The combinations were grouped into sets to assess the effect that each variable had on the effectiveness of the cream preparations
against the relevant microorganisms and to determine whether the variables interacted or acted independently. For example,
the effect of increasing a particular variable such as T on the in vitro activity of the cream formulations against the microorganism involved was assessed by adding all the zone of inhibition values
of combinations containing a high level of T and subtracting the sum of all zones of inhibition for combinations containing a low level of T, as illustrated by the following equation:
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.
To determine whether any two variables interacted with each other, the authors added the zone of inhibition results of the
combinations in which the variables appeared and subtracted the corresponding sum of other combinations. For example, the
authors used the following equation to find the interaction between T and C:
A result of zero indicated no interaction, but a difference implied that the two variables had interacted. Similar equations
were used to estimate the interactions between T and S and between C and S.
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 The temperature was maintained at 37 ± 0.5 °C. The samples were withdrawn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.
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 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.