EVALUATION OF HYPOGLYCEMIC POTENTIAL OF PLANT EXTRACT(S) USING NORMAL AND GLUCOSE INDUCED HYPERGYCEMIC RATS

                                                                       STUDY PROTOCOL

EVALUATION OF HYPOGLYCEMIC POTENTIAL OF PLANT EXTRACT(S) USING NORMAL AND GLUCOSE-INDUCED HYPERGLYCEMIA RATS

1.0  INTRODUCTION:

The glucose tolerance test is a standard procedure that addresses how quickly exogenous glucose can be cleared from the blood. Specifically, the uptake of glucose from the blood by cells is regulated by insulin. Impairment of glucose tolerance (i.e., longer time to clear a given amount of glucose) indicates problems with the maintenance of glucose homeostasis (insulin resistance, carbohydrate metabolism, diabetes, etc.). 

According to the WHO, in a standard oral glucose tolerance test (OGTT), glucose level should be below 7.8 mmol/L (140 mg/dl) at 2 h. Levels between this and 11.1 mmol/L (200 mg/dl) indicate “impaired glucose tolerance”, and any level above 11.1 mmol/L (200 mg/dl) confirms a diagnosis of diabetes.

2.0  TEST SYSTEM DETAILS:

Species             : Rattus norvegicus (Rat)

Strain                : Wistar

Age                   : 8-10 weeks

Body Wight        : 180-200 g

Sex                   : Male

No. of animals    : 8 /Group

 

3.0  ALLOCATION OF GROUPS:

Groups	Treatment	Dose (mpk); ROA (p.o.)*	No. of Animals
			Initial Screening -Hypoglycemic activity in normal rats	Hypoglycemic activity by OGTT in Normal rats	Hypoglycemic activity by OGTT in Diabetic animals#
G1	Normal Control	Normal saline or 0.25% Na-CMC	8	8	8
G2$	Diabetic Control	Normal saline or 0.25% Na-CMC	-	-	8
G2	Reference Drug- Metformin	250	8	8	8
G3	Plant Extract-1	X	8	8	8
G4	Plant Extract-2	XX	8	8	8
G5	Plant Extract-3	XXX.	8	8	8

*The doses and ROA (Routes of administration) will be decided based on the type of reference drug

# 10-20% animals will be taken extra due to variability in the diabetes development and possibilities of animal mortalities 

Group G2$ will be incorporated only in the study of hypoglycemic activity by OGTT in diabetic animals

Notes-

o   The experiment can also be executed in mice (C57BL/6, Swiss albino) with alloxan single dose at 70 mpk; iv. Glucose dose will be 2 g/k; p.o..

o   Additional BGL time points 4, 6, 8 and 24 hr can be incorporated as per the study protocol

4.0 METHODOLOGY:

4.1 Initial Screening of Hypoglycemic Activity of Plant Extract(s) in Normal Rats

·       All the animals will be overnight fasted (14-16 hours) before the experiment.

·       Healthy rats (180-200 g) will be randomized based on the body weight and/or basal blood glucose level (0 min- before drug administration) into five groups of 8 animals each.

·       Group G1 and G2 animals will be administered with normal saline or 0.25% Na- CMC and reference drug Metformin at the dose of 150 mg/kg.

·       Similarly, animals of group G3, G4 and G5 will be administered with plant extract(s) at different doses.

·       Blood sample will be collected by snip-cut at the tip of the tail under mild anesthesia and glucose will be measured by glucometer at 0 min (before drug administration), 30, 60, and 120 min.

 

4.2 Screening of Hypoglycemic Activity of the Plant Extract by OGTT in Normal Rats

·       All the animals will be overnight fasted (14-16 hours) before the experiment.

·       Healthy rats (180-200 g) will be randomized based on the body weight and/or basal blood glucose level at 0 min (before drug administration) into five groups of 8 animals each.

·      All the group of animals will be treated with vehicle, reference drug, or plant extracts as shown in the table above (section 4.1).

·      After 30 min, all the animals will be orally administered with D- glucose (3g/kg) at the dose volume of 10 ml/kg.

·       Blood sample will be collected by snip-cut at the tip of the tail under mild anesthesia and glucose level will be measured by glucometer at 0 (before drug administration), 30, 60, and 120 min of glucose administration.

 

4.3 Screening of Hypoglycemic Activity of Plant(s) Extract by OGTT in Diabetic rats

4.3.1     Induction of Diabetes

Ø  Diabetes will be induced in Wistar rats by a single intraperitoneal injection of alloxan monohydrate in sterile normal saline to overnight fasted animals at a dose of 120 mg/kg b.w.

Ø  The fasting blood glucose level will be determined after 2-3 days of alloxan injection.

Ø  The rats having blood glucose levels above 200 mg/dl will be used for the study.

Ø  The diabetic animals will be allowed free access to water, pellet diet, and will be maintained at room temperature in polycarbonate cages. 

·       Overnight-fasted diabetic rats will be randomized into different groups based on the BGL at 0 min (before drug administration) as shown in the table above (#).

·       Normal control animals (group G1) will be treated with normal saline or 0.25% Na-CMC.

·       After 30 min, all the animals will be orally administered with D- glucose (3g/kg) at the dose volume of 10 ml/kg.

·       Blood sample will be collected by snip-cut at the tip of the tail under mild anesthesia and glucose level will be measured by glucometer at 0 (before drug administration), 30, 60, and 120 min of glucose administration.

 

5.0 END POINT PARAMETER(S):

·       Body Weight

·       Blood glucose level 

6.0  REFERENCE(S):

6.1 Zhang, P. (2011). Glucose Tolerance Test in Mice. Bio-protocol Bio101: e159. DOI: 10.21769/BioProtoc.159;

6.2 Sze Han Ng, Mohd Shazwan Mohd Zain, Fatariah Zakaria, Wan Rosli Wan Ishak, and Wan Amir Nizam Wan Ahmad. Hypoglycemic and Antidiabetic Effect of Pleurotus sajor-caju Aqueous Extract in Normal and Streptozotocin-Induced Diabetic Rats. Biomed Res Int. 2015; 2015: 214918.

6.3 Sornalakshmi V, Tresina Soris P, Paulpriya K, Packia Lincy M and Mohan V.R. Oral Glucose Tolerance Test (OGTT) in Normal Control and Glucose Induced Hyperglycemic Rats with Hedyotis leschenaultiana DC. International Journal of Toxicological and Pharmacological Research 2016; 8(1); 59-62.

6.4 Fadoua El Amrani, Abdeljalil Rhallab, Tajelmolk Alaoui, Khalid El Badaoui and Said Chakir. Hypoglycaemic effect of Thymelaea hirsuta in normal and streptozotocin-induced diabetic rats. Journal of Medicinal Plants Research Vol. 3(9), pp. 625-629, September, 2009.

6.5 Maithili V, Dhanabal S P, Mahendran S, Vadivelan R. Antidiabetic activity of ethanolic extract of tubers of Dioscorea alata in alloxan-induced diabetic rats. Indian J Pharmacol 2011;43:455-9.

6.6 Source Research Needs YouTube Channel: Diabetes-Induced Erectile Dysfunction Video

                        END OF DOCUMENT

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