Studies On Isoflavone Constituents Of Puerariae Flos And Their Protect Activity On Primary Microglia

Puerariae Flos that is botanically from the dried flowers of Pueraria lobata（Willd.） Ohwi and P.thomsonii Benth（Leguminosae）is the most representative traditional Chinese medicine used to alleviate hangover.The isoflavonoid constituents and their pharmacological activities,as well as HPLC method for the quantitative determination of isoflavones in Puerariae Flos extracts were systematically investigated in this study.The main contents are as follows.1.Eleven polyhydroxy-isoflavones were isolated from ethanolic extracts of Puerariae Flos by means of solvent partition and column chromatographic methods,including Diaion HP-20,silica gel,polyamide gel,Sephadex LH-20 and MDS.On the basis of chemical evidence and spectral analysis,their structures were identified as genistein（1）,tectorigenin （2）,irisolidone（3）,gehuain（4）,genistin（5）,6-hydroxygenistein-7-O-β-D-glucopyranoside（6）, tectoridin（7）,6"-O-xylosyltectoridin（8）,kakkalide（9）,6-hydroxygenistein-6,7-di-O-β-D-glucopyranoside （10）and 6-hydroxybiochanin A-6,7-di-O-β-D-glucopyranoside（11）.Among them,compounds 10 and 11 were two new components,compound 6 was isolated as a natural product for the first time.2.The inhibitory activity of eleven isolated isoflavones on NO release in LPS-activated primary rat microglial cells was assayed.The results indicated that the potency(IC₅₀,μM)of the inhibition was in order of compound 1（1.3）＞3（2.3）＞2（9.3）＞10（38）＞4（43）＞8（45）＞7（62）,and compounds 5,6,9,11 showed no significant inhibition.The structure-activity relationships of the isoflavones on NO release in LPS-activated microglial cells are elucidated as follows.The glycosylation of 7-hydroxy group tends to reduce or loss the activity.The substituion of 6-methoxyl group decreases the activity of isoflavone aglycones.As for isoflavonoid glycosides,however,methylation or glycosylation of 6-hydroxy group tends to increase the activity.The methylation of 4’-hydroxyl group enhance the activity of isoflavone aglycones,in contrast,it makes their glycoside isoflavones loss the activity.3.The quantitative methods that included the determination of total isoflavones of Puerariae Flos extracts by UV method and that of four marker isoflavonoids by HPLC were established for the first time,which were convenient,accurate and adequate for the standardization of Puerariae Flos extracts.Furthermore,the extractive efficacy of isoflavonoids of Puerariae Flos using water-ethanol in various proportion was investagated, with the extract yield and the content and availability of four marker isoflavones being used as value parameters.The results indicated that the content as well as the availability of marker components in the extract using 80%EtOH as an extractive solvent were the highest,which provided basic experimental reference for the further study on the preparative technology of Puerariae Flos extracts.