Phytochemical resources on Thymus stojanovii degen. Species (labiatae)

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1 Phytochemical resources on Thymus stojanovii degen. Species (labiatae) M. ARCUŞ, V. SCHRÖDER Faculty of Pharmacy, Ovidius University, Romania, Constanta *Corresponding author Arcus Mariana Ovidius University Constanta Pharmaceutical sciences 1 1 Al. Universitatii, Campus B, Constanta , Romania T: arcusmariana@yahoo.com Key words: Thymus, histological study, labiatae Summary Thymus stojanovii Degen, is a perennial herbaceous plant, part of a botanical group called labiates, most of these plants being renowned for their high content in volatile oils, flavonoids and tannins, as well as for their uses against various human ailments. Starting from this idea, we initiated a pharmacognostic and phytochemical study in order to identify the species and differentiate the active principles synthesized by its vegetative organs.as a result of the chemical study, the following groups of compounds were emphasized: volatile oil, triterpens, carotenoids, flavonoid aglycones and flavonoids, catehic tannins, reductive compounds, triterpenic heterosides, proanthocyanidins, polyuronides, oses and polyoses. bracts similar to leaves. The persistent calyx is tubular and zygomorphic, five-toothed, with prickly superior ciliated teeth. The superior labium of the violet bilabiate corolla is as long as the calyx tube. The androecium is made up of four epipetalous stamens, while the gynoecium is superior, bicarpellate. The fruit is a tetranutlet (tetranucule) (Tutin et al., 1972). The genus Thymus includes approximately species distributed in Europe, Africa and temperate Asia. Thymus stojanovii Degen is an endemic species with limited area in the Balkan Peninsula (Fig. 1), but it does not vegetate in the Romanian flora (Savulescu et al., 1961). The specialized literature brings numerous data regarding the active principles contained by the genus Thymus, but Thymus stojanovii Degen has not yet been studied from a histological and chemical point of view. Introduction Thymus stojanovii Degen is a creeping perennial herbaceous plant, with lignified basal part and herbaceous superior part. It is 5-7 cm tall, while its stem is four-edged at the superior side and cylindrical at the inferior side. The leaves are whole, alternate, 5-7 mm long and 1-2 mm wide, elliptic - lanceolate, slightly fleshy, often hairy and ciliated at the base. The lateral veins are slightly visible. The hermaphrodite and zygomorphic flowers are grouped in spherical inflorescences, slightly cylindrical, each joined at the base by Fig. 1. Thymus stojanovii Degen. 1

Material and method Thymus stojanovii Degen. was used as study material. It was collected in full bloom from the Divčibare plateau, located in the Valjevo Mountains (Fig.

Divčibare plateau, located in the Valjevo Mountains Serbia.

2 Material and method Thymus stojanovii Degen. was used as study material. It was collected in full bloom from the Divčibare plateau, located in the Valjevo Mountains (Fig. 2), which are situated in the central part of Serbia and have a height of 900 m. This is where the species vegetates on thin soils developed in Jurassic peridotites. Fig. 2. Divčibare plateau, located in the Valjevo Mountains Serbia. The plant was identified and determinated in the Laboratory of General, Vegetal and Animal Biology within the Faculty of Pharmacy, using volume III of the Flora Europaea (Tutin et. al., 1972). For the purpose of the histo-anatomical analysis of the vegetative organs, namely stem and leaves, the work material was processed thus: - fixation and preservation of the fresh material in ethyl alcohol 70%; - manual transversal sectioning, by means of hand microtome and botanical razor, using elder pith for support; - javellization of the sections with sodium hypochlorite for minutes; - washing of the sections with acetic water and with distilled water; - staining of the sections with iodine green (for one minute and washing with ethyl alcohol 90%) and then with ruthenium red (for one minute and washing with distilled water); - setting of the stained sections in drops of glycero-gelatin, between slide and cover slip in order to generate permanent preparations (Sebanescu-Jitariu et al., 1983, Toma et al., 2000, Andrei et al., 2003). Color photos were taken of the preparations thus obtained under the OPTIKA photonic microscope, with a digital Canon A540 camera. Photo scale = 100 μm. In order to determine the chemical composition of the vegetal product Thymii stojanovii herba, all the stages of the pharmacognostic analysis were followed as research methodology. The dry plant was ground and the powder thus obtained was successively extracted with solvents of various polarities, namely with a non-polar solvent for the emphasis of the lipophilic chemical compounds, with a polar solvent (ethanol) and finally with water in order to emphasize the hydrophilic chemical compounds (Ciulei et al., 1995). In order to identify the chemical compounds in the three extracts, these were analyzed separately, using methods appropriate to the physic-chemical properties of each group of active principles. To estimate the quality of the vegetal product studied, it was determined the lost weight produced by draying, the soluble in water and alcohol quantity substances (Ciulei et al., 1995). Results and discussions The transversal section through the stem emphasized the following at the superior region: - square contour with attenuated ribs (Fig.3); Fig. 3. Square contour of the cross section through the superior region of the stem. 2

- epidermis made up of isodiametric cells, with thin cellulosic walls, with convex outer wall, covered by a relatively thick and striated cuticle (Fig. 4); collenchyma (Fig.

here and there, it is disorganized, resulting in small cavities; Fig. 4. Cross section through the superior region of the stem; a epidermis; b- cuticle.

cell located between the epidermal cells, pedicel and pluricellular gland), and secretory hairs, smaller, with a unicellular gland (Fig. 5); Fig. 6.

- the last layer of the bark is an endodermis made up of big, isodiametric cells with thin walls, slightly suberized (Metcalfe et al., 1972) (Fig. 6-c); Fig. 5.

3 - epidermis made up of isodiametric cells, with thin cellulosic walls, with convex outer wall, covered by a relatively thick and striated cuticle (Fig. 4); collenchyma (Fig. 6) which continue outside the ribs with one-two layers, immediately under the epidermis; for the rest, the bark is relatively reduced, made up of several layers of cells with thin cellulosic walls; here and there, it is disorganized, resulting in small cavities; Fig. 4. Cross section through the superior region of the stem; a epidermis; b- cuticle. - at the epidermis level there are numerous, relatively long pluricellular uniseriate tectorial hairs, spread along the stem, and secretory hairs of two kinds: pluricellular (formed from the basal cell located between the epidermal cells, pedicel and pluricellular gland), and secretory hairs, smaller, with a unicellular gland (Fig. 5); Fig. 6. Cross section through the superior region of the stem; a angular collenchyma; b small cavities; c endodermis; d the bark. - the last layer of the bark is an endodermis made up of big, isodiametric cells with thin walls, slightly suberized (Metcalfe et al., 1972) (Fig. 6-c); Fig. 5. Cross section through the superior region of the stem; a - pluricellular uniseriate tectorial hair; b pluricellular secretory hair; c secretory hair with a unicellular gland. - in the all four ribs of the stem there are regions of very well developed angular Fig. 7. Cross sections through the superior region of the stem; a - the central cylinder; b the pith. - the central cylinder includes ring-type transport tissues (Fig. 7 and 8): a ring of phloem located towards the outside (Fig. 8-a) and a ring of xylem located towards the center of the stem (Fig. 8-b), the xylem ring is 3

closing a relatively thick pith whose cells are small, cellulosic, in contact with large and woody elements, with thickened and lignified walls in its central part (Fig.7); Fig. 9.

Cross sections through the superior region of the stem, the central cylinder detail; a phloem ring; b xylem ring; c xylem vessels in radial rows; d woody parenchyma.

8-c); cells of woody parenchyma can be seen between the xylem vessels, also with thickened and lignified walls (Fig. 8-d).

4 closing a relatively thick pith whose cells are small, cellulosic, in contact with large and woody elements, with thickened and lignified walls in its central part (Fig.7); Fig. 9. Cross section through the inferior region of the stem. Fig. 8. Cross sections through the superior region of the stem, the central cylinder detail; a phloem ring; b xylem ring; c xylem vessels in radial rows; d woody parenchyma. - the xylem includes xylem vessels with thickened and lignified walls, disposed very orderly, in radial rows (Fig. 8-c); cells of woody parenchyma can be seen between the xylem vessels, also with thickened and lignified walls (Fig. 8-d). The transversal section through the inferior region of the stem emphasized the following: - the four ribs are considerably attenuated, so that the stem profile is almost circular (Fig. 9); - the epidermis (Fig. 10-b) includes isodiametric cells with cellulosic walls; the outer ones are covered by a thin cuticle (Fig. 10-a), much more striated than the one in the previously analyzed region; - the collenchyma (Fig. 10-e) is present in the ribs; for the rest, the bark (Fig. 10-c) is relatively reduced, made up of several layers of cells with thin cellulosic walls; - the last layer of the bark is an endodermis made up of large, tangentially elongated cells with thin walls, slightly suberized (Fig. 10-d); Fig. 10. Cross section through the inferior region of the stem; detail - 40x; a cuticle; b epidermis; c the bark; d endodermis; e collenchyma; f phloem ring; g xylem ring; h woody parenchyma. - cells of woody parenchyma with thickened and lignified walls are present between the rows of xylem vessels (Fig.10-h); - the central cylinder is much wider. - the xylem and phloem are ring-type; - the phloem ring has approximately the same size as the previously analyzed level, but the xylem ring includes several radial rows of longer xylem vessels (Fig. 11); - the pith includes several layers of cells with cellulosic walls; it is mostly disorganized causing the formation of a relatively narrow aeriferous canal (Fig.11); 4

Fig. 11. Central cylinder of the stem, inferior region; a phloem ring; b xylem ring; c xylem vessels; d - the pith; e aeriferous canal. Fig. 13.

Study of the leaf - seen on the front: the leaves are small, coriaceous, with shining adaxial side; Fig. 14.

Upper epidermis; a elongated waved cells; b diacytic stomata.

13-a); - diacytic-type stomata are present in both epidermises (Toma et al., 2000), thus the foliar blade is amphistomatic (Fig. 12-b and Fig.

5 Fig. 11. Central cylinder of the stem, inferior region; a phloem ring; b xylem ring; c xylem vessels; d - the pith; e aeriferous canal. Fig. 13. Lower epidermis; a elongated cells with higher waved walls; b - stomata diacytic type. Study of the leaf - seen on the front: the leaves are small, coriaceous, with shining adaxial side; Fig. 14. Upper epidermis cells towards the edge of the foliar blade with thickened walls and fitted with narrow punctuations. Fig. 12. Upper epidermis; a elongated waved cells; b diacytic stomata. - seen on the front both epidermises include elongate cells with waved walls, the waves has higher amplitude in lower epidermis (Fig. 12-a and Fig. 13-a); - diacytic-type stomata are present in both epidermises (Toma et al., 2000), thus the foliar blade is amphistomatic (Fig. 12-b and Fig. 13-b); - in the upper epidermis, towards the edges of the foliar blade the stomata are not present; it is very visible that the cell walls are thickened there and contain numerous narrow punctuations (Fig. 14); - sparse uniseriate pluricellular tectorial hairs (Fig. 15) can be seen, as well as two types of secretory hairs: some small with unicellular gland (Fig. 16 and 17) and others big, located in excavations of the 5

Fig. 15. Upper epidermis - uniseriate tectorial hair. Fig. 18.

Upper epidermis secretory hair with unicellular gland. Fig. 19.

elongated, covered by a very thick striated cuticle; Fig. 17.

6 Fig. 15. Upper epidermis - uniseriate tectorial hair. Fig. 18. Upper epidermis - secretory hair with pluricellular gland. Fig. 16. Upper epidermis secretory hair with unicellular gland. Fig. 19. Lower epidermis secretory hair with pluricellular gland and elongated cells. Study of the leaf - in transversal section: - the foliar blade is a V-shaped narrow ribbon (Fig. 20); both epidermises include large cells, usually tangentially elongated, covered by a very thick striated cuticle; Fig. 17. Lower epidermis secretory hair with unicellular gland. epidermis, pluricellular, with pluricellular gland (Fig. 18 and 19), the epidermal cells around them are much elongated. Fig. 20. Transversal section through the foliar blade. 6

Fig. 21. Lower epidermis, secretory hair with pluricellular gland. Fig. 23. Secretory hair with unicellular gland. Fig. 22.

- at the epidermis level there are sparse uniseriate pluricellular tectorial hairs, pluricellular secretory hairs, with

(Fig.23); - also, small stomata can be seen in both epidermises, located slightly above the level of the neighboring

- the mesophyll is differentiated into palisade tissue (towards the superior epidermis), made up of 2-3 layers of

7 Fig. 21. Lower epidermis, secretory hair with pluricellular gland. Fig. 23. Secretory hair with unicellular gland. Fig. 22. Excavation of the lower epidermis. - at the epidermis level there are sparse uniseriate pluricellular tectorial hairs, pluricellular secretory hairs, with pluricellular gland, located in excavations of the epidermis (Fig 21 and 22), and secretory hairs with unicellular gland (Fig.23); - also, small stomata can be seen in both epidermises, located slightly above the level of the neighboring epidermal cells (Fig. 24); Fig. 24. a upper epidermis stomata; b lower epidermis stomata; c - palisade tissue; d - lacunar tissue. - the mesophyll is differentiated into palisade tissue (towards the superior epidermis), made up of 2-3 layers of elongated cells, and lacunar tissue (towards the inferior epidermis), made up of several layers of cells that leave among them large meatus or lacunae; the blade is bifacial-heterofacial, normally dorsoventral (Fig.24); 7

Fig. 25. Numerous xylem phloem fascicle of different size.

paranchymatous - the median vein includes a single large xylem-phloem fascicle (Fig. 26), rarely two fascicles (Fig.

8 Fig. 25. Numerous xylem phloem fascicle of different size. - the transport tissue form different size, numerous xylem phloem fascicle, the median one being the biggest (Fig. 25); Fig. 27. The median vein with two xylem phloem fascicles; a sclerenchyma bands; b sclerenchymatous elements; c paranchymatous sheath. - all the fascicles are surrounded by a parenchymatous sheath made up of large cells with cellulosic walls (Fig 26 and 27); Fig. 26. The median vein with single large xylem phloem fascicle; a- xylem cordon; b phloem cordon; c band of sclerenchyma; d paranchymatous sheath. - the median vein includes a single large xylem-phloem fascicle (Fig. 26), rarely two fascicles (Fig. 27); the xylem is represented by xylem vessels with small diameter, with thickened and lignified walls, while the phloem includes holed tubes and annex cells; - the large fascicles display a thick band of sclerenchyma at the phloem pole, made up fibers with uniformly thickened and lignified walls; only few sclerenchymatous elements can be seen at the xylem pole; (Fig. 27) Fig. 28. Foliar blade; a cuticle; b upper epidermis; c palisade tissue; d lacunar tissue; e xylem phloem fascicle. Fig. 29. Median veins; a angular collenchyma. 8

9 - the smallest transport fascicles contain only a few xylem vessels and 2-3 sclerenchymatous fibers. - the fascicles that belong to the median veins are anchored by the two epidermises through pillars of angular collenchyma (Fig. 29); The chemical global analysis meant the gradual and selective extraction of the vegetal products with different polarity solvents and their separation based on physical - chemical properties with the help of chemical methods followed by the making of the specific reactions which help to identify groups of active principle or chemicals constituents. After effecting the identification reactions to the Thymus stojanovii vegetal product the following results were obtained: 1. In the extractive ethereal solution: - volatile oil; - sterol (triterpens); - carotenoids; - flavonoid aglycones; 2.1 In the extractive non-hydrolyzed alcoholic solution: - catehic tannins; - reductive compounds; 2.2 In extractive hydrolyzed alcoholic solution: - triterpenic heterosides; - flavonoids; - cumarines; - proanthocyanidins; 3. In the extractive hydrous solution - polyuronides; - oses and polyoses (Ciulei et al., 1995). Conclusions The macroscopic exam of the Thymus stojanovii confirmed the description of the species in the European Flora guides. The hysto - anatomical analysis highlighted typical characters of the Labiatae family, respective: angular collenchyma in the ribs of stem, diacytic stoma type and pluricellular secretory hairs, with pluricellular gland, located in excavations of the epidermis and secretory hairs with unicellular gland. After effecting the identification reactions to the Thymus stojanovii vegetal product the following results were obtained: volatile oil, sterol (triterpens), carotenoids, flavonoid aglycones, catehic tannins, reductive compounds, triterpenic heterosides, flavonoids, cumarines, proanthocyanidins, polyuronides, oses and polyoses; Three preliminary determinations of lost through drying were made and their values were situated between the limits 14,4943g - 15,9902g. Three determinations for soluble in ethanol substances were made and the values situated between 16,2459g - 16,5601g. Substances soluble in water found out between 12,1589g 13,1589g. References Andrei M. & Paraschivoiu Roxana Maria, Microtehnică botanică, Ed. Niculescu, București, Ciulei I., Istudor Viorica, Palade Madalena, Niculete Elena, Gârd Cerasela Elena, Analiza farmacognostică şi fitochimică a produselor vegetale, vol. I (Oze, poliholozide, heterozide, lipide), Ed. Tehnoplast Company S.R.L., Bucureşti, p , Ciulei I., Istudor Viorica, Palade Madalena, Niculete Elena, Gârd Cerasela Elena, Analiza farmacognostică şi fitochimică a produselor vegetale, vol. II (Aetherolea, rezine, principii amare, carotenoide, alcaloizi, protoalcaloizi, amine, vitamine, varia), Ed. Tehnoplast Company S.R.L., Bucuresti, , Metcalfe C. R., Chalk L., Lamiaceae, in Anatomy of the Dicotyledons, Clarendon Press, Oxford, 2: , Savulescu Tr., Beldie Al., Buia Al., Grintescu Gh., Grintescu I., Gusuleac M., Morariu I., Nyarady E. I., Pauca A., Prodan I., Ravarut M., Topa Em., Flora R.P.R., Vol. VII, Ed. Acad. R.P.R., p , Bucurest, Serbanescu-Jitariu Gabriela, Andrei M., Mitroiu- Radulescu Natalia, Petria Elena. Practicum de biologie vegetală, Ed. Ceres, Bucureşti, Toma C., Gostin Irina, Histologie vegetală, Editura Junimea, Iaşi, Tutin T.G., Heywood V.H., Burges N.A., Flora Europea, vol. 3, p. 174,

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