S. aureus, a Gram-positive bacterium, is associated with skin ailments such as boils, lesions, and infected burn sites. C. albicans, a fungus, is responsible for about 70–80% of all candidal infections. Infections can occur anywhere and are most common in skin folds and web spaces, on the penis, and around fingernails (11).

Preparation of test cream formulations. Cream formulations containing five different concentrations of BRB (0.5%, 1.0%, 2.0%, 5.0%, and 10% w/w BRB) were prepared based on aqueous cream BP. Two concentrations, 5% and 10% w/w, of the two emulsifiers were used to prepare the creams. Various concentrations of menthol (2.5%, 5.0%, 10%, and 12.5% w/w) also were incorporated in the cream formulation that contained 10% w/w BRB and the optimum concentration of emulsifier (Apifil 10%) as a permeation enhancer. Control formulations containing no BRB or permeation enhancers were also prepared for skin-irritation and anti-inflammatory studies. Samples of the test cream formulations were stored at room temperature and shielded from light for a period of three months to determine the stability of the creams.

Table Ib: Composition of topical cream formulations of BRB prepared with permeation enhancer (%w/w).

Viscosity measurements. A rotational digital viscometer (DV II RVTDV-II, Brookfield Engineering, Brookfield, MA) was used to measure the viscosity (in cps) of the creams. The spindle was rotated at 10 rpm. Samples of the creams were allowed to settle over 30 min at the temperature of test (25±1 °C) before the measurements were taken.

Preparation of microorganisms. S. aureus and C. albicans were grown in tryptone soy broth for 24 h at 37 °C and for 48 h at 28 °C, respectively. Bacteria (100 mL of a 1-in-100 dilution) of the overnight culture were used to inoculate 20 mL of molten sterile nutrient agar (Hi-Media Laboratories, Mumbai) at 45 °C and allowed to set. For the fungi, 20 mL of molten sterile sabouraud dextrose agar (Hi-Media Laboratories) containing 0.05 mg/mL cycloheximide and 0.5mg/mL chloramphenicol was poured into 9-cm sterile plates and allowed to set. The surface was inoculated with 0.2 mL of the culture. The surface of the set agar was allowed to dry at 37 °C and equidistant 6-mm wells were cut into the agar. Tetracycline and nystatin were used as positive controls for the bacteria and fungi, respectively.

Antimicrobial analysis of cream formulations. One gram of each formulation was mixed with 1 mL of sterile water using a whirl mixer until a slurry was formed. One hundred μL of each slurried cream was placed into prelabeled wells. The creams were allowed to diffuse for 1 h before incubating the bacteria plates at 37 °C for 24 h, and the fungal plates were incubated at 25 °C for a minimum of 48 h. The diameter of zone of inhibition was measured after the incubation period for each formulation.

Formulation experimental design. Experiments were designed separately on formulations containing different concentrations of BRB. To study how well the various cream formulations inhibited S. aureus and C. albicans growth, each of the three variables—type of emulsifier (T), concentration of emulsifier (C), and time of storage (S)—were used at a high level (denoted by a subscript H) and a low level (denoted by a subscript L) in a 2³ (= 8) factorial experimental design (12–14). Using the above nomenclature, the eight combinations between the variables were T _H C _L S _L , T _H C _L S _H , T _H C _H S _L , T _H C _H S _H , T _L C _L S _L , T _L C _L S _H , T _L C _H S _L , and T _L C _H S _H , where T _L is Plurol, T _H is Apifil, C _L is 5% w/w of emulsifier, C _H is 10% w/w of emulsifier, S _L is zero time in storage of cream preparations, and S _H is 12 months of storage of cream preparations. Samples of the test cream formulations were stored at room temperature and shielded from light for a period of 12 months to test their stability.