Rajiv Gandhi University Of Health Sciences

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

SYNOPSIS FOR

M.PHARM DISSERTATION

SUBMITTED TO

RAJIV GANDHI UNIVERSITY OF HEALTH SCIENCES,

KARNATAKA.

SUBMITTED BY

VEERA JYOTHSNA.M

I M.PHARM

DEPARTMENT 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.M
LOCAL ADDRESS

P.E.S. COLLEGE OF PHARMACY

HANUMANTHANAGAR,
50 FT. ROAD,
BENGALURU -560050.

PERMANENT ADDRESS

D/O M.UMA MAESHWARA REDDY
1/504,SMIT ROAD,
NAGARAJUPET,
DIST-KADAPA,
PIN-516001, ANDHRA PRADESH.
2 / NAME OF THE INSTITUTION /

P.E.S. COLLEGE OF PHARMACY

HANUMANTHANAGAR,
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.
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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More than 900 drugs have been implicated in causing liver injury1 and it is the most common reason for a drug to be withdrawn from the market.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.

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.
TREATMENT / DRUG / DOSE
Group-1 / Vehicle Control / -
Group-2 / CCl4 treated (positive control) / 0.5 ml/kg, i.p.
Group-3 / CCl4 + Silymarin / 100 mg/kg, p.o
Group-4 / CCl4 + ethanolic extract of E.A / Low dose# , p.o
Group-5 / CCl4 + ethanolic extract of E.A / High dose#, p.o
Group-6 / CCl4 + aqueous extract of E.A / Low dose#, p.o
Group-7 / CCl4 + aqueous extract of E.A / High dose#, p.o
# Dose of extracts will be fixed after the outcome of acute oral toxicity test.
2: Effect of aqueous and ethanolic extract of Evolvulus alsinoides on Paracetamol induced hepatotoxicity in rats23.
Animal used :-Albino Wistar Rats
Sex :- Either sex
Weight :- 150-200g
Number of animal in each group:- 06
Procedure:
The evaluation of hepatotoxicity is followed accordingly. Rats are divided into seven groups of 6 animals each. Group I animals will receive vehicle for one week (vehicle control). Group II animals will receive vehicle for one week and paracetamol 2g/kg orally on 5th day (positive control). Group III animals will receive standard reference silymarin (100 mg/kg p.o.) .Groups IV, V& VI,VII will be administered with low and high doses of ethanolic extracts and aqueous extracts of Evolvulus alsinoides once a day for seven days respectively. On the fifth day, after the administration of the respective treatments, all the animals of groups III, IV, V,VI & VII will be administered with paracetamol 2 g/kg orally. On the seventh day after 2 hrs of respective treatments the blood samples are collected for the estimation of biochemical marker enzymes serum ALT, AST, ALP and Bilirubin. Animals are then sacrificed under ether anesthesia and 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 and Glutathione reduction. The other portion of liver will be used for histopathological studies.
TREATMENT / DRUG / DOSE
Group-1 / Vehicle Control / -
Group-2 / Paracetamol treated (Positive Control) / 2 g/kg p.o.
Group-3 / Paracetamol + Silymarin / 100 mg/kg p.o.
Group-4 / Paracetamol + Etanolic extract of E.A / Low dose#, p.o
Group-5 / Paracetamol + Ethanolic extract of E.A / High dose#, p.o
Group-6 / Paracetamol + Aqueous extract of E.A / Low dose#, p.o
Group-7 / Paracetamol + Aqueous extract of E.A / High dose#, p.o
# Dose of extracts will be fixed after the outcome of acute oral toxicity test
STATISTICAL ANALYSIS :-
All the values will be expressed as mean± SEM. The data will be analyzed by using one way ANOVA followed by suitable post-hoc test. Statistical significance will be set at p< 0.05.
7.3 Does the study require any investigation to be conducted on patients
or other humans or animals?
Yes, the experimental models require usage of laboratory animals.
7.4 Has ethical clearance been obtained from your institution in case of
7.3?
Yes, ethical clearance has been obtained [copy of IAEC clearance has been attached]
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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