http://informahealthcare.com/phb ISSN 1388-0209 print/ISSN 1744-5116 online Editor-in-Chief: John M. Pezzuto Pharm Biol, 2014; 52(6): 688–697 ! 2014 Informa Healthcare USA, Inc. DOI: 10.3109/13880209.2013.865239

ORIGINAL ARTICLE

Chemical composition of the essential oil and botanical study of the flowers of Mentha suaveolens

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El-Sayeda A. El-Kashoury, Hesham I. El-Askary, Zeinab A. Kandil, and Mohamed A. Salem Department of Pharmacognosy, Faculty of Pharmacy, Cairo University, Cairo, Egypt

Abstract

Keywords

Context: Herbal medicines play a paramount role in the treatment of wide range of diseases, so there is a growing need for their quality control and standardization. Traditionally, histological and morphological inspections have been the usual methods to authenticate herbs intended for medicinal applications. Mentha suaveolens Ehrh. (Lamiaceae) is native to Africa Temperate Asia and Europe and it’s cultivated in Egypt. Objective: The macro- and micromorphology of the flowers of M. suaveolens Ehrh. cultivated in Egypt were studied to find the diagnostic characters of this species. In addition, the chemical composition of the essential oil of the flowers was also studied to define the chemotype of the plant. Materials and methods: Photographs of macro- and micromorphology were taken using Casio and Leica DFC500 digital cameras, respectively. In addition, the essential oil was prepared by hydrodistillation followed by gas chromatographic/mass spectrometric (GC/MS) analysis for identification of its components. Results: The macro- and micromorphological characteristics of M. suaveolens were determined. The yield of the essential oil obtained by hydrodistillation from M. suaveolens flowers was 1.7% calculated on dry weight basis. GC/MS analysis of the oil resulted in identification of 29 components, which amounted to 99.77% of the total oil composition. The major component was carvone (50.59%) followed by limonene (31.25%). Discussion and conclusion: The results obtained herein revealed for the macro, micromorphological and chemical composition characteristics of the flowers. The results of GC/MS analysis of the essential oil supported that M. suaveolens cultivated in Egypt could be categorized as carvone-rich chemotype since this compound pertained its high relative percentile.

Apple mint, botanical study, carvone, inflorescence, taxonomy

Introduction Mints are perennial aromatic herbs that are cultivated for their essential oils used both for medicinal and aromatic purposes (McKay & Blumberg, 2006). The taxonomy of mints is a complex problem and several classifications, varying, in the number of recognized species have been proposed in the past. The systematics of the genus Mentha (Lamiaceae) are especially difficult because of frequent hybridization occurring both in wild populations and in cultivation (Sˇaric´Kundalic´ et al., 2009). Within the genus Mentha, it has been suggested that the five basic species; Mentha arvensis L., Mentha aquatica L., Mentha spicata L., Mentha longifolia (L.) Huds and Mentha suaveolens Ehrh. (apple mint) have given rise to 11 naturally occurring and named hybrids (Tucker & Naczi, 2007). The present literature suggests the classification of genus Mentha into the three basic lines named as, capitatae, spicatae and verticillatae, based on inflorescence characters. The line Correspondence: Mohamed A. Salem, Department of Pharmacognosy, Faculty of Pharmacy, Cairo University, Kasr El-Ainy St., Cairo 11562, Egypt. Tel: +20 1007515754. Fax: +20 25320005. E-mail: [email protected]

History Received 17 March 2013 Revised 4 September 2013 Accepted 8 November 2013 Published online 6 February 2014

capitatae includes all species with compact, head-like inflorescence; the type species is M. aquatica. The spicatae species have a spike as shown by M. spicata, M. longifolia and M. suaveolens. The third line is represented by M. arvensis having an inflorescence vertically partitioned into whorls (Sˇaric´-Kundalic´ et al., 2009). However, alternatively mints were classified based on the dominant monoterpene compound prevailing in the essential oil reflected by three metabolic pathways. Thus, the production of linalool and linalyl acetate is typical for the linalool pathway; menthol, menthone and menthofuran are constituents of the menthol pathway; and carvone, dihydrocarvone and carveol characterize the carvone pathway (Lawrence, 2007; Sˇaric´-Kundalic´ et al., 2009; Schalk & Croteau, 2000). Nothing was found in the available literature concerning the chemical composition of the essential oil of M. suaveolens Ehrh. cultivated in Egypt flowers. Few reports were found concerning their taxonomical features. Little information was reported on the macromorphology of flower and nothing was found regarding their micromorphology. Therefore, the macroand micromorphology of the flowers of the plant cultivated in Egypt is carried out to find its diagnostic characters, which help in identification and differentiation of this species.

DOI: 10.3109/13880209.2013.865239

In addition, the chemical composition of the essential oil of the flowers is presented to define its chemotype.

Materials and methods

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Plant material The fresh flowers of M. suaveolens cultivated in Egypt were collected during the summers of 2009–2010 from plants cultivated in The Experimental Station of Medicinal and Aromatic Plants, Pharmacognosy Department, Faculty of Pharmacy, Cairo University, Giza, Egypt. The plant was kindly authenticated by Dr Gemma L. C. Bramley, Curator of the Lamiaceae collections, Herbarium Department, Library, Art & Archives, Royal Botanic Gardens, Kew, Richmond, Surrey, United Kingdom. Botanical profiling Specimens for morphological studies were dried according to standard herbarium techniques and voucher samples (M-20/313) were kept at the Herbarium of the Department of Pharmacognosy, Faculty of Pharmacy, Cairo University. Photographs were taken using a Casio digital camera. Anatomical investigations were performed on cross sections of the flowers, which were preserved in 70% ethyl alcohol containing 5% glycerin and air-dried finely powdered samples. The photographs were taken using a Leica DFC500 digital camera (Leica Company, Wetzlar, Germany) attached to a light microscope. Preparation of the essential oil Samples of fresh flowers (1 kg, each) were collected and subjected to hydrodistillation for 3 h and the oils obtained were dried over anhydrous sodium sulfate. The yield was calculated on dry weight basis and the oils were saved in a refrigerator for further analysis. The specific gravity and refractive index were determined according to the Egyptian Pharmacopoeia (E.P., 1984) procedures. All stated values were the average of three determinations. GC/MS analysis The hydrodistilled oil was subjected to gas chromatographic/ mass spectrometric (GC/MS) analysis on an Agilent (Santa Clara, CA) GC–MS system, model 6890, fitted with an Agilent mass spectroscopic detector (MSD), model 5937, as well as a 30 -m long, cross-linked 5% phenyl polysiloxane (HP5-MS capillary column, Hewlett Packard, San Jose, CA) fused-silica column (i.d. 0.25 mm, film thickness 0.25 mm). The initial temperature was 60  C, kept isothermal for 3 min, then increased to 260  C at 8  C/min, and the final temperature was kept isothermal for 15 min. The ion source temperature was 230  C and the quadrupole temperature was 150  C. The carrier gas was helium adjusted at a flow rate of 0.1 mL/min. Ionization energy was 70 eV, and scan range was 40–500 m/z at 3.62/scan. Identification of the oil components Library search for identification of the oil components was carried out using a Willey and Nist (6th ed.) 275 L GC-MS

Botanical study and taxonomy of Mentha suaveolens flowers

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data base. A series of authentic n-alkanes (C8-C22, Poly Science Inc., Niles, IL) were subjected to GLC analysis under the same experimental conditions. The retention indices (Kovat’s indices, KI) of the oil constituents were computed by logarithmic interpolation between bracketing alkanes (Jennings & Shibamoto, 1980). Identification of the individual components was confirmed by comparison of their retention indices and MS fragmentation patterns with published data (Adams, 2004).

Results Essential oil composition The yield of the essential oil obtained by hydrodistillation from flowers of M. suaveolens cultivated in Egypt was 1.7%, calculated on dry weight basis. The oil sample was colorless, with spearmint-like odor and readily soluble in ethanol 70%. The specific gravity determined at 25  C was determined to be 0.815 at 25  C. Refractive index recorded at 20  C was 1.491. GC/MS analysis of the oil (Table 1) resulted in identification of 29 components with relative percentage equal or exceeding 0.01%, which amounted to 99.77% of the total oil Table 1. Chemical composition of the essential oil of flowers of M. suaveolens Ehrh. cultivated in Egypt. Peak

Kovat’s index

Identified component

1 1021 Limonene 2 1035 g-Terpinene 3 1088 Linalool 4 1150 Borneol 5 1168 cis-Dihydrocarvone 6 1175 trans-Dihydrocarvone 7 1193 cis-Carveol 8 1235 Carvone 9 1296 Dihydrocarveol acetate 10 1305 trans-Carvyl acetate 11 1324 Piperitenone 12 1333 cis-Carvyl acetate 13 1357 b-Bourbonene 14 1380 cis-Jasmone 15 1397 trans-b-Caryophellene 16 1407 b-Cedrene 17 1423 b-Copaene 18 1435 a-Humulene 19 1447 g-Muurolene 20 1465 Germacrene D 21 1483 Bicyclogemacrene 22 1502 g-Cadinene 23 1511 trans-Calamenene 24 1527 a-Cadinene 25 1577 Caryophyllene oxide 26 1603 Cubenol-1,10-di-epi 27 1627 epi-a-Cadinol 28 1640 epi-a-Muurolol 29 1965 Manoyl oxide Number of identified compounds % of identified components Classes of the essential oil components A-Hydrocarbons  Monoterpenes  Sesquiterpenes  Aromatic hydrocarbons B-Oxygenated constituents  Monoterpenes  Sesquiterpenes  Other oxygenated constituents

Percentage 31.25 0.01 0.32 0.68 0.15 0.85 2.31 50.59 0.36 0.35 0.12 0.44 1.27 1.27 2.56 0.32 0.28 1.24 0.61 2.04 0.35 0.13 0.45 0.09 0.30 0.31 0.50 0.34 0.28 29 99.77 40.60 31.26 8.89 0.45 59.17 56.17 1.45 1.55

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composition. The amount of identified oxygenated constituents was 59.17%, while that of identified hydrocarbons was 40.60%. The overall chromatographic profile of the oil sample was dominated by the oxygenated constituents (59.17%), dominated by ketones (51.90%) among which carvone (50.59%) was the major constituent in the oil. Other classes of the oxygenated constituents as alcohols, esters and oxides were minor. Monoterpenes was the major class of hydrocarbons (31.26%) detected, while sesquiterpenes constituted only 8.89%. Limonene was the main monoterpene hydrocarbon (31.25%).

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Macromorphological study M. suaveolens (Figure 1A) is an herbaceous, perennial plant. It grows to 40–60 cm tall and spreads by creeping suckers. Stems are erect, quadrangular and tomentose. Leaves are sessile, nearly ovate and green in color. Flowers are arranged in verticillasters forming a terminal spike-like inflorescence. The flowering season begins in July and lasts until September. The inflorescence (Figure 1B) is verticillaster of many flowers. These verticillasters are crowded at the apex of the main stem and lateral branches, with the subtending leaves being reduced to bracts, thus appearing as a spike-like inflorescence and measuring about 3.5–6 cm in length and 0.4–1 cm in width. It has aromatic odor and taste. The bract (Figure 1C) is oval with acuminate apex, broadly dentate margin, two teeth in each side, green in color, hairy, measuring 0.2–0.4 cm in length and 0.1–0.2 cm in width at the middle region. The flower (Figure 1D) is zygomorphic, hermaphrodite and sessile with the general floral formula %, , K(5), C(5), A4, G(2). It has aromatic odor and taste, measuring about 2–5 mm in length and 1–3 mm in diameter. The calyx is green in color, hypogenous, pubescent, tubular (A)

gamosepalous, consists of equally five-toothed sepals. The calyx tube measures 1–2 mm in length and 0.5–1 mm in diameter. The corolla is white in color, hypogenous, pubescent, and formed of five petals sympetalous. It is differentiated into tube and a limb that widens towards the mouth, the tube is straight, paler in color than the limb. The limb is markedly divided into two lips, the two posterior petals forming the upper lip, while the three anterior petals form the lower lip. The corolla tube measures 2–4 mm in length and 1– 2 mm in diameter at the mouth. The androecium (Figure 5) consists of four short and equal epipetalous stamens. The filaments are white in color while the anthers are yellowish white. The filament measures 0.5–0.8 mm in length and 0.3 mm in diameter. The anthers are two celled, each is dehiscing longitudinally and measuring 0.2–0.3 mm in length and 0.1 mm in width. The gynaecium consists of a syncarpous, bicarpillary, tetraocular and superior ovary which measures 0.4–0.8 mm in length and about 0.4 mm in diameter. Style is cylindrical, gynobasic, white in color, long measuring 3–5 mm in length and about 0.1–0.2 mm in diameter. Stigma is bifid and measures 0.2–0.3 mm. Micromorphological study A transverse section in the rachis (Figure 2) is more or less quadrangular in outline. It shows an outer epidermis surrounding 6–9 rows of collapsed collenchymatous cells followed by 3–4 rows of parenchymatous cells. The cortex is lined by almost differentiated endodermis enclosing the pericycle and vascular tissue. The ring of the vascular bundle (A)

(B)

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(C)

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(D)

Figure 1. Photographs of Mentha suaveolens Ehrh. (A) A flowering branch of Mentha suaveolens Ehrh. (X ¼ 0.6). (B) The inflorescence (X ¼ 1). (C) The bract (X ¼ 10). (D) The flower (X ¼ 12).

Figure 2. Transverse section of the rachis of the inflorescence of Mentha suaveolens Ehrh. (A) Low power view (X ¼ 30). (B) High power view (X ¼ 215). (C) High power view from the corners (X ¼ 315). col., collenchyma; c.par., cortical parenchyma; end., endodermis; ep., epidermis; m.r., medullary ray; n.gl.t., non-glandular trichomes; per., pericycle; ph., phloem; pi., pith; xy., xylem.

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surrounds a wide parenchymatous pith. The epidermal cells (Figures 2 and 4A) are polygonal with straight beaded anticlinal walls and covered with thin striated cuticle. Trichomes are of both glandular and non-glandular types (Figure 8). The glandular trichomes are of two types: trichomes with unicellular stalk and unicellular head, covered with thin cuticle (capitate type) and trichomes with unicellular stalk and multicellular head of eight radiating cells (liliaceous type). The non-glandular trichomes are abundant and arise from one or more epidermal cells. They are either simple unicellular or multicellular (5–10), uniseriate, conical, with acute or blunt apices and covered with warty cuticle. The apical cells are sometimes curved and rarely collapsed. The cortex (Figure 2) is slightly narrow and consists of 5–7 rows of collapsed collenchymatous cells followed by 3–4 rows of parenchymatous cells. At the corners, the cortical tissue is formed of 8–10 rows of collenchymatous cells followed by 4–5 rows of parenchymatous cells. The endodermis is almost differentiated by casparian thickenings (stained red with Sudan III on the radial walls). The pericycle (Figure 2) is formed of a continuous ring of about 2–3 rows of collapsed parenchymatous cells surrounding the vascular tissue. The vascular tissue (Figure 2) is moderately wide, concentrated at the four corners. The phloem (Figure 2) is comparatively narrow and consists of thin-walled cellulosic phloem elements: sieve tubes, companion cells and phloem parenchyma with no fibers. The medullary rays (Figures 2 and 8) are uni- to biseriate, being cellulosic, radially elongated, nonlignified in the phloem and slightly lignified in the xylem region. The cambium (Figure 2) is almost indistinguishable. The xylem (Figures 2 and 8) is lignified, forming a continuous ring and composed of fibers, vessels and wood parenchyma. The fibers (Figure 8) have moderately thin-lignified walls, wide lumina and acute apices. The vessels (Figures 2 and 8) are lignified, diffused either isolated or in radial rows with spiral thickenings. Wood parenchyma (Figures 2 and 8) are diffused and formed of moderately thin-walled, pitted, lignified and axially elongated cells. The pith (Figure 2) is wide, formed of moderately large rounded, sometimes pitted cells with thin cellulosic walls and narrow intercellular spaces. A transverse section in the bract (Figure 3) shows upper and lower epidermises enclosing a dorsiventral mesophyll with one row of palisade cells being interrupted in the midrib region with parenchymatous cells. The midrib is strongly prominent on the lower surface of the lamina. It shows a single crescent-shaped collateral vascular bundle, accompanied with a parenchymatous pericycle. The upper epidermal cells (Figures 3 and 4B) are polygonal somewhat elongated having straight anticlinal walls and covered with thin striated cuticle. The lower epidermal cells (Figures 3 and 4C) are similar to those of the upper epidermis, differing only in having slightly wavy walls and covered with thin smooth cuticle. The neural cells have similar structure. Stomata are few of diacytic type. Trichomes (Figures 3, 4 and 8) are very numerous on both surfaces. They are of glandular and nonglandular types. The glandular trichomes are more frequent on the lower epidermis than the upper one. They are similar to those present on the rachis and differ only in size. The mesophyll (Figure 3) is dorsiventral with an upper palisade

Botanical study and taxonomy of Mentha suaveolens flowers (A)

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(B)

(C)

Figure 3. Transverse section of the bract of Mentha suaveolens Ehrh. (A) Low power view (X ¼ 40). (B) High power view of the midrib (X ¼ 100). (C) High power view of the lamina (X ¼ 333). c.par., cortical parenchyma; ca., cambium; gl.t., glandular trichomes; l.ep., lower epidermis; m.r., medullary ray; n.gl.t., non-glandular trichomes; pal.t., palisade tissue; per., pericycle; ph., phloem; sp.t., spongy tissue; u.ep., upper epidermis; xy., xylem.

Figure 4. Epidermal cells of the rachis and bract of Mentha suaveolens Ehrh. (A) Epidermal cells of the rachis (X ¼ 450). (B) Upper epidermal cells of the bract (X ¼ 433). (C) Lower epidermal cells of the bract (X ¼ 433). cap.gl.t., capitate glandular trichomes; cic., cicatrix; lab.gl.t., labiaceous glandular trichomes; str.cu., striated cuticle.

formed of one row of radially elongated columnar cells with straight anticlinal walls and containing green chloroplasts. The spongy tissue is formed of irregular shaped parenchymatous cells with wide intercellular spaces. The cortex (Figure 3A and B) consists of upper and lower cortical tissues. The upper cortical tissue of the midrib consists of 9–10 rows of polygonal parenchymatous cells with thin cellulosic walls. The lower cortical tissue of the midrib consists of 2–3 rows of collenchymatous cells followed by 6–8 rows of more or less rounded parenchymatous cells with thin cellulosic walls. The endodermis is indistinct. The pericycle (Figure 3A and B) consists of parenchymatous cells below the vascular bundle. The cells are small, polygonal rounded with thick cellulosic

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Figure 5. Transverse section of the flower of Mentha suaveolens Ehrh. (At the upper part of the calyx) (X ¼ 60). ant., anther; pet., petals; p.gr., pollen grains; sep., sepals; sty., style.

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(F)

(H)

walls. The vascular tissue (Figure 3A and B) shows a single crescent-shaped collateral vascular bundle. The phloem (Figure 3A and B) is comparatively narrow and consists of thin-walled cellulosic phloem elements: sieve tubes, companion cells and phloem parenchyma with no fibers. The medullary rays (Figure 3A and B) are uniseriate, formed of polygonal cells with thin cellulosic walls. The cambium (Figure 3A and B) is almost distinguishable, formed of about 2–3 rows of thin-walled small and radially arranged cells. The xylem (Figures 3A and B and 8) consists of xylem vessels and wood parenchyma with no fibers. The vessels are lignified, diffused in radial rows and show spiral thickenings. A transverse section in the flower (at the upper part of the calyx) representing the sepals, petals, anthers and style is shown in Figure 5. A transverse section in the sepal (Figure 6A) is composed of outer and inner epidermises, enclosing in-between collapsed cortical parenchymatous cells. The vascular strand is embedded within the cortical tissue. The inner epidermis at the base (Figure 6B) consists of axially elongated cells with straight anticlinal walls and covered with thin striated cuticle. At the tip (Figure 6C), the cells are more or less isodiametric. The outer epidermises at the base and tip (Figure 6D and E) consist of slightly axially elongated cells with straight anticlinal walls, covered with thin striated cuticle and showing diacytic stomata. Trichomes

(E)

(G)

(I)

(J)

Figure 6. Micromorphology of the calyx and corolla of Mentha suaveolens Ehrh. (A) Transverse section of the sepal (X ¼ 60). (B) Inner epidermal cells of the calyx at the base (X ¼ 450). (C) Inner epidermal cells of the calyx at the tip (X ¼ 450). (D) Outer epidermal cells of the calyx at the base (X ¼ 450). (E) Outer epidermal cells of the calyx at the tip (X ¼ 450). (F) Transverse section of the petal (X ¼ 140). (G) Inner epidermal cells of the corolla at the base (X ¼ 775). (H) Inner epidermal cells of the corolla at the tip (X ¼ 775). (I) Outer epidermal cells of the corolla at the base (X ¼ 775). (J) Outer epidermal cells of the corolla at the tip (X ¼ 775). c.par., cortical parenchyma; l.ep., lower epidermis; n.gl.t., non-glandular trichomes; u.ep., upper epidermis; v.b., vascular bundle.

Botanical study and taxonomy of Mentha suaveolens flowers

DOI: 10.3109/13880209.2013.865239

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Figure 7. Micromorphology of the androecium and gynaecium of Mentha suaveolens Ehrh. (A) Detailed transverse section of the anther (X ¼ 160). (B) Detailed transverse section of the filament (X ¼ 125). (C) Detailed transverse section of the ovary (X ¼ 160). (D) Ovary wall (X ¼ 333). (E) Transverse section of the style (X ¼ 267). (F) Transverse section of the stigma (X ¼ 217). c.par., cortical parenchyma; c.t., connective tissue; ep., epidermis; f.l., fibrous layer of anther; i.ep., inner epidermis; n.gl.t., non-glandular trichome; o.ep., outer epidermis; ov., ovule; o.w., ovary wall; p.gr., pollen grains; p.s., pollen sac; v.b., vascular bundle.

(A)

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(Figure 8) are of both glandular and non-glandular types. The glandular trichomes are similar to those present on the bract and differ only in size. The non-glandular trichomes are bi- to tricellular, conical, with blunt apices and covered with warty cuticle. A transverse section in the petal (Figure 6F) is composed of an outer and inner epidermises enclosing inbetween a narrow homogenous mesophyll formed of several rows of more or less rounded parenchymatous cells traversed by vascular strands. The inner epidermis at the base (Figure 6G) consists of axially elongated cells with straight anticlinal walls and covered with thin striated cuticle. At the tip (Figure 6H), the cells are more or less isodiametric. The outer epidermises at the base and tip (Figure 6I and J) consist of axially elongated cells with straight anticlinal walls and covered with thin smooth cuticle. Trichomes (Figure 8) are of both glandular and non-glandular type. The glandular trichomes are similar to those previously mentioned on the rachis and bract but differ only in size. The non-glandular trichomes are simple unicellular, conical, with blunt apices and covered with warty cuticle. A transverse section in the anther (Figure 7A) shows two anther lobes attached by the connective tissue, which has a vascular strand in the center; each lobe contains numerous pollen grains. The epidermis (Figure 8) consists of elongated cells, with straight anticlinal walls and covered with thin faintly striated cuticle. Stomata and trichomes are absent. The fibrous layer (Figures 7A and 8) is formed of one row of radially elongated cells, nearly polygonal in surface view showing lignified bar-like thickening. The pollen grains (Figures 7A and 8) are more or less oval to rounded in shape with smooth surface. A transverse section in the filament (Figure 7B) is kidney shaped and shows homogenous

693

(F)

parenchymatous cells within which a vascular strand is embedded. The epidermal cells (Figure 8) are formed of elongated cells with straight anticlinal walls, covered with striated cuticle and devoid of stomata and trichomes. A transverse section in the ovary (Figure 7C) is more or less rounded in outline, showing four locules with central placentation. The ovary wall is followed by a parenchymatous mesophyll showing scattered vascular bundles. The outer and inner epidermal cells of the ovary (Figure 8) are polygonal in shape, more or less axially elongated, having straight anticlinal walls and covered with thin smooth cuticle. Stomata are absent. Trichomes (Figures 7D and 8) of only non-glandular type are present. They are bicellular, conical, with blunt apices and covered with warty cuticle. A transverse section in the syle (Figure 7E) is more or less rounded in outline. It shows vascular strands surrounded by homogenous parenchymatous cells. The epidermal cells of the style (Figure 8) are polygonal, with straight anticlinal walls and covered with thin smooth cuticle. A transverse section in the stigma (Figure 7F) is more or less rounded in outline and shows vascular strands surrounded by homogenous parenchymatous cells. The epidermal cells of the stigma (Figure 8) are polygonal, papillosed with straight anticlinal walls. The microscopical measurements of the different elements of the bract, rachis and flower are listed in Table 2. The powdered inflorescence (Figure 8) is yellowish-green in color with aromatic characteristic odor and taste. It is characterized microscopically by the following features: (1) Fragments of epidermis of the rachis showing polygonal cells with straight beaded anticlinal walls and covered with striated cuticle.

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Figure 8. Powdered inflorescence of Mentha suaveolens Ehrh. ep.an, epidermis of anther (X ¼ 650); ep.br, epidermis of bract (X ¼ 433); ep.ca, epidermis of calyx (X ¼ 450); ep.cr, epidermis of corolla (X ¼ 775); ep.fil, epidermis of filament (X ¼ 650); ep.ov., epidermal cells of the ovary (X ¼ 375); ep.ra, epidermis of rachis (X ¼ 450); ep.stg., epidermal cells of the stigma (X ¼ 450); ep.sty., epidermal cells of the style (X ¼ 525); f.l., fibrous layer of anther (X ¼ 650); gl.t., glandular trichomes (X ¼ 433); m.r., medullary ray (X ¼ 550); n.gl.t., non-glandular trichomes (X ¼ 433); p.gr., pollen grains (X ¼ 650); pal., palisade cells (X ¼ 400); v.o.dp., volatile oil droplets (X ¼ 300); w.f., wood fibers (X ¼ 233); w.p., wood parenchyma (X ¼ 500); x.v., xylem vessel (X ¼ 750).

(2) Fragments of elongated cells of the lower epidermis of the bract with straight anticlinal walls covered with smooth cuticle. Few diacytic stomata are present. (3) Fragments of the outer epidermis of the calyx at the base, consists of slightly axially elongated cells with straight anticlinal walls. They are covered with thin striated cuticle and showing few diacytic stomata. (4) Fragments of the inner epidermis of the corolla at the base consist of axially elongated cells with straight anticlinal walls and covered with thin striated cuticle. (5) Fragments from the epidermis of the anther consist of elongated cells, with straight anticlinal walls and covered with thin faintly striated cuticle. (6) Fragments from the epidermis of the filament formed of elongated cells with straight anticlinal walls, covered with striated cuticle and devoid of stomata and trichomes. (7) Fragments from the epidermis of the ovary consist of polygonal, more or less axially elongated cells, with straight anticlinal walls, covered with thin smooth cuticle and devoid of stomata.

(8) Fragments from the epidermis of the syle formed of polygonal cells, with straight anticlinal walls, and covered with thin smooth cuticle. (9) Fragments from the epidermis of the stigma consist of polygonal, papillosed cells with straight anticlinal walls. (10) Numerous glandular trichomes of capitate and labiaceous types. (11) Numerous non-glandular trichomes, unicellular and multicellular uniserate (2–6) with thin walls, wide lumina, acute or blunt apices and covered with warty cuticle. The apical cells are sometimes curved and rarely collapsed. (12) Fragments of xylem vessels with spiral thickenings. (13) Numerous fragments of fusiform wood fibers of the rachis having moderately thin-lignified walls, wide lumina and acute apices. (14) Fragments of pitted and lignified axially elongated wood parenchyma and medullary ray cells with cellulosic and non-lignified walls of the rachis. (15) Fragments of the fibrous layer of the anther are formed of radially elongated cells, nearly

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Figure 8. Continued.

(16) (17) (18) (19)

polygonal in surface view showing lignified bar-like thickening. Numerous pollen grains which are more or less oval to rounded in shape. Numerous volatile oil droplets (stained red with Sudan III). Fragments of columnar, thin walled palisade cells of the bract containing green chloroplasts. Absence of calcium oxalate crystals and starch granules.

Discussion Previous investigations on the chemical composition of the essential oil from M. suaveolens different populations collected in various regions showed high percentage of oxides. These include piperitone oxide (Hendriks et al., 1976; Holeman et al., 1985; Oumzil et al., 2002) and piperitenone oxide (Koliopoulos et al., 2010; Holeman et al., 1985; Oumzil et al., 2002; Sutour et al., 2010). Other chemotypes of M. suaveolens showed high percentage of alcohols such as menthol (Velasco-Negueruela

et al., 1996). Some chemotypes of M. suaveolens showed high percentage of ketones such as pulegone (Oumzil et al., 2002; Sutour et al., 2008; Velasco-Negueruela et al., 1996), piperitenone (Sutour et al., 2010) and dihydrocarvone (Hendriks et al., 1976). Chemical analysis of the essential oil showed that M. suaveolens contained mostly monoterpenes bearing oxygen function at C-3 such as piperitenone, pulegone and menthol which indicates that it is more closely related to M. x piperita (Lupein et al., 1999). However, the essential oils from M. x piperita are rich in menthone or menthol depending on the age of the plant (Gershenzon et al., 2000; McConkey et al., 2000). Most M. suaveolens chemotypes contained no menthone or menthol. Such divergence may be due to both developmental and environmental factors as well as to a difference in levels of biosynthetic enzymes in particular the NADPH-dependent reductases that convert pulegone to menthone or menthol (Gershenzon et al., 2000). M. suaveolens cultivated in Egypt contained, however, monoterpenes bearing oxygen function at C-2 such as carvone

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Pharm Biol, 2014; 52(6): 688–697

Table 2. Microscopical measurements of the different organs of the inflorescence M. suaveolens Ehrh.

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Element 1. The rachis Epidermal cells Glandular trichomes Capitate type  The stalk  The head Labiaceous type  The head Non-glandular trichomes Medullary rays Wood fibres Wood parenchyma Xylem vessels Volatile oil droplets 2. The bract Upper epidermal cells Lower epidermal cells Upper neural cells Lower neural cells Glandular trichomes Capitate type  The stalk  The head Labiaceous type  The head Non-glandular trichomes Xylem vessels Palisade cells Volatile oil droplets 3. The flower (a) The calyx Inner epidermis at the base Inner epidermis at the tip Outer epidermis at the base Outer epidermis at the tip Stomata Glandular trichomes Capitate type  The stalk  The head Labiaceous type  The head Non-glandular trichomes Xylem vessels Volatile oil droplets (b) The corolla Inner epidermis at the base Inner epidermis at the tip Outer epidermis at the base Outer epidermis at the tip Glandular trichomes Capitate type  The stalk  The head Labiaceous type  The head Non-glandular trichomes (c) The androecium Epidermis of filament Epidermis of anther Fibrous layer of anther Pollen grains (d) The gynaecium Epidermis of ovary Epidermis of style Epidermis of stigma Non-glandular trichomes

Length

Height

Width

Diameter

17–33–45

15–26–33

2–4–6



4–9–11 20–22–25

8–11–13 16–20–22

– –

– –

– 100–105–111 11–23–27 278–280–386 55–62–67 – –

– 6–8–12 14–16–18 8–9–13 31–33–37 – –

– – – – – – –

35–37–40 – – – – 17–20–23 4–5–8

22–31–34 26–33–48 15–26–40 13–22–41

13–22–25 11–17–34 6–13–23 8–13–23

35–40–42 25–32–40 – –

– – – –

11–18–46 15–23–34

11–18–27 15–23–29

– –

– –

– 115–138–162 – 12–17–28 –

– 4–6–9 – 5–7–10 –

– – – – –

40–43–49 – 21–25–28 – 3–4–5

13–22–33 15–22–35 15–20–37 9–11–33 17–19–23

6–8–12 13–18–31 6–13–15 6–11–13 13–15–18

– – – – –

– – – – –

3–5–11 11–13–20

3–4–7 10–13–15

– –

– –

– 24–33–46 – –

– 6–9–23 – –

– – – –

41–44–45 – 15–18–20 3–4–5

19–21–32 15–16–33 21–23–38 6–13–20

11–13–15 10–13–23 3–5–7 3–5–7

– – – –

– – – –

9–13–15 19–20–23

7–8–10 13–16–18

– –

– –

– 57–69–80

– 18–23–30

– –

30–31–34 –

17–23–48 6–10–22 14–15–20 –

11–14–18 3–5–9 5–7–10 –

9–15–21 8–9–11 – –

– – – 7–11–15

18–20–35 8–11–30 2–10–18 44–46–48

12–15–20 7–8–12 1–3–6 8–10–13

– 7–11–13 9–13–16 –

– –

The underlined numbers indicate average values.



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DOI: 10.3109/13880209.2013.865239

which indicates that it is more closely related to M. spicata not M. x piperita. The absence of menthone or menthol in M. suaveolens cultivated in Egypt may be due to the absence of pulegone, which is the precursor of menthone or menthol. The biosynthesis of monoterpenes in some oils of Mentha species is produced through the formation of diosphenolene which carries central oxygen function both at C-2 and C-3. Enzymatic reduction and loss of water at C-3 of diosphenolene result in the formation of carvone followed by further reduction of other components of carvone series such as dihydrocarvone, cis-carveol, trans-carveol, neo-dihydrocarveol and their acetates. Enzymatic reduction and loss of water at C-2 of diosphenolene result in the formation of piperienone series such as piperitone oxide, piperitenone, pulegone, piperitone, menthone, menthol and their acetates (Hendriks et al., 1976 and references cited therein). This explains the variation in the constituents of essential oil of M. suaveolens in different regions. We can expect that the presence of carvone as a major constituent of essential oil of M. suaveolens cultivated in Egypt may be due to different developmental and environmental factors as well as to a difference in levels of biosynthetic enzymes in the biosynthesis of different constituents. So the presence of carvone and its derivatives such as cis-dihydrocarvone, trans-dihydrocarvone, cis-carveol, dihydrocarveol acetate and transcarvyl acetate can be explained by occurrence of carvone pathway via enzymatic reduction and loss of water at C-3 of diosphenolene. Therefore, M. suaveolens cultivated in Egypt could be categorized as carvone-rich type.

Conclusion Flowers of M. suaveolens cultivated in Egypt are presented with the aim of finding the important selective characteristics for authentication of plant. In addition, the plant could be categorized as carvone-rich type after a study of the flowers essential oil.

Declaration of interest The authors report no conflicts of interest. The authors alone are responsible for the content and writing of this article.

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Chemical composition of the essential oil and botanical study of the flowers of Mentha suaveolens.

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