EVALUATION OF TEST FORLMULATION IN HYPERGLYCEMIA AND HYPERLIPIDEMIA ACTIVITY OF STREPTOZOTOCIN-INDUCED DIABETIC RATS

PROTOCOL 

EVALUATION OF TEST FORLMULATION IN HYPERGLYCEMIA AND HYPERLIPIDEMIA ACTIVITY OF STREPTOZOTOCIN-INDUCED DIABETIC RATS’’

1.0   TEST SYSTEM DETAILS:

Species                  : Rattus norvegicus (Rat)

Strain                    : Wistar or Sprague Dawley

Age                       : 8-10 weeks

Body Wight          : 200-250 g

Sex                        : Male/Female

No. of animals      : 10 /Group

Total Animals       : Model (60+10 Extra= 70)

2.0   ALLOCATION OF GROUPS:

Model :

Groups	Treatment	Dose; ROA	No. of Animals
G1	Normal Control	Normal saline or 0.25% Na-CMC	10
G2	Negative Control	0.25% Na-CMC	10
G3	Reference Drug- Glibenclamide (GLB)	10 mg/kg; p.o.	10
G4	Test Formulation-1	X1 mg/kg; p.o.	10
G5	Test Formulation-1	X2 mg/kg; p.o.	10
G6	Test Formulation -2	XX1 mg/kg; p.o.	10
G7	Test Formulation -2	XX2 mg/kg; p.o.	10

 

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

# 10 extra (~20%) animals will be taken extra due to STZ Induction possibilities of animal mortalities

 

3.0  METHOD:

·       Effect of extracts in normoglycemic rats :

·       The rats will be randomized into seven groups (10 animals in each group). They are Normal control (NC), NC with lower and higher doses of Test Formulation-1, NC with lower and higher doses of and NC with a standard drug.

Single-Dose One-Day Study :

Oral Glucose Tolerance Test (OGTT) :

             The calculated doses of extracts and standard drug will be administered orally to normoglycemic rats which are fasted for 18 h. 30 min later, glucose (2 g/kg b.w) will be administered orally. Serum glucose (SG) will be estimated at 0 min (i.e. immediately after glucose load), 30, 60 and 120 min after glucose administration.  

 

Oral Sucrose Tolerance Test (OSTT) :

             The calculated doses of extracts and standard drug will be administered orally to normal rats which are fasted for 18 h. 30 min later, sucrose (1 g/kg b.w) will be administered orally. SG will be estimated at 0 min (i.e. immediately after sucrose load), 30, 60 and 120 min, after sucrose load. 

 

Effect of Test Forlmulations on stz-induced diabetic rats :

             A single intraperitonial injection of STZ (55 mg/kg body weight) will be administered to 10 animals per group. After seven day post STZ administration, SG levels will be estimated and the extent of glucosuria will be estimated using Diagnostic kit. Rats showing glucose level > 300 mg/dl will be considered as diabetic and included in the study.

These diabetic rats will be randomized into seven groups (Ten animals in each group) based on their glucose levels.

They are Diabetic control, rats treated with lower and higher doses of Test Formulation-1, rats treated with lower and higher doses of  Test Formulation-2 and rats treated with a standard drug. They are treated for 30 consecutive days and SG will be estimated at weekly intervals. At the end of the study, following endpoint parameters will be estimated.  

 

4.0  END POINT PARAMETER(S):

·       Clinical observation

·       Feed water consumption

·       Body Weight.

·       Oral glucose tolerance test (OGTT)

·       Insulin tolerance test (ITT)

·       Serum Insulin

·       Serum Triglyceride (TG)

·       Serum Total Cholesterol (TC)

·       HDL-c, VLDL-c, LDL-c

·       TC/HDL-c & LDL-c/HDL-c

Histopathological studies :

The pancreas will be collected from the animals and fixed in 10% formalin, and subjected to histopathological examination.

Endogenous enzymatic and non-enzymatic antioxidant levels :

In-vitro free radical scavenging activity :     

Ø  2,2-diphenyl-1-picrylhydrazyl [DPPH] Scavenging activity

Ø  Hydroxyl radical scavenging activity 

In-vivo free radical scavenging activity :   

Animals will be sacrificed by cervical dislocation, livers excised and the following parameters will be estimated

Ø  Glutathione

Ø  Total thiol

Ø  Lipid peroxidation

Ø  Catalase 

5.0   REFERENCE(S):

5.1 Deb L, Dutta A. Diabetes mellitus its possible pharmacological evaluation techniques and naturopathy. Int J Green Pharmacy 2006;1:7-28.

5.2  Kaneto H, Matsuoka T, Nakatani Y, Kawamori D, Miyatsuka T, Matsuhisa M, et al. Oxidative stress, ER stress and the JNK pathway in type 2 diabetes.  J Mol Med 2005;83:429-39.

5.3 Sakai K, Matsumoto K, Nishikawa T, Suefuji M, Nakamaru K, Hirashima Y, et al. Mitochondrial reactive oxygen species reduce insulin secretion by pancreatic β-cells. Biochem Biophys Res Commun 2003;300:216-22.

5.4 Kasiviswanath R, Ramesh A, Kumar KE. Hypoglycemic and antihyperglycemic effect of Gmelina asiatica Linn. in normal and Alloxan-induced diabetic rats. Biol Pharm Bull 2005;28:729-32.

5.5  Li Y, Wen S, Kota BP, Peng G, Qian Li GQ, Yamahara J, et al.  Punica granatum flower extract, a potent a-glucosidase inhibitor, improves postprandial hyperglycemia in Zucker diabetic fatty rats. J Ethnopharmacol 2005;99:239-44.

5.6  Gandhipuram PS, Palanisamy A, Durairaj SK, Sorimuthu PS. Antidiabetic activity of fruits of Terminalia Chebula on Streptozotocin induced Diabetic Rats.                        J Health Sci 2006;52(3):283-91.

5.7  Singh SN, Vats P, Suri S, Shyam R, Kumria MM, Ranganathan S, et al. Effect of an antidiabetic extract of Catharanthus roseus on enzymatic activities in streptozotocin induced diabetic rats. J Ethnopharmacol 2001;76:269-77.

5.8 Palsamy P, Subramanian S. Resveratrol, a natural phytoalexin, normalizes hyperglycemia in streptozotocin-nicotinamide induced experimental diabetic rats. Biomed Pharmacother 2008;62:598-605.

5.9 Takada J, Fonseca-Alaniz MH, Campos TB, Campos TB, Andreotti S, Campana AB, et al. Metabolic recovery of adipose tissue is associated with improvement in insulin resistance in a model of experimental diabetes. J Endocrinol 2008;198:51-60.

                                   END OF DOCUMENT

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