EVALUATION OF THE EFFICACY OF A NOVEL TEST FORMULATION IN RAT MODEL OF HIGH-FAT DIET AND STREPTOZOTOCIN-INDUCED TYPE-2 DIABETES MELLITUS

  EVALUATION OF THE EFFICACY OF A NOVEL TEST FORMULATION IN RAT MODEL OF HIGH-FAT DIET AND STREPTOZOTOCIN-INDUCED TYPE-2 DIABETES MELLITUS

1.0  TEST SYSTEM DETAILS:

Species                   : Rattus norvegicus (Rats)

Strain                     : Sprague Dawley or Wistar

Age                        : 5 weeks

Sex                        : Male

No. of animals        : 7/Group

Total animals         : 84

 

2.0  TEST ARTICLES DETAILS

 

HF-1: A polyherbal formulation prepared from the plant extracts.

 

3.0  VEHICLE DETAILS

 

The test articles will be formulated by utilizing 0.5% methylcellulose as the vehicle.

 

4.0   ALLOCATION OF GROUPS:

Group No.	Group Description	Disease Induction procedure	Treatment administered	Dose Volume and Route
G1	Normal Control	Normal Diet × 52 days and 0.1 M citrate buffer, pH 4.5 administered by intraperitoneal route on Day 15 (14 days after initiation of normal diet)	0.5% MC, b.i.d.	5 ml/kg, p.o.
G2	Disease Control	High Fat Diet (with 60Kcal% from fat) × 52 days and Streptozotocin (40 mg/kg) administered intraperitoneally on Day 15 (14 days after initiation of high-fat diet offering)	0.5% MC, b.i.d.
G3	Reference Control		Metformin-200 mg/kg, q.d. (morning) + 0.5 % MC (evening)
G4	Treated with a low dose of HF1		HF1: 5-15 mg/kg, b.i.d.
G5	Treated with intermediate dose 1 of HF1		HF1: 15-50 mg/kg, b.i.d.
G6	Treated with intermediate dose 2 of HF1		HF1: 50-150 mg/kg,   b.i.d.
G7	Treated with a high dose of HF1		HF1: 150-500 mg/kg,  b.i.d.

Abbreviations: MC: Methylcellulose, p.o.-per os. q.d.: quaque die; bid: bis in die.

5.0  METHOD:

·    Healthy animals will be selected for the study, randomized based on body weight, and will be assigned to 7 groups consisting of 12 animals each.

·      Animals of Group G1 will be designated as normal-control and administered 0.5% MC, p.o., b.i.d.

·      Disease control animals (assigned to group G2) will receive 0.5% MC, p.o., b.i.d.

·      Animals of group G3 will be treated with reference drug Metformin at the dose of 200 mg/kg, p.o., q.d.

·      Animals of group G4-G7 will be treated with HF1, at different incremental dose levels as outlined in Section 4.0 of Annexure-I, twice daily.

·      The animals allocated to groups G2-G7 will be fed a commercially available high-fat diet for 14 days. Subsequently, they will be administered streptozotocin (40 mg/kg), dissolved in 0.1 M Citrate buffer, by intraperitoneal route.

·       The animals allocated to group G1 will be fed with standard pelleted chow for 14 days. Then they will be injected 0.1 M Citrate buffer by intraperitoneal route.

·    Five days post-streptozotocin administration, the fasting blood glucose of the animals will be measured and animals exhibiting a fasting blood glucose of greater than 250 mg/dL will be considered as diabetic animals. Fasting insulin levels will also be measured in the animals. These values will be categorized as Day 0 values.

·     Diabetic animals allocated to G2-G7 will again be randomized on the basis of their blood glucose level.

·         Test article administration will be initiated subsequently.

·        Blood will be withdrawn from the lateral tail vein of the diabetic animals on Days 14, 21, and 28. Fasting blood glucose will be estimated on Days 14 and 21, whereas fasting blood glucose and insulin levels will be measured on Day 28.

·        On day 29 after metformin and compound administration in the morning blood will be withdrawn at different time points viz. 0 hour (prior to metformin and compound administration) and at 10 minutes, 30 minutes, 1 hour, 2 hours, 4 hours, 8 hours, 12 hours and 24 hours (post- metformin and compound administration) for estimation of the pharmacokinetics of Metformin. 

·    On day 31, an oral glucose test will be conducted, wherein, overnight fasted animals will be administered glucose at the dose of 2 g/kg and blood will be withdrawn prior to and at 15 minutes, 30 minutes, 1 hour, 1.5 hours and 2 hours after glucose loading, for estimation of blood glucose.

·     On Day 33, intraperitoneal insulin tolerance test will be conducted where, overnight fasted animals will be administered commercially available human insulin intraperitoneally at the dose of 1.2 U/kg. Blood will be withdrawn prior to and at 15 minutes, 30 minutes, 1 hour and 1.5-hours post-insulin injection for the estimation of blood glucose.     

·        On Day 34, the animals will be sacrificed under overdose of thiopentone anaesthesia (150 mg/kg) administered intraperitoneally. Subsequently, the liver, adipose tissue, pancreas and skeletal muscle will be excised from the animals. One portion of the liver, pancreas, and adipose tissue will be transferred to 10% neutral buffered formalin for histopathological evaluation. The remaining portions will be snap-frozen in liquid nitrogen and stored at -80°C for biochemical and molecular evaluations.

6.0  PARAMETERS TO BE EVALUATED:

·         Body weight: Twice a week

·         Fasting blood glucose on days 0, 14, 21 and day 28 post-disease induction; Fasting insulin levels on days 0 and 28

·         Serum biochemical parameters. Alanine transaminase, aspartate transaminase, triglycerides, total cholesterol, HDL-c and LDL-c terminally.

·        Glycosylated hemoglobin terminally

·         Hepatic triglycerides and cholesterol content.

·        Enzyme activities of hexokinase, glucose-6-phosphatase and fructose-1,6-bisphosphatase in liver.

·        Quantitative real-time PCR in liver, adipose tissue and skeletal muscle: Expression of PPARγ, GLUT4, GLUT1, PI3K, p-AKT.

·         Histology of liver, adipose tissue and pancreas sections stained with Hematoxylin and Eosin.

 

7.0  REFERENCE(S):

1.  Stalin, A. et al. Synthesis of a 1,2,3-bistriazole derivative of embelin and evaluation of its effect on high-fat diet fed-streptozotocin-induced type 2 diabetes in rats and molecular docking studies. Bioorg. Chem. 103579 (2020) doi:10.1016/j.bioorg.2020.103579.

2.  Rajiv, G., Jothi, G. & James, P. Gallic acid attenuates high-fat diet fed-streptozotocin-induced insulin resistance via partial agonism of PPAR γ in experimental type 2 diabetic rats and enhances glucose uptake through translocation and activation of GLUT4 in PI3K / p-Akt signaling pathway. Eur. J. Pharmacol. 1–16 (2014) doi:10.1016/j.ejphar.2014.10.044.

3.  Huang, M. et al. Biological activities of salvianolic acid B from Salvia miltiorrhiza on type 2 diabetes induced by high-fat diet and streptozotocin. Pharm. Biol. 00, 1–8 (2015).

 

                                             END OF THE DOCUMENT

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