Study On Rapid Mass Spectrometric Analytical Method And Correlation Spectroscopy Of NMR/RRLC-MS Of Constituents In The Crude Extract Of Anoectochilus Roxburghii

Mass spectrometric analytical method was used to identify main constituents including flavonoids and organic acids in the rare endangered plant Anoectochilus roxburghii (A. roxburghii), and to compare constituents and their content in the wild and artificial ones bred with biotechnologies. First of all, constituents in the crude extract of A. roxburghii were identified using RRLC-MS/MS and RRLC-MSE in both positive and negative ion modes. Consequently, combined with RRLC-MS/MS, the NMR/RRLC-MS PDS and NMR/RRLC-MS HCS were used to identify the main constituents in the crude extract. Three types of natural products were identified successfully, which exploited the application of correlation spectroscopy of NMR/RRLC-MS. In addition, comparison of constituents and their content between the wild and artificial breeding A. roxburghii were implemented using RRLC-MS/MS. Overall, the results of this paper have provided important basis for sustainable utilization of A. roxburghii and evalution of the cultivated species.1. Fast identification of constituents in the crude extract of Anoectochilus roxburghii using RRLC-MS/MS and MSEIn order to establish RRLC-MS/MS and MSE analytical methods to identify the constituents in the crude extract of A. roxburghii, the analytical condition of liquid chromatography and mass spectrometry analysis have been investigated systematically. RRLC-MS/MS and RRLC-MSE analytical methods were then established and allowed the identification of seventeen constituents, including four aromatic acids, five flavonoid aglycones, five flavonoid mono-O-glycosides, two aromatic acids and p-hydroxybenzaldehyde, of which three constituents were discovered in the plant for the first time. The results indicated that rapid identification of flavonoids, aromatic acids and other constituents in A. roxburghii could be achieved using RRLC-MS/MS and RRLC-MSE2. Study of NMR/RRLC-MS PDS and NMR/RRLC-MS HCS on crude extract of Anoectochilus roxburghiiBased on the incomplete separation analysis strategy, the crude extract of A. roxburghii was separated into a series of fractions with the concentration of constituent dynamic variation using reversed-phase preparative chromatography. The RRLC-MS and 1H NMR data of series of fractions were acquired and then processed by MATLAB to form NMR/RRLC-MS PDS and NMR/RRLC-MS HCS. The correlation relationship between 1H NMR and the extracted ion chromatogram (XIC) signals deriving from the same individual constituent could be correlated and extracted simultaneous from the mixture spectra. Combined with RRLC-MS/MS spectra, three kinds of natural products were then successfully identified, including eight flavonoids, four aromatic acids and p-hydroxybenzaldehyde, five of which have not previously been reported in A. roxburghii. The results highly proved that this approach should be of benefit in unequivocal structural determination of a variety of classes of compounds from an extremely complex mixture, such as herbs and their active extracts.3. Comparison on the main constituents and their contents between the wild Anoectochilus roxburghii and the artificial breeding speciesAn RRLC-MS/MS method was established to compare the wild A. roxburghii with the artificial breeding species on main constituents and their contents. First of all, sensitivity, precision, calibration curves and accuracy of the methods have been studied systematically. The results indicated that RRLC-(-)ESI-MS/MS analysis methods in negative ion mode could quantify the main constituents accurately and exactly. Nine artificial breeding species (J1-J9) were then compared with wild A. roxburghii (JO)on constituents using this method, and it was found that Jl and J5 is almost the same with JO on constituents. Furthermore, qualitiation of twenty constituents and quantification of six constituents in wild and artificial breeding A. roxburghii were implemented, and the results indicated J5 and J6 were found to have almost the same constituents and their constants with JO, while J2 showed great difference from that of JO. In all, analytical method for content assessment of artificial breeding species was established, and the results provided important scientific basis for screening and evaluation of artificial cultivation technologies.