EVALUATION OF ANTIHYPERTENSIVE ACTIVITY OF THE TEST FORMULATION IN EXPERIMENTAL ANIMALS

PROTOCOL

EVALUATION OF ANTIHYPERTENSIVE ACTIVITY OF THE TEST FORMULATION IN EXPERIMENTAL ANIMALS

TEST SYSTEM DETAILS:

Species                  : Rattus norvegicus (Rat)

Strain                    : Wistar or Sprague Dawley

Age                       : 8-10 weeks

Body Wight          : 200-250 g

Sex                        : Male

No. of animals      : 10 /Group

Total Animals       : 10×8= 80

1.0   ALLOCATION OF GROUPS:

Model – 1: One Kidney One Clip (1K1C) Hypertension

Groups	Treatment	Dose; ROA	No. of Animals
G1	Normal Control	Normal saline or 0.25% Na-CMC	10
G2	Disease Control	0.25% Na-CMC	10
G3	Reference Control: Captopril	50 mg / kg; p.o.	10
G4	Test Formulation	X1 mg / kg; p.o.	10

 

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

Model – 2: Dexamethasone Induced Hypertension In Rats

Groups	Treatment	Dose; ROA	No. of Animals
G1	Normal Control	Normal saline or 0.25% Na-CMC	10
G2	Disease Control  (Dexamethoson)	0.25% Na-CMC,    1.5 mg / kg; s.c.	10
G3	Reference Control: Captopril	50 mg / kg; p.o.	10
G4	Test Formulation	X2 mg / kg; p.o.	10

 

2.0  METHOD:

Methods :

1.     One Kidney One Clip (1K1C) Hypertension

·   Albino Wister rats weighing 200-250 g will be anaesthetized with 50 mg/kg i.p pentobarbital and the fur on the back will be shaved and the skin disinfected.

·        In the left lumbar area of flank incision will be made parallel to a long axis of the rat.

·        The renal pedicel will be exposed with the kidney retracted to the abdomen.

·     The renal artery will be traced out and a U-shaped silver clip will be clipped around it near the aorta.

·       Using a special forceps the size of the clip will be adjusted so that the internal gap ranges from 0.25-0.38 mm.

·      The right kidney will be removed after tying of the renal pedicle. The skin incisions will be closed and appropriate treatment will be given to prevent infection to the wounds.

·     4-5 weeks after clipping blood pressure will be measured and rats with values higher than 150 mmHg selected for the experiments.

·      Blood pressure readings will be taken on each of 3 days prior to drug treatment.

2. Dexamethasone Induced Hypertension In Rats

·     Albino wistar rats of either sex weighing 200-250 g in a group of 10 each will be used. Rats will be given subcutaneous injections of 1.5 mg Dexamethasone every alternate day, resulting in an increase in systolic blood pressure within 1 wk.

·   Tail-Cuff method will be used to measure blood pressure in experimentally induced hypertensive rats. The rats will be trained in the apparatus [animal restrainer] several times before BP measurement.

·       Systolic Blood Pressure will be measured by Tail-Cuff Method by Harvard non-invasive blood pressure monitor [Harvard apparatus with MP -100 data acquisition system] after rats will be pre–warmed at 40ºC for 10 min.

·     The tail cuff pulse sensor will be on the tail and will be connected to the blood pressure analyzer. Since rats will be pre-conditioned to the BP measurement procedure, they become calm during measurement. Three readings will be taken and average of three will be taken as individual systolic blood pressure.

3.0  END POINT PARAMETER(S):

·       Clinical observation

·       Feed water consumption

·       Body Weight

·       Measurement of in vitro ACE inhibitory activity

Ø  Microscopic evaluation (Histopathology) Heart and Kidney

 

7.0  REFERENCE(S):

7.1  Mizuno S, Nishimura S, Matsuura K, Gotou T, Yamamoto Y. Release of short and proline-rich antihypertensive peptides from casein hydrolysate with an Aspergillus oryzae protease. J Dairy Sci 2004;87:3183-8.

7.2  Li GH, Qu MR. Wan JZ, You JM. Antihypertensive effect of rice protein hydrolysate with in vitro angiotensin I converting enzyme inhibitory activity in spontaneously hypertensive rats. Asia Pac J Clin Nutr 2007;16(1):275-80.

7.3  Lopez FR, Otte J, Camp V.  Physiological, chemical and technological aspects of milk-protein-derived peptides with antihypertensive and ACE-inhibitory activity. Int Dairy J 2006;16:1277-93.

7.4  Miguel M, Aleixandre A. Antihypertensive peptides derived from Egg proteins.  J Nutr 2006;136:1457-60.

7.5  Yang Y, Marczak ED, Usui H, Kawamura Y, Yoshikawa M. Antihypertensive Properties of Spinach Leaf Protein Digests. J Agric Food Chem 2004; 52(8):2223-5.

7.6  Vogel GH. Drug discovery and evaluation – Pharmacological Assay. 2nd ed. Verlag Springer; 2002: p. 172-9.                                                                       

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