Moxibustion prevents tripterygium glycoside-induced oligoasthenoteratozoospermia in rats via reduced oxidative stress and modulation of the Nrf2/HO-1 signaling pathway.

Oligoasthenoteratozoospermia (OAT) decreases male fertility, seriously affecting the production of offspring. This study clarified the preventive impact of different moxibustion frequencies on OAT and selected the optimal frequency to elucidate the underlying mechanism. An OAT rat model was constructed by gavage of tripterygium glycosides (TGS) suspension. Daily moxibustion (DM) or alternate-day moxibustion (ADM) was administered on the day of TGS suspension administration. Finally, we selected DM for further study based on sperm quality and DNA fragmentation index, testicular and epididymal morphology, and reproductive hormone level results. Subsequently, the oxidative stress (OS) status was evaluated by observing the OS indices levels; malondialdehyde (MDA), 8-hydroxy-deoxyguanosine (8-OHdG), total antioxidant capacity (T-AOC), and total superoxide dismutase (T-SOD) in testicular tissue using colorimetry and enzyme-linked immunosorbent assay. Furthermore, heme oxygenase 1 (HO-1) and nuclear factor erythropoietin-2-related factor 2 (Nrf2) were evaluated using Western blotting. Immunohistochemistry was employed to locate and assess the expression of HO-1 and Nrf2 protein, while quantitative real-time polymerase chain reaction was utilized to detect their mRNA expression. MDA and 8-OHdG levels decreased following DM treatment, while T-SOD and T-AOC increased, suggesting that DM may prevent TGS-induced OAT in rats by decreasing OS in the testis. Furthermore, protein and mRNA expression of Nrf2 and HO-1 in the testis were elevated, indicating that DM may reduce OS by activating the signaling pathway of Nrf2/HO-1. Therefore, DM could prevent OAT in rats via the Nrf2/HO-1 pathway, thereby presenting a promising therapeutic approach against OAT.

Tripterygium wilfordii Hook.f. ameliorates paraquat-induced lung injury by reducing oxidative stress and ferroptosis via Nrf2/HO-1 pathway.

Paraquat (PQ) poisoning can induce acute lung injury and fibrosis and has an extremely high mortality rate. However, no effective treatments for PQ poisoning have been established. In this study, the potential efficacy of Tripterygium wilfordii Hook.f. (TwHF) in alleviating PQ-induced lung injury and fibrosis was investigated in a mouse model. Mice were randomly assigned to the control, PQ, PQ + TwHF1 (pretreatment before inducing poisoning), and PQ + TwHF2 (treatment after poisoning) groups. The mice in the PQ + TwHF1 group were pretreated with TwHF for 5 days before receiving one dose of PQ (120 mg/kg) and then received a daily oral gavage of the indicated dosages of TwHF until sacrifice. The mice in the PQ + TwHF2 group were treated with TwHF 2 h after PQ exposure until sacrifice. The pathological analysis and Fapi PET/CT showed that treatment with TwHF attenuated lung injury. And TwHF reduced pulmonary oxidative stress, as indicated by the reduction in, malondialdehyde (MDA), glutathione (GSH), and reactive oxygen species (ROS) levels, as well as by the increase in superoxide dismutase (SOD) levels. Accordingly, the Perls DAB staining showed increased iron concentrations and western blotting revealed a decreased GPX4 expression after PQ exposure, as well as the mitigation of the overexpression of Nrf2 and HO-1 induced by PQ. In conclusion, our study demonstrated the potential of TwHF as a treatment for PQ-induced lung injury and fibrosis. The protective mechanism of this medicinal herb may involve the regulation of ferroptosis.

Identification of a cytochrome P450 from Tripterygium hypoglaucum (Levl.) Hutch that catalyzes polpunonic acid formation in celastrol biosynthesis.

Tripterygium hypoglaucum (Levl.) Hutch, a traditional Chinese medicinal herb with a long history of use, is widely distributed in China. One of its main active components, celastrol, has great potential to be developed into anti-cancer and anti-obesity drugs. Although it exhibits strong pharmacological activities, there is a lack of sustainable sources of celastrol and its derivatives, making it crucial to develop novel sources of these drugs through synthetic biology. The key step in the biosynthesis of celastrol is considered to be the cyclization of 2,3-oxidosqualene into friedelin under the catalysis of 2,3-oxidosqualene cyclases. Friedelin was speculated to be oxidized into celastrol by cytochrome P450 oxidases (CYP450s). Here, we reported a cytochrome P450 ThCYP712K1 from Tripterygium hypoglaucum (Levl.) Hutch that catalyzed the oxidation of friedelin into polpuonic acid when heterologously expressed in yeast. Through substrate supplementation and in vitro enzyme analysis, ThCYP712K1 was further proven to catalyze the oxidation of friedelin at the C-29 position to produce polpunonic acid, which is considered a vital step in the biosynthesis of celastrol, and will lay a foundation for further analysis of its biosynthetic pathway.

Probing the functions of friedelane-type triterpene cyclases from four celastrol-producing plants.

Triterpenes are among the most diverse plant natural products, and their diversity is closely related to various triterpene skeletons catalyzed by different 2,3-oxidosqualene cyclases (OSCs). Celastrol, a friedelane-type triterpene with significant bioactivities, is specifically distributed in higher plants, such as Celastraceae species. Friedelin is an important precursor for the biosynthesis of celastrol, and it is synthesized through the cyclization of 2,3-oxidosqualene, with the highest number of rearrangements being catalyzed by friedelane-type triterpene cyclases. However, the molecular mechanisms underlying the catalysis of friedelin production by friedelane-type triterpene cyclases have not yet been fully elucidated. In this study, transcriptome data of four celastrol-producing plants from Celastraceae were used to identify a total of 21 putative OSCs. Through functional characterization, the friedelane-type triterpene cyclases were separately verified in the four plants. Analysis of the selection pressure showed that purifying selection acted on these OSCs, and the friedelane-type triterpene cyclases may undergo weaker selective restriction during evolution. Molecular docking and site-directed mutagenesis revealed that changes in some amino acids that are unique to friedelane-type triterpene cyclases may lead to variations in catalytic specificity or efficiency, thereby affecting the synthesis of friedelin. Our research explored the functional diversity of triterpene synthases from a multispecies perspective. It also provides some references for further research on the relative mechanisms of friedelin biosynthesis.

Network Pharmacology to Uncover the Molecular Mechanisms of Action of LeiGongTeng for the Treatment of Nasopharyngeal Carcinoma.

BACKGROUND Nasopharyngeal carcinoma (NPC) is a common head and neck cancer epidemic in southern China and southeast Asia. LeiGongTeng has been widely used for the treatment of cancers. The purpose of this study was to determine the pharmacological mechanism of action of LeiGongTeng in the treatment of NPC using a network pharmacological approach. MATERIAL AND METHODS The traditional Chinese medicine systems pharmacology (TCMSP) database was used to identify active ingredients and associated target proteins for LeiGongTeng. Cytoscape was utilized to create a drug-disease network and topology analysis was conducted to analyze the degree of each ingredient. The Search Tool for the Retrieval of Interacting Genes/Proteins (STRING) online tool was applied for the construction and analysis of the protein-protein interaction (PPI) network, while Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment and Gene Ontology (GO) functional analyses were utilized to determine drug-disease common genes. RESULTS 22 active ingredients including kaempferol, nobiletin, and beta-sitosterol, and 30 drug-disease common genes including VEGFA, CASP3, ESR1, and RELA were identified. GO analysis indicated that 94 biological processes, including RNA polymerase II, apoptotic process, response to drug, cell adhesion, and response to hypoxia, were found to be associated with NPC. The KEGG enrichment analysis showed that 58 pathways, including the PI3K-Akt signaling pathway, microRNAs in cancer, tumor necrosis factor (TNF) signaling pathway and pathways in cancer were found to be associated with NPC. CONCLUSIONS LeiGongTeng exerts its therapeutic effect through various biological processes and signaling pathways since it acts on several target genes. Systematic pharmacology can be used to predict the underlying function of LeiGongTeng and its mechanism of action in NPC.

Therapeutic targets of thunder god vine (Tripterygium wilfordii hook) in rheumatoid arthritis (Review).

Celastrol and triptolide, chemical compounds isolated from Tripterygium wilfordii hook (also known as thunder god vine), are effective against rheumatoid arthritis (RA). Celastrol targets numerous signaling pathways involving NF­κB, endoplasmic reticulum Ca2+­ATPase, myeloid differentiation factor 2, toll­like receptor 4, pro­inflammatory chemokines, DNA damage, cell cycle arrest and apoptosis. Triptolide, inhibits NF­κB, the receptor activator of NF­κB (RANK)/RANK ligand/osteoprotegerin signaling pathway, cyclooxygenase­2, matrix metalloproteases and cytokines. The present review examined the chemistry and bioavailability of celastrol and triptolide, and their molecular targets in treating RA. Clinical studies have demonstrated that T. wilfordii has several promising bioactivities, but its multi­target toxicity has restricted its application. Thus, dosage control and structural modification of T. wilfordii are required to reduce the toxicity. In this review, future directions for research into these promising natural products are discussed.

Genome of Tripterygium wilfordii and identification of cytochrome P450 involved in triptolide biosynthesis.

Triptolide is a trace natural product of Tripterygium wilfordii. It has antitumor activities, particularly against pancreatic cancer cells. Identification of genes and elucidation of the biosynthetic pathway leading to triptolide are the prerequisite for heterologous bioproduction. Here, we report a reference-grade genome of T. wilfordii with a contig N50 of 4.36 Mb. We show that copy numbers of triptolide biosynthetic pathway genes are impacted by a recent whole-genome triplication event. We further integrate genomic, transcriptomic, and metabolomic data to map a gene-to-metabolite network. This leads to the identification of a cytochrome P450 (CYP728B70) that can catalyze oxidation of a methyl to the acid moiety of dehydroabietic acid in triptolide biosynthesis. We think the genomic resource and the candidate genes reported here set the foundation to fully reveal triptolide biosynthetic pathway and consequently the heterologous bioproduction.

Integrated metabolomics and network toxicology to reveal molecular mechanism of celastrol induced cardiotoxicity.

Celastrol (CS), an active triterpene derived from traditional Chinese medicine Tripterygium wilfordii Hook. f, has been used to treat chronic inflammation, arthritis and other diseases. However, it has been reported that CS can trigger cardiotoxicity and the molecular mechanism of heart injury induced by CS is not clear. Considering the wide application of Tripterygium wilfordii Hook. f in clinics, it is necessary to develop an accurate and reliable method to assess the safety of CS, and to elucidate as much as possible the mechanism of cardiotoxicity induced by CS. In this study, Ultra-performance liquid chromatography coupled with quadrupole time of flight mass spectrometry (UPLC-Q-TOF/MS)-based metabolomics revealed clues to the mechanism of CS-induced heart injury. Palmitic acid significantly increased in plasma from CS-treated rats, and this increase resulted in oxidative stress response in vivo. Excessive ROS further activate TNF signaling pathway and caspase family, which were obtained from the KEGG enrichment analysis of network toxicology strategy. Protein expression level of caspase-3, caspase-8, bax were significantly increased by western blot. Q-PCR also showed the similar results as western blot. It means that apoptosis plays a key role in the process of celastrol induced cardiotoxicity. Blocking this signal axis may be a potential way to protect myocardial tissue.

Differential expression of the TwHMGS gene and its effect on triptolide biosynthesis in Tripterygium wilfordii.

3-Hydroxy-3-methylglutaryl-CoA synthase (HMGS) is the first committed enzyme in the MVA pathway and involved in the biosynthesis of terpenes in Tripterygium wilfordii. The full-length cDNA and a 515 bp RNAi target fragment of TwHMGS were ligated into the pH7WG2D and pK7GWIWG2D vectors to respectively overexpress and silence, TwHMGS was overexpressed and silenced in T. wilfordii suspension cells using biolistic-gun mediated transformation, which resulted in 2-fold increase and a drop to 70% in the expression level compared to cells with empty vector controls. During TwHMGS overexpression, the expression of TwHMGR, TwDXR and TwTPS7v2 was significantly upregulated to the control. In the RNAi group, the expression of TwHMGR, TwDXS, TwDXR and TwMCT visibly displayed downregulation to the control. The cells with TwHMGS overexpressed produced twice higher than the control value. These results proved that differential expression of TwHMGS determined the production of triptolide in T. wilfordii and laterally caused different trends of relative gene expression in the terpene biosynthetic pathway. Finally, the substrate acetyl-CoA was docked into the active site of TwHMGS, suggesting the key residues including His247, Lys256 and Arg296 undergo electrostatic or H-bond interactions with acetyl-CoA.

Celastrol suppresses nitric oxide synthases and the angiogenesis pathway in colorectal cancer.

The thunder god vine (Tripterygium wilfordii Hook. F) is traditionally used for inflammation-related diseases in traditional Chinese medicine. In recent years, celastrol (a natural compound from the root of the thunder god vine) has attracted great interest for its potential anticancer activities. The free radical nitric oxide (NO) is known to play a critical role in colorectal cancer growth by promoting tumour angiogenesis. However, how celastrol influences the NO pathway and its mechanism against colorectal cancer is largely unknown. In this study, we investigated the effects and mechanism of celastrol on nitric oxide synthase (NOS) and the angiogenesis pathway in colorectal cancer. Our data show that celastrol inhibited HT-29 and HCT116 cell proliferation, migration, and NOS activity in the cytoplasm. The antiproliferation activity of celastrol was associated with the inhibition of iNOS and eNOS in colorectal cancer cells. Treatment with celastrol inhibited colorectal cancer cell growth and migration, and was associated with suppression of the expression of key genes (TYMP, CDH5, THBS2, LEP, MMP9, and TNF) and proteins (IL-1b, MMP-9, PDGF, Serpin E1, and TIMP-4) involved in the angiogenesis pathway. In addition, combinational use of celastrol with 5-fluorouracil, salinomycin, 1400 W, and L-NIO showed enhanced inhibition of colorectal cancer cell proliferation and migration. In sum, our study suggests that celastrol could suppress colorectal cancer cell growth and migration, likely through suppressing NOS activity and inhibiting the angiogenesis pathway.

ITRAQ-Based Proteomics Analysis of Triptolide On Human A549 Lung Adenocarcinoma Cells.

BACKGROUND/AIMS: Triptolide (TP) is a diterpenoid triepoxide extracted from the traditional Chinese medical herb Tripterygium wilfordii that exerts prominent broad-spectrum anticancer activity to repress proliferation and induce cancer cell apoptosis through various molecular pathways. We previously observed that TP inhibits the progression of A549 cells and pancreatic cancer cells (PNCA-1) in vitro. However, the complex molecular mechanism underlying the anticancer activity of TP is not well understood. METHODS: To explore the molecular mechanisms by which TP induces lung cancer cell apoptosis, we investigated changes in the protein profile of A549 cells treated with TP using a proteomics approach (iTRAQ [isobaric tags for relative and absolute quantitation] combined with NanoLC-MS/MS [nano liquid chromatography-mass spectrometry]). Changes in the profiles of the expressed proteins were analyzed using the bioinformatics tools OmicsBean and the Kyoto Encyclopedia of Genes and Genomes (KEGG) and were verified using western blotting. Apoptosis and cell cycle effects were analyzed using flow cytometry. RESULTS: TP induced apoptosis in A549 cells and blocked A549 cells at the G2/M phase. Using iTRAQ technology, we observed 312 differentially expressed proteins associated in networks and implicated in different KEGG pathways. Gene Ontology (GO) analysis showed the overviews of dysregulated proteins in the biological process (BP), cell component (CC), and molecular function (MF) categories. Moreover, some candidate proteins involved in PARP1/AIF and nuclear Akt signaling pathways or metastasis processes were validated by western blotting. CONCLUSION: TP exerted anti-tumor activity on non-small cell lung cancer (NSCLC) A549 lung adenocarcinoma cells by dysregulating tumor-related protein expression. Herein, we provide a preliminary study of TP-related cytotoxicity on A549 cells using proteomics tools. These findings may improve the current understanding of the anti-tumor effects of TP on lung cancer cells and may reveal candidate proteins as potential targets for the treatment of lung cancer.

Efficacy and safety of Tripterygium wilfordii Hook F for CKD in Mainland China: A systematic review and meta-analysis.

Tripterygium wilfordii Hook F (TwHF) is a promising Chinese traditional medicine used to significantly reduce proteinuria and improve renal function. However, its efficacy and safety in treatment of chronic kidney disease need to be further explored in order to promote its application in clinics. This review compared the efficacy and safety of TwHF with the placebo, conventional Western medicine and other immunosuppressive medicine in a range of kidney disorders. One hundred three randomized controlled trials were included. TwHF therapy decreased 24-hr proteinuria by 0.59 g/day (95% confidence interval [CI; -0.68, -0.50]), serum creatinine level by 1.93 µmol/L (95% CI [-3.69, -0.17]), and blood urea nitrogen level by 0.24 mmol/L (95% CI [-0.41, -0.07]); increased the total effective rate by 27% (95% CI [1.24, 1.30]); and decreased the incidence of adverse reactions by 19% (95% CI [0.68, 0.96]) overall. Meta regression results showed that the duration of therapy and mean age of participants were the major sources of high heterogeneity. Sensitivity analysis demonstrated that our statistic results were relatively stable and credible. The present findings suggested that TwHF possibly has nephroprotective effects by decreasing proteinuria, serum creatinine level, and blood urea nitrogen level and no more adverse reactions compared with control group in most kidney disorders. However, these findings still need to be further confirmed by high-quality trials.

Triptolide inhibits proliferation, differentiation and induces apoptosis of osteoblastic MC3T3­E1 cells.

Ankylosing spondylitis (AS) is characterized by the formation of bony spurs. Treatment of the resulting ankylosis, excessive bone formation and associated functional impairment, remain the primary therapeutic aims in research regarding this condition. Triptolide is the primary active component of the perennial vine Tripterygium wilfordii Hook. f., and has previously been demonstrated to exert anti­tumor activities including inhibition of cell growth and the induction of apoptosis, however, the effect of triptolide on osteoblasts remains to be elucidated. In the present study, the MC3T3­E1 mouse osteoblast cell line was treated with differing concentrations of triptolide for various intervals. Cell proliferation was detected using the bromodeoxyuridine assay, cell cycle and apoptosis were measured by flow cytometry, nuclear apoptosis was observed by Hoechst staining and associated proteins were determined via western blot analysis. The cells were then further incubated with osteogenic induction medium supplemented with triptolide for 7 or 12 days and the differentiation to osteoblasts was examined by picrosirius staining, observation of alkaline phosphatase activity and a calcium deposition assay. It was demonstrated that treatment with triptolide significantly inhibited osteoblast proliferation and induced cell cycle arrest and apoptosis of the osteoblasts. Furthermore, treatment with triptolide reduced collagen formation, alkaline phosphatase activity and calcium deposition. The present study demonstrated an inhibitory effect of triptolide on osteoblast proliferation and differentiation, and therefore suggests a potential therapeutic agent for the treatment of AS in the future.

Functional characterization of NES and GES responsible for the biosynthesis of (E)-nerolidol and (E,E)-geranyllinalool in Tripterygium wilfordii.

Triptolide and celastrol, two principal bioactive compounds in Tripterygium wilfordii, are produced from geranylgeranyl diphosphate (GGPP) and farnesyl diphosphate ((E,E)-FPP) through terpenoid biosynthesis pathway. However, little is known about T. wilfordii terpene synthases which could competitively utilize GGPP and (E,E)-FPP as substrates, producing C15 and C20 tertiary alcohols. Here we firstly cloned the genes encoding nerolidol synthase (NES) and geranyllinalool synthases (GES1, GES2), which are responsible for the biosynthesis of (E)-nerolidol and (E,E)-geranyllinalool. In vitro characterization of recombinant TwNES and TwGES1 revealed both were functional enzymes that could catalyze the conversion of (E,E)-FPP and GGPP to (E)-nerolidol and (E,E)-geranyllinalool, which were consistent with the results of yeast fermentation. Biochemical characterization revealed TwNES and TwGES1 had strong dependency for Mg2+, Km and Kcat/Km values of TwNES for (E,E)-FPP were 12.700 µM and 0.029 s-1/µM, and TwGES1 for GGPP were 2.039 µM and 0.019 s-1/µM. Real-time PCR analysis showed the expression levels of NES and GES1 increased by several fold in the suspension cells treated with alamethicin, indicating TwNES and TwGES1 are likely to utilize GGPP and (E,E)-FPP to generate tertiary alcohols as precursor of plant volatiles, which play important roles in the ecological interactions between T. wilfordii and other organisms.

Molecular Analysis of Chinese Celastrus and Tripterygium and Implications in Medicinal and Pharmacological Studies.

Celastrus and Tripterygium species, which are used in traditional Chinese medicine, have attracted much attention due to their anti-tumor promoting and neuroprotective activities, in addition to their applications in autoimmune disorders. However, systematic relationships between them and among species are unclear, and it may disturb their further medicinal utilization. In the present study, the molecular analysis of combined chloroplast and nuclear markers of all Chinese Celastrus and Tripterygium was performed, and clear inter- and intra-genus relationships were presented. The result suggests that Tripterygium constitute a natural monophyletic clade within Celastrus with strong support value. Fruit and seed type are better than inflorescence in subgeneric classification. Chinese Celastrus are classified for three sections: Sect. Sempervirentes (Maxim.) CY Cheng & TC Kao, Sect. Lunatus XY Mu & ZX Zhang, sect. nov., and Sect. Ellipticus XY Mu & ZX Zhang, sect. nov. The phylogenetic data was consistent with their chemical components reported previously. Owing to the close relationship, several evergreen Celastrus species are recommended for chemical and pharmacological studies. Our results also provide reference for molecular identification of Chinese Celastrus and Tripterygium.

Integrative Approaches for the Identification and Localization of Specialized Metabolites in Tripterygium Roots.

Members of the genus Tripterygium are known to contain an astonishing diversity of specialized metabolites. The lack of authentic standards has been an impediment to the rapid identification of such metabolites in extracts. We employed an approach that involves the searching of multiple, complementary chromatographic and spectroscopic data sets against the Spektraris database to speed up the metabolite identification process. Mass spectrometry-based imaging indicated a differential localization of triterpenoids to the periderm and sesquiterpene alkaloids to the cortex layer of Tripterygium roots. We further provide evidence that triterpenoids are accumulated to high levels in cells that contain suberized cell walls, which might indicate a mechanism for storage. To our knowledge, our data provide first insights into the cell type specificity of metabolite accumulation in Tripterygium and set the stage for furthering our understanding of the biological implications of specialized metabolites in this genus.

Development and application of a comprehensive lipidomic analysis to investigate Tripterygium wilfordii-induced liver injury.

Lipid metabolic pathways play pivotal roles in liver function, and disturbances of these pathways are associated with various diseases. Thus, comprehensive characterization and measurement of lipid metabolites are essential to deciphering the contributions of lipid network metabolism to diseases or its responses to drug intervention. Here, we report an integrated lipidomic analysis for the comprehensive detection of lipid metabolites. To facilitate the characterization of untargeted lipids through fragmentation analysis, nine formulas were proposed to identify the fatty acid composition of lipids from complex MS (n) spectrum information. By these formulas, the co-eluted isomeric compounds could be distinguished. In total, 250 lipids were detected and characterized, including diacylglycerols, triacylglycerols, glycerophosphoethanolamines, glycerophosphocholines, glycerophosphoserines, glycerophosphoglycerols, glycerophosphoinositols, cardiolipins, ceramides, and sphingomyelins. Integrated with the targeted lipidomics, a total of 27 inflammatory oxylipins were also measured. To evaluate the aberrant lipid metabolism involved in liver injury induced by Tripterygium wilfordii, lipid network metabolism was further investigated. Results indicated that energy lipid modification, membrane remodeling, potential signaling lipid alterations, and abnormal inflammation response were associated with injury. Because of the important roles of lipids in liver metabolism, this new method is expected to be useful in analyzing other lipid metabolism diseases.

[Effects of elicitors on growth of adventitious roots and contents of secondary metabolites in Tripterygium wilfordii Hook. f].

To study the effects of the extract of fungal elicitor, AgNO3, MeJA and yeast on the growth and content of secondary metabolites of adventitious roots in Tripterygium wilfordii. The above elicitors were supplemented to the medium, the growth and the content of secondary metabolites were measured. When the medium was supplemented with the elicitor Glomerella cingulata or Collectotrichum gloeosporioides, the content of triptolide was increased by 2.24 and 1.93-fold, the alkaloids content was increased by 2.02 and 2.07-fold, respectively. The optimal concentration of G. cingulata was 50 µg/mL for accumulation of triptolide, alkaloids and for the growth of adventitious roots. AgNO3 inhibited the growth of adventitious roots and the accumulation of the alkaloids, whereas it (at 25 µmol/L) increased the accumulation of triptolide by 1.71-fold compared to the control. The growth of adventitious roots, the contents of triptolide and alkaloids were increased 1.04, 1.64 and 2.12-folds, respectively when MeJA was at 50 µmol/L. When the concentration of yeast reached 2 g/L, the content of triptolide increased 1.48-folds. This research demonstrated that supplementation of AgNO3 and yeast enhanced the biosynthesis of triptolide in adventitious roots and the synergism of G. cingulata and MeJA could promote the biosynthesis of both triptolide and alkaloids.

[Different metabolites of leaves between Tripterygium wilfordii and Tripterygium hypoglaucum based on UPLC-Q-TOF-MS].

To analysis the differences between Tripterygium wilfordii and T. hypoglaucum, specimens of their leaves were collected from five production regions and analyzed by ultra performance liquid chromatography coupled with quadrupole time of flight mass spectrometry (UPLC-Q-TOF-MS). The data were analyzed by multivariate statistical method, such as hierarchical cluster analysis (HCA) principal component analysis (PCA) and orthogonal signal correction partial least square discrimination (OPLS-DA). Potential markers with VIP values above 5.0 and corresponding r values above 0.85, were selected and further tested by combining mann-Whitney nonparametric. Those with P < 0.001 and AUC = 1 were confirmed as metabolite markers to discriminate them from each other. Results revealed that the two species were obviously different in their leaf metabolites. Based on their mass spectra, 23 potential metabolite markers were identified to distinguish T. wilfordii from T. hypoglaucum.