Antimicrobial Screening of Mentha piperita Essential Oils GO ¨ KALP I ˙ S ¸ CAN,NES ¸ E KI ˙ RI ˙ MER,MI ˙ NE KU ¨ RKCU ¨ OG ˇ LU, K. HU ¨ SNU ¨ CAN BAS ¸ ER, AND FATI ˙ H DEMI ˙ RCI ˙ * Medicinal and Aromatic Plant and Drug Research Centre (TBAM), Anadolu University, 26470-Eskis ¸ ehir, Turkey Essential oils of peppermint Mentha piperita L. (Lamiaceae), which are used in flavors, fragrances, and pharmaceuticals, were investigated for their antimicrobial properties against 21 human and plant pathogenic microorganisms. The bioactivity of the oils menthol and menthone was compared using the combination of in vitro techniques such as microdilution, agar diffusion, and bioautography. It was shown that all of the peppermint oils screened strongly inhibited plant pathogenic microorganisms, whereas human pathogens were only moderately inhibited. Chemical compositions of the oils were analyzed by GC and GC/MS. Using the bioautography assay, menthol was found to be responsible for the antimicrobial activity of these oils. KEYWORDS: Mentha piperita; essential oils; antimicrobial activity; bioautography; TLC; GC; GC/MS INTRODUCTION The well-known and widely used peppermint (Mentha piperita L.) (Lamiaceae) is a cultivated natural hybrid of Mentha aquatica L. (water mint) and Mentha spicata L. (spearmint). Although a native genus of the Mediterranean region, it is cultivated all over the world for its use in flavor, fragrance, medicinal, and pharmaceutical applications. Peppermint oil is one of the most widely produced and consumed essential oils (1-3). Besides its uses in food, herbal tea preparations, and confectioneries, the medicinal uses of mint, which date back to ancient times, include carminative, antiinflammatory, antispas- modic, antiemetic, diaphoretic, analgesic, stimulant, emmena- gogue, and anticatharrhal application. It is also used against nausea, bronchitis, flatulence, anorexia, ulcerative colitis, and liver complaints. Mint essential oils are generally used externally for antipruritic, astringent, rubefacient, antiseptic, and antimi- crobial purposes, and for treating neuralgia, myalgia, headaches, and migraines (2-8). With the development and wide use of synthetic and semi- synthetic antibiotics, pros and cons have been experienced throughout the last 50 years which have directed research back to natural antimicrobial products as indispensable resources (9, 10). Another remaining problem for mankind is plant pathogens and their damage to agriculture. Furthermore, total yield and food quality has to be guaranteed by controlling fungi that produce mycotoxins which affect human health. Filamentous fungi can cause opportunistic systemic mycoses and other complications. Consequently, a large demand exists for fungi- cides, as well as antibacterial agents, in agriculture, food protection, and medicine (9, 11). Mentha spp. have been previously investigated for their essential oil compositions by our group (12, 13). Peppermint oil has been the subject of numerous other studies (13-21). This investigation concerns the antimicrobial screening of M. piperita oils using different bioassay techniques in combination. The antimicrobial activities of the oils were determined and compared by using techniques such as agar diffusion, microdi- lution, and bioautographic agar overlay methods (22-24). The bioautography method aided in the identification of the anti- fungal active component. The chemical compositions of the peppermint oils were evaluated and compared using simple techniques such as thin-layer chromatography (TLC) and gas chromatography (GC). Gas chromatography/mass spectroscopy (GC/MS) analyses were also performed. MATERIALS AND METHODS Plant Materials. Peppermint oils ex Mentha piperita L. were obtained from four commercial sources: (A), Evcin Company, Turkey (plant material was supplied by this company from their farm in Adana, and the oil was obtained in TBAM by hydrodistillation (3.5%, w/v)); (B), Jet-Farms, Yakima, WA; (C), Mari-Linn Farms, Oregon; (D), Erdog ˇmus ¸ Perfume Industry, imported from India. The oils of samples B-D were grown specifically for commercial peppermint oil produc- tion. Major constituents (-)-menthol (Polarome, Jersey City, NJ) (M1) and (-)-menthone (Fluka, Germany) (M2) were purchased from commercial sources. Gas Chromatography. The GC analysis was carried out using a Shimadzu GC-9A with CR4A integrator. A Thermon 600T fused silica capillary column (50 m × 0.25 mm i.d., 0.20-µm film thickness) was used. Carrier gas was nitrogen. Oven temperature was kept at 70 °C for 10 min and programmed to 180 °C at a rate of 2 °C/min, then kept at 180 °C for 30 min. Injector and detector temperatures were 250 °C. Relative percentage amounts of the separated compounds were calculated from FID chromatograms. Gas Chromatograpy-Mass Spectrometry. The oils were analyzed by GC/MS using a Hewlett-Packard GCD system. HP-Innowax FSC * To whom correspondence should be addressed. E-mail: fdemirci@ anadolu.edu.tr. Phone: +90 (222) 335 29 52. Fax: +90 (222) 335 01 27. J. Agric. Food Chem. 2002, 50, 3943-3946 3943 10.1021/jf011476k CCC: $22.00 © 2002 American Chemical Society Published on Web 05/30/2002