Development of a new spectrophotometric method for the determination of flavonoids in herb of red clover (Trifolium pratense L) and zigzag clover (Trifolium medium L)
Main Article Content
Keywords
Clover herb, total flavonoid, gallium (III) nitrate, spectrophotometry
Abstract
Objective: Medicinal plants containing flavonoids are attracting increasing interest of researchers due to their complex therapeutic effects on the human body. Such medicinal plants include representatives of the genus Clover - red clover (Trifolium pratense L.) and zigzag clover (Trifolium medium L.). Methods: However, spectrophotometric techniques for the determination of the total flavonoids have numerous disadvantages, which prompted the development of a new analytical technique. As a result of our research, such a technique using gallium (III) nitrate as a staining reagent instead of the traditionally used aluminium chloride was developed, and a standard for recalculation (luteolin) was substantiated. Results: It has been shown that the new methodology is capable of giving satisfactory results, is characterised by excellent metrological characteristics, meets the requirements of SP RF XV ed. and the leading pharmacopoeias of the world (EP, USP, JP). Conclusion: The proposed methodology can be recommended for use not only for scientific purposes, but also in the field of standardisation of plant raw materials at pharmaceutical industries specialising in the production of medicinal preparations of plant origin.
References
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21. Al-Shami AS, Essawy AE, Elkader HE. Molecular mechanisms underlying the potential neuroprotective effects of Trifolium pratense and its phytoestrogen-isoflavones in neurodegenerative disorders. Phytother Res. 2023;37(6):2693-2737. doi:10.1002/ptr.7870
22. Błaszczuk A, Barańska A, Kanadys W, et al. Role of phytoestrogen-rich bioactive substances (Linum usitatissimum L., Glycine max L., Trifolium pratense L.) in cardiovascular disease prevention in postmenopausal women: a systematic review and meta-analysis. Nutrients. 2022;14(12):2467. doi:10.3390/nu14122467
23. Quah Y, Park NH, Lee EB, et al. Trifolium pratense ethanolic extract alters the gut microbiota composition and regulates serum lipid profile in the ovariectomized rats. BMC Complement Med Ther. 2022;22(1):5. doi:10.1186/s12906-021-03486-w
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25. Fedoseeva AA, Lebedkova OS, Kanibolotskaya LV. Antioxidant activity of tea infusions. Khimiya Rastitel’nogo Syr’ya. 2008;(3):123-127. (In Russian)
26. Sundaresan A, Radhiga T, Deivasigamani B. Biological Activity of Biochanin A: A Review. Asian J Pharm Pharmacol. 2018;4(1):1-5. DOI: 10.31024/ajpp.2018.4.1.1
27. Tay KC, Tan LTH, Chan CK, Hong SL, Chan KG, Yap WH, Pusparajah P, Lee LH, Goh BH. Formononetin: A review of its anticancer potentials and mechanisms. Front Pharmacol. 2019;10:820. doi:10.3389/fphar.2019.00820
28. Xiao H, Qin X, Wan J, Li R. Pharmacological Targets and the Biological Mechanisms of Formononetin for Alzheimer’s Disease: A Network Analysis. Med Sci Monit. 2019;25:4273-4277. doi:10.12659/MSM.916662
29. Jiang D, Rasul A, Batool R, Sarfraz I, Hussain G, Tahir MM, Quin T, Selamoglu Z, Ali M, Li J, Li X. Potential Anticancer Properties and Mechanisms of Action of Formononetin. Biomed Res Int. 2019;2019:11 p. doi:10.1155/2019/5854315
30. Zaprometov MN. Phenolic Compounds: Distribution, Metabolism and Functions in Plants. Moscow; 1993. 119 p. (In Russian)
31. Maciejewska-Turska M, Zgorka G. In-depth phytochemical and biological studies on potential AChE inhibitors in red and zigzag clover dry extracts using reversed–phase liquid chromatography (RP-LC) coupled with photodiode array (PDA) and electron spray ionization-quadrupole/time of flight-mass spectrometric (ESI-QToF/MS-MS) detection and thin-layer chromatography-bioautography. Food Chem. 2021;375:131846. doi:10.1016/j.foodchem.2021.131846
32. Dluhošová J, Řepková J, Jakešová J, Nedělník H. Impact of interspecific hybridization of T. pratense × T. medium and backcrossing on genetic variability of progeny. Genet Plant Breed. 2016;52:125-131. doi:10.17221/115/2016-CJGPB
33. Butkutė B, Lemežienė N, Padarauskas A, Norkevičienė E, Taujenis L. Chemical Composition of Zigzag Clover (Trifolium medium L.). In: Breeding Grasses and Protein Crops in the Era of Genomics. 2018:83-87. doi:10.1007/978-3-319-89578-9_15
34. Dmitrienko VA, Kudrinskaya VA, Apyari VV. Methods of isolation, condensation and determination of quercetin. Zhurn Analit Khimii. 2012;67(4):340-353. (In Russian). DOI:10.1134/S106193481204003X
35. Beketov EV, Abramov AA, Nesterova OV. Identification and quantitative assessment of flavonoids in bird cherry fruits. Vestn Mosk Univ Khimiya. 2005;46(4):259-262. (In Russian)
36. Belova OA, Kurkin VA, Egorov MV. Method for quantitative determination of the sum of flavonoids in Glycyrrhiza glabra herb. Farmatsiya i Farmakologiya. 2023;11(2):127-136. doi:10.19163/2307-9266-2023-11-2-127-136
37. State Pharmacopoeia of the Russian Federation XV Edition [Electronic Edition]. Available from: https://pharmacopoeia.regmed.ru/pharmacopoeia/izdanie-15/
38. Marakhova AI. Unification of physico-chemical methods for analysis of medicinal plant raw materials and complex preparations based on plant materials [Abstract of the dissertation for the degree of Doctor of Pharmaceutical Sciences]. Samara; 2017. 48 p. (In Russian)
2. Gounden T, Moodley R, Jonnalagadda SB. Elemental analysis and nutritional value of edible Trifolium (clover) species. J Environ Sci Health B. 2018;53(8):487-492. doi:10.1080/03601234.2018.1462923
3. Zhang H, Zhao J, Shang H, Guo Y, Chen S. Extraction, purification, hypoglycemic and antioxidant activities of red clover (Trifolium pratense L.) polysaccharides. Int J Biol Macromol. 2020;148:750-760. doi:10.1016/j.ijbiomac.2020.01.194
4. Kaurinovic B, Popovic M, Vlaisavljevic S, Schwartsova H, Vojinovic-Miloradov M. Antioxidant profile of Trifolium pratense L. Molecules. 2012;17(9):11156-11172. doi:10.3390/molecules170911156
5. Silveira D, Boylan F. Medicinal plants: advances in phytochemistry and ethnobotany. Plants (Basel). 2023;12(8):1682. doi:10.3390/plants12081682
6. Kolodziejczyk-Czepas J. Trifolium species-derived substances and extracts-biological activity and prospects for medicinal applications. J Ethnopharmacol. 2012;143(1):14-23. doi:10.1016/j.jep.2012.06.048
7. Krenn L, Paper DH. Inhibition of angiogenesis and inflammation by an extract of red clover (Trifolium pratense L.). Phytomedicine. 2009;16(12):1083-1088. doi:10.1016/j.phymed.2009.05.017
8. Malca-Garcia GR, Zagal D, Graham J, et al. Dynamics of the isoflavone metabolome of traditional preparations of Trifolium pratense L. J Ethnopharmacol. 2019;238:111865. doi:10.1016/j.jep.2019.111865
9. Syutkina AI. Clover herb as a promising source of medicinal plant raw materials. Young Scientist. 2015;11(91):719-721. (In Russian)
10. Chaudhary N, Tripathi S. A review on chemical and biological activity of Trifolium Pretense. PharmaTutor. 2014;2(3):93-101.
11. Bajaj M, Bahri S, Sinha R. Phytoestrogens of Trifolium pratense L. as therapeutics: A review. South Asian J Exp Biol. 2023;13:252-262. doi:10.38150/sajeb.13(4).p252-262
12. Myers S, Vigar V. Effects of a Standardised Extract of Trifolium Pratense (Promensil) at a Dosage of 80 Mg in the Treatment of Menopausal Hot Flushes: A Systematic Review and Meta-Analysis. Phytomedicine. 2017;24:141-147. doi:10.1016/j.phymed.2016.12.003
13. Les F, Cásedas G, López V. Bioactivity of Medicinal Plants and Extracts. Biology (Basel). 2021;10(7):63. doi.org/10.3390/biology10070634
14. Dyshlyuk L, Osintseva M, Kozlova O, Fotina N, Prosekov A. Antiradical and oxidative stress release properties of Trifolium pratense L. extract. J Exp Biol Agric Sci. 2022;10:852-860. doi:10.18006/2022.10(4).852.860
15. Renda G, Yalçın FN, Nemutlu E, et al. Comparative assessment of dermal wound healing potentials of various Trifolium L. extracts and determination of their isoflavone contents as potential active ingredients. J Ethnopharmacol. 2013;148(2):423-432. doi:10.1016/j.jep.2013.04.031
16. Kolodziejczyk-Czepas J. Trifolium species — the latest findings on chemical profile, ethnomedicinal use and pharmacological properties. J Pharm Pharmacol. 2016;68(7):845-861. doi:10.1111/jphp.12568
17. Lee JH, Dean M, Austin JR, et al. Irilone from red clover (Trifolium pratense) potentiates progesterone signaling. J Nat Prod. 2018;81(9):1962-1967. doi:10.1021/acs.jnatprod.8b00131
18. Lagari V, Levis S. Phytoestrogens for menopausal bone loss and climacteric symptoms. Steroids Biochem Mol Biol. 2014;139:294-301. doi:10.1016/j.jsbmb.2012.12.002
19. Lemežienė N, Padarauskas A, Butkutė B, Ceseviciene J, Taujenis L, Norkevičienė E. The concentration of isoflavones in red clover (Trifolium pratense L.) at flowering stage. Zemdirbyste. 2015;102(4):443-448. doi:10.13080/z-a.2015.102.057
20. Vlaisavljevic S, Kaurinovic B, Popovic M, et al. Trifolium pratense L. as a potential natural antioxidant. Molecules. 2014;19(1):713-725. doi:10.3390/molecules19010713
21. Al-Shami AS, Essawy AE, Elkader HE. Molecular mechanisms underlying the potential neuroprotective effects of Trifolium pratense and its phytoestrogen-isoflavones in neurodegenerative disorders. Phytother Res. 2023;37(6):2693-2737. doi:10.1002/ptr.7870
22. Błaszczuk A, Barańska A, Kanadys W, et al. Role of phytoestrogen-rich bioactive substances (Linum usitatissimum L., Glycine max L., Trifolium pratense L.) in cardiovascular disease prevention in postmenopausal women: a systematic review and meta-analysis. Nutrients. 2022;14(12):2467. doi:10.3390/nu14122467
23. Quah Y, Park NH, Lee EB, et al. Trifolium pratense ethanolic extract alters the gut microbiota composition and regulates serum lipid profile in the ovariectomized rats. BMC Complement Med Ther. 2022;22(1):5. doi:10.1186/s12906-021-03486-w
24. Konopleva MM. Farmakognoziya: prirodnye biologicheski aktivnye veshchestva [Pharmacognosy: natural biologically active substances]. Vitebsk; 2007. 273 p. (In Russian)
25. Fedoseeva AA, Lebedkova OS, Kanibolotskaya LV. Antioxidant activity of tea infusions. Khimiya Rastitel’nogo Syr’ya. 2008;(3):123-127. (In Russian)
26. Sundaresan A, Radhiga T, Deivasigamani B. Biological Activity of Biochanin A: A Review. Asian J Pharm Pharmacol. 2018;4(1):1-5. DOI: 10.31024/ajpp.2018.4.1.1
27. Tay KC, Tan LTH, Chan CK, Hong SL, Chan KG, Yap WH, Pusparajah P, Lee LH, Goh BH. Formononetin: A review of its anticancer potentials and mechanisms. Front Pharmacol. 2019;10:820. doi:10.3389/fphar.2019.00820
28. Xiao H, Qin X, Wan J, Li R. Pharmacological Targets and the Biological Mechanisms of Formononetin for Alzheimer’s Disease: A Network Analysis. Med Sci Monit. 2019;25:4273-4277. doi:10.12659/MSM.916662
29. Jiang D, Rasul A, Batool R, Sarfraz I, Hussain G, Tahir MM, Quin T, Selamoglu Z, Ali M, Li J, Li X. Potential Anticancer Properties and Mechanisms of Action of Formononetin. Biomed Res Int. 2019;2019:11 p. doi:10.1155/2019/5854315
30. Zaprometov MN. Phenolic Compounds: Distribution, Metabolism and Functions in Plants. Moscow; 1993. 119 p. (In Russian)
31. Maciejewska-Turska M, Zgorka G. In-depth phytochemical and biological studies on potential AChE inhibitors in red and zigzag clover dry extracts using reversed–phase liquid chromatography (RP-LC) coupled with photodiode array (PDA) and electron spray ionization-quadrupole/time of flight-mass spectrometric (ESI-QToF/MS-MS) detection and thin-layer chromatography-bioautography. Food Chem. 2021;375:131846. doi:10.1016/j.foodchem.2021.131846
32. Dluhošová J, Řepková J, Jakešová J, Nedělník H. Impact of interspecific hybridization of T. pratense × T. medium and backcrossing on genetic variability of progeny. Genet Plant Breed. 2016;52:125-131. doi:10.17221/115/2016-CJGPB
33. Butkutė B, Lemežienė N, Padarauskas A, Norkevičienė E, Taujenis L. Chemical Composition of Zigzag Clover (Trifolium medium L.). In: Breeding Grasses and Protein Crops in the Era of Genomics. 2018:83-87. doi:10.1007/978-3-319-89578-9_15
34. Dmitrienko VA, Kudrinskaya VA, Apyari VV. Methods of isolation, condensation and determination of quercetin. Zhurn Analit Khimii. 2012;67(4):340-353. (In Russian). DOI:10.1134/S106193481204003X
35. Beketov EV, Abramov AA, Nesterova OV. Identification and quantitative assessment of flavonoids in bird cherry fruits. Vestn Mosk Univ Khimiya. 2005;46(4):259-262. (In Russian)
36. Belova OA, Kurkin VA, Egorov MV. Method for quantitative determination of the sum of flavonoids in Glycyrrhiza glabra herb. Farmatsiya i Farmakologiya. 2023;11(2):127-136. doi:10.19163/2307-9266-2023-11-2-127-136
37. State Pharmacopoeia of the Russian Federation XV Edition [Electronic Edition]. Available from: https://pharmacopoeia.regmed.ru/pharmacopoeia/izdanie-15/
38. Marakhova AI. Unification of physico-chemical methods for analysis of medicinal plant raw materials and complex preparations based on plant materials [Abstract of the dissertation for the degree of Doctor of Pharmaceutical Sciences]. Samara; 2017. 48 p. (In Russian)
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Jan 9, 2026
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How to Cite
1.
Development of a new spectrophotometric method for the determination of flavonoids in herb of red clover (Trifolium pratense L) and zigzag clover (Trifolium medium L). Pharm Pract (Granada) [Internet]. 2026 Jan. 9 [cited 2026 Jan. 25];23(4):1-10. Available from: https://www.pharmacypractice.org/index.php/pp/article/view/3384
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Original Research
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Author Biographies
Nikulin Alexander Vladimirovich, Dr.Sc. in Pharmaceutical Sciences, Associate Professor at I.P. Alimarin Department of Analytical Chemistry of MIREA – Russian Technological University (M.V. Lomonosov Institute of Fine Chemical Technologies).
Dr.Sc. in pharmaceutical sciences
Radus Fedor Valerievich, Assistant at I.P. Alimarin Department of Analytical Chemistry of MIREA – Russian Technological University (M.V. Lomonosov Institute of Fine Chemical Technologies).
Assistant at I.P. Alimarin Department of Analytical Chemistry
Fedorina Ludmila Ilyinichna, Senior lecturer at I.P. Alimarin Department of Analytical Chemistry of MIREA Russian Technological University (M.V. Lomonosov Institute of Fine Chemical Technologies)
senior lecturer at I.P. Alimarin Department of Analytical Chemistry
Efimova Yulia Alexandrovna, PhD in Chemical Sciences, Associate Professor at I.P. Alimarin Department of Analytical Chemistry of MIREA – Russian Technological University (M.V. Lomonosov Institute of Fine Chemical Technologies
PhD in chemical sciences
Lazov Mikhail Alexandrovich, PhD in Chemical Sciences, Associate professor at I.P. Alimarin Department of Analytical Chemistry of MIREA – Russian Technological University (M.V. Lomonosov Institute of Fine Chemical Technologies).
PhD in chemical sciences
Mironova Elena Valeryevna, Senior lecturer at I.P. Alimarin Department of Analytical Chemistry of MIREA – Russian Technological University (M.V. Lomonosov Institute of Fine Chemical Technologies)
senior lecturer at I.P. Alimarin Department of Analytical Chemistry
Dorovskikh Ekaterina Anatolevna, PhD in Pharmaceutical Sciences, Maître Assistant at the Pharmaceutical Natural Sciences Department of the First Sechenov Moscow State Medical University (Sechenov University).
PhD in Pharmaceutical Sciences
Sergunova Ekaterina Vyacheslavovna, Dr.Sc. in Pharmaceutical Sciences, Professor at the Department of Pharmaceutical Natural Sciences of Institute of Pharmacy, Sechenov First Moscow State Medical University (Sechenov University)
Dr.Sc. in pharmaceutical sciences
Bobkova Natal’ya Vladimirovna, Dr.Sc.in Pharmaceutical Sciences, Professor at the Department of Pharmaceutical Natural Sciences of Institute of Pharmacy, Sechenov First Moscow State Medical University (Sechenov University)
Dr.Sc. in pharmaceutical sciences
Kostikova Elena Nikolaevna, PhD in Pharmaceutical Sciences, Associate Professor at the Department of Pharmaceutical Natural Sciences of Institute of Pharmacy, Sechenov First Moscow State Medical University (Sechenov University)
PhD in pharmaceutical Sciences
Lutsenko Sergey Viktorovich, Dr.Sc in Biological Sciences, Professor, Head of Department of Biotechnology, Sechenov First Moscow State Medical University (Sechenov University)
Dr.Sc. in biological sciences
Savina Valeriya Alexandrovna, PhD Student, Assistant of the Department of Biotechnology, Sechenov First Moscow State Medical University (Sechenov University).
PhD student
Potanina Olga Georgievna, Dr.Sc. in Pharmaceutical Sciences, Dr. Habil. Head of the Pharmacognosy and Industrial Pharmacy Department of Medicine Faculty of Lomonosov Moscow State University
Dr.Sc. in pharmaceutical sciences
Abramovich Rimma Aleksandrovna, Dr.Sc. in Pharmaceutical Sciences, Dr. Habil. Professor of the Pharmacognosy and Industrial Pharmacy Department Faculty of Medicine Faculty of Lomonosov Moscow State University
Dr.Sc. in pharmaceutical sciences
Kovaleva Tatiana Yurievna, PhD in Pharmaceutical Sciences, Associate Professor at the Department of Pharmaceutical Natural Sciences of Institute of Pharmacy, Sechenov First Moscow State Medical University (Sechenov University).
PhD in pharmaceutical Sciences
How to Cite
1.
Development of a new spectrophotometric method for the determination of flavonoids in herb of red clover (Trifolium pratense L) and zigzag clover (Trifolium medium L). Pharm Pract (Granada) [Internet]. 2026 Jan. 9 [cited 2026 Jan. 25];23(4):1-10. Available from: https://www.pharmacypractice.org/index.php/pp/article/view/3384