| Cortex Lycii, the root bark of Lycium chinense Mill. or Lycium barbarum L., is documented in Chinese Pharmacopoeia (2010 version). It has been widely used in China as an antipyretic and for the treatment of pneumonia, night-sweats, cough, hematemesis, inflammation, and diabetes mellitus. In the present thesis, the chemical constituents and quality control methods of the titled herb were investigated.Using various chromatographic methods, a total of 52 compounds were isolated from the root bark of L. chinense. On the basis of their spectral, the structures of 48 compounds were identified, including 8 phenolic acids, 9 phenylpropanoids, 3 flavonoids, 16 phenolic amides, 4 cyclic peptides and 8 other types of compounds. Among them, compounds 1-8 are identified as new compounds, named (E)-3-{ (2,3-trans)-2-(4-hydroxy-3-methoxyphenyl)-3-hydroxymethyl-2,3-dihydro-benzo[b][1,4]dioxin-6-yl}-N-(4-hydroxyphenethyl)acrylamide (1), (Z)-3-{(2,3-trans)-2-(4-hydroxy-3-methoxyphenyl)-3-hydroxymethyl-2,3-dihydrobenzo[b] [ 1,4]dioxin-6-yl}-N-(4-hydroxyphenethyl)acrylamide (2), (2,3-trans)-3-(3-hydroxy-5-methoxy-phenyl)-N-(4-hydroxyphenethyl)-7- { (E)-3-[(4-hydroxyphenethyl)amino]-3-oxoprop-1-en-1-yll-2,3-dihydrobenzo[b][1,4]dioxine-2-carboxamide (3), (2,3-trans)-3-(3-hydroxy-5-methoxyphenyl)-N-(4-hydroxyphenethyl)-7- { (Z)-3-[(4-hydroxy-phenethyl)amino]-3-oxoprop-1-en-1-yl } -2,3-dihydrobenzo[b] [ 1,4]dioxine-2-carboxamide (4). (E)-2-(4,5-dihydroxy-2-{3-[(4-hydroxyphenethyl)amino]-3-oxopropyl)phenyl)-3-(4-hydroxy-3,5-dimethoxyphenyl)-N-(4-hydroxyphenethyl)-acrylamide (5). (E)-2-(4,5-dihydroxy-2-{3-[(4-hydroxyphenethyl)amino]-3-oxopropyl }phenyl)-3-(4-hydroxy-3,5-dimethoxyphenyl)-N-(4-acetamidobutyl)-acrylamide (6), (E)-2-(4,5-dihydroxy-2-{3-[(4-hydroxyphenethyl)amino]-3-oxopropyl )phenyl)-3-(4-hydroxy-3-methoxyphenyl)-N-(4-acetamidobutyl)acrylamide (7). (1,2-trans)-N3-(4-acetamidobutyl)-1-(3,4-dihydroxyphenyl)-7-hydroxy-N2-(4-hydroxyphenethyl)-6,8-dimethoxy-1,2-dihydro-naphthalene-2,3-dicarboxamide (8). Twelve compounds were firstly isolated from the genus. The cinnamic acid amides, neolignanamides, and lignanamides possess moderate radical-scavenging activity against the DPPH (2,2-diphenyl-l-picrylhydrazyl) and superoxide radicals. No hypoglycemic activity was observed among cyclic peptides with AMP-activated protein kinase (AMPK) and protein tyrosine phosphatase 1B model.The fragmentation behaviors of phenolic amides and cyclic peptides from the Cortex Lycii were systematically studied by using LTQ-Orbitrap mass spectrometer. Several characteristic pathways were summarized, which were useful in distinguishing the compounds and unknowns with similar structures. Using UHPLC-PDA-ESI-HRMS,39 compounds including 12 new ones were tentatively characterized from the phenolic amides extract of Cortex Lycii, of which 16 were confirmed by comparison with the standards. In addition, using the fragmentation pathways of cyclic peptides,4 structures with the same type were deduced.A fast HPLC-ESI-MS/MS method was developed for the determination of 24 components including 11 phenolic compounds,9 phenolic amides, and 4 cyclic peptides from Cortex Lycii. The analytes were quantified by a triple quadrupole instrument in multiple reaction monitoring (MRM) mode. This assay method was fully validated and applied to analyze 28 batches of Cortex Lycii. The results indicated that cyclic peptides and phenolic amides were not only the abundant constituents, but also the characteristic components for Cortex Lycii to distinguish with the adulterants. Principle component analysis (PCA) was used to discriminate samples from different geographical regions of China, and cyclic peptides were considered to be the chemical markers responsible for the classification. The highest content of lyciumin A was found in most samples except for those from Anhui and Henan provinces, while the content of lyciumin D in these samples were obviously higher than in others especially from Ningxia and Gansu provinces. With respect to lyciumin B, it was quantified in all the certified samples and the content was relatively higher in the samples from Shanxi province. The systematic and integrated assessment of Cortex Lycii provides sufficient evidence for the establishment of the quality standard.Based on the systematic isolation and analysis of chemical composition of Cortex Lycii, as well as a comprehensive investigation of the pharmacological references, a scientific and rational quality control method was established in the present study. Qualitative analysis was developed by HPTLC identification and HPLC characteristic profiling methods, taking cyclic peptides and phenolic amides as the chemical markers, respectively. Quantitative analysis was established by HPLC-UV method using lyciumins A and B (two cyclic peptides with large amount) as the target analytes. Based on the analysis of the contents in different samples, the quality standard was recommended by the total content of lyciumins A and B. We believed that the proposed methods could control the quality of Cortex Lycii effectively in the routine use.Counter current chromatography (CCC) method was used in the isolation of lyciumins A-D, the markers for the quality evaluation of Cortex Lycii. The four compounds were directly isolated in one step from the crude extract with HPLC purity of above 90%. The present method is much convenient and easier than the conventionally separative methods.In conclusion, through a comprehensive research, we are now in better situation to understand the chemical constituents of Cortex Lycii. The scientific and rational quality control method would play an important role in the routine analysis and reasonable use in the herbal medicine market. |
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