EVALUATION OF EFFICACY OF TEST FORMULATIONS IN RAT MODEL OF CROTON-OIL-INDUCED HEMORRHOIDS

 EVALUATION OF EFFICACY OF TEST FORMULATIONS IN RAT MODEL OF CROTON-OIL-INDUCED HEMORRHOIDS

 

1.0  TEST SYSTEM DETAILS:

Species                   : Rattus norvegicus (Rat)

Age                        : 6-7 weeks

Sex                        : Male/Female

No. of animals        : 8 /Group

Total animals         : 56

 

2.0   ALLOCATION OF GROUPS:

 

Group No.	Group Description	Disease-Induction agent administered	Treatment administered	Dose Volume and Route
G1	Sham Control	Normal saline applied to anorectal region for three consecutive days	0.5% MC, p.o., b.i.d.	5 ml/kg, p.o. + 20 mg topical
G2	Disease Control	Application of 6% croton oil prepared in di-ethyl ether, to the anorectal region for three consecutive days	0.5% MC, p.o., b.i.d.
G3	Reference Control		Prednisolone-1 mg/kg, p.o., q.d. in 0.5% MC
G4	Treated orally with low dose of TF-1 + TC-1 topically		TF-1-X1 mg/kg, in 0.5% MC., b.i.d. + 20 mg of TC-1 topically, b.i.d.
G5	Treated orally with intermediate dose 1 of TF-1 + TC-1 topically		TF-1-X2 mg/kg, in 0.5% MC., b.i.d. 20 mg of TC-1 topically, b.i.d.
G6	Treated orally with intermediate dose 2 of TF-1 + TC-1 topically		TF-1-X3 mg/kg, in 0.5% MC., b.i.d. 20 mg of TC-1 topically, b.i.d.
G7	Treated orally with high dose of TF-1 + TC-1 topically		TF-1-X4 mg/kg, in 0.5% MC., b.i.d. 20 mg of TC-1 topically, b.i.d.

Abbreviations: MC-Methyl Cellulose, p.o.-per os. q.d.: quaque die; bid: bis in die. X1, X2, X3, X4 and Y1, Y2, Y3, Y4 are defined as the incremental doses of the Test formulations. The dose range will be from 10 mg/kg to 1000 mg/kg, b.i.d.

 

3.0  METHOD:

·         After completion of quarantine, healthy animals will be selected for the study. Subsequently, they will be randomized based on body weight and allocated into 7, different groups consisting of 8 animals each.

·         Post-randomization, animals will be acclimatized for 5 days in an experimental room earmarked for the experiment.

·         For the induction of hemorrhoids, animals allocated to groups G2-G7 will be applied 6% croton oil prepared in diethyl ether, to the ano-rectal area, by using a cotton swab, for three consecutive days. The animals assigned to the sham control group (G1) will be applied normal saline for three consecutive days.

·         Treatments to be administered:

Ø Animals of the Group G1 and G2, designated as sham-control and disease-control respectively, will be administered 0.5% MC, p.o., b.i.d.

Ø Animals of group G3 will be treated with reference drug, Prednisolone, orally at the dose of 1 mg/kg, q.d.

Ø Animals of group G4-G7 will be treated with TF-1 orally at different dose levels ranging from 10-1000 mg/kg, b.i.d. Additionally, the animals will also receive 20 mg of TC-1 topically on the hemorrhoids.  

Ø Ø Animals will continue to receive the test compounds for seven days after the third application of croton oil.

·         On day 11, i.e. eight days after the final application of croton oil animals will be euthanized under overdose of thiopentone anaesthesia. After suitable anaesthesia but before the animal dies, blood will be collected and serum will be separated for estimation of cytokines.  

·      Immediately after the animal dies, the anorectal portion of the animal will be excised and weighed to calculate the Recto-anal coefficient (RAC) and then one portion will be fixed in 10% neutral buffered formalin for histopathological analysis. Further, the remaining tissue will snap-frozen in liquid nitrogen and immediately stored at -80°C for the subsequent biochemical and molecular evaluations.

 

4.0  PARAMETERS TO BE EVALUATED:

·         Body weight: Twice a week

·         Weight of the harvested ano-rectal portion.

·         Recto-anal coefficient

·         Cytokines in serum: IL-1β, IL-6, IL-8, TNF-α.

·         Gene expression analysis by Real-time PCR: PGE1, IL-1β, IL-6, IL-8, TNF-α, iNOS, COX-2, VEGF, MMP-9.

·         Histological analysis of the anorectal tissue (Hematoxylin & Eosin-stained).

 

5.0  REFERENCES:

1.        Zhang, H. et al. Anti-hemorrhoidal activity of Lian-Zhi-San, a traditional Chinese medicine, in an experimental hemorrhoidal model in rats. J. Integr. Med. 19, 42–49 (2021).

2.        Apaydin Yildirim, B. et al. Antihemorrhoidal activity of organic acids of Capsella bursa-pastoris on croton oil-induced hemorrhoid in rats. J. Food Biochem. 44, 1–12 (2020).

3.        Dey, Y. N., Wanjari, M. M., Kumar, D., Lomash, V. & Jadhav, A. D. Curative effect of Amorphophallus paeoniifolius tuber on experimental hemorrhoids in rats. J. Ethnopharmacol. 192, 183–191 (2016).

4.        Faujdar, S., Sati, B., Sharma, S., Pathak, A. K. & Paliwal, S. K. Phytochemical evaluation and anti-hemorrhoidal activity of bark of Acacia ferruginea DC. J. Tradit. Complement. Med. 9, 85–89 (2019).

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