25

Analele ştiinţifice ale Universităţii „Al. I. Cuza” IaşiTomul LVII, fasc. 2, s. II a. Biologie vegetală, 2011

STUDIES CONCERNING THE HISTO-ANATOMY AND BIOCHEMISTRYOFMENTHA LONGIFOLIA (L.) HUDS. DURING VEGETATIVE PHENOPHASE

ANCA RALUCA ANDRO*, DOINA ATOFANI*, IRINA BOZ*, MARIA-MAGDALENA 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 oftectorial 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 chemicalcompounds. It was recorded the presence of 25 chemical compounds, five of them representing 75.42% of thetotal obtained oil.

Keywords: volatile oil, Mentha longifolia, medicinal plant.

Introduction

The genus Mentha L. comprises herbaceous, perennial plants, common intemperate climates in Europe, Australia and South Africa [9]. Due to their properties theseplants are used in pharmaceutical, cosmetics and food industries. These properties are mostoften offered by volatile oils produced by the secretory hairs. The representatives of thegenus Mentha have capitates and peltate secretory hairs, provided by a single secretorygland, having eight secretory cells [4; 10; 14]. They are initiated very early and begin toaccumulate 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 inglandular peltate hairs [15].

The chemical composition of volatile oil derived from M. longifolia wasinvestigated by Mkaddem et al. (2009) [12]. Their research has been conducted onpopulations from different regions of Gabes, Tunisia. Compounds identified in significantquantities 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 thevolatile oil extracted from M. longifolia obtaining as main components trans- and cis-dihydrocarvone (23.64% and 15.68%), piperitone (17.33%), 1,8-cineole (8.18%) andneoisodihydrocarveol (7.87% ) [3].

In this context we decided to analyze the histo-anatomical aspects of the speciesM. longifolia and to identify the chemical composition of the volatile oil during thevegetative phenophase of the plant.

Materials and methods

The plant material used consisted of aerial organs from the species M. longifoliacollected in June of 2010, when the plant was in vegetative phenophase.

* Alexandru Ioan Cuza University, Faculty of Biology, Bd. Carol I, no. 11, Iasi - 700506 Romania,anca_andro@yahoo.com** University of Agronomical Sciences and Veterinary Medicine Bucharest, Romania, Faculty of Horticulture

26

The plants were collected from the banks of Bârlad River in Negreşti, Vaslui County, anddetermined 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 crosssections were made through these organs using a microtome and a botanical razor. Theobtained sections were colored with green iodine and ruthenium red to obtain a samplewhich was subsequently analyzed with optical microscope.

Volatile oils were extracted with a Clevenger device, according with the EuropeanPharmacopoeia Standards, in the Vegetal Physiology Laboratory of the Faculty of Biology,University “Al. I. Cuza” of Iaşi. Inside the balloon was introduced chopped plant materialand water. The distillation process was three hours long. Segregation and identification ofchemical compounds of essential oils were performed with GC_MS (Gas Chromatographycoupled with mass spectrometry), within the Research Center for Food and Agriculturefrom the Faculty of Horticulture, University of Agronomic Sciences and VeterinaryMedicine in Bucharest. Chemical compounds were identified by comparing retention timerecorded 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 withrelatively prominent ribs. It was observed an early transition to secondary structure, butonly 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. Thehairs are of two categories: tectorial and secretory. Tectorial hairs are numerous, long,multicellular, uni-seriated, with a sharp point (Fig. 5). Secretory hairs, although relativelyshort, presents a bicellular or tricellular pedicel and a unicellular gland; the hairs withunicellular pedicel and octo-cellular gland are very rare. The stomata protrude visible aboveexternal level of epidermic cells (Fig. 6). The cortex contains cords of angular collenchymein the ribs, a layer of hypodermic tangential collenchyme and assimilator parenchymabetween the ribs (4-5 layers). The cortex ends with a casparian endoderm.

In pericyclic position, the central cylinder has only on the periphery of largebeams, one very thin cord, unistratified, made of sclerenchymatous fiber, with moderatelythickened and lignified walls. Conductive tissues consist of four large libero-ligneousbundles (in the rib area) and four small ones between them, all with secondary structure. Atligneous level (thicker than libero) rows of vessels are separated by libriforme fibres withmoderately thickened but highly lignified walls. At the ligneous level medullary rays aresclerificate and lignified, forming together a sinuous ring. The pith is thick, with largeparenchymal cells; the perimedullar area has small cells with lignified walls. The middlelevel of the stem shows similar structure to that described above, the differences occurringin the epidermis level where that the tectorial and secretory hairs are more numerous perunit 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 centralcylinder there are ring-shaped conductive tissues: a continuous thin ring of secondaryphloem and a thicker ring (especially in the rib area) of secondary xylem, continuously dueto 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

27

vessels have lignified walls. The foliar limb is thin, with prominent median rib on thebottom and with a ditch on the top. This rib is comprised of a libero-ligneous bundle, littlehypodermic collenchyma and meatic parenchyma. On the both sides there are many longtectorial multicellular hairs, but higher are the short unicellular ones. Secretory hairs arerelatively rare, with bicellular pedicel and unicellular gland. The very short ones, with octo-cellular gland are very rare. The superior epidermis has cells bigger than the lowerepidermis. 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 palisadetissue (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 andRugină (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 aresimilar to ours. Studies on the genus Mentha species revealed the presence of secretorycapitate hairs, provided with a single secretory gland, and peltate hairs, provided with eightsecretory cells [4; 10; 15]. Cross sections made through the lamina of Mentha x piperita byMihaela Niţă et al. (2000) captured the presence of short secretory hairs, consisting of: abasal cell, a unicellular short pedicel and an octo-celullar gland [14].

Our analysis on the volatile oil obtained from M. longifolia revealed the presenceof 25 chemical compounds representing 98.59% of the total obtained, as presented in TableI. The main chemical compounds recorded (in terms of percentage) in this volatile oil testare: 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%, thusproviding the plant's aromatic character.

High amounts of piperitone - oxide (14.7%) were reported in the volatile oil fromM. longifolia analyzed Gulluce et al. (2007) [7]. This compound was found in the oilsample analyzed by us, but the percentage is much higher (36.74%). Research conducted byKokkini et al., 1987, identified the presence of large amounts of carvone (58%) in thevolatile oil from M. longifolia, a compound which was not present in the oil that we haveanalyzed. The menthol, a monoterpenic alcohol characteristic to genus Mentha species [5],was not identified in the analyzed oil sample. Piperitone oxide is a natural monoterpenepresent in the composition of certain volatile oils. Biological effects of this compound werestudied by Damien et al. (2003) [2]. They have shown the active antibacterial andantifungal 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 %1 α - Pinene 2.082 β - Pinene 2.43 β – Mircene 7.384 Sabinene 2.065 Limonene 17.616 Trans-β-Ocimene 5.647 Cis-β-Ocimene 0.738 Ocimene 0.229 Geranil izo butirat 1.38

10 Linaool 2.5

28

11 Cis – Hexenil - Valerat 0.8712 N – Hexenil –izovalerat 0.3513 Piperitone - Oxide 36.7414 Timol 0.6415 Dihydrocarvil acetate 0.2516 β – Burbonene 0.6117 β – Elemene 2.1818 β – Cariophyllene 3.219 Cariophyllene Oxide 0.2420 β–Cubebene 8.0521 Germacrene D-4-ol 0.4422 β – Cadinene 0.5123 r - elemene 1.9524 t - Murolol 0.2525 α – Cadinol 0.31

Other compounds 1.41

Conclusions

Histo-anatomical investigations conducted have revealed that the aerial stem hasquadratic shape in cross-section, with proeminent rounded ribs. The epidermis hasisodiametric cells with the external wall slightly thicker than others and covered by asupport cuticle and stomata thrust forward above it. Long, multicellular, uniseriated, sharppointed tectorial hairs are numerous per unit area and secretory hairs are multicellular, butshort. The cortex is collenchymatous in the ribs and parenchymatic assimilatory elsewhere,with both meatus and aerial gaps between cells. Conductive tissues form large libero-ligneous bundles of open collateral type in the 4 ribs; between them there are fourintermediate bundles. The leaf has a thrust forward median rib to the underside of laminawith a libero-ligneous bundle. Tectorial hairs are uni-, bi-, multicellular and secretory hairsare relatively rare, especially the ones with unicellular gland. The limb has bifacial-heterofacial structure.

The volatile oil contains mainly piperitone oxide (36.74%), limonene (17.61%), β-mircene (7.38%) which is compounds with antibacterial and antifungal properties.Knowing the exact composition of the vegetal material taken into study is very importantfor later using them into obtaining pharmaceutical products and some useful products forhuman use and in taking advantage of their antimicrobial and antifungal properties also.

AcknowledgementsThis work was supported by the the European Social Fund in Romania, under the

responsibility of the Managing Authority for the Sectoral Operational Programme forHuman Resources Development 2007-2013 [grant POSDRU/88/1.5/S/47646] and byprogram „Developing the innovation capacity and improving the impact of researchthrough post-doctoral programmes” POSDRU/89/1.5/S/4994.

29

REFERENCES

1. APROTOSOAIE C., FLORIA V., RUGINĂ R., HANCIANU M., MIRON A., STĂNESCU U., 2009 – Histo-anatomical 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: 31 – 38.

2. DERWICH E., BENZIANE Z., BOUKIR A., 2010 – Antibacterial activity and chemical composition of the leafessential oil of Mentha rotundifolia from Marocco. Elec. J. Environ., Agric. Food Chem. 9, 1: 19 – 28.

3. 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: 57 – 61.

4. 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: 145 – 148.6. 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: 1351 – 1357.

7. 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 andmethanol extract from Mentha longifolia L. ssp. longifolia. Food Chem., 103: 1449-1456.

8. KOKKINI S., KAROUSOU R., LANARAS T., 1987 – Essential oils of Spearmint (Carvone rich) Plants fromthe Island of Crete (Greece). Biochem. Syst. Ecol., 23, 4: 425 – 430.

9. LANGE B., CROTEAU R., 1999 – Genetic engineering of essential oil production in mint. Curr. Opinion inPlant Biotechnology, 2: 139 – 144.

10. MAFFEI M., BERTEA C., MUCCIARELLI M., 2006 – Anatomy, Physiology, Biosynthesis, MolecularBiology, Tissue Culture, and Biotechnology of Mint Essential Oil Production, in Lawrence B. (Ed.),Mint. The Genus Mentha. CRC Press, Boca Raton: 41 – 85.

11. MAGIATIS P., MELLIOU E., SKALTSOUNIS A. L., CHINOU I. B., MITAKU S., 1999 – Chemicalcomposition and antimicrobial activity of the essential oils of Pistacia lentiscus var. chia. Planta Med.,65, 8: 749 – 752.

12. MKADDEM M., BOUAJILA J., ENNAJAR M., LEBRIHI A., MATHIEU F., ROMDHANE M. 2009 –Chemical Composition and Antimicrobial and Antioxidant Activities of Mentha (longifolia L. andviridis). Essential Oils. J. Food Sci., 74, 7: 358 – 363.

13. 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: 100 – 108.15. TURNER G., GERSHENZON J., CROTEAU R. 2000 – Development of Peltate Glandular Trichomes of

Peppermint. Plant Physiol., 124: 215 – 223.

Explanation of Plate IFigure 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 limbFigure 5. Tectorial hairs from the stem of M. longifolia.Figure 6. Stomata from the stem of M. longifolia.

30

A. R. Andro, D. Atofani, I. Boz, M.-M. Zamfirache, I. Burzo, C. Toma PLATE I

Figure 3 Figure 4

Figure 2Figure 1

Figure 5 Figure 66