Download EVALUATION OF ANTI-DIABETIC ACTIVITY OF ETHANOLIC

EVALUATION OF ANTI-DIABETIC ACTIVITY OF
ETHANOLIC EXTRACT OF HOLARRHENA ANTIDYSENTRICA
LEAVES.
MASTER OF PHARMACY DISSERTATION PROTOCOL
SUBMITTED TO THE
RAJIV GANDHI UNIVERSITY OF HEALTH SCIENCES KARNATAKA,
BANGALORE
BY
JAISAL .K .K
Under The Guidance of
MR. MOSES SAMUEL RAJAN
M.PHARM
DEPARTMENT OF PHARMACOLOGY,
SRINIVAS COLLEGE OF PHARMACY
MANGALORE – 574 143
2012 – 2013
RAJIV GANDHI UNIVERSITY OF HEALTH SCIENCES
BANGALORE, KARNATAKA.
ANNEXURE-II
PROFORMA FOR REGISTRATION OF SUBJECT FOR DISSERTATION
1.0
NAME AND ADDRESS OF
THE CANDIDATE
2.0
NAME OF THE
INSTITUTION
3.0
COURSE OF STUDY AND
SUBJECT
4.0
SRINIVAS COLLEGE OF PHARMACY
VALACHIL, MANGALORE- 574 143
MASTER OF PHARMACY IN
PHARMACOLOGY
DATE OF ADMISSION TO
COURSE
5.0
JAISAL . K .K
KARIKULAKKATTIL HOUSE
CHERIYAMUNDAM (P.O)
MACHINGAPPARA
MALAPPURAM- 676 106
KERALA
TITLE OF THE TOPIC
26-06-2012
“EVALUATION OF ANTI-DIABETIC
ACTIVITY OF ETHANOLIC EXTRACT
OF HOLARRHENA ANTIDYSENTRICA
LEAVES”.
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6.0
BRIEF RESUME OF THE INTENDED WORK :
6.1 NEED FOR THE STUDY
Diabetes mellitus has been known to medical sciences longer than any other hereditary
metabolic diseases. It is a chronic metabolic disorder characterized by hyperglycemia
caused by insulin deficiency often combined with insulin resistance. Two main types
of diabetes mellitus: type I and type II.
In type I diabetes, there is an absolute deficiency of insulin resulting from autoimmune
destruction of β cells. Without insulin treatment, such patients will ultimately die with
diabetic ketoacidosis. Type II diabetes is accompanied by both insulin resistance and
by impaired insulin secretion. Such patients are often obese and usually present in
adult life, the incidence rising progressively with age as β cell function declines. In
conventional therapy, type I diabetes is treated with exogenous insulin and type II with
oral hypoglycemic agents 1.
India is the diabetic capital of the world, predicted to have 57.2 million diabetic
populations by the year 2025 2. Diabetes mellitus is a disease in which homeostasis of
carbohydrate, protein and lipid metabolism is improperly regulated by hormone
insulin resulting in elevation of fasting and postprandial blood glucose levels 3. The
major chronic complications associated with diabetes include retinopathy, neuropathy,
nephropathy, atherosclerotic coronary artery disease and peripheral atherosclerotic
vascular diseases. Besides hyperglycemia, several other factors like hyperlipidemia
and enhanced oxidative stress play a major role in diabetic pathogenesis. Despite the
great strides that have been made in the understanding and management of this
disease, the graph of diabetes-related mortality is rising unabated. Although a number
of synthetic drugs are available in the market, diabetes and its related complications
still remain uncontrolled 4.
The existing methods of treatment of diabetes are not completely satisfactory owing to
low efficacy, associated adverse effects and compliance issues. Among the therapies
non pharmacologic therapy (e.g. diet, exercise and weight loss) remains to be critical
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component in diabetes treatment. Dietary management includes the use of traditional
medicines mainly derived from plants.
Traditional medicinal plants have been used successfully since ancient times by
physicians to treat diabetes and its related complications, presenting a stirring prospect
for the expansion of an alternative way of treatment of diabetes. The multiple
activities of plant based medicinal preparations offer an enormous scope for not only
to control the hyperglycemia but also its late complications. Moreover, WHO study
groups have emphasized strongly the optimal rational uses of traditional medicines
because the theory of polyherbal formulations having synergistic, potentiative,
agonistic, antagonistic pharmacological actions within themselves work together in a
dynamic way 5.
Glibenclamide is an anti-diabetic drug coming under the class 2nd generation
sulfonylurea. As of 2007, it is one of only 2 oral anti-diabetic drugs in the World
Health Organization Model list of Essential Medicines. It is one of the major drugs
used in diabetic therapy extensively due to its abundant availability and low cost. It
acts by inhibiting ATP sensitive K+ channel in pancreatic β cell which finally leads to
stimulation of insulin release. It is 150 times more potent than 1st generation
sulfonylurea like tolbutamide, hence may cause severe hypoglycemia and may lead to
even coma state 6. The drug is poorly soluble or practically insoluble in water.
Advanced studies show that it is associated with a marginal decrease in circulating
lipids. Also cause rare side effects like weight gain due to fluid retention and edema,
photosensitization reaction, cholestatic jaundice 7.
Holarrehena antidysentrica: belonging .to family Apocynaceae, commonly known
as kutaja, or kurchi. Distributed in Asia, tropical area of Africa. Madagascar, India. .
Growing up to an altitude of 1300 m in the Himalayas, often gregariously in
deciduous forests. Open waste land and abundant in sub Himalaya tract. The plant has
been employed for long time in folkfore therapy. Kurchi bark is a important traditional
drug used in various ailments. The drug is astringent, anthelmentic, stomachic,
antipyretic and also anti diabetic. Kurchi is given either alone or with other astringent
drugs. A hot decoction of the drug is used as a gargle in toothache. It consist certain
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biochemical constituent namely alkaloid, glycoside, phenolic compounds and tannins.
the drug is collected from the wild source and varies in constituent and efficacy due to
the geographical diversity. Holarrhena antidysentrica is an important plant employed
in various indigenous system of medicine against several disease, and almost every
part of plant has diverse medicinal properties, the results of the study could be useful
in setting some diagnostic indices for the identification and preparation of a
monograph of the plant8.
6.2 REVIEW OF LITERATURE:
Holarrhena antidysentrica:
Description:
. H. antidysenterica is a deciduous laticiferous shrub or small tree, up to 13 m high and 1.1 m
in girth with clear bole of 3-7 m. Leaves 15-30 x 4-12 cm, base obtuse, rounded or acute,
nerves 10-14 pairs, opposite, sessile, elliptic or ovate, oblong, membranous, strong, arched;
petiole up to 1.5 cm; cymes 3-6 cm diameter. Corymbose sessile terminal; bracts small, ciliate;
pedicels slender. Flowers inodorous white, in terminal corymbose cyme. Calyxlobe 2.5-3 mm
long, oblong-lanceolate, acute, ciliate.Corolla puberulous outside; tube 8-13 mm long . A large
shrub or small tree grows up to 13 meters in height. Leaves simple, opposite, ovate,
acute-acuminate, membranous; flowers white in axillary or terminal corymbose
cymes; fruits long are pair of long follicles seeds long, brown, with silky hairs, aided
in dispersion.,
Is an important plant used in indigenous system of medicine as remedy for
bronchitis, hematuria spermatorrhoea, epilepsy, asthma, piles, leprosy eczema,
diarrhea, fever and jaundice.
Various part of holarrhena antidysentrica have been reported to possess antibacterial
activity. The bark has been reported to possess astringent and anti diarrheal properties.
leaves of the plant are used to cure scabies.
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However, a major constituent which has hindered the acceptance of alternative
medicine
is the lack of documentation and stringent quality control with this
backdrop, it is of the prime important to make an effort towards standardization of
the plant material. The process of standardization is achieved
pharmacognostic
by step wise
studies which in turn help to identify and authenticate plant
material9.
Chemical constituent:
Conessydjene, connessimine, conkurchine, holadiene, holarrhejne, holarrhimine,
kurchine, holarrhine, kuchisyne. Kurchi phylammine.
Synonyms:
Kuda (Hindi)
Kurchi (English)
Kodipla (Telungu)
Kodagapaia (Malayalam)
Veppajaj (Tamil)
Part used : Seed, Leaf, Stem bark,
Reported activities: Blood purifier, Anti-pyretic, Skin diseases, Abdominal disorders,
Anti-diabetic activity
6.3 OBJECTIVES OF THE STUDY:
The main objectives of the present study are as follows:1. To prepare the ethanolic extract of the leaves of kurchi (Holarrhena antidysentrica)
2. To study the anti-diabetic activity of Kurchi by using following animal models
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I. Alloxan Induced Diabetes In Rats
The antidiabetic activity will be evaluated by using following parameters
a) Serum glucose level will be checked for every alternate day
b) Body weight will be measured weekly once.
c) Water and s intake will be measured once in a week.
Biochemical Estimation:
After the completion of treatment, blood will be withdrawn from retro orbital sinus
and estimated for following parameters
i) Serum glutamic pyruvate transaminase (SGPT)
ii) Serum glutamic oxaloacetate transaminase (SGOT)
II. Streptozotocin Induced Diabetes In Rats
The antidiabetic activity will be evaluated by using following parameters
a) Serum glucose level will be checked for every alternate day
b) Body weight will be monitored weekly once.
c) Water and food intake will be measured once in a week.
Biochemical Estimation:
After the completion of treatment, blood will be withdrawn from retro orbital sinus
and estimated for following parameters
i) Serum glutamic pyruvate transaminase (SGPT)
ii) Serum glutamic oxaloacetate transaminase (SGOT)
III Oral Glucose Tolerance Test in rats
The blood glucose concentrations will be determined at 30, 60, 120 and 180 minutes
after dosing
7.0
MATERIALS AND METHODS:
7.1 SOURCE OF DATA:
Experiment will be performed as described in the standard bibliography, literatures
and text books. The reputed journals and publications are obtained from college
library and through web search.
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7.2 ANIMALS
Wistar albino rats (180-200 g) of either sex will be used for the study. They will be
maintained under standard conditions (temperature 22 ± 2oC, relative humidity 50±5%
and 12 h light/dark cycle) and have free access to standard pellet diet and water ad
libitum. All experimental protocols will be reviewed and approved by the Institutional
Animal Ethical Committee (IAEC) prior to the initiation of the experiment and the
care of the laboratory animals will be taken as per the CPCSEA regulations.
7.3 CHEMICALS
Alloxan monohydrate, Streptozotocin will be procured from Loba chemie, Mumbai,
India. Glibenclamide will be purchased from Cipla ltd ,mumbai, India. Diagnostic kits
to estimate serum parameters will be obtained from Mannheim diagnostic kits India
Ltd. All other chemicals used in the experiment will be of analytical grade obtained
from local suppliers.
7.4 COLLECTION OF THE DRUGS AND PREPARATION OF THE
EXTRACTS:
The leaves of holarrhena antidysentrica is procured either from commercial herbal
stores or from natural source. Fresh Leaf
of holarrhena antidysentrica will be
cleaned, cut in to small pieces and shade dried or, in an incubator for 2-3 days at
400 C. Crushed it in an electric grinder and then powdered. Make extracts of powder
by using Ethanol. Extracts will be prepared by over night maceration technique. The
concentration of this kurchi preparation was considered to have 300mg/ml on the basis
of the weight of the starting material according to the formula.
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7.5 EXPERIMENTAL METHODS :
1.
I. ALLOXAN INDUCED DIABETES IN RATS 9
Diabetes will be induced in overnight fasted animals by single intraperitoneal injection
of alloxan monohydrate prepared in normal saline (100mg/kg body weight). After a
fortnight, diabetes will be confirmed by hyperglycemia. The animals showing fasting
blood glucose level more than 250mg/dl will be considered to be diabetic and included
for the study.
EXPERIMENTAL DESIGN :
Animals will be randomly divided into 4 groups of 5 each. The different groups will
be assigned as follows
Group I: Vehicle control (normal saline).
Group II: Diabetic control (Alloxan100mg/Kg).
Group III: Diabetic + Glibenclamide (1mg/Kg)
Group IV: Diabetic + Kurchi(300mg/kg13)
EVALUATION:
Starting from the first day of treatment, blood will be collected from all animals and
blood glucose level will be estimated every alternate day. Daily body weight, food and
water intake will be monitored for 30 days. On 30th day post treatment blood will be
collected and serum will be separated by centrifugation and evaluated for biochemical
parameters.
II. STREPTOZOTOCIN INDUCED DIABETES IN RATS 10
Diabetes will be induced in overnight fasted animals by single intraperitoneal injection
of Streptozotocin (65 mg/kg) prepared in normal saline. After 48 hours of STZ
injection, blood samples will be taken and glucose estimation is done. Animals having
fasting blood glucose level more than 250mg/dl will be chosen for studies. In spite of
diabetes developed after 48h of STZ injection, the animal will be stabilized up to 6
days. The treatment will be started from 7th day of STZ injection once daily post orally
for next 30 days.
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EXPERIMENTAL DESIGN :
Animals will be randomly divided into 4 groups of 5 each and assigned as below.
Group I: Vehicle control (normal saline).
Group II: Diabetic control (Streptozotocin 65mg/Kg).
Group III: Diabetic + Glibenclamide (1mg/Kg)
Group IV: Diabetic + Kurchi(300mg/kg13)
EVALUATION:
Starting from the first day of treatment, blood will be collected from all animals and
blood glucose level will be estimated. Daily body weight, food and water intake will
be monitored for 30 days. On 30th day post treatment blood will be collected and
serum will be separated by centrifugation and evaluated for biochemical parameters.
2.
III. ORAL GLUCOSE TOLERANCE TEST (OGTT) IN RATS 11
After overnight fasting (16h) the blood glucose level of rats will be determined. The
animals will be treated with test samples after administrating oral glucose of 2g/Kg
body weight in 0.2ml water.
EXPERIMENTAL DESIGN :
The animals will be randomly divided into 3 groups of 5 each and assigned as below
Group I: Glucose control.
Group II: Glucose load + Glibenclamide (1mg/Kg).
Group III: Glucose load+ Kurchi (300mg/kg13)
EVALUATION:
The blood glucose concentration will be determined at 30, 60, 120 and 180 min after
dosing.
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7.5 STATISTICAL ANALYSIS:
The data will be expressed as Mean value + SEM and will be analyzed by the one-way
ANOVA.
7.6
DOES
THE
STUDY
REQUIRE
ANY
INVESTIGATIONS
OR
INTERVENTIONS TO BE CONDUCTED ON PATIENTS OR OTHER
HUMANS OR ANIMALS? IF SO PLEASE DESCRIBE BRIEFLY.
Yes. Study requires investigation on Wistar rats.
7.7 HAS ETHICAL CLEARANCE BEEN OBTAINED FROM YOUR
INSTITUTION?
Yes. Ethical clearance has been obtained. (Copy enclosed)
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8.0
REFERENCES:
1)
Rang HP, Dale MM, Ritter JM, Flower RJ. Rang and Dale’s pharmacology. 6th
ed. Churchill Livingstone Elsevier Ltd; 2007. p. 402-4.
2)
Pradeepa R, Deepa R, Mohan V. Epidemiology of diabetes in India- current
perspective and future projections. J Indian Med Assoc;100: 144-8.
3)
Tiwari AK, Rao JM. Diabetes mellitus and multiple therapeutic approaches of
phytochemicals: Present status and future prospects. Curr Sci 2002;83: 30-8
4)
Kaczmar T. Herbal support for diabetes management. Clin Nutr Insights
1998;6: 1-4
5)
Sundaram EN, Reddy PUM, Singh KP. Effect of alcoholic extracts of Indian
medicinal plants on the altered enzymatic activities of diabetic rats. Indian J
Pharm Sci 2009;71(5): 594-8
6)
Sharma HL, Sharma KK. Principles of pharmacology. Paras Medical
publisher; 2007. p. 652-3
7)
David EG, Armen H, Tashijan N, Ehrin JA, April WA. Principles of
pharmacology. 2nd ed. Lippincott’s, William & Wilkins; 2008. p. 540
8)
Parwaiz Akhtar, Mohd ali, M P Sharma, Humaira Farooqi, Showkat R. Mirand
Hmid Nawaz Khan.
Development of quality standard of Holarrena
antidysentrica ( linn) Bark. RRST 2011; 3(1):73-80
9)
P.S. Sujan ganapathy,Y.L Ramachandran, H.v sudeep. Pavan kumar
bellamokondi. pharmacognostic and phytochemicalevaluation of Holarrhena
antidysentrica wall. AAJPSB 2009; 3(1): 47-50
10) Sharma N, Garg V. Anti-hyperglycemic and anti-oxidative potential of hydro
alcoholic extract of Butea monosperma Lam flowers in alloxan-induced
diabetic mice. Indian J Exp Biol 2009;47: 571-6
11) Bajaj S, Srinivasan BP. Investigations into the anti-diabetic activity of
Azadirachta indica. Indian J Pharmacol 1999;31: 138-41
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12) Bhavana S, Santosh K, Satapathy N. Hypoglycemic and hypolipidemic effect
of Aegle marmelos leaf extract on STZ induced diabetic mice. Int J Pharmacol
2007;3(6): 444-52.
13) Keshri umarshankar pd, satish Chandra, janardan Sharma. Antidiabetic
activityof
ethanolic
extract
of
holarrhena
antidysentrica
seeds
in
streptoztocine-induced diabetic rats and its influence in certain biochemical
parameters. JDDT 2012;2(4):159-162.
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9.
SIGNATURE OF THE
CANDIDATE
10.
REMARKS OF THE GUIDE
11.
NAME AND DESIGNATION
Recommended and Forwarded
MR. MOSES SAMUEL RAJAN
ASSO. PROFESSOR
DEPARTMENT OF PHARMACOLOGY
SRINIVAS COLLEGE OF PHARMACY
11.1 GUIDE
11.2 SIGNATURE OF GUIDE
11.3 CO - GUIDE (IF ANY)
Not Applicable
11.4 SIGNATURE
-
11.5 HEAD OF THE
DEPARTMENT
MR. MOSES SAMUEL RAJAN
ASSO. PROFESSOR
DEPARTMENT OF PHARMACOLOGY
SRINIVAS COLLEGE OF PHARMACY
MANGALORE- 574 143.
11.6 SIGNATURE OF HEAD OF
DEPARTMENT
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12.1 REMARKS OF THE
PRINCIPAL
12.2 NAME & SIGNATURE OF
PRINCIPAL
DR. A. R. SHABARAYA
PRINCIPAL,
SRINIVAS COLLEGE OF PHARMACY
MANGALORE- 574 143.
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