Natvar J. Patel is a principal at S.K. Patel College of Pharmaceutical Education and Research, Ganpat University in India.

Natvar J. Patel

	Berberine hydrochloride (BRB) is an isoquinoline-alkaloid derivative that can be isolated from medicinal herbs such as 

 huangbai and huanglian are described as heat-removing agents for fever reduction (2). BRB, the major ingredient of these herbs, possesses antimicrobial activity against Gram-positive and Gram-negative bacteria, as well as against other microorganisms (3, 4). BRB inhibits the growth of streptococci and appears to prevent them from adhering to host cells (5). BRB also exhibits antimalarial, antisecretory, anti-inflammatory, and anticancer activities with relatively low cytotoxicity to human cells (6).

	Topical and transdermal products are important classes of drug-delivery systems, and their use is becoming more widespread. The purpose of topical dosage forms is to deliver drugs conveniently to a localized area of the skin (7). Topical creams (e.g., cold cream), which are oil-in-water (O/W) emulsions, are less greasy and more acceptable to patients. Patients generally prefer creams for the treatment of mild or short-duration conditions (8).

	Many topical antifungals are now available, but not all are equally effective. Only a few topical antibiotics are available for treating skin diseases (9, 10). The authors investigated cream formulations of BRB because it possesses antifungal, antibacterial, and anti-inflammatory activity.

	Apifil (PEG-8 Beeswax, Gattefossé, St. Priest, France) and Plurol Stearique WL 1009 (polyglyceryl-6-distearate, Gattefossé) were selected as O/W emulsifiers. Both emulsifiers can be used to formulate creams with various concentrations of the oil phase without phase inversion. At higher concentrations (e.g., 5–15%), they form stable creams with a firm texture and a smooth, glossy appearance. The agents emulsify vegetable oils by as much as 15% are particularly well-suited to the emulsification of short-chain or fatty-acid esters. They perform well with other fatty-acid esters, silicone oils, mineral oils, and their substitutes.

	The authors attempted to develop safe topical formulations of BRB that could deliver the drug locally in an effective concentration for its antimicrobial and anti-inflammatory effects. The effectiveness of the cream formulation would likely depend on the nature and concentration of the emulsifier used, on the concentration of BRB, and on the storage time of the cream formulations. The authors designed an experiment to investigate the effects of these variables on the formulation of BRB as a topical drug-delivery system.

 BRB HCl powder (90% purity) was received as a gift from Yucca Laboratories (Mumbai) and used in the study without further purification procedures.

 The authors chose the emulsifiers Apifil and Plurol Stearique WL 1009 for formulating the BRB creams. Both emulsifiers were obtained from Gattefossé. The formulations were based on aqueous cream BP (

). The formulation for 100 g of aqueous cream BP was the following:

		Boiled and cooled purified water (100 g).

	Emulsifying ointment BP consists of emulsifying wax BP (30%), white soft paraffin BP (50%), and liquid paraffin BP (20%), and acts as a hydrophobic vehicle, structural-matrix former, and emulsifying agent. Menthol BP was used as a permeation enhancer. The detailed composition of different cream formulations is shown in Tables Ia and Ib.

 a Gram-positive bacterium, is associated with skin ailments such as boils, lesions, and infected burn sites. 

 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).

 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.

 Drug content of the cream formulations was determined by dissolving an accurately weighed quantity of cream in about 50 mL of pH 7.2 phosphate buffer. These solutions were transferred quantitatively to volumetric flasks, and appropriate dilutions were made with the same buffer solution. The resulting solutions were passed through 0.45-mm membrane filters before undergoing spectrophotometric analysis for BRB at 345 nm (λ

 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.

 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.

 Experiments were designed separately on formulations containing different concentrations of BRB. To study how well the various cream formulations inhibited 

 growth, each of the three variables-type of emulsifier (

)-were used at a high level (denoted by a subscript 

) and a low level (denoted by a subscript 

 (= 8) factorial experimental design (12–14). Using the above nomenclature, the eight combinations between the variables were 

 is zero time in storage of cream preparations, and 

 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.

	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 

 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 

 and subtracting the sum of all zones of inhibition for combinations containing a low level of 

 as illustrated by the following equation:

	This value, whether positive or negative, was a quantitative measure of the effect of 

 on the activity of the cream formulation against the microorganism involved. Similar expressions were used to find the effects of 

 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 

	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 

 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 cm

. 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 (

=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.

 All prepared cream formulations of BRB contained 99.2–100.1% of the indicated amount of BRB. As the concentration of menthol increased in the formulations, the viscosity decreased in both gel formulations (see Table III).

 The results of the experiments on the antimicrobial effectiveness of the various cream formulations of BRB, as measured by the diameter of zone of inhibition (mm), are presented in Figure 1. The combination of variables significantly influenced the effectiveness of the cream formulations. All the creams generally showed considerable effectiveness against 

 creams with a BRB concentration of 0.5% and 1.0% w/w had little effectiveness. The effectiveness of the creams, however, increased considerably with an increase in BRB concentration.

	The relevant values of the zones of inhibition were used to calculate the independent and interaction coefficients of the variables' influence on the creams' effectiveness. The values of the coefficients are presented in Table II. The variables had positive and negative influences on the cream formulations' effectiveness. A positive influence was increased inhibition of bacterial growth, and a negative influence was a decreased inhibitory effect on bacterial growth.

	The independent coefficient values show the influence of individual variables on the effectiveness of the cream formulations (see Table II). The effects of the variables were highly dependent on the concentration of BRB in the formulation.

	The type of emulsifier had by far the greatest effects on the inhibitory value of the creams, especially at higher concentrations of BRB. The effects of 

 were generally positive, indicating that a change in emulsifier from Plurol, the low level of 

 increased the creams' antimicrobial effectiveness. 

 had a much greater effect on the creams' effectiveness against 

 generally had moderate effects on the inhibitory value of the creams, especially at higher concentrations of BRB, where these effects were generally positive. This result indicates that changing the concentration of emulsifier from 5 to 10% would increase the effectiveness of the creams that contained higher concentrations of BRB.

 generally had the least effects on the inhibitory value of the creams, and thus the time of storage should be the least influential variable. The effects were all negative, thereby showing that the creams' effectiveness decreases with time. This reduction in effectiveness is negligible, however, because the creams stored for 12 months showed a similar zone of inhibition to that of creams stored for no time.

	The interaction coefficient values indicated the effects of the variables in combination (see Table II). The authors had difficulty ranking the interaction effects, although the strongest interactions appeared to be between 

 is more susceptible to cream formulations that contain a high concentration of Apifil than is 

 More importantly, the various interactions appeared to be generally weak, suggesting that the variables were, to a large extent, acting independently of each other. This observation therefore implies that 

 is the most influential variable in this particular work because it had the largest effects.

 The authors investigated the penetration-enhancing effect of menthol on the permeability of cream formulations of BRB through the excised rat epidermis. Permeation parameters for BRB in the cream formulations are shown in Table III. The cumulative amount of drug permeation through the rat epidermis from cream formulations that contained various amounts of menthol is shown in Figure 2.

) of BRB that permeated during the 24 h of the study was 6.20 ±0.23 mg/cm

 from Formulation P prepared without menthol. The flux was obtained by dividing the cumulative amount of drug permeated per cm

 of the skin by time. The corresponding flux of BRB was 260.21 ±9.76 μg/cm

	The authors observed a marked effect of menthol on BRB skin permeation. The cumulative amounts (

) of BRB that permeated over 24 h increased from 9.43 ±0.34 to 35.07 ±0.95 mg cm

 for cream formulations containing 2.5 and 12.5 %w/w of menthol, respectively. The corresponding flux values ranged from 391.73 ±12.58 to 1485.75 ±42.93 μg cm

 . However, a lag period of 1 h was observed for both formulations. The drug-permeation data were consistent with zero-order kinetics from 2 to 24 h with a lag period of about 1 h for all cream formulations (see Table III).

As the menthol concentration increased from 0 to 12.5% w/w, the permeability of BRB also increased, as indicated by an increase in both the permeability coefficient and enhancement ration (ER) (see Figure 2 and Table III). The authors observed a fivefold increase in the permeability of the drug from the cream containing 12.5% w/w of menthol (see Table III). Terpenes increase the drug's percutaneous permeation mainly by disrupting the intercellular packing of the subcutaneous lipids (18–20).

	Formulation M5 (12.5% w/w menthol), prepared using Apifil, showed the highest 

 permeability of BRB, hence this formulation was selected for skin-irritation and anti-inflammatory studies.

 The skin-irritation test of Formulation M5 resulted in a score of less than 2 (see Table IV). Compounds that have scores of 2 or less are considered to be nonirritants (21). Hence, both types of topical cream formulations may be categorized as nonirritants.

 The results of anti-inflammatory activity studies after topical application are reported in Table V. For the control formulation, the paw volume increases arithmetically with time. Formulation M5 and the standard diclofenac gel formulation showed better reduction in paw-volume measurement than did the control formulation. Statistical analysis showed that the edema inhibition of Formulation M5 and the standard diclofenac gel formulation were much better than that of the control group (P < 0.05). However, there was no statistical difference between the anti-inflammatory effects of formulation M5 or the standard diclofenac gel (

	Topical applications have a great potential as an effective and safe way to administer BRB for local antimicrobial and anti-inflamatory effects. An antimicrobial study of a prepared cream formulation of BRB indicated good antibacterial activity against 

 permeation study using rat epidermal membranes and anti-inflammatroy studies by the carrageenan-induced rat-paw edema method showed that menthol enhanced the transdermal absorption of BRB formulations based on aqueous cream BP. The topical-cream formulations of BRB developed in this study have great utility and are a viable option for the effective and controlled management of inflammation and skin infections caused by 

 is an assistant professor in the department of pharmacognosy, 

 is a professor, all at S.K. Patel College of Pharmaceutical Education and Research, Ganpat University, Kherva, 382711 Mehsana, Gujarat, India, fax +91 2762 286082, shailyrakesh@yahoo.com.

	*To whom all correspondence should be addressed.

	Submitted: Feb. 20, 2009. Accepted: Mar. 23, 2009.

	2. K.C. Huang and W.M. Williams, "Antibacterial, Antiviral, and Antifungal Herbs," in 

 K.C. Huang, Ed. (CRC Press, New York, 2nd ed., 1999), pp. 381–383.

	3. A.H. Amin, T.V. Subbaiah, and K.M. Abbasi, 

	5. D. Sun, S.N. Abraham, and E.H. Beachey, 

 A.O. David and H.A. Anton, Eds. (Marcel Dekker, New York, 1st ed., 1990), p. 1.

	11. G. L. Darmstadt, J. G. Dinulos, and Z. Miller, 

	21. J.H. Draize, G. Woodward, and H.O. Calvery, 

Articles

The authors sought to prepare a topical formulation of berberine hydrochloride for the effective and controlled management of inflammation and skin infections.