Evaluation of Anti-Diabetic Activity of Ethanolic Extract of Citrus Maxima Stem Bark

EVALUATION OF ANTI-DIABETIC ACTIVITY OF ETHANOLIC EXTRACT OF CITRUS MAXIMA STEM BARK

MASTER OF PHARMACY DISSERTATION PROTOCOL

SUBMITTED TO THE

RAJIV GANDHI UNIVERSITY OF HEALTH SCIENCES KARNATAKA, BANGALORE

BY

ABDUL MUNEER M.T

Under The Guidance of

MR. MOSES SAMUEL RAJAN M PHARM

DEPARTMENT OF PHARMACOLOGY,

SRINIVAS COLLEGE OF PHARMACY

MANGALORE – 574 143

2012– 2014
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 / ABDUL MUNEER M.T,
VANIYAKOL HOUSE,
THAVANOOR (P.O.),
KUZHIMANNA VIA,
MALAPPURAM (DIST)
PIN-673641
2.0 / NAME OF THE INSTITUTION / SRINIVAS COLLEGE OF PHARMACY
VALACHIL, MANGALORE- 574 143
3.0 / COURSE OF STUDY AND SUBJECT / MASTER OF PHARMACY IN PHARMACOLOGY
4.0 / DATE OF ADMISSION TO COURSE /
26 -06-2012
5.0 / TITLE OF THE TOPIC / “EVALUATION OF ANTI-DIABETIC ACTIVITY OF ETHANOLIC EXTRACT OF CITRUS MAXIMA STEM BARK”.
6.0 / BRIEF RESUME OF THE INTENDED WORK :
6.1 NEED FOR THE STUDY
Introduction
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
component in diabetes treatment. Dietary management includes the use of traditional medicines mainly derived from plants.
Despite considerable progress in the treatment of diabetes by oral hypoglycemic agents, search for newer drugs continues because the existing synthetic drugs have several limitations. Pharmacotherapy of diabetes without any side effects is still a challenge. So there is the need for complementary and alternative medicine with anti-diabetic activity and less side effects. Alternative therapies with anti-diabetic activity have been researched extensively. Ideal therapies should have a similar degree of efficacy without the troublesome side effects associated with conventional treatments Alternative treatments for diabetes have become increasingly popular in the last several years, including medicinal herbs, nutritional supplementation, acupuncture and hot tub therapy5.
Glibenclamide is an anti-diabetic drug coming under the class 2nd generation. 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 jaundice7.
Citrus maxima have been recommended in traditional herbal medicine as the source of diabetic medication or remedy for diabetes. The citrus maxima are well recognized for their various ethno medicinal uses. These properties are attributed to their various chemical constituents. Citrus fruits contains flavonoids and limonoids which are proven to possess anti-inflammatory and antitumor effects. The peels are rich in pectin which is known to possess blood sugar lowering and cholesterol lowering properties. Sriparna et al., . demonstrated the antihyperglycemic effect and anti-oxidant property of C. maxima leaf in streptozotocin induced diabetic rats. Fruit juice of Shaddock (C.maxima) possess hypoglycemic and hypocholesterolemic activities5.
In view of the various effects of C. maxima , this study was designed to assess the effect of Citrus maxima stem bark on alloxan induced streptozotocin-induced type –II diabetic rats and glucose tolerance test.
6.2 REVIEW OF LITERATURE:
Description :
The pummelo tree may be 16 to 50 ft (5-15 m) tall, with a somewhat crooked trunk 4 to 12 in (10-30 cm) thick, and low, irregular branches. Some forms are distinctly dwarfed. The young branchlets are angular and often densely hairy, and there are usually spines on the branchlets, old limbs and trunk. Technically compound but appearing simple, having one leaflet, the leaves are alternate, ovate, ovate-oblong, or elliptic, 2 to 8 in (5-20 cm) long, 3/4 to 4 3/4 in (2-12 cm) wide, leathery, dull-green, glossy above, dull and minutely hairy beneath; the petiole broadly winged to occasionally nearly wingless. The flowers are fragrant, borne singly or in clusters of 2 to 10 in the leaf axils, or sometimes 10 to 15 in terminal racemes 4 to 12 in (10-30 cm) long; rachis and calyx hairy; the 4 to 5 petals, yellowish-white, 3/5 to 1 1/3 in (1.5-3.5 cm) long, somewhat hairy on the outside and dotted with yellow-green glands; stamens white, prominent, in bundles of 4 to 5, anthers orange. The fruit ranges from nearly round to oblate or pear-shaped; 4 to 12 in (10-30 cm) wide; the peel, clinging or more or less easily removed, may be greenish-yellow or pale-yellow, minutely hairy, dotted with tiny green glands; 1/2 to 3/4 in (1.25-2 cm) thick, the albedo soft, white or pink; pulp varies from greenish-yellow or pale-yellow to pink or red; is divided into 11 to 18 segments, very juicy to fairly dry; the segments are easily skinned and the sacs may adhere to each other or be loosely joined; the flavor varies from mildly sweet and bland to subacid or rather acid, sometimes with a faint touch of bitterness. Generally, there are only a few, large, yellowish-white seeds, white inside; though some fruits may be quite seedy. A pummelo cross-pollinated by another pummelo is apt to have numerous seeds; if cross-pollinated by sweet orange or mandarin orange, the progeny will not be seedy8.
Classification:
Kingdom : Plantae
Order : Sapindales
Family : Rutaceae
Genus : Citrus
Scientific names : Citrus maxima Merr, Citrus glandis, Citrus decumana Linn, Aurantium maximum Burm, Citrus aurantium Linn, Citrus pamplemos Risso.
Synonyms : (English)Chinese grape fruit, Pomelo, Jabong, Pummelo, (Hindi) Sadaphal and (Sanskrit) Madhukarkatika, Shaddock8.
Geographic Distribution:
The Pomelo is native to southeastern Asia and all of Malaysia and grows wild on river banks in the Fiji and Friendly Islands.It may have been introduced into China around
100 B.C. It is much cultivated in southern China (Kwang-tung, Kwangsi and Fukien Provinces) and especially in southern Thailand on the banks to the Tha Chine River. Also in Taiwan and southernmost Japan, southern India, Malaya, Indonesia, New Guinea and Tahiti.
Chemical constituents : Alkaloid, Amino acids, Benzenoids, Carbohydrates, Carotenoids, Coumarins, Flavanoids, Monoterpens, Sesquiterpenes, Saponin Glycosides, Triterpenes, and Steroids.
Parts used : Fruit peel, Fruit juice, Leaves and Stem Bark
Reported activities: Antioxidant, anti-inflammatory, antidiabetic, antihyperlipeadimic and antitumour 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 Stem bark of citrus maxima Merr.
2. To study the anti-diabetic activity of citrus maxima Merr. by using following animal models
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 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)
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.
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 Aventis pharma Ltd, Goa, 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 PREPERATION OF ETHANOLIC EXTRACT OF CITRUS MAXIMA STEM BARK :
The citrus maxima stem bark collected,pulverized and dried at 450 C. The oven-dried (450C) stem bark powder of C. maxima was refluxed three times in ethanol. And the methanol extract was obtained9.
7.5 EXPERIMENTAL METHODS :
I. ALLOXAN INDUCED DIABETES IN RATS 10
Diabetes was induced in rats by intraperitoneal administration of Alloxan monohydrate (150 mg/kg b. w,) in normal saline. After 72 hrs, rats with hyperglycemia (more than150 mg/dl) were selected and used for anti-diabetic evaluation. Blood Glucose was measured by Glucometer.
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 + Citrus maxima (200mg/Kg)11
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 12
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.
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 + Citrus maxima (200mg/Kg)
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.
1.  III. ORAL GLUCOSE TOLERANCE TEST (OGTT) IN RATS 13
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+ Citrus maxima(200mg/Kg)
EVALUATION:
The blood glucose concentration will be determined at 30, 60, 120 and 180 min after dosing.
7.6 STATISTICAL ANALYSIS:
The data will be expressed as Mean value + SEM and will be analyzed by the one-way ANOVA.
7.7 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.
7.8 HAS ETHICAL CLEARANCE BEEN OBTAINED FROM YOUR INSTITUTION?
Yes. Ethical clearance has been obtained. (Copy enclosed)
Ref:SCP/CPCSEA/P16/F150/2012
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)  Oyedepot A, Babarinde SO. Effects of Shaddock (Citrus maxima) Fruit Juice on Glucose. Chem Sci Trans 2012;2(1):19-24.
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)  Julia F. Morton, Miami, FL. Fruits of warm climates:Pummelo. [Online]. 1987 [cited 1987];[5 pages]. Available from: URL: http://www.hort.purdue.edu/newcrop/morton/pummelo.html
9)  Wen-Yuh Tenga,Yu-Ling Huangb, Chien-Chang Shenb,Ray-Ling Huangb, Ren-Shih Chunga and Chien-Chih Chenb. Cytotoxic Acridone Alkaloids from the Stem Bark of Citrus maxima. JCCS 2005;52:1253-1255.
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)  Parixit Bhandurge, Rajarajeshwari N, Alagawadi KR, Saurabh Agrawal. Antidiabetic and hyperlipaemic effects of citrus maxima linn fruits on alloxan-induced diabetic rats. IJDDR 2010; 2(1):273-278.
12)  Bajaj S, Srinivasan BP. Investigations into the anti-diabetic activity of Azadirachta indica. Indian J Pharmacol 1999;31: 138-41
13)  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.
9. / SIGNATURE OF THE
CANDIDATE
10. / REMARKS OF THE GUIDE / Recommended and Forwarded
11. / NAME AND DESIGNATION
11.1 GUIDE / MR. MOSES SAMUEL RAJAN
ASSO. PROFESSOR
DEPARTMENT OF PHARMACOLOGY
SRINIVAS COLLEGE OF PHARMACY
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
12 / 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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