver tissue with typical lobular arrangement. Heptocytes show variable size. There is a mild increase in fibrous connective tissues. Impression: Liver with mild sign of hepatotoxicity.
Methanolic Extract(D) 800 mg/kg	Observed under 100 x H.E of magnification, showed liver tissue with typical lobular arrangement. Few hepatocytes show steabotic accumulation. Impression: Liver with minimal sign of hepatotoxicity.

Table-3: Effect of methanolic extracts of VQ on Paracetamol induced hepatotoxicity in rats

Group	SGOT (IU/L)	SGPT (IU/L)	ALKP (IU/L)	TBL (mg/dL)	CHL (mg/dL)	TPTN (g/dL)	ALB (g/dL)
Control	110.6 ±1.94	99.93 ±0.95	202.5 ±1.67	0.96 ±0.01	123.7 ±2.1	6.39 ±1.68	4.56 ±0.42
Paracetamol	455.1 ±4.57	315.1 ±1.90	612.0 ±2.54	4.26 ±0.22	340.6 ±1.87	3.37 ±0.28	1.75 ±0.22
Silymarin	118.4 ±2.14*	103.6 ±1.92*	213.4 ±2.20*	1.22 ±0.09*	127.1 ±1.33*	6.34 ±0.25**	4.46 ±0.30**
VQM 200 mg/kg	406.5 ±2.35	286.3 ±4.01	404.6 ±2.11	4.36 ±0.06	232.5 ±2.74	3.07 ±0.03	2.07 ±0.06
VQM 400 mg/kg	154.1 ±1.55*	91.24 ±0.87*	224.9 ±2.28*	1.77 ±0.18*	113.1 ±2.09*	5.81 ±0.13**	4.68 ±0.13**
VQM 800 mg/kg	149.1 ±2.13*	89.02 ±3.62*	199.5 ±2.87*	2.05 ±0.03*	119.2 ±2.35*	5.58 ±0.22**	3.69 ±0.05**

Data expressed in mean±s.e.m, n=5

* Significant reduction compared to hepatotoxic group (P<0.05); ** Significant increase compared to hepatotoxic group (P<0.05)

Fig 1: Histopathological Microphotograph of Rat liver tissu

Fig-2: Effect of the crude extract of VQM at 200, 400 and 800mg/kg along with silymarin 25 mg/kg body wt. on percentage protection of various biochemical parameters against thioacetamide induced hepatotoxicity.

RESULTS:

Acute toxicity studies:

The ethyl acetate fraction of methanolic extract did not cause any mortality up to 2000 mg/kg and was considered as safe.

Paracetamol-induced hepatotoxicity

Table-3 and figure-2 shows Silymarin the standard drug at the dose of 25 mg/kg significantly reduced the increased levels of SGOT, SGPT, ALKP, TBL and CHL with the values 118.4 ± 2.14, 103.6 ± 1.92, 213.4 ± 2.20, 1.22 ± 0.09, and 127.1± 1.33 respectively and increased the levels of TPTN and ALB 6.34 ± 0.25 and 4.46 ± 0.30 respectively. Methanolic extract of vitex quinata at 200 mg/kg produced 406.5±2.35, 286.3±4.01, 404.6±2.11,4.36±0.06, 232.5±2.74,3.07±0.03, 2.07±0.06, whereas methanolic extract of vitex quinata at 400 mg/kg produced 154.1 ± 1.55, 91.24 ± 0.87, 224.9 ± 2.28, 1.77± 0.18, 113.1 ± 2.09, 5.81 ± 0.13 and 4.68 ± 0.13, where as methanolic extract of vitex quinata at 800mg/kg produced 149.1 ± 2.13, 89.02 ± 3.62, 199.5 ± 2.87, 2.05± 0.03, 119.2 ± 2.35, 5.58 ± 0.22 and 3.69 ± 0.05 respectively.

DISUSSION:

The results clearly depicted that paracetamol intoxication in normal rats elevated the serum levels of SGOT, SGPT, ALKP, TBL and CHL, where as decreased the levels of TPTN, ALB significantly when compared to control indicating acute hepatocellular damage and centrilobular necrosis. The rats treated with the methanolic extracts of vitex quinata and silymarin showed a significant (P<0.05) decrease in all the elevated SGOT, SGPT, ALKP, TBL, CHL and significant increase (P<0.05) in TPTN and ALB levels at 200,400 and 800 mg/kg.

ACKNOWLEDGMENT:

We would like to thank Dr. J.Rajasekhar, Andhra University, for his guidance and support throughout this project.

REFERENCES:

1. Mossa JS, Tariq M, Mohsin A, Aqeel AM, al-Yahya MA, al-Said MS, et al. Am J Chin Med 1991;19:223.

2. Mascolo N, Sharma R, Jain SC, Capasso F. J Ethnopharmacol 1998; 22:211.

3. Wolf PL. Biochemical diagnosis of liver diseases. Ind J Clin Biochem 1999; 14:59-90.

4. Handa SS, Sharma A, Chakraborti KK. Natural products and plants as liver protecting drugs. Fitoterapia 1986; 57:307-51.

5. Sharma SK, Ali M, Gupta J. Evaluation of Indian Herbal Hepatoprotective Drugs. Recent Progress in Medicinal Plants (Phytochemistry and Pharmacology), Vol II, Research Periodicals and Book Publishing House, Houston, Texas (USA), ISBN No. 0-9656038-7-3; 2002. p. 253-70.

6. Chatterjee TK. Medicinal plants with hepatoprotective properties. In: Herbal options, 3 ^rd ed. Calcutta: Books and Allied (P) Ltd.; 2000 p. 135-7.

7. Chattopadhyay RR. Possible mechanism of hepatoprotective activity of Azadirachta indica leaf extract: Part II. J Ethnopharmacol 2003; 89:217-9.

8. Achuthan CR, Babu BH, Padikkala J. Antioxidant and hepatoprotective effects of rosa damascena. Pharma Biol 2003; 41:357-61.

9. Aniya Y, Miyagi C, Nakandakari A, Kamiya S, Imaizumi N, Ichiba T. Free radical scavenging action of the medicinal herb Limonium wrightii from the Okinawa islands. Phytomedicine 2002; 9:239-44.

10. Gupta K, Chitme H, Dass SK, Misra N. Antioxidant activity of Chamomile recutita capitula methanolic extracts against CCl4-induced liver injury in rats. J Pharma Toxicol 2006;1:101-7.

11. Krishna Rao RV, Ranjit J. (1996) Indian J. Nat. Prod. 12(2): 26-28.

12. Rakesh. K, Sharma and Rajesh Arora., 2006, Herbal Drugs. Jaypee Brothers, Medical Publishers, 1st Edn.

13. Rao. K.S and Mishra. S.H., Journal of Ethnopharmacology, 60, 207-213, 1998

14. Schwartz MK, de Cediel N, Curnow DH, Fraser CG, Porter CJ, Worth HG, et al. International federation of clinical chemistry, education committee and international union of pure and applied chemistry, division of clinical chemistry: Definition of the terms certification, licensure and accreditation in clinical chemistry. J Clin Chem Clin Biochem 1985;23:899-01.

15. McComb RB, Bowers GN Jr. Study of optimum buffer conditions for measuring alkaline phosphatase activity in human serum. Clin Chem 1972;18:97-04.

16. Jendrassik L, Grof P. Quantitative determination of total and direct bilirubin in serum and plasma. Biochem Z 1938;297:81-9.

17. Richmond W. Preparation and properties of a cholesterol oxidase from Nocardia species and its application to the enzymatic assay of total cholesterol in serum. Clin Chem 1973;19:1350-6.

18. Peters T Jr. Proposals for standardization of total protein assays. Clin Chem 1968;14:1147-59.

19. Webster D. A study of the interaction of bromocresol green with isolated serum globulin fractions. Clin Chim Acta 1974;53:109-15.

20. Allain CC. Clin Chem 1974;20:470.

21. Gennaro AR. The Science and Practice of Pharmacy, 19 ^th ed, Vol I, (Mack publishing company, Easton PA; 1995. p. 111.

22. Dunnet CW. New tables for multiple comparisons with a control. Biometrics 1964;20:482-91.

Received on 16.12.2010

Accepted on 26.12.2010

© A&V Publication all right reserved

Research Journal of Pharmacognosy and Phytochemistry. 3(2): March- April 2011, 77-81