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47Table 3
The content of phenolic compounds in Sanícula europaea L. herb
№ Group of phenolic compounds Method of analysis Wavelength, nm Content, %, x ± Ax n = 9
1. Total polyphenols Spectrophotometry in terms of pyrogalol 760 10,43±0,03
2. Tannins Spectrophotometry in terms of pyrogalol 760 7,28±0,03
3. Hydroxycinnamic acids Spectrophotometry in terms of rosma-rinic acid 325 1,26±0,01
4. Flavonoids Spectrophotometry in terms of rutin 408 2,37±0,01
Thus, it was found that the content of the total polyphenols in Sanícula europaea L. herb was 10.43±0.03%, tannins - 7.28±0.03%, hydroxycinnamic acids - 1.26±0.01%, flavonoids - 2.37±0.01%.
Conclusions.
1. The composition of phenolic compounds of Sanicula europaea L. herb was studied using paper chromatography and HPLC methods.
2. The content of 14 phenolic compounds, which were represented by tannins, hydroxycinnamic acids, flavonoids and coumarins, was determined using HPLC method.
3. It was established that in Sanicula europaea L. herb the dominant compound among hydroxycinnamic acids was rosmarinic acid, among flavonoids -kaempferol, among tannins - epicatechin gallate, among coumarins - coumarin.
4. The content of total polyphenols, tannins, flavo-noids, hydroxycinnamic acids was established.
References
1. Vyznachennia kilkisnoho vmistu hidroksyko-rychnykh kyslot u syrovyni dyvyny zvychainoi/ A. A. Voloshyna, V. S. Kyslychenko, I. O. Zhuravel [ta in.].
Ukrainskyi medychnyi almanakh. 2012. Tom 15, № 5. S. 39 - 40.
2. Katsuba I. K., Kyslychenko V. S., Novosel O. M. Doslidzhennia fenolnykh spoluk lystia maty y ma-chukhy. Ukrainskyi medychnyi almanakh. 2011. Tom 14, № 6. S. 92 - 94.
3. Legin N. I. Vyznachennia vmistu okysniuvanykh fenoliv v syrovyni pidlisnyka yevro-peiskoho. Inovatsii v medytsyni: materialy 84 nauk.-prak. konf., studentiv i molodykh vchenykh z mizhnar. uchas., 12 - 13 berez. 2015 r. Ivano-Frankivsk, 2015. S. 170 - 171.
4. Investigation of phenolic compounds of the herbs of Veronica Genus / I. Milian, S. Marchyshyn, S. Kozachok [and et al.] // The Pharma Innovation Journal. 2016. No. 5(7). P.41 - 46.
5. Krivoruchko E. V. Carboxylic Acids from Cornus mas. Chemistry of Natural Compounds. 2014. 50, № 1. P. 112 - 113.
6. Temerdashev Z. A., Frolova N. A., Kolychev I. A. Determination of Phenolic Compounds in Medicinal Herbs by Reversed-Phase HPLC. Journal of Analytical Chemistry. 2011. Vol. 66, No. 4. P. 407-414.
MACROSCOPIC AND MICROSCOPIC MORPHOLOGICAL FEATURES OF VERBENA L. SPECIES
Grytsyk A.,
Doctor in Pharmaceutical Sciences, Professor
Posatska N., Master of Pharmacy, Assistant Svirska S.
PhD in Pharmaceutical Sciences, Associate Professor Ivano-Frankivsk National Medical University.
Ivano-Frankivsk, Ukraine
ABSTRACT
Verbena L. species are herbaceous plants or shrubs. They have strong root system and are able to grow up to 100 cm. Their stems are erect or branched, quadrangular and covered with hairs that are pressed to the surface. The leaves are small, opposite, oblong. They are located on the petioles. Inflorescences are at the top of the plants and are collected in panicles, long and spike-shaped, grow in the axils of the middle and upper leaves. Verbena L. flowers have different colors and shades. The fruits have a convex dihedral shape. They are linear, brown, slightly oblong and usually wrinkled.
The light microscopy and scanning electron microscopy are used in modern scientific and applied researches to perform in-depth study of the micromorphological structure of leaves, stems and flowers of plants. These methods give more accurate images of plant diagnostic features. Anatomical features of leaves, stems and flowers of Verbena officinalis L. were used to specify the microscopic characteristics of medicinal plant raw materials.
Key words: epidermis, leaf, stem, flower, Verbena officinalis L.
There are 250 species that belong to the genus Verbena L. Three species grow in Ukraine: Verbena officinalis L., Verbena supina L., Verbena hybrida Hort. [1].
Verbena L. species are herbaceous and ligneous plants of the family Verbenaceae. They have opposite leaves and gynandrous, irregular flowers. The flowers are collected in many-flowered spikes or umbels that form panicles. A corolla is cylindrical or funnel-shaped tubes and a four- or five-separate, indistinctly double-lipped bends. Usually there are four stamens that are attached to the corolla tube. An ovary is upper, four-nested; the stigma starts from the top of the ovary. The fruit is a drupe or it falls apart into four nuts [1 - 2].
Verbena officinalis L. is a perennial herbaceous rough-haired plant. A stem is quadrangular, erect, ascending, 25 - 100 cm tall, mostly branched. Leaves are 2 - 10 cm long, short petiolate, ovate-oblong or oblong, pinnate, middle leaves are pinnate or trifoliate with unequally serrated lobes, upper leaves are sessile, oblong and pinnate. Flowers are numerous. They are located in the axils of leaf-like bracts and collected in upright spike-shaped inflorescences. A tubular calyx has 5 pointed blades. A corolla is pale pink or purple, twice as long as the calyx. Fruits are nuts. Verbena officinalis L. blooms from April to October [1 - 4].
Verbena supine L. is a herbaceous plant with an erect, branched, pubescent stem. It is 70 cm tall. Leaves
are ovate-oblong, pubescent, short-petiolate, upper leaves are sessile, incised-serrated or entire. Inflorescences are oblong-spike, thin. The teeth of the calyx are short, sharp, pale purple, twice as long as the calyx. Fruits fall apart into four nuts. Verbena supine L. blooms from April to October. It grows on wet sandy places [1, 5 - 7].
Verbena hybrida Hort. is mostly used in Ukraine in ornamental horticulture [8].
Name Verbena hybrida Hort. combines numerous garden forms and varieties of complex hybrid origin, that formed after 1830. They are perennial herbaceous planst but are used as annual plants. The stems are 20 -50 cm tall, branched, creeping or erect from the base. Shoots are quadrangular. Leaves are located opposite. They are oblong or elongated-triangular with uneven edges, on short petioles. Upper leaves are almost sessile, lower leaves are heart-shaped. Stems and leaves are pubescent with rough, colorless hairs. The flowers are regular, carnation-like, from white to light red or dark purple in various shades, often with a light eye in the center, fragrant. Inflorescences are complex, consisting of 30 - 50 flowers, umbellate, that are extended into multi-flowered spikes. The fruit is a nut. Verbena hybrida Hort. blooms from June to October and is a self-seeding plant [1, 5 - 7].
Distinctive morphological features of Verbena L. species of Ukrainian flora are given in table. 1.
Table 1
Distinctive morphological features of Verbena L. species
Species name Diagnostic features
Leaves Inflorescences, flowers Stem References
Verbena officinalis L. Opposite, alternate 2-10 cm long, egg-shaped, dark green, pubescent, toothed, short-petiolate, sometimes entire Sessile, gynandrous,fused, irregular, small 1.5-2.5 cm in diameter, long spike-shaped inflorescences; corolla is 5-lobed, funnel-shaped, pale purple Quadrangu-lar, erect, pubescent, 75-100 cm tall 1 - 2, 4, 9
Verbena supine L. Medium leaves are twice pinnately dissected, lower leaves are cordate at the base, pinnate, short-petio-late, upper leaves are sessile, ovoid-wedge-shaped, 2-4 cm long Spike-shaped, short, 2-4 cm long, dense inflorescences of blue flowers, corolla is slightly longer then calyx, calyx teeth are short and sharp Branched, horizontal, quadrangular, short-haired, 10-40 cm long 1, 3, 9
Verbena hybrida Hort. Opposite, ovate, oblong-pubescent, short-petiolate; upper leaves are sessile; lower leaves are cordate at the base, incised-toothed or entire Correct, from white to purple-red or dark purple color of different shades, fragrant with complex umbrella-shaped inflorescences of 30-50 flowers, 5-6 cm in diameter Branched, rough-pubescent with colorless hairs on the faces, 20-50 cm tall 5 - 7
Verbena L. species can be identified by the following common features: the height of their shoots varies from 20 cm to 1,0 m; their stems are often erect, but there are some branched species; they are covered with the hairy green skin; they have extremely strong branched root system, opposite short-petiolate leaves with serrated edges and oval shape and spike-shaped inflorescence of different colors.
The State Pharmacopoeia of Ukraine presents the identification of Verbena officinalis L. herb using
methods of macro- and microscopic analysis. It is proposed to use whole or fragmented, dried aboveground parts of Verbena officinalis L. that were collected during flowering season [10]:
Identification A: Stem is grayish-brown, quadrangular, longitudinally grooved and roughly pubescent, especially along the faces. Larger leaves are petiolate and deeply pinnate, with obtusely serrated or serrated edges; leaf surfaces are rough and covered with bristly hairs, especially along the veins that are protruding on
the lower surface. The flowers are numerous and located in the axils of leaf-like bracts, gathered in an upright spike-shaped inflorescence; tubular calyx has 5 pointed blades, corolla is from pale pink to purple, almost twice as long as the calyx.
Identification B: To grind the raw material to a greenish-brown powder. Examine the powder under a microscope using a solution of chloral hydrate. In the powder should be found: long, polygonal or rectangular stem epidermal cells, with thickened outer shells; fragments of leaves (surface view) from the tortuous main cells of the epidermis and the stomata apparatus of the anomocytic or anisocytic types, more numerous on the lower epidermis; integumentary hairs are short, unicellular, thick-walled, about 500 ^m long, wide at the base and rising from the center of a separate rosette of dome-shaped spherical cells of the epidermis; occasionally one can notice glandular head hairs with a multicellular stalk and a flat, 4- to 8-cell head, about 25 ^m in diameter; essential oil glands with a unicellular stalk and an enlarged oval, about 65 ^m in diameter head, consisting of 8 radially arranged cells; fragments of the fibrous layer of anther with well-marked thickenings; pollen grains triangular or ovoid or round, about 30 ^m in diameter, with 3 pores and a smooth exine; separate groups of fibers, porous vessels and parenchymal tissue of the stem [10].
To clarify the anatomical structure of Verbena of-ficinalis L. herb, we conducted an additional study of the ultrastructure of the epidermal surface of the leaf, stem and flower petal. The images, that are obtained using the scanning electron microscope, are three-dimensional and convenient for studying the surface structure. It allows to establish some features of the plant structure that are necessary for plant taxonomy [11 -13].
For the study we used Verbena officinalis L. herb that was harvested in 2017 on the outskirts of Zalukva village, Halyts'kyi district, Ivano-Frankivsk region.
The external features of the ultrastructure of the epidermis surface of Verbena officinalis L. leaf are presented in Fig. 1 and Fig. 2.
The leaf is amphistomatic, stomata apparatus is anisocytic or anomocytic, stomata evenly distributed on both surfaces of the leaf blade, slightly above the level of the main epidermal cells. The adaxial surface of the leaf blade is collicular (Fig. 1). The projections and outlines of epidermal cells vary: prosenchymal cells with rectangular projections and straight outlines are observed above the mesophile; cells with elongated projections and straight outlines are observed in the area of conducting bundles.
Fig. 1. The ultrastructure of the adaxial epidermis of Verbena officinalis L. leaf blade: 1 - trichoma, 2 - glandular hair; 3 - basal cell of a simple hair; 4 - stomata; 5 - simple hair, 6 - periclinal walls of epidermal cells,
7 - epicuticular wax.
The anticline walls of epidermal cells are not complex, formed by simple (hooks) and glandular thickened. The boundaries of the cells are clear. The hairs. On the upper surface of the leaf there is a slight cuticle is well formed. The pubescence is scattered, pubescence (4.5 - 5 trichomes per 1 mm2) represented
by 0.4 - 0.5 mm long trichomes. Trichomes are observed both on the vein and on the entire surface of the leaf blade.
In the studied species, the abaxial surface differs from the adaxial (Fig. 2). It is characterized by dense ubescence.
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Fig. 2. The ultrastructure of the abaxial epidermis of Verbena officinalis L leaf blade: 1 - central vein; 2 - distal trichoma cell; 3 - outer periclinal walls of trichoma cells; 4 - glandular trichomes; 5 - basal cell of the thorn; 6 - distal thorn cell; 7 - stomata; 8 - basal cell of glandular hair; 9 - the head of the glandular hair; 10
- striatal cuticle.
The central vein of the leaf is densely pubescent with long thorny trichomes with a distinct basal cell and long distal trichomes. Thorns are observed on all surface of a leaf blade. Unlike the adaxial epidermis, the abaxial epidermis has a striatal cuticle. The epicuticular wax is found on the periclinal walls of the epidermal cells of both epidermis. The wax is represented by granules.
On the surface of the stem there are two distinct zones (costal and intercostal), that differ slightly in the ultrastructure of the surface. The stomata are anisocytic or anomocytic with clear cuticular rollers, evenly
spaced with epidermal cells. The relief of the stem surface is slightly collicular to smooth. Epidermal cells are prosenchymal with rectangular projections and straight outlines. The anticlinal walls of epidermal cells are not thickened, the outer periclinal walls are slightly convex. The boundaries of the cells are not clear. The cuticle is well formed. There is no pubescence on the surface of the stem. The epicuticular wax is found on the periclinal walls of the epidermal cells.
External features of the ultrastructure of the epidermis surface of Verbena officinalis L. stem are presented in Fig. 3.
Puc. 3. The ultrastructure of the epidermis surface of Verbena officinalis L. stem: 1 - costal area, 2 - intercostal area; 3 - stomata; 4 - cuticle (intercostal area); 5 - periclinal walls of epidermal cells, 6 - cuticle (costal area); 7 - epicuticular wax.
External features of the ultrastructure of the epidermis surface of Verbena officinalis L. flower petal are presented in Fig. 4.
Puc. 4.
The ultrastructure of the epidermis surface of Verbena officinalis L. flower petal: 1 - cuticle; 2 - papillae.
Internal (adaxial) surface. Cell boundaries are not visible. The cuticle of wrinkled type is well-formed. The surface relief is papillary (each epidermal cell carries papillae). There is no wax. When the inspection of
the specimens of Verbena officinalis L. flower petals a solid golden pellicle of cuticle which covers a single layer of epidermis can be seen. The cells of the epidermis are elongated, slightly tortuous, with tangential
outer and inner walls. The stomata sink deeper into the tissue of the hypodermis, under which is a large air cavity.
Thus, the epidermal cells of Verbena officinalis L. leaf are isodiametric. There are non-glandular and glandular trichomes on both epidermis. Non-glandular tri-chomes have a unicellular body and two multicellular and radially arranged basal cells. The body of these tri-chomes is slightly smooth, elongated with a pointed tip. It is distributed over both epidermal surfaces of the leaf.
The stems have quadrangular cross sections. Non-glandular and glandular trichomes were observed on the unradicated leaf epidermis. The type of glandular trichoma of the stem is the same as of the leaf of this species. There is a layer of subcutaneous cells under the epidermis. Collenchyma and sclerenchyma caps are present in the corners of the stem. There is a hypoder-mis in the stem. The endoderm is located around the perimeter of the stem.
Flower petals are characterized by a solid golden pellicle of cuticle, which covers a single layer of epidermis. The cells of the epidermis are elongated, slightly tortuous, with tangential outer and inner walls.
Conclusions
1. The study of morphological features of leaves, stems and flowers structure of Verbena L. species is conducted. These plants, depending on the species and growing conditions, can be annual and perennial herbaceous plants or shrubs. Stems are erect, creeping or spreading, rough or smooth. The leaves are opposite, pubescent, sometimes alternate or annular, dark green, toothed, dissected or pinnately incised, and in some species they are entire. There are hairy or corymbose inflorescences, spikelets or tassels consist of 30 - 50 small flowers from 1.5 cm to 2.5 cm in diameter. The range of colors of the flowers is extremely wide: pale purple, white, dark blue, blue monochromatic, yellow, cream, dark red, as well as with a cream or white eye. The fruit is a prefabricated nut that consists of four parts. Verbena L. species blooms from June to November depend on the species.
2. Microscopic features of the stem surface of Verbena officinalis L. were established. It is investigated that it is possible to distinguish two zones (costal and intercostal), which differ slightly in the ultrastructure of the surface. The stomata are anomocytic with distinct cuticular rollers, evenly spaced at the same level as the epidermal cells. Leaves are amphistomatic, their sto-mata are anisocytic, evenly distributed on both surfaces of the leaf blade, slightly above the level of the main epidermal cells. The adaxial surface of the leaf blade is collicular. Stem epidermal cells are prosenchymal with rectangular projections and straight outlines. The anticlinal walls of epidermal cells are not thickened, the outer periclinal walls are slightly convex. The boundaries of the cells are not distinct. The cuticle is well
formed. There is no pubescence. Periclinal walls of the epidermal cells contain epicuticular wax. The petals of Verbena officinalis L. flowers have entire golden pellicles of the cuticle, that cover a single layer of epidermis.
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