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Background: The present study aimed to investigate the protective effect of the water extract of Amydrium sinense (Engl.) H. Li (ASWE) against hepatic fibrosis (HF) and clarify the underlying mechanism.Methods: The chemical components of ASWE were analysed by a Q-Orbitrap high-resolution mass spectrometer. In our study, an in vivo hepatic fibrosis mouse model was established via an intraperitoneal injection of olive oil containing 20% CCl4. In vitro experiments were conducted using a hepatic stellate cell line (HSC-T6) and RAW 264.7 cell line. A CCK-8 assay was performed to assess the cell viability of HSC-T6 and RAW264.7 cells treated with ASWE. Immunofluorescence staining was used to examine the intracellular localization of signal transducer and activator of transcription 3 (Stat3). Stat3 was overexpressed to analyse the role of Stat3 in the effect of ASWE on HF.Results: Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) analyses showed that candidate targets of ASWE, associated with protective effects against hepatic fibrosis, were related to inflammation response. ASWE ameliorated CCl4-induced liver pathological damage and reduced the liver index and alanine transaminase (ALT) and aspartate transaminase (AST) levels. ASWE also decreased the serum levels of collagen Ⅰ (Col Ⅰ) and hydroxyproline (Hyp) in CCl4-treated mice. In addition, the expression of fibrosis markers, including α-SMA protein and Acta2, Col1a1, and Col3a1 mRNA, was downregulated by ASWE treatment in vivo. The expression of these fibrosis markers was also decreased by treatment with ASWE in HSC-T6 cells. Moreover, ASWE decreased the expression of inflammatory markers, including the Tnf-α, Il6 and Il1β, in RAW264.7 cells. ASWE decreased the phosphorylation of Stat3 and total Stat3 expression and reduced the mRNA expression of the Stat3 gene in vivo and in vitro. ASWE also inhibited the nuclear shuttling of Stat3. Overexpression of Stat3 weakened the therapeutic effect of ASWE and accelerated the progression of HF.Conclusion: The results show that ASWE protects against CCl4-induced liver injury by suppressing fibrosis, inflammation, HSC activation and the Stat3 signaling pathway, which might lead to a new approach for preventing HF.

Pogostemon cablin is an important aromatic medicinal herb widely used in the pharmaceutical and perfume industries. However, our understanding of the phytochemical compounds and metabolites within P. cablin remains limited. To our knowledge, no integrated studies have hitherto been conducted on the metabolites of the aerial parts of P. cablin. In this study, twenty-three volatile compounds from the aerial parts of P. cablin were identified by GC-MS, predominantly sesquiterpenes. Quantitative analysis showed the highest level of patchouli alcohol in leaves (24.89 mg/g), which was 9.12 and 6.69-fold higher than in stems and flowers. UHPLC-QTOFMS was used to analyze the non-volatile compounds of leaf, stem and flower tissues. The differences in metabolites between flower and leaf tissues were the largest. Based on 112, 77 and 83 differential metabolites between flower-leaf, flower-stem and leaf-stem, three tissue-specific biomarkers of metabolites were identified, and the differential metabolites were enriched in several KEGG pathways. Furthermore, labeling differential metabolites in the primary and secondary metabolic pathways showed that flowers accumulated more lipids and amino acids, including proline, lysine and tryptophan; the leaves accumulated higher levels of terpenoids, vitamins and flavonoids, and stems contained higher levels of carbohydrate compounds. Based on the role of acetyl coenzyme A, the distribution and possible exchange mechanism of metabolites in leaves, stems and flowers of P. cablin were mapped for the first time, laying the groundwork for future research on the metabolites in P. cablin and their regulatory role.

Farnesyl pyrophosphate synthase (FPPS) plays an important role in the synthesis of plant secondary metabolites, but its function and molecular regulation mechanism remain unclear in Pogostemon cablin. In this study, the full-length cDNA of the FPP synthase gene from P. cablin (PcFPPS) was cloned and characterized. The expressions of PcFPPS are different among different tissues (highly in P. cablin flowers). Subcellular localization analysis in protoplasts indicated that PcFPPS was located in the cytoplasm. PcFPPS functionally complemented the lethal FPPS deletion mutation in yeast CC25. Transient overexpression of PcFPPS in P. cablin leaves accelerated terpene biosynthesis, with an ~47% increase in patchouli alcohol. Heterologous overexpression of PcFPPS in tobacco plants was achieved, and it was found that the FPP enzyme activity was significantly up-regulated in transgenic tobacco by ELISA analysis. In addition, more terpenoid metabolites, including stigmasterol, phytol, and neophytadiene were detected compared with control by GC-MS analysis. Furthermore, with dual-LUC assay and yeast one-hybrid screening, we found 220 bp promoter of PcFPPS can be bound by the nuclear-localized transcription factor PcWRKY44. Overexpression of PcWRKY44 in P. cablin upregulated the expression levels of PcFPPS and patchoulol synthase gene (PcPTS), and then promote the biosynthesis of patchouli alcohol. Taken together, these results strongly suggest the PcFPPS and its binding transcription factor PcWRKY44 play an essential role in regulating the biosynthesis of patchouli alcohol.

Ilex asprella is a plant from Aquifoliaceae. Its root is commonly used as folk medicinal materials in southern China. The chemical compositions of I. asprella are rich in pentacyclic triterpenoids, which show various biological activities and demonstrate a good prospect for drug development. The elucidation of biosynthesis mechanism of triterpenoids in I. asprella could lay important foundations for the production of these precious plant secondary metabolites by metabolic engineering. Our previous studies have revealed IaAO1 (a CYP716A210 homolog) responsible for the C-28 oxidation of α- and β-amyrin. Herein, we reported the identification of three more cytochrome P450 monooxygenase genes IaAO2 (a CYP716A212 homolog), IaAO4 (CYP714E88), IaAO5 (CYP93A220), and a cytochrome P450 reductase gene IaCPR by using Saccharomyces cerevisiae eukaryotic expression system and gas chromatography-mass spectrometry. Among them, the protein encoded by IaAO2 can catalyze the C-28 oxidation of α-amyrin and β-amyrin, IaAO4 can catalyze the C-23 oxidation of ursolic acid and oleanolic acid, while IaAO5 is responsible for the C-24 oxidation of β-amyrin. By introducing three genes IaAO1, IaAO4 and IaCPR into S. cerevisiae. We constructed an engineered yeast strain that can produce C-23 hydroxyl ursane-type triterpenoid derivatives. This study contributes to a thorough understanding of triterpenoid biosynthesis of medicinal plants and provides important tools for further metabolic engineering.

Bornyl acetate (BA) is known as a natural aromatic monoterpene ester with a wide range of pharmacological and biological activities. Borneol acetyltransferase (BAT), catalyzing borneol and acetyl-CoA to synthesize BA, is alcohol acetyltransferase, which belongs to the BAHD super acyltransferase family, however, BAT, responsible for the biosynthesis of BA, has not yet been characterized. The seeds of Wurfbainia villosa (homotypic synonym: Amomum villosum) are rich in BA. Here we identified 64 members of the BAHD gene family from the genome of W. villosa using both PF02458 (transferase) and PF07247 (AATase) as Hidden Markov Model (HMM) to screen the BAHD genes. A total of sixty-four WvBAHDs are distributed on 14 chromosomes and nine unanchored contigs, clustering into six clades; three WvBAHDs with PF07247 have formed a separated and novel clade: clade VI. Twelve candidate genes belonging to clade I-a, I-b, and VI were selected to clone and characterize in vitro, among which eight genes have been identified to encode BATs acetylating at least one type of borneol to synthesize BA. All eight WvBATs can utilize (−)-borneol as substrates, but only five WvBATs can catalyze (+)-borneol, which is the endogenous borneol substrate in the seeds of W. villosa; WvBAT3 and WvBAT4 present the better catalytic efficiency on (+)-borneol than the others. The temporal and spatial expression patterns of WvBATs indicate that WvBAT3 and WvBAT4 are seed-specific expression genes, and their expression levels are correlated with the accumulation of BA, suggesting WvBAT3 and WvBAT4 might be the two key BATs for BA synthesis in the seeds of W. villosa. This is the first report on BAT responsible for the last biosynthetic step of BA, which will contribute to further studies on BA biosynthesis and metabolism engineering of BA in other plants or heterologous hosts.

Ilex asprella is a medicinal plant that is used extensively in southern China. The plant contains ursane-type triterpenoids and triterpenoid saponins which are known to be responsible for its pharmacological activities. Previously, a transcriptomic analysis of I. asprella was carried out and the gene IaAS1, which is important in the formation of the core structure α-amyrin, was identified. However, the genes related to the subsequent derivatization of the core structures of the triterpenoid remain largely unknown. Herein, we describe the cloning and functional characterization of an amyrin 28-carboxylase IaAO1 (designated as IaCYP716A210) and a glycosyltransferase IaAU1 (designated as UGT74AG5), based on transcriptomic data. The expression of IaAO1 in an α-amyrin producing yeast strain led to the accumulation of ursolic acid. An enzyme assay using recombinant protein IaAU1 purified from E. coli revealed that IaAU1 can catalyze the conversion of ursolic acid to ursolic acid 28-O-β-D-glucopyranoside. IaAU1 has regiospecificity for catalyzing the 28-O-glucosylation of ursane-/oleanane-type triterpene acids, as it can also catalyze the conversion of oleanolic acid, hederagenin, and ilexgenin A to their corresponding glycosyl compounds. Moreover, co-expression of IaAO1 and IaAU1 in the α-amyrin-producing yeast strain led to the production of ursolic acid 28-O-β-D-glucopyranoside, although in relatively low amounts. Our study reveals that IaAO1 and IaAU1 might play a role in the biosynthesis of pentacyclic triterpenoid saponins in I. asprella and provides insights into the potential application of metabolic engineering to produce ursane-type triterpene glycosides.

Native to Southern China, Ilex pubescens and Ilex asprella are frequently used in traditional Chinese medicine. Both of them produce a large variety of ursane-type triterpenoid saponins, which have been demonstrated to have different pharmacological effects. However, little is known about their biosynthesis. In this study, transcriptomic analysis of I. pubescens and comparison with its closely related specie I. asprella were carried out to identify potential genes involved in triterpenoid saponin biosynthesis. Through RNA sequencing (RNA-seq) and de novo transcriptome assembly of I. pubescens, a total of 68,688 UniGene clusters are obtained, of which 32,184 (46.86%) are successfully annotated by comparison with the sequences in major public databases (NCBI, Swiss-Prot, and KEGG). It includes 128 UniGenes related to triterpenoid backbone biosynthesis, 11 OSCs (oxidosqualene cyclases), 233 CYPs (cytochrome P450), and 269 UGTs (UDP-glycosyltransferases). By homology-based blast and phylogenetic analysis with well-characterized genes involved in triterpenoid saponin biosynthesis, 5 OSCs, 14 CYPs, and 1 UGT are further proposed as the most promising candidate genes. Transcriptomic comparison between two Ilex species using blastp and OrthoMCL method reveals high sequence similarity. All OSCs and UGTs as well as most CYPs are classified as orthologous genes, while only 5 CYPs in I. pubescens and 3 CYPs in I. asprella are species-specific. One of OSC candidates, named as IpAS1, was successfully cloned and expressed in Saccharomyces cerevisiae INVSc1. Analysis of the yeast extract by gas chromatography (GC) and gas chromatography–mass spectrometry (GC-MS) shows IpAS1 is a mixed amyrin synthase, producing α-amyrin and β-amyrin at ratio of 5:1, which is similar to its ortholog IaAS1 from I. asprella. This study is the first exploration to profile the transcriptome of I. pubescens, the generated data and gene models will facilitate further molecular studies on the physiology and metabolism in this plant. By comparative transcriptomic analysis, a series of candidate genes involved in the biosynthetic pathway of triterpenoid saponins are identified, providing new insight into their biosynthesis at transcriptome level.

Native to Southern China, Ilex pubescens and Ilex asprella are frequently used in traditional Chinese medicine. Both of them produce a large variety of ursane-type triterpenoid saponins, which have been demonstrated to have different pharmacological effects. However, little is known about their biosynthesis. In this study, transcriptomic analysis of I. pubescens and comparison with its closely related specie I. asprella were carried out to identify potential genes involved in triterpenoid saponin biosynthesis. Through RNA sequencing (RNA-seq) and de novo transcriptome assembly of I. pubescens, a total of 68,688 UniGene clusters are obtained, of which 32,184 (46.86%) are successfully annotated by comparison with the sequences in major public databases (NCBI, Swiss-Prot, and KEGG). It includes 128 UniGenes related to triterpenoid backbone biosynthesis, 11 OSCs (oxidosqualene cyclases), 233 CYPs (cytochrome P450), and 269 UGTs (UDP-glycosyltransferases). By homology-based blast and phylogenetic analysis with well-characterized genes involved in triterpenoid saponin biosynthesis, 5 OSCs, 14 CYPs, and 1 UGT are further proposed as the most promising candidate genes. Transcriptomic comparison between two Ilex species using blastp and OrthoMCL method reveals high sequence similarity. All OSCs and UGTs as well asmost CYPs are classified as orthologous genes, while only 5 CYPs in I. pubescens and 3 CYPs in I. asprella are species-specific. One of OSC candidates, named as IpAS1, was successfully cloned and expressed in Saccharomyces cerevisiae INVSc1. Analysis of the yeast extract by gas chromatography (GC) and gas chromatography-mass spectrometry (GC-MS) shows IpAS1 is amixed amyrin synthase, producing α-amyrin and β-amyrin at ratio of 5:1, which is similar to its ortholog IaAS1 from I. asprella. This study is the first exploration to profile the transcriptome of I. pubescens, the generated data and gene models will facilitate further molecular studies on the physiology and metabolism in this plant. By comparative transcriptomic analysis, a series of candidate genes involved in the biosynthetic pathway of triterpenoid saponins are identified, providing new insight into their biosynthesis at transcriptome level. [ABSTRACT FROM AUTHOR]