INTRODUCTION:

Ziziphora L. genus of Lamiaceae family includes about 30 species. The following species of the Ziziphora genus grow in Kazakhstan: Ziziphorabungeana, Ziziphora pamiroalaica and Ziziphora clinopodidoides¹.

Ziziphora clinopodioides Lam.(Lamiaceae) is a perennial herb with numerous erect, simple and rarely branched stems. Leaves are petiolate, oblong-ovate or elliptic, to 1.0-2.5 cm long and 0.3-1.0 cm wide, smooth-edged, glandular, glabrous and covered in short hairs. Flowers were collected in the apical capitate inflorescences. The calyx is pubescent, narrowly cylindrical, 1.0-1.2 cm long. Corolla is 1.0-1.2 cm long, light purple; tube twice as long as limb².

It grows on the rocky, sandy, gravelly and steppe slopes of the Central Kazakhstan small hills. It is found in practically all floristic areas, except for deserts in Kazakhstan³. The natural areas of Ziziphora clinopodioides Lam. and Ziziphora bungeana overlap with each other. Thus, the errors may be in diagnosis of the raw materials. Therefore, the microdiagnostic features should be identified⁴.

The essential oils were identified in Ziziphora leaves, and their main constituents are pulegone (72.8%), neomenthol (23.1%), menthone (13.3%), piperitenone (2.6%) and piperitone (1.2%)⁵. These essential oils are often rich in various chemical compounds including monoterpenes and sesquiterpenes. Some of these compounds may have the antibacterial properties. The main component of the essential oil of all studied types of ziziphora is pulegone, which has bactericidal properties against many pathogenic bacteria that infect the oral cavity and upper respiratory tract⁶. Some data can be found to use the essential oil of Ziziphora clinopodioides Lam. in the agriculture as an antifungal agent for the pathogenic fungus of Sclerotiniasclerotiorum which parasitizes plants. Today, a huge number of microorganisms and bacterial strains pose a threat to human life and health^7-8.Extracts from Z. clinopodioides are recommended to treat arrhythmia in case of weakening of heart muscle activity. Aqueous extracts of Ziziphora clinopodioides Lam. showed the activity against COX-1 (cyclooxygenase) for Bacillus subtillis and E. coli.^9-10.

The extract and essential oil have an antimicrobial effect including microorganisms isolated from the genitourinary tract¹¹, they have high fumigant activity, anthelmintic action against Anopheles stephensi and Culex pipiens¹², antioxidant effect, it has a positive gastroeffect, the flavonoids found in the extract have vasodilatory activity¹³. Some new biologically active derivatives were synthesized using the isolated compounds from Z. clinopodioides¹⁴.

Pharmacognostic studies help to determine the quality and purity of medicinal plant or animal raw materials, identify the presence of possible impurities and evaluate the biological activity of raw materials^15-16.This is important to ensure the safety, effectiveness and quality of medicines^17-18.

The aim of our research is to analyze the macro- and microscopic, histochemical parameters of Ziziphora clinopodioides Lam. and to identify the diagnostic characters of the plant.

MATERIALS AND METHODS:

The research object was the Ziziphora clinopodioides Lam. aerial parts (leaves, inflorescences and shoots). This species was collected in the flowering phase. The collecting place was Kerney settlement (the Karaganda region) on August 20, 2023. The analysis of the morphological parameters investigated the appearance, surface structures, color of shoots, leaves, inflorescences and flowers. These raw material specimens were examined using a Levenhuk DTX 30 Digital Microscope.

The plant specimens collected in the flowering phase were applied for the anatomical study of Ziziphora clinopodioides Lam. The air-dried raw materials were softened in a mixture of the distilled water : glycerol : alcohol 96 % (in the ratio of 1:1:1), then boiled in 5 % sodium hydroxide solution. The microscope slide and sections taken from surface were prepared. For microscopy of the objects, the slides were investigated on a BIOLAM microscope using magnification of 16*10 and 16*4. Microphotographs were obtained using the Altamy Studio software. The processing was performed in Paint 10.1 software. Explanatory drawings were made manually for better understanding of the tissue localization.

For the histochemical analysis we used the following reagents: methylene blue (essential oil); 10% alcohol solution of K₂Cr₂O₇ (phenolic compounds) and 3% alcohol solution of FeCl₃ (flavonoids). The microscopic photographs of the cross sections of the aerial organs were taken using a Biomed-4 microscope, 10x/20 mm eyepieces, 4 ×, 10 ×, 20 ×, 40 × lens. We photographed using a camera and Altamy Studio software.

The histochemical reactions were applied to detect and identifythe essential oils, phenolic compounds and flavonoids. In particular, the histochemical analysis can determine the presence and location of the biologically active substances in the individual tissues and organs^19-21.

RESULTS AND DISCUSSION:

Table 1: The morphological parameters of the aerial organs

Parameters	Description
Leaves	Leaves are narrow-lanceolateor elliptic, 6-8 mm long and 3-3.5 mm wide, on short petioles. Surface is pubescent, slightly hairy on both sides, with a well-marked medvein. The edge is flat.
Structure of the adaxialsurface of leaf	The adaxialsurface of leaves is dark green, gland-dotted, dark brown or reddish.
Structure of the abaxial surface of leaf	The abaxial surface of leaves is pubescent, green, with well pinnate venation. Veins protrude above the surface of epidermis.
Stems	Stems are simple, hard, thin, straight, tetrahedral and to 3 mm in diameter. Surface is green or brown, usually smooth, sometimes with sparse trichomes. Stem is white at the break and a core is light green.
Flowers	The flowers are small, short, collected at the top of the stems in capitateor sub-globose inflorescences.
Calyx	The calyx is short and narrow, 5-6 mm long and to 1 mm in diameter. Its leaves are joined at the base, with teeth along the margins. The calyx teeth are pointed. The surface is finely ribbed. The color is purple-green.
Corolla	The corolla is small and pale purple.
Smell	Smell is persistent, specific and pleasant.Flavour is minty and spicy.

The following diagnostic characters were identified at the macroscopic level: leaf structure, stem shape, structure and color of the calyx and corolla of flower.

The anatomical structure of leaf:

The investigation of the leaf showed that the cells of the upper and lower epidermis are slightly twisted-walled, erect-walled, and they are smaller on the lower side. (Fig. 1).

Figure 1. The microscope slide of Ziziphoraclinopodioides Lam. leaf taken from surface. Scheme. Magnifications 16x10: A – upper epidermis, B – lower epidermis; 1 – essential oil gland, 2 – simple trichomes, 3 – stomata, 4 – main cells of the epidermis, 5 – glandular trichomes

Stomata are numerous. They lie at a general level or in epidermal tissue, have the diacytic type.

In the epidermal complex of the leaf, 8-celled essential oil glands elevated above the surface and 3 types of hairs were found: simple trichome hairs of the adaxial and abaxial surfaces of the leaf, consisting of 1-3 cells with different lengths; glandular hairs represented by a 1-celled pedicel with a rounded head; and geniculate 2-celled curved hairs, well visible along the leaf margin. The epidermis is covered by a thick layer of cuticle, which is rugose around the stomata and essential oil glands.

On cross section the lamina is transcurrent, of dorsoventral structure. The main cells of the epidermis are large with thickened walls on the cross section. The palisade parenchyma is a dense, arranged in 1-2 rows. Spongy parenchyma is multirow, and it consists of loose cells which have round or slightly elongated shape (Fig. 2). Drops of essential oil were observed in the individual cells of the assimilative tissue of the leaf. The conducting bundles of the main and lateral veins of the leaf were determined in the center. The bundle is closed and collateral (phloem - below, xylem - above), strongly sclerified. The covering of the bundle is parenchymal. A single-layered collenchyma lies along the periphery of the vein on the abaxial surface.

Figure 2: Cross median section of the ZiziphoraclinopodioidesLam. leaf. Magnifications 16x4: A - schematic drawing, B - microscope slide; 1 - upper epidermis, 2 - lower epidermis, 3 - palisade mesophyll, 4 - spongy mesophyll, 5 - conducting bundle, 6 - simple trichomes, 7 - glandular trichome, 8 - essential oil gland

On cross section, the Ziziphora stem is 4-angled and has an anatomical structure typical for bilobular ones (Fig. 3). On the outer side, the stem is covered with one-layer epidermis consisting of straight-walled and elongated cells. Their length is 1.5-2 times their width. The epidermis of the stem has the simple and glandular formations of the same types as those of the leaf.

Figure 3. Cross section of the Ziziphora clinopodioides Lam. stem. Magnifications 16x4: A – schematic drawing, B – microphotograph; 1 – epidermis, 2 – collenchyma, 3 – cortical parenchyma, 4 – endodermis, 5 – phloem, 6 – cambium, 7 – xylem, 8 – sclerenchyma, 9 – medullary parenchyma

The angular collenchyma is localised at the corners with large oblong cells of the cortical parenchyma between themadjacent to 1-row endoderm.The conductive system has the bundle type in the upper part and the ring type in the lower part for stems.The central cylinder is peripherally represented by 2-3 rows of small phloem cells and centrally by xylem chains. The cambial zone is weakly expressed. The conducting zone consists of sclerenchyma cells. The midrib of the stem has the large rounded and loosely arranged cells.

The calyx is narrow-cylindrical with 5 small pointed teeth. The flower is zygomorphic and pubescent. The microscope slide taken from surface showed that the epidermis of the calyx has various structures in different parts. Thus, Figure 4 illustrates thatabove the conducting bundles the epidermis has the small and slightly elongated thin-walled cells and some cells have bristly cuticular outgrowths.

Figure 4: The microscope slide of the Ziziphora clinopodioides Lam. calyx taken from the surface. Magnifications 16x10: A - epidermis above vein, B - main epidermis, C - epidermis of margin; D - microphotograph of calyx; 1 - epidermis cells, 2 - essential oil gland, 3 – hairs

The main cells of the epidermis are slightly sinuous, oriented along the axis of the leaf, with rare essential oil glands, densely pubescent with the simple and glandular hairs.

The main cells of the epidermis are weakly convoluted, orientated along the leaf axis, with sparse essential oil glands, densely pubescent with simple and glandular hairs. The marginal cells of the epidermis are erect-walled, small in the form of essential oil glands and simple hairs. The superficial examination determined that the epidermis of the flower corolla is composed of large thin-walled cells of the isodiametric shape (Fig. 5). The epidermal formations included 1-2-celled simple andgeniculate hairs. The 8-celled essential oil glands and glandular hairs are found occasionally.

Figure 5. The microscope slide of the epidermis of the Ziziphora clinopodioides Lam. corolla.Schematic drawing. Magnifications 16x10: 1 – epidermis, 2 – types of hairs: a – simple unicellular and multicellular, b – glandular, c – geniculate

The number and topography of stomata, essential oil glands and 3 types of hairs were identified as the diagnostic characters.

The histochemical analysis: The histochemical study was conducted for cross sections of stem, leaf and corolla of flower of Ziziphora clinopodioides Lam. We used the following reagents for the histochemical analysis: methylene blue (essential oil); 10% alcoholic solution of K₂Cr₂O₇ (phenolic compounds) and 3-% alcoholic solution of FeCl₃ (flavonoids).

The study revealed the characteristic staining of various cell types. It was characterized by the result of the interaction of reagents with the detectable metabolites. The results of the histochemical analyses of aerial organs of Ziziphora clinopodioides Lam. are presented in Table 1 and Figure 1.

Table 2. The histochemical analyses of underground organs of ZiziphoraclinopodioidesLam.

Component	Reagent	Staining	Presenceofcharacteristicstaining
Component	Reagent	Staining	Stem	Leaf	Corollaofflower
Essentialoil	Methyleneblue	Blue	+	+	+
Flavonoids	1-% alcoholic solution of FeCl3	Black-blue-green	+	+	-
Phenoliccompounds	10% alcoholic solution of K₂Cr₂O₇	Brown, yellow	+	+	+
“+” - positive reaction and “-“ - negative reaction

The results showed that localization of essential oil, flavonoids and phenolic compounds were observed on leaf, stem cross section and flower corolla preparations.

Thus, accumulation of the essential oil was observed in the epidermis of leaves, mesophyll and conductive bundles, and also in the epidermis of flower corolla. On a cross section of the stem, the essential oil was localized in the epidermis, angular collenchyma and xylem region.

The accumulation of flavonoids was detected on the cross section of the stem in the cortical parenchyma and xylem vessels. The accumulation of flavonoids in the cortical and medullary parenchyma of the conductive zone was determined for microscope slides of leaf from cross section. The phenolic compounds were defined for all the organs investigated. In the corolla of the flower, the phenolic compounds are localized in the epidermis above the veins. In the stem, they were found in the cortical parenchyma. Inthe leaf, they were determined in the mesophyll.

CONCLUSION:

The research results determined the macro- and microscopic, histochemical features of raw materials of Ziziphora clinopodioides.

For leaf: the macroscopic features: The surface is rough, pubescent on both sides, with well-marked midvein, especially on the lower side, and margin is flat. Microscopic features: Stomata are numerous.

The microscopic features: Shape of the epidermal cells, tortuosity of walls, stomata of the diacytic type, large essential oil glands and rare trichomes.

For stem: Form of stem growth, color, opposite arrangement of leaves. The microscopic features: form of stem on the cross section, structure of bundles of 2 types.

For inflorescence: The macroscopic features: peculiarities of arrangement of flowers and bract leaves in inflorescence,

For calyx of flower: The macroscopic features: shape of calyx and teeth of calyx, color, presence of small ribs on the surface. The color is violet-green.

The microscopic features: The epidermal cells with elongated thin-walled cells and bristly outgrowths of cuticle.

For corolla of flower: The macroscopic features: shape and size of corolla, andcolor is pale violet or pinkish. The microscopic features: shape of epidermal cells, presence of essential oil glands and hairs.

A histochemical study: For leaf: The accumulation of the essential oils was found in epidermis of leaves, and accumulation of flavonoids was defined in the cortical and medullary parenchyma of the conductive zone. The phenolic compounds in the mesophyll.

For stem: The essential oils are in epidermis, angular collenchyma and xylem region. Flavonoids are in the cortical parenchyma and xylem vessels, and the phenolic compounds are in the cortical parenchyma.

For flower: The essential oils are in the epidermis of the corolla of the flower. The phenolic compounds in the corolla of flower are localized in the epidermis above the veins.

CONFLICT OF INTEREST:

We declare that we have no conflict of interest.

ACKNOWLЕDGMENTS:

This work was supported by the Science Committee of the Ministry of Science and Higher Education of the Republic of Kazakhstan under Grant No. AP AP19677164.

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Received on 22.04.2024 Modified on 15.07.2024

Research J. Pharm. and Tech 2024; 17(11):5254-5260.

DOI: 10.52711/0974-360X.2024.00804


Leaf, ferricchloride	Leaf, methyleneblue

Stem, potassiumbichromate	Stem, methyleneblue

Flower, methyleneblue	Flower, potassiumbichromate