ANTIMICROBIAL PROPERTIES OF NATURAL LIMONENE 141 (ACCT 25922) and Gram-positive bacteria S. epidermidis (ACCT 49461), yeast C. albicans, and fungi T. rubrum, A. niger, P. commune, Alternaria alternata, T. viride, and C. cladosporioides. The fungi were obtained from the collection of the Department of Biotechnology, Insti- tute of Inorganic Chemical Technology and Environmental Engineering (IIChTEE), West Pomeranian University of Technology in Szczecin. The microorganisms were iso- lated from air of habitable buildings by the sedimentation method and identifi ed by morphology and biochemical tests. Twenty-four–hour bacterial cultures on plate control agar (BioMaxima, Lublin, Poland) for E. coli and brain heart infusion (BioMaxima) for S. epidermidis were used. Solutions of fungal spores were prepared from 7-d cultures carried out at 37°C (yeast) and 25°C (fungi) on agar slants. Sabouraud agar (BioMaxima) for yeast and dermatophytic fungi T. rubrum and malt extract agar (Merck, Darmstadt, Germany) for fi lamentous fungi were used in the tests. The concentration of the microorganism was determined by the spectrophotometric method at wavelength λ = 550 nm for fungi and λ = 600 nm for bacteria. A fungal suspension at a concentration of 1.76 × 107 CFU (colony-forming units) × cm-3 and a bacterial suspension at 1.4 × 108 (approximately 0.5 according to McFarland standard) in 0.85% NaCl solution were prepared. Diffusion disk method was used for the assessment of microbial growth inhibition. Sterile paper disks (Whatman No. 1, diameter 5 mm) impregnated with tested compounds (10 μL/disk) were placed at different locations on the surface of agar plates. Incubation of microorgan- isms was carried out under the following conditions: bacteria and yeasts at the tempera- ture of 37°C for 24 h and fungi at 25°C for 72 h. After incubation, the growth inhibition zones (mm) around the paper disks were measured. METHOD OF PREPARATION OF THERAPEUTIC CREAMS To prepare the therapeutic cream, an oil phase consisting of 12.5 g saffl ower oil and 3 g of beeswax was weighed. Next, the water phase with the following composition was weighed: 3.5 g of urea (pure, Chempur, Pikary Śląskie, Poland), 0.5 g of allantoin (pure, Chempur), and 21.25 g of water. In the next stage to the aqueous phase, terpinolene was added in an amount of 0.2 or 0.83 g [concentration of terpinolene 0.5 wt% (cream named KRT1) and 2 wt% (cream named KRT2), respectively]. The beakers with the water phase and the oil phase were placed in a water bath at the temperature of 80°C. After dissolving the oil phase ingredients, the beakers were taken out of the bath and the oil phase was added to the water, with intense mixing. The whole mixture was stirred until a slightly yellow cream with a homogeneous consistency was obtained (Figure 2). RESULTS AND DISCUSSION Natural limonene, the postreaction mix ture of compounds obtai ned after the isomeriza- tion of natural limonene (limonene, α-terpinene, γ-terpinene, terpinolene, and p-cymene), and each of the products of isomerization of limonene separately were tested for their antimicrobial properties (Table I). The pure compounds (standards) showed the highest antimicrobial activity. The growth inhibition zones around the paper disks impregnated with limonene and terpinolene were signifi cantly larger for all tested microorganisms (Table I). The mean inhibition zone for

JOURNAL OF COSMETIC SCIENCE 142 the bacteria and dermatophytic organisms (C. albicans and T. rubrum) ranged from 9.0 to 25.2 mm, indicating a remarkable effect of natural limonene. The terpinolene exerted a particularly strong antifungal effect against the mold fungi (A. niger, P. commune, A. alter- nata, T. viride, and C. cladosporioides). The mean inhibition zone ranged from 18.4 to 52.2 mm (Table I). Interestingly, the reaction mixture obtained after limonene isomerization presented the best antimicrobial activity against dermatophytes C. albicans and T. rubrum and fi lamentous fungi T. viride. T. rubrum can be considered as the most sensitive organ- ism because growth inhibition zones were present around disks impregnated with almost all tested substances (the only exception was p-cymene). Therefore, the antifungal effects of all tested substances are presented in Figure 3. By contrast, the most resistant organism was the mold fungi P. commune. Growth inhibi- tion zones were observed only around the disk impregnated with terpinolene (Table I). On the basis of obtained results, terpinolene was assigned as t he substance with the best antimicrobial properties against a broad spectrum of microorganisms. For these reasons, terpinolene was chosen as a representative compound for the preparation of therapeutic creams. Two therapeutic creams containing 0.5 and 2 wt% of terpinolene, respectively, were prepared. The performed studies showed that both creams showed activity only against the Gram-negative bacteria E. coli and Gram-positive bacteria S. epidermidis. The growth inhibition zone around disks impregnated with both creams was not dose dependent and ranged from 8.0 mm for E. coli to 10.2 mm for S. epidermidis (Table II). It was concluded that 0.5 wt% of terpinolene in a cream is suffi cient to inhibit the growth of these microorganisms. The growing number of multidrug-resistant microorganisms and pe rsons with allergy has become a worldwide public health problem. This concerns in particular to producers of products such as creams, ointments, and toothpastes. Therefore, there is need to fi nd a new effective antimicrobial agent that kills or inhibits the growth of microbes but is safe to use on humans. The best candidates meeting these conditions appear to be natural substances such as monoterpenes or products of their isomerization. In the presented research, the antimicrobial activity of the po streaction mixture obtained after the isomerization of limonene and its individual components was investigated. Only terpinolene showed the highest activity against all species of tested bacteria and fungi. The considerable inhibition zone obtained for this compound against all dermatophytes Figure 2. Pictures of prepared creams: KRT1 (A) and KRT2 (B).