Screening Of Bioactive Glycosides And Their Metabolites By UPLC/MS~n

The global "return to nature" trend would be bound to bring the unprecedented opportunities for the natural medicine. To make the traditional Chinese medicine recognized and accepted in the whole world, we must evaluate its quality accurately, confirm its pharmacological mechanism and ensure its efficacy. Therefore, the researchers in the pharmacological analysis field are required to establish a sensitive and reliable analytical method for rapid screening of active ingredients in complex traditional Chinese medicine, which would build the foundation for the further scientific research.In this thesis, a powerful ionic liquid-based ultrasonic-assisted extraction (ILUAE) method combined with ultra-performance liquid chromatography coupled to electrospray ionization quadrupole time-of-flight tandem mass spectrometry (UPLC/ESI-QTOFMSn) was employed in the rapid simultaneous screening of iridoid glycosides, phenylethanoid glycosides, and cucurbitacin glycosides from P. Scrophulariiflora. Meanwhile, it successfully developed electrospray ionization tandem mass spectrometry coupled with ultra performance liquid chromatography method to investigate on the chemical stabilities of C21steroidal glycosides from the stems of Stephanotis mucronata and the integral metabolism of Stemucronatoside K in rats. The concrete content is as follows.1. A green, convenient, and effective screening method based on ILUAE and UPLC/ESI-QTOFMSn was successfully developed for screening bioactive glycosides from P. Scrophulariiflora. The ILUAE procedure was optimized over several ultrasonic parameters, including the ultrasonic power, concentration of the ionic liquid, and solid-liquid ratio. A comparison with conventional heat-reflux extraction and regular UAE demonstrated that the optimized approach yielded a high extraction efficiency within a short extraction time of30min. The separation time of UPLC decreased by a factor of2-3relative to that of HPLC. Negative ion mode ESI-QTOFMS2analysis of the fragmentation reactions of the [M-H]-ions was conducted to characterize the diagnostic ions related to the glycosyl moieties, aglycone units, and the type and substituted position of the ester groups. Interestingly, the positional isomers of the iridoid glycosides could be easily discriminated based on the characteristic ions. A total of15glycosides, including three groups of iridoid glycoside isomers and two groups of phenylethanoid glycoside isomers, were conveniently identified within13.5min. Moreover,6’-O-vanilloyl catalpol was identified in P. scrophulariiflora for the first time. The method developed here was further validated by measuring the recovery, correlation coefficient (R2), and reproducibility (RSD, n=5) of three iridoid glycosides:89.60%-109.02%,0.9991-0.9998, and0.93%-1.44%, respectively. This study demonstrated the capabilities of ILUAE combined with UPLC/ESI-QTOFMSn for the rapid screening of glycosides in P. Scrophulariiflora.2. The chemical stabilities of C21steroidal glycosides which have the high immunological activities in the Stephanotis mucronata were investigated in detail by ESI-MS/MS. The samples were damaged under the conditions of acid solution, base solution, high temperature or1%H2O2. Then the decomposition pathways and decomposition products were discussed by UPLC/ESI-MS/MS. The experimental results revealed that the C21steroidal glycosides are unstable in acidic and basic condition. The long-chain glycosides hydrolyzed to produce the short-chain glycosides, which have higher-immunological activities. This conclusion would play a great part in studying the immune drugs in the future.3. The metabolism biotransformation of Stemucronatoside K obtained from the stems of Stephanotis mucronata in rats was investigated systematically using HPLC/ESI-MSn. The strategy for the rapid identification of the metabolites for Stemucronatoside K was proposed. This experiment provided the important foundation for the immune mechanism study of C21steroidal glycosides in vivo and brought a new idea for the development of new immune drugs.