Download annexure ii

PRECLINICAL EVALUATION OF HEPATOPROTECTIVE ACTIVITY OF THE
POLYHERBAL FORMULATION “LIVOPICK”.
M. PHARM DISSERTATION PROTOCOL
Submitted to the
Rajiv Gandhi University of Health Sciences, Karnataka
Bangalore – 560 041
By
RIYAZ AHMED
Under the Guidance of
Mr. LICTO THOMAS
Assistant professor
Department of Pharmacology,
Shree Devi College of Pharmacy
Airport Road, Kenjar
Mangalore – 574 142.
1
RAJIV GANDHI UNIVERSITY OF HEALTH SCIENCES,
BANGALORE, KARNATAKA,
ANNEXURE II
PROFORMA FOR REGISTRATION OF SUBJECTS FOR
DISSERTATION
1.
NAME OF THE CANDIDATE
RIYAZ AHMED
AND ADDRESS
S/o Abdul Razak
4-167(A) New House,
Fareed Nagar,
Harekala post.
Mangalore 574199
2.
Shree Devi College of Pharmacy,
NAME OF THE INSTITUTE
Airport road,
Kenjar village,
Mangalore, 574142
3.
COURSE
OF
STUDY
AND
Master of Pharmacy in Pharmacology
SUBJECT:
4.
DATE
OF
ADMISSION
COURSE:
5.
TO
December 2012
TITLE OF THE TOPIC:
“PRECLINICAL EVALUATION OF HEPATOPROTECTIVE ACTIVITY OF
THE POLYHERBAL FORMULATION “LIVOPICK”.
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6.
BRIEF RESUME OF THE INTENDED WORK:
6.1. NEED OF STUDY:
Liver disease is considered as the major problem worldwide, as liver has supreme
importance due to its functional involvement in metabolism, detoxification of xenobiotics, drugs, chronic alcoholism, in the alteration of homeostatis1.
Liver plays a important role in detoxification and excretion of many endogenous
and exogenous compounds. Any impairment in the functions of liver may lead to many
implications on health. Management of liver disease is still a challenge to the modern
scientific community2.
Liver injury is caused due to infections, certain drugs, environmental and social factors such as alcoholism3 resulting in severe pathological conditions such as hepatitis,
liver cirrhosis, hepatosis4. Conventional or synthetic drugs used in the treatment of liver
diseases are often inadequate and can have serious adverse effects. As a result, there is a
worldwide trend to go back to traditional medicinal plants. Many natural products of
herbal origin are in use for the treatment of liver ailments5.
There are number of polyherbal formulations which have been widely marketed,
claiming to be highly effective in the treatment of above mentioned organ disorder.
Many polyherbal formulations are widely used but most of them are not scientifically
validated yet.
COMPOSITION OF LIVOPICK:
Each capsule contains processed extracts of:
Latin Name
Quantity
Rheum emodi
50mg
Picrorhiza kurroa
50mg
Plumbago zeylanica
50mg
Phyllanthus niruri
50mg
Andrographis paniculata
50mg
Eclipta alba
100mg
Boerhaavia diffusa
50mg
Tinospora cordifolia .
50mg
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Triphala (Equal parts of
Terminalia chebula Extract
Terminalia Bellerica Extract
Emblica Officinalis Extract)
50mg
The above medicinal plants possess antioxidants, hepatoprotective, antidiabetic,
antihistaminic, antibacterial properties. Rheum emodi7, 8- anthelmentic, hepatoprotective, laxative. Picrorrhiza kurroa9, 10-antipyretic, hepatoprotective, cathartic, tonic, laxative, anti-allergic, anti-anaphylactic, anti-epileptic, anti-paralytic, anthelmentic, appetizer, anti-ulcer. Plumbago zeylanica10- anti diabetic, antioxidants, hepatoprotective activity, anticancer, antifungal activity. Phyllanthus niruri11, 12- dysentery, influenza, vaginitis, tumours, diabetes, diuretics, jaundice, kidney stones and hepatoprotective. Andrographis paniculata20, 21- hepatoprotective and hepatostimulative agent. Eclipta alba13, 20-inflammation, anthelmentic, digestive, anti-hepatotoxic. Boerhavia diffusa13, 20asthma, dyspepsia, tumours, abdominal pain, diuretic and liver disorders. Tinospora
cordifolia14,15-hepatoprotective,
immunomodulatory
activity.
Triphala16-anti-
inflammatory, anticancer, hepatoprotective, anti oxidant property.
LIVOPICK is the polyherbal formulation contains the extract of the above medicinal
plant. Individually all the above is proved to be hepatoprotective but no scientific report
is available for its combined hepatoprotective effect. Hence the present study is aimed
to investigate the hepatoprotective activity of the Polyherbal formulation “LIVOPICK”.
6.2. REVIEW OF LITERATURE:
Liver diseases have become one of the major causes of morbidity and mortality in
man and animals in the world. Liver is an important target of the toxicity of drugs, xenobiotics, and oxidative stress. The major causes for the liver disorders are excessive
alcohol consumption, viral influenced liver diseases, drug induced, environmental toxins like lead, phosphorus, petrochemicals, etc17. The major liver disorders are hepatitis,
fatty liver, liver cirrhosis, and genetic diseases.
The mechanism behind the Carbon tetrachloride toxicity is in the liver. CCl4 is metabolized to the highly reactive trichloromethyl radical. The free radical generated
would lead to auto-oxidation of the fatty acids present in the cytoplasmic membrane
phospholipids and causes functional and morphological changes in the cell
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membrane22. In acetaminophen toxicity, nitric oxide (NO) scavenges superoxide to produce peroxynitrite, which then causes protein nitration and tissue injury18. In thioacetamide induced hepatotoxicity is mediated by oxidative stress caused by the action of
cytokines through lipid peroxidation23.
Livopick is a polyherbal formulation, developed by Wilson Drugs and Pharmaceutical
Pvt. Ltd. India and is intended to use in jaundice, infective hepatitis, cirrhosis and also
loss of appetite, indigestion, habitual alcoholism.
 Rheum emodi belongs to the family Polygonaceae. The most important constituents of the Rheum emodi are anthraquinone derivatives such as chrysophanol,
aloe-emodin, emodin, physcion, rhein and its glycosides. This reportedly to have
purgative, anthelmentic and hepatoprotective actions6 and its tuber as bitter tonic, laxative, appetite stimulant, diuretic, anti-bilious, and curative for sore eyes
and bruises. It has also been reported to have antifungal7 and hypoglycaemic
properties 8. Rheum emodi root is being commonly used in traditional medicine
as hepatoprotective .
 Picrorrhiza kurroa belongs to the family Scrophulariacea contains picrorrhizin,
kutkin, picroside, and apocynin. The plant is used for anti-pyretic, hepatoprotective9, cathartic, laxative, anti-allergic, anti-anaphylactic, anti-epileptic, antiparalytic, anthelmentic, apetizer, anti-ulcer, skin diseases, antidote for dog bite
and as a anti-histaminic activity.
 Plumbago zeylanica belongs to the family Plumbagenaceae. It is having anti diabetic, antioxidants, hepatoprotective activity10 , anticancer, antifungal activity.
 Phyllanthus niruri 12 belongs to family Euphorbiaceae. It has been claimed to be
an excellent remedy for jaundice and infective hepatitis. The whole plant is used
as remedies for many conditions such as dysentery, influenza, vaginitis, tumours, diabetes, diuretics, jaundice, and kidney stones. The plant is also useful
for treating hepatotoxicity, hepatitis B, hyperglycaemia and viral and bacterial
diseases 11.
 Andrographis paniculata20 (Kalmegh) belongs to the family Acanthaceae. It is
used extensively in the Indian traditional system of medicine as a hepatoprotective and hepatostimulative agent12. The aqueous extract of the
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leaves of this plant has traditionally been used for treatment of various liver disorders and jaundice.
 Eclipta alba13 belongs to the family Astragenaceae contains wedelolactone, thiophene, furanocoumarins, demethylwedelolactone, ecalbutin, echinocystic acid,
flavones: luteoline, oleanic acid and ursolic acid. The plant used for antiinflammation, anthelmentic, digestive, anti-hepatotoxic20.
 Boerhavia diffusa
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belongs to the family Nyctagenaceae contains sitosterol,
tetracosanoic, arachidic acid, hentriacontane, ecdysone and triacontanol. The
plant is used for blood impurities, asthma, dyspepsia, tumours, abdominal pain,
diuretic and liver disorder.
 Tinospora cordifolia belongs to the family Menispermaceae. It has been reported
that the extract of this is has free radical scavenging and antioxidant properties14.
It is also reported to have hepatoprotective, immunomodulatory activity15.
 Triphala16,
19
is the most commonly used in the Indian Ayurvedic formulation
comprising the equal parts of Terminalia chebula(Combretaceae) Extract, Terminalia bellerica (Combretaceae) Extract, Emblica Officinalis (Euphorbiaceae)
Extract. It possess antiinflamatory, hepatoprotective, anti oxidant property.
To check Combined effect of hepatoprotective activity of the polyherbal formulation LIVOPICK, the evaluation will be conducted using various animal models.
6.3. OBJECTIVE OF STUDY:
To evaluate the hepatoprotective effect of the polyherbal formulation “LIVOPICK”
7.
MATERIALS AND METHODS:
7.1. SOURCE OF DATA:
The source of data for this study is based on the following. Mainly wistar rats of
either sex will be used during the course of the evaluation studies.
Various animal models selected for the study include
Carbon tetrachloride induced liver toxicity

Paracetamol induced liver toxicity

Thioacetamide induced liver toxicity
Biochemical and histopathological investigation will be carried out in each animal
model. Experiment will be performed as described in the standard bibliography,
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literature, text book, etc.
7.2 METHOD OF COLLECTION OF DATA:
MATERIALS:
Some of the main materials required are: Polyherbal formulation LIVOPICK (Wilson Drugs and Pharmaceutical Pvt Ltd, India), Carbon tetrachloride, paracetamol, thioacetamide, silymarin. Chemicals and reagents will be procured from standard companies.
DOSE SELECTION STUDY:
The doses which will be administered to rats will be calculated accordingly from human
dose by using human equivalent dose (HED) method24.
7.3. EXPERIMENTAL MODEL :
7.3.1.CARBON TETRACHLORIDE INDUCED ACUTE HEPATITIS IN
RATS25,26,29:
PROCEDURE:
Carbon tetrachloride (CCl4) induced liver injury. The animals will be divided into 5
groups consisting of six animals. The animals will be then subjected to either one of the
following treatments for 9 days. The CCl4 will be administered after dilution with liquid
paraffin the ratio of 1:1. Food will be withdrawn 12 hrs before CCl4 administration to
enhance liver damage in animals of groups 2, 3, 4 and 5. The animals will be sacrificed
24 hrs after the administration of CCl4. Blood samples will be collected by retro-orbital
puncture method and serum is used for assay of marker enzymes.
The liver will be isolated and washed with normal saline, dried using filtered paper and
weighed immediately. The liver will be then subjected to histopathological examination.
GROUPING:
Rats of either sex were divided into 5 treatment groups of six animals each.
Group-I :Vehicle (Normal control)
Group-II :CCl4(Diseased control)
Group-III : Low dose of LP + CCl4
Group-IV: High dose of LP + CCl4
Group-V : Silymarin (Standard) + CCl4
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The parameters to be estimated are:
1. Serum glutamate pyruvate transaminase (SGPT), Serum glutamate oxaloacetate
transaminase (SGOT), Alkaline phosphate (ALP) and Bilirubin (Total and Direct) activity in serum.
2. Histopathological Studies: Scoring in hepatocytes will be determined from
H-E transverse stain.
7.3.2 PARACETAMOL INDUCED LIVER TOXICITY IN RATS: 25,27
PROCEDURE:
The animals will be divided into 5 groups consisting of six animals. The animals
are then subjected to either one of the following treatments for 9 days. The paracetamol
(p.o.) will be administered after dilution with 40% w/v sucrose. Paracetamol will be
administered in 3 divided doses on day 9. Food will be withdrawn for 12 hrs before paracetamol will be administration to enhance liver damage in animals of groups 2, 3, 4
and 5. The animals will be sacrificed in 48 hrs after the administration of paracetamol.
Blood samples will be collected by retro-orbital puncture method and serum is used for
assay of marker enzymes. The liver will be then isolated and washed with normal saline,
dried using filtered paper and weighed immediately. The liver will be then subjected to
histopathological examination.
GROUPING:
Rats of either sex will be divided into 5 treatment groups of six animals each.
Group-I : Vehicle (Normal control).
Group-II : PCM (Diseased control).
Group-III: Low dose of LP+ PCM.
Group-IV: High dose of LP+ PCM.
Group-V : Silymarin (standard) + PCM.
The parameters to be estimated :
1. Serum glutamate pyruvate transaminase (SGPT), Serum glutamate oxaloacetate
Transaminase (SGOT), Alkaline phosphate (ALP) and Bilirubin (Total and Direct) activity in serum.
2. Histopathological Studies: Scoring in hepatocytes will be determined from
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H-E transverse stain.
7.3.3 THIOACETAMIDE INDUCED LIVER NECROSIS IN RATS25,28:
PROCEDURE:
The animals will be divided into 5 groups consisting of six animals. The animals
will be then subjected to either one of the following treatments for 8 days. The TAA
(s.c.) will be administered after dilution with distilled water. Food will be withdrawn 12
hrs before TAA administration to enhance liver damage in animals of groups 2, 3, 4 and
5. The animals will be sacrificed 48 hrs after the administration of TAA. Blood samples
will be collected by retro-orbital puncture method and serum is used for assay of marker
enzymes. The liver will be isolated and washed with normal saline, dried using filtered
paper and weighed immediately. The liver will be then subjected to histopathological
examination.
GROUPING:
Rats of either sex will be divided into 5 treatment groups of six animals each.
Group-I : Vehicle (Normal control).
Group-II : Distilled water + TAA. (Diseased control)
Group-III: Low dose of LP+ TAA.
Group-IV: High dose of LP+ TAA.
Group-V: Silymarin (standard) + TAA.
The parameters to be estimated :
1. Serum glutamate pyruvate transaminase (SGPT), Serum glutamate oxaloacetate
transaminase (SGOT), Alkaline phosphate (ALT) and Bilirubin (Total and Direct) activity in serum.
2. Histopathological Studies: Scoring in hepatocytes will be determined from
H-E transverse stain.
7.4 Does the study require any investigation or interventions to be conducted on
patients or the human or animals? If so please describe briefly:
YES
Study requires investigation on animals. The effects of the drug will be studied on various parameters using rats as experimental animals.
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7.5 Has ethical clearance been obtained from your institute:
Ethical Committee approval letter is enclosed.
LISTS OF REFERENCES:
1. Dhuley JN, Naik SR. Protective effect of herbal formulation against CCl4-induced
liver injury and survival in rats. J Ethnopharmacol 1997;56:159-164.
2. Sunilson JAJ, Muthappan M, Das A, Suraj A. Int J Pharmacol 2009;5(3):222-227.
3. Smuckler EA. Alcoholic Drink: Its Production and Effects. Fed Proe 1975;34:203844.
4. Kumar CH, Ramesh A, Kumar JNS, Ishaq BM. A Review on Hepatoprotective Activity of Medicinal Plants. Int J Res Pharm Sci 2011;2(3):501-11.
5. Sreelatha S, Padma PR, Umadevi M. Food and Chem Toxicol 2009;(47):702–708.
6. Singh AK, Mani H, Seth P. Picroliv preconditioning protects the rat liver against ischemia-reperfusion injury. Eur J Pharmacol 2000;395:229-239.
7. Agarwal SK, Singh SS, Verma S, Kumar S. Antifungal activity of anthraquinone derivatives from Rheum emodi. J Ethnopharmacol 2000;72:43-46.
8. Li J & Wang Z. Studies on hypoglycemic action of Rheum emodi. J Chin Med Materials 1997;20:249-250.
9. Visen PKS, Shukla B, Patnaik GK, Kaul S, Kapoor NK, Dhawan BN. Hepatoprotective activity of picroliv, the active principle of Picrorrhiza kurrooa, on rat hepatocytes against paracetamol toxicity. Drug Dev Res 1991;23:209-14.
10. Kanchana N, Sadiq A, Mohamed. Hepatoprotective effect of Plumbago zeylanica
on paracetamol induced liver toxicity in rats. Int J Phar Pharm Sci 2011;(1):151.
11. Bagalkotker G., Sagineedu S R, Saad M S, Stanslas J. Phytochemicals from Phyllanthus niruri linn. and their pharmacological properties: a review. J Pharm Pharmacol 2006;58:1559-70.
12. De Araujo JRF, Desouza TP, Pires JG, Soares LA, de Araujo AA, Petrovick PR. A
dry extract of Phyllanthus niruri protects normal cells and induces apoptosis in human liver carcinoma cells. Exp Biol Med 2012;237(11):1281-85.
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13. Murthy VN, Reddy BP, Venkateshwarlu V, Kokate CK, Antihepatotoxic activity of
Eclipta alba, Tephrosia purpurea and Boerhaavia diffusa. Anc Sci Life 1992;11(34):182-86.
14. Sankhala LN, Saini RK, Saini BS. A review on chemical and biological properties
of Tinospra cordiofolia. Int J Med Arom plants 2012;2(2):340-44.
15. Singh J, Bagla A, Pahal A. Hepatoprotective Activity of HerbalExtracts in Carbon
Tetrachloride Intoxicated Albino Rats by Measuring Anti-oxidant Enzymes. Int J
PharmTech Res 2010;2(3):2112-15.
16. Rasool MK, Sabina EP, Kumar L. Therapeutic effect of Indian Ayurvedic herbal
formulation Triphala on acetaminophen indused hepatotoxicity in mice. J Pharmacol
Toxicol 2007;2(8):725-731.
17. Mohamed TSS, Madhusudhana CS, Ramakanth S, Rajan VST, Mahesh KK, Gouthaman K. Hepatoprotective herbs-a review. Int J Res Pharm sci 2010;1(1):1-5.
18. Hartmut J, Gregory JG, Arthur IC, Hinson JK, Dominique PJ, Lemasters. Mechanisms of Hepatotoxicity. Toxicol Sci 2002;65(2):166-176.
19. Baliga MS, Meera S, Mathai B, Rai MP, Pawar V, Palatty PL. Scientific validation
of the ethanomedicinal properties of the Ayurvedic drug Triphala: a review. Chin J
Integr Med 2012;18(12):946-54.
20. Koh PH, Mokhtar RA, Iqbal M. Andrographis paniculata ameliorates carbon tetrachloride (CCl(4))-dependent hepatic damage and toxicity: diminution of oxidative
stress. Redox Rep 2011;16(3):134-43.
21. Trivedi N, Rawal UM. Hepatoprotective and toxicological evaluation of Andrographis paniculata on severe liver damage. Indian J Pharmacol 2000;32:288-293.
22. Pandith S, Sur TK, Jana U, Debnath PK, Sen S, Battacharyya D. Prevention of carbon tetrachloride induced hepatotoxicity in the rats by Adhatoda vasica leaves. Indian J Pharmacol 2004;36(5):312-320.
23. Edmund CS, Wong K, Huang TC, Tasi SC, Liu CS. Tetra methyl pyrazine protects
mice against thioacetamide induced acute hepatotoxicity. J Biomed Sci 2002;9:410414.
24. Shaw SR, Minakshi N, Ahmed N. Dose translation from animal to human studies
revisited. FACEB J Life Sci Forum. 2007;22:659-661.
25. Pachpute AP, Dr.Tushar, Deshmukh A. Antioxidant and hepatoprotective activity of
Piper cubeba. Int J Pharm World Res 2012;3(40):1-12.
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26. Matsuda H, Samukawa K, Kubo M, Antihepatotoxic activity of Ginsenoside Ro.
Plant Med 1991;57:523-26.
27. Jyoti Y, Kamath JV, Asad M. Effect of hexane extract of Boswelia serrata oleo gum
resin on chemically induced liver damage. Pak J Pharm Sci 2006;19(2):129-33.
28. Roy CK, Kamath JV, Asad M. Hepatoprotective activity of Psidium gujava Linn.
Leaf extract. Indian J Exp Biol 2006;44:305-12.
29. Sengottuvelu S, Duraisami S, Nandhakumar J, Duraisami R, Vasudevan M. Hepatoprotective Activity of Camellia sinensis and its Possible Mechanism of Action. Iranian J Pharmacol Ther 2008;7(1):9-14.
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9.
SIGNATURE OF THE CANDIDATE :
10.
REMARKS OF THE GUIDE:
The above information and literature has been extensively investigated, verified and was
found to be correct. The Project is viable and is recommended for clearance and approval.
11.
11.1 NAME AND DESIGNATION OF Mr. Licto Thomas
GUIDE:
Assistant Professor
Dept. of Pharmacology
Shree Devi College of Pharmacy,
Mangalore-574 142.
11.2 SIGNATURE:
11.3 HEAD OF THE DEPARTMENT:
Dr. Jagadish V Kamath
Professor
Dept. of Pharmacology
Shree Devi College of Pharmacy,
Mangalore-574 142.
11.4 SIGNATURE:
12.
12.1 REMARKS OF THE PRINCIPAL:
The project work has potential implication in the field of pharmacology. The above
mentioned information is correct and I recommend the same for approval.
12.2 SIGNATURE:
Dr. Jagadish V Kamath
Principal
Shree Devi College of Pharmacy
Mangalore, Karnataka
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