Study On The Chemical Constituents Of Chimonanthus Nitens Oliv.Leaf Granule

Chimonanthus nitens Oliv is a unique medicinal plant in China,often used in medicine with its leaf.As a commonly used traditional Chinese medicine,Chimonanthus nitens Oliv leaf has a variety of pharmacological activities.Researches on the chemical constituents of the leaf demonstrated that coumarins,flavonoids and alkaloids are its main constituents.In recent years,Chimonanthus nitens Oliv.leaf granule has been widely used to prevent and control COVID-19.However,studies on the chemical compounds of granule were rarely reported.Besides,the chemical components of traditional Chinese medicine are complex.Different kinds of compounds often have different transfer efficiency and some substances even undergo chemical transformation in the pharmaceutical process.Therefore,the chemical components of Chinese patent medicines are often different from that of their original medicinal materials.In this study,in order to explore the main chemical components of Chimonanthus nitens Oliv.leaf granule and the transfer rule of chemical components in the process of preparing them into granule,the following researches were carried out:firstly,based on electrospray high-resolution mass spectrometry,the gas-phase cleavage reactions of coumarins and flavonoids were explored under collision-induced activation,which laid a theoretical foundation for the multi-component study of chemical components of Chimonanthus nitens Oliv.leaf granule by LC-MS.Secondly,a set of multicomponent identification strategy based on mass spectrometry was constructed,involving unsaturation calculation,characteristic neutral loss,diagnostic ions,chromatographic retention time（RT）alignment,biosynthesis pathways analysis,theoretical calculation of physicochemical properties such as hydrophobic constant（Clog P）and acid-base.Thirdly,the systematic analysis of aromatic compounds in Chimonanthus nitens Oliv.leaf granule was carried out.Fourthly,the chemical component differences between leaf and granule were explored using a non-targeted metabolomics analysis platform,which revealed the impact of pharmaceutical processes on chemical components transfer.Finally,the alkaloid compounds in the granule were separated by using multiple column chromatography separation and high performance liquid chromatography,and the inhibitory activity of compounds on acetylcholinesterase were explored.In the study of the gas phase cleavage reactions of coumarin compounds,firstly,a novel gas phase cleavage reaction of coumarin was established,a competing cleavage reaction mechanism involving the"ring-opening"and"ring-closing"of the coumarinolide ring.Secondly,OH-7 on coumarin was found to be able to induce RDA reaction,and a new diagnostic ion m/z 81.0331 for the identification of coumarin was established,which quickly determined the presence of OH-7 and C-8 substituents on the coumarin skeleton.On the other hand,in the study of gas phase cleavage reactions of flavonoids,firstly,diphenyl ethyl diketone rearrangement reactions were first detected in flavonol compounds.In addition,m/z 85.0289 and 127.0396 were found in the product ions of flavonol glycosides,which can be used as diagnostic ions to identify such compounds.In the systematic analysis of chemical compounds,114 aromatic compounds,including 60 coumarins and 54 flavonoids,were identified in granule based on multi-component identification strategies.Mc Lafferty rearrangement occurring in coumarin glycosides while sugar group locating at C-4 was first observed during the identification process,and new diagnostic ions at m/z 147.0440,119.0488,and 91.0543 were constructed.By comparing the chemical components in the leaf and granule,29 differentiating components were found.It was found that flavonoid glycosides had significant delivery differences in the pharmaceutical process due to structural differences.Finally,in the separation study of alkaloids in granule,two compounds were separated and isomers of each other,which have inhibitory activity on acetylcholinesterase,IC₅₀ were 31.05μM and 2.53 m M,respectively.The novel fragmentation patterns were established of coumarins and flavonoids in this paper,which were applied to the identification of chemical compounds in Chimonanthus nitens Oliv.leaf granule.Of the 114 aromatic compounds identified in granule,85 of which were first discovered in this plant,including 36 new compounds.When studying the transfer rule of chemical components during granule preparation,we found that the granule preparation process effectively transferred most of the compounds with obvious biological activities and the content of 12 compounds in granule was significantly higher than that of leaf.In particular,kaempferfer-7-O-neohesperidoside and its congeners were obviously enriched in the granule.Studies have shown that they have biological activities such as anti-inflammatory,antimicrobial and hepatoprotective,therefore,we believe that these compounds may be an effective material basis for the clinical efficacy of Chimonanthus nitens Oliv.leaf granule.