“ANTI-DIABETIC ACTIVITY OF Caralluma attenuata AGAINST STREPTOZOTOCIN INDUCED OXIDATIVE STRESS IN EXPERIMENTAL DIABETIC RATS”

M. Pharmacy Dissertation Protocol Submitted to the

Rajiv Gandhi University of Health Sciences, Karnataka

Bangalore– 560 041

In partial fulfillment

Of the requirement for the Degree of

MASTER OF PHARMACY

IN

PHARMACOLOGY

BY

RUPAPARA MAYUR S.

I M.PHARM

DEPARTMENT OF PHARMACOLOGY

DAYANAND SAGAR COLLEGE OF PHARMACY

BANGALORE-560078

(2009-2010)

RAJIV GANDHI UNIVERSITY OF HEALTH SCIENCES

KARNATAKA, BANGALORE.

ANNEXURE-II

PROFORMA FOR REGISTRATION OF SUBJECTS FOR DISSERTATION

1. / Name of the candidate and address / Mr. RUPAPARA MAYUR SHAMBHUBHAI
S/o SHAMBHUBHAI A. RUPAPARA
“MAYUR” DOBARIYA WADI,
AMARNAGAR ROAD,
JETPUR-360370, DIST. – RAJKOT, GUJARAT.
2. /

Name of the institution

/ DAYANANDA SAGAR COLLEGE OF PHARMACY,
SHAVIGE MALLESHWARA HILLS,
KUMARASWAMY LAYOUT,
BANGALORE – 560078
3. /

Course of study and subject

/

MASTER OF PHARMACY IN

PHARMACOLOGY

4. /

Date of the admission to course

/ 19th June 2009
5. /

Title of the topic

“Anti-Diabetic Activity Of Caralluma attenuate against Streptozotocin Induced Oxidative Stress In Experimental Diabetic Rats”
6.0 /

Brief resume of the intended work

6.1
6.2
6.3
7.0
7.1
7.2
7.3
7.4 /
Need of the study
Diabetes mellitus is a chronic disease characterized by high blood glucose levels due to absolute or relative deficiency of circulating insulin levels. Though different types of oral hypoglycemic agents are available along with insulin for the treatment of diabetes mellitus, there is increasing demand by patients to use the natural products with antidiabetic activity. Insulin cannot be used orally and continuous use of the synthetic drugs causes side effects and toxicity1-3. Herbal drugs are prescribed widely even when their biologically active compounds are unknown, because of their effectiveness, less side effects and relatively low cost4.
Diabetes mellitus is considered one of the five leading causes of death in the world. The World Health Organization (WHO) has predicted that the worldwide number of patients with diabetes will be double by the year 2025, from the current number of approximately 150 million to 300 million5. It has been assumed that the etiology of the complication of diabetes involves oxidative stress perhaps as a result of hyperglycemia6. The elevated levels of blood glucose in diabetes produce oxygen free radicals which cause membrane damage due to peroxidation of membrane lipids and protein glycation7. As the diabetogenic action can be prevented by the SOD, CAT, and other hydroxyl radical scavengers such as ethanol, dimethyl urea, there is evidence to suggest that the incidence of diabetes involves superoxide anion and hydroxyl radicals can be counteracted by antioxidant enzymes such as SOD, CAT, and glutathione peroxides. There is clear cut evidence to show the role of free radicals in diabetes and studies indicates that tissues injury in diabetes may be due to free radicals8. Thus Antioxidant and free radical scavengers may help in the regeneration of beta cells and protect pancreatic islets.
Caralluma attenuata Wight. [Syn.: C. fimbriata Hook.] (Asclepiadaceae) is a thick, succulent perennial herb growing wild in and around Warangal and several other districts of Andhra Pradesh, India. Locally it is known as ‘Kundaetikommu’, and is eaten raw as a cure for diabetes (personal information from users). The juice of the plant along with black pepper is recommended in the treatment of migraine9. It reported the presence of luteolin-49-O-neohesperidoside10, with a significant anti-inflammatory and Antinociceptive activity11.
Review of literature
M. Ramesh et al10 reported presence of Flavone glycoside from three Caralluma species. In this study, Concentrated methanolic extracts prepared from the fresh whole plants of Caralluma attenuata, Caralluma umbellata and Caralluma lasiantha (5 kg each) were successively fractionated with toluene, ethyl acetate, butanone and n-butanol. A comparative TLC examination of the butanone fractions of the three plant materials showed the presence of a flavonoid glycoside (colour reactions) with identical Rf value. This flavonoid glycoside was isolated from all the three extracts by flash chromatography on 40-60 µ silica gel G [Isocratic solvent system - ethyl acetate : methanol : water (150 : 11 : 8)] and was identified as luteolin-4’-O-neohesperidoside or luteolin-4’-O-[α-(L-rhamnopyranosyl-(1→2)-β-D-glucopyranoside)] by UV, IR, HRFABMS, 1H NMR, 13C NMR, APT, HETCOR. The yield of luteolin-4@-O-neohesperidoside was the highest in Caralluma attenuata (0.5%), followed by Caralluma lasiantha (0.01%) and Caralluma umbellata (0.001%).
M. Ramesh et al11 studied anti-inflammatory activity of a flavonoid isolated from Caralluma attenuata and also reported antinociceptive activity. Extraction of the fresh whole plants (5 kg) was performed with ethyl alcohol by maceration process for 3 days. It was found that Luteolin-4’-O-neohesperidoside, i.e. luteolin-4’-O-[α-(L-rhamnopyranosyl-(1→2)-β-D-glucopyranoside)] has significant anti-inflammatory action. It is more potent than ibuprofen. Its antinociceptive activity is less pronounced when compared with its anti-inflammatory activity.
S. Venkatesh et al12 studied antihyperglycemic activity of Caralluma attenuata in 2003. It has been reported that Caralluma attenuata possess significant Antihyperglycemic activity. All three extracts, even if with a different degree, have shown significant reduction in blood glucose levels in both glucose loaded and alloxan-induced diabetic rats. The butanol extract has shown maximum effect in all the three tests compared to the other two extracts.
Jayakar et al13 reported effect of Caralluma attenuata in normal and alloxon induced rats.
Dr K. Madhava Chetty et al14, published “Flowering plants of chittoor district Andhra Pradesh”, India.
Alok Sharma et al15 reported action of Portulaca oleracea against Streptozotocin Induced Oxidative Stress in Experimental Diabetic Rats.
D. Taleb-Senouci et al16studied and reported Antioxidant effect of Ajuga iva aqueous extract in streptozotocin-induced diabetic rats.
Objective of Study:
The objective of the present investigation is to evaluate the antioxidant and antidiabetic activity of Caralluma attenuata.
Plan of work
Ø  Collection of plant
Ø  Plant Extraction
Ø  Preliminary phytochemical studies
Ø  Acute toxicity studies
Ø  Induction of diabetes by streptozotocin followed by plant extracts treatment to the sprague-dawley rats and albino mice.
Ø  Evaluate the extract on diabetic animals by using following parameters i.e.
ü  Fasting blood glucose,
ü  Oral glucose tolerance test,
ü  Total cholesterol, triglycerides, HDL cholesterol,
ü  Enzymatic antioxidant studies i.e. lipid peroxidation, superoxide dismutase, catalase etc.
MATERIAL AND METHODS
Source of Data:
Ø  Whole work is planned to generate data from laboratory studies i.e. experiments are performed as described in references. Experimental studies in journals and in text books available with college and various institutions.
NBRI library, Lucknow.
DSCP library, Bangalore
RGUHS digital library(Helinet), Bangalore
Web sites: www.scienencedirect.com
www.pubmed.com
www.google.com
www.ijp-online.com
Method of collection of data:
Plant materials - Plant material of Caralluma attenuata will be collected from Andhra Pradesh, India. The plant material will be identified taxonomically and authenticated by National Botanical Research Institute, Lucknow. A voucher specimen of the collected sample will be deposited in the departmental herbarium for future reference.
The data and related literatures will be collected from various scientific national and international journals i.e. Indian Journal Pharmacology, Diabetic Care, Diabetic Research and Clinical Practice, Journal of Ethnopharmacology, Planta Medica, etc.
Drug material: The drug will be procured from Department of Pharmacognosy and Ethnopharmacology, National Botanical Research Institute, Lucknow.
Evaluation of Antidiabetic activity will be done using following methods:
METHODS
Preparation of extract –
50% ethanolic17 extract of Caralluma attenuate will be extracted using a mechanical stirrer till exhaustion and it will be processed for the detailed anti-diabetic activity.
Preliminary phytochemical screening – The extract will be screened for the presence of various secondary metabolites like alkaloids, flavones glycosides, tannins and triterpenes etc.
Animals - Sprague-Dawley rats (150-175g) will be obtained from the animal colony of National Laboratory Animal Centre, Lucknow. They will be randomly distributed into various groups and housed in cages (6per cage) and will be maintained under standard conditions i.e. 26 ± 2 °C and relative humidity 44 - 56% and 10 h light: 14 h dark cycles each day for one week before and during the experiments. All animals will be fed standard rodent pellet diet (Amrut, India) and drinking water ad libitum.
Induction of experimental diabetes - A freshly prepared solution of STZ (50 mg/ kg, body weight) in 0.1 M citrate buffer pH 4.5 will be injected intraperitonealy in a volume of 1 ml/ kg. STZ injected animals will be exhibited massive glycosuria and hyperglycaemia within two days18. Diabetes will be confirmed in STZ rats by measuring the fasting blood glucose concentration after 96 h after the injection of STZ. The rats with blood glucose level above 200 mg/ dl will be considered to be diabetic and will be used in the experiment.
Experiment design and treatment - After induction of diabetes, the rats will be divided into 5 groups of 6 animals each as follows: Group I animals will be served as control rats will be received 2% gum acacia as vehicle solution. Group II constituted STZ diabetic rats and will be received 2% gum acacia. Group III and IV animals are STZ diabetic rats will be received Caralluma attenuata extracts (100 and 250 mg/kg b.w) and Group V will be received tolbutamide (10mg/kg b.w). The vehicle and the drugs will be administered orally using an intragastric tube daily for three weeks. After three weeks of treatment the rats will be fasted overnight and sacrificed by cervical decapitation. Blood samples will be analyzed for serum glucose content by commercial glucose kit (Qualigens Diagnostics, Mumbai, India). The liver and kidney will be exposed and perfused with cold phosphate buffer saline of pH 7.4. Blood free liver will be taken out and homogenized in a glass Teflon homogenizer (10%w/v). Incubation will be done at 370C under controlled conditions. Liver and kidney homogenates will be subjected for antioxidant enzymes estimation15.
Antioxidant activities - Lipid peroxidation in liver and kidney will be estimated colorimetrically by thiobarbituric acid reactive substances (TBRAS), Okhawa et al19 (1979) and hydroperoxides by the method of Jamall and smith20 (1985). Glutathione (GSH) level will be estimated using Butler et al21 (1967), Glutathione reductase (GR) activity will be estimated using standard method of Horn22 (1963). CuZn-superoxide dismutase (CuZn-SOD) will be measured by using the methods of Kakkar et al23 (1984). Catalse (CAT) activity will be measured by using the rate of decomposition of H2O2 by the method of Aebi24 (1974). All these estimations will be made in both liver and kidney.
Statistical analysis - All the data will be presented as mean ± S.E.M. and oneway analysis of variance (ANOVA) followed by Newman-Keuls Multiple Comparison Test will be applied for determining the statistical significance between different groups.
DOES THE STUDY REQUIRE ANY INVESTIGATION TO BE CONDUCTED ON PATIENTS OR ANIMALS? Yes,
In diabetic research, animal usage is most to reveal the findings and conclusion. In present study Sprague-Dawley rats are required.
HAS ETHICAL CLEARANCE BEEN OBTAINED FROM YOUR INSTITUTION IN 7.3?
Yes, Approved by CPCSEA.
8.0 / REFERENCES:
1.  Holmann RR, Turner RC. Text book of diabetes. Oxford: Blackwell, 1991.
2.  Chattopadhyay RR. Indian J Exp Bio 1993; 31:893.
3.  Kameswara Rao B, Giri R, Kesavulu MM, Apparao C. Herbal medicine: In the management of diabetes mellitus, Manphar vaidhya patrika. 1997;1(4-5):33-5.
4.  Valiathan MS. Curr Sci 1998; 75(10-11):1122.
5.  World Health Organization, Diabetes action Now: An Initiative of the World Health Organization and International Diabetes Federation, WHO Publication, Geneva; 2004 May 5.
6.  Hunt JB, Smith CCT, Wolf SP. Auto-oxidative glycosylation and possible involvement of peroxides and free radicals in LDL modification by glucose. Diabetes 1990; 39:420-1424.
7.  Baynes, JW. Perspectives in diabetes, role of oxidative stress on development of complications in diabetes. Diabetes 1991; 40: 405-12.
8.  Grankvist K, Markuld S, Teljedal IB. Superoxide dismutase is prophylactic against alloxan diabetes. Nature 1981; 294:158-61.
9.  Srinivasacharyulu, Y. Yogarathnakaram, Swatantra Press, Nellore, Andhra Pradesh, India 1931; 2:678.
10.  Ramesh M. , Y. N. Rao, M. Rama Kumar, G. Krishna Mohan, B. Ravi Kumar, A.V.N. Appa Rao et al. Flavone glycoside from three Caralluma species. Biochem. Syst. Ecol. 1999; 27:85-6.
11.  M. Ramesh , Y. N. Rao, A.V.N. Appa Rao, M.C. Prabhakar, C. Seshagiri Rao, N. Muralidhar, B. M. Reddy. Antinociceptive and anti-inflammatory activity of a flavonoid isolated from Caralluma attenuata. J. Ethnopharmaco 1998; 62:63–6.
12.  S. Venkatesh, G. D. Reddy, B. M. Reddy, M. Ramesh, A.V.N.Appa Rao. Antihyperglycemic activity of Caralluma attenuata. Fitoterapia 2003; 74:274-9.
13.  Jayakar et al. Effect of Caralluma attenuata in normal and alloxon induced rats. J. Herb Pharmacother 2004; 4(1):35-40.
14.  Dr. K. Madhava Chetty et al. Flowering plants of chittoor district Andhra Pradesh”, India. 2008.
15.  Sharma A., M. Vijayakumar, Ch. V. Rao, M.K.Unnikrishnan, G.D. Reddy. Action of Portulaca oleracea against Streptozotocin-Induced Oxidative Stress in Experimental Diabetic Rats. J Complement Integr Med 2009; 6(1): Article 1.
16.  D. Taleb-Senouci, H.Ghomari, D.Krouf, S.Bouderbala, J.Prost, M.A. Lacaille Dubois, M.Bouchenak. Antioxidant effectof Ajuga iva aqueous extract in streptozotocin-induced diabetic rats. Phytomedicine 2009; 16(6-7):623–31.
17.  P. Shokeen, P. Anand, Y. K. Murali, V. Tandon. Antidiabetic activity of 50% ethanolic extract of Ricinus communis and its purified fractions. Food Chem. Toxicol. 2008; 46: 3458–66.
18.  Vijayakumar M, Govindarajan R, Rao GMM, Rao ChV, Shirwaikar A, Mehrotra S, Pushpangadan P. Action of Hygrophila auriculata against streptozotocin induced oxidative stress. J. Ethnopharmacol. 2006; 104:356-61.
19.  Okhawa H, Oshishi N, Yagi K. Assay for lipid peroxised in animal’s tissues by thiobarbituric acid reaction. Anal. Biochem.1979; 95: 351-5.
20.  Jamall IS, Smith JC. Effect of cadmium on glutathione peroxides, super oxide dismutase and lipid peroxidation in the rat heart: a possible mechanism of cadmium cardiotoxicity. Toxicol. Appli. Pharmacol. 1985; 80: 33-42.
21.  Beutler E, Duron O, Kelly BM. Improved method for determination of blood glutathione. J.Lab. Clin. Med.1967; 61:882-8.
22.  Horn HD. Glutathione reductase, in Bergmyer, H.U. (ed.). Methods in enzymatic analysis, Academic press. 1963; 875-9.
23.  Kakkar P. Das B, Viswanathan PN. Modified spectrophotometric assay of SOD. Indian J. Biochem Biophys.1984; 2:130-2.
24.  Aebi H. Catalase, in Bergmeyer HU (ed.). Methods in enzymatic analysis, Academic Press, New York. 1983; 276-86.