Pharmacobotanical study of Hypericum thymopsis

ABSTRACT

Hypericum thymopsis Boiss., Hypericaceae, is an endemic herb which generally grows at the calcareous steppe regions of Central Turkey. In flowering stage, the aerial parts of this species are used for wound-healing and sedation, and its infusions are used against stomach diseases and throat infections by local people. The aim of this study, to examine and to reveal of the morphology, anatomy and histology of the aerial vegetative and reproductive organs of the H. thymopsis, which are used in popular medicine and thus contributing to the pharmacognostic evaluation of the species. In comparison with previous published morphological description of the species, some different findings about plant height and leaf length were found. In addition, some morphological characteristics such as dimensions of sepals and ovaries, length of filaments, anthers, pistils and pedicels were examined here for the first time. The anatomical characteristics of stem, leaf, sepal, filament and pistil were studied using light microscopy and additionally for stem and leaf using scanning electron microscopy. The stem has the secondary growth, and circular shape. The leaves are amphistomatic and the mesophyll is dorsiventral. Stomata are anisocytic and sunken. The glandiferous emergences are present on stem and translucent glands exist in leaf mesophyll. Type A secretory canals are present in stem, leaf, sepal and petal cross section. While type B secretory canals are observed in sepal and petal, type C secretory canals are observed solely in ovary cross section. All the structural features herein found can assist the diagnosis of H. thymopsis.

Keywords:
Anatomy; Clusiaceae; Hypericum thymopsis; Medicinal plant; Morphology; Turkey

Introduction

Hypericaceae is a small cosmopolite dicot family represented by nine genera and about 500 species of tree, shrub and herb in the world (Simpson, 2006; APG III, 2009; Carine and Christenhusz, 2010). Hypericum is the largest genus of Hypericaceae by comprising about 400 species throughout the world except the poles, deserts and low altitude areas of tropical regions (Lewis, 2003; Meseguer and Sanmartín, 2012). In Turkey, Hypericum is represented by 96 species, 104 taxa in which 45 of them are endemic with the endemism ratio 43% (Güner et al., 2012). The genus Hypericum characterized by possess generally pale to dark yellow flowers and transparently dotted leaves with red or black colored glands (Davis, 1966, 1988; Güner et al., 2000). The genus Hypericum is known worldwide with its wide folk medicinal usage (Avato, 2005; Assadi et al., 2011). Hypericum species are known under the names "binbirdelik otu", "kanotu", "peygamber çiçeği" and by a majority "sarı kantaron", and have been used as sedatives, antiseptics, anti-inflammatory, anti-astmatic, antispasmodics, wound-healing and to against stomach diseases, ulcers and diabetes in Turkish folk medicine (Bingol et al., 2011; Özkan and Mat, 2013). The taxa of Hypericum characterized by having extensively secretory structure such as translucent glands, black nodules and secretory canals which synthesizing and accumulating biologically active substances, so they have great phytochemical potential and composition (Siersch, 1927; Metcalfe et al., 1950; Curtis and Lersten, 1990; Ciccarelli et al., 2001a, 2001b; Onelli et al., 2002). Some of these phytochemical compositions of the Hypericaceae are naphthodianthrones (notedly hypericin and pseudohypericin), acylphloroglucinol derivatives (notedly hyperforin and adhyperforin), flavonoids (notedly quercetin, quercitrin, hyperoside and biapigenin), tannins, n-alkanes, xanthones and essential oils (Bombardelli and Morazzoni, 1995; Bruneton, 1995; Kitanov, 2001; Maggi et al., 2004). Due to their phytochemical composition and waste usage in traditional folk medicine, many studies were made on biological activities of different species extracts of Hypericum in Turkey and in the world. For example, analgesic activity (Öztürk, 2001), wound healing activity (Öztürk et al., 2007), hepatoprotective activity (Öztürk et al., 1992), anti-inflammatory activity (Öztürk et al., 2002), anti-Helicobacter pylori activity (Yeşilada et al., 1999) and antidepressant activity (Öztürk, 1997) of well-known Hypericum, H. perforatum L. were studied by different researchers. In addition, antioxidant activity of H. venustum Fenzl was studied by Spiteller et al. (2008), antimicrobial activity of H. hyssopifolium Chaix var. microcalycinum (Boiss. & Heldr.) Boiss. was studied by Toker et al. (2006), and myeloperoxidase activity of H. empetrifolium Willd. was studied by Kültür (2007). In addition there are some studies focused morphology, anatomy and secretory structure of some Hypericum species (Metcalfe et al., 1950; Toma and Rugină, 1998; Hong-Fei and Zheng-Hai, 2001; Yaylacı et al., 2013; Perrone et al., 2013a, 2013b).

Hypericum thymopsis Boiss. is an endemic species for Turkey Flora and naturally growing in Sivas, Kayseri, Malatya and Kahramanmaraş provinces (Davis, 1966). Essential oil composition of H. thymopsis was studied by Özkan et al. (2009, 2013). Özkan et al. (2013) studied composition of essential oils of five endemic Hypericum species for Turkey (H. uniglandulosum Hausskn. ex Bornm., H. scabroides Robson and Poulter, H. kotschyanum Boiss., H. salsugineum Robson and Hub.-Mor. and H. thymopsis). According to their study, α-pinene, baeckeol, limonene and spathulenol were identified as major components of H. thymopsis. Furthermore, H. thymopsis has greatest potential by having so much more α-pinene, within these five species. α-Pinene is one of the active substance which has bronchodilator and anti-inflammatory activity (Russo, 2011) and has bacteriostatic and bactericide effect against especially gram (+) and gram (-) bacteria with broad spectrum (Nissen et al., 2010). In addition, it has acetylcholinesterase inhibitor activity, thus it has effect that strengthens the memory of human (Russo, 2011). α-Pinene is also reported as one of the main components of the essential oils of other some Hypericum species (Joulain and König, 1998; Santos et al., 1999; Zeng and Zhou, 2001; Gudžić et al., 2002, 2004).

In the field works, by interviewing local peoples under an ethnobotanical studies, it was learned that above ground parts of H. thymopsis are used for wound-healing and sedation, and its infusions and decoctions as tea are used also against stomach diseases and throat infections. By considering great usage of various Hypericum taxa in folk medicine, their anatomical features are not well-known except H. perforatum. In Turkey, there are some anatomical studies on other Turkish Hypericum taxa (Yaylacı et al., 2013; Altıntaş and Akçin, 2015). The morphological characteristics of all Turkish Hypericum taxa are more or less known. But there is some deficiency on some endemic species such as H. thymopsis. For example, in Flora of Turkey (Davis, 1966), dimensions of sepals and ovaries, length of filaments, anthers, pistils and pedicels of H. thymopsis are not reported. Despite of some essential oil studies on H. thymopsis (Özkan et al., 2009, 2013), there is no report on anatomical and histological structure of the species. The aim of this study, revealed entirely morphological characteristics, and anatomical and histological structure in all above ground parts and their secretory structures which make up biologically active substances of H. thymopsis.

Materials and methods

Plant material

The specimens of Hypericum thymopsis Boiss., Hypericaceae, were collected during the flowering and fruiting time from different natural populations in Sivas province of Turkey. Field works were carried out in between the years 2012–2014. Locality 1: B6 Sivas: Ulaş district, Ziyarettepe, 1444 m, 39º33'06.7″ N; 37º01'11.9″ E, 26.06.2013; Locality 2: B6 Sivas: Ulaş district, Kurtlukaya village to Boğazdere village, 1458 m, 39º23'02.1″ N; 36º55'48.3″ E, 05.06.2014; Locality 3: B6 Sivas: Sivas-Kangal-Gürün road intersection, 1560 m, 39º07'53.1″ N; 37º14'32.9″ E, 06.08.2012, ibid. 05.06.2014, ibid. 05.07.2014.

These were registered under collector numbers M. Tekin 1309, 1470, 1562, 1566, 1625 and are conserved at the Cumhuriyet University, Faculty of Science Herbarium (CUFH), Department of Biology, Sivas, Turkey. Taxonomical identification was made according to Flora of Turkey (Davis, 1966) by author.

Structural analysis

Morphological description of H. thymopsis was made using both fresh and herbarium samples collected from different localities. Parts of the some fresh material were stored in 70% alcohol–water solution for later histo-anatomical procedures. For this reason, hand-made transverse sections of stem, leaf, sepal, petal, filament, ovary and style were taken. Also superficial hand-made sections of adaxial and abaxial surface of the leaf blade were taken with razor blade. The sections were stained in 1% Alcian blue (Sigma) and 1% Safranine O (Sigma), in a ratio 3/2 (Davis and Barnett, 1997). The sections were kept about 5 min in the dye. Semi-permanent slides were mounded using glycerin-gelatine (Jensen, 1962). The structural investigations of vegetative and reproductive parts of H. thymopsis were made using Olympus BX22 light microscopy. Photomicrographs were taken using Olympus BX51 light microscopy coupled with Olympus DP70 digital camera. The anatomical terminology used is that according to Esau (1965). For secretarial analyses, the study of Ciccarelli et al. (2001b) was carried out: (a) Translucent glands. These are pale glands or glandular pockets (Robson, 1981) and are spherical or oblong glands delimited by two layers. (b) Three types of secretory canals. Type A: the lumen of the canals is usually surrounded by four (or more in especially flowers parts) polygonal cells which have very thin wall toward canal lumen. Type B: they have a same structure with translucent glands with their wide lumen, but they seem elongated and pale which differ from the translucent glands. Type C: type C canals composed of wide cavity surrounded by one or more cell layers which have densely stained and thin walls.

For scanning electron microscopy (SEM) analyses, the some plant parts were mounted on aluminum stubs and coated with gold. The micromorphological and anatomical observations were made, and micrographs were taken at different magnifications by using LEO 440 SEM.

Results

Morphological aspects

Perennial herbs. Stems 3–14 cm, erect, numerous, glabrous, scabrid with glandiferous emergences (Fig. 1B). Leaves 5–18 × 0.6–1.6 mm, linear, revolute with obtuse or rounded apex, glabrous, usually papillose at adaxial surface and especially at margins. Black glands present at the margins of small leaves which positioned at the base of pedicel on stem. Inflorescence corybose, up to 17 cm in fruiting time, soliter or 20 flowered. Sepals 1.9–2.8 × 0.8–1.1 mm, united at base, lanceolate to lanseolate-elliptic with sessile or subsessile black glands at margins and acute apex (Fig. 1D). Sepals reddish when flowers in bud stage, green in flowering time (Fig. 1A, D). Petals 4.5–7.5 × 2–3.8 mm, pentamerous, elliptic-oblanceolate to obovate and without black glands (Fig. 1C, E). Filaments 4–5.5 mm, thin and numerous. Anthers 0.35–0.45 mm, dorsifixed and longitudinal dehiscence (Fig. 1F). Pistils 4–5 mm, ovaries 1.5–2.2 × 0.9–1.4 mm, 3 loculed and ovoidal shaped. Style 3, free and selender, 2.5–3.2 mm (Fig. 1G). Placentation axillar, ovules anatropous. Pedicels 2–8 mm. Fruit 4.5–5.5 × 2.7–3.1 mm, ovoid, reddish-brown, septicidal capsule rostrate and with 3 locule, each locule bear usually 2, occasionally 1 seed. Seeds 2–2.3 × 0.7–1 mm, light brown, oblong-cylindrical and covered by densely short thick white hairs (Fig. 1).

Fig. 1
Morphology of Hypericum thymopsis. In natural habitat at flowering stage (A); stem under SEM, arrows indicate glandiferous emergences (B); flower (C); calyx, arrow indicate black glands (D); petal (E); stamen (F); pistil (G).

Anatomical and histological aspects

Stem

The stem is in the secondary growth stage and circular shaped in transverse section (Fig. 2A, E). The epidermis is uniseriate and its cells are small, oval or usually irregular shaped, and are surrounded by thick cutinized wall at all around the cell. Cuticle layer is thick. There are two small wings and glandiferous emergences on stem (Fig. 2A). Cortex consists of 3–7 parenchymatous cell layers. Cortex cells are oval or circular-shaped and there are some intercellular spaces between them. There is a thin collenchyma which compose a ring in cortex. Endodermis is uniseriate and cells are dorsoventrally depressed. The pericycle is conspicuous with thickened laterally cell walls, just beneath the endodermis (Fig. 2B, F). Vascular bundles are located in the major portion of the stem transverse section. In phloem, there are lots of type A secretory canals which are composed of four secretory cells. Cambium is indistinguishable. Secondary xylem is ring-porous. Pith rays are parenchymatous and uniseriate, occasionally biseriate (Fig. 2C, G). Pith cells are parenchymatous and are oval or circular shaped. Size of pith cells enlarge toward center and the cell walls are lignified and prominently thickened in this region (Fig. 2D, H).

Fig. 2
Photomicrographs of stem transverse section of Hypericum thymopsis. Under light microscopy (LM) (A–D); under scanning electron microscopy (SEM) (E–H). General view (A, E), red arrows indicate small wings in A; detail of cortex region (B, F); detail of vascular bundles (C, G); detail of pith region (D, H). Abbreviations: cl, collenchyma; cx, cortex; ep, epidermis; en, endodermis; ge, glandiferous emergence; pe, pericycle; ph, phloem; pt, pith; pt-lg, pith cells-lignified; pt-nlg, pith cells-nonlignified; pr, pith ray; sc, secretory canal-type A; sl, secretory cell; ve, vessel element; xy, xylem.

Leaf

Leaf is "3" shaped in transverse section. There are uniseriate epidermises on both surfaces of the leaves. Adaxial epidermis cells are very large, oval or rectangular shaped and usually papillose especially near lamina margins, conversely, abaxial epidermis cells are small, rectangular or occasionally squarish shaped and are all non-papillose (Fig. 3A, D). Epidermal cells have a smooth cuticle on both sides. There is conspicuous epicuticular wax on abaxial epidermis while it is lack of on adaxial surface (Fig. 4C–F). The cell wall of adaxial epidermis is very thick, despite that it is thin in abaxial epidermis cells (Fig. 3D). In addition, margins of adaxial epidermis cells are distinct and slightly wavy, while margins of abaxial epidermis cells are obscure. Stomata are anisocytic and sunken, occurring in both on the adaxial and predominantly abaxial sides of the leaf lamina (Figs. 3F and 4). The mesophyll is dorsiventral and is formed by 2–3 layers of palisade parenchyma and 2–3 layers of spongy parenchyma. While, the palisade parenchyma cells are generally cylindrical, occasionally rectangular, squarish or ovoid-shaped, the spongy parenchyma cells are usually cylindrical occasionally ovoid or irregular-shaped (Fig. 3B, D). There are large translucent glands that positioned between just beneath the adaxial epidermis and the middle area of spongy parenchyma (Fig. 3A, D). Small collateral vascular bundles are observed immersed into the mesophyll, associated with one or a few type A secretory canals in the phloem (Fig. 3E). The midrib is oval-shaped, particularly large and protrude from the abaxial surface. The midrib surrounded by a uniseriate parenchymatous bundle sheath. There are a collenchyma cap which cells are usually ovoid or circular-shaped and which make the midrib even more prominent and conspicuous (Fig. 3B). The cambium is clearly distinguishable between xylem and phloem, so vascular bundle type is open collateral. In vascular bundle, there are numerous type A secretory canals which are composed of four secretory cells and are immersed the phloem (Fig. 3C).

Fig. 3
Photomicrographs of the leaf trasnverse sections of Hypericum thymopsis. General view (A); detail of midrib region (B); detail of vascular bundle in midrib (C); detail of lamina with translucent gland (D); detail of small vascular bundle with a type A secretory canal in mesophyll (E); detail of sunken stomata in adaxial epidermis (F). Abbreviations: ab, abaxial epidermis; ad, adaxial epidermis; as, air space; bs, bundle sheath; ca, cambium; cx, cortex; gc, guard cells; ph, phloem; pp, palisade parenchyma; sc, secretory canal-type A; sl, secretory cell; sp, spongy parenchyma; st, stoma; tg, translucent gland; vb, vascular bundle; xy, xylem.

Fig. 4
Photomicrographs of the leaf blade superficial sections on light microscopy (A and B); micrographs of the leaf blade surface on scanning electron microscopy (C–F) of Hypericum thymopsis; general view of adaxial surface (A, C); detail of adaxial surface (B, D); general view of abaxial surface (E); detail of abaxial surface (F). Abbreviations: wx, epicuticular wax; adc, adaxial epidermis cell; gc, guard cell; st, stoma; tg, leaf surface view of translucent gland.

Sepal

The transverse section of sepal was taken from calyx tube. Epidermis is uniseriate in both sides. Adaxial epidermis cells are rectangular or rectangular ovoid-shaped, but they are larger and irregular shaped especially above the main vascular bundles region. Abaxial epidermis cells are rectangular, squarish or oval shaped and are usually larger than adaxial epidermis cells (Fig. 5A). Mesophyll consists of 7–12 parenchymatous cell layers. These cells are usually oval, rarely circular-shaped and they have intercellular spaces. The midrib is almost circular-shaped and lacks bundle sheath (Fig. 5B). There are type A secretory canals in phloem of vascular bundles and these canals consist of 4–6 secretory cells (Fig. 5C). Beneath the abaxial epidermis, there are large type B secretory canals which alternate with the vascular bundles and immersed in mesophyll (Fig. 5D).

Fig. 5
Photomicrographs of calyx tube transverse section of Hypericum thymopsis. General view (A); detail of sepal midrib region with type A and type B secretory canals (B); detail of vascular bundle and type A secretory canal in sepal (C); detail of type B secretory canal (D). Abbreviations: ab, abaxial epidermis; abw, abaxial epidermis cell; ad, adaxial epidermis; cu, cuticle; pc, parenchymatous cell; ph, phloem; scA, secretory canal-type A; scB, secretory canal-type B; sl, secretory cell; st, stoma; vb, vascular bundle; xy, xylem.

Petal

Adaxial and abaxial epidermises are uniseriate in petal transverse section. While abaxial epidermis cells are rectangular or squarish-shaped, adaxial epidermis cells are usually irregular shaped. The mesophyll consists of 5–8 layered parenchymatous cells which are usually oval-shaped and have intercellular spaces between them (Fig. 6A, C). Vascularization is made by a few small vascular bundles in the center of mesophyll. Type A and type B secretory canals may occur in same section, as they can be seen in separate sections as in Fig. 6A and C. Type A secretory canals are present in vascular bundles and they consist of four or more secretory cells (Fig. 6B). Type B secretory canals occur just beneath the abaxial epidermis and they alternate with the veins (Fig. 6C, D).

Fig. 6
Photomicrographs of petal transverse section of Hypericum thymopsis. General view with type A secretory canals (A) and detail view of vascular bundle and type A secretory canal (B). General view with type B secretory canals (C) and detail view of type B secretory canal (D). Abbreviations: ab, abaxial epidermis; ad, adaxial epidermis; cu, cuticle; pc, parenchymatous cell; ph, phloem; scA, secretory canal-type A; scB, secretory canal-type B; sl, secretory cell; vb, vascular bundle; xy, xylem.

Filament

Filaments are very thin and triangular-shaped in transverse section. Uniseriate epidermal cells are large and usually irregular-shaped. Cuticle is thick and rugose. There is small single vascular bundle in the center of transverse section. Between epidermis and vascular bundle, there are irregular-shaped parenchymatous cells. The members of secretory structure are absent (Fig. 7A).

Fig. 7
Photomicrographs of the reproductive parts transverse section of flower of Hypericum thymopsis. General view of filament (A); general view of style (B); general view of ovary (C); detail of carpel (D). Abbreviations: ab, abaxial epidermis; ad, adaxial epidermis; cp, carpel wall; cu, cuticle; ep, epidermis; pc, parenchymatous cell; ph, phloem; pt, pollen-tube transmitting tract; scC, secretory canal-type C; sl, secretory cell; vb, vascular bundle; xy, xylem.

Pistil

The style is rectangular-shaped in transverse section. Epidermis is uniseriate, with rectangular, squarish or irregular-shaped cells. Outer walls of epidermal cells are thick. Cuticle is rugose and thick. There is single vascular bundle which positioned close to epidermis. There is pollen tube transmitting tract (PTTT) in the center of transverse section. Between the region of epidermis and PTTT are filled by oval, circular or irregular shaped parenchymatous cells. The members of secretory structure are absent in transverse section of style (Fig. 7B). The ovaries have 3 locules (Fig. 7C). Adaxial epidermis cells of carpels are usually irregular, occasionally rectangular or squarish shaped. Abaxial epidermis cells of carpels are rectangular or squarish shaped. The mesophyll cells are parenchymatous and usually irregular-shaped. Just beneath the adaxial epidermis, there are numerous type C secretory canals which immersed in the parenchymatic mesophyll of each locules (Fig. 7D).

Discussion

Despite of its usage in folk medicine, the morphology of H. thymopsis is not entirely known. There are some general knowledge on morphology of the species in Flora of Turkey (Davis, 1966), but there are no report on the shape of petals, dimensions of sepals and ovaries, length of filaments, anthers, pistils, styles and pedicels of the species. These features are examined in present study. In Flora of Turkey (Davis, 1966), stems height and petals length of H. thymopsis are given as 3–11 cm and 5–7 mm, respectively. In present study, stems height and petals length of H. thymopsis were found as 3–14 cm and 4.5–7.5 mm, respectively. In addition, black glands were observed at the margins of small leaves which are positioned at the base of pedicel on stem. It is not reported in Flora of Turkey (Davis, 1966) (Fig. 1).

According to Metcalfe et al. (1950), pith rays are uniseriate, the xylem and phloem are relatively narrow, whilst there is a greater development of pith in stem of herbaceous Hypericum. Conversely, in H. thymopsis stem, phloem and especially xylem fill major part of the transverse section, pith located in the small region of the transverse section center (Fig. 2A, E). Pith rays of H. thymopsis are commonly uniseriate, occasionally biseriate. In addition, pith ray cells of H. thymopsis are upright and usually oval or rectangular-shaped, whereas the examined Hypericum species of Metcalfe et al. (1950), they are upright and entirely square-shaped (Fig. 2C, G). The outlines of stem transverse section are circular-shaped, vascular bundles of the H. thymopsis stems always formed a circle which is reported for some other Hypericum species by Arda (1989) and Tokur and Mısırdalı (1989) (Fig. 2A, E).

According to Metcalfe et al. (1950), the leaves of Hypericaceae are dorsiventral with hypoderm in certain species of Hypericum and stomata of Hypericum species are commonly surrounded by three or more cells. The leaves of H. thymopsis are dorsiventral, without hypodermis and stomata are anisocytic (Figs. 3B, D and 4A, B). The level of stomatal guard cells is lower than epidermis cells, so stomata are sunken type which is known as a special feature of the xerophytes (Fig. 3F). Prominent epicuticular waxes are present on abaxial side of the leaf and thus, the capacity of reduce the water loss is increase (Fig. 4E, F). Furthermore, presence of the wax reduces mechanical damage and prevents from fungal and insect attack (Eglinton and Hamilton, 1967). In addition, according to Briquet (1919) leaves of certain species of Hypericum growing arid regions in Brasil have thickened margins consisting of enlarged epidermal cells or collenchyma. The margin epidermal cells of H. thymopsis leaves are enlarged, the cuticle and outer cell walls of these cells are more thickened than other epidermal cells (Fig. 3A, D) so, the leaves of H. thymopsis show the same feature of mentioned knowledge.

Gîtea et al. (2011) were studied four Hypericum (H. perforatum, H. tetrapterum Fries, H. maculatum Crantz and H. hirsutum L.) in point of secretory structure of some vegetative organs. In their study, while type A canals are present in the phloem of all four species stems, type B secretory canals are present solely, in subepidermic area of H. perforatum and H. tetrapterum, and they are absent in H. maculatum and H. hirsutum. In stem of H. thymopsis, type A canals and glandiferous emergences are present, but type B canals are absent as well as H. maculatum and H. hirsurum (Fig. 2). However, there are no reports on presence of glandiferous emergences, in stems of their studied four Hypericum species. According to Gîtea et al. (2011), all examined species have 1–2 layered palisade parenchyma in leaf mesophyll, whereas it was observed as 2–3 layered in H. thymopsis (Fig. 3B, D). According to Shields (1950), having more strongly developed palisade tissue is one of the characteristic of the xerophytic plants, due to being the xeromophic plant, H. thymopsis has similar feature of mentioned literature.

Recently, Perrone et al. (2013a) were studied the leaf and stem anatomy of eight Hypericum species (H. aegypticum L., H. androsaemum L., H. hircinum L., H. perfoliatum L., H. perforatum, H. pubescens Boiss., H. tetrapterum Fr. and H. triquetrifolium Turra) which grow heterogeneous environments of Sicily Island. General anatomical characteristics of H. thymopsis are similar with those in their study. At the same time, there are some evident different characteristics for H. thymopsis especially in leaf anatomy. In stem anatomy, there are numerous glandiferous emergences outer of the H. thymopsis stem (Figs. 1B and 2A, E). There is no report on the presence of this characteristic in stems of their studied eight species. Transverse section shape of the stem and presence of slightly two wings in H. thymopsis are common characteristics with some species studied by Perrone et al. (2013a). Secondary xylem of H. thymopsis is ring-porous (Fig. 2C, G) such as their studied six species, except H. pubescens and H. aegypticum which have diffuse-porous secondary xylem. The leaves of H. thymopsis are dorsiventral (Fig. 3A, B, D) as with H. perforatum, H. perfoliatum, H. tetrapterum, H. androsaemum and H. hircinum, but the leaves of H. pubescens, H. triquetrifolium and H. aegypticum are isobilateral (Perrone et al., 2013a). The leaves of H. thymopsis are amphistomatic (Fig. 4) as with H. pubescens, H. tetrapterum, H. triquetrifolium and H. aegypticum while the leaves of the H. perforatum, H. perfoliatum, H. androsaemum and H. hircinum are hypostomatic. All stomatal complexes are anisocytic in the studied eight species by Perrone et al. (2013a), and this feature is the same for H. thymopsis (Fig. 4A, B). Among their studied eight species, solely H. triquetrifolium is xerophyte as H. thymopsis and they have many common characteristics, such as possess two small wings on stem, amphistomatic leaves and ring-porous secondary xylem. One of the prominent differences between two species is the leaf mesophyll which is dorsiventral in H. thymopsis while it is isobilateral in H. triquetrifolium.

There are some studies on morphology and anatomy of the Turkish Hypericum (Arda, 1989; Tokur and Mısırdalı, 1989; Erkaya and Tokur, 2004; Altıntaş and Akçin, 2015). Erkaya and Tokur (2004) were studied morphology and anatomy of H. montbretii Spach., H. origanifolium Willd. and H. perforatum growing in and around Eskişehir province of Turkey. Many anatomical characteristics of stems and leaves of these three species and H. thymopsis are similar such as presence of collenchyma in stem cortex, amphistomatic leaves, anisocytic stomata and translucent glands in leaf mesophyll. However, H. thymopsis has dorsivental leaves while all their studied three species have equifacial leaves which is the main difference for H. thymopsis. Yaylacı et al. (2013) were studied anatomical characteristics of recently described new Hypericum species, H. sechmenii Ocak & Koyuncu for Turkey. According to their study, presence of amphistomatic leaves and xeromorphic stomata in H. sechmenii are the same features as for those in H. thymopsis. But, having dorsiventral leaves, anisocytic stomata (Fig. 4A, B) and 2–3 layered palisade parenchyma (Fig. 3B, D) are distinctive features for H. thymopsis, because H. sechmenii has equifacial leaves, anomocytic stomata and 1–2 layered palisade parenchyma. In addition while stem pith region is made of solely xylem elements in H. secmenii, there is clearly parenchymatous pith cells some of which have lignified cell wall in H. thymopsis (Fig. 2D, H).

There are several studies focused on secretory structure of some Hypericum species (Ciccarelli et al., 2001a, 2001b; Gîtea et al., 2011). One of these, Ciccarelli et al. (2001b) were studied secretory structure of H. perforatum, considering the translucent glands and secretory canals of vegetative and floral parts. They observed translucent glands, type A and type B secretory canals in sepals and petals. While, they were observed translucent glands and type A secretory canals in leaves, they were observed type B secretory canals with type A secretory canals in stem transverse section. In addition, there were only type A and type C secretory canals in transverse section of ovaries of H. perforatum. When compare these results with those in H. thymopsis, there are some differences. There are present type A and type B secretory canals in sepals and petals of H. thymopsis, but translucent glands are absent (Figs. 5 and 6). In ovary, while H. thymopsis has numerous type C secretory canals, type A secretory canals are absent (Fig. 7C, D). In stem transverse section of H. thymopsis, type A canals are present in phloem, but type B canals are absent. One of the other differences for H. thymopsis is presence of glandiferous emergences (Figs. 1B and 2A, E, B, F) which is not reported on the stem of H. perforatum by Ciccarelli et al. (2001b). The secretory structures are absent in filament transverse section of H. thymopsis (Fig. 7A) and this feature is similar with H. perforatum (Ciccarelli et al., 2001b).

As a conclusion, as can be seen from the literature survey, this study seems to be the first extensive research on morphology and first research on anatomy of the stem, leaf, sepal, petal, filament and pistil of H. thymopsis. In addition some micromorphological characteristics are revealed for the first time. In morphological studies some different results and new characters were determined, and by using these characters, morphological description of the H. thymopsis was possibly extended. The anatomy of all aerial parts used in folk medicine and their secretory structures were investigated in detail. The presence of glandiferous emergences, translucent glands, type A, type B and type C secretory canals which produce biologically active substances were shown anatomically and histologically in vegetative and reproductive organs of H. thymopsis. Therefore, some results given in this research were compared with the results of other members of Hypericum in the literature records and differences between them were stated in detail. All this results can be used to evaluate of the diagnostic features of H. thymopsis as well as the genus Hypericum.

References

• Altıntaş, M.Y., Akçin, Ö.E., (M.Sc. thesis) 2015. Ordu ve Giresun'da yayılış gösteren bazı Hypericum L. (Hypericaceae, Guttiferae) türleri üzerinde morfolojik, anatomik ve mikromorfolojik bir çalışma. Ordu University, Ordu.
• APG III, 2009. An update of the Angiosperm Phylogeny Group classification for the orders and families of flowering plants: APG III. Bot. J. Linn. Soc. 161, 105-121.
• Arda, H., (M.Sc. thesis) 1989. Edirne Çevresindeki Hypericum L. (Guttiferae) Türleri Üzerinde, Morfolojik, Anatomik ve Karyolojik Araştırmalar. Trakya University, Edirne.
• Assadi, A., Zarrindast, M.R., Jouyban, A., Samini, M., 2011. Comparing of the effects of hypericin and synthetic antidepressants on the expression of morphine-induced conditioned place preference. Serv. Iran. J. Pharm. Res. 10, 619-626.
• Avato, P., 2005. A Survey of the Hypericum genus: secondary metabolites and bioactivity. Stud. Nat. Prod. Chem. 30, 603-634.
• Bingol, U., Cosge, B., Gurbuz, B., 2011. Hypericum species in flora of Turkey. In: Odabas, M.S., Cirak, C. (Eds.), Hypericum Medicinal and Aromatic Plant Science and Biotechnology, vol. 5 (Special Issue 1), pp. 86–90.
• Bombardelli, E., Morazzoni, P., 1995. Hypericum perforatum Fitoterapia 66, 43-68.
• Briquet, J., 1919. La structure foliaire des Hypericum à feuilles scléro-marginées. C. R. Soc. Phys. Hist. Nat. Genève 36, 75-90.
• Bruneton, J., 1995. Pharmacognosy, Phytochemistry, Medicinal Plants. Lavoisier Publishing, Paris, pp. 367–370.
• Carine, M.A., Christenhusz, M.J.M., 2010. About this volume: the monograph of Hypericum by Norman Robson. Phytotaxa 4, 1-4.
• Ciccarelli, D., Andreucci, A.C., Pagni, A.M., 2001a. The 'black nodules' of Hypericum perforatum L. subsp. perforatum: morphological, anatomical and histochemical studies during the course of ontogenesis. Israel J. Plant Sci. 49, 33-40.
• Ciccarelli, D., Andreucci, A.C., Pagni, A.M., 2001b. Translucent glands and secretory canals in Hypericum perforafum L. (Hypericaceae): morphological, anatomical and histological studies during the course of ontogenesis. Ann. Bot. 88, 637-644.
• Curtis, J.D., Lersten, N.R., 1990. Internal secretory structures in Hypericum (Clusiaceae): H. perforatum L. and H. balearicum L.. New Phytol. 114, 571-580.
• Davis, P.H., 1966. Flora of Turkey and the East Aegean Islands, vol. 2. Edinburgh University Press, Edinburgh, UK, pp. 355.
• Davis, P.H., 1988. Flora of Turkey and the East Aegean Islands, vol. 10. Edinburgh University Press, Edinburgh, UK, pp. 96.
• Davis, A.P., Barnett, J.R., 1997. The leaf anatomy of the genus Galanthus L. (Amaryllidaceae J. St.-Hil.). Bot. J. Linn. Soc. 123, 333-352.
• Eglinton, G., Hamilton, R.J., 1967. Leaf epicuticular waxes. Science 156, 1322-1335.
• Erkaya, İ.P., Tokur, S., 2004. Morphological and anatomical investigations on some Hypericum L. species growing naturally in and around Eskişehir. Trakya Univ. J. Sci. 5, 97-105.
• Esau, K., 1965. Plant Anatomy. John Wiley and Sons, New York.
• Gîtea, D., Şıpoş, M., Tămaş, M., Paşca, B., 2011. Secretory structures at species of Hypericum genera from Bihor county, Romania. Note I. Vegetative organs. Famacia 59, 424-431.
• Gudžić, B., Đordević, S., Vajs, V., Palić, R., Stojanović, G., 2002. Composition and antimicrobial activity of the essential oil of Hypericum maculatum Crantz. Flavour Fragr. J. 17, 392-394.
• Gudžić, B., Đordević, S., Nedeljković, J., Šmelcerović, A., 2004. Essential oil composition of Hypericum atomarium Boiss.. Chem. Ind. 58, 413-415.
• Güner, A., Özhatay, N., Ekim, T., Başer, K.H.C., 2000. Flora of Turkey and the East Aegean Islands, vol. 11. Edinburgh University Press, Edinburgh, UK, pp. 71.
• Güner, A., Aslan, S., Ekim, T., Vural, M., Babaç, M.T. (Eds.), 2012. Türkiye Bitkileri Listesi (Damarlı Bitkiler). Nezahat Gökyiğit Botanik Bahçesi ve Flora Araştırmaları Derneği Yayını, İstanbul.
• Hong-Fei, Lü, Zheng-Hai, Hu, 2001. Comparative anatomy of secretory structures of leaves in Hypericum L.. Acta Phytotaxon. Sin. 39, 393-404.
• Jensen, W.A., 1962. Botanical Histochemistry: Principles and Practice. W.H. Freeman and Company, London.
• Joulain, D., König, W.A., 1998. The Atlas of Spectra Data of Sesquiterpene Hydrocarbons. E.B.-Verlag, Hamburg, Germany.
• Kitanov, G.M., 2001. Hypericin and pseudohypericin in some Hypericum species. Biochem. Syst. Ecol. 29, 171-178.
• Kültür, S., 2007. Medicinal plants used in Kırklareli Province (Turkey). J. Ethnopharmacol. 111, 341-364.
• Lewis, L.W., 2003. Medical Botany. John Wiley and Sons, Inc., Hoboken, NJ, pp. 654.
• Maggi, F., Ferretti, G., Pocceschi, N., Menghini, L., Ricciutelli, M., 2004. Morphological, histochemical and phytochemical investigation of the genus Hypericum of the Central Italy. Fitoterapia 75, 702-711.
• Meseguer, A.S., Sanmartín, I., 2012. Paleobiology of the genus Hypericum (Hypericaceae): a survey of the fossil record and its palaeogeographic implications. An. Jard. Bot. Madr. 69, 97-106.
• Metcalfe, C.R., Chalk, L., Chattaway, M.M., Hare, C.L., Richardson, F.R., Slatter, E.M., 1950. Hypericaceae. In: Metcalfe, C.R., Chalk, L. (Eds.), Anatomy of the Dicotyledons, I. Leaves, Stem and Wood in Relation to Taxonomy with Notes on Economic Uses. Clarendon Press, Oxford, pp. 165–169.
• Nissen, L., Zatta, A., Stefanini, I., Grandi, S., Sgorbati, B., Biavati, B., Monti, A., 2010. Characterization and antimicrobial activity of essential oils of industrial hemp varieties (Cannabis sativa L.). Fitoterapia 81, 413-419.
• Onelli, E., Rivetta, A., Giorgi, A., Bignami, M., Cocucci, M., Patrignani, G., 2002. Ultrastructural studies on the developing secretory nodules of Hypericum perforatum Flora 197, 92-102.
• Özkan, A.M.G., Demirci, B., Başer, K.H.C., 2009. Essential oil composition of H. thymopsis Boiss.. J. Essent. Oil Res. 21, 149-153.
• Özkan, E.E., Demirci, B., Gürer, Ç.Ü., Kültür, Ş., Mat, A., Başer, K.H.C., 2013. Composition of essential oils from five endemic Hypericum species of Turkey. Org. Chem. Curr. Res. 2, 1.
• Özkan, E.E., Mat, A., 2013. An overview on Hypericum species of Turkey. J. Pharmacogn. Phytother. 5, 38-46.
• Öztürk, Y., Aydın, S., Başer, K.H.C., Kirimer, N., Kurtar-Özturk, N., 1992. Hepatoprotective activity of H. perforatum L. alcoholic extract in rodents. Phytother. Res. 6, 44-46.
• Öztürk, Y., 1997. Testing the antidepressant effects of Hypericum species on animal models. Pharmacopsychiatry 30, 125-128.
• Öztürk, Y., 2001. Possible mechanism of the analgesic effect of St. John's Wort. Fundam. Clin. Pharmacol. 15, 113-153.
• Öztürk, B., Apaydın, S., Goldeli, E., İnce, I., Zeybek, U., 2002. H. triquetrifolium Turra. extracts exhibits antiinflammatory activity in the rat. J. Ethnopharmacol. 80, 207-209.
• Öztürk, N., Korkmaz, S., Öztürk, Y., 2007. Wound-healing activity of St. John's Wort (H. perforatum L.) on chicken embryonic fibroblasts. J. Ethnopharmacol. 111, 33-39.
• Perrone, R., Rosa, P.D., Castro, O.D., Colombo, P., 2013a. Leaf and stem anatomy in eight Hypericum (Clusiaceae). Acta Bot. Croat. 72, 269-286.
• Perrone, R., Rosa, P.D., Castro, O.D., Colombo, P., 2013b. A further analysis of secretory structures of some taxa belonging to the genus Hypericum (Clusiaceae) in relation to the leaf vascular pattern. Turk. J. Bot. 37, 847-858.
• Robson, N.K.B., 1981. Studies in the genus Hypericum L. (Guttiferae). 2. Characters of the genus. Bull. Br. Museum (Nat. Hist. Bot. Ser.) 8, 55-226.
• Russo, E.B., 2011. Taming THC: potential Cannabis synergy and phytocannabinoid–terpenoid entourage effects. Br. J. Pharmacol. 163, 1344-1364.
• Santos, P.A.G., Figueiredo, A.C., Barroso, J.G., Pedro, L.G., Scheffer, J.J.C., 1999. Composition of the essential oil of Hypericum foliosum Aiton from five Azorean Islands. Flavour Fragr. J. 14, 283-286.
• Shields, L.M., 1950. Leaf xeromorphy as related to physiological and structural influences. Bot. Rev. 16, 399-447.
• Siersch, E., 1927. Anatomie und Mikrochemie der Hypericumdruesen (Chemie des Hypericins). Planta 3, 481-489.
• Simpson, M.G., 2006. Plant Systematics, 1st edition. Elsevier-Academic Press, Amsterdam.
• Spiteller, M., Özen, T., Smelcerovic, A., Zuehlke, S., Mimica-Dukic, N., 2008. Phenolic constituents and the in vitro antioxidant activity of the flowers of H. venustum Fitoterapia 79, 191-193.
• Toker, Z., Kızıl, G., Özen, H.C., Kızıl, M., Ertekin, S., 2006. Compositions and antimicrobial activities of the essential oils of two Hypericum species from Turkey. Fitoterapia 77, 57-60.
• Tokur, S., Mısırdalı, H., 1989. Bazı Hypericum Türleri Üzerinde Anatomik Çalışmalar Anadolu Üniv. Fen Ed. Fak. Derg. 2, 1-8.
• Toma, C., Rugină, R., 1998. Atlas de anatomia plantelor medicinale. Ed. Acad. Rom. 98, 142-145.
• Yaylacı, Ö.K., Özgişi, K., Sezer, O., Orhanoğlu, G., Öztürk, D., Koyuncu, O., 2013. Anatomical studies and conservation status of rare endemic Hypericum sechmenii Ocak & Koyuncu (Sect: Adenosepalum) from Eskişehir-Turkey. J. Selçuk Univ. Nat. Appl. Sci. 2, 1-11.
• Yeşilada, E., Gürbüz, I., Shibata, H., 1999. Screening of Turkish antiulcerogenic folk remedies for anti-Helicobacter pylori activity. J. Ethnopharmacol. 66, 289-293.
• Zeng, H., Zhou, P., 2001. Analysis of the chemical constituents of the essential oils from the leaves of two Hypericum plants. Nat. Sci. J. Xiangtan Univ. 23, 52-54.

Publication Dates

• Publication in this collection
  Mar-Apr 2017

History

• Received
  16 June 2016
• Accepted
  19 Sept 2016