The Study Of Screening Bioactive-Chemical Quality Marker Combination Of Pollen Typhae And Plasma Pharmacokinetics Of Viticis Fructus

Quality control is the key safeguard for the traditional Chinese medicine to exert clinical efficacy.Therefore,due to its characteristics of multi-component and multi-target,it is particularly important to choose the bioactive components as indicators for the quality control of TCM.However,the existing quality control indicators mainly based on chemical markers which are weakly related to medicinal effects.It failed to reflect the relationship between the quality of TCM and active ingredients and efficacy.Traditional Chinese medicine extraction methods such as ultrasonic method and heating reflux method have limitations with long extraction time,large reagent consumption,and complicated steps,which require more green and novel quality evaluation methods.Therefore,it is of great significance to rationally develop a method for evaluating the quality of traditional Chinese medicines.Establishing a quantitative marker system based on screening different active ingredients in TCM and combining the green extraction methods.In this study,the structure-activity relationship of natural flavonoids in TCM against thrombin was explored by molecular docking virtual screening technology combined with anti-thrombin activity determination method.Thus,a bioactive-chemical quality marker combination of Pollen typhae inhibit thrombin was screening by using metabolomics strategy coupled with UHPLC-MS/MS.At the same time,a green extraction and quantification technology of six flavonoids in Pollen typhae was established based on industrial MCM-41-miniaturized matrix solid-phase dispersion extraction and UHPLC.Finally,a UHPLC-MS/MS method was developed to investigate the rat plasma pharmacokinetics after oral administration of aqueous and ethanolic extract of viticis fructus.The specific results are as follows:1.In this study,virtual screening using molecular docking technique was used to screen103 flavonoids.Out of this number,42 target compounds were selected to test their inhibitory effects on thrombin by chromogenic substrate method.The results showed that there were 22compounds showed inhibition on the thrombin.It indicated that the C2,C3 and C4 sites on C ring and C5,C7 sites on A ring as well as C3′,C4′and C5′sites on B ring were the key sites of flavonoids for activity against thrombin.Moreover,C=C,C=O bonds and OH groups were the indispensable substituent group for flavonoids against thrombin.In conclusion,these are the key aspects revealed through the SAR analysis of natural flavonoids on anti-thrombin activity.These findings would promote the study on activity mechanism of flavonoid-type compounds as well as design and develop more potent flavonoid-type inhibitors against thrombin.2.A method was developed for the thrombin-based discovery strategy of bioactive-chemical marker combination based on metabolomics coupled with chemometrics and anti-thrombin activity determination in vitro for the quality evaluation of Pollen typhae.Firstly,acquisition of metabolic features and component characterization of 23 batches Pollen typhae were obtained using UHPLC-Q-TOF/MS;secondly,assessment of direct inhibitory effects of Pollen typhae on thrombin using chromogenic substrate method together with HPLC.Thereafter,bioactive-chemical marker combination associated with anti-thrombin segregation was screened using supervised classifiers.Lastly,quantitative assay and prediction-model of selected markers were established.Results showed that a total of 22compounds were identified from Pollen typhae and 19 batches samples had inhibitory effect on thrombin.Citric acid and linolenic acid inhibited thrombin activity with IC₅₀ values,0.52±0.02 and 0.51±0.02 mg/m L,respectively.A bioactive-chemical marker combination including citric acid,linolenic acid,typhaneoside,and isorhamnetin-3-O-neohespeidoside were discovered and selected as quality markers for evaluation of Pollen typhae according to inhibitory thrombin.3.In this study,six flavonoids in Pollen tyohae including L-epicatechin,typhaneoside,isorhamnetin-3-O-neohespeidoside,naringenin,kaempferol and isorhamnetin were choose as markers.Thus,several variables were optimized in detail for the extraction,including mesh number of sieves,type of adsorbent,mass ratio of sample to adsorbent,grinding time,methanol concentration and elution volume.Central composite design was applied to optimize the best conditions for the maximum yields of the total flavonoids.A method was established based on industrial MCM-41-miniaturized matrix solid-phase dispersion extraction with ultra-performance liquid chromatography for the quality evolution of Pollen typhae.The 25 mg dried Pollen typhae powder passed over 200 meshes homogeneous was mixed with 35 mg industrial MCM-41 and grinded for 2 min.the elution solution was 1.25 m L 70%methanol/water（7:3,v/v）.The advantage of the established method was the less sample and organic reagent as also as less extraction duration in the extraction process and significantly superior to conventional heating reflux extraction in China Pharmacopoeia.It was proved that the developed method was conformed to the concept of green chemistry and provided the new technology for the quality control of traditional Chinese medicine.4.An ultra-performance liquid chromatography-tandem mass spectroscopy（UHPLC-MS/MS）was developed and used to detect the blood concentration of four active compounds（agnuside,10-O-vanilloylaucubin,luteolin,and casticin）in rat after oral administration of aqueous and ethanolic extract of Viticis fructus.Liquid-liquid extraction with ethyl acetate was used to pretreat the plasma samples,and formononetin and geniposide were used as the internal standards.Plasma pharmacokinetics parameters of the target compounds were analyzed after oral administration of both extracts.Good linearity was achieved with correlation coefficient（r²）of 0.990.With the exception of10-O-vanilloylaucubin which was not detected in the ethanolic extract of Viticis fructus,all other compounds were detected in both extracts in plasma.The plasma pharmacokinetic parameters of the four compounds were significantly different between the two extracts.The optimized,sensitive and validated UHPLC-MS/MS method was successfully applied for the plasma pharmacokinetics comparison analysis of the two Viticis fructus extracts.