STUDIES CONCERNING THE HISTO-ANATOMY AND BIOCHEMISTRY OF MENTHA LONGIFOLIA (L.) HUDS. DURING VEGETATIVE PHENOPHASE. Introduction

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1 Analele ştiinţifice ale Universităţii Al. I. Cuza Iaşi Tomul LVII, fasc. 2, s. II a. Biologie vegetală, 2011 STUDIES CONCERNING THE HISTO-ANATOMY AND BIOCHEMISTRY OF MENTHA LONGIFOLIA (L.) HUDS. DURING VEGETATIVE PHENOPHASE ANCA RALUCA ANDRO*, DOINA ATOFANI*, IRINA BOZ*, MARIAMAGDALENA ZAMFIRACHE*, I. BURZO**, C. TOMA* Abstract: The article analyzed the histo-anatomy of the vegetative organs of species Mentha longifolia (L.) Huds collected from Negreşti (Vaslui). On the surface of the vegetative organs was identified the presence of tectorial and secretory hairs. Volatile oil was extracted from the aerial parts of the plant using a Clevenger device, and with a GC-MS (Gas Chromatography coupled with mass spectrometry) there were identified the chemical compounds. It was recorded the presence of 25 chemical compounds, five of them representing 75.42% of the total obtained oil. Keywords: volatile oil, Mentha longifolia, medicinal plant. Introduction The genus Mentha L. comprises herbaceous, perennial plants, common in temperate climates in Europe, Australia and South Africa [9]. Due to their properties these plants are used in pharmaceutical, cosmetics and food industries. These properties are most often offered by volatile oils produced by the secretory hairs. The representatives of the genus Mentha have capitates and peltate secretory hairs, provided by a single secretory gland, having eight secretory cells [4; 10; 14]. They are initiated very early and begin to accumulate these substances when the leaves are 5 mm long [6]. Turner et al. (2000 a) considered that the most monoterpenes of the mint volatile oil are produced and stored in glandular peltate hairs [15]. The chemical composition of volatile oil derived from M. longifolia was investigated by Mkaddem et al. (2009) [12]. Their research has been conducted on populations from different regions of Gabes, Tunisia. Compounds identified in significant quantities in the essential oil were pulegone (54.41%), isomenthone (12.02%), 1,8-cineole (7.41%), borneol (6.85%), and piperitone oxide (3.19%). Džamić et al. (2010) analyzed the volatile oil extracted from M. longifolia obtaining as main components trans- and cisdihydrocarvone (23.64% and 15.68%), piperitone (17.33%), 1,8-cineole (8.18%) and neoisodihydrocarveol (7.87% ) [3]. In this context we decided to analyze the histo-anatomical aspects of the species M. longifolia and to identify the chemical composition of the volatile oil during the vegetative phenophase of the plant. Materials and methods The plant material used consisted of aerial organs from the species M. longifolia collected in June of 2010, when the plant was in vegetative phenophase. * Alexandru Ioan Cuza University, Faculty of Biology, Bd. Carol I, no. 11, Iasi Romania, anca_andro@yahoo.com ** University of Agronomical Sciences and Veterinary Medicine Bucharest, Romania, Faculty of Horticulture 25

2 The plants were collected from the banks of Bârlad River in Negreşti, Vaslui County, and determined by Dr. Ciprian Mânzu, taxonomist at the Faculty of Biology, University Al. I. Cuza of Iaşi. In order to observe the anatomical structure of the vegetative organs cross sections were made through these organs using a microtome and a botanical razor. The obtained sections were colored with green iodine and ruthenium red to obtain a sample which was subsequently analyzed with optical microscope. Volatile oils were extracted with a Clevenger device, according with the European Pharmacopoeia Standards, in the Vegetal Physiology Laboratory of the Faculty of Biology, University Al. I. Cuza of Iaşi. Inside the balloon was introduced chopped plant material and water. The distillation process was three hours long. Segregation and identification of chemical compounds of essential oils were performed with GC_MS (Gas Chromatography coupled with mass spectrometry), within the Research Center for Food and Agriculture from the Faculty of Horticulture, University of Agronomic Sciences and Veterinary Medicine in Bucharest. Chemical compounds were identified by comparing retention time recorded at a spectra library, located in the device database. Results and discussions In the superior level of stem the contour of the cross-section is quadratic with relatively prominent ribs. It was observed an early transition to secondary structure, but only in the central cylinder, in cambium area, even in terminal internodes of the stem (Fig. 1). The epidermis presents isodiametric cells, covered by a thin and striatum cuticle. The hairs are of two categories: tectorial and secretory. Tectorial hairs are numerous, long, multicellular, uni-seriated, with a sharp point (Fig. 5). Secretory hairs, although relatively short, presents a bicellular or tricellular pedicel and a unicellular gland; the hairs with unicellular pedicel and octo-cellular gland are very rare. The stomata protrude visible above external level of epidermic cells (Fig. 6). The cortex contains cords of angular collenchyme in the ribs, a layer of hypodermic tangential collenchyme and assimilator parenchyma between the ribs (4-5 layers). The cortex ends with a casparian endoderm. In pericyclic position, the central cylinder has only on the periphery of large beams, one very thin cord, unistratified, made of sclerenchymatous fiber, with moderately thickened and lignified walls. Conductive tissues consist of four large libero-ligneous bundles (in the rib area) and four small ones between them, all with secondary structure. At ligneous level (thicker than libero) rows of vessels are separated by libriforme fibres with moderately thickened but highly lignified walls. At the ligneous level medullary rays are sclerificate and lignified, forming together a sinuous ring. The pith is thick, with large parenchymal cells; the perimedullar area has small cells with lignified walls. The middle level of the stem shows similar structure to that described above, the differences occurring in the epidermis level where that the tectorial and secretory hairs are more numerous per unit area; an exception are secretory hairs with octo-cellular gland, which are very rare (Fig. 2). In the inferior level of the strain the ribs remain visible. The hairs frequency (tectorial and secretory) is the same as for the levels discussed above (Fig. 3). In the central cylinder there are ring-shaped conductive tissues: a continuous thin ring of secondary phloem and a thicker ring (especially in the rib area) of secondary xylem, continuously due to medullary rays strongly sclerificate and lignified with vessels scattered here and there. Only a few of primary xylem is observed, because the parenchyma cells between the 26

3 vessels have lignified walls. The foliar limb is thin, with prominent median rib on the bottom and with a ditch on the top. This rib is comprised of a libero-ligneous bundle, little hypodermic collenchyma and meatic parenchyma. On the both sides there are many long tectorial multicellular hairs, but higher are the short unicellular ones. Secretory hairs are relatively rare, with bicellular pedicel and unicellular gland. The very short ones, with octocellular gland are very rare. The superior epidermis has cells bigger than the lower epidermis. The stomata, very prominent above the skin, are located only on the bottom side, the lamina being hypostomatic. The mesophyll is differentiated in the unilayer palisade tissue (50% of the assimilatory parenchyma thickness) and in multilayer lacunous tissue, with small cells so the lamina has a dorsal-ventral bifacial structure (Fig. 4). Anatomical investigations on the taxon Mentha x piperita were made by Toma and Rugină (1998) [13]. M. longifolia was anatomical researched by Aprotosoaie et al. in 2009 [1]. The plant material investigated was part of an experimental batch, but their results are similar to ours. Studies on the genus Mentha species revealed the presence of secretory capitate hairs, provided with a single secretory gland, and peltate hairs, provided with eight secretory cells [4; 10; 15]. Cross sections made through the lamina of Mentha x piperita by Mihaela Niţă et al. (2000) captured the presence of short secretory hairs, consisting of: a basal cell, a unicellular short pedicel and an octo-celullar gland [14]. Our analysis on the volatile oil obtained from M. longifolia revealed the presence of 25 chemical compounds representing 98.59% of the total obtained, as presented in Table I. The main chemical compounds recorded (in terms of percentage) in this volatile oil test are: piperitone-oxide (36.74%), limonene (17.61%), β-cubebene (8.05%), β-mircene (7.38%), trans-β-ocimene (5.64%) and β-cariophyllene (3.20%), totalizing 78.68%, thus providing the plant's aromatic character. High amounts of piperitone - oxide (14.7%) were reported in the volatile oil from M. longifolia analyzed Gulluce et al. (2007) [7]. This compound was found in the oil sample analyzed by us, but the percentage is much higher (36.74%). Research conducted by Kokkini et al., 1987, identified the presence of large amounts of carvone (58%) in the volatile oil from M. longifolia, a compound which was not present in the oil that we have analyzed. The menthol, a monoterpenic alcohol characteristic to genus Mentha species [5], was not identified in the analyzed oil sample. Piperitone oxide is a natural monoterpene present in the composition of certain volatile oils. Biological effects of this compound were studied by Damien et al. (2003) [2]. They have shown the active antibacterial and antifungal activity of the compound. According to Magiatis et al. 1999, the compounds α pinene, β - pinene limonene and linalool show strong antimicrobial activity [11]. Table I. Chemical composition of volatile oils from M. longifolia (vegetative phenophase) No Compounds α - Pinene β - Pinene β Mircene Sabinene Limonene Trans-β-Ocimene Cis-β-Ocimene Ocimene Geranil izo butirat Linaool 27 %

4 Cis Hexenil - Valerat N Hexenil izovalerat Piperitone - Oxide Timol Dihydrocarvil acetate β Burbonene β Elemene β Cariophyllene Cariophyllene Oxide β Cubebene Germacrene D-4-ol β Cadinene r - elemene t - Murolol α Cadinol Other compounds Conclusions Histo-anatomical investigations conducted have revealed that the aerial stem has quadratic shape in cross-section, with proeminent rounded ribs. The epidermis has isodiametric cells with the external wall slightly thicker than others and covered by a support cuticle and stomata thrust forward above it. Long, multicellular, uniseriated, sharp pointed tectorial hairs are numerous per unit area and secretory hairs are multicellular, but short. The cortex is collenchymatous in the ribs and parenchymatic assimilatory elsewhere, with both meatus and aerial gaps between cells. Conductive tissues form large liberoligneous bundles of open collateral type in the 4 ribs; between them there are four intermediate bundles. The leaf has a thrust forward median rib to the underside of lamina with a libero-ligneous bundle. Tectorial hairs are uni-, bi-, multicellular and secretory hairs are relatively rare, especially the ones with unicellular gland. The limb has bifacialheterofacial structure. The volatile oil contains mainly piperitone oxide (36.74%), limonene (17.61%), βmircene (7.38%) which is compounds with antibacterial and antifungal properties. Knowing the exact composition of the vegetal material taken into study is very important for later using them into obtaining pharmaceutical products and some useful products for human use and in taking advantage of their antimicrobial and antifungal properties also. Acknowledgements This work was supported by the the European Social Fund in Romania, under the responsibility of the Managing Authority for the Sectoral Operational Programme for Human Resources Development [grant POSDRU/88/1.5/S/47646] and by program Developing the innovation capacity and improving the impact of research through post-doctoral programmes POSDRU/89/1.5/S/

5 REFERENCES 1. APROTOSOAIE C., FLORIA V., RUGINĂ R., HANCIANU M., MIRON A., STĂNESCU U., 2009 Histoanatomical researches regarding the influence of Topsin M treatments on Mentha longifolia L. (Huds.) (Lamiaceae). An. Şt. Univ. Al. I. Cuza Iasi, s.ii a. Biol Veget., 55, 1: DERWICH E., BENZIANE Z., BOUKIR A., 2010 Antibacterial activity and chemical composition of the leaf essential oil of Mentha rotundifolia from Marocco. Elec. J. Environ., Agric. Food Chem. 9, 1: DŽAMIĆ A. M., SOKOVIĆ M. D., RISTIĆ M., NOVAKOVIĆ M., GRUJIĆ-JOVANOVIĆ S., TEŠEVIĆ V., MARIN P., 2010 Antifungal and antioxidant activity of Mentha longifolia (L.) Hudson (Lamiaceae) essential oil. Botanica Serbica, 34, 1: FAHN A., 1979 Secretory Tissues in Plants. Academic Press London. 5. GALEOTTI N., MANNELLI L., MAZZANTI G., BARTOLINI A., GHELARDINI C., 2001 Menthol: a natural analgesic compound. Neurosci. Lett., 322: GERSHENZON J., MAFFEI M., CROTEAU R., 1989 Biochemical and Histochemical Localization of Monoterpene Biosynthesis in the Glandular Trichomes of Spearmint (Mentha spicata). Plant Physiol., 89: GULLUCE M., SAHIN F., SOKMEN M., OZER H., DAFERERA D., SOKMEN A., POLISSIOU M., ADIGUZEL A., OZKAN H., 2007 Antimicrobial and antioxidant properties of the essential oils and methanol extract from Mentha longifolia L. ssp. longifolia. Food Chem., 103: KOKKINI S., KAROUSOU R., LANARAS T., 1987 Essential oils of Spearmint (Carvone rich) Plants from the Island of Crete (Greece). Biochem. Syst. Ecol., 23, 4: LANGE B., CROTEAU R., 1999 Genetic engineering of essential oil production in mint. Curr. Opinion in Plant Biotechnology, 2: MAFFEI M., BERTEA C., MUCCIARELLI M., 2006 Anatomy, Physiology, Biosynthesis, Molecular Biology, Tissue Culture, and Biotechnology of Mint Essential Oil Production, in Lawrence B. (Ed.), Mint. The Genus Mentha. CRC Press, Boca Raton: MAGIATIS P., MELLIOU E., SKALTSOUNIS A. L., CHINOU I. B., MITAKU S., 1999 Chemical composition and antimicrobial activity of the essential oils of Pistacia lentiscus var. chia. Planta Med., 65, 8: MKADDEM M., BOUAJILA J., ENNAJAR M., LEBRIHI A., MATHIEU F., ROMDHANE M Chemical Composition and Antimicrobial and Antioxidant Activities of Mentha (longifolia L. and viridis). Essential Oils. J. Food Sci., 74, 7: TOMA C., RUGINĂ R., 1998 Anatomia plantelor medicinale. Atlas. Edit. Acad. Rom., Bucureşti. 14. TOMA C., NIŢĂ M., RUGINĂ R., IVĂNESCU L., COSTICĂ N., 2000 Morfologia şi anatomia plantelor, (Manual de lucrări practice). Edit. Univ. Al. I. Cuza Iaşi: TURNER G., GERSHENZON J., CROTEAU R Development of Peltate Glandular Trichomes of Peppermint. Plant Physiol., 124: Explanation of Plate I Figure 1. Cross section through the superior level of Mentha longifolia stem. Figure 2. Cross section through the middle level of M. longifolia stem. Figure 3. Cross section through the inferior level M. longifolia stem. Figure 4. Cross section through the main rib of M. longifolia foliar limb Figure 5. Tectorial hairs from the stem of M. longifolia. Figure 6. Stomata from the stem of M. longifolia. 29

6 A. R. Andro, D. Atofani, I. Boz, M.-M. Zamfirache, I. Burzo, C. Toma Figure 1 PLATE I Figure 2 Figure 3 Figure 4 Figure 6 Figure

A COMPARATIVE STUDY REGARDING THE MORPHOLOGY AND ANATOMY OF THE VEGETATIVE APPARATUS IN TWO OCIMUM BASILICUM L. BREEDS.

Analele ştiinţifice ale Universităţii Al. I. Cuza Iaşi Tomul LIV, fasc. 2, s.ii a. Biologie vegetală, 2008 A COMPARATIVE STUDY REGARDING THE MORPHOLOGY AND ANATOMY OF THE VEGETATIVE APPARATUS IN TWO OCIMUM