PROTECTIVE EFFECT OF TEST FORMULATION AGAINST DIABETIC NEPHROPATHY INDUCED BY STREPTOZOTOCIN IN EXPERIMENTAL ANIMALS

 

STUDY PROTOCOL

 

PROTECTIVE EFFECT OF TEST FORMULATION AGAINST DIABETIC NEPHROPATHY INDUCED BY STREPTOZOTOCIN IN EXPERIMENTAL ANIMALS

1.0   TEST SYSTEM DETAILS:

Species                  : Rattus Norvegicus (Rat)

Strain                    : Wistar

Age                       : 8-10 weeks

Body Wight          : 200-220 g

Sex                        : Male

No. of animals      : 10 /Group

Total Animals       : Model (50+10 Extra= 60)

 

2.0   ALLOCATION OF GROUPS:

Groups	Treatment	Dose; ROA	No. of Animals
G1	Normal Control	Normal saline or 0.25% Na-CMC	10
G2	Negative Control (Streptozotocin 60 mg /kg) q.d	0.25% Na-CMC	10
G3	Reference Drug- Metformin  (Streptozotocin 60 mg /kg) q.d	500 mg/kg; p.o.	10
G4	Test Formulation-1 (Streptozotocin 60 mg /kg) q.d	X1 mg/kg; p.o.	10
G5	Test Formulation-1 (Streptozotocin 60 mg /kg) q.d	X2 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 Streptozotocin Induction possibilities of animal mortalities

 

3.0  METHOD:

3.1  The bodyweight of individual animals will be taken daily for each group. If the death of any animal occurs in between the study time, its weight will be recorded. Food intake will be measured on daily basis.

3.2  Diabetes will be induced in overnight fasted rats by a single intraperitoneal injection of freshly prepared  STZ solution, STZ was dissolved in 0.1 M citrate buffer ((0.1M, pH 7.4) for the stock solution of at 60 mg/kg body weight. A subset of the STZ-induced diabetic rats intragastrically administered with test formulation (TF) after the onset of nephropathy, whereas other diabetic rats will receive sod. CMC as the same volume of TF. After diabetes induction if the blood glucose level is down immediatlely after STZ induction, 5 % DNS or insulin intramuscular injection will be given to maintain the blood glucose level. The development of diabetic nephropathy will be confirmed by blood glucose, blood urea nitrogen, uric acid, serum creatinine, and serum urea on the weekly basis.

3.3  All treatments doses will be given very next day of confirmation of the diabetes till the end of the study. At the end of the study, all animals will be sacrificed by overdose of thiopendal sodium after the final day treatment. While kidneys will be isolated and washed with ice-cold saline, then homogenate will be prepared and stored at –80°C till further analysis for histopathological, and biochemical assessments.

 

4.0  END POINT PARAMETER(S):

·       Clinical observation

·       Feed water consumption

·       Body Weight.

·       Blood glucose level (Weekly)

·       Blood urea nitrogen (Weekly)

·       Blood uric acid (Weekly)

·       Serum creatinine (Weekly)

·       Serum urea (Weekly)

·       Biochemical estimation(TBARS , GSH, SOD, CATALASE)

·       Histopathological studies : The one kidney will be collected from the animals and fixed in 10% formalin, and subjected to histopathological examination.

 

5.0   REFERENCE(S):

5.1  Krishna RN, Anitha R, Ezhilarasan D. Aqueous extract of Tamarindus indica fruit pulp exhibits antihyperglycaemic activity. Avicenna Journal of Phytomedicine. 2020 Sep;10(5):440.

5.2  Akbarzadeh A, Norouzian D, Mehrabi MR, Jamshidi Sh, Farhangi A, Verdi AA, Mofidian SM, Rad BL.(2007) Induction of diabetes by Streptozotocin in rats. Indian J Clin Biochem.;22(2):60-4

5.3  Parvin A , Md. Alam M, Md. Haque A ,Bhowmik A  (2013) Study of the Hypoglycemic Effect of Tamarindus indica Linn. Seeds on Non-diabetic and Diabetic Model Rats, British Journal of Pharmaceutical Research, 3(4): 1094-1105.

5.4  Rajkumar M, Das UK, Ghosh DG (2004) Antidiabetic effect of aqueous extract of seed of Tamarindus indica streptozotocin-induced diabetic rats Journal of Ethnopharmacology, 92(1): 85-91

5.5  Yerima M (2015) Antidiabetic Effect Of The Saponin-Rich Fraction Of the Extract of Tamarindus indica L. On Experimental Hyperglycemia, International Journal of Pharmaceutical Sciences And Research, Vol. 6(2): 733-738.

5.6  Sole SS, Srinivasan B.P, Akarte A.S, (2013) Anti-inflammatory action of Tamarind seeds reduces hyperglycemic excursion by repressing pancreatic β-cell damage and normalizing SREBP-1c concentration 51(3): 350–360

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