A) BRIEF RESUME OF THE INTENDED WORK: Need of Study:
Liver is one of the heaviest organs in the body. It performs a wide variety of functions such as metabolism of bilirubin, bile salts, drugs, lipids, storage and release of substrates, vitamins and minerals1.
Liver diseases are acute in nature or chronic. Elevated occurance of etiological factors like viral infections, hepatitis, alcohol consumption, immune disorders, vascular abnormalities, inherited metabolic disorders, biliary track diseases, infectious diseases, drugs and toxins leads to tremendous rise in hepatic ailments2. Hence liver diseases have emerged as global problem3.
Traditionally, numerous drugs from the plant kingdom have been used for the treatment of various hepatic ailments.
Subsequently they were scientifically proved for their efficacy and safety in number of experimental models in animals and patients by either the manufacturing industry or institutional research scholars. Later, these plants were incorporated in number of formulations available for management of hepatic complications. Most commonly used plants in herbal formulation are;- Andrographis paniculata, Boerhaavia diffusa, Eclipta alba, phyllanthus niruri,
Picrorrhiza kurroa and Tinospora cordifolia.
Our interest in the present research is to compare the hepatoprotective activity of conventional hepatoprotective plant viz., Androgrophis paniculata, phyllanthus niruri and Picrorrhiza kurroa in their standardized extract form using different hepatotoxic models of animal.
Review of Literature:
It is reported that phyllanthus niruri possess De-obstruent, diuretic, astringent, cooling and anti viral property. It is also been used in edema, gonorrhea, menorrhagia, urogenital diseases, dysentery, diabetes, dyspepsia. Recent studies suggest that this naturally occurring plant has potent effect against jaundice and hence considered as the main herb for the management of liver diseases4.
In literature, Andrographis paniculata plant extract is reported to possess anticancer and immunomodulatory activities and hence has the potential for being developed as a cancer therapeutic agent. It is also found to strongly stimulate phagocytosis and production of specific antibodies. Further, this herb is used as digestant, vermicidal, anti- acne, laxative, analgesic, anti-inflammatory, sedative and hypoglycemic agent and forms the principal ingredient of a reputed household medicine ('alui'), used as a bitter tonic and febrifuge. Furthermore, it is shown to increase biliary flow and liver weight in rat. Andrographolide, principle active ingredient, produces a significant dose dependent choleretic effect5.
Picrorrhiza kurroa is reported to show anti-bacterial, anti-fungal, anti-pyretic, cholagogue, stomachic, immune stimulant, anti-inflammatory, anti-coagulant, anti-parastatic activities. It is also been used for indigestion, constipation, diarrhea, dysentery, malaria, conjunctivitis, hemorrhoids, infantile seizures, immune system tonification, intestinal parasites, bladder infections, viral and yeast infections. Further, it is used as anti-allergic, bitter tonic, laxative, emetic, abortifacient, epilepsy, improves eye sight, fever, malaria, paralysis, scorpion sting and skin diseases. Furthermore, it possesses potent activities against jaundice, hepatitis and cirrhosis6.
Objective of Study:
The present work deals with the comparative study of hepatoprotective efficacy of few standardised plant extracts in different experimental models of hepatotoxicity with the following specific objectives:
To study the hepatoprotective activity of standard extract of Andrographis paniculata, phyllanthus niruri
and Picrorrhiza kurroa using following animal models.
o Chronic hepatitis model by inducing CCl4 hepatic injury.
o Acute hepatitis by using following hepatotoxic models
. CCl4 induced acute hepatic injury.
. Paracetamol induced liver toxicity.
. Thioacetamide induced liver necrosis.
B) Materials & Methods:
Source of Data: Data will be obtained from CD-Rom, Internet facilities, Literatures and related articles from libraries of Krupanidhi
College of Pharmacy, Indian Institute of Sciences, Government College of Pharmacy etc., and other Research
Publications and Journals.
Method of Collection of Data (including sampling procedure, if any): The data collected will be based on animal experiments as per the parameters studied under each model, which are mentioned under the objectives of the study.
Methods:
The animal will be grouped as follows in each model (n=6)
Group I: Control
Group II: Hepatotoxic control
Group III: Standard drug (Silymarin 100mg/kg/day,p.o)
Group IV: Phyllanthus niruri extract in Hepatotoxic model(142.5 mg/kg,p.o)7
Group V: Andrographis paniculata extract in Hepatotoxic model(300mg/kg p.o)8
Group VI: Picrorrhiza kurroa extract in Hepatotoxic model (200mg/kg,p.o)9
CHRONIC HEPATITIS MODEL
Carbon tetra chloride induced chronic hepatic injury
Animals will be treated with the standard extract of the plant daily for eight weeks. Dose of CCl4 (0.2 ml/kg, p.o) diluted with liquid paraffin (1:1) will be administred twice weekly to groups 2, 3, 4, 5 and 6. Rats will be sacrificed
24hrs after the last treatment10. Blood samples will be collected by retro orbital puncture method and serum will be subjected for marker enzyme estimations like Alanine aminotransferase (ALT), Aspartate aminotransferase (AST),
Serum alkalinephosphatase (ALP) and bilirubin. The liver will be isolated and washed with normal saline, blotted with filter paper and will be weighed immediately10. Histopathological examinations will be done to confirm biochemical findings.
ACUTE HEPATITIS MODEL
Carbon tetrachloride induced acute hepatic injury:
th The single dose of CCl4 (0.5 ml/kg, p.o.) diluted with liquid paraffin (1:1) will be administered on 10 day and sacrificed 24 hours after the administration of CCl4. Blood samples will be collected by retro orbital puncture method and separated serum will be subjected for marker enzyme estimations like Alanine aminotransferase (ALT),
Aspartate aminotransferase (AST), Serum alkaline phosphatase (ALP) and bilirubin. The liver will be isolated and washed with normal saline, blotted with filter paper and will be weighed immediately11. Biochemical studies will be further confirmed by histopathological examinations.
Paracetamol induced liver toxicity:
Paracetamol (2g/kg, p.o.) diluted with sucrose solution (40%w/v) will be administered in three divided doses and animals will be sacrificed 48 hrs after administration of paracetamol12. Food will be withdrawn 12 hrs before paracetamol administration to enhance the acute liver toxicity in animals of group 2, 3, 4,5 and 6. Blood samples will be collected and separated serum will be subjected for marker enzyme estimations like Alanine aminotransferase (ALT), Aspartate aminotransferase (AST), Serum alkaline phosphatase(ALP) and bilirubin. The liver will be isolated and washed with normal saline, blotted with filter paper and will be weighed immediately 12.
Biochemical studies will be further confirmed by histopathological examinations.
Thioacetamide induced liver necrosis:
Single dose of thioacetamide (100 mg/kg s.c) diluted with distilled water (5% solution) will be given at the end of ten days treatment to groups 2, 3, 4,5 and 6 and sacrificed 48 hrs after administration of thioacetamide. Blood samples will be collected by retro orbital puncture method and separated serum will be subjected for marker enzyme estimations like Alanine aminotransferase (ALT), Aspartate aminotransferase (AST), Serum alkaline phosphatase
(ALP) and bilirubin12. The liver will be isolated and washed with normal saline, blotted with filter paper and will be weighed immediately. Biochemical studies will be further confirmed by histopathological examinations.
Does the study require any investigations or interventions to be conducted on patients or other human or animals? If so please describe briefly:
YES,
Study requires investigation on animals. The comparative studies will be done on various parameters using rats as experimental animals.
Has the Ethical Clearance been obtained from your Institution ?
Ethical Committee approval letter is enclosed
C) LIST OF REFERENCES:
1. Christopher H, Edwin RC, Nicholas AB, Nicki RC. Davidson’s principles and practice of medicine.19th edition, 2002:831-888.
2. Roger W, Clive E.Clinical pharmacy and therapeutics.3rd edition, 2002:209-223.
3. Rajesh MG, Lata MS. Preliminary evalution of the antihepatotoxic activity of kamilari,a polyherbal
formulation. J.Ethnopharmacol 2004; 91:99-1043.
4. Sarkar MK, Sil PC. Hepatocytes are protected by herb Phyllanthus niruri protein isolate against
thioacetamide toxicity. Pathophysiology. 2007; 14:113-20.
5. Singha PK, Roy S, Dey S. Protective activity of andrographolide and arabinogalactan proteins from
Andrographis paniculata Nees against ethanol-induced toxicity in mice. J Ethnopharmacol. 2007; 111:13-21.
6. Singh M, Tiwari V, Jain A, Ghoshal S. Protective activity of picroliv on hepatic amoebiasis associated with
carbon tetrachloride toxicity. Indian J Med Res. 2005;121:676-82.
7. Umarani D, Devaki T,Govindaraju P,Shanmugasundaram KR Ethanol induced metabolic alteration and the
effects of phyllantus niruri in their reversal.Ancient Sci Life 1985; 43:174-180.
8. Rana AC, Avadhoot Y. Hepatoprotective effects of Andrographis paniculata against carbon tetrachloride-
induced liver damage. Arch Pharm Res. 1991; 14:93-5.
9. Vaidya AB, Antarkar DS, Doshi JC, Bhatt AD, Ramesh V, Vora PV, Perissond D, Baxi AJ, Kale PM.
Picrorhiza kurroa (Kutaki) Royle ex Benth as a hepatoprotective agent--experimental & clinical studies. J
Postgrad Med. 1996; 42:105-8.
10. McLean EK, McLea AEM, Sutton PM. An improved method for producing cirrhosis of liver in rats by
stimulus administration of carbon tetrachloride and pentobarbitone Br J Exp path 1969; 50:502-503.
11. Matsuda H,Samukawa K,Kubo M. Anti-hepatic activity of Ginsenoside Ro.Planta Med 1991; 57:523-526.
12. Asha VV,Pushpangadan P.Preliminary evaluation of the antihepatoprotective activity of Phyllantus
kozhikodianus,P.maderaspatensis and Solanum indicum Fitoterapia 1998; LXIX,3:255-259.