Rajiv Gandhi University of Health Sciences Karnataka, Bengaluru

“EVALUATION OF ANTIHEPATOTOXIC AND ANTIOXIDANT ACTIVITY OF PARACALYX SCARIOSA (ROXB.) ALI. IN CELL LINES & STUDY OF THEIR POSSIBLE MECHANISM OF ACTION”

A Protocol submitted to Rajiv Gandhi University of Health Sciences Karnataka, Bengaluru In partial fulfillment of the requirement for the award of

MASTER OF PHARMACY IN PHARMACOLOGY

RAMOJI ALLA

Department of Pharmacology, National College of Pharmacy, Balraj-Urs Road, Shimoga-577 201 Karnataka-INDIA RAJIV GANDHI UNIVERSITY OF HEALTH SCIENCES, KARNATAKA, BENGALURU

Annexure – II

PROFORMA FOR REGISTRATION OF SUBJECTS FOR DISSERTATION

RAMOJI ALLA S/O GOPALA KRISHNA RAO ALLA Name and Address of the 01 RANGANNAGUDEM Candidate BAPULAPADU (MD), KRISHNA (DT) A.P. 521110.

NATIONAL COLLEGE OF PHARMACY, 02 Name of the Institution BALRAJ-URS ROAD, SHIMOGA-577201, KARNATAKA.

Course of the Study 03 M. Pharm. (Pharmacology) Branch 04 Date of Admission to course 01-10-2010 EVALUATION OF ANTIHEPATOTOXIC AND ANTIOXIDANT ACTIVITY OF 05 Title of the Topic PARACALYX SCARIOSA (ROXB.) ALI. IN CELL LINES & STUDY OF THEIR POSSIBLE MECHANISM OF ACTION. Brief resume of the intended work Enclosure – I 6.1. Need for the Study 06 6.2. Review of the Literature Enclosure – II

6.3. Objective of the Study Enclosure – III

Materials and Methods Enclosure – IV 7.1. Source of data 7.2. Methods of collection of data Enclosure – V 07 7.3. Does the study require any Investigations on animals? Enclosure – VI If yes give details 7.4. Has ethical clearance been obtained form your institution Yes – Enclosure – VI – A in case of 7.3. 08 List of References (About 4 – 6) Enclosure – VII

09 Signature of the Candidate

The present research work is original and not 10 Remarks of the Guide published in any of the journals. This work can be carried out in our Pharmacology Department laboratory.

Name and Designation of (in Block Letters)

11.1. Guide Dr. K. L. MANKANI. Ph.D. PROFESSOR DEPARTMENT OF PHARMACOLOGY, NATIONAL COLLEGE OF PHARMACY, BALRAJ-URS ROAD, SHIMOGA-577201, KARNATAKA-INDIA.

11.2.Signature

11.3.Co-Guide (if any) N. A. 11

11.4.Signature N. A.

11.5. Head of the Department Dr. K. L. MANKANI. Ph.D HEAD OF THE DEPARTMENT, DEPARTMENT OF PHARMACOLOGY, NATIONAL COLLEGE OF PHARMACY, SHIMOGA-577201, KARNATAKA-INDIA.

11.6. Signature

Remarks of the Principal The present study is permitted to perform in the Pharmacology Department laboratory of our institution and the study protocol has been 12 approved by IAEC.

12.1. Signature Principal ENCLOSURE I

Brief resume of intended work:

6.1 Need for the study:

Medicinal plants have been acknowledged and are extremely valued all over the world as a prosperous source of bioactive for the prevention and treatment of ailments.

Herbal medicines are being used by about 80% of the world population primarily in the developing countries for primary health care. They have stood the test of time for their safety, efficacy, cultural acceptability and minimal side effects. Ancient literatures also mention herbal therapy for age-related diseases namely memory loss, osteoporosis, diabetic wounds, immune and liver disorders, etc. for which no modern medicine or only palliative therapy is yet available. The herbal drug products are prepared from renewable resources of raw materials by ecofriendly processes and will bring economic prosperity to the masses growing these raw materials1. Recently, World Health

Organization (WHO) defined traditional medicine (including herbal drugs) as therapeutic practices that have been in existence, often for hundreds of years, before the development and spread of modern medicine and are still in use today. These practices incorporated ancient beliefs and were passed on from one generation to another by oral tradition and/or guarded literature. Therefore, these plant drugs deserve detailed studies in the light of modern science. Naturopaths and others, who look beneath the symptoms of an illness to its underlying cause, often discover that the liver has a role to play.

More than 500 vital functions have been identified with the liver and a person’s nutritional level is not only determined by what he eats, but also by what the liver processes1. Unfortunately, it is extremely difficult to detect early warning symptoms specific to liver metabolic imbalances and a person may suffer for a long time from a liver disorder without knowing it. The incredible complexity of liver chemistry and its fundamental role in human physiology is so daunting to researchers that they visualize that perhaps simple plant remedies might have something to offer is astonishing and incredible. In fact, the nature has bestowed some plants with the property to prevent, treat and cure hepatic disturbances with interception of fewer side effects.

Hepatoprotectives are a class of therapeutic agents that includes synthetic as well as natural product which offer protection to liver from damage or help in regeneration of hepatic cells. Medicinal herbs are significant source of hepatoprotective drugs. It has been reported that about 170 phytoconstituents isolated from 110 plants belonging to

55 families do possess hepatoprotective activity2. Liver protective herbal drugs contain a variety of chemical constituents like phenols, coumarins, curcuminoids, lignans, essential oils and terpenoids. Clinical research has also shown that herbals have genuine utility in the treatment of liver diseases. Only a small portion of hepatoprotective plants as well as formulations used in traditional medicine are pharmacologically evaluated for its efficacy3. Free radical initiating auto oxidation of cellular membrane lipids can lead to cellular necrosis and is now accepted to be important in connection with a variety of pathological conditions4. Liver is an aerobic organ which generates reactive oxygen species that induce oxidative tissue damage.

These radicals react with cell membranes and induce lipid peroxidation or cause inflammation, which may result as important pathological mediators in many clinical disorders such as heart disease, hepatitis, diabetes, gout and cancer. Reduction of these radicals by antioxidant molecules is crucial to the protection of cells against various disorders. Development of life threatening diseases like cancer and also liver disorders are linked to the availability of these antioxidants5.

Paracalyx scariosa (Roxb.) Ali. (Family: Fabaceae) is a woody twiner with tomentose branches and stems finely downy. Leaves are 3-foliate with rhachis prolonged 6-13 mm. between the insertion of the leaflets and stipels of the terminal one. Petioles are 1.3-5 cm. long, downy. Stipules are 4-4.5 mm. long, triangular, acute and downy. Leaflets are 5-10 by 3.8-5.7 cm. acute, clothed with soft velvety pubescence above, densely downy and prominently reticulately veined beneath, base is subcordate.

Petiolules are 3 mm. long, densely downy. Stipels are 3 mm. long, subulate. Flowers are in copius axillary peduncled racemes or panicles. Pedicles are very short, downy.

Bracts are large, ovate, and caduceus. Calyx is finely 2.5-3.2 cm. long. Pods are small, oblique, downy, enclosed in the calyx. It is distributed in Central Provinces, West &

South India, and Upper Burma.

Common name: Kadle naaru.

The fruit is acrid and bitter, improves taste, appetizer, and astringent to the bowels. The plant enriches the blood, cures biliousness, liver disorders and “kapha”. The root is useful for treating dysentery and leucorrhoea and also applied externally along with other drugs to reduce tumors6, 7.

Till date, Hepatoprotective activity was not reported regarding the whole plant

Paracalyx scariosa (Roxb.) Ali. (Family: Fabaceae). In the view of above facts, the evaluation of hepatoprotective activity becomes essential. ENCLOSURE II

6.2 Review of Literature:

 Two new prenylated flavonoids from Paracalyx scariosa8.

 Two new prenylated flavonoids from Paracalyx scariosa9.

 Hepatoprotective effects of aqueous leaf extract and isolates of Murraya

Koenigii against in vitro ethanol-induced hepatotoxicity model10.

 Alaternin and emodin with hydroxyl radical inhibitory and/or scavenging

activities and hepatoprotective activity on tacrine-induced cytotoxicity in

HepG2 cells11.

 Protective effect of some antioxidant against CCl4-induced toxicity in liver cells

from BRL3A cell line12.

 Interaction of silver nanoparticles with primary mouse fibroblasts and liver

cells13.

 Pyruvate modulates antioxidant status of cultured human lens epithelial cells

under hypergalactosemic conditions14.

 The influence of cell growth, detoxifying enzymes and DNA repair on

hydrogen peroxide-mediated DNA damage (measured using the COMET assay)

in human cell lines15.

 Expression of superoxide dismutase, catalase and glutathione peroxidase in

glioma cells16.

 Antioxidant enzymes in malignant prostate cell lines and in primary cultured

prostatic cells17.

 Influence of quercetin and rutin on growth and antioxidant defense system of a

human hepatoma cell line (HepG2)18.

 The roles of glutathione and antioxidant enzymes in menadione-induced

oxidative stress19. WAF1  Modulation of antioxidant enzymes p21 and p53 expression during

proliferation and differentiation of human cell lines20.

 Mitochondrial manganese superoxide dismutase prevents neural apoptosis and

reduces ischemic brain injury: suppression of peroxynitrite production, lipid

peroxidation and mitochondrial dysfunction21.

 Evaluation of hepatoprotective activity of Ocimum Sanctum in HepG2 cell

lines22.

 In vitro antioxidant and hepatoprotective activity of ethanolic extract of Bacopa

Monnieri Linn. Aerial parts23.

 Screening of various Swertia species extracts in primary monolayer cultures of

rat hepatocytes against carbon tetrachloride and paracetamol induced toxicity24.

 Antioxidant and hepatoprotective effects of Acanthus ilicifolius25. ENCLOSURE III

6.3 Objectives of present study:

1. Phytochemical investigation of “Paracalyx scariosa (Roxb.) Ali”.

2. In vitro evalution of antioxidant activity.

3. In vitro evaluation of “Paracalyx scariosa (Roxb.) Ali" for hepatoprotective

activity in galactosamine HCl induced hepatotoxicity in hepatic cell lines. ENCLOSURE IV

Materials and methods:

7.1 Source of data: The required data will be obtained from:

1. Electronic data (Internet).

i. Online journals

ii. Wikipedia.in

iii. PubMed

iv. Google

v. Helinet

2. Published research papers.

3. Review and research articles from Journals.

4. Library, National College of Pharmacy, Shimoga. ENCLOSURE V

7.2 Methods of collection of data:

1. The whole plant “Paracalyx scariosa (Roxb.) Ali” will be collected from local

areas of Shimoga, Karnataka.

2. Authentication: Renowned botanist will authenticate the plant.

3. The plant material will be subjected to phytochemical studies.

4. In vitro evaluation of antioxidant property.

5. The in vitro hepatoprotective activity will be done by galactosamine HCl

induced hepatotoxicity in hepatic cell lines.

6. Hepatoprotection will be evaluated by biochemical parameters and cell

viability.

7. The results will be analyzed by ANOVA test.

ENCLOSURE-VI

7.3 Does the study require any investigation or intervention to be conducted on patients or other humans/animals?

The above study requires investigation on Wistar Albino rats for the procurement

of cell lines to screen antihepatotoxic effects and planned in accordance with the procedure

reported in the literature.

7.4 Has ethical clearance been obtained from your institution?

Ethical clearance is provided by the IAEC.

Clearance number: NCP/IAEC/CL/20/05/2011-12.

ENCLOSURE VII

List of References: 1. Brower V. Nutraceuticals, Nutritional Therapy, Phytonutrients and Phytotherapy for

Improvement of Human Health. Nat Biotechnol. 1998; 16:728-31.

2. Handa SS. Plants as drugs. The Eastern Pharmacist. 1991; 34:79-85.

3. Trease GE and Evans WC. Pharmacognosy. London: Balliere Tindall Press; 1983.

p.56-57.

4. Pyor W. Oxy radical and related species, their formation, Life times and reactions.

Ann Rev Physiol. 1986; 48:657-67.

5. Back NL, Kim YS, Kyung JS and Park KH. Isolation of anti-hepatotoxic agent from

the roots of Astragalus membraceous Korean. Korean J. Phamacog. 1996; 27:111-16.

6. Yoganarasimhan SN. Medicinal plants of India. Vol-1. Karnataka: Interline

publishing pvt ltd; 1996. p.349.

7. Kirtikar KR and Basu BD. Indian medicinal plants. 2nd ed. Vol-1. Periodical expert’s

book agency; 1991. p.812-13.

8. Mokhtar AN, Rao KV, Sreeramulu K and Gunasekhar D. Two new prenylated

flavonoids from Paracalyx scariosa. J Nat Prod. 1992; 55(8):1152-54.

9. Ali Nia Asli Rodsary and Gunasekhar D. Two new prenylated flavonoids from

Paracalyx scariosa. Iran J Chem Chem Eng. 1992; 11(1):1120-22.

10. Sathaye S, Bagul Y, Gupta S, Kaur H, Redkar R. Hepatoprotective effects of

aqueous leaf extract and isolates of Murraya Koenigii against in vitro ethanol

induced hepatotoxicity model. Exp Toxicol Pathol. Forthcoming 2010 May 18.

doi:10.1016/j.etp.2010.04.012. 11. Hyun AH Jung, Hae YC, Takako Y, Youn CK, Sook KH and Jae SC. Alaternin

and Emodin with hydroxyl radical inhibitory and/or scavenging activities and

hepatoprotective activity on tacrine-induced cytotoxicity in HepG2 cells. Arch

Pharm Res. 2004; 27(9):947-53.

12. Kamel HH, Azza H Abd-El-Rahman, Walaa MS Ahmed and Amira H Mohamed.

Protective effect of some antioxidants against CCl4-induced toxicity in liver cells

from BRL3A cell line. J Am Sci. 2010; 6(10):992-03.

13. Arora S, Jain J, Rajwade JM and Paknikar KM. Interaction of silver nanoparticles

with primary mouse fibroblasts and liver cells. Toxicol Appl Pharm. 2009;

236:310-18.

14. Ipseeta M, Sujatha J, Deepa T, Sushma S, Radhika T and Suresh KG. Pyruvate

modulates antioxidant status of cultured human lens epithelial cells under

hypergalactosemic conditions. Mol Cell Biochem. 2002; 238:129-35.

15. Susan JD and Andrew RC. The influence of cell growth, detoxifying enzymes and

DNA repair on hydrogen peroxide-mediated DNA damage (measured using the

COMET assay) in human cell lines. Free Radical Bio Med. 1997; 22(4):717-24.

16. Weixiong Z, Tao Y, Ramon L and Larry WO. Expression of superoxide dismutase,

catalase and glutathione peroxidase in glioma cells. Free Radical Bio Med. 1999;

27(11-12):1334-45.

17. Klaus J, Beate S, Birgit R, Michael L, Marcus VC, Wolfgang H, Gerhard H,

Dietmar S and Stefan AL. Antioxidant enzymes in malignant prostate cell lines and

in primary cultured prostatic cells. Free Radical Bio Med. 1997; 23(1):127-33. 18. Alia M, Mateos R, Ramos S, Lecumberri E, Bravo L and Goya L. Influence of

quercetin and rutin on growth and antioxidant defense system of a human hepatoma

cell line (HepG2). Eur J Nutr. 2006; 45:19-28.

19. Tzeon-Jye C and Woan-Fang T. The roles of glutathione and antioxidant enzymes

in menadione-induced oxidative stress. Toxicology. 2000; 154:75-84.

20. Bravard A, Petridis F and Luccioni C. Modulation of antioxidant enzymes

p21WAF1 and p53 expression during proliferation and differentiation of human

melanoma cell lines. Free Radical Bio Med. 1999; 26(7-8):1027-33.

21. Jeffrey NK, Mark SK, Fredrick WH, Daret K and Hsiu-Chuan Y. Mitochondrial

manganese superoxide dismutase prevent neural apoptosis and reduce ischemic

brain injury: Suppression of peroxynitrite production, lipid peroxidation and

mitochondrial dysfunction. J Neurosci. 1998; 18(2):687-97.

22. Ginpreet K, Meena C, Veeranjaneyulu A and Agrawal SS. Evaluation of

hepatoprotective activity of Ocimum Sanctum in HepG2 cell line. Arch Pharm Sci

Res. 2009; 1(1):25-30.

23. Tirtha G, Tapan kumar M, Mrinmay D, Anindya and Deepak kumar D. In vitro

antioxidant and hepatoprotective activity of ethanolic extract of Bacopa monnieri

linn. Aerial parts. Iran J Pharmacol Therp. 2007; 6:77-85.

24. Reen RK, Karan M, Kuldip S, Karan V, Jihri RK and Jaswanth S. Screening of

various Swertia species extracts in primary monolayer cultures of rat hepatocytes

against carbon tetrachloride and paracetamol induced toxicity. J Ethnopharmacol.

2001; 75(2-3):239-47. 25. Babu BH, Shylesh BS and Padikkala J. Antioxidant and hepatoprotective effect of

Acanthus ilicifolius. Fitoterapia. 2001; 72:272-77.