Essential Oils of Lamiaceae with High Content of α-, β-pinene and Limonene Enantiomers

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1 ISSN Print: X ISSN Online: Essential Oils of Lamiaceae with High Content of α-, β-pinene and Limonene Enantiomers Hanna G. Shutava 1 *, Natallia A. Kavalenka 2, Halina N. Supichenka 2, Victor N. Leontiev 2 and Tatsiana G. Shutava 3 1 Department of Biochemistry and Biotechnology, Central Botanical Garden of Belarus, Minsk, Belarus, , 2 Department of Analytical Chemistry, Belarusian State Technological University, Minsk, Belarus, , 3 IfM, Louisiana Tech. University, Ruston, Louisiana, 71272, USA Abstract: The essential oils from Korean mint (Agastache rugosa (Fisch. et Mey)) and Lemon mint (Mentha piperita var. citrata (Ehrh.) Briq.) have a high content of chiral terpene compounds that reaches 16.1 % and 10.5 % accordingly with the predominance of limonene, while the oils are obtained from preliminary dried plant material. The content of the enantiomers in peppermint (Mentha x piperita L) oil does not exceed 3.8 %. In each of three investigated Lamiacea species, one specific enantiomeric form of α-pinene, β-pinene, and limonene prevails during all vegetation period; the differences in accumulation of these three monoterpene enantiomers in essential oils obtained from the plants on different ontogenetic stages and after different preprocessing history (fresh and dried material) are discussed for the first time. (-)-α-pinene was found as the dominating form in the essential oils from all investigated species. (-)-β-pinene and (+)-limonene with a % enantiomeric excess were detected in the oil from Agastashe, while the (+)-enantiomer of β-pinene and the (-)-limonene prevails in both essential oils from the Mentha kind. The analysis of terpene enantiomer composition in the plant essential oil confirms the morphological classification of M. citrata as a variant of M. piperita with a high accumulation of citral B. Key words: Enantiomer, chirality, aromatic plants, herbs, essential oils. Introduction The Lamiaceae (Lindl.) family consists of about 200 kinds (up to 3500 species) of herbaceous plants broadly distributed all over the world and particularly widespread in the Mediterranean. Several plants of the family are of interests as a source of essential oils; they differ by high level of accumulation and variety of fragrant substances. The differences in odor and remedial properties displayed by plant essential oils are strongly connected to their overall composition as well as to the structure of chiral constituents, as optical isomers of the same compound can have various effect on organism. As commercial application of essential oils for flavor and fragrance increases, the optimization of profits by using synthetic compounds or cheaper natural essential oils from another plants has became a logical consequence. Utilization of enantiomer composition as a proof of phytoproduct authenticity is a perspective method of elimination of adulterated essential oils The composition of essential oils originated from plants depends on many factors, such as *Corresponding author (Hanna G. Shutava) < anna_shutova@mail.ru > 2013, Har Krishan Bhalla & Sons

2 difference in chemotypes, plant growing conditions, technology of production and storage of the vegetative raw materials 15. Here we report composition and enantiomeric terpene distribution of essential oils from Lamiaceae plants cultivated in the central region of Belarus: korean mint (Agastache rugosa (Fisch. et Mey)), peppermint (Mentha x piperita L.), lemon mint (Mentha piperita var. citrata (Ehrh.) Briq.) grown in the Central Botanical Garden, Minsk (the Herbs and Spice Collection). As to the authors knowledge, despite the data on composition of essential oil from A. rugosa and its bioactive properties are widespread 3,18,19,21, the distribution of terpene enantiomers in the oils obtained from the plant on different ontogenetic stages and after different preprocessing history (fresh and dried material) has been never reported. Enantiomeric ratios of three terpenes, such as α-, β-pinenes, and limonene, in several commercial samples of Mentha kind essential oils have been previously investigated 13. However, the M. citrata species from the Collection differs from other essential oil sources, like M. piperita, in the major constituents composition, more specifically by the predominance of citral B instead of menthol and menthon 12,14,16,17. The enantiomeric analysis of most common monoterpenes in this essential oil is of interest as an indication of the activity of terpene synthesizing enzymes. The evaluation of constituents of essential oils obtained from three Lamiaceae species cultivated and processed under similar conditions allows for better understanding of biosynthesis and transformation of chiral secondary metabolites. Materials and methods Plant material The aerial parts of Agastache rugosa, Mentha piperita, Mentha piperita var. citrata plants were collected in 2009 from the Herbs and Spice Collection of the Central Botanical Garden, National Academy of Sciences of Belarus. A half of the collected sample was immediately investigated and the other part was dried in shade at room temperature for two days and then analyzed. Extraction and isolation of oils The plant materials were subjected to hydrodistillation in a Ginsburg type apparatus (0.2 kg each loading) for 2 h 6. The collected samples of essential oils were dried with anhydrous Na 2 SO 4 for 24 h and stored at 4-8 C until analyzed. Chromatography A Tsvet-800 gas chromatograph (TsvetChrom LTd., Russia) equipped with a HP-5 column (Agilent Technologies Inc., (5 %-phenyl) methylpolysiloxane: equivalent to USP Phase G27, 30 m x 0.25 mm, film thickness 0.25 μm) with N 2 as a carrier gas was used. The column temperature was ranged from 50 to 200 C with an increase programmed at 3 C/min. Characterization of essential oil composition was achieved on the basis of retention indices (RI) using a homologous series of n-alkanes (Supelco) and standard substances. A flame ionization detector (FID) was used through the method. The enantioselective capillary GC analysis was conducted on a Cyclosil B (Agilent Technologies Inc.) capillary column (30 m x 0.32 mm, film thickness 0.25 μm, 30 % hepatkis (2,3-di-Omethyl-6-O-t-butyl dimethylsilyl)-β-cyclodextrin in DB-1701). The column temperature was ranged from 70 to 200 C in five consecutive steps: a 5 min isotherm at 70 C, an 3 C/min increase to 115 C, an isotherm for 20 min, a 4 C/ min increase to 200 C and an isotherm for 10 min. Nitrogen was used as a carrier gas at the linear speed of 30 cm/s, the split ratio was 1:50. The identification of terpene enantiomers was performed on the basis of retention indices of the substances: ( (-)-α-pinene (P7408, Sigma), (+)- α-pinene (W290238, Kosher, Aldrich), (-)-βpinene (402753, Aldrich), (+)-β-pinene (80607, Fluka), (-)-limonene (62128, Fluka), (+)- limonene (62118, Fluka)) in a standard mixture. Triplicate analyses of each oil sample were performed and quantitative results were presented as a mean of the data derived from the GC-FID analyses. The relative amounts of individual components (in %) were calculated on the basis of GC peak areas without using correction factors. Limited experimental data available on retention indices (RI) of terpene enantiomers and

3 the uncertainty in the order a pair of +/- optical isomers exit various stationary phases 7,8,13 do not allow unequivocal theoretical assignment of a specified peak to a particular optical isomer. Therefore, a standard mixture of the terpene enantiomers was preliminary separated on the Cyclosil B capillary column and retention times for three most common enantiomeric compounds were determined (Table 1). Results and discussion High qualitative and quantitative differences exist in the compositions of the oils extracted from the plants collected upon mass blooming (Table 2). Estragol and isomenthon were found to be the most abundant constituents in the essential oil of A. rugosa; their summarized content goes over 60 %. Estragol was completely absent in oils from both Mentha species, while 3-4 % of isomenthon was found in M. piperita. Menthon and menthol are two major components in M. piperita (up to 75 % total), while citral B reaches 83 % in the essential oil from M. piperita var. citrata. The essential oils of A. rugosa and M. piperita var. citrata show a high content of chiral terpene compounds, α-, β-pinenes, and limonene (Figure 1.), that exceeds 10 % with limonene being the most abundant among them. For comparison, M. piperita oil contains less than 3.8 % of the compounds (Table 2). A simultaneous analysis of essential oil Table 1. Retention times and RI of terpene enantiomers on a Cyclosil B capillary column Compound Retention time, min RI 1S-(-)-α-Pinene R-(+)-α-Pinene R-(+)-β-Pinene S-(-)-β-Pinene S-(-)-Limonene R-(+)-Limonene Figure 1. Typical chromatogram of M. piperita var. citrata essential oil on a Cyclosil B capillary column and the structures of selected terpenes with chiralic centers indicated with asterisks

4 composition and most common enantiomeric terpenes in the oils gives insights in the activity of enzymes involved in terpenes biosynthesis in the selected plants. α-, β-pinenes, and limonene are synthesized in plants from geranyl diphosphate as a nonracemic pair of dextrorotary (+) / levorotary (-) optical isomers, depending on specific pinene and limonene synthases occurring in the plant of interest 2,5,10. The pinene synthase from grand fir produces (-)-α- and (-)-β- pinene at a ratio of 42:58 2, and the (-)-pinene synthase from sage (Salvia officinalis) produces an assortment of five monoterpenes: (-)- camphene, (-)-α-pinene, (-)-β-pinene, myrcene, and (-)- limonene 5. The enzyme cloned from Artemisia annua revealed in vitro a great preference for Table 2. Percentage composition of the essential oils from selected plants of the Lamiaceae family Compounds RI* Mentha Mentha piperita Agastache piperita var. citrata rugosa Fresh Dried Fresh Dried Fresh Dried α-thujene α-pinene Camphene Sabinene β-pinene Myrcene Phellandrene ,8-Cineole Limonene (E)-β-Ocimene (E)-Sabinenhydrate Linalool Isomenthon Menthon Menthofuran Menthol Terpinene-4-ol Estragol Isomenthol α-terpineol Citral B Pulegone Piperiton Carvone Menthylacetate β-elemene β-burbonene β-caryophyllene Aromadendren α-elemene Total identified *retention indices of individual compounds on a HP-5 column

5 (-)-β-pinene, which represented 94 % of the product, much higher than that produced by its counterpart in grand fir or sage 10. Furthermore, the β-/α-ratios detected from the in vitro assay and from the needles of grand fir trees were different, and this may suggest the presence of another pinene synthase that produces more α- pinene in this plant 2. From the most of Lamiaceae plants, including M. spicata and M. longifolia, (-)-limonene synthase genes have been cloned. Only in Schizonepeta tenuifolia (Japanese catnip) and A. rugosa (+)-limonene synthases occurs 11. The essential oil of A. rugosa from the collection was characterized by a low concentration of α-pinene through all flowering period, with the substance being mainly in the form of (-)-isomer (Table 3). A low concentration of the (+)-isomer was observed on the termination of blooming stage in fresh processed samples, but not confirmed by the analysis of oil from preliminary dried material. The concentration of β-pinene in the oil obtained from fresh A. rugosa plant material was negligible, an accounted amount of (-)-β-pinene was observed only on the termination of blooming stage (Table 3). However, the oils produced from the dried plant had the β-/α-ratios between 0.54 and 3.0 accompanied by a decrease in α-pinene concentration. In oils from the dried plants, (-)-β-pinene dominated through all ontogenetic stages, traces of (+)-β-pinene were observed only on the full blooming stage. One can conclude that in A. rugosa the (-)-α-isomer is preferably synthesized. Furthermore, (-)-β-pinene appeared only after drying and can be due to activation of a α β transition process on the drying step 4. The difference with Artemisia annua 10 in pinene synthase activity is clearly seen. The highest limonene concentration irrespective of its optical activity was noticed in the essential oils from the vegetative raw material collected by the end of mass flowering of A. rugosa. It reached 15.6 % by weight (Table 2). The analysis of limonene enantiomeric distribution confirmed the domination of (+)-limonene biosynthesis in A. rugosà (Table 3). The finding is in good accordance with Pino, et. al., 17 and Rohloff, et. al. 18 and differs A. rugosà from M. piperita and M. piperita var. citrata, the plants with dominating synthesis of (-)-limonene. According to Mastelic, et. al., 12 and Murray, et. al., 14, the main components of essential oil of M. piperita var. citrata are linalool (10-55 %) and linalylacetate (3-21 %). The essential oil of lemon mint from the collection principally differed from the samples described in literature. Achiralic citral B was the main compound of the essential oil obtained at different ontogenetic stages (Table 4). Insignificant content of all other components was found, including another aliphatic monoterpene linalool (less than 0.4 %). Linalylacetate was completely absent in the oil. The limonene content in the oil was high and reached % on different ontogenetic stages (Table 4). Limonene was found in the Table 3. Enantiomeric distribution of α-pinene, β-pinene and limonene in Agastache rugosa essential oil (%) Enantiomer Material Ontogenetic stage preprocessing Budding Blooming Termination of blooming (-) (+) E.e.* (-) (+) E.e. (-) (+) E.e. α-pinene fresh dried β-pinene fresh dried Limonene fresh dried * enantiomeric excess.

6 Table 4. Percentage composition of the essential oil from Mentha piperita var. citrata Compounds Ontogenetic stage Vegetation Budding Early blooming Blooming Termination of blooming α-pinene β-pinene B-Myrcene ,8-Cineole t t t Limonene Ocimene Linalool t 0.40 Menthone 0.45 t Menthophyrane 0.41 t Terpinene-4-ol α-terpineol Citral B Piperitone β-elemene Caryophyllene investigated oil sample as its (-)-enantiomer with a 40 % excess (Table 5). The level of biosynthesis of α- and β-pinenes in M. piperita var. citrata was generally low. In the essential oil the (-)-form of α-pinene prevailed while β-pinene was presented to a slightly higher degree by the dextrorotatory isomer (Table 5). Such a terpene enantiomers accumulation confirmed the classification of the plant as a variant of M. piperita made on the basis of its morphological characteristics but complete excluded the possibility to consider it as one of M. aquatic x M. viridis hybrids that produces essential oil with pronounce citric flavor 13. Typical composition of peppermint essential oil is described in details elsewhere 17,18. The GC analysis of the essential oil of Mentha x piperita L. grown in the collection showed that it contains menthol as a major component along with a large amount of menthon and small amounts of limonene, isomenthon, menthofuran, isomenthol, and pulegone. The constituent compositions of the oils obtained from fresh and dried phytomass of M. piperita are similar (Table 2). However, Table 5. Enantiomeric distribution of α-, β-pinene and limonene in Mentha piperita var. citrata and Mentha piperita essential oil (%) Enantiomer Material Mentha piperita var. citrata Mentha piperita preprocessing Ontogenetic stage Budding Termination of blooming Blooming (-) (+) E.e.* (-) (+) E.e. (-) (+) E.e. α-pinene fresh dried β-pinene fresh dried Limonene fresh dried

7 higher concentrations of β-pinene, limonene, 1,8- cineol, and menthylacetate were found in the oil from preliminary dried material. The essential oil from M. piperita cultivated in the collection had a typical for this species composition with high menthol content. In the investigated sample of essential oil obtained from the dried M. piperita plant material collected on the blooming stage, enantiomeric excesses of (-)-α-pinene and (+)-β-pinene were observed. The levorotatory form of limonene was found in much greater excess (83 %) than in the essential oil of M. piperita var. citrata (Table 5). The last observation is typical for peppermint essential oils 9. Thus, the essential oils from A. rugosa and M. citrata were characterized by a high content of chiral terpene compounds that reaches 16.1 % and 10.5 % accordingly with the dominance of limonene, while obtained from preliminary dried plant material. The content of the enantiomers in the M. piperita oil processed under similar conditions does not exceed 3.8 %. In each of three investigated Lamiacea species, one specific enantiomeric form of α-pinene, β- pinene, and limonene prevailed during all vegetation period. Such dominance was more prominent in the essential oil of korean mint (a % enantiomeric excess for each constituent). In peppermint and lemon mint, one of the forms was synthesized to a greater extend. (-)-α-pinene was found as the dominating form in the essential oils from all investigated species. For β-pinene, the (-)-enantiomer was detected in trace amounts in Agastashe, while the (+)- enantiomer was prevalent in both Mentha specimens. Limonene exclusively exists as the (+)-form in Agastashe, while in the essential oils from the Mentha kind the (-)-form is abundant. These differences in the occurrence of one or another form of limonene and β-pinene, along with overall constituents composition, can be used for detection of korean mint raw plant material admixed to phytopreparations of Mentha including essential oils. Since only one enantiomeric form of limonene which is synthesized in high amounts in korean mint, the pant raw material can be used as a natural source of the isomer. Acknowledgment The authors are grateful to Dr. Lidiya V. Kuchareva, the curator of the Herbs and Spice Collection, Central Botanical Garden, NAS of Belarus for the plants given for research. References 1. De Sousa, D.D., De Farias, F.F. and De Almeida, R.N. (2007). Influence of the chirality of (R)-(-)- and (S)-(+)-carvone in the central nervous system: A comparative study. Chirality. 19: Bohlmann, J., Steele, C.L. and Croteau, R. (1997). Monoterpene synthases from grand fir (Abies grandis). cdna isolation, characterization, and functional expression of myrcene synthase, (-)-(4S)-limonene synthase, and (-)-(1S,5S)-pinene synthase. J. Biol. Chem. 272(35): Charles, D.J., Simon, J.E. and Widrlechner, M.P. (1991). Characterization of the essential oil of Agastache species. J. Agric. Food Chem. 39 (11): Diaz-Maroto, M.C., Cabezuclo, M.D. and Perez-Coello, M. (2002). Effect of Drying Method on the Volatiles in Bay Leaf (Laurus nobilis L.). J. Agric. Food. Chem. 50(16): Gambliel, H. and Croteau, R. (1984). Pinene cyclases I and II. Two enzymes from sage (Salvia officinalis) which catalyze stereospecific cyclizations of geranyl pyrophosphate to monoterpene olefins of opposite configuration. J. Biol Chem. 259(2): Godovalnikov, G.V. (2006). Pharmacopoeia of the Republic of Belarus: General methods for drug quality control. Minsk, 650 p. 7. Hener, U., Kreis, P. and Mosandl, A. (1990). Enantiomeric distribution of α-pinene, β-pinene and limonene in essential oils and extracts. Part 2. Oils, perfumes and cosmetics. Flavour and

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