Ayanin, a natural flavonoid inhibitor of Caseinolytic protease, is a promising therapeutic agent to combat methicillin-resistant Staphylococcus aureus infections.

Antimicrobial resistance (AMR) is a global health threat. The dramatic increase of Methicillin-resistant Staphylococcus aureus (MRSA) infections emphasizes the need to find new anti-infective agents with a novel mode of action. The Caseinolytic protease (ClpP) is a central virulence factor in stress survival, virulence, and antibiotic resistance of MRSA. Here, we found ayanin, a flavonoid isolated from Callicarpa nudiflora, was an inhibitor of MRSA ClpP with an IC50 of 19.63 µM. Using quantitative real-time PCR, ayanin reduced the virulence of Staphylococcus aureus (S. aureus) by down-regulating the level of some important virulence factors, including agrA, RNAⅢ, hla, pvl, psmα and spa. The results of cellular thermal shift assay and thermal shift assay revealed a binding between ayanin and ClpP. Molecular docking showed that ASP-168, ASN-173 and ARG-171 were the potential binding sites for ClpP binding to ayanin. ClpP mutagenesis study further indicated that ARG-171 and ASN-173 were the main active sites of ClpP. The affinity constant (KD) value of ayanin with ClpP was 3.15 × 10-5 M measured by surface plasmon resonance. In addition, ayanin exhibited a significant therapeutic effect on pneumonia infection induced by S. aureus in mice in vivo, especially in combination with vancomycin. This is the first report of ayanin with in vivo and in vitro efficacy against S. aureus infection. In conclusion, ayanin is a promising therapeutic agent to combat MRSA infections by targeting ClpP.

The Spectrum of Pharmacological Actions of Syringetin and Its Natural Derivatives-A Summary Review.

Mono- and poly-O-methylated flavonols and their glycoside derivatives belong to the group of natural plant polyphenols with a wide spectrum of pharmacological activities. These compounds are known for their antioxidant, antimutagenic, hepatoprotective, antidiabetic, and antilipogenic properties. Additionally, they inhibit carcinogenesis and cancer development. Having in mind the multidirectional biological activity of methylated flavonols, we would like to support further study on their health-promoting activities; in this review we summarized the most recent reports on syringetin and some of its structural analogues: laricitrin, ayanin, and isorhamnetin. Natural sources and biological potential of these substances were described based on the latest research papers.

Vascular interactions of Croton schiedeanus major flavonoids in isolated aortic rings from Wistar rats / Interacciones vasculares de los principales flavonoides de Croton schiedeanus en anillos aislados de aorta de ratas Wistar

Background: Ayanin (3,7,4'-Tri-O-methylquercetin) and 3,7-Di-O-methylquercetin (DMQ) are the main active metabolites isolated by bioguided fractionation from Croton schiedeanus, species known popularly in Colombia as "almizclillo", which has been studied in experimental models in rats, exerting vasodilator and antihypertensive effects. Also, when the effect of these flavonoids was studied separately, important vasodilation was observed. Objective:To evaluate whether flavonoids from Croton schiedeanus have synergistic vasodilator properties when different combinations are used in isolated aorta rings. Methods: Cumulative concentrations of ayanin (10-8 M - 6x10-5 M or 0.01 µM - 60 µM) were assayed in the absence and presence of an increasing concentration of 3,7-Di-O-methylquercetin (DMQ) (10-8 ­ 3x10-5M or 0.01­30 µM) in isolated rings from Wistar rats, pre-contracted with phenylephrine. The concentration-response curve with the maximal effect was compared with that obtained by Croton schiedeanus whole ethanolic extract (10-6 ­ 3x10-4 g/mL). Also, this combination was assayed in the presence of the nitric oxide synthetase inhibitor L-NAME (10-4 M) and the guanylate cyclase inhibitor methylene blue (10-4 M) to assess the role of the NO/cGMP pathway in this interaction. Results: Ayanin and DMQ display a dual interaction in vascular relaxant response: agonism at higher concentration ranges (10-6 ­ 3x10-5 M or 1­30 µM) and antagonism at lower concentration ranges (10-8 ­ 3x10-7 M or 0.01­0.3 µM). The efficacy at the highest concentration was greater than that obtained by the whole extract (Emax: 98.4% vs. 33.9%). This response was decreased but not reverted in the presence of L-NAME and methylene blue. Thus, the vasodilator effect of this combination does not depend entirely on the NO/cGMP cyclic pathway. Conclusion: The combined use of appropriate concentrations of these flavonoids could represent an advantage over Croton schiedeanus whole extract for vasodilator purposes

Antecedentes: Ayanina (3,7,4'-Tri-O-metilquercetina) y 3,7-Di-O-metilquercetina (DMQ) son los principales metabolitos activos aislados mediante fraccionamiento bioguiado, a partir de Croton schiedeanus, especie conocida popularmente en Colombia como "almizclillo", la cual ha sido estudiada en modelos experimentales en ratas, ejerciendo efectos antihipertensivos y vasodilatadores. Además, al estudiar por separado el efecto de los flavonoides, se observó importante vasodilatación. Objetivo:Evaluar si los principales flavonoides de Croton schiedeanus tienen propiedades vasodilatadoras sinérgicas al utilizar diferentes combinaciones de ellos en anillos de aorta aislados. Metodología: Se analizaron concentraciones acumulativas de ayanina (10-8 M - 6x10-5 M o 0,01 µM - 60 µM) en ausencia y en presencia de concentraciones crecientes de DMQ (10-8 M - 3x10-5 M o 0,01 µM ­ 30 µM) en anillos aislados de ratas Wistar, pre-contraídos con fenilefrina. La curva concentración respuesta obtenida con el efecto máximo, fue comparada con la obtenida con el extracto etanólico de Croton schiedeanus (10-6 - 3x10-4 g/mL). Adicionalmente, esta combinación fue ensayada en presencia del inhibidor de óxido nítrico sintetasa L-NAME (10-4 M) y el inhibidor de guanilato ciclasa, azul de metileno (10-4 M) para evaluar el papel de la vía NO/GMPc en esta interacción. Resultados: Ayanina y DMQ muestran una interacción dual en la respuesta vascular relajante: agonismo en el rango más alto (10-6 M ­ 3x10-5 M o 1 µM ­ 30 µM), y antagonismo en el rango más bajo (10-8 M ­ 3x10-7 M o 0.01 µM ­ 0,3 µM). A altas concentraciones, la eficacia de los flavonoides fue mayor que las obtenidas por el extracto completo (Emáx: 98,4% vs 33,9%). Esta respuesta disminuyó, pero no se revirtió en presencia de L-NAME y azul de metileno. Por lo tanto, el efecto vasodilatador de esta combinación no depende enteramente de la vía NO/GMPc. Conclusión: El uso combinado de las concentraciones apropiadas de estos flavonoides podría representar una ventaja sobre el extracto de Croton schiedeanus, con propósitos vasodilatadores

Mechanisms of relaxation induced by flavonoid ayanin in isolated aorta rings from Wistar rats

Introduction: This study shows the relaxant effect induced by ayanin in aorta rings from Wistar rats linked to nitric oxide/cyclic-GMP pathway. This flavonoid is the prevalent compound obtained from Croton schiedeanus Schlecht (Euphorbiaceae), specie used in Colombian folk medicine for the treatment of arterial hypertension. Objectives: To identify possible action mechanisms of vascular relaxation induced by ayanin (quercetin 3,4',7-trimethyl ether).Methodology: Isolated aorta rings from Wistar rats obtained at the Animal House of the University of Salamanca were contracted with KCl (80 mM) or phenylephrine (PE, 10-6 M) and exposed to ayanin (10-6-10-4 M). Then, the effect of ayanin was assessed in deendothelized rings contracted with PE and in intact rings contracted with PE previously incubated with: ODQ (10-6 M), L-NAME (10-4 M), L-NAME plus D- and L-arginine (10-4 M), indomethacin (5x10-6 M), dipyridamole (3x10-7 M), glibenclamide (10-6 M), propranolol (10-6 M), verapamil (10-7 M) or atropine (3x10-5 M). In addition, the relaxant effect of acetylcholine (Ach, 10-8-3x10-4 M), and sodium nitroprusside (SNP, 10-9-3x10-5 M) was assessed in the presence and absence of ayanin (10-6 M).Results: Ayanin induced a greater concentration-dependent relaxation in vessels contracted with phenylephrine (pEC50: 5.84±0.05), an effect significantly reduced by deendothelization and by both ODQ and L-NAME. L-arginine was able to reverse the effect of L-NAME. Indomethacin weakly inhibited ayanin response. Dipyridamole, glibenclamide, propranolol, verapamil, and atropine did not affect ayanin relaxation. Ayanin did not have any effect on the relaxation elicited by acetylcholine (ACh), while weakly decreasing the relaxation induced by sodium nitroprusside (SNP).Conclusion: Ayanin induces endothelium-dependent relaxation in the rat aorta mainly related to nitric oxide/cGMP pathway, according to the response observed in the presence of L-NAME, L-arginine and ODQ.

Introdución: Este estudio muestra el efecto vasodilatador inducido por ayanina en anillos de aorta de ratas Wistar vinculado con la vía óxido nítrico/GMP-cíclico. Este flavonoide es el compuesto mayoritario aislado de Croton schiedeanus Schlecht (Euphorbiaceae), especie utilizada en la medicina popular colombiana para el tratamiento de la hipertensión arterial. Objetivos: Identificar los posibles mecanismos vasodilatadores inducidos por la ayanina (quercetin 3,4',7-trimetileter). Metodología: Se adicionó ayanina (10-6 - 10-4 M) a anillos aislados de aorta procedentes de ratas Wistar contraídos con KCl (80 mM) o fenilefrina (10-6 M). Luego se evaluó el efecto de la ayanina en anillos sin endotelio contraídos con fenilefrina y en anillos íntegros, contraídos con fenilefrina, previamente incubados con: ODQ (10-6 M), L-NAME (10-4 M), L-NAME más L- o D-arginina (10-4 M), indometacina (5x10-6 M), dipiridamol (3x10-7 M), glibenclamida (10-6 M), propranolol (10-6 M), verapamilo (10-7 M) o atropina (3x10-5 M). Además se examinó la relajación inducida por acetilcolina (Ach, 10-8-3x10-4 M) y nitroprusiato de sodio (SNP, 10-9-3x10-5 M) en presencia y ausencia de ayanina (10-6 M). Resultados: La ayanina produjo una mayor relajación en los anillos contraídos con fenilefrina (pEC50: 5.84±0.05), efecto que se redujo en anillos sin endotelio o en anillos íntegros preincubados con ODQ y L-NAME. L-arginina fue capaz de revertir la respuesta inducida por L-NAME. La indometacina inhibió discretamente la relajación generada por la ayanina. El dipyridamol, la glibenclamida, el propranolol, el verapamilo y la atropina no modificaron el efecto de la ayanina. La ayanina no afectó la relajación inducida por la acetilcolina y débilmente disminuyó la inducida por el nitroprusiato de sodio...