Histochemical Analysis of Raw Materials of Linaria vulgaris Mill. Growing in Central Kazakhstan

 

Itzhanova Kh.I1, Nurkadirov D.K.1*, Ishmuratova M. Yu.2, Kosherbek A.B.1,

Baidullayeva S.A.3, Ewa Poleszak4

1NJSC “Karaganda Medical University”, 100000, Kazakhstan, Karaganda, Gogol Street, 40.

2NJSC “Buketov Karaganda University”, 100028, Kazakhstan, Karaganda,

Universitetskaya St., 28, Building No. 3.

3Department of Drugs Expertise, National Centre for Expertise of Medicines and Medical Devices, Kazakhstan, Astana, Imanov Street, 13.

4Medical University of Lublin, 20-059, Poland, Lublin, St. Al. Racławickie 1.

*Corresponding Author E-mail: darhan_2295@mail.ru

 

ABSTRACT:

The article presents the results of a histochemical study of the aerial part of Linaria vulgaris Mill., collected during the period of mass flowering (July-August 2023) in the Bukhar-Zhyrau district, Karaganda region (Republic of Kazakhstan). Goal. To ascertain the specific localization patterns of various groups of biologically active substances within the aerial organs of Linaria vulgaris Mill., as found in the Central Kazakhstan region. Materials and methods. For the histochemical analysis, aerial parts of Linaria vulgaris Mill. were collected during the flowering stage. These plant samples were fixed in Strauss-Fleming solution, followed by preparation of cross sections. Histochemical reactions were then carried out to pinpoint and characterize various biologically active compounds, including essential oils, starch, flavonoids, polysaccharides, sesquiterpene lactones, and alkaloids. The microscopic examination and histochemical tests adhered to the protocols outlined in the State Pharmacopoeia of the Republic of Kazakhstan. Results. Histochemical techniques employing light microscopy were used to identify the distribution of biologically active compounds within the plant raw material of Linaria vulgaris Mill. The analysis confirmed the presence of flavonoids, phenolic compounds, alkaloids, and polysaccharides. However, essential oils and sesquiterpene lactones were not observed. Conclusion. This marks the inaugural application of histochemical tests to examine the distribution of biologically active substances within the tissues of Linaria vulgaris Mill. These findings hold significant potential for verifying the authenticity, identifying, and establishing standards for the aerial components of Linaria vulgaris Mill.

 

KEYWORDS: Histochemical analysis, Linaria vulgaris Mill., medicinal plant, biological active substances, flavonoids, alkaloids.

 

 


INTRODUCTION: 

Chemical and physicochemical analyses play a crucial role in identifying both active and accompanying substances present in plants. Histochemical analysis is among the methods used for this purpose. By utilizing histochemical reactions, researchers can gather supplementary data to authenticate medicinal plant raw materials. This method also facilitates the identification of biologically active compounds and determines their specific distribution within the tissues and organs of the examined plant.

 

Linaria vulgaris Mill. (Scrophulariaceae family) is a perennial herbaceous plant widely distributed in the territory of the Republic of Kazakhstan and is common in Europe, Asia, Western Siberia, and the Far East1,2.

 

The herb of Linaria vulgaris Mill. contains alkaloid peganin, flavonoid glycosides - linarin, neolinarin and pectolinarin (one of the glycosides is nitrogenous and contains hydrocyanic acid), carotene, tannins, phytosterol, paraffin, ascorbic acid, and organic acids. Due to its chemical composition, herb of Linaria vulgaris Mill. is used in medicine as a diuretic and laxative, as well as a remedy for worms and for chronic skin diseases3. The aerial part of Linaria vulgaris Mill. is used in folk medicine as an anti-inflammatory, diuretic, antimicrobial, and antihemorrhoidal agent4,5.

The widespread use of Linaria vulgaris Mill. in folk medicine indicates the need for an in-depth study of the chemical composition and pharmacological properties of this plant. In this regard, a comprehensive study of Linaria vulgaris Mill. herb with the aim of introducing it into scientific medicine is relevant6.

 

MATERIALS AND METHODS:

The focus of this study was on the aerial parts of Linaria vulgaris Mill., Scrophulariaceae family (Fig. 1), collected during the flowering period in the steppe regions of the Bukhar-Zhyrau area within the Karaganda region, Republic of Kazakhstan. Collection took place between July and August 2023, with species verification carried out by staff members of the Department of Botany at E.A. Buketov Karaganda University in Karaganda.

 

Figure 1. Appearance of Linaria vulgarisMill. in the flowering phase

 

Drying of the raw materials was carried out at 25-30°C, in the shade, for 3 days. The humidity of the studied raw materials after drying was 10-12%.

 

Histochemical tests were carried out according to the methods of the State Pharmacopoeia of the Republic of Kazakhstan and methodological manuals on pharmacognosy7-9.

 

Samples of dry raw materials (shoots, leaves, inflorescences) were soaked and fixed in a mixture of ethyl alcohol (70%), glycerin and distilled water (Strauss-Fleming solution) in a ratio (1:1:1).

 

The distribution of specific biological compounds within Linaria vulgaris Mill. tissues was assessed by observing color changes. Histochemical preparations were examined under a Biomed-4 microscope equipped with ×10 and ×20 eyepieces, along with ×4, ×10, ×20, and ×40 lenses. Photographic documentation was accomplished using the Altamy Studio 7.1 software, followed by image processing in Paint 10.1.

 

Micropreparation descriptions and analysis of biologically active substance localization were carried out, incorporating information from relevant literature sources10-15.

 

RESULTS AND DISCUSSION:

Histochemical tests revealed distinct staining patterns in specific tissues, indicating the interaction between reagents and the substances present. Consequently, qualitative responses to flavonoids, phenolic compounds, alkaloids, and polysaccharides were identified, while essential oils and sesquiterpene lactones were absent. For instance, applying an iron (III) chloride solution resulted in strong staining of leaf epidermal cells and conducting bundles, as depicted in Table 1 and Figure 2. Mesophyll and conceptacle cells exhibited comparatively lighter staining.

 

Table 1: Results of histochemical analysis of the aerial and underground parts of Linaria vulgaris Mill.

S. No.

Component

Shoots

Leaves

1

Flavonoids

+

+

2

Alkaloids

+

+

3

Essential oil

+

+

4

Sesquiterpene lactones

-

+

5

Phenolic compounds

+

+

6

Starch

-

-

Note: «-» negative reaction;

«+» positive reaction


 

 

 

Leaf fragment, stained with iron (III) chloride

Fragment of a cross section of a stem, stained with iron (III) chloride

 

 

Leaf fragment, staining with Dragendorff's reagent

Fragment of a cross section of a stem, staining with Dragendorff's reagent

 

 

Leaf fragment, staining with methylene blue solution

Fragment of a cross section of a stem, staining with methylene blue solution

 

 

Leaf fragment, staining with a solution of vanillin in sulfuric acid

Fragment of a cross section of a stem, staining with a solution of vanillin in sulfuric acid

 

 

Leaf fragment, staining with Lugol's solution

Fragment of a cross section of a stem, staining with Lugol's solution

 

 

Leaf fragment, staining with potassium bichromate solution

Fragment of a cross section of a stem, staining with potassium bichromate solution

Figure 2. Photographs of microslides of Linaria vulgaris Mill. with different types of histochemical staining

 


On a cross section of the stem, intense staining was noted for the epidermis, collenchyma cells, and xylem elements; less intense staining was observed for the cells of the cortex and pithy parenchyma.

 

The use of Dragendorff's reagent made it possible to establish trace amounts of alkaloids with characteristic staining on a cross section of a leaf of xylem elements; on a cross section of the stem - bark parenchyma and xylem ring.

The essential oil was established for leaf epidermal cells and columnar mesophyll, as well as for the cortical parenchyma of the stem.

 

Sequiterpene lactones were not identified in the epidermal cells of leaves or in stem tissues.

 

The use of Lugol's solution did not show characteristic staining, which confirms the absence of starch in the leaf and stem tissues of this plant.

 

Using a solution of potassium dichromate, staining of leaf mesophyll tissue and conducting bundles was obtained on a cross section of the stem, as well as epidermal tissue, cortical and pithy parenchyma.

 

CONCLUSION:

Thus, based on the results of a histochemical study, the localization of flavonoids, phenolic compounds, trace indicators for the presence of essential oil, alkaloids and sesquiterpene lactones in the leaf and stem tissues was revealed. The studies carried out did not reveal the accumulation of starch.

 

The results of the study can be an addition to confirming the authenticity of the raw materials and will be included in the set of regulatory documents for medicinal plant raw materials of Linaria vulgaris MILL.

 

CONFLICT OF INTEREST:

The authors declare no conflict of interest.

 

ACKNOWLEDGMENTS:

The authors express their gratitude to the management and staff of the NJSC E.A. Buketov Karaganda University, Department of Biotechnology and Ecomonitoring for the opportunity to carry out histochemical studies.

 

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18.   Dipsundar Sahu, Rajesh Bolleddu, Manosi Das, Debajyoti Das, Tusar Kanti Mandal, Saroj Kumar Debnath, Laxmidhar Barik, Jyoti Dahiya, Suvendu Mandal, P V V Prasad. Pharmacognostical and Phytochemical Studies of Leaves of Plectranthus amboinicus (Lour.) Spreng. (Parnayavani). // Research Journal of Pharmacy and Technology. 2022; 15(2): 717-2. DOI: 10.52711/0974-360X.2022.00119.

19.   Prakash Chandra Gupta, Ashish Kar, Nisha Sharma, Nikunj Sethi, Prashant Pandey, Dipankar Saharia, Naba Kumar Goswami. Pharmacognostic and Physicochemical Studies of Garcinia lanceifolia Roxb. Var. Oxyphylla. // Research J. Pharm. and Tech 2019; 12(2): P.706-710. DOI: 10.5958/0974-360X.2019.00125.2.

 

 

 

Received on 22.02.2024            Modified on 06.05.2024

Accepted on 17.06.2024           © RJPT All right reserved

Research J. Pharm. and Tech 2024; 17(9):4477-4480.

DOI: 10.52711/0974-360X.2024.00692