Study of the anatomical structure of the aboveground vegetative organs of hybrid clover (Trifolium hybridum L.)

The genus of Clover (Trifolium L.) of Fabaceae family is numerous; it has up to 250 species. In Central Russia and Kazakhstan, one of the fairly common species of this genus is hybrid clover (pink clover) - Trifolium hybridum L. This species is widely used in traditional medicine around the world as an anti-inflammatory, antiseptic, wound healing, sedative, analgesic, expectorant, diuretic. The antibacterial and antioxidant activity of the extract has been experimentally established. Hybrid clover is not included in the State Pharmacopoeia of Russia and Kazakhstan; the pharmacopoeia article for medicinal plant raw materials has not been approved. The possibility of using a new species in scientific medicine makes it necessary to study the anatomical structure of hybrid clover. Objective - the study of the anatomical structure of the aboveground vegetative organs of hybrid clover and the identification of microdiagnostic signs of medicinal plant raw materials. Materials and methods. The study of anatomical features of aboveground vegetative organs (stem, leaf) of hybrid clover was carried out in accordance with the articles of the State Pharmacopoeia of the Russian Federation of the XIV edition, as well as the State Pharmacopoeia of the Republic of Kazakhstan. Freshly harvested, dried and fixed raw materials were used for the study. Results. Microdiagnostic signs of aboveground vegetative organs (stem, leaf) of hybrid clover have been described. Conclusion. Microdiagnostic signs have been established that allow for reliable identification of aboveground vegetative organs (stem and leaf) of hybrid clover from other representatives of the Clover genus having a similar structure. The results obtained can be used in the preparation of regulatory documents (pharmacopoeia article) under the section "Authenticity" ("Microscopic signs") for a new type of medicinal plant raw material of hybrid clover.

1. Mayevskiy P.F. Flora of the middle zone of the European part of Russia. Moscow: Tovarishchestvo nauchnykh izdaniy KMK, 2014. 635 p. (in Russ.)

2. Budantsev A.L., editor. Plant resources of Russia: Wild flowering plants, their component composition and biological activity. Vol. 3. Families of Fabaceae-Apiaceae. Saint-Petersburg, Mosow: Tovarishchestvo nauchnykh izdaniy KMK, 2010. 601 p. (in Russ.)

3. Kiseleva K.V., Mayorov S.R., Novikov V.S. Flora of Russia middle zone. Moscow: ZAO «Fiton+», 2010. 544 p. (in Russ.)

4. Peskova I.M. Herbaceous plants of the central part of Russia. Moscow: Fiton XXI, 2023. 448 p. (in Russ.)

5. Gripas′ M.N., Arzamasova E.G., Popova E.V., Onuchina O.L. A new variety of hybrid Flamingo clover. Agrarnaya nauka Yevro-Severo-Vostoka, 2016;52(3):10-14. (in Russ.). EDN: VWPDNF.

6. Spaner D., Todd A.G. The impact of underseeding barley (Hordeum vulgare L.) on timothy (Phleum pratense L.)-clover (Trifolium pratense L.; Trifolium hybridum L.) forage production in a cool maritime climate. Journal of Agronomy and Crop Science. 2003;189(5):273-279.

7. British Herbal Pharmacopoeia 1996. Published by British Herbal Medicine Association, 1996. 212 p.

8. Oleszek W., Stochmal A. Triterpene saponins and flavonoids in the seeds of Trifolium species. Phytochemistry. 2002;61(2):165-170.

9. Honkanen, E., Moisio, T., Karvonen, P. Studies on the volatile flavour substances in some clover species. Suomen Kemistilehti. 1969;42(12):448-451.

10. Mestechkina N.M., Anulov O.V., Smirnova N.I., Shcherbukhin V.D. Study of galactomannan from seeds of Trifolium hybridum L. Applied Biochemistry and Microbiology. 1996;32(6):590-592.

11. Wu Q., Wang M., Simon J.E. Determination of isoflavones in red clover and related species by high-performance liquid chromatography combined with ultraviolet and mass spectrometric detection. Journal of Chromatography A. 2003;1016(2):195-209. DOI: 10.1016/j.chroma.2003.08.001.

12. Andreyeva V.Yu., Kalinkina G.I., Poluektova T.V., Gulyayeva V.A.Comparative study of phenolic compounds of species of the genus clover (Trifolium L.) of the flora of Siberia. Khimiya rastitel′nogo syr′ya. 2018;(1): 97-104 (in Russ.). DOI: 10.14258/jcprm.2018011846. EDN: YRVTLT.

13. Renda G., Yalçın F.N., Nemutlu E., Akkol E.K., Süntar I., Keleş H., Ina H., Çalış I., Ersöz T.Comparative assessment of dermal wound healing potentials of various Trifolium L. extracts and determination of their isoflavone contents as potential active ingredients. Journal of Ethnopharmacology. 2013;148(2):423-432. DOI: 10.1016/j.jep.2013.04.031.

14. Kolodziejczyk-czepas J., Nowak P., Kowalska I., Stochmal A. Antioxidant action of six Trifolium species in blood platelet experimental system in vitro. Molecular and Cellular Biochemistry. 2015;410(1): 229-237. DOI: 10.1007/s11010-015-2556-2.

15. Drozdova I.L., Trembalya Ya.S., Minakova E.I. The study of morphological and anatomical structure of Lunaria annua L. flowers of the family Brassicaceae. Meditsinskiy vestnik Bashkortostana. 2018; 13(3-75):58-61 (in Russ.). EDN: XYLGUH

16. Trembalya Ya.S., Drozdova I.L., Kuzkina E.E. Anatomical structure of vegetative organs of wood geranium (Geranium Sylvaticum L.). Mezhdunarodnyy nauchno-issledovatel’skiy zhurnal. 2018;1-1(67):176-180 (in Russ.). DOI: 10.23670/IRJ.2018.67.109. EDN: YNVWWE.

17. Fedotova V.V., Konovalov D.A. Anatomical characteristics of leaves and flowers of Cornus sanguine subsp. australis (C.A. Mey.) Jav. Pharmacognosy Journal. 2020;12(3):448-451. DOI: 10.5530/pj.2020.12.69.

18. Fedotova V.V., Konovalov D.A., Kruglaya A.A. Anatomical study of Pachyphragma macrophyllum (Hoffm.) N. Busch. Pharmacognosy Journal. 2019;11(6s):1587-1590. DOI: 10.5530/pj.2019.11.242.

19. Serebryanaya F.K., Konovalova I.D. Microscopical investigation of Sideritis taurica. Borneo Journal of Pharmacy. 2020;3(3):162-169. DOI: 10.33084/bjop.v3i3.1524.

20. Serebryanaya F.K., Nasuhova N.M., Konovalov D.A. Morphological and anatomical study of the leaves of Laurus nobilis L. (Lauraceae), growing in the introduction of the Northern Caucasus Region (Russia). Pharmacognosy Journal. 2017;9(4):519-522. DOI: 10.5530/pj.2017.4.83.

21. Serebryanaya F.K., Orlov A.N., Konovalov D.A., Nasuhova N.M.Comparative morphological and anatomical research of leaves 6 sorts of laurels noble (Laurus nobilis L.), growing in the conditions of an introduction in the Nikitsky Botanical Garden. Pharmacognosy Journal. 2018;10(1):172-178. DOI: 10.5530/pj.2018.1.29.

22. State Pharmacopoeia of the Russian Federation. XIV edition. Vol. II. [Electronic resource]. Federal’naya elektronnaya meditsinskaya biblioteka (in Russ.). URL: http://femb.ru/femb/pharmacopea.php

23. State Pharmacopoeia of the Republic of Kazakhstan. Vol.1. Almaty: Izdatel’skiy dom «ZHibek zholy», 2008. 592 p. (in Russ.)

24. Samylina I.A., Potanina O.G. Pharmacognosy. Atlas in 3 volumes. Volume 1. The general part. Terms and techniques of microscopic analysis in pharmacognosy. Moscow: GEOTAR-Media, 2022. 192 p. (in Russ.)

25. Belashova O.V.Comparative morphological and anatomical study of the stem of the genus Trifolium L. Mir nauki, kul′tury, obrazovaniya. 2011:31(6-2):402-404 (in Russ.). EDN: PHHSRZ.

26. Drozdova I.L., Trembalya Ya.S., Minakova E.I. Morphological and anatomical study of Genista tinctoria flowers, us ed in traditional medicine in Russia. Traditsionnaya meditsina. 2018;1(52):33-37 (in Russ.). EDN: YWPKMA.

27. Serebryanaya F.K., Sharenko O.M., Serkov M.A. Morphological and anatomical investigations of Vicia truncatula Fish. ex Bieb. Pharmacognosy Journal. 2015;7(3):205-209. DOI:10.5530/PJ.2015.3.10.