A Global Analysis of Alternative Splicing of Dichocarpum Medicinal Plants, Ranunculales.

Background: The multiple isoforms are often generated from a single gene via Alternative Splicing (AS) in plants, and the functional diversity of the plant genome is significantly increased. Despite well-studied gene functions, the specific functions of isoforms are little known, therefore, the accurate prediction of isoform functions is exceedingly wanted. Methods: Here we perform the first global analysis of AS of Dichocarpum, a medicinal genus of Ranunculales, by utilizing full-length transcriptome datasets of five Chinese endemic Dichocarpum taxa. Multiple software were used to identify AS events, the gene function was annotated based on seven databases, and the protein-coding sequence of each AS isoform was translated into an amino acid sequence. The self-developed software DIFFUSE was used to predict the functions of AS isoforms. Results: Among 8,485 genes with AS events, the genes with two isoforms were the most (6,038), followed by those with three isoforms and four isoforms. Retained intron (RI, 551) was predominant among 1,037 AS events, and alternative 3' splice sites and alternative 5' splice sites were second. The software DIFFUSE was effective in predicting functions of Dichocarpum isoforms, which have not been unearthed. When compared with the sequence alignment-based database annotations, DIFFUSE performed better in differentiating isoform functions. The DIFFUSE predictions on the terms GO:0003677 (DNA binding) and GO: 0010333 (terpene synthase activity) agreed with the biological features of transcript isoforms. Conclusion: Numerous AS events were for the first time identified from full-length transcriptome datasets of five Dichocarpum taxa, and functions of AS isoforms were successfully predicted by the self-developed software DIFFUSE. The global analysis of Dichocarpum AS events and predicting isoform functions can help understand the metabolic regulations of medicinal taxa and their pharmaceutical explorations.

Metabolite Profiling Based on UPLC-Q-TOF-MS/MS and the Biological Evaluation of Medicinal Plants of Chinese Dichocarpum (Ranunculaceae).

The genus Dichocarpum is endemic to East Asia, and many species have been used to treat various diseases. However, phytochemical researches of this genus have been limited to date. In the present study, a metabolomic approach based on UPLC-Q-TOF-MS/MS was used to explore the phytochemical profiles of 10 Chinese Dichocarpum species, and cannabinoid receptor (CB1/CB2) agonistic activities evaluation of these plants was performed. A total of 128 features were putatively annotated, belonging to alkaloids, flavonoids, triterpenes saponins, phenolic acids, and others. Semi-quantitative statistics demonstrated that alkaloids and flavonoids were widely distributed, with the former the most abundant, whereas triterpenes saponins were mainly distributed in D. fargesii and D. wuchuanense. The phylogenetic results obtained from DNA sequencing assigned the 10 species to three groups. Further results of in silico annotation revealed three chemical families and helped determine the characteristic features of the three groups. In addition, the plant extracts of nine species from this genus showed agonistic activity on CB2 receptors. This comprehensive analysis revealed the chemotype distribution and pharmacophylogenetic relationship, to provide clues for the prospective resource utilization of the medicinal plants from the genus Dichocarpum.

A molecular phylogeny of Dichocarpum (Ranunculaceae): Implications for eastern Asian biogeography.

East Asia is characterized by high levels of species diversity and endemism. However, the biogeographical patterns and processes underlying the distribution of biodiversity within the area are still poorly known. In this study, we used plastid (matK, trnL-F, and trnH-psbA) and nuclear (ITS) DNA sequences to investigate the historical biogeography of Dichocarpum (Ranunculaceae), an eastern Asian endemic genus throughout warm-temperate and subtropical forests of the area. Phylogenetic analyses strongly support Dichocarpum as monophyletic, which contains two major clades. Clade I corresponds to section Hutchinsonia, and clade II includes sections Dichocarpum and Fargesia. Section Dichocarpum and its subsections Dalzielia and Dichocarpum are not recognized as monophyletic. Our results suggest that the most recent common ancestor of Dichocarpum occurred in central China and Japan in the earliest Early Miocene, and thus support an ancient vicariance event between Japan and China. Within mainland China, three migrations at the species level were hypothesized to explain the expansion of Dichocarpum from central China to southeastern Yunnan, Hengduan mountains, and eastern Himalaya. These migration events occurred in the Late Miocene to Early Pliocene, which may be associated with the uplift of the southeastern Qinghai-Tibetan Plateau and accordingly the expansion of subtropical forests in China around that period. A migration or dispersal from central China to Taiwan was inferred in the Early Pleistocene, which supports the close floristic affinity between Taiwan and mainland China. This study contributes to our knowledge on the historical biogeography of plants in eastern Asia.

Dissection of full-length transcriptome and metabolome of Dichocarpum (Ranunculaceae): implications in evolution of specialized metabolism of Ranunculales medicinal plants.

Several main families of Ranunculales are rich in alkaloids and other medicinal compounds; many species of these families are used in traditional and folk medicine. Dichocarpum is a representative medicinal genus of Ranunculaceae, but the genetic basis of its metabolic phenotype has not been investigated, which hinders its sustainable conservation and utilization. We use the third-generation high-throughput sequencing and metabolomic techniques to decipher the full-length transcriptomes and metabolomes of five Dichocarpum species endemic in China, and 71,598 non-redundant full-length transcripts were obtained, many of which are involved in defense, stress response and immunity, especially those participating in the biosynthesis of specialized metabolites such as benzylisoquinoline alkaloids (BIAs). Twenty-seven orthologs extracted from trancriptome datasets were concatenated to reconstruct the phylogenetic tree, which was verified by the clustering analysis based on the metabolomic profile and agreed with the Pearson correlation between gene expression patterns of Dichocarpum species. The phylogenomic analysis of phytometabolite biosynthesis genes, e.g., (S)-norcoclaurine synthase, methyltransferases, cytochrome p450 monooxygenases, berberine bridge enzyme and (S)-tetrahydroprotoberberine oxidase, revealed the evolutionary trajectories leading to the chemodiversity, especially that of protoberberine type, aporphine type and bis-BIA abundant in Dichocarpum and related genera. The biosynthesis pathways of these BIAs are proposed based on full-length transcriptomes and metabolomes of Dichocarpum. Within Ranunculales, the gene duplications are common, and a unique whole genome duplication is possible in Dichocarpum. The extensive correlations between metabolite content and gene expression support the co-evolution of various genes essential for the production of different specialized metabolites. Our study provides insights into the transcriptomic and metabolomic landscapes of Dichocarpum, which will assist further studies on genomics and application of Ranunculales plants.

Screening of acetylcholinesterase inhibitors and characterizing of phytochemical constituents from Dichocarpum auriculatum (Franch.) W.T. Wang & P. K. Hsiao through UPLC-MS combined with an acetylcholinesterase inhibition assay in vitro.

ETHNOPHARMACOLOGICAL RELEVANCE: The genus Dichocarpum is endemic to East Asia, and many of them are traditionally used folk medicine in China. Dichocarpum auriculatum (Franch.) W. T. Wang et P. K. Hsiao has the effect of clearing away heat, removing toxicity, and relieving swelling in southwestern China. Intriguingly, its root and whole herb also used as remedy for the neurological disease epilepsy. However, there are not any scientific reports on the phytochemistry and pharmacological activities of D. auriculatum. AIM OF STUDY: Traditional and folk medicinal knowledge would be useful for finding new pharmaceutical resources. There are many evidences over the years reported that an interaction probably exists between epilepsy and Alzheimer's disease (AD). The aim of the study was to investigate the potential AChE inhibitors and the phytochemical profiles of the specie D. auriculatum. MATERIALS AND METHODS: The AChE inhibitory activity of plant extracts of D. auriculatum and other 6 species from different regions of the genus Dichocarpum were evaluated in vitro assays and the UPLC-Q-TOF-MS technique was used to analyze the chemical constituents. Moreover, UPLC-ESI-MS/MS was used to determine the distribution of 12 standard compounds in samples. RESULTS: As a preferred source of potential acetylcholinesterase inhibitors of the genus Dichocarpum, D. auriculatum has been further investigated. The screening results show that the ability of root extracts from D. auriculatum (IC50 = 0.15 mg·mL-1) to inhibit AChE was better than other samples, it is consistent with traditional medicinal records. The phytochemical constituents of D. auriculatum was surveyed firstly by UPLC-Q-TOF-MS analysis, and 36 compounds, including 14 alkaloids, 16 flavonoids, 6 others, were identified tentatively. Further experiments showed that five compounds (columbamine, palmatine, dauricine, jatrorrhizine and berberine) from D. auriculatum were confirmed the potential inhibition of AChE activity in vitro (IC50: 0.24-6.37 µM) and UPLC-ESI-MS/MS results showed that the content of most active compounds in roots was much higher than in aerial parts. Palmatine (IC50 = 0.34 µM) and columbamine (IC50 = 0.24 µM) showed prominent AChE inhibitory activity among the tested compounds. CONCLUSIONS: This is the first report about the evaluation of AChE inhibitory activity and phytochemical profiles of D. auriculatum, led to the identification of 36 compounds including alkaloids and flavonoids, and five alkaloids exhibited a significant AChE inhibitory activity and had the potential as AChE inhibitors. This study provided scientific experimental basis for the traditional efficacy of neurological disease of the plant.