Pharmacological Evaluation of Combination of Plant Steroids and Low Dose Glucocorticoid for Treatment of Inflammatory Disorders in Experimental Animals

Abstract

Aim and Objective Hecogenin is a steroidal sapogenin obtained from the leaves of species such as Agave genus, including A. cantala, A. sisalana, A. avellanidens, A. cerulata, A. cocui, A. goldmaniana, and A. aurea. That plays important role in the treatment of variety of inflammatory diseases. The management of inflammatory disorders is very difficult because of the development of drug resistance after their chronic and prolonged treatment. These treatments are ineffective and associated with variety of side effects on long term use. Therefore, currently research has been thrown on searching effective anti-inflammatory treatments of herbal origin associated with minimal side effects. We have investigated the anti-inflammatory effects of Hecogenin and combination of Hecogenin with low dose Fluticasone on various inflammatory models such as Croton oil induced ear edema, Cotton pellet induced granuloma, Tri-Nitro-Benzene-Sulphonic acid induced colitis, 2, 4-dinitrofluorobenzene induced contact dermatitis,Ovalbumin induced lung edema and Complete Freund Adjuvant induced arthritis Material and Methods The selection of plant steroid was done by comparing all the phytosteroids (Hecogenin, Diosgenin, Solasodine, Glycyrrhizin, Boswellic acid, Guggulsterone, Sarsasapogenin or Withaferin-A) by using croton oil induced ear edema model. The ear edema was induced by topical, single administration of croton oil (20 μl of 2.5% v/v) on the inner surface of right ear, while the left ear received 20 μL of vehicle acetone only. After 15 minutes the drug treatments were topically applied on the right ear. Ear edema was evaluated 6 h after croton oil application and was expressed as increase in ear weight (mg). The anti-inflammatory effect of Hecogenin was evaluated by measuring the ear weight, % inhibition of edema and histopathological analysis of ear tissues. The cotton pellets induced granuloma model was used to assess anti-inflammatory activity of Hecogenin in rats. The autoclaved cotton pellets of 10 ± 1.0 mg were aseptically implanted sub-cutaneously in the back region of anesthetized rats. On day 8 cotton pellets ware removed surgically & dried in hot air oven at 60°C to a constant weight. Mean weight of the granuloma tissue in each group was recorded and percentage inhibition was calculated by comparing the mean weight of test group with the control group. Blood samples were collected from rats for tumor necrosis factor-α and interleukin-12 cytokines analysis. The adrenal gland and thymus gland were removed from rats and weighed. The colitis was induced by instillation of 10 mg of tri-nitro-benzene-sulphonic acid dissolved in 0.25 ml of 50 % ethanol (v/v) in rats by trans-rectal route. Hecogenin and combination were orally administered 48, 24 and 1 h by using catheter prior to the induction of colitis and 24 h after colitis induction. The rats were killed after 72 h of colitic induction colon weight, colon weight to length ratio were recorded. The inflammatory response was assessed by macroscopic, microscopic, myeloperoxidase activity, tumor necrosis factor alpha and Interleukin-12 levels and a histological study of rat colon tissue. The dermatitis was induced by repeated application of 25 μL of 0.15% 2, 4- dinitrofluorobenzene in acetone/olive oil (3:1) to the inner and outer surfaces of the ears, and 100 μL of the same solution applied to shaved back skin once on days 1 and 4. On days 7, 10, and 13, sensitized mice were challenged by applying 0.2% 2, 4-dinitrofluorobenzene to the back and ear skin surfaces. On day 7 to 13 drug treatment was done. Normal control group was treated with the same volumes of vehicle (Acetone). On day 14 the animal were sacrificed and ear thickness, ear weight, dermatitis score, myeloperoxidase activity, tumor necrosis factor-α, interleukin-12 and histological study of ear tissue was performed Balb/c mice were injected intra-peritoneal with a mixture of ovalbumin (50 μg) and alum (1 mg) in 0.2 ml of normal saline except for the NC group on 0 and 7 days. At 14 and 21 days, the mice were challenged with 2.5 % (w/v) ovalbumin aerosol through a nebulizer for 20 min. The normal control mice were exposed to saline aerosol for 20 min on days 14 and 21 (Epstein, 2006). 1 h before each Ovalbumin sensitization and challenge on 14 and 21 day after the initial sensitization 20 μl of drug treatments were administered by intra-nasal route on once daily on 14 to 21 days respectively (Vasconcelos et al, 2008). The animals were sacrificed 48 h after the last challenge on day 22 and total, differential cell count, spleen, thymus weight, body weight, blood glucose level, Myeloperoxidase, Tumor Necrosis Factor- α, Interleukin-12 levels and histopathological analysis of ear tissue was performed. Male Wistar rats were immunized by intradermal injection of 0.1 ml of complete freund adjuvant into the left hind paw on day 1. The dosing of Hecogenin and combination was started from day 12 once daily, topically. Anti-arthritic activity was evaluated by measuring paw volume, arthritic score, joint diameter, body weight, spleen and thymus weight, serum biochemical parameters, haematological parameters, myeloperoxidase, serum cytokines levels, X-rays of hind paw joints, histological analysis of joint tissue and COX-2 expressions study was done. In acute toxicity study, the Swiss albino mice were orally administered with single dose of HG 2000 mg/kg body weight and were continuously observed for behavioral and autonomic profile for 2 h and for any signs of toxicity or mortality up to 48 h. In safety pharmacological study, the Swiss albino mice were orally administered with HG (50 μg/mice) once daily for 14 days and control groups mice were received with acetone, orally. The mice were observed for any indications of toxicity effect within the first six hours after the treatment period, and daily further for a period of 14 days. Surviving animals were weighed and visual observations for mortality, behavioral pattern, changes in physical appearance, injury and signs of illness were conducted daily during the period.