2.6. Skin irritation study
The skin irritation potential of triptolide-loaded MBH was conducted in rabbits. The backs of rabbits (2.0–2.5 kg, Experimental Animal Center of Jiangxi University of Traditional Chinese Medicine, Jiangxi, China), were clipped free of fur with scissors 24 h prior to application of a patch. Just prior to the test, the rabbits were randomly divided into two groups, one intact-skin group and one skin-injury group, obtained by scarifying intact skin until capillary hemorrhage. The two groups were subdivided into a single-application subgroup and a multiple-applications subgroup. The skin surface was further divided into four regions for triptolide-loaded microemulsion, triptolide-loaded MBH, blank microemulsion and blank MBH applications. For the single application, 0.8 g of both preparations described above was applied on the designated region (3×3 cm2). Polyethylene was used to cover the applications and gauze tape was used to fasten the polyethylene for 24 h until the drug was removed with warm water. Inspection on the administration site for the presence of erythema and edema began 1 h after removal of the drug. For multiple applications, two preparations were applied on the same skin regions for 6 h and 1 h after removal of the drug, then the sites were assessed for signs of skin irritation. The test procedure was repeated for 6 consecutive days. The irritation scores of the test area were determined by judging the extent of erythema and edema according to the criteria proposed by Chen et al.38. Erythema and edema were graded as follows: zero for no visible reaction, one for slight reaction, two for moderate reaction, three for severe reaction, and four for extremely serious reaction. The total scores for the irritation test in each condition were calculated using the following equation.
Average irritation score=(Erythema reaction score+Dropsy reaction score)/Amount of animal