“EVALUATION OF HEPATOPROTECTIVE ACTIVITY OF AQUEOUS AND ETHANOLIC EXTRACTS OF EVOLVULUS ALSINOIDES IN RATS ”

SYNOPSIS FORM.PHARM DISSERTATION

SUBMITTED TORAJIV GANDHI UNIVERSITY OF HEALTH SCIENCES,

KARNATAKA.

SUBMITTED BYVEERA JYOTHSNA.M

I M.PHARMDEPARTMENT OF PHARMACOLOGY,

PES COLLEGE OF PHARMACY,BENGALURU-560050.

(2010-12)

RAJIV GANDHI UNIVERSITY OF HEALTH SCIENCES,

KARNATAKA, BENGALURU -560050

1 NAME OF THE CANDIDATE AND ADDRESS

VEERA JYOTHSNA.MLOCAL ADDRESSP.E.S. COLLEGE OF PHARMACYHANUMANTHANAGAR,50 FT. ROAD,BENGALURU -560050.

PERMANENT ADDRESSD/O M.UMA MAESHWARA REDDY1/504,SMIT ROAD,NAGARAJUPET,DIST-KADAPA,PIN-516001, ANDHRA PRADESH.

2 NAME OF THE INSTITUTION

P.E.S. COLLEGE OF PHARMACYHANUMANTHANAGAR,50 FT. ROAD,BENGALURU -560050.

3 COURSE OF STUDY AND SUBJECT MASTER OF PHARMACY IN PHARMACOLOGY.

4 DATE OF THE ADMISSION 31st MAY 2010

5 TITLE OF THE TOPIC: “Evaluation of Hepatoprotective activity of aqueous and ethanolic

extracts of Evolvulus alsinoides in rats.”

ANNEXURE-II

PROFORMA FOR REGISTRATION OF SUBJECTS FOR DISSERTATION

6. Brief resume of the intended work:

6.1 Need for the study:

Liver, the key organ of metabolism and excretion has an immense task

of detoxification of xenobiotics, environmental pollutants and chemotherapeutic

agents. Hence, this organ is subjected to variety of diseases and disorders. The liver

plays a central role in transforming and clearing chemicals and is susceptible to the

toxicity from these agents. Hepatotoxicity implies chemical-driven liver damage.

Certain medicinal agents when taken in overdoses and sometimes even when

introduced within therapeutic ranges may injure the organ. Other chemical agents used

in laboratories and industries, natural chemicals can also induce hepatotoxicity.

Chemicals that cause liver injury are called hepatotoxins.

More than 900 drugs have been implicated in causing liver injury1 and it is

the most common reason for a drug to be withdrawn from the market. Chemicals often

cause subclinical injury to liver which manifests only as abnormal liver enzyme tests.

Drug induced liver injury is responsible for 5% of all hospital admissions and 50% of

all acute liver failures2, 3.

India’s system of medicine offers a number of traditional herbs to improve liver health.

Besides directly helping the body cope with liver problems, including jaundice,

hepatitis, and cirrhosis, these herbs help the liver eliminate toxins and microbial

infections4.

In the Indian traditional system of medicine, Several hundreds of plants have been

examined for use in a wide variety of liver disorders. The plant extracts of Evolvulus

alsinoides contains alkaloids: betaine, shankhapushpine and evolvine. Fresh plant

contains volatile oil. It also contains a yellow neutral fat, an organic acid and saline

substances5, 6.

Antioxidants play an important role in inhibiting and scavenging free

radicals and thus providing protection against infections and degenerative diseases. The

ethanolic extract and water infusions of Evolvulus alsinoides shows strong antioxidant

activity7.

The recent studies shows that betaine may protect liver from fibrogenesis by

maintaining the cellular antioxidant capacity8. It may protect the liver against ethanol-

induced oxidative injury most probably via its effects on the sulfur-amino acid

metabolism9. Oral betaine either improves recovery or reduces the toxic effects of CCl4

on cell organelles in liver cells of male Han-Wistar rats10.

The present study is therefore being carried out to investigate the

hepatoprotective activity using the aqueous and ethanolic extracts of Evolvulus

alsinoides.

6.2 Review of the Literature:

Evolvulus alsinoides was found to have adaptogenic and anti-amnesic properties in

rodents11. Natural substances from Evolvulus alsinoides L has shown antioxidant

potency12. Evolvulus alsinoides and Convulvulus pluricaulis were found to have

anxiolytic activity in rodents13. Flavonoid glycosides from Evolvulus alsinoides found

to have antioxidant property 15. Evolvulus alsinoides Linn. was found to have memory

enhancement activity in rodents16. Evolvulus alsinoides has shown Immunomodulatory

activity17. Betaine supplementation was alleviated the dimethylnitrosamine-induced

liver injury and fibrosis in rats8. Betaine supplementation was alleviated the acute

ethanol-induced liver injury and impaired metabolomics of S-containing substances9.

Oral administration of betaine has shown the reduction of carbon tetrachloride-induced

hepatotoxic in male Han-Wistar rats10. Betaine has shown the recovering effect on

carbon tetrachloride-induced liver injury18.

PLANT PROFILE:

Plant name: Evolvulus alsinoides

Family: Convulvulaceae6

Vernacular names14:-

Sanskrit-: Vishnu krantha, Harikrantha, Shankha pushpi,

English:- Dwarf morning glory,

Telugu:- Vishnukranta,

Malayalam:- krishnakranti,

Hindi:- Shankhauli,

Kannada:- Shankhapushpi.

Distribution:-Widely distributed in tropical and subtropical regions throughout the

world. It grows commonly as a weed in open and grassy places throughout India,

ascending at 6000ft6.

Parts used: whole plant6

Description:

Leaves: Leaves are small, entire, elliptic to oblong, obtuse, apiculate, base acute and

densely hairy. Petiole is minute or nearly absent. Bracts are linear and persistent5.

Flowers: Flowers mostly solitary in upper axils. Corolla blue rotate and broad funnel

shaped, Calyx 4 is lobed, lanceolate and the tip acute. Peduncle is long and axillary5.

Branches: Its branches are annual, numerous, more than 30 cm long, often prostrate,

slender and wiry with long hairs5.

Chemical constituents: The plant contains an alkaloid-shankhapushpine. Fresh plant

contains volatile oil and potassium chloride. It also contains a yellow neutral fat, an

organic acid and saline substances. Three alkaloids- evolvine, betaine, and an

unidentified compound have been isolated6.

Uses: Nootropic agent, Chronic bronchitis, General weekness. Used in fever, nervous

debility, loss of memory, also in syphilis and scrofula. It is used as Rasayan. Whole

plant is used as hepatoprotective6, 19.

Traditional medicinal uses:-

1. The whole herb is used medicinally in the form of decoction with cumin and

milk in fever, nervous debility, loss of memory and syphilis.

2. Decoction of the drug, with Ocimum sanctum is administered in fevers

accompanied by indigestion or diarrhea. Decoction was given in cases of

malarial fever.

3. The root is used by the santals, for intermittent childhood fever,

4. The leaves are made into cigarettes and smoked in chronic bronchitis and

asthma.

5. The oil promotes the growth of hair5.

6.3 Objectives of the study:

1. To investigate hepatoprotective activity of aqueous and ethanolic extract

of Evolvulus alsinoides in CCL4 induced hepatotoxicity.

To investigate hepatoprotective activity of aqueous and ethanolic extract of

Evolvulus alsinoides in paracetamol induced hepatotoxicity.

Parameters to be evaluated:

Parameters measured in this study will be serum Aspartate aminotransferase

(AST), Alanine aminotransferase (ALT), Alkaline phosphatase (ALP),

Bilirubin, and antioxidant studies such as lipid peroxidation and Glutathione

reduction (GSH).

Histopathological studies of liver.

7. Materials and methods

7.1 Source of data

Whole experiment is planned to generate data from laboratories studies. Experiment

will be performed as described in the standard bibliography, may be obtained from

standard journals and text books available within the college or from other pharmacy

colleges or from libraries of National Institutes or through internet.The following

various sources will be utilized to obtain related information regarding this research

protocol.

IISc library, Bengaluru.

PESCP library, Bengaluru.

RGUHS digital library (Helinet), Bengaluru.

Websites: www.sciencedirect.com

www.pubmed.gov

www.google.com

7.2 Method of collection of data:

The whole study is divided into following phases;

Phase I: Collection of plant material: The whole plant will be procured from the

authenticated supplier from Bangalore market and it will be authenticated by

Taxonomist.

Phase II: Preparations of extracts:

Dried powder of the plant will be extracted with water and alcohol in a soxhlet

apparatus for 72 hours. The residue obtained after extraction is dried. The residue

mass obtained is evaporated and reduce temperature to dryness. A % yield of the

extracts will be determined.

Phase III: Preliminary Phytochemical investigation:

Preliminary phytochemical investigation will be done as described in Practical

Pharmacognosy- Techniques and Experiments20

Phase IV: Acute oral toxicity (as per OECD guidelines):

Female Swiss albino mice (18-20 g) are individually identified and allowed to acclimate

to the laboratory condition for 7 days before the start of the study. Only one mouse

receives single dose at a particular time. First animal receives a dose of 175 mg/kg and

is observed for any toxicity signs, survival or death up to 48 hrs. If the first animal died

or appeared moribund, the second animal receives a lower dose (55mg/kg). The dose

progression or reduction factor is 3.2 times of the previous dose. If no mortality is

observed in the first animal then the second animal receives a higher dose (55 mg/kg).

Dosing of the next animal is continued depending on the outcome of the previously

dose for a fixed time interval (48 hours). The test is stopped when one of the stopping

criteria is observed.

5 reversals occur in any 6 consecutive animals tested.

3 consecutive animals died at one dose level.

Survived animals are observed for long-term outcomes for a period of 14 days. The

acute oral toxicity values are calculated using AOT 425 software (Environmental

Protection Agency, USA) based on the short term (48 hours) and long term out come

(14 days) 21.

Phase V: Pharmacological evaluation:For determination of hepatoprotective activity the following experiments are to be

performed.

All the models used in the pharmacological experiments will consist of the below 5

groups consisting of 6 animals in each group

1: Effect of aqueous and ethanolic extract of Evolvulus alsinoides on

CCl4 induced hepatotoxicity in rats22.

Animal used : - Albino Wistar Rats

Sex :- Either Sex

Weight :- 150-200g

Number of animal in each group:- 06

Procedure:

Rats are divided into seven groups of six animals each. Group I will serve

as normal control and receives only the vehicle. Group II animals will receive CCl4 (0.5

ml/kg) i.p. once daily for 7 days. Group III animals will receive CCl4 0.5 ml/kg i.p. and

reference standard silymarin 100 mg/kg orally (p.o.) for 7 days. Groups IV, V & VI,VII

will be administered with low and high doses of ethanolic extracts and aqueous extracts

of Evolvulus alsinoides and CCL4 at a dose of 0.5 ml/kg i.p. for 7 days respectively.

After 24hours of the last treatment, the blood samples will be collected for the

estimation of biochemical marker enzymes serum ALT, AST, ALP, and Bilirubin. Then

animals will be sacrificed under ether anesthesia. The livers from all the animals will be

collected. A portion of the liver will be homogenized and used for the antioxidant

studies such as lipid peroxidation, Glutathione reduction. The other portion of liver will

be used for histopathological studies.

8. References:-

1. Friedman, Scott E, Grendell, James H, McQuaid, R. K. Current diagnosis &

treatment in gastroenterology. New York: McGraw-Hill; 2003.

2. McNally, PeterF. GI/Liver Secrets: with student consult Access. Saint Louis:

Mosby, CV.

3. Ostapowicz G, Fontana RJ, Schiodt F. Results of a prospective study of acute liver

failure at 17 tertiary care centers in the United States. 12 ed: Ann. Intern. Med; 2002.

4. http://www.tasteforlife.com/content/default.asp?artid=1711&title=AyurvedicHerbs

5. Singh A. Review of Ethnomedicinal Uses and Pharmacology of Evolvulus

alsinoides Linn. Ethanobotanical leaflets. 2008;12:734-40.

6. Prajapati, Purohit, Shrama, Kumar. A hand book of medicinal plants; 2003.

7. Auddy B, Ferreira M, Blasina F, Lafon L, Arredondo F, Dajas F, et al. Screening of

antioxidant activity of three Indian medicinal plants, traditionally used for the management

of neurodegenerative diseases. J Ethnopharmacol. 2003 Feb;84(2-3):131-8.

8. Sang K. Kim, JMS, Yu R. Chae, Young S. Jung, Jae H, Park and Young C, et al.

Alleviation of dimethylnitrosamine-induced liver injury and fibrosis by betaine

supplementation in rats Chemico-Biological Interactions. 2009 12 February;177(3):204-11

9. Kim SJ, Jung YS, Kwon do Y, YC. K. Alleviation of acute ethanol-induced liver

injury and impaired metabolomics of S-containing substances by betaine supplementation.

Biochem Biophys Res Commun. 2008 Apr 18;368(4):893-8.

10. Junnila M, Rahko T, Sukura A, LA. L. Reduction of carbon tetrachloride-induced

hepatotoxic effects by oral administration of betaine in male Han-Wistar rats: a

morphometric histological study. Vet Pathol. 2000 May;37(3):231-8.

11. Kiran Babu Siripurapua, Prasoon Guptab, Gitika Bhatiac, Rakesh Mauryab,

Chandishwar Natha, Palita. G. Adaptogenic and anti-amnesic properties of Evolvulus

alsinoides in rodents. Pharmacology Biochemistry and Behavior. 2005 July;81(3):424-32.

12. Cervenka F, Koleckar V, Rehakova Z, Jahodar L, Kunes J, Opletal L, et al.

Evaluation of natural substances from Evolvulus alsinoides L. with the purpose of

determining their antioxidant potency. J Enzyme Inhib Med Chem. 2008 Aug;23(4):574-8.

13. Alok Nahata, U.K. Patil, Dixit. VK. Anxiolytic activity of Evolvulus alsinoides and

Convulvulus pluricaulis in rodents. Pharmacopsychiatry. 2009 May;47(5):444-51

14. F. Daniel, Austin. Evolvulus alsinoides (Convolvulaceae): An American herb in

the Old World. Journal of Ethnopharmacology. 2008 8 May;117(2):185-98

15. Kumar M, Ahmad A, Rawat P, Khan MF, Rasheed N, Gupta P, et al. Antioxidant

flavonoid glycosides from Evolvulus alsinoides. Fitoterapia. 2010 Jun;81(4):234-42.

16. Nahata A, Patil UK, VK. D. Effect of Evolvulus alsinoides Linn. on learning

behavior and memory enhancement activity in rodents. Phytother Res. 2010

Apr;24(4):486-93.

17. Ganju L, Karan D, Chanda S, Srivastava KK SR, W. S. Immunomodulatory effects

of agents of plant origin. Biomed Pharmacothe. 2003 Sep;57(7):296-300.

18. Murakami T, Nagamura Y, K. H. The recovering effect of betaine on carbon

tetrachloride-induced liver injury. J Nutr Sci Vitaminol. 1998 Apr;44(2):249-55.

19. Dr.K.Madhava chetty, K.Sivaji, rao. KT. Flowering plants of Chittor district.

A.P,India; 2008.

20. KR. Kandelwal. Practical Pharmacognosy-techniques and experiments. Pune:

Nirali Prakashan; 1996.

21. Acute oral toxicity studies [online].Dec 2001[cited 2009 Dec 09]; Available from:

URL: http://www.oecd.org/dataoecd/17/51/1948378.pdf

22. Ranawata L, Bhattb J, J. P. Hepatoprotective activity of ethanolic extracts of bark

of Zanthoxylum armatum DC in CCl4 induced hepatic damage in rats. J Ethnopharmacol.

2009.

23. Ramachandra S.S, Quereshi. AA, Viswanath Swamy AHM, Patil. T, Prakash T, K.

P. Hepatoprotective activity of Calotropis procera flowers against paracetamol-induced

hepatic injury in rats. Fitoterapia. 2007;78:451-4.

NAME OF THE CANDIDATE

VEERA JYOTHSNA.M

10. SIGNATURE OF THE CANDIDATE

11.1 REMARKS OF THE GUIDE

11.2 NAME AND DESIGNATION OF THEGUIDE

Mr. R.Srinath. HOD & Asst.Professor Department of Pharmacology P.E.S College of Pharmacy Bengaluru-560050.

NAME OF THE CANDIDATE

VEERA JYOTHSNA.M

10. SIGNATURE OF THE CANDIDATE

11.1 REMARKS OF THE GUIDE

11.2 NAME AND DESIGNATION OF THEGUIDE

Mr. R.Srinath. HOD & Asst.Professor Department of Pharmacology P.E.S College of Pharmacy Bengaluru-560050.