RAJIV GANDHI UNIVERSITY OF HEALTH SCIENCES KARNATAKA, BANGALORE

M. PHARM SYNOPSISYEAR OF ADMISSION-NOVEMBER 2011

TITLE OF THE SYNOPSIS

“In- vivo Evaluation of Anti diarrheal Activities of Sapindus Laurifolia Vahl Fruit Extract in Rats”.

BY

Ms.VARSHITHA.C

DEPARTMENT OF PHARMACOLOGY

UNDER THE GUIDANCE OF

Dr. B.M. Vrushabendra Swamy M.Pharm, Ph.d, FICCP.

Professor

Department of Pharmacology

INSTITUTIONGAUTHAM COLLEGE OF PHARMACY

R. T. NAGAR, BANGALORE-32KARNATAKA

RAJIV GANDHI UNIVERSITY OF HEALTH SCIENCES KARNATAKA, BANGALORE

ANNEXURE - II

PROFORMA FOR REGISTRATION OF SUBJECTS FOR DISSERTATION

1. Name of the Candidate And Address

Ms.VARSHITHA.C

PERMANENT ADDRESSD/o H.N.Chandrashekar.#551,Shivajyothi NilayaFort street,DevanahalliBangalore-562110

PRESENT ADDRESSGautham College of PharmacyBhuvaneswari nagar, R.T.Nagar Post,Bangalore- 32.

2. Name of the Institution Gautham College of Pharmacy Bhuvaneswari nagar, R.T.Nagar Post, Bangalore- 32.

3. Course of Study and Subject Master of Pharmacy in Pharmacology.

4. Date of Admission 30-11-2011

5. Title of the Topic:

“In- vivo Evaluation of Anti diarrheal Activities of Sapindus Laurifolia Vahl Fruit Extract in Rats”.

6.0 BRIEF REVIEW OF THE INTENDED WORK:

6.1 INTRODUCTION:

Diarrhea has long been recognized as one of the most important health problems in

developing countries. It is defined as an increase in the frequency, fluidity, or volume of

bowel movements and characterized by increased frequency of bowel sound and

movement, wet stools, and abdominal pain. In clinical terms, it is used to describe

increased liquidity of stools, usually associated with increased stool weight and frequency.

In Nigeria, diarrhea remains the number one killer disease among children aged 1-5 years,

and worldwide the disease accounts for 4-5 million deaths among humans annually.

Treatment of diarrhea is generally non-specific and usually aimed at reducing the

discomfort and inconvenience of frequent bowel movements. To overcome the menace of

diarrheal diseases in developing countries, the World Health Organization (WHO) has

included a programme for the control of diarrhea, which involves the use of traditional

herbal medicine. Several plants have been reported to be used in treating and managing

diarrheal diseases [1].

The antimotility agents such as Loperamide, Diphenoxylate, Codeine, Morphine,

Bismuth Salicylate, anti-infective agents like Ciprofloxacin, Tinidazole, Metronidazole,

Chloroquine, Emetine, Dehydroemetine and adsorbents such as Kaolin, Pectin, Chalk,

Charcoal, Methylcellulose etc. are used to treat diarrhea and these drugs produces side

effects such as anorexia, nausea, drowsiness, rashes, neutropenia,metallic taste, abdominal

cramps[2, 3].

Diarrhea may be defined as a situation in which an adult daily stool exceeds 200 g

and contains 60-95% water [4]. It is the world's third highest killer disease, contributing

substantially to pediatric morbidity and mortality, especially in the malnourished [5]. This

disease is common in the tropics. Indeed, in certain parts of the world, diarrhea produces

more illness and causes death of more infants and children than all other diseases

combined (Weber, 1976). Diarrhea ranges from a mild to a socially inconvenient illness to

a major cause of malnourishment among children of developing countries, and causes 4-5

million deaths throughout the world, annually [4]. Most people are affected by diarrhea at

some time in their lives. It is often accompanied by stomach pains, feeling sick and

vomiting. It is usually due to consumption of drinking water contaminated with bacteria,

undercooked meat and eggs or inadequate kitchen hygiene-in other words-an infection.

According to WHO estimates for 1998, about 7.1 million deaths were caused by diarrhea.

In Nigeria, diarrhea remains the number one killer disease among children aged 1-5 years,

and worldwide the disease accounts for 4-5 million deaths among humans annually.

Treatment of diarrhea is generally non-specific and usually aimed at reducing the

discomfort and inconvenience of frequent bowel movements [6].

The use of herbal drugs in the treatment of diarrhea disease is common practice in

many countries [4].The medicinal value of plants lies in some chemical substances that

produce a definite physiological action on the human body. The most important of these

bioactive compounds of plants are alkaloid, flavonoids, tannins and phenol compounds.

The use of medicinal plants as traditional medicines is well known in rural areas of many

developing countries. Traditional healers claim that their medicines are cheaper, more

effective and impart least side effects as compared to synthetic medicine. Medicinal plants

represent a rich source of antimicrobial agents. A wide range of medicinal plant parts is

used for extract as a raw drugs and they posses varied medicinal properties. The different

Parts are including bark, root, stem and fruit and modified plant organ. While some of

these raw drug are collected in smaller quantities by the local communities and folk

healers for local used many other raw drug are collected in larger quantities and traded in

the market as the raw material for many herbal industries.

Hence the purpose of study to know the safe and potent antidiarrheal effect of

sapindus laurifolia .On the contrary most of the herbal drugs reduce the offensive factors

and proved to be safe, clinically effective, better patient tolerant, relatively less expensive,

and globally competitive. Plant extracts, however, are some of the most attractive sources

of new drugs and have been shown to produce promising results in the treatment of

diarrhea

6.2 REVIEW OF LITERATURE [7, 8]:

Botanical name: Sapindus laurifolia Vahl.

Common names: Soap nut tree of South India

Hindi: Reetha

Kannada: Antawala, Kunkatekaye.

Tamil: Puvamkottai

Telugu: Kunkudu

Family: Sapindaceae

Distribution: India, South-West India, Srilanka, Burma.

Plant description:

Characteristics: It is a deciduous tree, reaching up to a height of 18m and

1.5m in girth.

Leaves: They are abruptly pinnate, 12-30cm long, with 2-3 pairs of leaflets.

Flowers: These are white, born in terminal, rusty-pubescent panicles, with

a few bisexual and numerous males.

Fruits: It is a fleshy drupe, which is 2-3 lobed, with a single pea-sized,

smooth, blackish seed in each lobe.

Bark: It is rough, shiny and grey.

Wood: It is not very durable, tough it is hard and heavy. It is bright yellow,

streaked with lighter and darker colours. It is medium and even textured.

Traditional uses:

The plant is used as in Ayurveda and Siddha medicine. The fruit of the plant is

used as emetic, tonic, astringent and anthelmintic, asthma, colic due to indigestion,

diarrhea, tubercular glands, paralysis of limbs and lumbago. Roots and bark expectorant

and demulcent.

Phytoconstituents:

Fruit contains saponin. Kernel contains oil. Saponin from defatted pericarp

hydrolysed to give hedaragenin and oleanolic and sapindic acids. Nuts contain kaempferol,

quercetin, and β-sitosterol.

6.3 OBJECTIVES OF THE STUDY:

Antidiarrheal Activity:

The main objective of the proposed work In-vivo evaluation of antidiarrheal

activities of sapindus laurifolia fruit extract in rats. The whole study is divided into two

phases.

Phase I:

Collection and authentication of plant material. The powdered fruit extracted with

hydroalcohol(70% v/v) by using Soxhlet apparatus.

To investigate preliminary phytochemical constituents present in the extract.

Determination of LD50 value and dose selection for proposed activities (selection of

an appropriate dose from LD50 value) those considered as respective doses.

Phase II:

To evaluate In- Vivo evaluation of antidiarrheal activity of fruit extract of Sapindus

laurifolia by using the experimental animal model like:

1. Castor oil induced diarrhea.

2. Gastrointestinal motility test.

3. PGE2 induced enteropooling.

4. Magnesium sulphate-induced diarrhea.

7.0 MATERIALS AND METHODS:

7.1 Source of Data:

The Source of data will be based on laboratory experiments on animals and also

the data obtained from the literature.

1. Standard Books:

Goodman and Gilmann’s: The Pharmacological basis Therapeutics.

Rang and Dale’s Pharmacology.

Wealth of India.

Indian Medicinal Plants.

Yoganarasimhan SN. Medicinal Plants of India, vol-1.

2. Internet sources:

Pub med Science Direct SCOPUS Helinet Ovid Open J gate DOAJ

Chemical Abstracts CABI International Pharmaceutical Abstracts Google Wikipedia

3. Journal sources:

Indian Journal of Pharmacology.

Journal of Pharmacology and experimental Therapeutics.

Journal of Ethanopharmacology.

Phytochemistry.

American Journal of Pharmacology and Toxicology.

7.2 Preparation of Extract [9]:

The fruits were dried in an incubator for two days at 40°C, reduced to a coarse

powder. The powdered fruits of Sapindus laurifolia are subjected to soxhlet extraction by

using Hydro-alcohol (70%v/v). The prepared extracts are concentrated to lesser volume

under reduced pressure and evaporate to dryness. .

7.3 ANTIDIARRHEAL ACTIVITY:

A. Experimental animals: Adult albino rats weighing approximately 150-200 g of

either sex will be used for antidiarrheal activity. And mice’s are used for the acute

toxicological studies. The animals will be fed with standard diet and will be given

water ad libitum.

B. Plant material: Naturally available fruit of Sapindus laurifolia vahl will be

collected, identified and extracted with hydro-alcohol(70%v/v).

C. Acute toxicity study of crude extract [10]: For this purpose female albino rats will

be used. Fixed dose method (OECD guideline no. 425) of CPCSEA will be

adopted.

D. Grouping of animals: The animals are divided into six groups, each group consists

of six rats.

8: Experimental Animal Group:

8.1: Castor oil induced diarrhea[ 11]:

Overnight fasted thirty six rats were divided into six groups equally as follows.

Group I: (Control group) Rats of this group received 1 ml 2% v/v aqueous Tween 80

orally. Group II: (Standard drug treated group). Rats of this group were treated with the

reference drug, diphenoxylate at the dose of 50 mg/kg body weight, orally. Group III, IV

and V: (HASL treated groups) Rats of these groups were treated with hydroalcoholic

extract of Sapindus laurifolia fruits at the doses of low, medium, high mg/kg body weight

by oral route respectively suspended in 2% v/v aqueous Tween 80. After one hr of dosing,

all the rats were treated with 1 ml of castor oil orally by gavage and observed for

consistency of faecal material. The numbers of wet faecal droppings were measured for

four hrs after castor oil administration. Characteristic diarrheal droppings were noted in

transparent plastic dishes placed beneath the individual perforated rat cages. The total

number of diarrheal faeces of the control group was considered 100%. The results were

expressed as percentage of inhibition of diarrhea.

Group I: Normal control (12% v/v aqueous Tween 80).

Group II: Standard drug (Diphenoxylate) will be supplied at a dose of 50 mg/kg orally.

Group IV: A dose of HASL (1/20th of LD50) will be supplied orally in distilled water.

Group V: A dose of HASL (1/10th of LD50) will be supplied orally in distilled water.

Group VI: A dose of HASL (1/5th of LD50) will be supplied orally in distilled water.

8.2: Gastrointestinal motility test [12].

The rats were divided into five groups of six animals each and fasted for 18 hr but

water was freely provided. The first group (control group) received orally normal saline (5

ml/kg body weight), The second group received orally the standard drug, atropine sulphate

(5 mg/kg body weight). While the third, fourth and fifth groups were given orally HASL in

doses of low, medium, high mg/kg body weight. Thirty (30) min later, each animal was

given 1 ml of charcoal meal (10% activated charcoal in 5% gum acacia) via the oral route.

All animals were sacrificed 30 min thereafter, and the distance covered by the charcoal

meal in the intestine, from the pylorus to the caecum was measured and expressed as

percentage of distance moved.

Group I: Distilled water will be supplied and serve as normal control

Group II: Standard drug (Atropine sulphate) will be supplied at a dose of 5 mg/kg i.p.

Group III: A dose of HASL (1/20th of LD50) will be supplied orally in distilled water

Group IV: A dose of HASL (1/10th of LD50) will be supplied orally in distilled water

Group V: A dose of HASL (1/5th of LD50) will be supplied orally in distilled water

8.3: PGE2 induced enteropooling [13]:

Albino rats (Wistar) weighing 150-200g rats were divided into five groups of six

animals each and fasted for 18 hr but water was freely provided. Which were placed in

five perforated cages. The first and second group received 1ml of 5% v/v ethanol in

normal saline (i.p). While the third, fourth and fifth groups of rats were treated with HASL

(low, medium, high mg/kg body weight, p.o) while the fourth and fifth group received 1ml

of 5% v/v ethanol in normal saline (i.p). The first group was then administered 1ml of

normal saline and used as control. Immediately afterwards, each rat was treated with PGE2

(100µg/kg body weight in 5% v/v ethanol in normal saline) administered orally. All the

rats were sacrificed under mild anesthesia after 30min. The entire length of intestine

from the pylorus to the caecum was dissected out, and its contents were collected and

measured

Group I: Distilled water will be supplied and serve as normal control

Group II: This group received 1ml of 5% v/v ethanol i.p

Group III: A dose of HASL (1/20th of LD50) will be supplied orally in distilled water

Group IV: A dose of HASL (1/10th of LD50) will be supplied orally in distilled water

Group V: A dose of HASL (1/5th of LD50) will be supplied orally in distilled water.

8.4 Magnesium sulphate-induced diarrhea[14]

Diarrhoea was induced by oral administration of magnesium sulphate at the dose of

2 g/kg to the animals 30 min after pre-treatment with vehicle (1% Tween 80 in water, 10

ml/kg, p.o.) to the first group, loperamide (3 mg/kg) to the second group, and While the

third, fourth and fifth groups of rats were treated with HASL (low, medium, high mg/kg

body weight, p.o)

Group I: This group received 1% Tween 80 in water

Group II: Standard drug (loperamide) will be supplied at a dose of 3 mg/kg orally

Group III: A dose of HASL (1/20th of LD50) will be supplied orally in distilled water

Group IV: A dose of HASL (1/10th of LD50) will be supplied orally in distilled water

Group V: A dose of HASL (1/5th of LD50) will be supplied orally in distilled water.

HASL: Hydro-alcoholic extract of Sapindus laurifolia

P.O: Post Oral.

i.p: Intra peritoneal.

8.5: Statistical Analysis:

All the values that are generated out of this study execution will be expressed as

mean ± SEM from six animals. Statistical difference in mean will be analyzed using one

way ANOVA (Analysis of Variance) followed by Dunnett’s‘t’ test. P values less than 0.05

were considered as indicative of significance.

8.5: Does the study require any investigation or intervention to be conducted on

patients or other humans or animals? If so, please mention briefly.

Yes, the above study requires In vivo screening techiniques on Wistar rats.

8.6: Does ethical clearance is obtained from your institution?

The copy of ethical clearance certificate is enclosed.

9. BIBLIOGRAPHY:

1. Praveen Sharma, Gali Vidyasagar, Sunder Singh, .et al. Antidiarrheal activity of

leaf extract of celosia argentea in experimentally induced diarrhea in rats. J Adv

Pharm Technol Res 2010; 1(1): 41–48.

2. Rang HP, Dale MM, Ritter JM, Flower RJ. Pharmacology. 6 ed. New Delhi,

British Library Cataloguing in Publication Data 2007; pp 394.

3. Tripathi KD. 5 ed. New Delhi. Jaypee brothers Medical publishers 2003;pp 663-

664.

4. Sini JM, Umar IA, Anigo, et al. Antidiarrheal activity of aqueous extract of

Combretum sericeum roots in rats. African Biotechnol 2008; 7 (17):3134-3137.

5. Gerald Ngo Teke, Jules-Roger Kuiate, Victor Kuete, et al. Antidiarrheal activity

of extracts and compound from Trilepisium madagascariense stem bark. Indian J

Pharmacol 2010;42(3):157-163.

6. Karanayil R Sini1, Barij N Sinha,Aiyolu Rajshekaran. Anti diarrheal activity of

capparis Zeyanica leaf extracts in rats. J Adv Pharm Tech Res 2011;2(1)39-42.

7. Yoganarasimhan SN. Medicinal Plants of India. Karnataka: Interline Publishing.

1996; (1):415-416.

8. Nursery Manual for Forest Tree Species By Ravindranath NH, Bhat DM, Vidya S.

Swamy. Universities press(India) Private Limited 2003; pp 244-246.

9. Lakshminarayana M, Shivkumar H, Rimaben P, Bhargava VK. Antidiarrheal

activity of leaf extract of Moringa Oleifera in experimentally induced diarrhea in

rats. International Journal of Phytomedicine 2011;3: 68-74.

10. OECD Guidelines for the Testing of Chemical. Acute Oral Toxicity – Up and Down

Procedure (UDP) [Internet]. 2008 [Cited 2011 September 25]. Available from:

http://iccvam.niehs.nih.gov/SuppDocs/FedDocs/OECD/OEC Dtg425.pdf(Accessed

on 18-6-2012).

11. Sekhar K Bose, Saikat Dewanjee, Avijit Sen Gupta et al. In -Vivo Evaluation of

Antidiarrheal Activity of Rhus Semialata Fruit Extract in Rats. Afr J Tradit

Complement Altern Med 2008; 5(1): 97–102.

12. Vrushabendra Swamy Bhyrapur Mathad, Sridhar Chandanam, Sreenivasa Rao

Thirumala Setty et al. Antidiarrheal Evaluation of Benincasa hispida (Thunb.)

Cogn. Fruit Extracts. Iranian J Pharmacol Ther 2005; 4(1):24-27.

13. Vrushabendra Swamy BM, Jayaveera KN, Ravindra Reddy KP. Antidiarrheal

activity of fruit extract of Momordica cymbalaria Hook. F. The Internet Journal of

Nutrition and Wellnes 2008; 5 (2):1-7.

14. Raushanara Akter, Raquibul Hasan SM, Mokarram Hossain MD, et al. In Vitro

Antioxidant and In Vivo Antidiarrhoeal Activity of Hydromethanolic Extract of

Xanthium Indicum Koenig. Leaves. European Journal of Scientific Research

2009;33(2):305-312.

9 SIGNATURE OF THE CANDIDATE:

10 REMARKS OF THE GUIDE:

“In- vivo Evaluation of Anti diarrheal Activities of Sapindus Laurifolia Vahl Fruit Extract in Rats”. To be carried out by Ms. Varshitha.C of M. Pharm has been discussed and worked out under my directions and supervision as an official guide. The project work envisaged is of great importance in the field of pharmacology. The work can be carried out in pharmacology laboratory of Gautham College of Pharmacy for which facilities are available. Hence the project is viable and is recommended for clearance.

11 NAME AND DESIGNATION OF:

11.1 GUIDE:

Dr. B M Vrushabendra Swamy M.Pharm,Ph.D, FICCP Director / Professor & Head,Department of Pharmacology.Gautham college of pharmacyBangalore-560032

11.2 SIGNATURE:

11.3 HEAD OF THE DEPARTMENT: Dr. B M Vrushabendra Swamy M.Pharm,Ph.D, FICCP Director / Professor & Head,Department of Pharmacology.Gautham college of pharmacyBangalore-560032

11.4 SIGNATURE

12 CLEARENCE FROM INSTITUTIONAL ETHICAL COMMITTEE:

The study is cleared from Animal Ethical Committee of the Institution.

(Approval no:491/01/c/CPCSEA)

13 13.1 REMARKS OF THE PRINCIPAL:

The program and research work that Ms. Varshitha.C is undertaking have

potential implication in the field of Pharmacology. The work can be carried out in the

Research Laboratories of Pharmacology Department at Gautham college of

Pharmacy.

Hence the project is recommended and requested for clearance and approval.

13.2 SIGNATURE

Prof. Archana Swamy. P M.Pharm (Ph.D)

PrincipalGautham College of PharmacyBhuvaneswari nagar,R.T.Nagar Post,Bangalore- 32.