ANTIMICROBIAL PROPERTIES OF NATURAL LIMONENE 139 antioxidants is used in the treatment of atherosclerosis because of its ability to pre- vent low-density lipoprotein (LDL) oxidation (14–17). Terpinolene is used as an additive to fruits, baked products, ice creams, nonalcoholic bev- erages, and candies. This compound has a calming effect. Terpinolene inhalations affect the functioning of the autonomic nervous system and the human psyche. As a conse- quence, terpinolene reduces tension and increases the feeling of relaxation. Therefore, this compound can be used in the treatment of mental disorders such as depression. In addition, terpinolene has the potential to treat atherosclerosis. This compound, in combination with β-carotene and α-tocopherol, effectively prevents oxidation of LDL, which plays a key role in the formation of artelectrosclerosis (18–21). p-Cymene is a product of dehydroaromatization of limonene isomers. This compound is a component of numerous essential oils and is becoming more and more popular among scientists. For example, it is a ligand of many catalysts applied in olefi n metathesis—one of the most frequently used reactions in modern organic synthesis. p-Cymene can also be used as a solvent because it is less harmful to the environment than commonly used organic solvents (22–25). Considering the presented antimicrobial properties of limonene, α-terpinene, γ-terpinene, terpinolene, and p-cymene, we decided to investigate whether a mixture of the mentioned compounds (the mixture obtained after the isomerization of limonene) exhibited an anti- microbial activity. Such use of the postreaction mixture of terpenes would lower the cost of preparing therapeutic formulations (e.g., creams), as it will not require a step of isolation of individual components from the postreaction mixture, e.g., by distillation methods. Such a mixture could be treated as an “artifi cial essential oil” with a very narrow composi- tion. The following microorganisms were selected for microbiological tests: Escherichia coli K12 (ACCT 25922) and Staphylococcus epidermidis (ACCT 49461), yeast fungi C. albicans, and fungi Trichophyton rubrum, Aspergillus niger, Penicillium commune, Trichoderma viride, and Cladosporium cladosporioides. With the compound having the highest antimicrobial activ- ity (terpinolene), therapeutic creams containing 0.5 and 2 wt% of this compound were prepared. The creams used in the research were as simple as possible, so that the com- pounds included in their composition did not infl uence the results of the microbiological tests. Therefore, we did not use emulsifi ers as ingredients of the investigated creams. These creams can be used as potential therapeutic and protective creams for relief of skin lesions and in the treatment of acne or atopic dermatitis. MATERIALS AND METHODS SEPAR ATION OF NATURAL LIMON ENE Natural orange oil containing up to 98% of limonene can be obtained from waste orange peels by the steam distillation method. This method does not require the application of organic solvents, which makes it environmentally friendly, and the water used for the process can be reused in subsequent processes. The method of obtaining essential orange oil from orange peels by the steam distillation method is based on passing water vapor through fresh orange peels shredded with the use of a food processor. A kilogram of orange peels prepared in this way was placed in a glass reactor and 2.5 L of distilled water was poured into the reactor. The steam passed through the plant material and volatile components

JOURNAL OF COSMETIC SCIENCE 140 in the steam were condensed in the condenser. The insoluble fraction of the orange oil remained on the surface of the water. The obtained water–oil mixture was then placed in a freezer, and after a few hours, the layer of essential oil was poured from above the ice layer. The essential oil obtained in this way constituted on average 1.06% by mass of the orange peels used (about 10.6 g of oil) and contained 95% of limonene, which was deter- mined on the basis of gas chromatography (GC) analysis. ISOMERIZATION OF NATURAL LI MONENE OVER TI-SBA-15 CATALYST Isomerization of the natura l limonene was carried over the Ti-SBA-15 catalyst. This catalyst belongs to the group of mesoporous materials. Their unquestionable advantages include high hydrothermal stability and neutrality toward living organisms. In the structure of this cata- lyst, hexagonally arranged cylindrical pores are present. In addition, the structure of this cata- lyst is stabilized by micropores, which connect the cylindrical mesopores. The isomerization of limonene was performed in a round-bottom fl ask with a capacity of 25 cm3. For isomeriza- tion, 5 g of the natural limonene and 0.75 g of the catalyst (content in the reaction mixture 15 wt%) were mixed up. The reaction was carried out at a temperature of 150°C for 24 h. The weight of the postreaction mixture amounted to 4.20 g. Before the microbiological tests, the catalyst was separated from the postreaction mixture by centrifugation. The composition of the postreaction mixture was determined by using the external standard method and GC method. For the GC analyses, a Thermo FOCUS chromatograph (Anchem, Warszawa, Poland) was used, which was equipped with a fl ame ionization detector and a Quadrex 007-5 capillary column (30 m × 250 μm × 0.25 μm). The chromatograph was also equipped with an autosampler. The parameters of the chromatographic analyses were as follows: helium pres- sure, 60 kPa sample chamber temperature, 240°C detector temperature, 250°C and ther- mostat temperature, changed according to the program: isothermally 60°C for 2 min, temperature increase 10°C/min, isothermally 240°C for 4 min, and cooling to 60°C. As standards for GC analyses, the follo wing were used: limonene (93%, Sigma-Aldrich, Poznań, Poland), γ-terpinene (97%, Sigma-Aldrich), α-terpinene (85%, Sigma-Aldrich), terpinolene ( 85%, Sigma-Aldrich), and p-cymene (99%, Sigma-Aldrich). Also, the same compounds were used in the microbial tests described in the following paragraph. The results of the analyses of the composit ion of the postreaction mixture (the mixture obtained after the isomerization of limonene) using the GC method showed that the postmixture contained 11.12 wt% (0.43 g) of limonene, 9.02 wt% (0.38 g) of γ-terpinene, 18.17 wt% (0.76 g) of α-terpinene, 13.44 wt% (0.56 g) of terpinolene, and 31.11 wt% (1.31 g) of p-cymene. The conversion of limonene (calculated as the amount of moles of limonene that was converted to isomerization products divided by the initial number of moles of limonene) after 24 h of the reaction amounted to 88.80 mol%, and the selectivities of the obtained products were as follows: 10.6 mol% of γ-terpinene, 21.36 mol% of α-terpinene, 15.80 mol% of terpinolene, and 37.12 mol% of p-cymene. MICROBIOLOGICAL TESTS The antimicrobial propert ies of natural and syn thetic limonene and products of the isom- erization of natural limonene were tested against the Gram-negative bacteria E. coli K12