Histochemical Analysis of Aerial part of Dracocephalum ruyschiana L. and Dracocephalum nutans L. growing in the Territory of Central Kazakhstan

Sabiyeva A.¹, Ishmuratova M. Yu.², Atazhanova G. A.¹, Smagulov M. K.², Zhuravel I. A.³

¹NCJSC “Karaganda Medical University”, 100000, Kazakhstan, Karaganda, Gogol Str. 40.

²NCJSC “Karaganda University named after E.A. Buketov”, 100028, Kazakhstan,

Karaganda, Universitetskaya Str 28, Block 3.

³National University of Pharmacy, 61168, Kharkov, Valentinivska Str. 4, Block 4.

*Corresponding Author E-mail: aseka9520@mail.ru

ABSTRACT:

Histochemical analysis of medicinal plants and raw materials is intended to be used to study the localization of various chemical substances and products of their metabolism in tissues. The article presents the results of histochemical analysis of leaves, flowers and stems of Dracocephalum ruyschiana L. and Dracocephalum nutans L. The genus Dracocephalum L. belongs to the family Lamiaceae Lindl. (Labiatae), whose plants are of interest as sources for medicinal preparations. These two species of Dracocephalum are related to Dracocephalum moldavica L., used in folk medicine for respiratory diseases, as an antipyretic agent, for asthenia, as a potency-increasing agent. Studies have shown that some species of Dracocephalum have antibacterial, antitussive, antidiarrheal, antioxidant, anti-cancer, anti-inflammatory, anti-diabetic and sedative properties. To determine the localization features of certain groups of secondary metabolites in the aerial part of Dracocephalum ruyschiana L. and Dracocephalum nutans L. growing in the territory of Central Kazakhstan at the microscopic level. Materials and methods. Cross-sections of stems, flowers, and D. ruyschiana and D. nutans were fixed in Strauss-Fleming solution, after which histochemical reactions were carried out to locate and identify essential oils, phenolic acids, flavonoids, sesquiterpene lactones, polysaccharides and alkaloids. The study of microscopic features, as well as histochemical tests were carried out according to the methods of the State Pharmacopoeia of the Republic of Kazakhstan using a light microscope Biomed-4. Results and Discussion. The localization of biologically active substances in the studied plant raw materials was determined by histochemical methods using light microscopy. The presence of phenolic acids, flavonoids and essential oil was established. The absence of sesquiterpene lactones, polysaccharides and alkaloids in the studied raw materials was established. Conclusion. The localization of secondary metabolites was studied for the first time by histochemical tests in the tissues of D. ruyschiana and D. nutans. The results of histochemical studies can be used to confirm the authenticity, identification and standardization of the aerial parts of D. ruyschiana and D. nutans.

KEYWORDS: Dracocephalum, histochemical analysis, plant structure, secondary metabolites, plant, essential oil, flavonioids, epidermis, trichomes, glands.

INTRODUCTION:

Establishing the authenticity of medicinal raw materials is the most important task for researchers. Today, there are many research methods that make it possible to assess the belonging of medicinal plants to a particular species, as well as to establish the localization of secondary metabolites. Such methods include histochemical analysis, which makes it possible to identify the presence and placement of biologically active substances in tissues and organs. Medicinal plants contain many secondary metabolites, such as alkaloids, tannins, phenolic substances, flavonoids, polysaccharides, etc. These secondary metabolites are in great demand in the pharmaceutical industry and have a variety of pharmacological activities^1-5.

Histochemical studies are based on the reactions between the test substance and a specially selected reagent. The result of this interaction is the formation of a colored or fluorescent complex. By the distribution of color (fluorescence) in the preparation, one can judge about the localization, and by the intensity - about the quantitative presence of the substance of interest in the tissues and cells^6,7.

Among them, the most famous are plants of the genus Dracocephalum, which are a rich and very widespread source of essential oils, and are widely used in folk medicine in many countries as a medicinal raw materials. According to The Plant List database (as of August 2016), the genus includes 74 species, 20 species of which grow in Kazakhstan⁸.

D. ruyschiana and D. nutans are used in folk medicine, however, they have prospects for inclusion in the State Pharmacopoeia of the Republic of Kazakhstan (SP RK)⁹, which requires pharmacognostic analysis, one of the elements of which is histochemical study.

As objects, we studied D. ruyschiana and D. nutans, which are widespread in the territory of the Republic of Kazakhstanand possess raw materials resources. In this regard, the study of the localization of biologically active substances of the aerial part of D.ruyschiana and D. nutans using histochemical study can become an important stage for the identification of raw materials and further research in the field of pharmacy and medicine^10-15.

Dracocephalum nutans L. contains a complex of biologically active substances and is used in the medicine of the peoples of Southeast Asia in the treatment of inflammation of the kidneys and gastrointestinal diseases such as hepatitis, gastritis, etc.

Dracocephalum ruyschiana L. is an essential oil bearing plant that is used in folk medicine. Medicinal properties are due to the content of biologically active substances in the aerial part of plants - cardenolides, alkaloids, tannins, coumarins, flavonoids. It is used in folk medicine for diseases of the respiratory channel, as an antipyretic agent, for asthenia, as a means that increases potency. D. ruyschiana nectar is active against gram-positive and gram-negative bacteria^16-21.

MATERIALS AND METHODS:

Raw materials:

The object of the study is the aerial parts of D. ruyschiana and D. nutans, collected during flowering phase, the collection site is the Karkaraly mountains (Karaganda region), July 2020 (Fig. 1).

Figure 1. D.ruyschiana (A) and D.nutans (B) during flowering

Histochemical study:

Freshly collected organs were fixed in a mixture of alcohol (70%), glycerin, and distilled water in a 1:1:1 ratio (Strauss-Fleming solution). Histochemical study was carried out for cross sections of the stem, leaves, petiole and whole flower²². To carry out histochemical analysis, we used the following reagents: methylene blue (essential oil); 10% solution of thymol and concentrated H₂SO₄ (polysaccharides), Lugol’s reagent (starch); vanillin solution in concentrated H₂SO₄ (sesquiterpene lactones); 10% alcoholic solution of K₂Cr₂O₇ (phenolic compounds); 1% alcohol solution FeCl₃ (flavonoids), Dragendorff’s reagent (alkaloids). A change in the color of certain tissues served as a sign of the localization of metabolite groups in tissues of both types. Microscopic photographs of cross sections of aerial organs were made using a Biomed-4 microscope with 10×, 20× eyepieces, 4×, 10×, 20×, 40× lenses. Photo processing was carried out in the Paint 10.1 program.

RESULTS AND DISCUSSION:

As a result of the study, a characteristic imblueing of cells of different types was revealed, which is characterized by the result of the interaction of reagents with detectable metabolites. The results of histochemical analysis in the aerial organs of D. ruyschiana and D. nutans are presented in Table 1 and in Figures 1-7.

Table 1. Histochemical analysis in the aerial parts of D. ruyschiana и D.nutans

The component being detected	Reagent	Imblueing	Type of aerial organs
			Stem		Leaf		Flower
			D. *ruyschiana*	D.nutans	D. *ruyschiana*	D. nutans	D. *ruyschiana*	D. nutans
Essential oil	Methylene blue	Blue	+	+	+	+	+	+
Sesquiterpene lactones	solution of vanillin in conc. H₂SO₄	Yellow	-	-	-	-	-	-
Flavonoids	1-% alcohol solution of FeCl₃	Black-blue-green	+	+	+	+	-	+
Phenolic compounds	10% alcohol solution of K₂Cr₂O₇	Brown, yellow	+	+	+	+	+	+
Polysaccharides	10% solution of thymol, conc. H₂SO₄	Orange-red	-	-	-	-	-	-
Starch	Lugol’s reagent	Blue	-	-	-	-	-	-
Alkaloids	Dragendorff’s reagent	Black	-	-	-	-	-	-

Note: – negative reaction; + positive reaction

А B C

Figure 2. The result of histochemical reactions with methylene blue (magnification ×10) for D. ruyschiana

А- cross section of a leaf, B- cross section of a stem, C-flower

А B C

Figure 3. The result of histochemical reactions with methylene blue (magnification ×10) for D. Nutans А-cross section of a leaf, B-cross section of a stem, C-flower

Identification of essential oil in aerial organs of D. ruyschiana.

Treatment of micropreparations of a leaf, stem and flower with a solution of methylene blue showed that a specific color appears. The main place of localization of essential oil is:

· In both species on the cross section of the leaf: conducting bundles, essential oil glands, trichomes, areas of collenchyma and cork parenchyma;

· In both species, on the surface preparation of flower petals, imblueing of essential oil glands and areas along the leaf veins was observed (Fig. 2-3).

Identification of phenolic acids in aerial organs:

After treatment of micropreparations of the studied samples with 1% alcohol solution of FeCl₃, an intense black-brown imblueing of areas of the stem, leaf and flower was observed (Fig. 4-5):

· On the cross section of the stem, the following structures were colored in both species: epidermis, chlorenchyma, cork parenchyma, vascular-conducting bundles (especially sclerenchyma and xylem);

· On the cross section of the leaf in both species: spongy and columnar mesophyll, conducting bundles and trichomes;

· In D. nutans in flower petals, imblueing was observed in the form of a diffuse distribution of flavonoids in the main cells, while in D. ruyschiana, imblueing was not observed.

Identification of phenolic acids in aerial organs.

To identify phenolic acids, the test material was placed in a 10% potassium dichromate solution and left for 7 days. The presence of phenolic acids was confirmed by intense yellow-brown staining in all studied organs of Dracocephalum, which indicates the presence of phenolic compounds in all cells. However, the staining of the micropreparations was uneven, which makes it possible to judge the varying degrees of accumulation of phenolic compounds in the cells. Thus, the areas with the maximum accumulation of phenolic compounds are the sclerenchyma and chlorenchyme of the leaf, the core and conductive zone of the stem, the conductive zone, and the mechanical tissues of the petiole (Fig. 6-7).

· On the cross section of the stem, the following structures were stained: core and conductive zone;

· On the cross section of the leaf: sclerenchyma, chlorenchyme, conducting zone and bundles;

· In the petals of the flower, staining is observed in the conductive zone of the petal.

А B

Figure 4. The result of histochemical reactions with a 1% alcohol solution of FeCl₃ (magnification ×10) for D. ruyschiana

А-cross section of a leaf, B-cross section of a stem

А B C

Figure 5. The result of histochemical reactions with a 1% alcohol solution of FeCl₃ (magnification ×10) for D. nutans

А-cross section of a leaf, B-cross section of a stem, C-flower

А B C

Figure 6. The result of histochemical reactions with a 10% alcoholic solution of potassium dichromate (magnification ×10) for D. ruyschiana

А- flower, B- cross section of a stem, c- cross section of a leaf

А B C

Figure 7. The result of histochemical reactions with a 10% alcoholic solution of potassium dichromate (magnification ×10) for D.nutans

А-cross section of a leaf, B-cross section of a stem, C-flower

The identification of sesquiterpene lactones, alkaloids, and polysaccharides was not accompanied by a imblueing effect, which indicates the absence of these compounds in the aerial organs of D.ruyschiana and D. nutans.

CONCLUSION:

For the first time, the study of the aerial organs of D. ruyschiana and D. nutans was carried out using the methods of light microscopy in combination with histochemical tests. As a result of histochemical tests, essential oils, phenolic acids, flavonoids were found on the cross sections of leaves, stems and a superficial section of flowers of D. ruyschiana and D. nutans were found, and their localization was also established:

· Essential oils - in essential oil glands and capitate hairs;

· Phenolic acids - in the sclerenchyme and chlorenchyme of the leaf, the cork and conducting zone of the stem, the conducting zone and the mechanical tissue of the petiole;

· Flavonoids - in the leaf mesophyll; angular collenchyma, epidermis, chlorenchyma and vascular-conducting bundles of the stem; chlorenchyme and vascular bundles of the petiole.

CONFLICT OF INTEREST:

The authors declare no conflict of interest.

ACKNOWLEDGMENTS:

The authors express their gratitude to the management and staff of the Department of Botany of NCJSC “Karaganda University named after E.A. Buketov” for the opportunity to carry out histochemical studies.

REFERENCES:

1. Rakesh Kumar Joshi. Chemical Constituents of Artemisia nilagirica (Clarke) from Western Himalaya of Uttrakhand, India. Asian J. Pharm. Ana. 2020; 10(4):182-184. doi: 10.5958/2231-5675.2020.00033.2

2. AK Meena, MM Rao, RP Meena, P Panda, Renu. Pharmacological and Phytochemical Evidences for the Plants of Wedelia Genus– A Review. Asian J. Pharm. Res. 1(1): Jan.-Mar. 2011; Page 07-12.

3. Sandeep B. Patil, Nilofar S. Naikwade, Chandrakant S. Magdum, Vikas B. Awale. Some Medicinal Plants Used By People of Sangli District, Maharashtra. Asian J. Pharm. Res. 1(2): April-June 2011; Page 42-43.

4. Mariyappan M., Bharathidasan R., Mahalingam R., Madhanraj P., Panneerselvam A. , Ambikapathy V.. Antibacterial Activity of Cardiospermum halicacabum and Melothria heterophylla. Asian J. Pharm. Res. 1(4): Oct. - Dec. 2011; Page 111-113.

5. Rita N. N. Rammo. Bactericidal and Anti-biofilm Formation of Aqueous Plant Extracts against Pathogenic Bacteria. Asian J. Pharm. Res. 2017; 7(1): 25-29. doi: 10.5958/2231-5691.2017.00005.3

6. P. Lalitha, V. Sachithanandam, N. S. Swarnakumar, R. Sridhar. Review on Anti-inflammatory Properties of Mangrove plants. Asian J. Pharm. Res. 2019; 9(4):273-288. doi: 10.5958/2231-5691.2019.00045.5

7. Narendra B. Patil, Ketan B. Patil, Sandip S. Kshirsagar. Medicinal plant is new Horizones in Spinocerebellar ataxia. Asian J. Pharm. Res. 2020; 10(1): 29-30. doi: 10.5958/2231-5691.2020.00006.4

8. The Plant List ,www.theplantlist.org

9. Flora of Kazakhstan. Vol. 7. – Alma-Ata: Publishing house of the Academy of Sciences of the Kazakh SSR; 1964.

10. Muhammad Hamza Ashfaq, Amna Siddique, Sammia Shahid. Antioxidant Activity of Cinnamon zeylanicum: (A Review). Asian Journal of Pharmaceutical Research. 2021; 11(2):106-6. doi: 10.52711/2231-5691.2021.00021

11. State Pharmacopoeia of the Republic of Kazakhstan. V. 2. - Almaty: Publishing House "Zhibek Zholy", 2009. - 792 p.

12. Sarmistha Rej, Madhurima Dutta, Shahid Jamal, Sumanta Das ,Sabyasachi Chatterjee. Study of Phytochemical Constituents and Antibacterial Activity of Clerodendrum infortunatum. Asian J. Res. Pharm. Sci. 4(4): Oct.-Dec. 2014; Page 187-195.

13. Olshanskaya L.N., Bakanova E.M., Yakovleva E.V. Histochemical studies of the localization of heavy metals in the tissues of higher plants in the process of phytoextraction. Proceedings of Universities. Chemistry and chem. Technology; 2016; 59 (5); 3-15.

14. State Pharmacopoeia of the Republic of Kazakhstan. V. 1. - Almaty: Zhibek Zholy Publishing House, 2008. - P. 563-564

15. Naeim H., El-Hawiet A., Raoufa A., Rahman A., Hussein A., Maha A. E Demellawy and Amira M. Embab. Antibacterial activity of Centaurea pumilio L. root and aerial part extracts against some multidrug resistant bacteria. Complementary Medicine and Therapies (2020) 20:79. https://doi.org/10.1186/s12906-020-2876-y

16. Khoshbakht T., Karami A., Tahmasebi A. and Maggi F. The Variability of Thymol and Carvacrol Contents Reveals the Level of Antibacterial Activity of the Essential Oils from Different Accessions of Oliveria decumbens. Antibiotics 2020; 9; 409; doi:10.3390/antibiotics9070409.

17. Silva de Jesus G., Micheletti Ana C., Gonçalves Padilha R., Jessica de Souza de Paula, Macedo Alves F., Rejane Brito Leal C., Rodrigues Garcez F., Silva Garcez W. and Cristiane Yoshida N. Antimicrobial Potential of Essential Oils from Cerrado Plants against Multidrug−Resistant Foodborne Microorganisms. Molecules; 2020; 25; 3296; doi:10.3390/molecules25143296.

18. Iseppi R., Tardugno R., Brighenti V., Benvenuti S., Sabia C., Federica Pellati and Patrizia Messi. Phytochemical Composition and In Vitro Antimicrobial Activity of Essential Oils from the Lamiaceae Family against Streptococcus agalactiae and Candida albicans Biofilms. Antibiotics; 2020; 9; 592; doi:10.3390/antibiotics9090592.

19. Kurmanova E.N., Ferubko E.V., Strelkova L.B., Kurmanov R.K., Sheichenko O.P. Study of the anti-inflammatory activity of Dracocephalum moldavica L. extract. Pathological physiology and experimental therapy; 2020; 64(1): 108-112.

20. Samylina, I.A.Pharmacognosy. Atlas. Volume 1; Samylina I.A., Anosova O.G. - Moscow: GEOTAR-Media; 2010. - ISBN 978-5-9704-1576-4.

21. Samylina, I.A.Pharmacognosy. Atlas. Volume 2; Samylina I.A., Anosova O.G. - Moscow: GEOTAR-Media; 2010. - ISBN 978-5-9704-1578-8.

22. Nikitina A.S., Popova O.I. Determination of the main morphological and anatomical diagnostic features of the Dracocephalum moldavica L. grass, cultivated in the conditions of the Stavropol Territory. Pyatigorsk state pharmacy academy. Development, research and marketing of new pharmaceutical products, 2006; 42-44.

Received on 07.07.2021 Modified on 13.11.2021

Research J. Pharm. and Tech 2022; 15(9):3831-3835.

DOI: 10.52711/0974-360X.2022.00642