Morphometric Study of Hyssopus ambiguus Growing in the Territory of Central Kazakhstan

Corresponding Author: Yekaterina Victorovna Lakomkina Department of Pharmaceutical Disciplines and Chemistry, NC JSC "Karaganda Medical University", Karaganda, Kazakhstan Email: yekaterina.yankovkskaya@inbox.ru Abstract: The article presents the results of the study of Hyssopus ambiguus herb, collected on the territory of Central Kazakhstan. We obtained data on diagnostic features of the studied species at morphological and anatomical levels. The purpose of the study is to determine the macroand microscopic diagnostic features of the aboveground part of Hyssopus ambiguus. Materials and Methods: Raw material was collected on the territory of the Republic of Kazakhstan, Karaganda region. The microscopic and morphological studies were carried out according to the methods of the Republic’s of Kazakhstan State Pharmacopoeia (RKSP) using a Biomed-4 light microscope and a Levenhuk macroscope. Results: The main diagnostic features of Hyssopus ambiguus had been determined: Trichomas and essential oil glandules. Analysis of the anatomical and morphological features of leaves, shoots and sepals was made; the characteristics of essential oil glandules were described. During the morphological and anatomical study of Hyssopus ambiguus, the diagnostically significant macroand microscopic features of raw materials were identified and biometric characteristics were established. The experimental data allowed to confirm the authenticity, identification and standardization of the aboveground part of Hyssopus ambiguus.


Introduction
The studies of unexplored medicinal plants aimed to identify their potential in traditional medicine are of great importance. Biologically active substances included in the compositions of medicinal plants determine their pharmacological activity. They affect more relative to the human body, on the various biochemical processes occurring in it and, therefore, are better absorbing than their synthetic analogues.
In particular, it is of interest to study vegetable raw materials with antimicrobial properties as an alternative to synthetic antibacterial drugs. A number of studies of scientists from different countries proved antibacterial properties of the plants from the genus Hyssopus. Kukaniev et al. (2012) detected excellent antibacterial activity of Hyssopus zeravshanicus against Bacillus cereus bacteria, Staphylococcus aureus, but weak activity against gram-negative bacteria and fungi. Diego Sampietro et al. (2017) revealed strong antifungal activity of Hyssopus ambiguus, which grows in West Kazakhstan, with activity to Fusarium strains, due to the camphen content and other oxygen-containing monoterpenes. Moulodi et al. (2018) proved Hyssopus officinalis essential oil activity in relation to Escherichia coli. Tomasz Baj et al. (2018) pointed to the difference between antibacterial activity of Hyssopus officinalis essential oil obtained from plants with white flowers and with pink flowers: The first one exhibits antimicrobial activity against Streptococcus pyogenes, S. pneumoniae, S. mutans, Candida albicans, C. parapsilosis, whereas the second one-against such gram-positive bacteria as Staphyllococcus aureus, S. epidermidis, Streptococcus pyogenes, S. pneumoniae, S. mutans, Bacillus subtilis.
According to the research of Kovalenko et al. (2019), essential oil of Hyssopus officinalis obtained from a rosecolor variety has antibacterial activity against Salmonella alony, Clostridium sp., Escherichia coli, but essential oil obtained from Zeva's variety inhibits the growth of bacteria Staphylococcus aureus and Pseudomonas aeruginosa.
As an object of the present study, we have chosen Hyssopus ambiguus, a plant, which widespread in the Karaganda region of the Republic of Kazakhstan. Essential oils, contained in Hyssopus ambiguus, represent perspective for the pharmaceutical industry as a source of antimicrobial and antifungal components. Previously, Hissopus ambiguus, growing on Kalbinsky Range (East Kazakhstan), has been studied and described by Kazakhstani scientists in several articles Tursynova et al., 2015;Myshabaliyeva et al., 2016;Diego. Sampietroa et al., 2017;Ishmuratova et al., 2020). Medicinal preparations based on Hyssopus ambiguus does not yet exist.
According N.V. Pavlov (1964), Hyssopus ambiguus (Trautv.) Iljin ex Prochorov & Lebel. (family Lamiaceae) refers to perennial plants; 25-40 cm height, consisting of numerous four-pointed stalks, rustic at the base; leaves without omitting, with whole edges, narrow core, dorsalventral type; inflorescences of Hyssopus ambiguus are thin, length from 5 to 12 cm, with narrowing to the top; the flowers are numerous; sepals exceed the sizes of the whorls, in which there are 5-6 flowers; a sepal consists of 5 almost identical teeth and has a length of 4 to 6 mm; the length of teeth are 1/3 of the sizes of the cup; sepals are blue or violet color, from 0.8 to 1 cm long, consists of two almost equal lips: The top lip is two-blade, the lower -three-blade; 4 stamens, of which 2 are longer than the sepal and 2 are equal to its by length; the pistil column coincides in length with longer stamens; fruits are tetra-achenes, oblong ovoid shape, have 3-sided. Hyssopus ambiguus grows on the slopes of steppe hills, mountains, lowland and pebbles. The species grows in East and Central Kazakhstan.
Objective: To determine macro-and microscopic diagnostic features of aboveground organs of Hyssopus ambiguus.

Plant Material
The vegetable raw material was collected in Karaganda region of the Republic of Kazakhstan: Karkaraly Mountains, June, 2020; phase-flowering (Fig. 1). The plant material was determined by professor of Botany Department M.Yu. Ishmuratova and specimen deposited in the herbarium of the faculty of biology and geography. The study of macro-and microscopic features was carried out according to the methods of the Republic's of Kazakhstan State Pharmacopoeia (RKSP) using a Biomed-4 light microscope and a Levenhuk macroscope (Musinov and Tulegenova, 2016).

Macroscopic Analysis
The study of macroscopic features of raw materials of Hyssopus ambiguus was carried out according to the methods of RKSP, Vol. I using a Levenhuk macroscope (Musinov and Tulegenova, 2016).

Microscopic Analysis
The study of microscopic features of raw materials was conducted according to the procedures of RKSP, Vol. 1 using a Biomed-4 microscope (Musinov and Tulegenova, 2016).
To investigate the above-ground part of Hyssopus ambiguous, dry plant material was softened in Straus-Fleming solution (a mixture of alcohol-glycerol-distilled water in a ratio of 1:1:1). Surface preparations and sections were prepared manually. Photos of micro preparations were made in Altami Studio and processed in Paint 10.0. To describe the anatomical structure, we relied on the works of, Vekhov and Filin (1980),; Nikitina et al. (2019) and Kovalev (2003).

Statistical Processing
The sample consisted of 10 measurements. The identified macro-and microscopic features of the studied raw materials were compared with existing data. Statistical data analysis was performed using the Statgraphics Centurion XVI software for Windows.

Macroscopic Features
We used a Levenhuk microscope when examining the composite components of the analytical sample to identify the characteristic external features of the above-ground organs of Hyssopus ambiguus (Fig. 2).
The surface of the leaf on both sides is rough, green, with numerous essential oil glands of orange-brown color. Trichomas are absent ( Fig. 2A).
A leaf sheet is narrow-lancing form, dorsal and ventral type, 10-20 mm long and 1-1.5 mm wide. The petiole is very short, 1-2 mm long. The edges of the leaf sheets are slightly bent on the lower side. The vein is expressed weak; the main vein on the bottom of the leaf is clearly observed (Fig. 2B, C).
A stalk is reprehensive, along the entire length of the green, in the nodes painted in a lilac-purple colour; on the cross-section 4-graded; white colour. The diameter of a stem is 2-4 mm (Fig. 3A, C).
The surface of the stalk is smooth, hardware and trichomas are not expressed (Fig. 3B).
A sepal is not clear two-lips, up to 5-8 mm long and up to 2-3 mm width; the shape is narrow-bell, the surface is ribbed. The upper part of sepal is expanded and has short and pointed teeth. Colour of teeth and vein is lilac-purple, the lower part of a sepal is green-painted (Fig. 4A, B).
Longitudinal veins are noted on the surface of the sepa. Along the veins, large essential oil glandules, amber or light brown, are placed rows. There are rare simple trichomas, white ones on the teeth of the sepal (Fig. 4C).

Microscopic Features
The upper and lower epidermis cells of Hyssopus ambiguus leaf, with thickened walls, 0.8 nm long and 1.0 mm wide (Fig. 5, 6). The stomata are diacytic type, 1.5-1.6 μm wide, are located on both sides of the leaf sheet. Over the entire surface of the leaf, numerous essential-oil glandules of a rounded form with a diameter of 0.5-0.7 μm are scattered. Trichomas are simple, located along the edge of the leaf sheet, have a length 1-1.2 μm and a width 0.2 μm.
The cross-section of leaf of Hyssopus ambiguus is dorsal and ventral type (Fig. 7), with a clearpronounced division of the mesophyll on the palisade and spongy tissues. A width of leaf sheet is 3.6-5 μm, the length is 12.5-13.6 μm. On both sides of the leaf are surrounded by a single-layer epidermis, a width of 0.3-0.4 μm, whose cells are rounded rectangular shape with thickened walls. Palisade mesophyll consists of three cell layers and has a width of 1-1.5 μm; spongy mesophyll is with a thickness of 1.4-1.8 μm.
A conductive bundles are collateral type, closed, consists from site of xylem and phloem. The diameter of the central conductive bundle is 2-2.2 μm, the diameter of the lateral conductive bundle is 1.2-1.3 μm. Rare simple trichomas are well observed, the length of which is 0.2-0.6 μm and the width of 0.2 μm. The essential-oil glandules are rounded shape, are raised above the epidermis surface, diameter is from 0.2 to 0.5 μm.
A stalk of Hyssopus ambiguus on the cross-section is a four-sided, rectangular-blade (Fig. 8). The perimeter of the stalk contains a single-layer epidermis with a width of 0.3-0.4 μm, with thickened outer cell walls. Rounded essential oil glandules have the diameter of 0.2-0.4 μm; and simple trichomes with a width of 0.2 to 0.4 μm and a length of 0.2-0.5 μm are detected on the epidermis of the stalk. Under the epidermis, the sites of the chlorenchima are located, 0.5-0.6 μm wide, interrupting the edges of the corner collenchymas with thickness of 2 μm. There is a core parenchyma with a thickness of 0.5-0.7 μm between the conductive zone and chlorenchima. The conductive zone is restrictive to a single-layer endoderm, which width is from 0.7 to 1.5 μm. The conductive system is represented by a phloem and xylem -oriented ring. The rows of xylem of 2-3.5 μm width are well expressed. In the center of the stalk is the pith parenchyma representing the lying thinwalled cells, the diameter of the pith is from 9.5 to 12 μm.
The main cell epidermis cells have a prosenchymal shape, the walls are slightly winding, 0.3x1 μm (Fig. 9). The entire surface of the cup is covered with numerous essential oil glandules of a rounded shape, a diameter of glandular is 0.5-1 μm. Through the surface of the cup, veins with a thickness of 0.6 to 1.1 μm are running. Trichomas are simple, long 0.9-1.5 μm and a width of 0.2 μm, are mainly located along the edge of the teeth.

Discussion
The results of microscopic diagnostic features study of Hyssopus ambiguus are the following: 1) A form of the leaf blade -narrowly lanceolate 2) A colour of stalk -green over the entire length in the nodes dyed lilac-violet colour, at the breakalmost white 3) A sepal shape -narrowly bell-shaped, surfaceribbed 4) Trichomas location -sepal teeth covered with sparse simple white trichomas 5) The arrangement of essential oil glandules are abundantly coated surface of the blade on both sides, are completely absent on the stem, arranged in rows along the veins of the sepal The diagnostic features of Hyssopus ambiguus on the microscopic level are: 1) A leaf: Form of the main cells of the epidermis, location, type of essential oil glandular and trichomes 2) A stalk: Conductive system of closed type, shape and location of glandules and trichomes 3) A sepal: The shape of the cells of the epidermis, the location of essential oil glandules over the surface of epidermis and simple trichomes on the teeth of the flower cup

Conclusion
The diagnostic features of Hyssopus ambiguus are the form and the structure of the epidermis cells of a leaf and a stalk, the shape and the arrangement of essential oil glandules and trichomes, the structure of a leaf and a stalk on the transverse cut.
The diagnostic features of Hyssopus ambiguus on the macroscopic level are: The shape of a leaf, the coloration of a stalk, the absent of trichomas on stalks, the form of a flower cup; the location of trichomas and essential oil glandules.
The diagnostic features of Hyssopus ambiguus on the microscopic level are: 1. A leaf: Form of the main cells of the epidermis, location, type of essential oil glandular and trichomes 2. A stalk: Conductive system of closed type, shape and location of glandules and trichomes 3. A sepal: The shape of the cells of the epidermis, the location of essential oil glandules over the surface of epidermis and simple trichomes on the teeth of the flower cup
Margarita Yulaevna Ishmuratova: Analysis, manuscript writing, data interpretation, identification of species, collecting of raw material.