Chemosystematic markers for the essential oils in leaves of Mentha species cultivated or growing naturally in Estonia

Abstract. The content and composition of essential oils in leaves of Mentha × piperita L., M. spicata L., M. arvensis L., and M. longifolia (L.) Huds. from Estonia were investigated using hydrodistillation and GC–MS analyses. Some chemosystematic markers for the identification of Mentha species are presented. The essential oil yield of the studied species was 2.4–3.0%, 0.9– 2.3%, 0.9–1.4%, and 0.7–2.5%, respectively. From the isolated essential oils, 75, 89, 67, and 69 compounds were identified and quantitatively evaluated, representing 92.2–99.5% of the total oil. The main constituents of M. × piperita oils were menthol (26.4– 47.7%) and menthone (13.6–31.9%); the ratio of their content was usually between 1 and 3. Four chemotypes of M. spicata were determined: rich in carvone (62.1–67.4%), rich in piperitenone oxide (61.9%), rich in α-terpinyl acetate + trans-β-caryophyllene (61.9% and 11.4%, respectively), and rich in acetic acid,1-methyl-1-(4-methyl-5-oxo-cyclohex-3-enyl) ethyl ether (6.5%). The last two chemotypes were determined for the first time. Only the essential oils of M. arvensis were found to contain trans-1-octen3-ol (3.8-7.7%) and neryl propionate (2.4% in both samples). Good chemosystematic markers for M. arvensis were also trans(16.0–19.1%) and cis-β-ocimene (16.0–20.5%). The ocimene-rich chemotype was found by us for the first time. The chemosystematics of M. longifolia was found to be the most complicated, as many chemotypes have been determined by us and several other authors. In this study, a high concentration of carvone (51.4–57.6%) was characteristic of all analysed M. longifolia samples.

Most Mentha species are characterized by great morphological variation, reflected in the high number of different taxonomic rank names.In spite of the rather stable qualitative oil composition of cultivated mints, in most wild-growing Mentha species a great chemical diversity is observed (Mimica-Dukic and Bozin, 2008).The findings of our earlier study showed that the qualitative and quantitative content of different groups of terpenoids could be used in the chemosystematics of plant species (Orav et al., 2011a).
The aim of the present study was to find chemosystematic markers for the identification of Mentha species.

Plant material
The content and composition of essential oils hydrodistilled from leaves of Mentha × piperita, Mentha spicata, Mentha arvensis, and Mentha longifolia cultivated or growing naturally in Estonia was studied.All leaves of cultivated or wild Mentha species studied were collected from July to August 2011 from different locations in Estonia (Table 1).A herbarium of the collected plant species was made.The plant material

Hydrodistillation of essential oil
The essential oil was isolated from dried plant material using the distillation method described in the European Pharmacopoeia (2010).To take up the essential oil 20 g of the whole drug and 0.50 mL of xylene were used.Distillation time was 2 h at a rate of 3-4 mL/min.
The components of the oils were identified by comparing their retention indices (RI) on two columns to the RI values of reference standards (from Sigma), to our RI data, and to literature data (Davies, 1990;Zenkevich, 1996Zenkevich, , 1997Zenkevich, , 1999)).The results obtained were confirmed by GC-MS.
The percentage composition of the oils was calculated using the normalization method without correction factors.The relative standard deviation of the percentages of oil components in three repeated GC analyses of the single oil sample did not exceed 5%.
Previously we found much more significant differences between the minimum and maximum yields of essential oils: 17-fold in Coriandrum sativum (Orav et al., 2011b), 16-fold in Levisticum officinale (Raal et al., 2008), 14-fold in Thymus serpyllum (Paaver et al., 2008), and 10-fold in Chamomilla recutita (Orav et al., 2010), collected or obtained as commercial samples from different retail pharmacies in various countries.Over years, the variance of oil yield in Juniperus communis branches was 14-fold (Raal et al., 2010).Therefore, instead of the content of essential oil, the yield of terpenoids should be the basis of the chemosystematics of the genus Mentha.Moreover, the content of oil can be affected by several factors such as climatic and vegetation variations.
Menthone, the second main constituent of M. × piperita oil, can also be used in the chemosystematics of peppermint.The oil of the M. × piperita sample from Pangodi village contained menthone even above the EP limit (32%).Menthone and menthol were the typical components of peppermint oils and can be used as chemical markers.The ratio between their content according to our current study was 0.05-3.4,but usually 1-3; the same ratio was found in our previous (Orav et al., 2004) and other studies (Pino et al., 2002;Schmidt et al., 2009;Sokovic et al., 2009).According to Rohloff and co-authors (2001), changes in menthone-menthol metabolism are related to reduced menthone content from the vegetative stage to full bloom.
The presence of trans-1-octen-3-ol and neryl propionate was specific only to essential oils from M. arvensis (Table 2).Good chemosystematic markers for M. arvensis were also trans-and cis-β-ocimene, both being present in the concentration of 16% and more.This mint species contained also significantly more β-pinene and α-pinene, as well as 1-octen-3-ol than other mint species studied.In other studies (Rao, 1999;Rao et al., 1999;Pandey et al., 2003;Hussain et al., 2010a;Pandey et al., 2010;Prasad et al., 2010) menthol was determined as a principal compound of M. arvensis oil but cis-β-ocimene was not found.The ocimenes-rich M. arvensis chemotype was determined by us for the first time.
The main results of our study are presented in Table 3.

Table 1 .
Mentha spp.samples studied from Estonia Table 1 for number and origin of samples.