“EVALUATION OF HEPATOPROTECTIVE ACTIVITY OF

JUSTICIA BETONICA LINN. EXTRACT IN RATS.”

SYNOPSIS FOR

M. PHARM DISSERTATION

SUBMITTED TO RAJIV GANDHI UNIVERSITY OF HEALTH SCIENCES

KARNATAKA, BANGALORE

BYMr. YOGESH H PATEL

DEPARTMENT OF PHARMACOLOGY

UNDER THE GUIDENCE OFMrs. RUBY .K. KOSHY M.PHARM (PhD)

ASST.PROFESSOR

DEPARTMENT OF PHARMACOLOGY

THE OXFORD COLLEGE OF PHARMACY

HOHGASANDRA, BANGALORE-68

KARNATAKA1

RAJIV GANDHI UNIVERSITY OF HEALTH SCIENCES,

BANGALORE, KARNATAKA,

ANNEXURE II

PROFORMA FOR REGISTRATION OF SUBJECTS FOR

DISSERTATION

1. Name of the Candidate

and Address

PATEL YOGESH HARESHBHAI

a. Permanent Address

S/o. Hareshbhai A Patel

A-14,Natraj Bankers Society,

Kheralu Road,Visnagar-384315

Gujarat.

b. Postal Address

The Oxford College of Pharmacy

No.6/9, 1st Cross,

Begur Main Road, Hongasandra

Bangalore- 68, Karnataka

2. Name of the Institute The Oxford College of Pharmacy

No.6/9, 1st Cross,

Begur Main Road, Hongasandra

Bangalore-68, Karnataka

3. Course of Study and Subject Master in Pharmacy

[Pharmacology]

4. Date of Admission to Course

25 September 2010

5. Title of the Topic:

“Evaluation of Hepatoprotective activity of Justicia betonica Linn. extract in rats.”

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6. BRIEF RESUME OF THE INTENDED WORK:

6.1 Need of study:

Any change in anatomy or function of liver is characterized as liver diseases.

Liver has tremendous capacity to detoxify toxic principle and synthesized useful

principle. Therefore damage to the liver inflicted by hepatic-toxic agents is of grave

consequences. Various type of liver disorder is characterized by cirrhosis, jaundice,

tumors, metabolic and degenerative lesions, liver cell necrosis and hepatitis etc.

besides, liver disorder can rise due to xenobiotics, excessive drug therapy,

environmental pollutions and alcohol intoxication. Management of liver diseases is

still challenge to modern medicine. The modern allopathic drugs have very little to

offer for alleviation of hepatic aliments and some of these drugs adversely affect the

liver function1,2. Drugs discovered from herbs will give good therapeutic medicine

with fewer side effects and lower cost. The present study will help the industry to

develop herbal medicine in the treatment of hepatotoxicity with fewer side effects. In

future the development of formulation by these plant constituents will give good

hepatoprotective medicine at lower cost. Herbal drugs or their extracts are prescribed

widely, even when their biological active compounds are unknown3.

ANTI OXIDANT ACTIVITY:

Reactive oxygen species (ROS) induced oxidative damage to tissues is

common end point in chronic pathological conditions such as atherosclerosis, diabetes

and rheumatoid arthritis and cardiovascular complications4. Oxidative stress induced

by reactive species plays a major role as endogenous initiators and promorters of DNA

damage and mutation. Degenerative and pathological process such as alzheimer’s

disease, rheumatoid arthritis, coronary artery disease, parkinson’s disease, cataracts,

inflammation, cancer, aging. Pancreas damage in diabetes are accompanied by the free

radicals and reactive oxygen species oxidative stress5. Oxidative damage is shown to

be extensive and has been proposed as a major cause of physiological changes

associated with ageing and degenerative diseases related to ageing such as cancer and

diabetes6. Oxidative stress is likely to play a causative role in tissue and cellular

damage in these diseases. Oxidative stress occurs by the source of stimulated

polymorph nuclear leukocytes, macrophages, peroxysomes and aerobic respiration,

causing intracellular and extracellular damage of macromolecular like proteins, lipids 3

and nucleic acid7. Superoxide dismutase, glutathione peroxidase (GPx) and catalase

are the antioxidant enzymes. Hydroxyl radical produced by metal catalyzing process

by cleavage of water and hydrogen peroxide, initiates the peroxidation of the cell

membrane Lipids (lipid peroxides), converts malondialdehyde into carcinogenic and

mutagenic compounds8. Many clinical and experimental studies have supported the

association of increased oxidative stress and hyperglycemia. Excessive ROS or their

inadequate neutralization by antioxidants leads to damage of proteins, lipids and

DNA9. Thus, ROS mediated oxidative stress plays a major part in the pathogenesis of

cellular dysfunctions involving hepatic, cardiovascular and reproductive systems

besides teratogenic and mutational defects10. Antioxidant agents form an effective

mechanism in preventing mutagenesis by scavenging the free radicals. They do this by

being oxidized themselves, so antioxidants are often  reducing agents such

as thiols, ascorbic acid or polyphenols11.

Natural antioxidants can protect the human body from free radicals and retard

the progress of many chronic diseases as well as lipid oxidative rancidity in foods.

Furthermore, the area of natural antioxidants developed enormously in the past decade

mainly because of the increasing limitations on the use of synthetic antioxidants.

Medicinal plants have been used for a large range of purposes, including medicine,

nutrition, flavourings, beverages, dyeing, repellents, fragrances, cosmetics, charms,

smoking and industrial uses. Now a day, medicinal plant extracts are very attractive

not only in the modern phytotherapy but also for food industry12.

As there is no scientific evidence for the use of Justicia betonica Linn. as a

hepatoprotective . So the present study was undertaken to explore its hepatoprotective

activity.

6.2 Review of Literature:

Justicia betonica Linn. is belonging to family acanthaceae. Justicia betonica Linn. is a

glabrous shrub that can grow upto 2 m high. The stem is threat, tumid and purple

above nodes. Leaves are oval-oblong-lanceolate, from an acute base tapering into

petiole, subcuminate. Spikes are terminal, simple, occasionally branched. Roots,

leaves and flowers are used13.

Chemical constituents of Justicia betonica are olean – 12 –ene -1beta, 11 alpha,

28-tetraol, 28-O-beta-D-glucopyranosyl- (1->2) - beta - D-glucopyranoside, 11alpha 4

methoxy-olean-12-ene-1beta, 3beta, 28-triol 28-O-beta – D – glucopyranosyl - (1->2)-

beta -D-glucopyranosize, jusbetonin, 10H-quindoline, 6Hquinindoline and 5H, 6H-

quinindolins-11-one13.

Justicia betonica is used in the treatment of various gastrointestinal complaints

and to relive stomachache14.

The Indians make use of the plant in the treatment of diarrhea. To treat vomiting

and constipation the Indian used the inflorescence given orally15.

The Indian and sriLankan community apply poultice made from crushed leaves of

the plant over abscesses toprovide relieve of pain and swelling.the leaves and

flower ash are used in the treatment of orchitis in Kenya. Other species of Justicia

betonica are used as hepatoprotective activity. Like Justicia ecbolium Linn. roots

are useful in jaundice16.

The antibacterial activity of Justicia betonica Linn. were tested against

pathogenic bacteria, viz. Aeromonas hydrophila, Escherichia coli, Klebsiella

pneumoniae, Pseudomonas aeruginosa, Salmonella typhi, Salmonella sp,

Staphylococcus aureus, Vibrio cholerae and V. parahemolyticus17.

Justicia paniculata, decoction or infusion of the leaves has been used in sluggish

liver18.

The leaves of Justicia adhatoda showed significant hepatoprotective effect at a

dose of 50-100mg/kg on the liver damage induced by d-galactosamine in rats19.

6.3 Objective of study:

The present study will be aimed at evaluating the hepatoprotective activity of

Justicia betonica Linn. Ethanolic leaf Extract in rats.

Experimental design for hepatoprotective activity.

Carbon tetrachloride induced liver fibrosis in rats20

Paracetamol Induced Liver toxicity in rats21

Isoniazid and Rifampacin induced liver necrosis in rats22

Biochemical estimation:

Alkaline phosphatase (ALP)23

Alanine transaminases (ALT)24

Aspartate transaminases (AST)24

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Gamma glutamate transpeptase (γ-GT)24

Total protein24

Bilirubin25

Super Oxide Dimutase(SOD)26

Histopathological studies27

7. MATERIALS AND METHODS:

7.1 Source of Data:

Data will be obtained from CD-rom, Internet facilities, Literatures and related

articles from libraries of The Oxford College of Pharmacy, Indian Institute of

Sciences, Government College of Pharmacy etc., and other Research Publications

and Journals.

7.2 Method of Collection of Data:

The data collected will be based on animal experimentation as per the parameters

studied under each animal model, which are mentioned under the objectives of the

study.

EXPERIMENTAL MODELS

Source of animal: Wistar albino rats will be obtained from animal house of

NIMHANS, IISC and Veterinary hospital.

Qualitative analysis:

The Ethanolic extract of Justicia betonica Linn. will be tested for different phyto

constituents like alkaloids, tannins, steroids, saponins, flavonoids, terpenoids, glycosides,

amino acids and carbohydrates28.

Preparation of Extract:

The leaf Justicia betonica Linn. will be collected from Thiruvallur (dt) Tamil Nadu

and dried. The shade dried leafs will be powdered. About 500g of dried powder was

extracted with ethanol by continuous hot percolation, using soxhlet apparatus. The

concentrated crude extracts will be lyophilized in to powder and used for the study.

6

Acute toxicity study29:

Procedure

Use over night fasted rats. Each dose group should consist of 10 rats. Inject the

drug by intraperitoneal route and observe the animal for 2 hours for death due to acute

toxicity. The percent mortality values are converted to probit values by reading the

corresponding probit units. Plot the probit values against log doses and read LD50 from

the graph.

HEPATOPROTECTIVE ACTIVITY:

1) Carbon tetrachloride induced liver toxicity in rats20

Test compounds:

The Ethanolic leaf extract of Justicia betonica Linn. and standard drug Silymarin

(100 mg/kg body weight) will be used.

Rats will be divided into four groups as following protocol.

Treatment Protocol:

GROUP I: Normal control (n=4), the animals will be given gum acacia (5mg/kg.

p.o.

for 5 Days + 6th Day to 14th days Normal Saline)

GROUP II: Hepatotoxic control (n=4, the animals will be given CCl4 for 5 Days)

GROUP III: Treatment group (n=4, the animals will be given CCl4 for 5 Days + 6th

Ethanolic leaf extract of Justicia betonica Linn. for 14 days)

GROUP IV: Standard group (n=4), the animals will be given CCl4 for 5 Days + from

6th Silymarin for 14 Days)

After 24 hours of drug treatment, the animals will be dissected under ether

anesthesia. Blood from each rat will be withdrawn from carotid artery at the neck

and collected in previously labeled centrifuging tubes and allowed to clot for 30 min

at room temperature. Serum will be separated by centrifugation at 3000 rpm for 15

minutes. The separated serum will be used for the estimation biochemical

parameters like Alanine transaminases (ALT)24, alkaline phosphatase (ALP)23, total

Bilurubin25, total protein24, will be analyzed.

Histopathological Studies

Liver slices fixed for 12hrs in Bovin’s solution will be processed for paraffin

embedding following standard micro technique27. 5µ section of the livers grained 7

with alum haematoxylin and eosin, will be observed microscopically for

histopathological changes i.e., normal liver, damaged and recovered liver will be

studied and compared.

3) Paracetamol induced hepatotoxicity in rats:21

Wistar rats of 150-200g will be divided into four groups with six each (n=4). Group

1 and group 2 were served as normal control and disease control respectively. Group

3 and 4 corresponded to reference standard (Silymarin -100mg/kg/day, p.o.), Justicia

betonica ethanolic leaf extract as drug. The treatment will be carried out for 7 days

and on seventh day at night all the animals will be fasted for 12 hrs and all the

animals except those in group 1 were treated with paracetamol (2g/kg, p.o.) in

sucrose solution (40 % v/v) in three divided doses. 48 hrs after paracetamol

administration, body weight was documented and blood samples were collected for

the estimation of biochemical parameters, namely, SGPT, SGOT, ALP, SOD and

total bilirubin. All the animals will be sacrificed and the liver weight will be

recorded. Liver tissues collected were subjected to histopathological studies30.

4) Isoniazid and Rifampicin induced liver necrosis in rats22

Isoniazid and Rifampicin (100 mg/kg) solution will be prepared separately in sterile

distilled water. Rats will be treated with Isoniazid, co-administered with Rifampicin

for 21 days by i.p route31. Silymarin (100mg/kg p.o) will be used as a standard drug

in this study32. Rats will be treated with Ethanolic Extract of Justicia betonica Linn.

Rats will be divided into four groups as following protocol.

Treatment Protocol:

GROUP I: Normal control (n=4, the animals will be given normal saline only)

GROUP II: Hepatotoxic control (n=4, the animals will be given INH+RIF for 21

Days)

GROUP III: Treatment group (n=4, the animals will be given INH+RIF+ Ethanolic

leaf Extract of Justicia betonica Linn. for 21 days)

GROUP IV: Standard group (n=4, the animals will be given INH+RIF+Silymarin

for 21 days)

Rats will be treated as per the treatment protocol. Body weights of these rats will be

8

monitored sequentially in control and experimental animals for a period of 21 days.

Biochemical estimation: Rats will be sacrificed 1 hour after administration on day 21.

The blood will be collected by retro orbital artery bleeding. Blood samples will be

centrifuged for 10 minutes at 3000 rpm to separate the serum. ALP, ALT, AST, γ-GT,

Total protein and Bilirubin levels will be estimated from the serum by using standard

kits.

Histopathological studies: The livers will be excised quickly and fixed in 10% formalin

and stained with haemotoxylin and eosin and then observed under microscope for

degeneration, fatty changes, necrotic changes and evidence of hepatotoxicity if any.

Statistical Analysis: All data will be expressed as mean ± SD. Student’s t-test will be

performed for each experimental group. Data will be compared by analysis of variance

(ANOVA) and only values with P<0.05 will be considered as significant.

7.3 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 / extract will be studied

on various parameters using rats as experimental animal model.

7.4 Has ethical clearance been obtained from your institute

Ethical Committee approval letter is enclosed.

8. List of References:

1. Meyer SA, Kulkarni AP, Hepatotoxicity. In:Hodgson E, Smart RC. Edition

Introduction to Biochemical Toxicology, New York: A. John Wiley and Sons Inc

2001; 3rd ed: pp 487 – 90.

2. Harsh Mohan. Text book of Pathology. New Delhi, Jaypee Publisher 2002; 4 th

ed: pp 569 – 630.

3. Gupta RK, Kesari AN, Murthy PS, Chandra R, Tandon V, Watal G.

Hypoglycemic and antidiabetic effect of ethanolic extract of leaves of Annona

squamosa L. in experimental animals. J Ethnopharmacol 2005; 99(1): 75-81.

4. Chauhan PS, Sundaram K. Genetic toxicology of environmental chemicals, A

perspective. Bulletin of Sciences1986; 2: 41-47.

5. Joseph LE, Goldfine IR, Betty AM, Gerold MG. Oxidative stress and stress 9

activated signaling pathway: A unifying Hypothesis of type 2 diabetes. Endocrine

review 2006; 23 (5): 599‐602.

6. Ceriello A. New insights on oxidative stress and diabetic disorders may lead to

casual antioxidant therapy. Diabetes care 2003; 26: 1589-1596.

7. Halliwell B, Gutteridge JM. Free radicals in Biology and Medicine. Oxford:

Oxford University, 1989; pp 96-98.

8. Black HS, Okotie-Eboh G, Gerguis J. Diet potentiate the UV- carcinogenic

response to beta-carotene. Nutr.Cancer 2003; 37: 173-178.

9. Shigenaga MK, Hagen TM , Ames BN. Oxidative damage and mitochondrial

decay in aging. Proc Natl Acad Sci USA 1994; 91: 10771- 8.

10. Blasiak J, Arabski M, Krupa K, Woziak K, Zadrozny H, Kasnik J, Zurawska M,

Derwoski J.DNA damage and repair in type 2 Diabetes mellitus. Mut Res 2004;

554: 297-304.

11. Sahoo TA. Antibacterial activity of Barringtonia acutangula Linn. against

selected urinary tract pathogens, Ind J Pharm Sci 2008; 70(5): 677‐680.

12. Srinivasan R, Chandrasekar M, Nanjan MJ and Suresh B. Antioxidant activity of

Caesalpinia digyna root. J Ethnopharmacol 2007; 25: 1‐8.

13. Khare CP. Indian Medicinal Plants: an illustrated dictionary springer-verlag

berlin 2007; pp 250.

14. Kokwaro JO. Timothy Johns Luo. Biological Dictionary East African Education

publishers Nairobi 1998; pp: 13.

15. Pascaline J, Catherine, George, Dennis O, Charles M. Herbal treatment in aldai &

kaptumo divisons in nandi district, rift nally province, kanya, African Journal of

Traditional, Complimantary and Alternative Medicines 2008; 5(1): 103-105.

16. Dr. K.M. Nadkarni. Indian Materia Medica Plant no: 1390 2000; (1): pp 714.

17. Sasikumar JM, Tha.thayumanavan, Subhashkumar R, Janardhanan K,

Lakshamanaperumalsamy P. Anti bacterial activity of Justicia betonica Linn

Natural Product Radiance 2007; vol 6(1): 34-39.

18. Dr. K.M. Nadkarni. Indian Materia Medica Plant no: 171 2000; volume 1: pp

103.

19. Bhattacharya D, Pundits, Jana U, Sens & Sar TK. Hepatoprotective activity of

adhatoda vasaka aq.leaf extract on d-galactosamine induced liver damage

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fitoterapia 2005; 76: 223-225.

20. Gerhard Vogel. H, Drug Discovery and Evaluation. 2nd ed. Springer; pp 940-41.

21. Devaraj VC, Gopala Krishna B, Viswanatha GL, Jagadish V, Sanjay Kumar.

Hepatoprotective activity of Hepax- A polyherbal formulation, Asian Pacific

Journal of Tropical Biomedicine 2011; 142-146.

22. Mohamed Saleem TS, Christina AJM, Chidambaranathan N, Ravi V, Gauthaman

K. International Journal of Applied Research in Natural Productst 2008; Vol.

1(3): 1-7.

23. Reitman S, Frankel S. In vitro determination of transaminase activity in serum.

Am. J. Clin. Path 1975; 28: 56 – 60.

24. Bat-Ami Israeli, Bogin E. Biochemical changes in liver, kidney and blood

associated with common bile duct ligation Clinica Chimica Acta 1986; Vol 160

(2): 211-221.

25. Richard MD, Wennberg P, Richard depp MD. Indications for early exchange

transfusion in patients with erythroblastosis fetalis, The journal of pediatrics1978;

volume 92 issue 5: 789-792.

26. Al-Harbi MM, Quershi S, Raza M, Ahmed MM, Afzal M, Shah AH. Gastric

antiulcer and cytoprotective effect Commiphora molmol in rats. J

Ethnopharmacol 1997; 55: 141-50.

27. Galigher AE, Kozloff, EN. Essential Practical Microtechnique, Lea and

Febiger,Philadelphia, 1971; 2nd edn: pp 77-210

28. Dongmei Yang ME1, Qiushuang Wang ME1, Leqin Ke BE1,2, Jianmei Jiang BE1, Tiejin Ying PhD. Antioxidant activities of various

extracts of lotus (Nelumbo nuficera Gaertn) rhizome, Asia Pac J Clin Nutr 2007;

16 (Suppl 1): 158-163.

29. Kulkarni S.K., Hand Book of Experimental Pharmacology (3rd ed. Revised and

Enlarged Edition) Delhi, Vallabh Prakashan 1999; pp.169-170.

30. Chanchal KR, Jagadish VK, Mohammed A. Hepatoprotective activity of Psidium

guajava Linn. Leaf extract. Ind J Exp Biol 2006; 44: 305-11.

31. Jiang Yue, Ren-xiu Peng, Jing Yang, Rui Kong, Juan Liu. CYP2E1 mediated

11

isoniazid-induced hepatotoxicity in rats. Acta Pharmacol Sin2004; 25(5): 699-

704.

32. Parthasarathy R, Nivethetha M, Brindha P. Hepatoprotective activity of

Caesalpinia bonducella seeds on paracetamol induced hepatotoxicity in male

albino rats. Indian Drugs 2007; 44(5): 401-404.

33. Rajesh KG, Achyut NK, Geeta W, Murthy PS, Ramesh C, Vibha T. Nutritional

and Hypoglycemic Effect of Fruit Pulp of Annona squamosa in Normal Healthy

and Alloxan-Induced Diabetic Rabbits. Ann Nutr Metab 2005; 49: 407–413.

9. SIGNATURE OF THE CANDIDATE:

[Patel Yogesh Hareshbhai]

10. REMARKS OF THE GUIDE: Recommended

11. Name and Designation of

12

11.1 Guide Mrs. Ruby K. Koshy

Asst. Professor

Department of Pharmacology

11.2 Signature

11.3 Head of the Department Dr. S. Jai Kumar

Professor & HOD

Department of Pharmacology

The Oxford College of Pharmacy

11.4 Signature

12. 12.1 Remarks of the Principal: Recommended and Forwarded

12.2 Signature

Dr.Padmaa M. Paarakh

Principal,

The Oxford College of Pharmacy

Bangalore-68

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