Vol. 58, No 4/2011

In vitro plantlets and callus of M. longifolia were established and their volatile constituents characterized by GC-MS analysis of their headspaces (HSs) and essential oils (EOs). Significant quali-quantitative differences were found in the aromatic fingerprints in comparison with the M. longifolia parent plants. In fact, limonene and carvone were the main constituents in the EOs of the mother plants, while the aroma of the in vitro plant material were especially enriched in oxygenated terpenes. In particular, huge amounts of piperitenone and piperitenone oxide (75 %) were found for in vitro plantlets, while trans-carvone oxide (19 %) and trans-piperitone epoxide (9 %) were found in callus EO. However, the established in vitro plant material showed lack of pulegone and menthofurane, thus preserving an important feature observed in the volatile fingerprint of the parent plants. In fact, because of their well-known toxicity significant amounts of pulegone and menthofurane may compromise the safety using of mint essential oil. Therefore the in vitro M. longifolia plantlets and callus may be regarded as a potential source of a safe flavouring agent.


InTROduCTIOn
Mentha longifolia L. (Lamiaceae) or wild mint is a perennial herb, extremely variable both morphologically and chemically.It comprises a number of isolated populations extending over the whole of Europe, and from African highlands down to the Cape of Good Hope (Lawrence, 1981).The aerial parts of adult plants are commonly used in folk medicine for the treatment of cold, cough, asthma, and chest inflammations, including pulmonary tuberculosis.It is also used externally to treat wounds and swollen glands (Ikram & Haq, 1980;Evans, 1996;Mimica-Dukic et al., 1996;2003).Mint extracts are commonly used as food flavoring additive and are generally considered safe to use as they provide good defense against oxidative damage and health benefits (Dorma et al., 2003).However, a revision of the safety aspects of some mint constituents such as pulegone and menthofurane has been discussed recently (Nair, 2001;JECFA, 2009).M. longifolia is known also under synonymous names as M. spicata var.longifolia L. or M. sylvestris.The majority of M. longifolia chemotypes and subspecies contain piperitenone oxide, piperitone oxide, carvone, menthone, and 1,8-cineol as the main constituents, even though major variation in the dominating compounds has been found in wild or cultivated plant material grown in different habitats (Maffei, 1988;Venskutonis et al., 1996;Fleisher & Fleisher, 1998;Karousou et al., 1998;Baser et al., 1999;Abu Al-Futuh et al., 2000;Mastelic & Jerkovic, 2002;Jaimand & Rezaee, 2002;Rasooli & Rezaei, 2002;Mathela et al., 2005;Oyedeji & Afolayan, 2006;Gulluce et al., 2007).A summary of literature data on the essential oil (EO) composition of M. longifolia is reported in Table 1.
In the present study, leaves and stems of M. longifolia selected from cultivated adult plants (Pulawy, Poland) were used as mother plants to establish in vitro plantlets and callus.Both the headspaces (HSs) and the essential oils (EOs) were studied to compare the complete aromatic fingerprint of in vivo and in vitro biomass.To our knowledge, no studies have been reported on the volatile profile of in vitro cultures of M. longifolia.

MaTERIal and METHOd
Plant Material.Seeds of Mentha longifolia (catalogue number: 239112) were obtained from the National Centre for Plant Genetic Resources at the Plant Breeding and Acclimatization Institut (Radzikow, Poland).Plants were cultivated in an experimental field of the Institute of Soil Science and Plant Cultivation in Pulawy, Poland.Plants were harvested at the beginning of flowering.
Seed sterilization and sowing.Seeds were washed in running tap water and sterilized with 70 % ethanol for 2 min, then transferred to 10 % perhydrol solution with shaking for 20 min, and finally rinsed three times with sterile distilled water.Sterilized seeds were placed for germination on half-strength LS basal medium (Linsmayer & Skoog, 1965) supplemented with 15 g L -1 sucrose and solidified with agar 6 g L -1 , adjusted to pH 5.8 followed by autoclaving at 121 °C and 0.1 MPa for 20 min.Seeds were sown on the medium in Petri dishes and then were incubated in a growth chamber at 25 °C, under 16 hours light /8 hours dark cycle provided by fluorescent lamps.Sprouting seeds were planted on LS medium enriched with 0.2 mg L -1 NAA (naphtaleneacetic acid) and 0.2 mg L -1 IAA (indole-3-acetic acid).Flasks were kept in the growth chamber under the same conditions.Young in vitro plants were used for callus induction.Some of those plants after 30 days of cultivation were harvested, freezedried and used for biochemical analysis.
Callus induction.Leaves from 3-week-old in vitro plants were used as explants.They were cut into small pieces and transferred on several media variants supplemented with dichlorophenoxyacetic acid (2,4-D) in combination with 6-benzylaminopurine (BA) or isopenthenyladenine (2iP) added at the concentration 0.5, 1 and 2 mg L -1 .As a control LS basal medium without any hormones was applied sucrose (30 g L -1 ) and agar (8 g L -1 ) were added to the media and pH was adjusted to 5.8.
Callus tissues were grown in Petri dishes.For each type of medium at least ten dishes containing explants from five seedlings were used for callus growth.The cultures were maintained in a growth chamber at 25 °C under a 16-h photoperiod, provided by cool white fluorescent lamps.After 30 days the mint callus induction and its characteristic features such as abundance, color, structure, tendency to form roots, shoots and necrosis were observed.Calli were subcultured at four-week intervals.For biochemical analysis calli from 7th 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 reference material after GC-MS grade purity determination (98-99 %).The samples and standard solutions were prepared using nhexane (Carlo Erba, HPLC-grade).
Extraction procedure.Freezedried 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 Et2O, dried over anhydrous MgSO4, filtered and the solvent removed by evaporation on a water bath.The essential oil yields are summarised in Table 1.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 run on an 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 μl; 10 % n-hexane solution.Identification of the essential oil constituents was performed for both columns by 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 nhydrocarbons (C9-C23) on the two columns.
GC-MS analysis.GC/EI-MS analyses were performed on a Varian CP-3800 gas chromatograph equipped with an 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 a 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 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.Identification of the constituents was based on a comparison of the retention times with those of authentic samples, comparing their LRI with those of a series of n-hydrocarbons (C9-C30).Computer matching of the mass spectra by two commercial data bases (NIST 2000, ADAMS) as well as a home-made library built up from pure substances or known oils were used to perform identification of the volatile constituents.Moreover, the molecular weights of the identified substances were confirmed by GC/CIMS, using MeOH as CI ionizing gas.

HS-SPME-GC-MS.
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. longifolia.Each aliquot was inserted separately into a 50-ml conic glass 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 the fiber was withdrawn into the needle and transferred to the injection port of the GC and GC-MS system, operating in the conditions described for the essential oils, apart from the splitless injection mode and the injector temperature (250 °C).
Quantitative analysis.Quantification of the essential oils was conducted using an internal standard (is, nundecane) added to the volatile oil under the conditions of the GCMS analysis used for standard mixtures.Calibration curves of the analytes were performed by using standards which have chemical similarity with the compounds of interest in the volatile oils (Table 2).The correspondent regression lines (five points) of each standard in Table 2 were obtained with chromatographic injections of solutions obtained by mixing accurate volumes of the standard stock solution and an accurate volume of 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 2).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 volatile fraction, 100 μL of internal standard solution (1 mg/mL) and n-hexane to 1 mL (three measurements for sample).
The quali-quantitative results are shown in Tables 3-5.

RESulTS and dISCuSSIOn
The volatile constituents emitted from field and in vitro biomass of M. longifolia were extracted both by hydrodistillation to obtain the essential oil (EO) and by solid phase microextraction (SPME) to sample the spontaneous aroma.The EO yields were 2.2 % v/w for stems and 1.5 % for leaves collected from adult plants of M. ongifolia.
The EO yields of in vitro plants and callus were much lower (0.2 and 0.4 % v/w, respectively) (Table 1).However, these yields from in vitro biomass were similar or higher than those obtained from air-dried or fresh wild M. longifolia reported in the literature (Table 1).The EO composition was similar for the stems and leaves of field-grown adult plants, even if significant quantitative   differences were observed.The main components were limonene and carvone both in stems (15.3 and 15.1 %, respectively) and leaves (5.8 and 7.9 %, respectively).The hydrocarbon monoterpenes such as α-and β-pinene, 1,8-cineole, as well as Zand E-ocimene were much more abundant in stems than in the leaf EOs (Table 4).On the other hand, the oxigenated pmenthene compounds, which are other typical constituents of mint spp., showed higher percentages in the leaf than in the stem EOs.
The menthol/menthone ratio in the stem (0.4) and in leaf EOs (0.1) of the parent plants were similar to those reported in the literature for airdried Cretan M. longifolia plants (0.3-0.1), but it was much lower than found in Italian fresh samples (3.0) (Maffei, 1988;Karousou et al., 1998).Previous studies on wild extra-European M. longifolia, such as Asian and Australian samples, showed a menthol/menthone ratio in favour of menthone, as those plants did not produce menthol.On the other hand, wild M. longifolia samples from Turkey, Iran, and South Africa, had a large amount of pulegone instead of menthone or menthol.
Regarding sesquiterpenes, β-caryophyllene (2.8 %) and germacrene D (2.0 %) were the most abundant volatiles in stem EOs, while the same compounds dropped under 1 % in the leaves of M. longifolia parent plants.Previous studies on M. longifolia EO reported that germacrene D is a typical sequiterpene of different varieties of this species (Maffei, 1988;Evans, 1996;Rasooli & Rezaei, 2002).SPME-GC-MS analyses confirmed these results for the EO extracted from adult plants (Table 5).
Both stems and leaves of Polish M. longifolia were used to establish in vitro plant material and the induction of callus tissue was noticed on all tested media (outside the control variant).The M. longifolia callus induction on the medium enriched with isopenthenyladenine and dichlorophenoxyacetic acid generally gave poor or very poor callus growth.A slightly better effect was observed when 6-benzylaminopurine was used as cytokinin, but the callus tissues obtained were also poor.Small necroses on explant surfaces were observed, and roots or shoots were not formed.The best effects were observed on media containing 1.0 mg L -1 BA plus 0.5 mg L -1 2,4-D.The callus obtained on this medium was creamy, fragile which is important in the case of suspension cultures.Moreover this tissue was characterized by good biomass production.The GC-MS analysis of in vitro plantlets and callus of M. longifolia showed a large variety of the typical volatile constituents already found in their parent plant material.Furthermore, most of these volatile constituents were also emitted spontaneously by in vitro biomass as confirmed by the SPME-GC-MS analysis carried out directly on this material (Table 5).However, significant quali-quantitative differences were observed between the in vivo and in vitro biomass.
The ratio of oxides/ketones was found to be an important parameter to indicate the efficiency of the conversion from piperitenone to piperitenone oxide in the in vivo plants.Piperitone and piperitenone were not detected in the callus EO, while their corrispondent oxides, cis-(2.4%) and trans-(8.0%) piperitone epoxide as well as piperitenone oxide (3.3 %) were present (Tables 3-5).
Furthermore, the huge amounts of carvone which were detected in the M. longifolia EO of adult plants (15.1 and 7.9 % in stems and leaves, respectively) were not pro-  duced in the callus EO in favour of cis-(2.3%) and trans-(18.9%) carvone oxides.
Although the M. longifolia callus did not show limonene and carvone as main constituents like their mother plants, they were characterized by the highest oxidative status of carvone and piperitone.In addition, the scarce amount of limonene (0.7 %) and the lack of terpinolene, which are the precursors of piperitenone and carvone, could be justified by the complete transformation of these two latter compounds into their corrispondent oxides (Fig. 1).Regarding the in vitro plantlets, they showed the specific terpenes of M. longifolia generally reported in the literature.P i p e r i t e n o n e (30.4 %) and piperitenone oxide (44.8 %) were the main constituents in their EOs (Table 4).However, as mentioned above, neither these compounds was not detected in the Polish M. longifolia mother plants.
Nevertheless, the established in vitro plantlets could be considered similar to a chemotype of M. longifolia rich in piperitenone oxide (75 %).In fact, a chemotype of M. longifolia Hudson (L.) particularly rich in piperitenone oxide (77 %) was selected from populations growing wild in Piedmont Valley (Italy) (Maffei, 1988) and Lithuania (Venskutonis, 1996).In our work, the in vitro plantles of M. longifolia showed an aromatic fingerprint characterized by huge amounts of piperitone and piperitenone oxide (75 %) with a clear reduction in other typical p-menth-compounds detected in their parent plants (Tables 3-4).Furthermore, the in vitro callus and plantlets did not show pulegone and its major metabolite menthofuran.The present study is the first report on the aromatic profiling of M. longifolia as in vivo and in vitro plant material.Both types of in vitro plant material, callus and plantlets, were characterized by a lack of pulegone and methofurane.It is important to point out that the EFSA Scientific Committee on Food was recently asked to provide scientific advice on the implications for human health of chemically defined flavouring substances used in or on foodstuffs in the EU Member States.According to EU Flavourings List, pulegone and menthofuran cannot be used as flavouring substances (Moorthy, 1991;Tisserand & Balacs, 1995;Nair, 2001;JECFA, 2009).Therefore, the in vitro plantlets and callus of M. longifolia which did not contain pulegone and menthofurane preserved an important safety feature of their parent plants and they may be qualified as alternative flavouring ingredients.