EVALUATION OF THE ANTIPYRETIC ACTIVITY OF HERBAL FORMULATIONS IN RAT MODEL OF LIPOPOLYSACCHARIDE (LPS)-INDUCED HYPERTHERMIA

  EVALUATION OF THE ANTIPYRETIC ACTIVITY OF HERBAL FORMULATIONS IN RAT MODEL OF LIPOPOLYSACCHARIDE (LPS)-INDUCED HYPERTHERMIA

1.0  TEST SYSTEM DETAILS:

Species                   : Rattus norvegicus (Rats)

Strain                     : Sprague Dawley or Wistar

Age                        : 6-7 weeks

Sex                        : Male

No. of animals        : 8/Group

Total animals         : 56

 

2.0  TEST ARTICLES DETAILS

Tinospora cordifolia-based herbal formulation.

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 saline administered by intraperitoneal route (i.p.), Single injection	0.5% MC, p.o., b.i.d.	5 ml/kg, p.o.
G2	Disease Control	LPS (E. coli 0111: B4)- 100 µg/kg, i.p.-Single injection	0.5% MC, p.o., b.i.d.
G3	Reference Control		Paracetamol-250 mg/kg, p.o., b.i.d.
G4	Treated with low dose		LD: 5-15 mg/kg, b.i.d.
G5	Treated with intermediate dose 1		ID-1: 15-50 mg/kg, b.i.d.
G6	Treated with intermediate dose 2		ID-2: 50-150 mg/kg,   b.i.d.
G7	Treated with high dose		HD: 150-500 mg/kg,  b.i.d.

Abbreviations: MC: Methylcellulose, p.o.-per os., i.p.-intraperitoneal 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 8 animals each.

·         Animals of the 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 paracetamol at the dose of 250 mg/kg, p.o., b.i.d.

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

·         Normal control group (G1) will be administered a single injection of sterile normal saline by intraperitoneal route, whereas animals allocated to groups G2 – G7 will be administered a single dose of LPS (100 µg/kg, i.p.) dissolved in sterile normal saline.

·         Vehicle/Reference compound/Test compounds will be given orally on the day of LPS-administration.

·         The rectal temperature of animals will be measured once prior to LPS-administration and then at 2, 4, 6, 12 and 24 hours post-LPS administration. Additionally, the feed consumption of animals will be measured at 4, 6, 12 and 24 hours post-LPS administration and water intake of animals will be recorded 24 hours post-LPS administration.

·         For estimation of the sickness behaviour in animals induced by hyperthermia, the forced swim test and elevated plus maze test will be conducted 4 hours post-LPS administration. For both the tests, the animals will be acclimatized to the experimental apparatus 24 hours prior to LPS administration.

·         To conduct the elevated plus maze test, animals will be placed at the centre of an apparatus containing two open arms perpendicular to two closed arms in a plus shape and a centre area. The time spent and number of entries of animals in different arms will be recorded for 5 min. The preference for being in open arms over closed arms will be calculated to measure anxiety-like behavior. Further, to conduct the forced swim test, animals will be placed in a cylinder of appropriate height and diameter containing water at a temperature of 25 ± 1°C and forced to swim for 5 min. The immobility time of the animals will be recorded and after completion of the test session, the animals will be dried and returned to their cages. An increase in the immobility time of the animals reflects depression-like behaviour.

·         For the estimation of pro-inflammatory markers, blood will be collected from the retro-orbital plexus of anaesthetised rats at 6 hours post LPS administration. After 24 hours of LPS administration, animals will be humanely sacrificed by an overdose of Thiopentone sodium (150 mg/kg). After suitable anaesthesia, but before the animal dies, blood will be collected from the retro-orbital plexus and serum will be separated for the estimation of pro-inflammatory markers. Immediately after the animal dies, the brain will be harvested, snap-frozen in liquid nitrogen, and stored at -80°C until processed for molecular analysis.

 

6.0  PARAMETERS TO BE EVALUATED:

·         Rectal temperature

·         Body weight

·         Feed and water intake

·         Elevated plus maze and Forced swim test

·         Serum pro-inflammatory markers

·         qPCR analysis in brain tissues for pro-inflammatory markers

 

7.0  REFERENCE(S):

1.  Taksande, B. G., Chopde, C. T., Umekar, M. J., & Kotagale, N. R. (2015). Agmatine attenuates lipopolysaccharide-induced anorexia and sickness behavior in rats. Pharmacology Biochemistry and Behavior, 132, 108-114.

2.  Vijayakaran, K., Kannan, K., Kesavan, M., Suresh, S., Sankar, P., Tandan, S. K., & Sarkar, S. N. (2014). Arsenic reduces the antipyretic activity of paracetamol in rats: modulation of brain COX-2 activity and CB1 receptor expression. Environmental toxicology and pharmacology, 37(1), 438-447.

3.  Wrotek, S., Jedrzejewski, T., Potera-Kram, E., & Kozak, W. (2011). Antipyretic activity of N-acetylcysteine. Journal of Physiology and Pharmacology, 62(6), 669.

4.  Jedrzejewski, T., Piotrowski, J., Kowalczewska, M., Wrotek, S., & Kozak, W. (2015). Polysaccharide peptide from Coriolus versicolor induces interleukin 6-related extension of endotoxin fever in rats. International Journal of Hyperthermia, 31(6), 626-634.

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