EVALUATION OF THE HEPATOPROTECTIVE EFFICACY OF HERBAL FORMULATIONS IN RAT MODEL OF HEPATOTOXICITY INDUCED BY ADMINISTRATION OF ANTI-TUBERCULOSIS DRUGS

  EVALUATION OF THE HEPATOPROTECTIVE EFFICACY OF HERBAL FORMULATIONS IN RAT MODEL OF HEPATOTOXICITY INDUCED BY ADMINISTRATION OF ANTI-TUBERCULOSIS DRUGS

1.0  TEST SYSTEM DETAILS:

Species                   : Rattus norvegicus (Rats)

Strain                     : Sprague Dawley or Wistar

Age                        : 6-7 weeks

Sex                        : Female

No. of animals        : 8/Group

Total animals         : 48

 

2.0  TEST ARTICLES DETAILS

 

Phyllanthus niruri-based herbal formulation, additionally enriched with vitamins extracted from natural sources.

  

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
G1	Normal Control	0.5 % MC administered orally thrice a week  for 16 weeks	0.5% MC, p.o., b.i.d.
G2	Disease Control	Oral administration of Pyrazinamide (175 mg/kg) + Ethambutol (140 mg/kg) + Isoniazid (70 mg/kg) + Rifampicin (52 mg/kg) thrice a week for 16 weeks.	0.5% MC, p.o., b.i.d.
G3	Reference Control		Silymarin-50 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 High Dose		HD: 50-150 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 6 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 silymarin at the dose of 50 mg/kg, p.o., b.i.d.

·         Animals of group G4-G6 will be treated with HF, at different incremental dose levels as outlined in Section 4.0 of Annexure-I, twice daily.

·           The animals allocated to groups G2-G6 will be administered Pyrazinamide (170 mg/kg) + Ethambutol (140 mg/kg) + Isoniazid (70 mg/kg) + Rifampicin (52 mg/kg), thrice a week for 16 weeks.

·         Animals assigned to Group 1 will be administered 0.5% methylcellulose, which is the vehicle for anti-tuberculosis drugs, thrice a week for 16 weeks.

·         Test article and silymarin administration will be initiated concurrently with the disease induction protocol.

·       Blood will be withdrawn from the retro-orbital plexus under isoflurane anaesthesia of the animals forty-eight hours after completion of Week 4, Week 8 and Week 12 of disease induction for estimation of clinical chemistry parameters.

·        Forty-eight hours after the last administration of anti-tuberculosis drugs, animals will be sacrificed under overdose of thiopentone anaesthesia. After suitable anaesthesia but before the animal dies, blood will be collected from the retro-orbital plexus for the estimation of biochemical parameters. Immediately after the animal dies, the liver of the animals will be excised, weighed and examined for gross pathological changes. Subsequently, one lobe of the liver will be fixed in 10% neutral buffered formalin for histopathological whereas the others will be stored at -80°C for biochemical and molecular evaluations.

 

6.0  PARAMETERS TO BE EVALUATED:

·         Body weight: Twice a week

·         Serum biochemical parameters: Aspartate transaminase, alanine transaminase, alkaline phosphatase, gamma glutamyl transferase, total bilirubin, total cholesterol, triglycerides, high density and low density-associated cholesterol, total protein, albumin, urea and creatinine.

·         Hepatic triglycerides and cholesterol content.

·         Cytokines in serum: TNF-α, IL-β, IL-6, IFN-γ and HMGB-1.

·         Oxidative and nitrosative stress parameters in the liver: Contents malondialdehyde, oxidised and reduced glutathione along with their ratio, total nitrite content, activities of myeloperoxidase, superoxide dismutase and catalase.

·         Quantitative real-time PCR in liver: Expression of Nrf2, HO-1 and NQO-1.

·         Histology of liver sections stained with Hematoxylin and Eosin as well as Masson’s Trichrome stain.

 

7.0  REFERENCE(S):

1.  Jaswal, A., Sinha, N., Bhadauria, M., Shrivastava, S. & Shukla, S. Therapeutic potential of thymoquinone against anti-tuberculosis drugs induced liver damage. Environ. Toxicol. Pharmacol. 36, 779–786 (2013).

2.  Liu, X. et al. Protective Effect of Bicyclol on Anti-Tuberculosis. (2017) doi:10.3390/molecules22040524.

3.  Wen Y, Zhang G, Wu X. The role of the farnesoid X receptor in quadruple anti-tuberculosis drug-induced liver injury. Toxicology. 2022 Jun 30;476:153256. doi: 10.1016/j.tox.2022.153256.

                                                      END OF THE DOCUMENT

You may like to read these links: 

1. List of All SOPs and Documents for the Microbiology Laboratory

2. List of All SOPs and Documents for In-vitro Laboratory

3. List of All SOPs and Documents for the In-vivo Laboratory.

4. List of All SOPs and Documents for Clinical Research

5. List of Instruments and Equipment Used In Laboratory Animal Facility

6. Animal  Facility Design - Small Laboratory Animals (Rat, Mice, Rabbit, Guinea Pig)

7. List of Chemicals Needed for In-vivo Laboratory

8. List of Physiological Data for Small Laboratory Animals 

9. Leading Lab-Diet Suppliers in India and Beyond

10. List of All SOPs and Documents for Animal House Facility