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“HEPATOPROTECTIVE AND ANTIOXIDANT ACTIVITY OF ALBIZIA
LEBBECK LINN ON THIOACETAMIDE INDUCED HEPATOTOXICITY IN
RATS”
By
DHARNENDRA KUMAR B.K
B.Pharm
M. Pharmacy Dissertation Protocol Submitted to the
Rajiv Gandhi University of Health Sciences,
Bangalore– 560 041, Karnataka.
In partial fulfillment
of the requirement for the Degree of
MASTER OF PHARMACY
IN
PHARMACOLOGY
Under the Guidance of
Mr. JOHN WILKING EINSTEIN M.PHARM [Ph .D]
ASSISTANT PROFESSOR
Dept. of Pharmacology and Toxicology
St. John’s Pharmacy College
St. John’s Educational Institutions
Bangalore – 560040.
2010-11
RAJIV GANDHI UNIVERSITY OF HEALTH SCIENCES,
BANGALORE, KARNATAKA.
ANNEXURE II
PROFORMA FOR REGISTRATION OF SUBJECT FOR DISSERTATION
DHARNENDRA KUMAR B.K
1.
Name of the candidate &
Address.
POST GRADUATE STUDENT,
DEPT OF PHARMACOLOGY AND TOXICOLOGY,
ST. JOHN’S PHARMACY COLLEGE,
BANGALORE- 560040
St. John’s Pharmacy College
2.
Name of the Institution.
No 6, 9th cross, 2nd main, Vijayanagar,
2nd stage (Hampinagar),
Bangalore- 560040
Tel: 91-80-23300958/23300668
Email: [email protected]
3.
Course of the study
& subject.
Master Of Pharmacy - Pharmacology
4.
Date of admission.
25TH-MAY-2009
5.
Title of the topic :“HEPATOPROTECTIVE AND ANTIOXIDANT ACTIVITY OF ALBIZIA LEBBECK
LINN. ON THIOACETAMIDE INDUCED HEPATOTOXICITY IN RATS.”
6.0
Brief resume of the intended work:
6.1 – Need for the study:
Liver diseases such as jaundice, cirrhosis and fatty liver are very common
worldwide. There are many factors for the development of these diseases, one of the important
factors being the use of drugs.1
Drug-induced liver injury is a major health problem that challenges not only health
care professionals but also the pharmaceutical industry and drug regulatory agencies.
According to the United States Acute Liver Failure Study Group, drug-induced liver injury
accounts for more than 50% of acute liver failure, including hepatotoxicity caused by overdose
of acetaminophen (39%) and idiosyncratic liver injury triggered by other drugs (13%). Drugs
are an important cause on liver injury. Approximately 75% of the idiosyncratic drug reaction
results in liver transplantation (or) death.2
In spite of tremendous advances in pharmaceutical field, there is no satisfactory drug
for the treatment of hepatotoxicity. Synthetic drugs used in the treatment of liver diseases are
sometimes less effective and cause serious adverse effects. Many formulations containing
herbal extracts are sold in the Indian market for liver disorders. But management of liver
disorders by a simple and precise herbal drug is still an intriguing problem.3 Therefore it is
necessary to search for alternative drugs for the treatment of liver disease in order to replace
currently used drugs of doubtful efficacy and safety. Hence world health organization (WHO)
is giving stress to develop the herbal medicines for the treatment of hepatotoxicity and there is
worldwide trend for use of traditional herbal drugs for the treatment of liver diseases.
Albizia lebbeck leaves is said to possess hepatoprotective activity and used in ayurveda
and other ancient system of medicine for the treatment of liver diseases4. Hence this study is
design to evaluate the hepatoprotective and antioxidant activity of Albizia lebbeck plant
extracts in rats.
6.2 -Review of Literature:-
Thioacetamide is a weak diuretic and acts by inhibiting carbonic anhydrase enzyme.
Major adverse effect of Thioacetamide is hepatotoxicity and Thioacetamide (TAA)
administration is an established technique for generating rat models of liver fibrosis and
cirrhosis. Oxidative stress is believed to be involved as TAA-induced liver fibrosis is initiated
by thioacetamide S-oxide, which is derived from the biotransformation of TAA by the
microsomal flavine-adenine dinucleotide (FAD)-containing monooxygense (FMO) and
cytochrome P450 systems.5
Kumar et al6 have reported The ethanolic extract of Trianthema portulacastrum L.
(Aizoaceae) showed a significant dose dependent protective effect against paracetamol and
thioacetamide induced hepatotoxicity in albino rats. Galisteo et al7 have reported the capability
of an ethanol extract of Rosmarinus tomentosus to protect rat liver in an experimental model of
cirrhosis induced by thioacetamide (TAA) has been evaluated.
Various plants are known to possess hepatoprotective activity in animals.8 Although, a
big list of hepatoprotective phytomolecules are reported in the scientific literature. silymarin,
andrographolide,
neoandrographolide,
curcumin,
picroside,
kutkoside,
phyllanthin,
hypophyllanthin, and glycyrrhizin have largely attracted the scientific community.4 Several
multi herbal formulations are available in the market for the treatment of liver diseases.9
Albizia lebbeck (L.) Benth. Belongs to the Family Mimosaceae.10 The leaves contains
Alkaloids, kaemferol, quercetin, caffeic acid, mature leaves contain keto acids.11,12
The plant is proved for
following activities like Antispermatogenic, Antiandrogenic,
Anticonvulsant13, Milk-clotting14,Antibacterial15,Anti-inflammatory16, Antioxidant activities.17
plant is also said to have other activities 11
6.3 – Objective of the Study:

Collection and authentication of Albizia lebbeck Linn (Mimosaceae) plant leaves.

Extraction of plant material using various solvents of increasing order of polarity ether,
alcohol and water.

Evaluation of Hepatoprotective activity of Albizia lebbeck extracts.
7.0 Materials and Methods:
7.1 Source of Data:
Whole work is planned to generate data from laboratory studies. Experiments are
performed as described in reference, experimental studies in journals and in textbooks
available with college, IISc library, Bangalore, RGUHS digital library (Helinet).
Web sites: www.sciencedirect.com
www.ncbi.nlm.nih.gov/pubmed
www.google.com
www.ijp-online.com
www.pubmedcentral.com
Will be used to obtain related information regarding this research protocol.
7.2 Method of collection of data:
The relevant data whatever related to the study will be collected from the internet.
Animal required

Species:

Age/Weight/Size:

Gender:

Numbers to be used: 6 animals in each group.
: Albino Wistar Rats.
: Rats 200±20g.
: male.
Healthy male Wister rats weighing (200±20g) maintained under controlled temperature
at 23°C ± 2°C and a 12 hr light-12 hr dark period will be employed for the experimentation.
Animals used for the study will be supplied by the St. Johns Pharmacy College, animal house.
Animals will be housed in large spacious polypropylene cages on clean paddy husk bedding
during the course of the experimental period.
7.3 Method:
Experimental design:
Rats will be divided into nine groups, each group consisting of six animals.
DRUG
Group-1
Saline(2ml/kg)
Group-2
Thioacetamide (TAA) (100 mg/kg b. w., s.c.) as a 2% w/v solution
in water for injection on day 1st and then normal saline for 21 days
Group-3
Thioacetamide (TAA) (100 mg/kg b. w., s.c.) as a 2% w/v solution
in water for injection on day 1st and then silymarin (25mg/kg per
day, p. o.) for 21 days
Group-4
Thioacetamide (TAA) (100 mg/kg b .w., s.c.) as a 2% w/v solution
in water for injection on day 1st and then ether
extract of
A.lebbeck (low dose) for 21 days
Group-5
Thioacetamide (TAA) (100 mg/kg b .w., s.c.) as a 2% w/v solution
in water for injection on day 1st and then ether
extract of
A.lebbeck (medium dose) for 21 days
Group-6
Thioacetamide (TAA) (100 mg/kg b .w., s.c.) as a 2% w/v solution
in water for injection on day 1st and then ethanolic extract of
A.lebbeck (low dose) for 21 days
Group-7
Thioacetamide (TAA) (100 mg/kg b .w., s.c.) as a 2% w/v solution
in water for injection on day 1st and then ethanolic extract of
A.lebbeck (medium dose) for 21 days
Group-8
Thioacetamide (TAA) (100 mg/kg b .w., s.c.) as a 2% w/v solution
in water for injection on day 1st and then aqueous extract of
A.lebbeck (low dose) for 21 days
Group-9
Thioacetamide (TAA) (100 mg/kg b .w., s.c.) as a 2% w/v solution
in water for injection on day 1st and then aqueous extract of
A.lebbeck (medium dose) for 21 days
At the end of experimental period, all the animals will be sacrificed by cervical dislocation.
Blood samples will be collected, allowed to clot. Serum will be separated by centrifuging at
2500 rpm for 15 min and analyzed by auto analyzer by using biochemical kits for the
following parameters.
(A) Biochemical Parameters. 18, 19

Aspartate amino transferase (AST)

Alanine amino transferase (ALT)

Alkaline Phosphatase (ALP)

Total bilirubin.

Total protein.
The liver samples will be collected from each animal and morphological changes will
be observed. Two lobes of liver will be used for histopathological examination.
A 10% of liver homogenate will be prepared and the following antioxidant parameters
will be estimated using standard procedure.

Lipid peroxidation (LPO).

Superoxide dismutase (SOD).

Catalase (CAT).

Glutathione peroxidase (GPx).
Acute oral toxicity study: 20 Acute toxicity studies for the ether, ethanolic and aqueous extract
of Albizia lebbeck (L.) DC will be done according to the OECD guidelines No: 423 and low
and high dose will be selected for treatment.
Statistical analysis:
The data obtained will be analyzed using one way ANOVA (Graph pad prism version
5.00 software) followed by suitable post test.
Number Of Animals Required
Total No. of animals required:
No of animals for acute oral toxicity
=18
No of animals in each group
=06
No of groups
=09
Total no. of animals required = (06 × 09) =54+18 =72
7.3 - Does the study require any investigations or interventions to be conducted on
Patients or other humans or animals? If so, Please describe briefly.
Yes, the experimental models require usage of laboratory animals (Male Wistar rats ).
7.4 – Has ethical clearance been obtained from your Institution in case of 7.3?
Yes, ethical clearance has been obtained (copy enclosed).
8.0
List of References:1. Jyothi Y, Jagadish V K, Asad M. Effect of Hexane extract of Boswellia serrata oleogum resinon chemically induced liver damage. Pak J Pharm Sci 2006;19:125-9.
2. Mohamed Saleem TS, Christina AJM, Chidambaranathan N, Ravi V, Gauthaman K.
Hepatoprotective activity of Annona squamosa Linn. on experimental animal model. Int
J Appl Res Nat Pro 2008;1:1-7.
3. Gupta AK, Misra N. Hepatoprotective Activity of Aqueous Ethanolic Extract of
Chamomile capitula in Paracetamol Intoxicated Albino Rats. Am J Pharmacol Toxicol
2006; 1:17-20.
4. Low TY, Leow CK, Salto-Tellez M and Chung CMM. A proteomic analysis of
thioacetamide-induced hepatotoxicity and cirrhosis in rat livers. Proteomics
2004;4:3960-74.
5. Madhava CK, Sivaji K, Tulasi RK. Flowering plants of chittoor district Andra
Pradesh,published by students offset printers(Tirupati), Ist Edition:552.
6. Kumar G, Banu S, Pappa PV, Sundararajan M, Pandian R. Hepatoprotective activity of
Trianthema portulacastrum L. against paracetamol and thioacetamide intoxication in
albino rats J Ethnopharmacol 2004;92: 37–40.
7. Galisteo M,Sua RA, Montilla MP, Fernandez MI, Gil A,Navarro MC. Protective effects
of Rosmarinus tomentosus ethanol extract on thioacetamide-induced liver cirrhosis in
rats. Phytomed 2006; 13:101–8.
8. Sharma B, Sharma U K. Hepatoprotective activity of some indigenous plants. Int J
PharmTech Res 2009;1:1330-4.
9. Girish C, Koner BC, Jayanthi S, Rao KR, Rajesh B, Pradhan SC. Hepatoprotective
activity of six polyherbal formulations in paracetamol induced liver toxicity in mice. .
Indian J Med Res 2009;129:569-78.
10. Sasya sampada digitized inventory of plants resources of india[online] 2009 [cited on
2009 Dec 14];[1 screen]. Available from
URL.http://221.135.191.194/sasya/showhtml.php?d=html_files/Albizia%20lebbeck1/Al
bizia%20lebbeck.htm
11. Prajapati ND, Purohit SS,Sharma AK, Kumar T.A Handbook of Medicinal Plants : A
Complete Source Book, publication Agrobios(India),Ist Edition: 26.
12. Yoganarasimhan SN. Medicinal Plants of India:vol:2: 30-1
13. Gupta RS, Kachhawa BS, Chaudhary R. Antispermatogenic, antiandrogenic activities of
Albizia lebbeck (L.) Benth bark extract in male albino rats. Phytomed 2006;13:277-83.
14. Egito AS, Girardet JM, Laguna LE, Poirson C, Molle D, Miclo L, et al. Milk-clotting
activity of enzyme extracts from sunflower and Albizia seeds and specific hydrolysis of
bovine k-casein. Int Dairy J 2007; 17:816-25.
15. Rashid RB, Chowdhury R, Jabber A, Hasan CM, Rashid MA. Constituents of albizzia
lebbeck and antibacterial activity of an isolated flavone derivative. Saudi pharm
J;2003:11:52-6.
16. Prakash Babu N, Pandikumar P, Ignacimuthu S. Anti-inflammatory activity of Albizia
lebbeck Benth, an ethnomedicinal plant, in acute and chronic animal models of
inflammation. J Ethnopharmacol 2009;125:356–6.
17. Resmi CR, Venukumar MR, Latha S. Antioxidant activity of Albizzia lebbeck
(Linn.)benth. in alloxan diabetic rats. Indian J Physiol Pharmacol 2006;50:297-302.
18. Aftab A, Pillai KK, Abul KN, Shibli JA, Pal SN, Balani DK. Evaluation of
hepatoprotective potential of jigrine post-treatment against thioacetamide induced
hepatic damage. J Ethnopharmacol 2002;79:35–41.
19. Kumar G, Sharmila Banu G, Vanitha Pappa P, Sundararajan M, Rajasekara Pandian M.
Hepatoprotective activity of Trianthema portulacastrum L. against paracetamol and
thioacetamide intoxication in albino rats.J Ethnopharmacol 2004;92:37-40.
20. The Organization of Economic Co-Operation and Development (OECD). The OECD
Guideline for Testing of Chemicals: 423 Acute Oral Toxicity, OECD, Paris 2001 ;1-14