Characterization of an Anti-Dermatophyte Cream from Zataria multiflora Boiss

1. Introduction

     The Labiatae family has several members with a significant content of essential oils, which have commercial and medical values. This family is generally known for its multiple pharmacological effects such as analgesic, anti-inflammatory [1], antioxidant [2], hepato-protective [3] and hypoglycemic actions [4]. Labiatae is widespread throughout the world as well as Iran [5-9].

     Zataria multiflora Boiss. is a plantbelonging to the Labiatae family that is geographically distributed only in Iran, Pakistan and Afghanistan. This plant with the vernacular name of "Avishan-e Shirazi" (in Iran) has several traditional uses such as antiseptic, anesthetic, antispasmodic and laxative [6]. It is used for the treatment of premenstrual pain, chronic catharsis, asthma, jaundice, sore throat, neuralgia and edema [6]. It is also prescribed for the treatment of premature labor pain [10, 11]. The aerial parts and leaves of Z. multiflora are reputed to be an effective remedy for treating pains and gastrointestinal disorders [12, 13]. Also, the extracts of the aerial parts of Z. multiflora show anti-inflammatory effects against acute and chronic inflammations in mice and rats [14]. Antibacterial and antifungal effects of Z. multiflora Boiss. have also been reported [15,16].

      Dermatophytes are the major cause of superficial mycoses and remain a public health problem. They have the ability to invade keratinized tissues and cause dermatophyto-sis, the most common human contagious fungal disease [17, 18]. The humid weather, overpopulation and poor hygienic conditions are conductive factors to the growth of dermatophytes. Even though it responds to treatment with conventional antifungal drugs, the diseases have a tendency to recur at the same or at different sites.

     There is a growing movement to find new medicines, or rediscover old ways of treating illness and improving general health. Throughout the history of mankind, many infectious diseases have been treated with plant extracts. As Z. multiflora has been shown to have antifungal effects [15, 16], the aim of this study was to prepare creams with different concentrations of Z. multiflora extract as anti-dermatophyte preparations, and evaluate the physicochemical properties and stability of the formulations.

2. Materials and methods

2.1. Plant materials

     Aerial parts of Z. multiflora Boiss. were collected in July 2005, from the Kolahghazi Mountains (Isfahan Province, Iran). The plant was identified by Department of Botany, Faculty of Agriculture, Shahid Chamran University, Ahwaz, Iran, and a voucher specimen was deposited in the herbarium of the Department of Pharmacognosy, Faculty of Pharmacy, Ahwaz Joundishapour University of Medical Sciences, Ahwaz, Iran. The collected material was air-dried at shade and the room temperature. To avoid degradation, the air dried plant material was ground just before extraction.

2.2. Preparation of plant extract

     Hydroalcoholic extract was prepared using the maceration method. Therefore, the powdered plants (500 g) were macerated in 1500 ml ethanol (80%, v/v) at the room temperature (25 °C) with occasional stirring for 3 days. After filtration with Whatman no.1 filter paper, the combined ethanolic extracts were evaporated in vacuo at 30 °C and stored in refrigerator for future use. The yield of the hydroalcoholic extract of Z. multiflora was 2% w/w.

2.3. Preparation of fungal inoculum

     The freeze-dried sealed glass ampoules of microorganisms used in this study were obtained from Persian Type Culture Collection (PTCC), the Iranian Research Organization for Science and Technology, Tehran, Iran. The microorganisms are: Microsporum canis PTCC no. 5069, Microsporum gypseum PTCC no. 5070, Trichophyton rubrum PTCC no. 5143, and Trichophyton verrucosum PTCC no. 5056. These microorganisms were activated on Saboraud Dextrose Broth (SDB) and then cultured on Saboraud Dextrose Agar (SDA) for 21 days at 22 °C to obtain adequate growth. Following the period of incubation, colonies were scraped with a sterile scalpel and macerated in 10 ml of sterile 0.05% tween 80 solution (in sterile distilled water). Fungal suspensions were adjusted to 10% absorbance at 580 nm using a spectrophotometer.

 Table 1. Amounts of the main ingredients used for making 100 g of the primary general base formulation.

2.4. Determination of minimum inhibitory concentration

     Minimum inhibitory concentration (MIC) of Z. multiflora was determined according to the method described by Evans and Richardson [9]. Various concentrations of the plant extract (0.5, 0.25, 0.1, and 0.05 g of dried extract per 100 ml of SDA) were prepared, transferred to sterile Petri plates and were allowed to solidify. Triplicate plates were used for each dilution. Each plate was inoculated with 0.2 μl fungal suspension and incubated at the room temperature for seven to fourteen days. Plates of T. verrucosum were incubated at 37 °C. Suitable controls were also included. SD broth with 0.2 μl of inoculum served as the positive control. Uninoculated SD broth served as negative control.

     Following the incubation period, plates were examined for growth, and colony diameters were precisely measured and expressed in millimeter. The MIC was regarded as the lowest concentration of the extract that did not show any viable growth after 21 days of incubation.

 Table 2. Inhibitory effect of different concentrations of the herbal extract (g/100 ml) on fungal growth in SDA medium after one and two weeks.

Table 3. Minimum inhibitory concentration of the Z. multiflora extract after 14 days.

2.5. Formulation design

     Water in oil emulsion base was chosen for its emollient and detergency properties. The base was mainly composed of bees wax, liquid paraffin, spermaceti, borax and water. Then, different amounts of the ingredients were incorporated together and formulations (F₁ to F₂₅) were compared regarding their extent of oil phase, the viscosity of the product, and the amount of the emulsifier added in the final preparation. 

      Different formulations were evaluated regarding their appearance, particle size and phase homogeneity, emolliency and viscosity, and the best one was chosen. Then, the required amount of the herbal extract was added to make a proper formula having the best antifungal effect. The composition and amounts of the ingredients are shown in Table 1. Regarding the effect of the formulations on selected dermatophytes in culture media, the best percentage of the herbal extract was determined and the physicochemical stability of the formulations were evaluated.

2.6. Homogeneity test

     Five hundred mg of each sample was spread on a clean slide and observed using an optical microscope (×10 and ×40).

2.7. Creaming and coalescence

     A 10 g sample of each formulation was placed in a beaker and stored at the room temperature for 3 months. Their physical stability was determined after one week and one and three month storage.

2.8. Centrifugation

      A 10 g sample of each formulation was placed in a centrifuge tube (1 cm diameter) and centrifuged at 2000 rpm for 5, 15, 30 and 60 min. Then the phase separation and solid sedimentation of the samples were evaluated.

2.9. Thermal cycle test

     The samples were stored at 5 °C for 48 h and then at 25 °C for 48 h. The procedure was repeated 6 times and then their stability and appearance were evaluated.

2.10. Thermal change test

     Three 20 g samples of each formulation (or base) were stored at 4-6 °C, 25 °C and 45-50 °C. After 24 h, one and three months, their stability and appearance were evaluated.

2.11. Freezing and thawing

      Twenty gram samples of each formula were stored periodically at 45-50 °C and 4 °C for 48 h each. The procedure was repeated six times and then the samples were checked regarding their appearance and stability.

2.12. Determination of pH

      A suspension of each sample in 1% potassium nitrate solution was prepared and its pH was determined. A magnetic stirrer was used to produce homogeneity. The pH was determined at 48 h, one and three weeks after preparation.

2.13. Viscosity determination

      Using a Brookfield viscometer (model DV-I with No. 6 spindle) the rheologic behavior of the samples were studied. Each sample was placed in a container and spindle velocity was raised gradually to maximum extent. Then the viscosity was determined at 0.3, 0.6, 3, 6 and 60 rpm.

2.14. Chemical controls

      The extent of volatile and non-volatile composition and water content of the formulations were determined using standard methods.

 Table 4. Amounts of ingredients (g) used in formulations 1 to 12 and the results of their pH and stability considerations (n=5).

*Distilled water was added to all of the formulations to make 100 ml volume.

Table 5. Amounts of ingredients (g) used in formulations 13 to 25 and the results of their pH and stability considerations (n=5).

*Distilled water was added to all of the formulations to make 100 ml volume.

Table 6. Results from viscosity monitoring of the final formulation after 3 months storage.

2.15. Antimicrobial preservative effectiveness determination

     To evaluate the effectiveness of the formulation preservation, the single microbial challenge test prescribed in USP30 was employed. The test was performed by adding 0.1 ml of 10⁸ cfu/ml Staphylococcus aureus (PTCC No. 1189) and Pseudomonas aeruginosa (PTCC No. 1074) to each of the20 ml prediluted sample of the formulation. Inoculated containers were kept at 22.5±2.5 °C for 28 days. At appropriate time intervals as specified in the USP(1, 7, 14, 21, and 28 days), aseptically, 1 ml portions from each sample was withdrawn and were subjected to the pour plate count procedure; and changes in microbial numbers at various intervals were recorded.

3. Results and discussion

     Many skin diseases such as tinea and ringworm caused by dermatophytes exist in tropical and semitropical areas. In general, these fungi live in the dead, top layer of skin cells in moist areas of the body, such as between the toes, the groin, and under the breasts. These fungal infections cause only a minor irritation. Other types of fungal infections could be more serious. They can penetrate into the cells and cause itching, swelling, blistering and scaling. The dermatophytes, Trichophyton, Epidermophyton and Microsporum canis are commonly involved in such infections. However, their clinical dif-ferentiation is difficult.

      The herbal extract total ash, insoluble ash percent and pH of a 2% sample of the extract were 11.75±0.07%, 2.89±0.06% and 5.76±0.06, respectively. The results from MIC determination are listed in Tables 2 and 3.

     One of the main problems with the preliminary base formula was the excess fat content which produced a greasy sense on usage. In addition, it was relatively turbid and its low consistency and rigid nature were the other problems with that. Therefore, the formula was modified to overcome the problems. At first, the proportions of the oily phase components were changed and three formulations were made (formulations F₁ to F₃; Table 4). F₁ and F₂ were very dilute and had low consistency, but the third was more consistent. These 3 formulations were rejected due to their greasy appearance. In the second step, solid paraffin was used to produce better flow properties (F₄ to F₇ formulations, Table 4). The results showed that by increasing the amount of solid paraffin, the stiffness and consistency of the formula were increased. The best concentration was chosen to be 1.2 percent of paraffin. Centrifugation and thermal tests showed that all of the formulations (F₄ to F₇) were unstable and a phase separation phenomenon occurred.

     To enhance the stability of the formula, borax was added in different quantities (F₈ to F₁₁; Table 4). F₈ was very solid and had a turbid and stiff appearance. F₉ had better flow properties but was broken after centrifugation. F₁₀ had a good appearance and consistency and was stable during centrifugation and after 2 weeks of storage. For F₁₁ formula, the amount of the borax was too low to produce a stable emulsion.

      To adequate stability and prevent the changes in pH, tween 80 was added in formulations F₁₂ to F₁₇ (Tables 4 and 5). All of the mentioned formulations had a proper consistency, while only F₁₄ and F ₁₅ had relatively stable emulsion characteristics during centrifugation and thermal cycle tests. Due to undesirable appearance of the formulations F₁₂ to F₁₇, different amounts of spermaceti were added to soften and brighten the product [19]. F₁₈ to F₂₅ (Table 5) were prepared using 3 to 7% of this material. The results showed that F₂₁ and F₂₂ had the best flow characteristics, of which F₂₂ was more homogenous, clear and bright; so it was chosen as the best base formula for incorporation with the Z. multiflora extract.

     As mentioned before, the minimum inhibitory concentration of the extract was 0.5%; therefore, three different percentages of the extract were incorporated with the base and their antifungal potency were determined. According to the findings, 2% formulation had enough requirements to prevent the fungal growth.

     Finally, since creams and other water containing dosage forms should be preserved from microbial contamination, a usual preservative mixture, methyl and propyl paraben (3:1 ratio) was added. Control experiments and stability determination showed a homogenous appearance during 3 month storage period and no creaming or coalescence occurred. Also, there was no significant change in the appearance of the samples and the base during centrifugation, thermal cycle and freeze and thawing tests. Finally there was no significant change in the viscosity of the samples in 3 month storage period (Table 6). Antimicrobial preservation efficacy test showed that the preservative used had the ability to preserve the formula completely.

     The amounts of ingredients used in the final formulation are listed in Table 7. Due to the good antidermatophyte activity of the herbal extract, it could be added in topical formulations in order to protect skin against damage caused by various dermatophytes. Nevertheless, evaluation of cutaneous permeation and in vivo efficacy of formulations added with plant extract are necessary in order to confirm their use for skin protection.

Table 7. The final composition and amount of ingredients used to make Zataria anti-dermatophyte cream.