Biochanin A, the Most Potent of 16 Isoflavones, Induces Relaxation of the Coronary Artery Through the Calcium Channel and cGMP-dependent Pathway Thomas Migkos, Jana Pourová, Marie Vopršalová, Cyril Auger, Valérie Schini-Kerth, Přemysl Mladěnka To cite this version: Thomas Migkos, Jana Pourová, Marie Vopršalová, Cyril Auger, Valérie Schini-Kerth, et al.. Biochanin A, the Most Potent of 16 Isoflavones, Induces Relaxation of the Coronary Artery Through the Calcium Channel and cGMP-dependent Pathway. Planta Medica, Georg Thieme Verlag, 2020, 86 (10), pp.708 - 716. 10.1055/a-1158-9422. hal-03033813 HAL Id: hal-03033813 https://hal-cnrs.archives-ouvertes.fr/hal-03033813 Submitted on 1 Dec 2020 HAL is a multi-disciplinary open access L’archive ouverte pluridisciplinaire HAL, est archive for the deposit and dissemination of sci- destinée au dépôt et à la diffusion de documents entific research documents, whether they are pub- scientifiques de niveau recherche, publiés ou non, lished or not. 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Published online: 2020-05-14 Original Papers Biochanin A, the Most Potent of 16 Isoflavones, Induces Relaxation of the Coronary Artery Through the Calcium Channel and cGMP-dependent Pathway Authors Thomas Migkos1, Jana Pourová 1,MarieVopršalová 1, Cyril Auger 2, Valérie Schini-Kerth 2,Přemysl Mladěnka 1 Affiliations Supporting information available online at 1 Department of Pharmacology and Toxicology, Faculty of http://www.thieme-connect.de/products Pharmacy in Hradec Kralove, Charles University, Hradec Kralove, Czech Republic ABSTRACT 2 INSERM (French National Institute of Health and Medical Research), UMR 1260, Regenerative Nanomedicine (RNM), The dietary intake of flavonoids seems to be inversely related FMTS, Université de Strasbourg, Faculté de Pharmacie, to cardiovascular mortality. The consumption of isoflavonoids Illkirch, France is increasing in the general population, especially due to the use of food supplements and a variety of isoflavonoid-rich Key words foods. However, detailed studies on the vascular influence of blood vessels, cardiovascular, ex vivo, glycitein, isoflavonoid individual pure isoflavonoids are mostly missing. For this study, 16 isoflavonoids were initially screened for their vaso- received February 3, 2020 relaxant properties on rat aortas. The 2 most potent of them, revised April 2, 2020 biochanin A and glycitein, were further tested for the mecha- accepted April 13, 2020 nism of action on porcine coronary arteries. They both in- duced an endothelium independent vascular relaxation, with Bibliography EC50 below 6 and 17 µM, respectively. Biochanin A, but not DOI https://doi.org/10.1055/a-1158-9422 glycitein, was able to block the vasoconstriction caused by published online | Planta Med © Georg Thieme Verlag KG KCl, CaCl2, serotonin, and U46619 in a dose-dependent man- Stuttgart · New York | ISSN 0032‑0943 ner. Another series of experiments suggested that the major mechanism of action of biochanin A was the inhibition of Correspondence L‑type calcium channels. Moreover, biochanin A in relatively Assoc. Prof. Přemysl Mladěnka, Pharm.D., Ph.D. small concentrations (2–4 µM) interfered with the cGMP, but Department of Pharmacology and Toxicology, Faculty of not cAMP, pathway in isolated coronary arteries. These results Pharmacy in Hradec Králové, Charles University indicate that some isoflavonoids, in particular biochanin A, Akademika Heyrovského 1203, 500 05 Hradec Králové, are able to have vasodilatory effects in micromolar concentra- Czech Republic Downloaded by: Université de Strasbourg. Copyrighted material. tions, which is of potential clinical interest for the manage- Phone: + 420495067295, Fax: + 420495067170 ment of cardiovascular pathologies. [email protected] Introduction sources are legumes and herbs, such as soybean (Glycine max), red clover (Trifolium pratense), milkvetch (Astragalus sp.), Prunus, Puera- Cardiovascular diseases remain the leading cause of mortality and ria, and Ononis sp. [5–8]. Isoflavonoid intake in humans is also in- morbidity worldwide. Among them, ischemic coronary disease creasing due to the use of a variety of dairy products, processed and stroke are major culprits of mortality. Hence, vascular func- food, and food supplements derived from the aforementioned tion can be considered one of the main targets when developing plants [9,10]. Most of these species are traditionally used in Chi- innovative drugs focused on both the treatment and prevention of nese medicine, among other things, for the treatment of cardio- cardiovascular disorders. vascular diseases [7]. The lower incidence of cardiovascular disor- Indeed, isoflavonoids appear to be related to a lower incidence ders in East Asian populations, compared to those from Western of myocardial and cerebral infarctions [1], reduced arterial stiffness countries, has been also attributed to the higher consumption of [2], and blood pressure normalization [3]. Due to their structural isoflavonoids, particularly soy isoflavones [11]. Despite these en- similarity to endogenous estrogens, they have affinity to estrogen couraging epidemiological observations, most of the conducted receptors [4] and are known as phytoestrogens. Their main natural human studies have investigated the effect of isoflavone-rich diets, Migkos T et al. Biochanin A, the … Planta Med Original Papers ▶ Table 1 The chemical structures of the 16 tested isoflavonoids. Isoflavonoid: R5 R6 R7 R8 R3′ R4′ aglycones daidzein HHOHHHOH genistein OHHOHHHOH biochanin A OHHOHHHOCH3 glycitein HOCH3 OHHHOH isoformononetin HHOCH3 HHOH formononetin HHOHHHOCH3 calycosin H H OH H OH OCH3 tectorigenin OH OCH3 OHHHOH cladrin HHOHHOCH3 OCH3 prunetin OH H OCH3 HHOH glycosides daidzin HHO‑GlcHHOH genistin OH H O‑GlcHHOH glycitin HOCH3 O‑GlcHHOH ononin HHO‑GlcHHOCH3 tectoridin OH OCH3 O‑GlcHHOH puerarin HHOHGlcHOH Glc: glucose. plant extracts, and mixtures of isoflavonoids [1–3]. The data on the was not significantly modified by the absence of the vascular en- effects of individual purified isoflavonoids themselves are hence dothelium (▶ Fig. 2, Fig. 3S, Supporting Information) in any of the largely missing, with some exceptions [12]. Additionally, ex vivo artery models with a small exception of higher concentration of and animal vascular studies have been performed for a limited glycitein in coronary rings. Interestingly, the EC50 of biochanin A number of compounds [6,11,13,14], and even less information is on coronary arteries (5.5 ± 0.6 µM) was lower than that on rat aor- available in respect to coronary vessels [15–17]. tas (25.4 ± 2.2 µM) and that of glycitein (16.2 ± 1.0 µM). For glyci- With the present study, we strove to directly compare the vas- tein, the difference between EC50s for the 2 types of vessels was cular effects of 16 pure isoflavones, several of which were tested smaller (EC50s = 27.5 ± 2.8 µM and 16.2 ± 1.0 µM on aortas and for the first time, and to further elucidate the mechanism of ac- coronary arteries, respectively). Since the effect on coronary ar- tion of the most active ones, biochanin A and glycitein. Within this teries was achieved in lower concentrations, and given the similar- framework, we also challenged the hypothesis that biochanin A ities between porcine and human hearts [18], this model was functions as a calcium channel blocker. used for further testing. To further elucidate the mechanism(s) of the selected isoflavo- Downloaded by: Université de Strasbourg. Copyrighted material. Results noids, endothelium-denuded coronary arteries pretreated with biochanin A (3, 10, and 30 µM) or glycitein (10, 15, and 30 µM) In the first series of experiments, a screening of the vasodilatory were exposed to increasing concentrations of 4 different vasocon- effects of 16 isoflavonoids (▶ Table 1) on norepinephrine precon- tricting stimuli. Contractions induced by both KCl and CaCl2 were tracted rat aortic rings was performed. All glycosides, namely dose-dependently inhibited by biochanin A. In the highest dose, genistin, daidzin, glycitin, ononin, tectoridin, and puerarin, had biochanin A equally blocked 73% of the maximal vasoconstriction clearly very low vasorelaxant effects, which were comparable to caused by both inducers (▶ Fig. 3 a). On the other hand, glycitein that of the control containing only the solvent (Fig. 1S, Support- only caused an approximate 25% reduction of the maximal effect ing Information) and hence much lower than that of their corre- of KCl and did not significantly alter the contraction induced by sponding aglycones (▶ Fig. 1, Fig. 2S, Supporting Information). the maximal concentration of CaCl2. However, 30 µM of glycitein In particular, biochanin A and glycitein appeared the most potent partly blocked the effect of lower concentrations of CaCl2 vasodilators, having EC50 below 30 µM, followed in ascending or- (▶ Fig. 3 b). Similarly, biochanin A demonstrated higher potency der by genistein, isoformononetin, formononetin, tectorigenin, against receptor-mediated contractions induced by serotonin or calycosin, cladrin, prunetin, and daidzein, with EC50 ranging from thromboxane-analogue U46619. In the highest concentration, it 30 to 110 µM (▶ Table 2). blocked 88% and 56% of their maximal effects, respectively Thereafter, the 2 most potent isoflavonoids, biochanin A and (▶ Fig. 3 a). On the contrary,