This value, whether positive or negative, was a quantitative measure of the effect of T on the activity of the cream formulation against the microorganism involved. Similar expressions were used to find the effects of C and S on the activity of the cream formulation against the relevant microorganisms.

In vitro skin-permeation studies. In vitro skin-permeation studies were performed using a Franz diffusion cell with a receptor compartment capacity of 21 mL and an effective diffusion area of 1.84 cm². Excised rat abdominal skin was mounted between the donor and receptor compartments of the diffusion cell. One gram of each cream formulation was placed on the skin. The receptor compartment of the diffusion cell was filled with phosphate buffer (pH 7.2). The whole assembly was fixed on a magnetic stirrer (Remi, Mumbai), and the solution in the receptor compartment was continuously stirred using magnetic beads at 50 rpm. The temperature was maintained at 37 ± 0.5 °C. The samples were withdrawn at different time intervals and analyzed for drug content spectrophotometrically at a wavelength of 345 nm. The concentration of BRB in each sample was determined from a previously calculated standard curve. The receptor phase was replenished with an equal volume of phosphate buffer after each sample withdrawal. The cumulative percents of drug permeation per square centimeter of skin were plotted against time.

Skin-irritation test. The authors followed the "Guidelines of the Institutional Animal Ethics Committee" for this experiment. The hair on the dorsal side of Wistar albino rats was removed by clipping one day before the start of the experiment (15). The rats were divided into three groups (n=6). Group I was the control (i.e., cream without drug and permeation enhancers or Formulation P), Group II received cream with permeation enhancers (M5), and Group III received a 0.8% v/v aqueous solution of formalin as a standard irritant (16). A new cream or new formalin solution was applied daily for seven days. Finally, the application sites were graded according to a visual scoring scale, always by the same investigator.

Anti-inflammatory studies. Anti-inflammatory studies of prepared formulations were compared by the carrageenan-induced rat-paw edema method in Wistar albino rats. The protocol was approved by the Institutional Animal Ethics Committee. Eighteen rats were divided into three groups of six rats each for the different treatments shown in Table IV. Group I served was the control (i.e., Formulation P), Group II received cream with permeation enhancers (M5), and Group III received diclofenac gel containing 1% w/w of diclofenac (Diclomol Gel, Win-medicare, Delhi) as a standard formulation. Animals were fasted for 24 h before the experiment and given free access to water. Approximately 0.1 mL of 1% carrageenan suspension in saline was prepared 1 h before each experiment and injected into the plantar side of the right hind paw of the rat. Next, 100 mg of cream containing 10% of BRB was applied to the plantar surface of the hind paw by gently rubbing 50 times with the index finger. Rats of the control groups received only the cream base. Diclofenac gel 1.0% w/w was applied in the same way as a reference. Active and placebo creams were applied 1 h before the carrageenan injection. The paw volume was measured initially and at 1, 2, 3, and 4 h after carrageenan injection using the plythesmographic method (17). The percentage of inflammation was calculated for the purpose of comparison.