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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.
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