J. Cosmet. Sci., 70, 137–147 (May/June 2019) 137 Microbiological Tests of Natural Limonene and the Compounds Obtained after Isomerization of Limonene in the Presence of Ti-SBA-15 Catalyst—α-Terpinene, γ-Terpinene, Terpinolene, and p-Cymene A GNIESZKA WRÓBLEWSKA, M. RETAJCZYK, D. KĄDZIOLKA, and A. MARKOWSKA-SZCZUPAK, Institute of Organic Chemical Technology, Faculty of Chemical Technology and Engineering, West Pomeranian University of Technology Szczecin, 70-322 Szczecin, Poland (A.W., M.R.) , Institute of Inorganic Chemical Technology and Environmental Engineering, Faculty of Chemical Technology and Engineering, West Pomeranian University of Technology Szczecin, 70-322 Szczecin, Poland (D.K., A.M.-S.) Acce pted for publication June 7, 2019. Synop sis The antimicrobial properties of natural limonene and the compounds obtained after isomerization of limonene (α-terpinene, γ-terpinene, terpinolene, and p-cymene) were studied. The following microorganisms were selected for the tests: Gram-negative bacteria Escherichia coli K12 (ACCT 25922), Gram-positive Staphylococcus epidermidis (ACCT 49461), yeast fungi Candida albicans, and fungi Trichophyton rubrum, Aspergillus niger, Penicillium commune, Trichoderma viride, and Cladosporium cladosporioides. During the studies, terpinolene showed the highest activity, and therefore, this compound was chosen for the preparation of therapeutic creams (content of terpinolene: 0.5 and 2 wt%). The obtained creams were active in the microbiological tests even at the lowest content of terpinolene. The mixture of products obtained after the isomerization of limonene also showed antimicrobial activity. Probably, in the future, this mixture of products can be used as a potential and relatively inexpensive ingredient in therapeutic and protective creams that can be applied for the relief of skin lesions and in the treatment of acne or atopic dermatitis. INTRODU CTION Essenti al oils containing terpene compounds can fi nd numerous applications in medicine. Studies conducted with the participation of terpenes isolated from various essential oils showed their bactericidal or bacteriostatic activity. This is connected with the high lipo- philicity of terpenes, thanks to which they easily penetrate the cell walls and membranes of various microorganisms, which in turn disrupts the integrity of these structures and Address all correspondence to Agnieszka Wróblewska at agnieszka.wroblewska@zut.edu.pl.

JOURNAL OF COSMETIC SCIENCE 138 leads to the disintegration of microbial cells. Toxic action of terpene compounds against microbes consists in coagulation of the cytoplasm and increased cell membrane permea- bility, resulting in excessive loss of hydrogen and potassium ions, which in turn causes reduction in membrane potential, which disturbs the functioning of proton pumps and, thus, reduces the amount of synthesized adenosine triphosphate (1–4). An example may be tea tree oil, which contains about 100 compounds (monoterpenes, sesquiterpenes, and their alcohol derivatives). The main components of tea tree oil are terpinene-4-ol, α-terpinene, and γ-terpinene (70%). In this oil, the following are also present: p-cymene, terpinolene, α-terpineol, and α-pinene (15%). Research on tea tree oil showed (3,5,6) that most of the ingredients in this oil inhibit the development of Candida albicans yeast—a commensal organism—that can become pathogenic in immunocompromised people. In addition, these studies showed that the antifungal activity of this oil can be infl uenced by all of its ingredients, even those that occur in small quantities or are con- sidered as inactive. Limonene is a terpene compound that is very valuable and of natural origin and can be obtained from waste orange peels (renewable waste from the food industry, biomass). By simple isomerization of this compound (over an appropriate catalyst, e.g., Ti-SBA-15, and without any solvent), it is possible to obtain from limonene other valuable compounds, such as α-terpinene, γ-terpinene, terpinolene, and p-cymene (Figure 1). These com- pounds, the same as limonene, have a lot of applications in medicine and also in cosmetic and food industries. Of particular interest are the cosmetic and medical applications of these compounds (7–9). α-Terpinene is a fragrant compound of natural origin that is present in various foods, such as oranges, coriander, and oregano. It can also be obtained as the product of isomerization of limonene. Thanks to its characteristic refreshing scent, it is widely used as a fragrance in cosmetic, household, and food products. α-Terpinene is a com- ponent of tea tree oil and is considered to be a component responsible for the anti- oxidant properties of this oil. The properties of tea tree oil allow the use of this oil in the treatment of skin problems, such as acne or mycosis. Studies on α-terpinene showed the effi ciency of this compound in the treatment of parasitic infections with auger in horses (10–14). γ-Terpinene is anot her product that can be obtained during the isomerization of limonene. In nature, this compound can be synthesized by a plant like rice (γ-terpinene destroys the cell membrane of the bacteria that causes plague among this plant). Generally, this com- pound exhibits antibacterial properties. Moreover, γ-terpinene in combination with routine Figure 1. Structure of limonene and the products of limonene isomerization and dehydroaromatization.