“EVALUATION OF HEPATOPROTECTIVE ACTIVITY OFEXTRACTS OFBALANITES AEGYPTIACAFRUITSIN EXPERIMENTALLY INDUCED HEPATIC DAMAGE IN RATS’’
SYNOPSIS FOR
M. PHARM DISSERTATION
SUBMITTED TO
RAJIVGANDHIUNIVERSITY OF HEALTH SCIENCES
BENGALOORU, KARNATAKA.
SUBMITTED BY
LEENA.KAVERIAPPA.B
I M. PHARM
DEPARTMENT OF PHARMACOLOGY
P.E.SCOLLEGE OF PHARMACY
BENGALOORU – 560050
(2009-2011)
RAJIVGANDHIUNIVERSITY OF HEALTH SCIENCES
BENGALOORU, KARNATAKA.
ANNEXURE-II
PROFORMA FOR REGISTRATION OF SUBJECT FOR
DISSERTATION
1.0 / NAME AND ADDRESS OF THE CANDIDATE. / LEENA.KAVERIAPPA.Bp.e.s.college of pharmacy HANUMANTHANAGAR, 50 FT. ROAD, BENGALOORU-560050.
PERMANENT ADDRESS
146,4th cross ,21st Main ,
H.S.R.Layout 1st sector,
Agara , Bangalore-560034.
2.0 / NAME OF THE INSTITUTION. / P.E.S.COLLEGE OF PHARMACY
Hanumanthanagar, 50 feet road, BENGALOORU – 560050.
3.0 / COURSE OF STUDY AND SUBJECT. / M.PHARM.
PHARMACOLOGY.
4.0 / DATE OF ADMISSION TO COURSE. / 20th MAY, 2009
5.0 / TITLE OF THE TOPIC:
“EVALUATION OF HEPATOPROTECTIVE ACTIVITY OF
EXTRACTS OF BALANITES AEGYPTIACA FRUITS IN EXPERIMENTALLY INDUCED HEPATIC DAMAGEIN RATS”
6.0
7.0
8.0 / 6.1. NEED FOR THE STUDY:
The Liver is one of the largest organ in the body carrying out most of the biochemical synthesis and secretory functions. Living in a world of inadequately controlled environment, pollution and expanding therapy with potent drugs, it is continuously exposed to variety of xenobiotics and therapeutic agents resulting in its structural or functional damage.
The major factors that contribute to Liver disease becoming a global problem because of increasing alcohol consumption in both developed and developing countries. Malnutrition, anemia, infection and availability of hepatotoxic drugs over the counter. The conventional drugs used in the treatment of liver diseases viz; Corticosteroids, anti-viral and immunosuppressant agents are sometimes inadequate and may lead to serious adverse effects. Paradoxically, they may themselves cause hepatic damage, as exemplified by cholestatic jaundice with azathioprine and elevation of serum transaminases by interferon and virazole. It is therefore, imperative to search for better drugs to treat liver diseases.
More than 900 drugs have been implicated in causing liver injury1 and it is the most common reason for a drug to be withdrawn from the market.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.
In the Indian traditional system of medicine, Several hundred plants have been examined for use in a widevariety of liver disorders.The methanolic extract of leaves ofBalanites roxburghii was found to have hepatoprotective activity against carbon tetrachloride (CCl4)-induced hepatic damage in rats.4The aqueous extract is used in Sudanese folk medicine in the treatment of jaundice.
The present study is therefore being carried out to investigate the hepatoprotective activity using the fruit extracts of Balanites Aegyptiaca.
6.2objective of the study:
- To investigate hepatoprotective activity of Balanites aegyptiacaein CCL4 induced hepatotoxicity.
- To investigate hepatoprotective activity of Balanites aegyptiacaein paracetamol induced hepatotoxicity.
- Parameters measured in this study will be serum Aspartate aminotransferase (AST), Alanine aminotransferase (ALT), Alkaline phosphatase(ALP),Bilirubin, andantioxidant studies such as lipidperoxidationand Glutathione reduction (GSH).
- Histopathological studies of liver.
Balanites aegyptiaca (L.) Del. is a Savannah treecharacterised by long straight green spines arrangedspirally along the branches. Each spine has a two leaflet compound leaf below it. All the branches (whether bearing flower or not) are armed with spines which are usually simple and practically straight. Various parts of the tree are traditionally used for pharmaceutical purposes4.
The aqueous extract is used in Sudanese folk medicine in the treatment of jaundice; fruits are used as an antidiabetic in Egyptian folk medicine.One of the studies reported that Balanites aegyptiaca fruit mesocarp was more efficient anthelmentic than Praziquantel in miceinfected with Sudanese strain of Schistosoma mansoni.5
It was reported that methanol extracts of fruit mesocarp of Balanites aegyptiaca and five fractions from the methanol extract which were tested against the Aedes aegypti mosquito larvae showed excellent larvicidal activity6.
The tree has many folk uses in various African countries and it is largely used as component of many popular preparations for its abortive, antiseptic, anti-malarial, antisyphilitic and anti-viral (Herpes zoster) activity7.
The fruits are commonly used to purge, toremove intestinal parasites and sometimes to treat Schistosomumjaponicum8.
The bark aqueous extractis traditionally used as anti-jaundice, while the one of fruit
mesocarps is administered as oral hypoglycemic and seems to be effective against theFasciola gigantica9
Some of the scientific reports on plant based preparations or agents having hepatoprotective activity are given here under:
The ethanolic extracts of bark of Zanthoxylum armatumreported to possess hepatoprotective activity against CCl4 induced hepatotoxicity in rats10. A study showed that the aqueous extracts of Woodfordia fruticosa significantly restore physiological integrity ofhepatocytes11. The antihepatotoxic effect of methanolic extract of seeds of Apium graveolensHygrophila auriculata was studied on paracetamol and thioacetamide induced liver damage in rats by assessing liver function tests12
PLANT PROFILE
Balanites aegyptiacae:
Family : Zygophyllaceae.
Vernacular name: English: desert date
Hindi: betu,engua,hingan,hingn, hingot, hingota, hongoti
Sanskrit: ingudi
Tamil : nanjunda
Arabic : lalob, hidjihi, and heglig
Description:The yellow, single-seededfruit is edible, but bitter. Many parts of the plant are used as famine foods in Africa; the leaves are eaten raw or cooked, the oily seed is boiled to make it less bitter and eaten mixed with sorghum, and the flowers can be eaten. The tree is considered valuable in arid regions because it produces fruit even in dry times. The fruit can be fermented for alcoholic beverage. The seed contains 30-40% seed oil and contains the sapogenins, diosgenin and yamogenin. Diosgenin can be used to produce hormones such as those in combined oral contraceptive pills and corticoid. The oil is used as cooking oil. The seed cake remaining after the oil is extracted is commonly used as animal fodder in Africa. The seeds of the Balanites aegyptiaca have molluscicide effect on Biomphalaria pfeifferi.
Medicinal uses of this plant are many. The fruit is mixed into porridge and eaten by nursing mothers, and the oil is consumed for headache and to improve lactation. Bark extracts and the fruit repel snails and copepods, organisms that host the parasites schistosome and guinea worm, respectively13.
Habitat: Commonly cultivated throughout India in the plains and up to an altitude of 1200m.
Part Used: Barks, leaves, fruits, seeds and thorns.
Chemical Constituents: Nitogenin glucoside isolated , diosgenin and stigmasterol from leaves , isolation and structure of saponins-balanitisines A,B,C,D and E from fruits , diosgenin content of fruits , pericarp , defatted seed and oil.14
Uses:
Root & Bark:Laxative, diarrhoea, haemorrhoid.
Bark :Vermifuge, stomach aches, sterility, jaundice, yellow fever, syphilis, coughs, epilepsy and anxiety
Root :Snake bites, anthrax
Thorns: Leprosy
Leaves :Weakness, anthrax
Fruit: Rheumatism
Seed Ointments, balms, magico-religious uses4
MATERIAL AND METHODS:
7.1 SOURCE OF DATA :The plant material will be procured from authenticated suppliers. Whole experiment is planned to generate data from laboratory 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 source.
7.2 METHODS OF COLLECTION OF DATA:
The whole study is divided into following phases:
PhaseI. Collection of plant material.
The fruitswill be collected from the fields and authenticatedwill be authenticated by Taxonomist.
Phase II: Preparations of extracts.
The fruits will be successively extracted with alcohol (70%) andwater in a soxhlet apparatus for 72 hours. The residue obtained after extraction will be dried andevaporated under reduced temperature to dryness. A percentage 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 experiments15.
Acute oral toxicity of fruitextract
Acute oral toxicity is carried out as per OECD 425 guidelines. 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 outcome (14 days)16.
II. PHARMACOLOGICAL EVALUATION:
1: Effect of extracts of Balanites aegyptiacaeon CCl4 induced hepatotoxicity in rats.
Animal used : - Albino Wistar Rats
Sex :- Either Sex
Weight :- 150-200g
Number of animal in each group:- 06
Procedure:
Thirty rats are divided into five groups of six animals each. Group I willserve as normal control and receives only the vehicle. Group II animals willreceive CCl4(0.5 ml/kg) i.p. once daily for 7 days. Group III animals willreceiveCCl4 0.5 ml/kg i.p. and reference standard silymarin 100 mg/kg orally (p.o.) for 7 days. Groups IV, V, VI and VII will be administered withaqueous, alcoholicextracts of Balanites aegyptiacae and CCL4 at a dose of 0.5 ml/kg i.p. for 7 days.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 studies10.
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 + aqueous extract of B.A / Low dose# , p.o
Group-5 / CCl4 + aqueous extract of B.A / High dose#, p.o
Group-6 / CCl4 + alcoholic extract of B.A / Low dose# , p.o
Group-7 / CCl4+ alcoholic extract of B.A / High dose#, p.o
#Dose of extracts will be fixed after the outcome of acute oral toxicity test
2: Effect of extracts ofBalanites aegyptiacaeon Paracetamol induced hepatotoxicity in rats.
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. Thirty rats are divided into five groups of 6 animals each. Group I animalswill receivevehicle for one week (vehicle control). Group II animals will receivevehicle for one week and paracetamol 2g/kg orally on 5th day (positive control). Group IIIanimals will receive standard reference silymarin (100 mg/kg p.o.) .Groups IV , V, VI and VII will be administered with low and high doses of aqueous and alcoholic extracts of sesamumindicumonce a day for seven days. On the fifth day, after the administration of the respective treatments, all the animals of groups III, IV, V, VI and 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, ALPand Bilirubin.Animals are then sacrificed under ether anesthesia andlivers 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 studies17.
TREATMENT / DRUG / DOSE
Group-1 / VehicleControl / -
Group-2 / Paracetamol treated(PositiveControl) / 2 g/kg p.o.
Group-3 / Paracetamol + Silymarin / 100 mg/kg p.o.
Group-4 / Paracetamol + aqueous extract of B.A / Low dose# , p.o
Group-5 / Paracetamol + aqueous extract of B.a / High dose#, p.o
Group-6 / Paracetamol + alcoholic extract of B.A / Low dose# , p.o
Group-7 / Paracetamol + alcoholic extract of B.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 expressedas mean±SEM. The data willbe analyzed by using one way
ANOVA followed by Dunett’s t-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]
REFERENCES :
1.Friedman, Scott E, Grendell, James H, McQuaid, Kenneth R. Current diagnosis & treatment in gastroenterology. New York: McGraw-Hill; 2003.
2.McNally., Peter F. 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.Thirupathi K, Krishna DR, Ravi Kumar B, Apparao AVN, Krishna Mohan G. Hepatoprotective effect of leaves of Balanites roxburghii against carbon tetrachloride-induced hepatic damage in rats. Current Trends Biotech Pharm.2009;3(2):219-24
5.Ojo OO, Nadro MS, Tella IO. Protection of rats by extracts of some common Nigerian trees against acetaminophen-induced hepatotoxicity. Afr J Biotechnol. 2006;5(9):755-60.
6.Kokoa WS, Abdallab HS, Galala M, Khalida HS. Evaluation of oral therapy on mansonial schistosomiasis using single dose of Balanites aegyptiaca fruits and PraziquantelFitoterapia 2005;76:30–4.
7.Zeev W, Bishnu PC. Larvicidal activity of saponin containing extracts and fractions of fruit mesocarp of Balanites aegyptiacaFitoterapia. 2006;77:420-4.
8.Duke J. Medirinul Plants of the Bible. New York; 1983.
9.Koko WS, Ahmed MM, Yousaf S, Galal M, Iqbal Choudhary M. In vitro immunomodulating properties of selected Sudanese medicinal plants. J Ethnopharmacol. 2008;118(1):26-34.
10.Kamel MS, Ohtani K, Kurokawa T, Assaf MH, El-Shanawany MA, Ali AA, et al. Studies on Balanites Aegyptiaca, fruits, an antidiabetic Egyptian folk medicineChem Pharm Bull. 1991;39:1229–33.
11.Ranawata L, Bhattb J, Patelb J. Hepatoprotective activity of ethanolic extracts of bark of Zanthoxylum armatum DC in CCl4 induced hepatic damage in rats. J Ethnopharmacol. 2009.
12.Chandana BK, Saxenaa AK, Shuklab S, Sharmaa N, Gupta DK, Singha K, et al. Hepatoprotective activity of Woodfordia fruticosa Kurz flowers against carbon tetrachloride induced hepatotoxicity. J Ethnopharmacol. 2008;119:218-24.
13.Singh A, Handa SS. Hepatoprotective activity of Apium graveolens and Hygrophila auriculata against paracetamol and thioacetamide intoxication in rats. J. Ethnopharmacol 1995;49:119-26.
14. "
15.Rastogi R, Mehrotra BN. Compendium of Indian medicinal plants. Lucknow: Central Drug Research Institute; 1985-1989.
16.Khandelwal KR. Practical Pharmacognosy-techniques and experiments. Pune: Nirali Prakashan; 1996.
17. Acute oral toxicity studies [online].Dec 2001[cited 2009 Dec 09]; Available from:
URL:
18.Ramachandra S.S, Quereshi. AA, Viswanath Swamy AHM, Patil. T, Prakash T, Prabhu K. Hepatoprotective activity of Calotropis procera flowers against paracetamol-induced hepatic injury in rats. Fitoterapia 78:451–4.
9.0 / NAME OF THE CANDIDATE / LEENA.KAVERIAPPA.B
10.0 / SIGNATURE OF THE CANDIDATE / (LEENA.K.B )
11.1 / REMARKS OF THE GUIDE / Recommended and forwarded for the approval
11.2
11.3
11.4
11.5
12.1
12.2 /
NAME AND DESIGNATION OF THE
GUIDE
SIGNATURE
HEAD OF THE DEPARTMENT
SIGNATURE
REMARKS OF THE PRINCIPAL
SIGNATURE / MR.MUKUND HANDRAL
ASST. PROFESSOR,
DEPT. OF PHARMACOLOGY
P.E.S.COLLEGE OF PHARMACY
Mr. SRINATH. R
HOD & ASST PROFESSOR
DEPT. OF PHARMACOLOGY
P.E.S.COLLEGE OF PHARMACY
FORWARDED FOR APPORVAL
Prof.Dr.S. MOHAN
PRINCIPAL AND DIRECTOR
P.E.S.COLLEGE OF PHARMACY
BENGALOORU -560050.