Menthol from the stem and leaf in-vitro Mentha piperita Linn.

The need for menthol in Indonesia is increasing annually, but it is not followed by increases the availability in the country, so the number of imports is increasing each year. Therefore, in this research conducted a study using plant tissue culture techniques to produce secondary metabolite especially menthol using shoot multiplication. A single node of Mentha piperita L. was inoculated on three kinds of growth medium, which are MS + 0.1 mg L-1 NAA + 0.1 mg L-1 BA, MS + 0.5 mg L-1 BA, and MS + 2 mg L-1 BA. Based on fresh weight and morphological observation, MS + 0.1 mg L-1 NAA + 0.1 mg L-1 BA was the best growth medium. Based on the previous experiment, the study was aiming to continue observing the concentration of menthol found in M. piperita shoot. Stems and leaves of M. piperita were dried in room temperature, the dried stems and leaves were extracted using steam distillation method with ratio 0.3 g of dried stems and leaves in 25 mL aquadest. The crude extract was analyzed by gas chromatography (HP 6890) using INNOWAX 19095N-123 column. The results showed that 2 wk was the best amount of time to obtain the highest concentration of menthol specifically (1 218.5 ± 47.1) mg L−1 (yield = 9.748 %).


Introduction
Mentha spp. belongs to Labiatae family, there are three main species which have high economic value like Mentha piperita L. The industrial demands of M. piperita product are high, but Indonesia cannot fulfill the demands without import. In 2006 Indonesia imported Mentha spp. product worth the USD 3.78 × 10 6 [1]. M. piperita is a first sterile hybrid generation between M. spicata and M. aquatica [2]. The main compound from Mentha essential oil is menthol; this compound is produced in peltate glandular trichome which located at leaf [3].
The purpose of this research is to increase the production of menthol using plant tissue culture technique. Cytokinin is phytohormone that induced shoot proliferation and cell division [4]. Plant tissue culture is known to produce plants that grow faster [5]. In addition, the concentration of secondary metabolites, especially monoterpenes in Lavandula pedunculata in vitro, is known to be greater than plants grown in their natural [6]. The growth medium used for multiplication of shoots with the aim of increasing menthol concentration is Murashige & Skoog media (MS) + 6-Benzylaminopurine (BAP) + Naphthaleneacetic acid (NAA) [7]. The advantage of in vitro shoot multiplication techniques is that it is able to produce target compounds under controlled conditions from changes in weather and soil conditions, plants obtained free from microorganisms and insects, all types of plants can be used in this technique and reduce labor costs and increase productivity [8].

Plant materials, treatment, and growing condition
This research was carried out at the Laboratory of Plant Biotechnology and Biopurification and Biomolecular Laboratory, Faculty of Biotechnology, University of Surabaya. The peppermint seeds (Biopot ® ) used in this study were pre-sterilized using bactericidal and fungicidal solution for an hour. The surface sterilization of explants was continued in Laminar Air Flow Cabinet with immersion in 70 % ethanol for 1 min, and 5.25 % NaOCl solution for 5 min followed by rinsing with sterile H2O at least three times. Furthermore, sterilization was carried out again by immersion in 1.75 % NaOCl solution for 10 min followed by rinsing with sterile water at least three times. Sterilized mint seeds are planted on MS media without hormones. The culture bottle was incubated under the white fluorescent lamps. Explants used are nodes from mint plants that are approximately 3 mo. The plant is cut to only one node. Furthermore, nodules cut into various growth media (table 1.) The culture was incubated in a culture room with a temperature of (25 ± 2) °C with the lighting of white fluorescent lamps and observed the formed tissue.

Extraction and determination of menthol
The extraction samples used were leaves and stems of mint plants obtained from the bud multiplication process that have been dried in room temperature under air conditioning. The solvent used was 25 mL of water (distilled water) to extract 0.3 g of dried stems and M. piperita leaves, the sample was extracted using the steam distillation method. Then the extract obtained was analyzed for menthol content using a gas chromatography device (HP 6890) with INNOWAX 19095N-123 column [9]. For the ex vitro sample used stem and leaves of M. piperita ex vitro at 3 mo.

2.3.Data analyses
The research conducted was an experimental study in the laboratory with a Completely Randomized Design (CRD). In this study two non-parametric data were obtained because the data wasn't normally distributed, namely the best media used to grow M. piperita plants in vitro, and data on menthol production monitored every week. To determine the best growth media, and the best time to harvest, need to obtain the fresh weight of M. piperita in vitro, and menthol concentration for 7 wk. The

Preliminary test
Preliminary tests were carried out to determine the three types of growth media to be tested. Further, the aspects considered were morphology of M. piperita grown in vitro. From this preliminary test selected, MS medium + 0.1 mg L -1 NAA + 0.1 mg L -1 BA, MS + 0.5 mg L -1 NAA and MS + 2 mg L -1 NAA to be used at the next research stage because it has the best morphology. The reason for choosing MS media was 0.5 mg L -1 NAA and MS + 2 mg L -1 NAA compared to MS + 1 mg L -1 BA and MS + 2 mg L -1 BA,because the hormones NAA and IAA (Indole-3-Acetic Acid) were able to increase the oil content of essential oils and menthol in M. piperita [9]. Then MS + 0.5 mg L -1 NAA and 2 mg L -1 NAA has a better morphology than MS + 2 mg L -1 BA + 1 mg L -1 NAA, the morphology in question is the appearance of the plant (abnormal).

Growth index curve
Growth Index Curve is obtained from the final weight of the stem and fresh leaves divided by fresh initial weight. The calculation of the growth index was done by weighing fresh initial weight before planting and after being cultivated for (1, 2, 3, 4, 5, 6 and 7) wk. Observation of the growth index stopped at week 7 because the index value had decreased compared to the 6th week.   Fresh weight of M. piperita grown on MS + 2 mg L -1 NAA is the largest among other growth media. This is due to the presence of callus at the base of the plant stem. Callus also found at the base of the plant stem that grown using MS + 0.5 mg L -1 NAA. M. piperita that grew at MS + 0.1 mg L -1 NAA + 0.1 mg L -1 BA obtained plants do not form callus and have a heavier mass than MS media + 0.5 mg L -1 NAA. Menthol compounds are stored in the peltate glandular trichome [3]. The peltate glandular trichome is found in parts of plants exposed to air [10]. So it can be said that the larger/, the heavier plant, the more peltate glandular trichome, in other words, the more menthol that can be extracted. In addition, other factors considered by the morphology of plants planted in vitro. There was no monoterpene found in Mentha spicata callus because the glandular oil gland or peltate glandular trichome had not been formed because callus cells were undifferentiated plant cells [11]. Therefore, the MS medium was selected + 0.1 mg L -1 NAA + 0.1 mg L -1 BA as the best growth medium.

Extraction and determination of menthol
Extraction of menthol compounds was carried out using the steam distillation method because it is commonly used to isolate essential oils. The distillation process is stopped until all the water in the flask has evaporated. From steam distillation results obtained ± 24 mL of crude menthol extract from steam distillation. The crude extract obtained was analyzed using a gas chromatography device (HP 6890) with the INNOWAX 19095N-123 column.   There is a difference between the crude extracts of M. piperita in-vitro and ex-vitro, the peaks that appeared at retention time was around 5 min in the crude extract profile of ex-vitro plants but were not found in crude plant extracts in-vitro. And the crude extracts of plants in-vitro found peaks at retention time about 8 min which were not found in crude extracts of ex-vitro plants. In other words, the M. piperita plant in-vitro is not capable of producing secondary metabolites that are exactly the same as their original plants and is able to form secondary metabolites that are completely different from the original plants. Several factors that influence the difference in results is nutrition availability and environmental conditions. The availability of nutrients includes the concentration of sugar, nitrate, phosphate and growth regulating substances whereas environmental conditions include temperature, light intensity and pH [12]. Plant tissue culture systems are able to produce secondary metabolites that are completely different from the original plants because of very different environmental conditions, while the levels can be the same, larger or smaller [13]. In-vitro plant growth media have been given nutrition, sufficient growth regulating substances, and the growth environment is conditioned stable with adequate lighting, the temperature of 20 °C and pH of 5.8. All treatments given to plants in-vitro are common treatments to optimize growth in plant tissue culture while the ex-vitro plants all of these factors are not controlled at all, or in other words depending on nature.  The highest concentration of menthol was 2 wk old M. piperita that have grown using MS + 0.1 mg L -1 NAA + 0.1 mg L -1 BA, and the least concentration of menthol was ex-vitro M. piperita. This phenomenon is not in accordance with the theory, that the production of secondary metabolites generally occurs at the end of the stationary period when the nutrient supply is depleted [4]. This may be caused by menthol has been further metabolized to menthyl acetate [14]. There are also indications that the terpenoids used for defense have decreased due to the absence of threats. Another factor that might cause the highest phenomenon of menthol concentration in the second week of culture is stress caused by injury at the beginning of cutting explants. This phenomenon was also showed on another research, where at 2 wk after the plant was injured there was a significant increase in the concentration of monoterpenes compared to non-injured plants [15]. This increase in the concentration of monoterpenes may be caused by an increase in the activity of the enzyme monoterpene cyclase [16]. Monoterpene cyclase plays a role in converting Geranyl pyrophosphate (GPP) to 4s-limonene [17]. GPP is the earliest compound in menthol biosynthesis pathway or it can be said that it is a compound that initiates menthol formation so that plants will tend to produce menthol in this condition. M. piperita grown in vitro is able to produce more menthol than ex vitro. This may be due to the availability of sufficient nutrients so that in vitro plants are able to produce more menthol. Besides that phytohormones are able to increase the levels of essential oil and menthol oil in Mentha spp. [18].

Conclusion
Based on this research, it can be concluded that the best growth medium for multiplying M. piperita shoots in-vitro was MS + 0.1 mg L -1 NAA + 0.1 mg L -1 BA. Chromatogram profile on crude extracts of M. piperita plant in vitro showed three peaks, namely at about two retention times: 4.5 min and 8 min. Chromatogram profile on crude extract of M. piperita ex-vitro showed three peaks, namely at about two retention times: 4.5 min and 5 min. The menthol concentration observed for 7 wk in the largest M. piperita plant in-vitro was at the culture time of the 2 nd week which was (1 218.5 ± 47.1) mg L -1 . The menthol concentration observed in the M. piperita ex-vitro plant was (97.21 ± 1.373) mg L -1 .