RAJIV GANDHI UNIVERSITY OF HEALTH SCIENCES,

BANGALORE, KARNATAKA

ANNEXURE-II

PROFORMA FOR REGISTRATION OF SUBJECT FOR DISSERTATION

1. / NAME OF THE CANDIDATE AND ADDRESS / Miss. APARNA B. GORE
DEPT.OF PHARMACOLOGY,
K.L.E.SOCIETY’S COLLEGE OF PHARMACY, VIDYANAGAR,
HUBLI-580031.
2. / NAME OF THE INSTITUTION / K.L.E.SOCIETY’S COLLEGE OF PHARMACY, VIDYANAGAR,
HUBLI-580031.
3. / COURSE OF STUDY AND SUBJECT / MASTER OF PHARMACY IN PHARMACOLOGY
4. / DATE OF ADMISSION TO COURSE / JUNE-2008
5. / TITLE OF THE TOPIC:
“EVALUATION OF PREVENTIVE EFFECTS OF Plectranthus amboinicus (LOUR.) SPRENG ON ALLOXAN INDUCED DIABETES MELLITUS IN ALBINO RATS”
6. / BRIEF RESUME OF INTENDED WORK:
6.1 Need for the study:
Diabetes mellitus (DM) is a commonest endocrine syndrome due to deficiency or absence of hormone insulin, which is main hormone responsible for the control of sugar in the blood. It represents a series of metabolic conditions associated with hyperglycemia and characterized by complications like retinopathy, nephropathy, neuropathy, microangiopathy, diabetic ketoacidosis.1
The total number of people with diabetes in India is approximately 33 million and likely to increase to 57.2 million by the year 2025.2 Currently available synthetic antidiabetic agents like oral hypoglycemic agents produce serious side effects like hypoglycemic coma, weight gain, hypersensitivity reactions.s3 Sulfonylurea and metformin are valuable treatment for hyperglycemia in NIDDM but they cannot be used in pregnancy, lactation and hepatorenal disturbances.3
So still there is a challenge and increased demand for research on natural products with antidiabetic activity. World Health Organization estimated that large portion of world’s population use botanical medicines for their primary health care needs because of their increased efficacy, minimal side effects and low cost.
Increased evidences from clinical and experimental studies suggests that oxidative stress plays a major role in pathogenesis of diabetes mellitus. Abnormally high levels of free radicals and simultaneous decline in antioxidant defense mechanism may cause tissue damage4 and develop insulin resistance.5 Hence, recently researcher suggested a different and innovative approach to a possible “causal” antioxidant therapy, eg. the flavonoids.5
Scientific reports are available that plants such as Securinega virosa6, Cleome droserifolia4, Setaria megaphylla7 contains phytochemical constituents like flavonoids, quarcetin, saponins, tannins, steroids shows antioxidant and antidiabetic activities.
The literature survey reveals the importance of leaf extract of Plectranthus amboinicus has an antioxidant property and phytochemical constituents like flavonoid, terpenoid, saponins, steroids, tannins and volatile oil.8 But no scientific report is available for antidibetic effect of Plectranthus amboinicus. Hence present study has been planned to evaluate the preventive effect of Plectranthus amboinicus leaf extract on alloxan induced diabetes mellitus in albino rats.
6.2 Review of literature:
A study of ancient literature indicates that Diabetes Mellitus was fairly well known and well conceived as an entity in India. It is a syndrome with disordered metabolism and inappropriate hyperglycemia ( high blood glucose level – fasting blood glucose> 7.o mmol/l or plasma glucose>11.1mmol/l 2hrs after glucose load) due to insulin deficiency or insulin resistance.1
Hyperglycemia occurs because of uncontrolled hepatic glucose output and reduced uptake of glucose by skeletal muscle with reduced glycogen synthesis. Once renal threshold for glucose reabsorption is exceeded, glucose spills out into urine (glycosuria) and causes osmotic diuresis (polyuria) which in turn results in dehydration, thirst. Insulin deficiency causes wasting due to increased breakdown and reduced synthesis of proteins.1
DM is of 2 types; in type 1 (Insulin Dependent DM or juvenile) pancreatic blood glucose β-cells are destroyed usually by autoimmune mechanism in the islets of Langerhans. Type 2 (Non Insulin Dependent DM) diabetes is associated with moderate reduction in no. of β-cells; insulin in circulation may be low, normal or even high. Recently it has been reported that even moderately elevated blood glucose level can increase the risk of cardiovascular diseases, morbidity and mortality.3
The long standing hyperglycemia with DM leads to advanced glycosylated end products involved in the generation of ROS and free radicals formed by glucose oxidation, non enzymatic glycation of protein and subsequent oxidative degradation of glucated proteins. These free radicals undergo dismutation to hydrogen peroxide with massive increase in cystolic calcium concentration which causes rapid destruction of pancreatic β-cells. Simultaneous decline in antioxidant defense mechanism may cause tissue destruction, enzyme increased lipid peroxidation and development of insulin resistance.
Plectranthus amboinicus (Lour) Spreng. syn: coleus amboinicus commonly known as Indian borage is a perennial herb, shrubby, hispidly villous. Stem is 30-90 cm in length, fleshy, leaves 2.5-5 cm long, petioled, broadly ovate, crenate and aromatic.10 The leaf extract of Plectranthus amboinicus contains chemical constituents like thymol, carvacrol, β-caryophylline, α-pinene, β-pinene, camphor, cymene, camphene, α-terpinene, p-cymene, limonene, α-phellandrene which are well known essential oils.11
The present plant has some folkloric uses like diuretic, asthma, cough, dyspepsia, convulsion, liver and urinary problems10. It has, also, previously established for antifungal, antileishmanial12, antileptospiral13, rheumatoid arthritis14 and epilepsy.15 But no scientific records mention the use of stated plant as an antidiabetic. Hence the present study has been undertaken to investigate the antidiabetic property of Plectranthus amboinicus in alloxan induced diabetes mellitus.
6.3 Objective of the present study:
The present study has been undertaken to investigate:
·  The hypoglycemic effect of ethanolic extract of leaves of Plectranthus amboinicus in normal and alloxan induced hyperglycemic rats to use Glibenclamide as the standard drug for comparison.
·  The effect of extract on plasma lipids, proteins, body weight and to carry out histopathology of pancreas in normal, control and hyperglycemic rats.
7. / MATERIALS AND METHODS
7.1 SOURCE OF DATA:
Data will be collected using laboratory experimental techniques on animals. In vivo evaluation such as antidiabetic activities will be carried out in Wistar albino rats. The results and data obtained from the present study will be analysed using the one way analysis of variance (ANOVA) test followed by Dunnet’s t-test.
7.2 METHOD OF COLLECTION OF DATA :
HYPOGLYCEMIC ACTIVITY :
Animals: Wistar albino rats weighing 150-200 g will be used.
Alloxan induced Hyperglycemia :
All rats will be fasted 24 h before administration of alloxan monohydrate. Alloxan will be given by intraperitoneal route in freshly prepared saline (0.9%w/v) solution at a dose of 150mg/kg body weight in two equally divided doses. After one hour of alloxan administration, the animals will be given feed and water ad libitum. Subsequently animals receive daily 1000 ml of 5% dextrose solution will be given in feeding bottle for a day to overcome the early hypoglycemic phase.16
72 h later, the blood (1 ml) will be collected from orbital sinus into capillary tubes and immediately used for determination of glucose. Only animals that presented with glucemic levels equal to or above 300mg/dL will be submitted to treatments.
Design of the study :
The 48 rats will be divided into eight groups (n=6). The first four groups will be nondiabetic group and last four groups will be diabetic group to receive treatment.
Non-diabetic group (Comprising Normal Rats) :
Group I (control) will be treated with vehicle (1ml/ 100g of rat) for 15 days.
Group II & III will be treated with different doses of Plectranthus amboinicus leaves extract for 15 days.
Group IV will be treated with standard drug Glibenclamide (GLB) [600µg / kg] for 15 days.
Diabetic Groups (Comprising Hyperglycemic Rats) :
Group V (control) will be treated with vehicle (1ml/ 100g of rat) for 15 days.
Group VI & VII will be treated with same doses of Plectranthus amboinicus leaves extract used in group II & III for 15 days.
Group VIII will be treated with Glibenclamide (GLB) [600µg / kg] for 15 days.
Treatment will be continued for 15 days. On 16th day, animals will be sacrificed after blood collection under diethyl ether anesthesia. Pancreas will be removed and used for histopathological studies.
Parameters monitored :
Fasting blood samples will be collected from the overnight fasted rats of all the groups by retro orbital puncture under diethyl ether inhalation anesthesia. Glucose level will be estimated using a glucose oxidase – peroxidase reactive strips and a glucometer after starting the treatment. The body weight of normal, control and all diabetic animals will be measured weekly.
Histopathological Studies :
At the end of the study, on 16th day, 48 rats will be sacrificed and pancreas will be collected. The tissue will be fixed in 10% formalin immediately after removal from the animal. Thin sections of the tissue will be embedded in paraffin wax and stained with hematoxylin-eosin. Finally will be treated with diphenyl xylene (DPX) and examined under microscope.
7.3 Does the study require any investigation or interventions to be conducted on patients or other humans /animals? If so please described briefly.
The above study requires investigation to be done on the albino rats of Wistar strain for the determination of antihyperglycemic property.
7.4 Has animal ethical committee clearance been obtained from your institution in case?
The study has been given for clearance from the animal ethical committee of the institution.
8. / LIST OF REFERENCES :
1.  Rang HP, Dale MM, Ritter JM, Moore PK. Pharmacology. 5th ed. Churchill Livingstone: Elsevier Science Ltd;2006. p. 385-6.
2.  Modak M, Dixit P, Londhe J, Ghaskadbi S, Devasagayam TPA. Indian herbs and herbal drugs used for treatment of diabetes. J Clin Biochem Nutr 2007 May;40:163-73.
3. Brunton LL, Lazo JS, Parker KL, editors. Goodman and Gilman’s The
pharmacological basis of therapeutics. 11thed. New York:McGraw
Hill;2006. p. 1634-44.
4. EL Naggar EMB, Bartosikova L, Zemlicka M, Svajdlenka E,
Rabiskova M, Strnadova V et al. Antidiabetic effect of Cleome
droserifolia aerial parts: Lipid peroxidation-induced oxidative stress in
diabetic rats. Acta Vet Brno 2005;74:347-52.
5. Lukasinova A, Mojzis R, Benacka R, Keller T, Maguth T, Kurila P et
al. Preventive effect of flavonoids on alloxan-induced diabetes mellitus
in rats. Acta Vet Brno 2008;77:175-82.
6. Tanko y, Okasha MA, Magoji GM,Yerima M, Yaro AH, Saleh MIA
et al. Antidiabetic properties of Securinega virosa (Euphorbiaceae)
leaf extract. African J Biotech 2008;7(1):022-4.
7. Okokon Je, Bassey AL, Nwidu LL. Antidiabetic and hypolipidaemic
effects of ethanolic root extract of Setaria megaphylla. Int J Pharmacol
2007;3(1):91-5.
8. Nirmala Devi K, Periyanayagam KV. Pharmacognostical studies on
the leaves of Plectranthus amboinicus (Lour) Spreng. Int J Green
Pharm 2008;2(3):182-4.
9. Lic.Rosa A.Menéndez CastilloSzudelski T. The mechanism of alloxan and streptozotocin action in β
cells of the rat pancreas. Physiology Research 2001;50:536-46.
10. Kirtikar KR, Basu BD. Indian Medicinal Plants. vol III Dehradun:
International Book Distributors; 1975. P. 1970-71.
11. Abdel-Mogib M, Albar HA, Batterjee SM. Chemistry of genus
Plectranthus. Molecules 2002;7:271-301.
12. Tempone AG, Sartorelli P, Teixeira D, Prado FO, Calixto IARL,
Lorenzi H et al. Brazilian flora extracts as source of novel
antileishmanial and antifungal compounds. Mem I Oswaldo
Cruz 2008 Aug;103.
13. Nirmala Devi K. In vitro Antileptospiral activity of Plectranthus
amboinicus (Lour) Spreng. Pharmaologyonline 2008;2:95-8.
14. Jia Ming Chang, Chun-Ming Cheng, Le-Mei Hung, Yuh-Shan
Chung,Rey-Yuh Wu. Potential use of Plectranthus amboinicus in the
treatment of rheumatoid arthritis. Evidence Based Complement and
Alternat Med 2007 Nov 23;1-6.
15. Salman JDG, Jimenez TEG, Castillo MR, Buznego MTR. Antioxidant
effect of excerpts-fluid and flavonoid Plectranthus amboinicus (Lour)
Spreng(Oregano French). Rev Cuban Plant Med 1996;1(2):27-30.
16. Ananthi J, Prakasam A, Pugalendi KV. Antihyperglycemic activity of
Eclipta alba leaf on alloxan induced diabetic rats. Yale J Biol Med
2003;76:97-102.
9. / SIGNATURE OF CANDIDATE
10. / REMARKS OF GUIDE / The topic which is assigned to
Miss. Aparna B. Gore
is under my supervision and guidance.
11. / 11.1 NAME AND DESIGNATION OF GUIDE / Mr. B.C. KOTI
PROFESSOR & HOD,
Dept. of Pharmacology,
K.L.E S’s College of Pharmacy
Vidyanagar, Hubli- 580031
11.2 SIGNATURE
11.3 NAME AND DESIGNATION OF CO-GUIDE / Mr. THIPPE SWAMY A.H.M.
LECTURER,
Dept. of Pharmacology,
K.L.E S’s College of Pharmacy
Vidyanagar, Hubli- 580031
11.4 SIGNATURE
11.5 HEAD OF THE DEPARTMENT / Mr. B.C. KOTI
PROFESSOR & HOD,
Dept. of Pharmacology,
K.L.E S’s College of Pharmacy
Vidyanagar, Hubli- 580031
11.6 SIGNATURE
12. / 12.1 REMARKS OF THE PRINCIPAL / The above mentioned information is correct and I recommend the same for approval.
12.2 SIGNATURE / Dr. B. M. PATIL
PRINCIPAL,
K.L.E S’s College of Pharmacy
Vidyanagar, Hubli- 580031