| Poria cocos (Schw.) Wolf is a widely used traditional Chinese medicine. It is a tonic according to Hunag Di Nei Jing. BFL (inner parts), FLP (epidermis) and FS (with pine root in the inner part) have different therapeutic applications in clinic according to the Chinese Pharmacopoeia (2010 edition). BFL is good at strengthening the spleen (according to Shen Nong Ben Cao Jing), FS can calm down the nerve (according to Ming Yi Bie Lu), FLP can increase urination (according to Ben Cao Gang Mu). What is the basis of their different clinical effects? It is worthwhile to be disclosed.This research provided a good model about disclosing the scientific basis for those traditional Chinese medicines which originate from the same plant but possessing different clinical therapy. In the present study, isolation of standard compounds, comparison study of lanostane acid type compounds and difference expression analysis of key unigene in transcriptome between FLP and BFL were carried out. Obvious difference of chemical constituent was observed between FLP and BFL, genes that played important role on generating 3,4-seco-lanostane-type triterpene acid were predicted and the pathway of its generation was proposed.The research work was carried out as follows:Firstly, fourteen standard compounds were isolated from FLP and BFL, and their structures were determined based on the analysis of MS,1H-13C-NMR analysis, they are pachymic acid, dehydrotumulosic acid,3-epi-dehydrotumulosic acid, dehydropachymic acid,3-epi-dehydropachymic acid, polyporenic acid C, trametenolic acid, dehydrotrametenolic acid, eburiconic acid, dehydroeburiconic acid, poricoic acid A, poricoic acid B, poricoic acid D and 5R,8R-peroxydehydrotumulosic acid.Then the MSn characteristic of lanostane-type triterpene acids was established as follows based on the research of the MS behaviour of standard compounds:(1) Determination of molecular weight (MW):precursor ion [M+H-18]+ was displayed in the positive ion mode, while in the negative ion mode, precursor ion [M-H]- was observed for those compounds with hydroxyl moieties at both C3 and C16 positions. While for compounds with only one hydroxy moiety at C3 position, precursor ion [M+H]+ was observed in the positive ion mode. (2) Determination of compound skeleton:characteristic ion with value of m/z:295 indicated the skeleton of △8-lanostane-type triterpene acids; characteristic ion with value of m/z:293 suggested the skeleton of △ 7,9(11)-lanostane-type triterpene acids; 3-oxo-△7,9(11)-lanostane-type triterpene acids (such as the skeleton of polyporenic acid C) displayed the characteristics ion with the value of m/z:309; while for all the 3,4-seco-lanostane-type triterpene acid displayed neutral loss of C3H6O2 or C4H8O2 (Am=74 or 88 Da) at C10 position in the negative ion mode. (3) Determination of substituent:neutral loss of C9H16O2/ C8H14O2 (Am=156/148 Da) indicated the substituent at C17 (△24(31) or △24 segments). (4) Determination of the stereoscopic position of the substituent at C3:for the compound with substituents at C3, C16, C17, if the ion produced via the ion transition [M+H-(C3-moiety)-(C16-moiety)]+ exhibited stronger ion intensity than the ion obtained from the ion transition [M+H-(C3-moiety)-(C17-moiety)]+, it could be proposed that the moiety at C3 position processed a configuration. While if the ion produced via ion transition [M+H-(C3-moiety)-(C17-moiety)]+ displayed stronger than the ion [M+H-(C3-moiety)-(C16-moiety)]+, moiety at C-3β was proposed.Comparison study of the chemical constituent array in FLP and BFL was carried out based on the compound identification and quantification analysis. UHPLC-DAD-FT/MSn had been used to identify the triterpene acids presented in the fungus based on a detailed analysis of the fragmentation behaviors of thirteen standard compounds. This analysis allowed for the identification of 27 triterpene acids, including five groups of isomers (15 compounds) and four potential new compounds. Furthermore, a UHPLC-MS/MS method had been developed for quantifying the amounts of nine bioactive triterpene acids in samples of the FLP and BFL, including three 3,4-seco-lanostane-type triterpene acids. These results revealed significant differences in the amounts of these compounds in the FLP and BFL samples (p<0.001). Principal component analysis (PCA) and partial least squares discriminant analysis (PLSA) of the results for the FLP and BFL samples clearly demonstrated that dehydrotumulosic acid, trametenolic acid, dehydrotrametenolic acid and poricoic acid A were the main compounds contributing to the clusters in FLP and BFL.Transcriptome data study of FLP and BFL were carried out to find out causes of chemical compositions diference in FLP and BFL. Based on the organic synthesis theory and the cases in biosynthesis, the key genes were proposed and the path way of the generation of 3,4-seco-lanostane-type triterpene acids was assumed. Then the target genes were selected from the transcriptome data, and semi-real time PCR (semi-RTPCR) was carried out about the target genes. The expression of the target genes in FLP and BFL was achieved and the relationship between the expression of the target genes and the contents of lanostane-type triterpene acids were studied. The result showed that when the content of polyporenic acid C was low, the expression of the target genes was high, while the content of poricoic acid B was high simultaneously. Thus, the target genes would be contributed to the product of 3,4-seco-lanostane-type triterpene acid. In this way, the pathway that the 3,4-seco-lanostane-type triterpene acids generating was disclosed in a certain degree. It is the first time to disclose the mechanism of the production of 3,4-seco-lanostane-type triterpene acids in Poria cocos (Schw.) Wolf.Summarized the above research work, obvious difference of chemical array in FLP and BFL was observed, key genes that played important role on the generation of 3,4-seco-lanostane-type triterpene acid were predicted and selected from transcriptome data. This work provided an example on how to carry out the research work for those traditional Chinese medicines which originate from the same plant but prossessing different clinical therapy. |
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