A novel method for ginkgolide biosynthesis elucidation based on MeJA induction and differential metabolomics.

Ginkgolides from Ginkgo Biloba have significantly therapeutic effect to cardiovascular and cerebrovascular diseases. However, the biosynthetic pathway of ginkgolides has not been fully elucidated until now. As ginkgolides are synthesized in the ginkgo roots, the accumulation of ginkgolides intermediate metabolites varies greatly between roots and leaves. As Methyl jasmonate (MeJA) can effectively enhance the biosynthesis of ginkgolides, a novel method based on MeJA induction and differential metabolomics was used to screen the differentially intermediate metabolites among ginkgo leaves, roots and roots-MJ-3. Two differential intermediate metabolites (dehydroabietadienal and 1, 2, 3, 4, 4a, 9, 10, 10a-Octahydro-6-hydroxy-7-isopropyl-1, 4a-dimethyl-1-phenanthrenemethanol) were identified in ginkgo roots by UPLC-QTOF-MS. Then, a new ginkgolides biosynthetic pathway was proposed based on differential metabolomics. This study provides a novel method for the elucidation of nature product precursor and is helpful to promote the clarification of ginkgolides biosynthetic pathway.

Comparative transcriptome analysis of MeJA-responsive AP2/ERF transcription factors involved in notoginsenosides biosynthesis.

Differential transcriptome analysis is an effective method for gene selection of triterpene saponin biosynthetic pathways. MeJA-induced differential transcriptome of Panax notoginseng has not been analyzed yet. In this study, comparative transcriptome analysis of P. notoginseng roots and methyl jasmonate (MeJA)-induced roots revealed 83,532 assembled unigenes and 21,947 differentially expressed unigenes. Sixteen AP2/ERF transcription factors, which were significantly induced by MeJA treatment in the root of P. notoginseng, were selected for further analysis. Real-time quantitative PCR (RT-qPCR) and co-expression network analysis of the 16 AP2/ERF transcription factors showed that PnERF2 and PnERF3 had significant correlation with dammarenediol II synthase gene (DS) and squalene epoxidase gene (SE), which are key genes in notoginsenoside biosynthesis, in different tissues and MeJA-induced roots. A phylogenetic tree was conducted to analyze the 16 candidate AP2/ERF transcription factors and other 38 transcription factors. The phylogenetic tree analysis showed PnERF2, AtERF3, AtERF7, TcERF12 and other seven transcriptional factors are in same branch, while PnERF3 had close evolutionary relationships with AtDREB1A, GhERF38 and TcAP2. The results of comparative transcriptomes and AP2/ERF transcriptional factors analysis laid a solid foundation for further investigations of disease resistance and notoginsenoside biosynthesis in P. notoginseng.

[Development of Tianma HPLC fingerprint and discriminant analysis].

Tianma(the tuber of Gastrodia eleta) is a widely used and pricy Chinese herb. Its counterfeits are often found in herbal markets, which are the plant materials with similar macroscopic characteristics of Tianma. Moreover, the prices of Winter Tianma(cultivated Tianma) and Spring Tianma(mostly wild Tianma) have significant difference. However, it is difficult to identify the true or false, good or bad quality of Tianma samples. Thus, a total of 48 Tianma samples with different characteristics(including Winter Tianma, Spring Tianma, slice, powder, etc.) and 9 plant species 10 samples of Tianma counterfeits were collected and analyzed by HPLC-DAD-MS techniques. After optimizing the procedure of sample preparation, chromatographic and mass-spectral conditions, the HPLC chromatograms of all those samples were collected and compared. The similarities and Fisher discriminant analysis were further conducted between the HPLC chromatograms of Tianma and counterfeit, Winter Tianma and Spring Tianma. The results showed the HPLC chromatograms of 48 Tianma samples were similar at the correlation coefficient more than 0.848(n=48). Their mean chromatogram was simulated and used as Tianma HPLC fingerprint. There were 11 common peaks on the HPLC chromatograms of Tianma, in which 6 main peaks were chosen as characteristic peaks and identified as gastrodin, p-hydroxybenzyl alcohol, parishin A, parishin B, parishin C, parishin E, respectively by comparison of the retention time, UV and MS data with those of standard chemical compounds. All the six chemical compounds are bioactive in Tianma. However, the HPLC chromatograms of the 10 counterfeit samples were significantly different from Tianma fingerprint. The correlation coefficients between HPLC fingerprints of Tianma with the HPLC chromatograms of counterfeits were less than 0.042 and the characteristic peaks were not observed on the HPLC chromatograms of these counterfeit samples. It indicated the true or false Tianma can be identified by either the similarity or characteristic peaks on HPLC fingerprint. Comparing the Winter Tianma with Spring Tianma showed that the HPLC chromatograms of 15 winter Tianma samples and 11 spring Tianma samples were similar at the mean correlation coefficient of 0.908. But the intensity of the characteristic peaks were different between the two groups of Tianma samples, i.e. the intensity of gastrodin, paishin A and C in winter Tianma was lower than those in spring Tianma. The Winter Tianma and Spring Tianma could be discriminated by either the Fisher unstandardized discrimination function or Linear discriminant function, based on the peak areas of 11 common peaks on HPLC chromatograms as variate.