Histochemical Analysis of Lophanthus schrenkii Above ground Parts growing in Central Kazakhstan
Ishmuratova M.Yu.1, Ramazanova А.2*, Atazhanova G.А.1,2, Smagulov M.K.1,
Kurmantayeva G. K.2, Khamidkyzy А.2
1NC JSC “Karaganda University named after E.A. Buketov”,
100028, Kazakhstan, Karaganda, Universitetskaya Str. 28, Campus 3.
2NC JSC “Karaganda Medical University”, Gogol Str. 40, 100000, Karaganda, Kazakhstan.
*Corresponding Author E-mail: sabievaa@qmu.kz
ABSTRACT:
Histochemical analysis of medicinal plants and raw materials is aimed at studying the localization of various chemical compounds and their metabolic products within plant tissues. This article presents the findings of a histochemical study of the leaves, inflorescences, and stems of Lophanthus shrenkii. Lophanthus shrenkii Levin (synonym: Nepeta schrenkii (Levin) Sennikov), a member of the Lamiaceae Lindl. family, is of particular interest as a potential source of medicinal preparations. This species has a relatively limited geographic range, occurring in Kazakhstan, Kyrgyzstan, and the Xinjiang Uygur Autonomous Region of China. In Kazakhstan, its natural habitat includes the Karkaraly and Bektauata Mountains, the Saur-Tarbagatai range, the Dzungarian and Kyrgyz Alatau, the Karatau, and the Western Tien Shan. In traditional medicine, Lophanthus shrenkii is used to treat respiratory conditions, reduce fever, combat fatigue (asthenia), and improve male potency. Research on various Lophanthus species has demonstrated a range of pharmacological effects, including antibacterial, antitussive, antidiarrheal, antioxidant, anticancer, anti-inflammatory, antidiabetic, and sedative activities. Objective. To determine the localization features of specific groups of secondary metabolites in the aerial parts of Lophanthus shrenkii growing in Central Kazakhstan at the microscopic level. Materials and Methods. Cross-sections of the stems and inflorescences of Lophanthus shrenkii were fixed in Strauss-Fleming solution, after which histochemical reactions were performed to determine the location and identify essential oils, phenolic acids, flavonoids, sesquiterpene lactones, polysaccharides, and alkaloids.Microscopic features and histochemical tests were studied according to the methods of the State Pharmacopoeia of the Republic of Kazakhstan using a binocular microscope NINGBO SUNNY Instruments Co EX30 with a digital camera Altami 8.5 Mpix. During photo processing, Altami Studio software was used. Results. Histochemical methods using light microscopy were employed to determine the localization of biologically active substances in the studied plant material.The presence of phenolic acids, flavonoids, and essential oil was confirmed. The absence of sesquiterpene lactones, polysaccharides, and alkaloids in the studied plant material was established. Conclusion. For the first time, the localization of secondary metabolites in the tissues of Lophanthus shrenkii was studied using histochemical tests.The results of histochemical studies can be used to confirm the authenticity, identification, and standardization of the aerial parts of Lophanthus shrenkii.
KEYWORDS: Lophanthus shrenkii, Histochemical analysis, Plant structure, Secondary metabolites, Dragonhead, Plant, Essential oil, Flavonoids, Epidermis, Trichomes, Glands.
INTRODUCTION:
Despite significant advances in the chemistry of artificial synthesis of organic medicinal substances, plant-based preparations still occupy a significant share in the treatment and prevention of many diseases. Many herbal remedies have advantages over synthetic drugs. They rarely cause allergic reactions, do not lead to the development of resistance in macro- and microorganisms, are low in toxicity, and are well tolerated by children. Many medicinal plants used in traditional domestic medicine no longer form industrial-scale populations, and some are considered rare or endangered1-3. The dynamics of environmental degradation and anthropogenic impact on natural phytocenoses of wild medicinal plant species are also catastrophically reducing their natural reserves. Therefore, one of the important directions in medical and pharmaceutical science is the search for new sources of biologically active compounds to further expand the production of phytopharmaceuticals4,5.
To verify the authenticity of medicinal plant materials, a variety of research methods are employed, including macroscopic, microscopic, chemical, physicochemical, and others. Each of these approaches provides unique and valuable information. Macroscopic analysis focuses on observable characteristics such as size, color, smell, and taste. Microscopic analysis examines the internal anatomical structure of the plant, helping to identify specific diagnostic features. Chemical and physicochemical methods assess the presence and composition of active and auxiliary substances, aiding in their identification. A key aspect of chemical research is histochemical analysis, which involves detecting and localizing biologically active compounds within plant tissues and organs. These studies are typically carried out on sections of fresh or specially prepared plant material6.
The basis of histochemical studies lies in the reactions between the substance being studied and a specifically selected reagent. The result of such an interaction is the formation of a colored or fluorescent complex.The distribution of color (fluorescence) in the preparation can be used to judge the localization, while the intensity indicates the quantitative presence of the substance of interest in the tissues and cells7.
Lophanthus shrenkii Levin is not a pharmacopoeial plant, and information about its macroscopic and microscopic characteristics, as well as the presence of biologically active substances, is unknown and not found in available literature. Therefore, the study of this plant is a relevant task for pharmaceutical science and determining the prospects of using various parts of the plant in medical practice8-10.
Lophanthus shrenkii belongs to the Lamiaceae family (mint family). This plant is widely distributed in South and East Asia and is actively used in traditional medicine. Lophanthus contains a complex of biologically active substances such as essential oils, flavonoids, saponins, coumarins, as well as traces of alkaloids, which determine its medicinal properties11.
The plant is used for the treatment of kidney inflammations, gastritis, hepatitis, and other gastrointestinal diseases. Essential oils possess antiseptic, anti-inflammatory, and antioxidant properties. Flavonoids and saponins improve metabolism and support the immune system. Coumarins are known for their anticoagulant properties12.
MATERIALS AND METHODS:
Plant Material.The object of the study was the aerial parts (leaves, inflorescences, and stems) of Lophanthus shrenkii. The plant material was collected during the flowering phase in the Kent Mountains (Karaganda region) in the 1st decade of June 2024 (Fig. 1).
Figure 1. Lophanthus shrenkii in the flowering phase
The species identification was confirmed by specialists of the Department of Botany of the Faculty of Biology and Geography at the Non-Profit Joint Stock Company ‘Karagandy University named after academician E.A. Buketov’. The type specimen is kept in the herbarium collection of the faculty.
Histochemical study. The dried plant material was soaked in a mixture of 96% ethyl alcohol, distilled water, and 40% aqueous glycerin (in a ratio of 1:1:1, Strauss-Fleming reagent). Transverse sections and surface preparations were made manually using a razor blade 13–15. For the stem, the middle part of the shoot was used; for the leaf, a section with the central vein; and for the petiole, the central part. The obtained micropreparations were examined using a NINGBO SUNNY Instruments Co EX30 binocular microscope with an Altami 8.5 MP digital camera. Altami Studio software was used for processing the photographs16.
When describing the anatomical structure, the principles outlined in the works of L.I. Lotova17, P.J. Rudall18.
Histochemical tests were conducted according to the methods outlined in the State Pharmacopoeia of the Republic of Kazakhstan19.
RESULTS AND DISCUSSION:
The histochemical study of the plant material was conducted using reagents that can give colored reactions for specific groups of biologically active substances (Table 1).
As a result of the study, characteristic staining of cells of different types was observed, which is the result of the interaction between reagents and the detectable metabolites. The results of the histochemical analysis in the aerial organs of Lophanthus shrenkii are presented in Table 1 and Figure 2.
Table 1. Histochemical reactions for groups of biologically active substances in transverse sections of the stem and leaf of Lophanthus shrenkii
|
Identified component |
Reagent |
Staining |
Presence of characteristic coloring |
|
|
stem |
leaf |
|||
|
Essential oil |
Methylene blue |
Blue |
+ |
+ |
|
Flavonoids |
1% - alcoholic solution of FeCl3 |
Black-blue-green |
+ |
+ |
|
Phenolic compounds |
10% alcoholic solution of K2Cr2O7 |
Brown, yellow |
+ |
+ |
|
Sesquiterpenes |
Vanillin solution in sulfuric acid |
Red-violet |
- |
- |
|
Starch |
Lugol’s reagent |
Blue |
- |
- |
|
Alkaloids |
Dragendorff’s reagent |
Black |
- |
- |
”+” - positive reaction, “-” - negative reaction
The results of staining with methylene blue showed that essential oils are widely present in the tissues of this species (Figure 2). A more intense staining indicates the localization of essential oils in the epidermis of the leaf and stem, the cortex and medullary parenchyma, while less intense staining was observed in the collenchyma and vascular bundles. The mesophyll of the leaf showed almost no characteristic staining.
When stained with an iron chloride solution, intense staining of the epidermis, angular collenchyma, and vascular bundles of the stem was observed, while the cells of the cortex and medullary parenchyma stained less intensely, indicating the presence of flavonoids. In the leaf tissues, the epidermis, mesophyll, and vascular bundles were stained.
Phenolic compounds were detected in the epidermal tissues, chlorenchyma, phloem, and xylem vessels of the stem. For the cross section of the leaf, the presence of phenolic compounds was noted in the trichomes, vascular bundles, and mesophyll.
Vanillin solution in sulfuric acid, Dragendorff’s reagent, and Lugol’s reagent did not result in characteristic staining, indicating the absence of starch, alkaloids, and sesquiterpene lactones in the tissues of this species.
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Fragment of a cross-section of the stem, staining – methylene blue.
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Fragment of a cross-section of the leaf, staining – methylene blue. |
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Fragment of a cross-section of the stem, staining – iron chloride. |
Fragment of a cross-section of the leaf, staining – iron chloride. |
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Fragment of a cross-section of the stem, staining – potassium dichromate. |
Fragment of a cross-section of the leaf, staining – potassium dichromate. |
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Fragment of a cross-section of the stem, staining – vanillin solution in sulfuric acid. |
Fragment of a cross-section of the leaf, staining – vanillin solution in sulfuric acid. |
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Fragment of a cross-section of the stem, staining – Dragendorff’s reagent. |
Fragment of a cross-section of the leaf, staining – Dragendorff’s reagent. |
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Fragment of a cross-section of the stem, staining – Lugol’s reagent. |
Fragment of a cross-section of the leaf, staining – Lugol’s reagent. |
Figure 2. Results of histochemical staining of the stem and leaf tissue sections of Lophanthus shrenkii
CONCLUSION:
For the first time, the above-ground organs of Lophanthus shrenkii were studied using light microscopy in combination with histochemical tests. As a result of the conducted histochemical tests on cross-sections of leaves, stems of Lophanthus shrenkii, essential oils, phenolic acids, flavonoids were identified, and their localization was determined.
ACKNOWLEDGMENTS:
The research was conducted within the framework of the program of the Science Committee of the Ministry of Science and Higher Education of the Republic of Kazakhstan – No. BR 28712367.
REFERENCES:
1. Sabiyeva Assel, Gayane Atazhanova, Zholdasbayev Mussa, Ashirbekova Bibigul, Aigul Medeshova, Kurmantayeva Gulnissa, Zhanel Turdiyeva, Sholpan Sarsembayeva, Ainur Ibraybekova, Mukasheva Gulnar. Development of Composition and Technology of Anti-microbial Gel with Dracocephalum nutans l. Essential Oil Growing in the Territory of Central Kazakhstan. Research Journal of Pharmacy and Technology. 2025; 18(5): 2343-8. doi: 10.52711/0974-360X.2025.00335
2. Gayane Atazhanova, Assel Sabiyeva, Saule Akhmetova, Marlen Smagulov, Aigul Medeshova, Sholpan Sarsembayeva, Aiman Sarsembayeva, Ulpan Aldabayeva, Gulnissa Kurmantayeva. Component Composition and Antimicrobial Activity of Dracocephalum nutans L. Essential Oil. Research Journal of Pharmacy and Technology 2023; 16(10): 4811-5. doi: 10.52711/0974-360X.2023.00791
3. Weremczuk-Jezyna I., Lisiecki P., Gonciarz W., Ku´zma Ł., Szemraj M., Chmiela M., and Grzegorczyk-Karolak I. Transformed Shoots of Dracocephalum forrestii W.W. Smith from Different Bioreactor Systems as a Rich Source of Natural Phenolic Compounds. Molecules. 2020; 25: Article ID 4533; doi: 10.3390/molecules25194533
4. Behnam A., Parvin R., Behrouz E., Talei, Reza G. Investigation on chemical composition, antimicrobial, antioxidant, and cytotoxic properties of essential oil from dracocephalum kotschyi check for this species in other resources BOISS. Ashrafi, Behnam; Ramak, Parvin; Ezatpour, Behrouz and Talei, Gholam Reza. Afr J Tradit Complement Altern Med. 2017; 14(3): 209-217 doi: 10.21010/ajtcam.v14i3.23
5. Flora of Kazakhstan. Vol. 7. Alma-Ata: Nauka, 1964. pp. 328.
6. Sabiyeva A. et al. Anatomical study of Dracocephalum ruyschiana L. and Dracocephalum nutans L. Research Journal of Pharmacy and Technology. 2023; 16(3): 1193-1198.
7. Ishmuratova М. Y. et al. Analysis of representatives of Lamiaceae family in the flora of the Central Kazakhstan. Bulletin of the Karaganda University. Biology. Medicine. Geographyseries. 2020; 98(2): 37-44.
8. Levaya Ya. K., Ishmuratova M. Yu., Atazhanova G. A., Zilfikarov I. N., Loseva I. V. Microscopic Evaluation of Salvia stepposa Des.- Schost above - ground part widespread in Kazakhstan. Research Journal of Pharmacy and Technology. 2021; 14(9): 4773-6. doi: 10.52711/0974-360X.2021.00830
9. Asadullaeva Z.M., Ishmuratova M. Yu., Atazhanova G.A., Smagulov M.K.. The macroscopic, microscopic and histochemical analyses of aerial parts of Achillea salicifolia growing in the Republic of Kazakhstan. Research Journal of Pharmacy and Technology. 2024; 17(11): 5463-8. doi: 10.52711/0974-360X.2024.00836
10. Dirmenci T. et al. Lophanthus (Lamiaceae) in Turkey: a new generic record and a new species. Turkish Journal of Botany. 2010; 34(2); 123-129.
11. Ishmuratova М. Y. et al. Analysis of representatives of Lamiaceae family in the flora of the Central Kazakhstan //Bulletin of the Karaganda University “Biology medicine geography Series”. 2020; 98(2): 37-44.
12. Butumbayeva M. K., Silant’eva M. M. Evaluation of successful introduction of plants from Lamiaceaefamily in the conditions of the Karaganda and Zhezkazgan cities. Bulletin of the Karaganda University “Biology medicine geography Series”. 2020; 100(4); 16-22.
13. Sabiyeva A., Ishmuratova M. Yu., Atazhanova G. A., Smagulov M. K., Zhuravel I. A. Histochemical Analysis of Aerial part of Dracocephalum ruyschiana L. and Dracocephalum nutans L. growing in the Territory of Central Kazakhstan. Research Journal of Pharmacy and Technology. 2022; 15(9): 3831-5. doi: 10.52711/0974-360X.2022.00642
14. Kenzhebek B. N., Ishmuratova M. Yu., Atazhanova G. A., Smagulov M. K.. The Pharmacognostic Research of Ziziphora clinopodioides Raw materials. Research Journal of Pharmacy and Technology. 2024; 17(11): 5254-0. doi: 10.52711/0974-360X.2024.00804
15. Kurmantayeva G.K., Ishmuratova M.Yu., Atazhanova G.A., Smagulov M.K., Sabiyeva A., Medeshova A.T., Makhmutova A.S.. Morphological and Anatomical Study of Aerial organs of Nepeta pannonica L.. Research Journal of Pharmacy and Technology. 2024; 17(11): 5311-6. doi: 10.52711/0974-360X.2024.00813
16. 16.Turdiyeva Zh. A., Ishmuratova M. Yu., Atazhanova G. A., Ramazanova A. Histochemical Analysis of The Aerial part of Ferula songarica growing in the Territory of the Karaganda Region (Central Kazakhstan). Research Journal of Pharmacy and Technology. 2023; 16(11): 5079-4. doi: 10.52711/0974-360X.2023.00823
17. Lotova L.I. Botany. Morphology and anatomy of higher plants. Moscow: KomKniga. 2007. 512 pp.
18. Rudall P.J. Anatomy of flowering plants. Cambridge: Cambridge University Press, 2007. - 159.
19. The State Pharmacopeia of Republic of Kazakhstan, Vol. 1-3. Astana, 2008-2015.
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Received on 18.04.2025 Revised on 15.08.2025 Accepted on 23.11.2025 Published on 20.05.2026 Available online from May 25, 2026 Research J. Pharmacy and Technology. 2026;19(5):2019-2023. DOI: 10.52711/0974-360X.2026.00289 © RJPT All right reserved
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