Open Chem., 2015; 13: 1326–1332 Open Access Research Article Mirosław A Hawrył*, Krystyna Skalicka-Woźniak, Ryszard Świeboda, Małgorzata Niemiec, Klaudia Stępak, Monika Waksmundzka-Hajnos, Anna Hawrył, Grażyna Szymczak GC-MS fingerprints of mint essential oils DOI: 10.1515/chem-2015-0148 received February 2, 2015; accepted October 21, 2015 Abstract: Essential oils from eleven Mentha species were obtained by Deryng hydrodistillation and analysed by GC–MS: 44 compounds were identified The most abundant were menthone, isomenthone, menthol, carvone, piperitone oxide, D-limonene and eucalyptol Chemometric similarity measures and principal component analysis were calculated, allowing comparisons based on secondary metabolite content The fingerprints may be helpful in chemotaxonomy Keywords: mint, Mentha, GC-MS, fingerprints, essential oils, chemometric, PCA Introduction The widely used Lamiaceae family includes about 220 genera and 3300 species They are rich in polyphenols and well known for their antioxidant properties The genus Mentha (Lamiaceae) comprises 25−30 species found in temperate regions of Eurasia, Australia, and South Africa [1-3] Mentha has been known for its carminative, stimulative, stomachic, diaphoretic, antimicrobial, antiseptic, anesthetic, antiemetic, anti-inflammatory, and strong antioxidant properties Mentha leaves, flowers and stems are used in traditional medicine as spasmolytics, antibacterial agents, promoters of gastric secretions, and relief for heavy colds Mint has been also used in *Corresponding author: Mirosław A Hawrył: Department of Inorganic Chemistry, Medical University of Lublin, Chodźki 4A St., 20-093 Lublin, Poland, E-mail: mirek.hawryl@umlub.pl Ryszard Świeboda, Małgorzata Niemiec, Klaudia Stępak, Monika Waksmundzka-Hajnos, Anna Hawrył: Department of Inorganic Chemistry, Medical University of Lublin, Chodźki 4A St., 20-093 Lublin, Poland Krystyna Skalicka-Woźniak: Department of Pharmacognosy and Medicinal Plant Units, Medical University of Lublin, Chodzki St., 20-093 Lublin, Poland Grażyna Szymczak: Botanical Garden, Maria Curie-Skłodowska University, Sławinkowska 3, 20-810 Lublin, Poland treatments of minor aches and sprains, as well as in nasal decongestants [3-8] Aromatic essential oils are complex mixtures of volatile secondary plant metabolites characterized by strong odor [7] They are primarily monoterpene and sesquiterpene hydrocarbons, their oxygenated derivatives, a variety of aliphatic oxygenated compounds, and a few aromatic compounds [10] Essential oil monoterpene compositions differ giving different or hybrid species different smells [11] Mentha essential oils possess antibacterial, antifungal, antiviral, antimicrobial and anticonvulsive actions and are suitable for food and cosmetics They have significant antioxidant and antitumor action, and some antiallergenic potential [7,9,10,12-15] One-dimensional GC has been the most common approach to unravelling food volatile and semi-volatile compositions It is effective in separation of simple samples With more complex mixtures many peaks result from two or more co-eluting compounds This problem is solved by linking the gas chromatograph to a mass spectrometer (GC-MS), which gives single-component peak assignment and quantitation [16] and identifies multicomponent peaks by deconvolution Obviously, library assignment of unknowns is more reliable (especially for more complex matrices) when high quality mass spectra of resolved compounds are obtained Total separations are always desirable but often difficult [16,17] The main aim of our work was to analyze terpenes and other compounds occurring in Mentha essential oils GCMS was used to construct fingerprints of Mentha species and varieties to facilitate their identification and assist in Mentha chemotaxonomy Experimental procedure 2.1 Plant material Mentha arvensis (L.), M piperita Huds cv Perpeta, M spicata var crispa (Benth.) Danert, M suaveolens var variegata, and M spicata were from the Botanical Garden of Maria Curie-Sklodowska University in Lublin; Mentha x © 2015 Mirosław A Hawrył et al., published by De Gruyter Open This work is licensed under the Creative Commons Attribution-NonCommercial-NoDerivs 3.0 License Unauthenticated Download Date | 2/25/17 6:45 PM GC-MS fingerprints of mint essential oils piperita cultivars Krasnodarskaja and Cernolistnaja were from the Pharmacognosy Garden of the Medical University of Lublin; and M longifolia was collected from a natural stand in Ostrowsko, southern Poland, during summer 2014 The study also included three ready-to-use Mentha species pharmaceutical mixtures (manufacturers 1−3) (Table 1) The identity of all plant materials was confirmed by Dr Grażyna Szymczak (Botanical Garden of Lublin) and voucher specimens were placed in the Botanical Garden of Lublin and the Medical University of Lublin Department of Pharmacognosy Medicinal Plant Unit All solvents were of analytical grade, purchased from Polish Reagents (POCH, Gliwice, Poland) Helium 5.0 was 99.999% pure (PGNiG, Poland) Forty grams of dried, fragmented aerial parts of each Mentha sp were placed in a 1000 mL round-bottom flask along with 600 mL distilled water and subjected to Deryng Table 1: Examined herbal substances and quantity of identified essentials oils Number of Plant material essential oil Mass (g) Oil volume (mL) Contents of oil (% v/m) Mentha piperita L 57.7867 (Manufacturer 1) 0.49 0.85 Mentha piperita L 40.0660 (Manufacturer 2) 0.47 1.17 Mentha arvensis L 40.0900 0.55 1.38 Mentha longifolia 80.0429 L (Essential oil obtained in 2014) 0.68 0.85 Mentha piperita L 52.4290 (Manufacturer 3) 0.45 0.86 Mentha piperita var Zgadka 39.5105 0.54 1.37 Mentha piperita var Perpeta 37.0229 0.60 1.62 Mentha spicata var Crispa 50.2947 0.79 1.57 Mentha suaveolens var Variegata 48.7064 0.56 1.15 10 Mentha piperita 46.3191 var Cernolistnaja 0.34 0.73 11 Mentha spicata L 30.5043 0.33 1.08 12 Mentha longifolia 49.5383 L (Essential oil obteined in 2013) 0.50 1.01 1327 hydrodistillation for three hours (distillation yields in Table 1) The essential oils were collected in small vials and dried over anhydrous sodium sulfate One drop of oil was dissolved in mL of ethyl acetate and refrigerated until analysis 2.2 GC-MS Analysis GC-MS was performed as previously described [18] A Shimadzu GC-2010 Plus GC coupled to a Shimadzu QP2010 Ultra mass spectrometer was used Compounds were separated on a fused-silica capillary column (30 m, 0.25 mm i.d.) with a film thickness of 0.25 µm (Phenomenex ZB-5 MS) The oven temperature program initiated at 50°C, held for min, then increased at 8–250°C min-1, and held for The spectrometer was operated in electron impact mode, the scan range was 40–500 amu, the ionization energy was 70 eV, and the scan rate was 0.20 s per scan The injector, interface, and ion source were kept at 250, 250, and 220°C, respectively Split injection (1 µL) was conducted with a split ratio of 1:20 and 1.0 mL min-1 helium was the carrier gas Retention indices were determined by comparison to a C8–C24 n-alkane homologous series under the same operating conditions Compounds were identified using the Mass Finder spectral library (http://www.massfinder com), and NIST MS data (http://webbook.nist.gov) [19] Individual isolated compound identifications were also performed by comparison of their mass spectra and retention indices with authentic compounds and literature data [18] 2.3 Chemometric and PCA analysis The GC-MS data were exported as jdx from Mass Finder ver 2.3 These were transformed by ImageJ software to JPG files, then saved as 8000 × pixel TIF pictures which allowed 32-bit precision All 12 TIF files were exported as CSV files into SpecAlign ver 2.3 [19] They were polynomial degree Savitzky-Golay smoothed with window halfwidth and denoised by the Symlet8 algorithm, threshold parameter = 0.5 Baselines were generated and subtracted from each chromatogram Finally, all were exported as csv files to ImageJ and transformed to TIF in which all 12 chromatograms were 8000 × pixel pictures The next stage was the calculation of similarity measures: correlation coefficient (R), R2, cosine angle (Cos), Euclidean distance (Euc), Manhattan distance (Man), Chebyshev’s distance (Che), Hausdorff’s distance Unauthenticated Download Date | 2/25/17 6:45 PM 1328 Mirosław A Hawrył et al Figure 1: GC-MS chromatogram of M piperita var Perpeta essential oil Figure 2: GC-MS chromatogram of M spicata var Crispa essential oil (Hau), average mean minimal distance (AMMD), new cosine based on ANND (nCos) and measures by Pieto et al [20] included in the MS-SSIM plug-in: luminance (Lum), contrast (Con), structure (Str) and MS-SSIM indexes Chromatogram alignment is an essential problem However, the SpecAlign RAFFT alignment algorithm decreased similarity between chromatograms of extracts derived from the same species Alignment was thus omitted Unaligned chromatograms were analyzed by PCA using Molegro Data Modeller software The input data was the matrix with 12 columns and 8000 lines Results and discussion Selected chromatograms are shown in Figs and Herb identitifications and essential oil yields are in Table The component relative concentrations (calculated as GC peak area percentages) are shown in Table Compounds present above 2% and their relative amounts are in Table All samples contain 23 compounds: α-pinene, sabinene, β-pinene, β-myrcene, 3-octanol, p-cymene, D-limonene, eucalyptol, β-ocymene, β-linalol, menthone, menthol, piperitone, pulegone, piperitone oxide, α-copaene, β-burbonene, β-cariophyllene, β-farnezene, Unauthenticated Download Date | 2/25/17 6:45 PM 1329 GC-MS fingerprints of mint essential oils Table 2: The relative percentage of the area of peak of essentials oils constituents Numbers as in Table Number of essentials oil (% of the area of peak) Oil component α-pinene NIST 933 sabinene 973 β-myrcene 989 β-pinene 3-octanol α-terpinene p-cymene MF 936 0.14 0.45 0.11 0.20 987 0.26 0.61 0.18 0.12 0.43 0.42 0.02 0.01 978 999 981 1025 973 978 1017 1013 1015 1.44 0.87 0.13 0.76 0.05 1.24 7.67 1.05 0.30 0.60 0.77 0.23 0.66 1.48 10 11 1.13 0.51 0.61 0.43 0.49 0.38 0.58 0.47 1.27 0.17 0.69 1.32 2.32 1.24 0.84 1.06 0.04 0.07 2.31 0.89 1.02 0.92 0.98 0.33 0.10 0.23 0.97 - - 0.01 0.02 - 0.61 1.14 0.06 - 8.38 0.03 0.03 0.10 0.50 1.98 0.17 1.89 2.57 3.67 1.63 0.57 3.23 2.44 2.63 0.86 5.02 4.94 0.78 0.33 0.22 3.58 β-ocymene 1036 1029 0.19 0.14 1.29 1.45 0.03 0.04 0.29 cis-sabinene hydrate 1072 1082 0.04 0.03 0.13 0.01 - 0.08 0.05 0.02 0.12 0.03 0.64 0.03 0.04 0.02 0.06 0.08 0.22 - β-linalool 1102 1086 0.28 0.23 0.89 0.12 0.33 0.25 2.53 0.04 0.12 0.03 0.06 0.04 0.06 0.07 trans-sabinol 1144 1120 0.09 0.21 menthon 1160 1136 1-octen-3-ethyl acetate izopulegol 1119 1151 1051 1093 1132 0.04 0.48 1.21 1059 - 0.74 0.34 γ-terpinene 0.63 - 0.36 0.04 6.83 0.08 0.05 3.82 0.05 0.80 2.40 0.67 0.15 1025 1024 0.52 3.99 1.59 1030 1032 0.85 12 8.14 D-limonene 1,8-cineol (eucalyptol) 0.08 0.76 1.79 31.53 - 0.42 0.14 0.32 0.12 0.33 0.06 - - - 3.11 0.41 0.89 0.10 0.18 0.02 0.05 0.16 0.04 0.03 0.20 0.02 0.10 0.05 - 0.07 0.17 0.37 - - 0.44 0.17 0.02 - 0.27 - - 18.81 17.52 1.22 0.17 10.71 9.23 0.69 0.36 20.57 0.75 2.57 - 0.01 0.02 20.87 0.19 - - - - 0.02 - 6.18 0.34 0.04 7.93 3.95 3.52 - 0.10 4.44 0.19 0.85 3.81 4.10 - 0.22 4.55 0.57 0.44 21.94 1.09 3.95 0.81 29.85 1.88 4.54 0.23 - 0.75 0.11 - 1.76 pinocarvon 1163 1137 - neomenthol 1174 1156 7.07 4.64 0.35 0.25 4.46 izomenthol 1193 1176 30.09 29.59 3.03 0.38 31.29 1.03 1.05 - - 0.79 0.49 0.15 - 0.33 - 0.27 - - 0.21 - - 1.87 - - - - 1.24 0.93 - 15.04 - 0.29 0.04 0.14 - 0.10 0.67 - 2.30 0.07 - - izomenthon menthol α-terpineol trans-dihydro carvon t-carveol 1167 1186 1199 1199 1224 1146 1172 1176 1177 1200 11.66 piperitone 1230 1226 - 0,02 - 0.10 0.03 0.07 carvon 1246 1214 1.60 0.93 - 0.64 0.03 5.76 1.48 0.37 pulegone piperitone oxide neomethyl acetate 1240 1257 1291 1215 1232 1263 13.59 - 1.58 0,05 - 0,13 11 0.07 0.08 0.07 0.33 1.84 0.39 0.82 0.07 0.04 0.03 1.13 15.76 3.26 0.04 42.83 0.15 3.29 1.72 0.31 9.79 5.06 0.61 53.61 0.36 - 2.10 0.94 1.28 0.39 0.39 0.10 - - - - 0.17 - 0.07 0.08 1.05 5.51 5.53 9.16 0.26 0.15 0.03 - 0.26 0.07 0.19 menthyl acetate 1306 1280 - 0.69 - - 1.76 - - - 0.44 0.01 0.52 0.17 0.17 0.34 - β-burbonen 1386 1386 0.03 0.07 0.10 0.07 0.04 0.02 0.06 0.07 0.24 0.54 0.90 0.36 0.34 0.20 0.41 0.41 α-copaen 1378 1379 0.71 - cis-jasmone 1393 1371 - - 0.34 0.13 - 0.09 0.27 0.35 β-farnezen 1452 1446 1.87 2.46 6.35 6.00 1.75 0.86 1.81 1.23 0.12 0.21 0.58 germakrane D 1484 1479 0.13 0.13 0.41 1520 0.92 2.31 3.21 0.08 0.18 1578 0,10 0,29 0.52 0.96 0.80 1.19 0.13 0.69 0.05 - β-cariophyllene α-humulene 1423 1459 δ-kadinen 1520 Cariophyllene oxide 1586 spatulenol globulol 1582 1600 1421 1455 1572 1589 0.05 2.19 1267 1278 0.15 - 0.03 58.98 1299 1300 1.17 - 0.79 0.88 thymol carvacrol 7.19 43.65 0.54 56.27 0.10 0.08 0.14 0.46 0.30 0.63 2.89 1.59 0.57 0.08 1.09 0.31 0.53 6.57 0.05 0.20 0.88 0.10 0.03 - 0.16 0.04 0.41 1.39 2.82 0.40 3.33 0.18 0.06 0.17 0.13 0.06 0.12 0.08 1.28 0.16 0.77 0.77 0.06 4.56 0.11 0.23 0.21 0.09 0.06 0.10 0.10 0,23 0,44 0,56 0,34 0,19 0,22 0,14 1.58 0.78 0.90 0.57 0.64 1.19 0.17 0.29 0.13 0.52 0.14 - 1.90 2.47 0.18 0.03 1,09 0.06 0.77 0.22 1.17 1.59 0.43 0.28 0.52 2.51 0.15 Unauthenticated Download Date | 2/25/17 6:45 PM 1330 Mirosław A Hawrył et al Table 3: The presence of the compounds (above 2%) and their relative concentrations + below 2%, ++ 2-10%, +++ above 10%, - absence Number of essential oil Compounds 10 11 12 β-pinene + + ++ + + + + ++ + + + + β-myrcene + + + + + + ++ + + + ++ + D-limonene + + ++ + + + ++ ++ ++ + ++ ++ eucalyptol + ++ ++ ++ + + ++ ++ + ++ ++ + β-linalool + + + + + + ++ +++ + + + + 1-octen-3-ethyl acetate + + + + + + + + ++ + + + menthon +++ +++ + + +++ +++ ++ + + +++ + ++ izomenthon +++ ++ + + ++ ++ ++ - + ++ + + neomenthol ++ ++ + + ++ ++ ++ - + ++ + + menthol +++ +++ ++ + +++ +++ +++ ++ + +++ + ++ trans-dihydrocarvone - - + - - - - + + - +++ - pulegone + + - - + + + + - ++ - - carvone + + +++ + + + + +++ ++ + +++ + piperitone oxide ++ + + +++ ++ ++ + + ++ ++ + +++ neomethyl acetate ++ +++ + + ++ ++ ++ + - ++ + + β-caryophyllene + ++ ++ ++ + + + + + ++ + ++ germacrene D + ++ ++ ++ + + + + + ++ + ++ globulol + + + - + + + + ++ + + + germakrane-D, δ-kadinene, spatulenol, and cariophylene oxide The compositions of the three finished pharmaceutical products not differ significantly, and each contains all 44 oil components (Table 2) Menthol is the most characteristic Mentha compound Its highest concentration was in M piperita var Krasnodarskaja and other piperita varieties The compound present in largest amount overall was menthone, representing over 10% of the total It was present at high concentrations in M piperita., piperita var Krasnodarskaja and piperita var Cernolistnaja The highest carvone quantities were in M arvensis and spicata varieties The most β-myrcene was in M piperita var Perpeta and M spicata M spicata var Crispa contained significantly more β-linalool The greatest content of 1-octen-3-ethyl acetate was in M suaveolens var Variegata Pulegone was present in seven species but its highest concentration was in M piperita var Cernolistnaja Piperitone oxide was the main component in M longifolia oil obtained in 2014 and 2013 Globulol was identified in all oils (highest concentration in M suaveolens var Variegata) except 2014 oil from M longifolia However, this oil contained more components than the 2013 oil Trans-dihydrocarvone was identified in M spicata and in small quantities in M arvensis, M spicata var Crispa, and M suaveolens var.Variegata Compared with other mint species, M piperita and its varieties contain significantly more menthol (> 20%), menthone (> 9%), isomenthone (> 3.5%), isomenthol (detected only in M piperita varieties), neomenthyl acetate, and β-cariophyllene M arvensis contains significant amounts of sabinene, β-pinene, D-limonene, carvone, and cariophyllene In addition to D-limonene and eucalyptol, M spicata contains significant amounts of β-myrcene, β-burbonene, and large amounts of trans-dihydrocarvone and carvone M spiccata var Crispa also contains a large amount of carvone, but differs from M spiccata in the level of β-linalool and its higher α-pinene, β-pinene, p-cymene and β-ocymene content M suaveolens contains about 4% D-limonene and about 3% 1-octen-3-acetate, which is present only in trace levels in other plants M longifolia differs from the other species; it contains more than 50% piperitone oxide, along with β-caryophyllene and carvacrol Table shows the similarity measures High values of R, R2, Cos, nCos, Lum, Con, Str and MS-SSIM mean better matching; low values of Euc, Man, Che, Hau and AMMD also show better matching Unauthenticated Download Date | 2/25/17 6:45 PM GC-MS fingerprints of mint essential oils 1331 Table 4: GC-MS chromatogram similarity measures and best matching Numbering as in Table 1; abbreviations as in Experimental section Number R of plant Rank R2 Rank Cos Rank nCos Rank Lum Rank Con Rank Str Rank MSSSIM Rank 0.9042 0.8176 0.9074 1.0000 0.9518 10 0.7069 10 0.6392 10 0.4301 10 0.9042 0.8176 0.9074 0.9818 0.9781 0.8249 0.7839 0.6325 0.8013 11 0.6421 11 0.8077 11 0.8794 11 0.9615 0.6995 0.5944 0.3955 0.7839 0.6325 10 11 12 0.9394 0.9008 12 0.8825 0.8115 0.6968 0.7527 12 0.9411 0.9043 0.4855 0.5665 0.3702 0.2638 12 0.9843 0.9994 0.7052 0.7625 0.1371 0.0696 0.8977 0.8013 0.9394 12 0.9603 0.9781 0.9822 10 0.9143 0.3931 0.2967 0.8058 0.6421 0.8825 12 12 0.7566 0.7058 12 0.5129 12 0.8249 0.9681 10 0.7705 10 0.6848 10 0.5109 10 0.9673 0.7268 10 0.6358 10 0.4453 10 0.7021 0.9615 0.6995 0.5569 0.3746 0.6095 0.9634 0.7160 0.5996 0.4137 0.9013 1.0000 10 0.9722 0.7764 0.7013 0.5294 0.8077 0.8794 0.9508 0.6600 0.5760 0.3584 0.9411 0.9843 0.9621 0.7566 0.7058 0.5129 Number Euc of plant Rank Man Rank Che Rank Hau Rank AMMD Rank 0.1089 2,0932 174 71.0282 1.3058 0.0970 1.3949 174 81.0987 0.8022 0.1183 11 2.2426 11 203 11 106.0189 1.8384 11 10 11 12 0.0705 0.0894 12 1.1169 10 1.3949 0.1635 0.1367 12 230 192 2.0410 10 10 2.3038 0.2042 11 0.2125 12 60.1332 62.0725 252 10 193 3.3748 3.9581 0.0894 0.1183 0.0705 12 0.8677 12 10 0.8022 58.4637 10 1.0368 10 10 133.0000 1.6755 200 173.4416 2.4123 200 172.1540 11 3.1209 1.4129 181 58.4637 0.9723 2.2426 203 124.7798 1.8384 1.1169 225 60.1332 0.8677 Oils and 12 have very similar compositions (good matches by all measures except Lum and Che) Table suggests that these analyses can be used for identification It also shows the M spp composition similarity The PCA results in Fig confirm these conclusions These similarities and differences can be used to identify mint species by fingerprinting Conclusions Figure 3: PCA graph for all samples Numbering as in Table The highest oil yields were from M piperita var Perpeta (1.62%), M spicata var Crispa (1.57%), M arvenis (1,38%), M piperita var Krasnodarskaja (1.37%), M.piperita (Manufacturer 2) (1.17%), M suaveolens var Variegata (1.15%), M spicata (1.08%), and M longifolia (2013) (1.01%) GC-MS successfully analyzed terpenoids from Mentha spp essential oils M piperita essential oils contain high amounts of pharmacologically active volatile compounds Unauthenticated Download Date | 2/25/17 6:45 PM 1332 Mirosław A Hawrył et al The similarity measures and PCA analysis show composition similarities and differences among Mentha species GC-MS gives comprehensive information about essential oil composition and can construct herb fingerprints References [1] Kamkar A., Javan J., Asadi F., Kamelinejad M., The antioxidative effect of Iranian Mentha pulegium extracts and essential oil in sunflower oil, Food Chem Toxicol, 2010,48, 1796-1800 [2] Dorman H J., Koşar M., Kahlos K., Holm Y., Hiltunen R., Antioxidant properties and composition of aqueous extracts from Mentha species, hybrids, varieties, and cultivars, J Agric Food Chem., 2003, 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A., Spectral transformations for deconvolution methods applied on gas chromatography–mass spectrometry data Anal Chim Acta, 2003, 488, 231–241 [18] Skalicka-Woźniak K., Walasek M., Preparative separation of menthol and pulegone from peppermint oil (Mentha piperita L.) by high-performance counter-current chromatography, Phytochem Lett., 2014, 10, 94 – 98 [19] Wong J W H., Cagney G., Cartwright H M., SpecAlign— processing and alignment of mass spectra datasets, Bioinformatics, 2005, 21, 2088-2090 [20] Prieto G., Chevalier M., Guibelalde E., http://www.ucm.es/info/ fismed/MSSIM/MSSIM.htm (Access 25 Aug 2015) Unauthenticated Download Date | 2/25/17 6:45 PM ... 1329 GC- MS fingerprints of mint essential oils Table 2: The relative percentage of the area of peak of essentials oils constituents Numbers as in Table Number of essentials oil (% of the area of. .. GC- MS fingerprints of mint essential oils piperita cultivars Krasnodarskaja and Cernolistnaja were from the Pharmacognosy Garden of the Medical University of Lublin; and M... 1328 Mirosław A Hawrył et al Figure 1: GC- MS chromatogram of M piperita var Perpeta essential oil Figure 2: GC- MS chromatogram of M spicata var Crispa essential oil (Hau), average mean minimal