Elemental Analysis Of Cordyceps Cicadae And Cordyceps Sinensis And Pharmacodynamic Material Basis Research Of Chitong Xiaoyanling Granules

Objective:1.To establish an inductively coupled plasma mass spectrometry（ICP-MS）method for the determination of 24 kinds of inorganic elements in Cordyceps cicadae and Cordyceps sinensis,pharmacodynamic material study and comparative analysis of the two traditional Chinese medicines in element level.2.To establish HPLC-MS/MS method for simultaneous quantification of multiple components in Chitong Xiaoyanling Granules and quality evaluation studies.3.To develop a rapid UHPLC-Q-TOF-MS/MS method for separating and indentifying metabolites of 5-O-methylvisammioside in vitro and in vivo,which provide a basis for the study metabolism study in vivo and pharmacodynamics of Chitong Xiaoyanling Granules.Methods:1.After microwave digestion treatment,the contents of 24 elements in Cordyceps cicadae and Cordyceps sinensis samples were determined by ICP-MS method and the results were statistically analyzed by combining PCA and OPLS-DA models of SIMCA-p 14.0 software.2.Analysis was performed on a COSMOSIL Packed Column 5C₁₈-PAQ in a gradient program.The multicomponent quantification of Chitong Xiaoyanling Granules was performed on multiple-reaction monitoring（MRM）mode and electrospray ionization source（ESI）transforming between positive and negative ion.3.Rats’plasma,urine,feces,bile and intestinal bacteria incubated samples at different times after administration of 5-O-methylvisammioside were collected and treated with ethyl acetate liquid-liquid extraction method.The structures and pathways of metabolites of 5-O-methylvisammioside were speculated using multiple mass loss（MMDF）and dynamic background deduction（DBS）dependenton-linedataacquistionin UHPLC-Q-TOF-MS/MS system combined with multiple post acquisition data processing.Results:1.The linear relationship,precision,repeatability,stability and recovery of 24 elements determined by ICP-MS method were good.The quantitative results showed that Cordyceps cicadae and Cordyceps sinensis were rich in macro elements K,Mg,P,Ca and essential trace elementsMn,Fe,Zn,and Se.But the statistical results displayed that there were differences between the two.The different elements were Al,Fe,Ca,K and Mn,and the content of Al,Fe,Ca and Mn in Cordyceps cicadae was significantly higher than that in Cordyceps sinensis.2.22 components of Chitong Xiaoyanling Granules determined by HPLC-MS/MS method have good linearity,precision,repeatability,stability and recovery.The quantitative results showed that there were statistically significant differences in 7 components among the samples from different manufacturers,and the contents of the other 6 components in Henan Furentang samples was significantly higher than that in Henan Zhongjie samples except hesperidin.3.26 metabolites in vivo and 7 metabolites in vitro of5-O-methylvisammioside were characterized,respectively.Metabolites in vitro can be detected in vivo biological samples except for M27.Additionally,themainmetabolicpathwaysincludeddeglycosylation,deglycosylation+demethylation,deglycosylation+oxidation,acetylation and sulfate conjugation.Conclusions:1.For the first time,an ICP-MS method after microwave digestion with high sensitivity,low detection limit and simultaneous detection of various elements was established for the determination and comparison of 24 trace elements in Cordyceps cicadae and Cordyceps sinensis.As a result,the two are rich in essential trace elements,and further clarify the pharmacological basis of Cordyceps cicadae and Cordyceps sinensis from the elemental point of view.Moreover,the effect of Cordyceps cicadae on blood tonic and calcium tonic,especially on anti-aging was better than Cordyceps sinensis,which could provide reference for the feasibility of replacing Cordyceps sinensis with Cordyceps cicadae.2.The HPLC-MS/MS method is accurate,simple,rapid,highly sensitive,and can be used for the simultaneous multicomponent quantification analysis of Chitong Xiaoyanling Granules,which will provide reliable data for its quality control and evaluation studies.3.In this study,UHPLC-QTOF-MS/MS method was applied to successfully identify the metabolites of 5-O-methylvisammioside in rats in vivo and in vitro,and the main metabolic pathways were summarized,which can be used to predict the overall metabolic characteristics of Chitong Xiaoyanling Granules,and is of great significance to the study of its pharmacodynamic material basis.