INTRODUCTION:

Determining the quality index of medicinal raw materials is an important task for the researcher. Currently, there are a number of research methods used to establish the authenticity of medicinal plants for the content of the main groups of biologically active substances.

These methods include histochemical analysis, which allows to detect the presence of biologically active substances, which include secondary metabolites, such as phenolic compounds, flavonoids, alkaloids, polysaccharides and sesquiterpene lactones^1-10. In the pharmaceutical industry, there is an increasing interest in biologically active substances from plant materials, since they have various pharmacological activities. Currently, herbal medicines used for the prevention and treatment of various diseases are of particular importance in the world market. The ever-increasing needs of society for biologically active substances with various pharmacological activities, including anti-inflammatory, antispasmodic, antiviral nature, determine the search for and introduction into medical practice of new drugs derived from plant materials¹¹.

More than 5,500 plants grow in Kazakhstan, of which at least 20% have medicinal properties^12-13. A valuable object for the search for new herbal remedies are species of the genus Nitraria from the family Nitrariaceae.

The structural features of some species of Nitraria are given in the article of Ak-Lama T.A.¹⁴, where interspecific differences are already visible at the stage of juvenile plants; when studying the composition of phenolic compounds in the leaves of plants N. schoberi, N. Sibirica, a total of 25 compounds were found, the entire spectrum of substances is not found in any populations. Two alkaloids were isolated from the aerial part of the plant Nitraria sibirica. Based on chemical transformations and spectral data, their structure is proposed (Allaberdiev F.Kh. et al.)¹⁵. In traditional folk medicine, the aerial part and fruits of N. schoberi were used to treat cardiovascular diseases, it was found that extracts of the fruits, leaves and roots of N. schoberi, collected in Iran (Zabol), have powerful antioxidant, antimicrobial, antifungicidal and anti-inflammatory properties¹⁶. Based on the extract of the roots of N. schoberi, which grows on the territory of the Russian Federation (Siberia), a method has been developed for obtaining an antiviral agent against influenza A viruses of subtypes H3N2 and H5N1¹⁷. Extracts containing flavonoids for use in medical practice^18-19, proanthocyanidins were obtained by ultrasonic extraction from Nitraria fruits²⁰.

According to literature sources, plants of the genus Nitraria are rich in secondary metabolites, such as organic acids, phytosterols, polyunsaturated fatty acids, sesquiterpene lactones, and alkaloids²¹. Two species of plants from the genus Nitraria (Nitraria schoberi L., Nitraria sibirica Pall.) grow on the territory of Kazakhstan. As a vicarious plant, we carried out a pharmacognostic study of the raw material Nitraria schoberi L., which grows in the territory of the Central Kazakhstan²² and has significant raw material resources. However, despite the practically valuable properties and prospects for the use of plants of the genus Nitraria in traditional medicine, the chemical composition and biological properties of the plant Nitraria schoberi L. remain unexplored. In this regard, the study of the localization of biologically active substances in the leaves, fruits, roots of Nitraria schoberi L. using a histochemical study is one of the important steps for identifying plant raw-materials and further research in the field of medicine.

MATERIALS AND METHODS:

Raw materials:

The object of the study is the aboveground and underground organs of Nitraria schoberi L., collected on the territory of the Karaganda region (near the village of Baimyrza) of the Republic of Kazakhstan. The studied raw materials were harvested during the fruiting period, August - September 2021. Histochemical study. Freshly harvested organs (leaves, stems, fruits, roots) were preserved in a mixture of alcohol (70%), glycerol and distilled water in a ratio of 1:1:1 (Strauss-Fleming solution).

A histochemical study was carried out for transverse sections of the stem, transverse and surface sections of leaves, surface preparations of fruits and transverse sections of roots in accordance with the requirements of the State Pharmacopoeia of the Republic of Kazakhstan (vol. 1. “Methods for testing medicinal plant materials”, “Technique for microscopic and microchemical examination of plant materials”)^13,23. When conducting histochemical analysis, we used the reagents listed in Table 1.

Table 1: Reagents for microchemical research

S. No.	Reagent	The component being defined	Coloration
1	Methylene blue	Essential oil	Blue
2	1 % Alcohol solution FeCl₃	Flavonoids	Black-blue-green
3	10 % Alcohol solution K₂Cr₂O₇	Phenolic compounds	Brown, yellow
4	Dragendorff's reagent	Alkaloids	Black
5	Concentrated H₂SO₄	Sesquiterpene lactones	Yellow
6	10 % Solution of thymol and concentrated H₂SO₄	Polysaccharides	Orange red
7	Lugol's reagent	Starch	Blue

The localization of individual groups of biological substances was judged by the change in the color of the tissues of Nitraria schoberi L. The study of anatomical preparations was carried out using a Biomed-4 microscope with eyepieces ×10, ×20, lenses ×4, ×10, ×20, ×40. Microscopic photographs of transverse sections of aerial organs and roots were taken using a Sony Siber Shot camera.

RESULTS AND DISCUSSION:

As a result of the study, staining of plant tissues was established, which showed the presence of phenolic compounds, flavonoids, alkaloids, polysaccharides and sesquiterpene lactones. The presence of essential oils, camphor, starch was not detected. The results of histochemical analysis of aboveground and underground organs of Nitraria schoberi L. are presented in Table 2 and in Figures 1-5.

Table 2: The results of histochemical analysis of the aboveground and underground parts of Nitraria schoberi L.

No	Reagent	Stem	leaf	root	fruits
1	Methylene blue	-	-	-	-
2	1 % alcohol solution FeCl₃	-	+	+	+
3	10 % alcohol solution K₂Cr₂O₇	+	+	+	+
4	Dragendorff's reagent	-	+	+	-
5	Concentrated H₂SO₄	-	+	+	-
6	10 % solution of thymol and concentrated H₂SO₄	-	-	+	-
7	Lugol's reagent	-	-	-	-

Note: "-" - negative reaction; "+" - positive reaction

Identification of flavonoids in aboveground and underground organs of Nitraria schoberi L.

After treatment of the micropreparation of the studied samples with 1% alcohol solution of FeCl₃, an intense black-blue-green staining of Nitraria schoberi L. was observed:

· On the transverse section of the leaf: the mesophyll is stained;

· On the surface preparation of the fruits, the color of individual cells of the epidermis is noted;

· On the transverse section of the root: intense staining of the core zone and the vascular bundle in the center of the root was noted (Fig. 1).

Fig.1: The result of histochemical reactions with 1% alcohol solution of FeCl₃ (magnification × 10).

A - transverse section of the leaf of Nitraria schoberi L., B - surface preparation of the fruit of Nitraria schoberi L., C - transverse section of the root of Nitraria schoberi L.

Identification of phenolic acids in aboveground and underground organs of Nitraria schoberi L.:

To identify phenolic acids, micropreparations were placed in a 10% potassium bichromate solution and left for 7 days. The presence of phenolic acids was confirmed by intense yellow-brown staining in all studied organs of Nitraria schoberi L. However, the staining of the micropreparations was uneven, which makes it possible to judge the different degree of accumulation of phenolic compounds in the cells. Thus, the areas with the maximum accumulation of phenolic compounds are:

· On the transverse section of the stem, the following structures were stained: the cortical parenchyma and the conducting zone;

· On the transverse section of the sheet: chlorenchyma, conductive zone and bundles;

· On the surface preparation of the fruits: separate areas of the epidermis are saturated;

· On the transverse section of the root: intense staining was noted for the cortical zone and parenchymal rays (Fig. 2).

Fig.2: The result of histochemical reactions with 10% alcohol solution of K₂Cr₂O₇ (mag. × 10).

A - transverse section of the stem of Nitraria schoberi L., B - transverse section of the leaf of Nitraria schoberi L., C - surface preparation of the fruit of Nitraria schoberi L., D-cross section of the root of Nitraria schoberi L.

Identification of alkaloids in aboveground and underground organs of Nitraria schoberi L.

The presence of alkaloids was confirmed by uneven staining in the form of black spots in the leaves and roots of Nitraria schoberi L. The areas of accumulation of alkaloids in the form of black spots are:

· On the transverse section of the leaf, the mesophyll stained;

· On the transverse section of the roots, the cortical parenchyma stained (Fig. 3).

Fig. 3: The result of histochemical reactions with Dragendorff's reagent (mag. × 10).

A-cross section of the leaf of Nitraria schoberi L., B-cross section of the root of Nitraria schoberi L.

Identification of sesquiterpene lactones in aboveground and underground organs of Nitraria schoberi L.

The presence of sesquiterpene lactones was confirmed by yellow staining on surface preparations of leaves and root cross sections. The sites of accumulation of sesquiterpene lactones are:

- on the surface preparation of the leaf, the mesophyll and more intensely the veins of the leaf stained;

- on the transverse section of the root, the cortical parenchyma stained (Fig. 4).

Fig. 4: The result of histochemical reactions with conc. H₂SO₄ (mag. × 10).

A-surface preparation of the leaf of Nitraria schoberi L., B-cross section of the root of Nitraria schoberi L.

Identification of polysaccharides in underground organs of Nitraria schoberi L.

The presence of polysaccharides was confirmed by intense yellow staining on the cross section of the root. Thus, the area with the maximum accumulation of polysaccharides is:

· On the transverse section of the root, the parenchyma of the conducting bundle was stained.

Fig. 5: The result of histochemical reactions with a 10% solution of thymol and concentrated sulfuric acid (mag. × 10).

A-cross section of the root of Nitraria schoberi L.

CONCLUSIONS:

For the first time, the study of the secretory structures of Nitraria schoberi L. were studied by light microscopy in combination with histochemical tests. As a result of histochemical tests on leaf cross sections, stem cross sections, fruit surface preparation, root cross sections, phenolic acids, flavonoids, alkaloids, polysaccharides and sesquiterpene lactones were found, and their localization was established:

· Phenolic acids - core parenchyma and conductive zone of the stem; chlorenchyma, conducting zone (bundles) in a leaf; intense staining of the core zone and the conducting bundle in the center of the root;

· Flavonoids - leaf mesophyll; staining of individual cells of the fruit epidermis, intense staining of the core zone and the vascular bundle in the center of the root;

· Alkaloids - leaf mesophyll; cortical parenchyma of roots;

· Sesquiterpene lactones - mesophyll and leaf veins; cortical parenchyma;

· Polysaccharides - the parenchyma of the vascular bundle of the root.

ACKNOWLEDGEMENTS:

The authors are grateful to the management and staff of the School of Pharmacy NCJSC «Karaganda medical university», NCJSC «Karaganda University named after Ye.A. Buketov» (Karaganda, Kazakhstan) and The Department of Botany for the opportunity to conduct histochemical studies.

This research is funded by the Science Committee of the Ministry of Education and Science of the Republic of Kazakhstan (Grant No. AP19176670).

CONFLICT OF INTEREST:

The authors declare no conflict of interest.

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Received on 27.12.2022 Modified on 19.02.2023

Research J. Pharm. and Tech 2023; 16(9):4188-4192.

DOI: 10.52711/0974-360X.2023.00685