In vitro production of M . × piperita not containing pulegone and menthofuran

The essential oils (EOs) and static headspaces (HSs) of in vitro plantlets and callus of Mentha x piperita were characterized by GC-MS analysis. Leaves were used as explants to induce in vitro plant material. The EO yields of the in vitro biomass were much lower (0.1% v/w) than those of the parent plants (2% v/w). Many typical mint volatiles were emitted by the in vitro production, but the callus and in vitro plantelet EOs were characterized by the lack of both pulegone and menthofuran. This was an important difference between in vitro and in vivo plant material as huge amounts of pulegone and menthofuran may jeopardise the safety of mint essential oil. Regarding the other characteristic volatiles, menthone was present in reduced amounts (2%) in the in vitro plantlets and was not detected in the callus, even if it represented the main constituent of the stem and leaf EOs obtained from the cultivated mint (26% leaves; 33% stems). The M. piperita callus was characterized by menthol (9%) and menthone (2%), while the in vitro plantlet EO showed lower amounts of both these compounds in favour of piperitenone oxide (45%). Therefore, the established callus and in vitro plantlets showed peculiar aromatic profiles characterized by the lack of pulegone and menthofuran which have to be monitored in the mint oil for their toxicity.

Identifying species in the genus Mentha is complicated due to extensive hybridization (Tucker & De Baggio, 2000).Furthermore, it may be assumed that the reported variability in peppermint oil is not due to genetic differences, since most of the commercial plantings, at least in North America and Europe, were propagated vegetatively from plants of the Black Mitcham variety, which originated in England.The European Pharmacopoeia reported a standard range of 30-55% menthol as target oil composition.High menthol content (44%) is the main criterion of peppermint oil quality according to ESCOP (1992).In fact, the acceptable commercial quality of peppermint oil is strictly related to the content of menthol, menthone, and menthyl acetate, with little or no pulegone and menthofuran (Burbott & Loomis, 1967;Murray et al., 1988).
Therefore, for some species, such as peppermint, a proportion of the oil yield must be sacrificed to ensure the required oil quality (Clark & Menary, 1979;1981).It is well known that mint of high commercial value can be produced only in certain geographic areas (Clark & Menary, 1981;Lawrence, 1985;Maffei, 1999).Additionally, the yields and the composition of peppermint essential oil are also strongly influenced by yearly weather conditions, harvest date, plant age (Weglarz & Zalecki, 1985, 1987;Murray et al., 1988;Kumar et al., 2000) as well as fertilization and planting time (Voirin et al., 1990;Marotti et al., 1994;Misra & Srivastava 2000).Furthermore, the oil composition is also related to leaf position with increasing menthol and decreasing menthone content in the basipetal direction (Maffei et al., 1994;Rohloff, 1999;Gershenzon et al., 2000).
Flower oil has much more pulegone and menthofuran than leaf oil and more than 50% of the flower oil may consist of pulegone and menthofuran, with less than half the oil is composed of menthone and menthol (Murray et al., 1988).Due to the high commercial value of peppermint EO, several efforts have already been made by plant biotechnological approaches as an alternative promising way to control the EO production and improve peppermint quality (Maffei et al., 2007).Callus, cell tissue cultures, biotransformation, immobilization bioreactors have produced considerable amounts of terpenoids, although in each case the choice of donor or parental plants was crucial (Banthorpe, 1996).Some cell lines or suspension cultures of M. piperita can synthesize essential oils and numerous efforts have been made to produce essential oil in vitro.Culture conditions such as pH, hormone concentration, seeding density, which affect cell growth and essential oil production, have been investigated (Kim et al., 1996;Tisserat & Silman, 2000;Maffei et al., 2007).
In particular, M. piperita shoot cultures are extremely sensitive to artificial light and temperature.In fact, menthofuran biosynthesis is favoured by long periods of relatively low light intensities and warm night temperatures, whereas menthone is accumulated under short light photoperiods and cold nights in peppermint cultures (Spencer et al., 1993).Agrobacterium-mediated and direct gene transfers into protoplasts of M. piperita cv Black Mitcham have been already successfully used to produce stable, transformed peppermint plants with the limonene synthase gene.This regenerated plant material was characterized by high menthone, menthofuran and pulegone content and low menthol level in comparison with the typical Midwest peppermint (Krasnyanski et al., 1999).Maximum accumulation of terpenoids has been found in the late exponential phase of the cell culture cycle and is higher in cell suspension than in callus cultures (Banthorpe, 1996).Furthermore, some studies have been undertaken to test the applicability of cell-recycled airlift bioreactors for high-density cultures of M. piperita cells (Maffei et al., 2007).However, most of these in vitro protocols are not yet of commercial relevance.Despite of all these different biotechnological attempts on M. piperita, the industrialization of mint cell cultures for the production of their essential oils is still limited by low productivity, low growth rate of cells, and high sensitivity to shearing (Kim et al., 1996;Banthorpe, 1996;Diemer et al., 1998;Tisserat & Silman, 2000;Bhat et al., 2002).
The aim of the present study, as part of a European Project (EC NUTRA-SNACK, 6 FP), was to investigate a selection of adult plants of M. x piperita cultivated in Poland as a starting raw material in order to establish callus and in vitro plantlets with a standardised volatile profile.

MATERIALS AND METHODS
Plant material.Stolons of M. x piperita were received from the National Centre for Plant Genetic Resources at the Plant Breeding and Acclimatization Institute (Radzikow, Poland).Field plant material was obtained in 2009 from the field experiments performed at the Experimental Farm, Pulawy, Poland.Plants were collected during the second vegetative year at the beginning of flowering, air dried and powdered.
Procedure of sterilization and planting.Apical buds of field grown plants were rinsed with tap water prior to surface sterilization with 70% ethanol for 1 min followed by 10% perhydrol (H 2 O 2 ) treatment for 5 min.Then they were rinsed three times with sterile distilled water and placed on half strength LS basal medium (Linsmayer & Skoog, 1965) supplemented with 15 g L -1 sucrose and solidified with agar at 6 g L -1 , adjusted to pH 5.8, and autoclaved at 121°C and 0.1 MPa for 20 min.The Sterilized material was placed on the medium in Petri dishes and was then were incubated in a growth chamber at 25°C, under a 16 h photoperiod and a light intensity of 140 μE m -2 s -1 .
After two weeks of growing apical buds were moved to LS medium enriched with 0.2 mg L -1 NAA (naphthaleneacetic acid) and 0.2 mg L -1 IAA (indole-3-acetic acid).NAA as well as IAA stock solutions were prepared by dissolving the substances in a drop of 1M NaOH and adjust adjusting to a desirable volume with deionized water.The Hormones were filter sterilized and added to the autoclaved LS medium.Young plantlets developed on this medium were propagated via cuttings.All plant material was kept in the growth chamber under the same conditions as above.
Callus cultures induction.30-day old leaves from in vitro plants were used as initial explants.They were cut into small pieces and transferred on several LS media variants supplemented with dichlorophenoxyacetic acid (2,4-D) in combination with 6-benzylaminopurine (BA) or isopenthenyladenine (2iP) at the concentration 0.5; 1 and 2 mg L -1 .Agar was added to each basal medium and pH was adjusted to 5.8.Callus tissues were grown in Petri dishes, which were incubated in a growth chamber at 25°C and a 16 h photoperiod, provided by cool white fluorescent lamps.The Tissue culture was subcultured every four weeks.For biochemical analysis callus tissues coming from the 7 th passage were used.

Phytochemical analysis
Chemicals.Commercial standards and isolated compounds from aromatic plant species were part of a homemade database of volatiles where each compound was used as a reference material after GC-MS grade purity determination (98-99%).The samples and standard solutions were prepared using n-hexane (Carlo Erba, HPLC-grade).
Extraction procedure.Freeze-dried plant samples were hydrodistilled (2 h, 2 L distilled water, flow 2.0 mL/min) by a Clevenger apparatus described in the European Pharmacopoeia V Ed.The essential oils were dissolved in Et 2 O, dried over anhydrous MgSO 4 , filtered and the solvent removed by evaporation on a water bath.The essential oils were diluted in n-hexane (HPLC solvent grade, 10%) and analysed by GC-FID (injection volume 1 μL, HP-WAX and HP-5 capillary columns) and GC-MS (injection volume 0.1 μL, DB-5 capillary column).
GC-FID analysis.GC-FID analyses were accomplished by a HP-5890 Series II instrument equipped with HP-WAX and HP-DB-5 capillary columns (30 m × 0.25 μm, 0.25 μm film thickness), working with the following temperature program: 60°C for 10 min, ramp of 5°C/ min up to 220°C; injector and detector temperatures 250°C; carrier gas nitrogen (2 mL/min); detector dual FID; split ratio 1:30; injection volume of 1 mL; 10% nhexane solution.Identification of the essential oil constituents was performed, for both columns, by the comparison of their retention times with those of pure authentic samples and by means of their Linear Retention Indices (L.R.I.) relative to a series of n-hydrocarbons (C 9 -C 23 ).
GC-MS analysis.GC/EIMS analyses were performed by a Varian CP-3800 gas chromatograph equipped with a HP DB-5 capillary column (30 m × 0.25 mm; coating thickness 0.25 μm) and a Varian Saturn 2000 ion trap mass detector.Analytical conditions: injector and transfer line temperatures 220 and 240°C, respectively; oven temperature programmed from 60°C to 240°C at 3°C/min; carrier gas helium at 1 mL/min; injection volume 0.1 μL (10% n-hexane solution); split ratio 1:30.The identifica-The aromatic profile of in vitro M. x piperita tion of the constituents was based on the comparison of the retention times with those of authentic samples, comparing their linear retention indices (LRI) relative to a series of n-hydrocarbons (C 9 -C 30 ).A computer matching of mass spectra by two commercial databases (NIST 2000; ADAMS) as well as a homemade library, built up from pure substances or known oils, were used to perform identification of the volatile constituents.Moreover, the molecular weights of the all identified substances were confirmed by GC/CIMS, using MeOH as CI ionizing gas.
HS-SPME-GC-MS analysis.The HS-SPME analyses were performed with Supelco SPME devices, coated with two different kinds of fibers (PDMS, PDMS-Carboxen, 100 μm) in order to sample the static headspace of a fixed portion of the freeze-dried plant material (stems, leaves, in vitro plantlets, callus) of M. piperita.Each aliquot was inserted separately into a 50 mL glass conic flask and allowed to equilibrate for 30 min.After the equilibration time, each fiber was exposed to the sample headspace for 5 min.at room temperature, and when the sampling was finished (5 min), the fiber was withdrawn into the needle and transferred to the injection port of the GC and GC-MS system, operating in the same conditions described for the essential oils, apart from the splitless injection mode and the injector temperature (250ºC).
Quantitative analysis.Quantification of the essential oils was conducted using an internal standard (is, n-undecane) added to the volatile oil under the conditions of the GCMS analysis used for standard mixtures.The calibration curves of the analytes were performed by using standards, which have chemical similarity with the compounds of interest in the volatile oils (Table 1).The correspondent regression lines (five points) of each standard were obtained with chromatographic injections of solutions obtained by mixing different accurate volumes of the standard stock solution and an accurate volume of an internal standard solution at 10 mg/mL (n-hexane as solvent).The limits of detection of the standard target compounds are given in mg/mL (Table 1).The qualiquantitative GC-MS results are given as a mass percentage composition (mg/100 mg) of each volatile sample which was determined by the injection of a solution (0.1 μL) obtained by mixing 10 μL of the volatile fraction, 100 μL of the internal standard solution (1 mg/mL) and n-hexane to 1 mL (three measurements for the same sample).All results of the quali-quantitative GC-MS analysis are in Tables 2-3.M. piperita calli were easily developed from the leaves of in vitro grown seedlings.The addition of 2 mg L -1 2iP and 0.5 mg L -1 2,4-D gave satisfying results.Callus induction was observed after 15 days.The callus obtained on this medium was light green and friable.Furthermore, small necroses on the explants surfaces were observed, but no roots or shoots were formed.The established tissues were successively subcultured and hydrodistilled by the same procedure used for the leaves and stems of the adult plants.The EO yields obtained from the openfield grown adult plants were found to be 1.8% v/w and 1.5% for stems and leaves, respectively.Therefore, this Polish mint selection showed an EO production similar to other European no-Mediterranean plant samples (Aflatuni et al., 2000;Stojanova et al., 2000).However, the EO yields dropped to 0.1% v/w in the in vitro plantlets and callus established from the selected Polish M. x piperita.
Regarding the EO composition, menthone, menthol, menthyl acetate, carvone, piperitone, 1,8-cineole, and pulegone were the major compounds in the M. x piperita adult plants.The hydrocarbon monoterpenes, α-pinene and sabinene (8 and 7%, respectively), were found especially in the stem EO (Table 2).Carvone and piperitone were present in similar amounts (3%) both in the stem and leaf EOs, while the other two characteristic constituents of mint spp., menthofuran and piperitenone, were not detected (Table 2).The HS-SPME-GC-MS analysis confirmed the absence of these two constituents in the aroma emitted spontaneously from the cultivated plant samples (Table 3).
Among the sesquiterpenes, β-caryophyllene and germacrene D were predominant both in stem and leaf EOs, but significant quali-quantitative differences were registered especially in the total sesquiterpene composition (3%, stems; 18% leaves; Table 2).A larger variety of sesquiterpenes was found in the leaf than in stem EO.This fact was confirmed also by HS-SPME-GC-MS analysis (Table 3).Many studies demonstrated that environmental conditions and geographical origins influenced greatly the EO production in M. piperita (Clark & Menary, 1981;Aflatuni, 1999;Maffei, 1999).Further- more, physiological features such as leaf/stem ratio and herb biomass are positively correlated with oil content, whereas plant height generally influences it negatively (Sharma & Tyagi, 1991).It is important to point out that the acceptable commercial quality of peppermint oil is strictly related to the content of menthone, menthol, and menthyl acetate in addition to traces or lack of pulegone and menthofuran (Burbott & Loomis, 1967;Lawrence, 1985).Regarding peppermint oils and the Pharmacopoeia requirements such as those of the British Pharmacopoeia (1968), menthol must exceed 45% and menthyl acetate must range from 4 to 9%.However, some European and extra-European peppermint oils may contain less than 45% of menthol (Maffei et al., 1994).It is well known that large variations in the menthol content are possible in some regional productions.However, these areas are generally capable of producing EOs with acceptable menthol concentration, even if some variability within the same area is possible due to the unevenness in plant maturity.However, although the menthol content is often below the required level, menthyl acetate concentration is generally satisfied (Shah & Gupta, 1989;Chalchat et al., 1997).
In the present study, menthol and menthyl acetate were both present in the leaf and stem EOs hydrodistilled from the adult plants cultivated in Poland.These EOs were richer in menthol than in menthyl acetate, even if menthol (7-12%, stems; 3-5%, leaves) was much lower than the quality parameters fixed by ESCOP (ES-COP, 1992).The established M. x piperita callus produced menthol (9%) in comparable amounts with its mother plant (12% leaves, 7% stems), while menthyl acetate was enhanced (8%) significantly in comparison with the stem and leaf EOs (3% and 5%, respectively).On the other hand, the in vitro plantlets showed much lower levels of menthol and menthyl acetate (2%) than callus.With respect to biosynthetic processes in mint spp., menthones derive from the reduction of menthylacetate, which is routinely found in measurable quantities in the peppermint oil (Croteau & Hooper, 1978).In the present study, the EOs obtained from adult plants cultivated in Poland showed a menthol-menthone ratio of 0.26 for stems and 0.36 for leaves.Lower values were determined in the correspondent static headspaces (0.16 stems; 0.12 leaves), but these results confirmed that menthone contributed more than menthol to the spontaneous aroma of the cultivated adult plants.In fact, menthone was predominant both in the stem (26%) and leaf (33%) EOs, while menthol showed lower levels (7 and 12% stems and leaves, respectively; Table 2) Pulegone was detected only in the leaf and stem EOs of adult plants (3-2%, respectively), while it was not observed in the EOs of the in vitro plant material.However, the composition of monoterpene alcohols and ketones showed interesting quali-quantitative differences especially in the in vitro biomass established from the different plant organs.
In fact, menthone and menthol were detected in the same amounts (2%) in the EOs of in vitro plantlets.On the other hand, menthone was not detected in the callus EOs that was rich especially in menthol (9%), 1,8-cineole (10%), methyl acetate (7%), and carvone (6%).The production of these typical mint oxygenated monoterpenes was enhanced in the callus tissues in comparison with the correspondent mother plants (Table 2).Otherwise, the biosynthesis of the hydrocarbon sequiterpenes was drastically reduced in the in vitro plant material of M. x piperita and only germacrene D was produced in significant amounts (2.5 and 2.9% in vitro plants, callus respectively).
The in vitro plantlets oil showed a completely different composition in comparison with the callus because it was characterized by especially huge amounts of the piperitenone oxide (45.3%, Table 2).As reported in the literature, the mint plants with low menthofuran content generally produce measurable amounts of trans-piperitone oxide and piperitenone oxide.These epoxyketones are reported infrequently in peppermint, and the total content very rarely exceeds a few percent in the distilled essential oil (Croteau, 1991;Wise & Croteau, 1999).
The EO of in vitro plantlets was also richer in limonene (10%) than callus.This compound is considered the precursor of pulegone, carvone, and piperitenone (Wise & Croteau, 1999;McConkey et al., 2000;Turner & Croteau, 2004).In the EOs of in vitro plantlets, pulegone was not detected and carvone (2%) was lower in comparison with the other samples.Therefore, limonene may be preferentially transformed to piperitenone, which is then oxidized to piperitenone oxide especially in the in vitro plantlets.Piperitenone oxide was also found in the HSs of M. x piperita callus and mother plants, but at much lower levels (2-3% and 0.1-2%, respectively; Table 3).
The commercial importance of peppermint mint depends on the percentage of menthol and menthone as well as the presence of some undesirable compounds such as pulegone and menthofuran (EMEA, 2004).In the present study, menthone and menthol characterised the EOs of the adult plants cultivated in Poland and in vitro plantlets.The callus tissue yielded an essential oil especially enriched in menthol and not containing menthone, menthofuran and pulegone.In the present study, the lack of menthofuran characterized both the in vivo and in vitro EO production.
This common feature was confirmed in all the analysed samples by the HSs analyses (Table 3).
It is worth to notice that pulegone, the precursor of menthofuran, was detected in the stem and leaf EOs of open-field plants (3 and 2%, respectively), but neither in the EOs and HSs of the correspondent in vitro plant material (Tables 2-3).As reported in the literature, pulegone and menthofuran are closely related to mint safety (Murray et al., 1988).The hepatoxicity of pulegone is believed to result from its metabolism to menthofuran (Thomassen et al., 1990).The Working Party on Herbal Medicinal Products of the EMEA Agency released a draft position paper on the use of herbal medicines containing pulegone and menthofuran (EMEA/HMPWP, 2004).More recently, the Scientific Committee on Food of EFSA was asked to provide scientific advice to the Commission on the implications for human health of chemically defined flavouring substances used in or on foodstuffs in the EU Member States.Pulegone and menthofuran were evaluated too and, accordingly, cannot be used as flavouring substances in the EU market (JECFA, 2009).
In conclusion, M. x piperita callus and vitro plantlets were established by sustainable biotechnological protocols using selected adult plants cultivated in Poland, which were already previously characterized by reduced amounts of pulegone as well as the lack of menthofuran.The essential oil obtained from the established callus did not contain pulegone or menthofuran.It was enriched in the typical mint volatiles such as 1,8-cineole, menthol, menthyl acetate, carvone, which were found also in the analysed parent plants.Although further studies will be needed to improve the EOs yields from the in vitro biomass, the established callus may be regarded as a potential source of a mint-type essential oil not containing pulegone or menthofuran.
piperita was collected during the second vegetative year (2009) when plants were very well established.Plants were harvested at the early flowering stage and dried in an open air in the shade.The leaves and stems were studied separately in order to evaluate the volatile constituents in different plant organs.

Table 1 . Calibration parameters of the standard compounds used for the GC-MS quantitative analysis.
y = C is /C s and x = A is /A s where C S , A S = concentration and peak area of standard, C is and A is = concentration and peak area of internal standard a

Table 3 . SPME-GC-MS analysis of adult plants and in vitro M. piperita plant material.
a PDMS polydimethylsiloxane fiber; CARB carboxen fiber.b Percentage average calculated as relative percentage composition (%) on DB-5 column without correction factors.c t = traces (%<0.1).d tentative identification