Development Of Analytical Methods For Osmanthus Fragrans Fruits By Liquid Chromatography-Mass Spectrometry

Traditional Chinese medicine(TCM)is a treasure of Chinese traditional culture,but what prevents TCM to be worldwild is the effectiveness and safty due to the complex components of themselves.The rapid identification and efficient separation of chemical constituents are the key technologies to the urgent need of TCM analysis.High performance liquid chromatography-mass spectrometry(LC-MS)has become the technology of choice for TCM analysis due to its high separation capacity,high selectivity,high sensitivity and high efficiency.In particular,the development of coupled mass spectrometry and high-resolution mass spectrometry technology can simultaneously obtain structural information of dozens of compounds on line,providing a simpler and faster approach for identification of chemical constituents of TCM.China is rich in the natural resources of Osmanthus fragrans(O.fragrans).The O.fragrans is a treasure,O.fragrans flowers,O.fragrans fruits,and O.fragrans roots are all of great medicinal value If these medicinal value could be organically integrated into food and cosmetics,it will have great development prospects and will bring greater economic benefits.However,all of the current analytical methods to characterize the active constituents of O.fragrans fruits and O.fragrans roots are based on traditional column chromatography,which is not only time-consuming and laborious,but also difficult to explore the integral chemical compounds profile,especially for those with low content or similar properties that cannot be completely separated and purified.So there is an urgent need to establish an analytical method to profile the chemical compounds more effectively and sencitively.At the same time,there is no efficient and effective quality control method for the effective utilization of O.fragrans fruits and O.fragrans roots.Furthermore,there is no method for analyzing the actual effective components in vivo of O.fragrans fruits.Therefore,this paper focus on O.fragrans fruits and O.fragrans roots which are not effectively utilized,and aim to develop the analytical methods of qualitative and quantitative analysis by LC-MS.Including the following contents:The first part is the new analytical method of active components in the nbutanol extract from O.fragrans fruits by HPLC-MS/MS.A total of 26 phenylethanoid glycosides were identified by LC-MS/MS.16 compounds were first discovered in O.fragrans fruits,and a compound was reported for the first time.LC-MS/MS methods for simultaneous detection of five phenylethanoid glycosides and two triterpenoids were established,respectively.The established methods were satisfactory in precision,repeatability and accuracy.The methods were applied to determine the content of active ingredients in different varieties of O.fragrans fruits,which provides an efficient and powerful means for quality analysis of O.fragrans fruits.The second part is the new analytical method of triterpenic acid and its esters in O.fragrans fruits by HPLC-MS.An efficient and sensitive HPLC-MS method for the analysis of triterpenic acid and triterpene ester compounds in O.fragrans fruits was established.Thirty-one compounds were identified from the ethyl acetate extract of O.fragrans fruits,including 27 compounds found in the O.fragrans fruits for the first time.The method can also be applied to the analysis of triterpenic acid and triterpene ester of O.fragrans flowers,and it is found that O.fragrans flowers are rich in natural resources of triterpenoids too.At the same time,semi-quantitative analysis of multiple groups of triterpenoids in O.fragrans fruits showed that triterpenic acid and triterpene esters exhibited three different trends,which provided a basis for us to formulate a reasonable fruit harvesting period.The third part is the study of the active ingredient in vivo of O.fragrans fruits by HPLC-MS.The absorption and metabolism profiles of the main active components of O.fragrans fruits were elucidated.The precursor compounds of these prototypic components and metabolites can be used as the important quality markers of O.fragrans fruits.At the same time,combining with the effective fraction study,the material basis of the traditional analgesic effect of O.fragrans fruits is clarified.On the other hand,according to the results of absorption and metabolism in vivo,the antifatigue effect of O.fragrans fruits is further explored,which provides a feasible direction for the development of O.fragrans fruits.The forth part is the study of the active constituents and network pharmacology of O.fragrans roots by HPLC-MS/MS.The phenylethanoid glycosides and lignans in the O.fragrans roots were identified by HPLC-MS/MS.36 chemical components in the O.fragrans roots were found quickly and effectively.Thirty-two compounds were first discovered in O.fragrans roots,and one compounds was reported for the first time.And the high sensitivity method by HPLC-MS/MS was established to detect the content of acteoside,isocacteoside,oleuropein and phillyrin in different varieties of O.fragrans roots,suggesting that there may be some differences in the active ingredients in different varieties.Using network pharmacology research methods,it was predicted that O.fragrans roots may play a role in inflammation,cardiovascular and cancer diseases.Rheumatoid arthritis is consistent with the traditional pharmacodynamics of O.fragrans roots.The related targets are serine/threonine protein kinase and matrix metalloproteinase(PRKCQ,MMP9,MMP3,MMP2,MMP1).The related compounds are mainly phenylethanoid glycosides such as oleuropein,ligstroside and 10-hydroxyligstroside.