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Leonurus cardiaca has a long history of use in western herbal medicine and is applied for the treatment of gynaecological conditions, anxiety, and heart diseases. Because of its botanical relationship to the primary Chinese species, L. japonicus, and extensive medical indications that go beyond the traditional indications for the Chinese species, it is a promising medicinal resource. Therefore, the features of genetic diversity and variability in the species have been prioritized. To explore these issues, we sequenced the chloroplast genomes of 22 accessions of L. cardiaca from different geographical locations worldwide using high-throughput sequencing. The results indicate that L. cardiaca has a typical quadripartite structure and range from 1,51,236 bp to 1,51,831 bp in size, forming eight haplotypes. The genomes all contain 114 distinct genes, including 80 protein-coding genes, 30 transfer RNA genes and four ribosomal RNA genes. Comparative analysis showed abundant diversity of single nucleotide polymorphisms (SNPs), indels, simple sequence repeats (SSRs) in 22 accessions. Codon usage showed highly similar results for L. cardiaca species. The phylogenetic and network analysis indicated 22 accessions forming four clades that were partly related to the geographical distribution. In summary, our study highlights the advantage of chloroplast genome with large data sets in intraspecific diversity evaluation and provides a new tool to facilitate medicinal plant conservation and domestication.

Background: Previous research suggested that Chinese Medicine (CM) Formula Huashibaidu granule might shorten the disease course in coronavirus disease 2019 (COVID-19) patients. This research aimed to investigate the early treatment effect of Huashibaidu granule in well-managed patients with mild COVID-19.Methods: An unblinded cluster-randomized clinical trial was conducted at the Dongxihu FangCang hospital. Two cabins were randomly allocated to a CM or control group, with 204 mild COVID-19 participants in each cabin. All participants received conventional treatment over a 7 day period, while the ones in CM group were additionally given Huashibaidu granule 10 g twice daily. Participants were followed up to their clinical endpoint. The primary outcome was worsening symptoms before the clinical endpoint. The secondary outcomes were cure and discharge before the clinical endpoint and alleviation of composite symptoms after the 7 days of treatment.Results: All 408 participants were followed up to their clinical endpoint and included in statistical analysis. Baseline characteristics were comparable between the two groups (P > 0.05). The number of worsening patients in the CM group was 5 (2.5%), and that in the control group was 16 (7.8%) with a significant difference between groups (P = 0.014). Eight foreseeable mild adverse events occurred without statistical difference between groups (P = 0.151).Conclusion: Seven days of early treatment with Huashibaidu granule reduced the likelihood of worsening symptoms in patients with mild COVID-19. Our study supports Huashibaidu granule as an active option for early treatment of mild COVID-19 in similar well-managed medical environments.Clinical Trial Registration:www.chictr.org.cn/showproj.aspx?proj=49408, identifier: ChiCTR2000029763.

Orobanche and Striga are parasitic weeds extremely well adapted to the life cycle of their host plants. They cannot be eliminated by conventional weed control methods. Suicidal germination induced by strigolactones (SLs) analogs is an option to control these weeds. Here, we reported two new halogenated (+)-GR24 analogs, named 7-bromo-GR24 (7BrGR24) and 7-fluoro-GR24 (7FGR24), which were synthesized using commercially available materials following simple steps. Both compounds strongly promoted seed germination of Orobanche cumana. Their EC50 values of 2.3±0.28×10−8M (7BrGR24) and 0.97±0.29×10−8M (7FGR24) were 3- and 5-fold lower, respectively, than those of (+)-GR24 and rac-GR24 (EC50=5.1±1.32–5.3±1.44×10−8; p

Interactions between colonizing microbiota and the host have been fully confirmed, among which the tongue-coating microbiota have a moderate rate of renewal and disease sensitivity and are easily obtained, making them an ideal research subject. Oral microbiota disorders are related to diabetes, obesity, cardiovascular disease, cancer, and other systemic diseases. As an important part of the oral cavity, tongue-coating microbiota can promote gastritis and digestive system tumors, affecting the occurrence and development of multiple chronic diseases. Common risk factors include diet, age, and immune status, among others. Metabolic regulatory mechanisms may be similar between the tongue and gut microbiota. Tongue-coating microbiota can be transferred to the respiratory or digestive tract and create a new balance with local microorganisms, together with the host epithelial cells forming a biological barrier. This barrier is involved in the production and circulation of nitric oxide (NO) and the function of taste receptors, forming the oral-gut-brain axis (similar to the gut-brain axis). At present, the disease model and mechanism of tongue-coating microbiota affecting metabolism have not been widely studied, but they have tremendous potential.

Commercial cultivation of the medicinal plant Atractylodes lancea is significantly restricted by low survival rates and reduced yields. Intercropping can reasonably coordinate interspecific interactions, effectively utilize environmental resources, and increase survival and yield. We conducted a field experiment from 2014 to 2016 to analyze the advantages and effects of intercropping on A. lancea survival, growth traits, individual volatile oil content, and total volatile oil content. In addition to A. lancea monoculture (AL), five intercropping combinations were planted: Zea mays L. (ZM) + A. lancea, Tagetes erecta L. (TE) + A. lancea, Calendula officinalis L. (CO) + A. lancea, Glycine max (Linn.) Merr. (GM) + A. lancea, and Polygonum hydropiper L. (PH) + A. lancea. The survival and average rhizome weight of A. lancea was higher in the ZM, CO, and TE treatments than in the monoculture treatment, and the average plant height was higher in all intercropping treatments than in the monoculture. The volatile oil content of A. lancea from the ZM and CO treatments was significantly improved relative to that of monoculture plants. The volatile oil harvest was higher in the ZM, CO, and TE treatments than in the monoculture. We conclude that intercropping is an effective way to increase the survival and yield of A. lancea. Furthermore, intercropping with ZM, CO, and TE increases the harvest of four volatile oils from A. lancea.

Abstract In recent years, mass spectrometry-based proteomics has provided scientists with the tremendous capability to study plants more precisely than previously possible. Currently, proteomics has been transformed from an isolated field into a comprehensive tool for biological research that can be used to explain biological functions. Several studies have successfully used the power of proteomics as a discovery tool to uncover plant resistance mechanisms. There is growing evidence that indicates that the spatial proteome and post-translational modifications (PTMs) of proteins directly participate in the plant immune response. Therefore, understanding the subcellular localization and PTMs of proteins is crucial for a comprehensive understanding of plant responses to biotic stress. In this review, we discuss current approaches to plant proteomics that use mass spectrometry, with particular emphasis on the application of spatial proteomics and PTMs. The purpose of this paper is to investigate the current status of the field, discuss recent research challenges, and encourage the application of proteomics techniques to further research.

Abstract Background Tripterygium wilfordii Hook. f. (T. wilfordii) is an important medicinal plant with anti-inflammatory, immunosuppressive and anti-tumor activities. The main bioactive ingredients are diterpenoids and triterpenoids, such as triptolide, triptophenolide and celastrol. However, the production of terpenoids from original plants, hairy roots and dedifferentiated cells (DDCs) are not satisfactory for clinical applications. To find a new way to further improve the production of terpenoids, we established a new culture system of cambial meristematic cells (CMCs) with stem cell-like properties, which had strong vigor and high efficiency to produce large amounts of terpenoids of T. wilfordii. Results CMCs of T. wilfordii were isolated and cultured for the first time. CMCs were characterized consistent with stem cell identities based on their physiological and molecular analysis, including morphology of CMCs, hypersensitivity to zeocin, thin cell wall and orthogonal partial least square-discriminant analysis, combination of transcriptional data analysis. After induction with methyl jasmonate (MJ), the maximal production of triptolide, celastrol and triptophenolide in CMCs was 312%, 400% and 327% higher than that of control group, respectively. As for medium, MJ-induced CMCs secreted 231% triptolide and 130% triptophenolide at the maximum level into medium higher than that of control group. Maximal celastrol production of induced CMCs medium was 48% lower than that of control group. Long-term induction significantly enhanced the production of terpenoids both in cells and medium. The reason for increasing the yield of terpenoids was that expression levels of 1-deoxy-d-xylulose-5-phosphate synthase (DXS), 1-deoxy-d-xylulose-5-phosphate reductoisomerase (DXR) and hydroxymethylglutaryl-CoA synthase (HMGS) were upregulated in CMCs after induction. Conclusions For the first time, CMCs of T. wilfordii were isolated, cultured, characterized and applied. Considering the significant enrichment of terpenoids in CMCs of T. wilfordii, CMCs could provide an efficient and controllable platform for sustainable production of terpenoids, which can be a better choice than DDCs.

Abstract Background The dry root and rhizome of Salvia miltiorrhiza Bunge, or Danshen, is a well-known, traditional Chinese medicine. Tanshinones are active compounds that accumulate in the periderm, resulting in red-colored roots. However, lines with orange roots have been observed in cultivated fields. Here, we performed metabolome and transcriptome analyses to investigate the changes of orange-rooted Danshen. Methods Metabolome analysis was performed by ultra-high-performance liquid chromatography-quadrupole time-of-flight mass spectrometry (UPLC/Q-Tof–MS) to investigate the metabolites variation between orange Danshen and normal Danshen. RNA sequencing and KEGG enrichment analysis were performed to analyzing the differentially expressed genes between orange-rooted and normal Danshen. Results In total, 40 lipophilic components were detected in metabolome analysis, and seven compounds were significantly decreased in the orange Danshen, including the most abundant active compounds, tanshinone IIA and tanshinone I in normal Danshen. Systematic analysis of transcriptome profiles revealed that the down-regulated genes related to catalytic dehydrogenation was not detected. However, two genes related to stress resistance, and four genes related to endoplasmic reticulum (ER)-associated degradation of proteins were up-regulated in orange Danshen. Conclusions Decreases in the content of dehydrogenated furan ring tanshinones such as tanshinone IIA resulted in phenotypic changes and quality degradation of Danshen. Transcriptome analysis indicated that incorrect folding and ER-associated degradation of corresponding enzymes, which could catalyze C15-C16 dehydrogenase, might be contributed to the decrease in dehydrogenated furan ring tanshinones, rather than lower expression of the relative genes. This limited dehydrogenation of cryptotanshinone and dihydrotanshinone I into tanshinones IIA and I products, respectively, led to a reduced quality of Danshen in cultivated fields.

Abstract Functional dyspepsia (FD) is one of the most prevalent functional gastrointestinal disorders, and more and more multicomponent drugs represented by traditional Chinese medicines have provided a favorable therapeutic effect in its treatment. However, their precise localization in the clinic, as well as corresponding mechanism, is ambiguous, thus hindering their widespread use. To meet this requirement, a precise and systematic approach based on a restriction of special disease-related molecules and the following network pharmacology analysis was developed and applied to a multicomponent conventional drug, XiaoErFuPi (XEFP) granules. Experimental verification of the results indicates that this approach can facilitate the prediction, and the precise and systematic efficacy of XEFP could be easily revealed, which shows that XEFP has an advantage over the positive control drug on lactate, gastrin, interleukin 4 and calcitonin gene-related peptide. Moreover, by the proteomics analysis, its superposition of multi-target effects was revealed and a new candidate target for the treatment of FD, striatin, was obtained and verified. This study provides a practicable precise approach for the investigation of the efficacy of multicomponent drugs against FD and offers a promising alternative for the systematical management of FD.

Abstract Background Polygonatum cyrtonema Hua (P. cyrtonema) is one of the most important herbs in traditional Chinese medicine. Polysaccharides in P. cyrtonema plants comprise a class of important secondary metabolites and exhibit a broad range of pharmacological functions. Results In order to identify genes involved in polysaccharide biosynthesis, we performed RNA sequencing analysis of leaf, root, and rhizome tissues of P. cyrtonema. A total of 164,573 unigenes were obtained by assembling transcripts from all three tissues and 86,063 of these were annotated in public databases. Differentially expressed genes (DEGs) were determined based on expression profile analysis, and DEG levels in rhizome tissues were then compared with their counterparts in leaf and root tissues. This analysis revealed numerous genes that were either up-regulated or uniquely expressed in the rhizome. Multiple genes encoding important enzymes, such as UDP glycosyltransferases (UGTs), or transcription factors involved in polysaccharide biosynthesis were identified and further analyzed, while a few genes encoding key enzymes were experimentally validated using quantitative real-time PCR. Conclusion Our results substantially expand the public transcriptome dataset of P. cyrtonema and provide valuable clues for the identification of candidate genes involved in metabolic pathways.

Dioscorea oppositifolia (Chinese yam) (DO) and Dioscorea hamiltonii (DH) are the plants of the genus Dioscorea and they are used as health food and traditional Chinese medicine. In order to development of the available DO and DH source, a comparative study of principal components and activities were carried on. DO and DH was qualitatively and quantitively profiled, and we comparatively evaluated their antioxidant (reducing power assay), anti-inflammatory (xylene-induced ear oedema test) and nonspecific immune regulating (carbon clearance test) activities. The 42 and 38 compounds were detected from DO and DH by the method UPLC-DAD-Q-TOF-MS/MS, respectively, as well as 14 compounds are quantified by HPLC and the methodology was examined. This is the first study of the composition and activities of DH, it also indicates that DO and DH consumption would be beneficial to the health. They may be useful in the production of functional food containing an efficacious dose.

Cyclophosphamide (CTX), used in cancer chemotherapy, a high dose of which would cause immunosuppressive effect and intestinal mucosa damage. American ginseng (Panax quinquefolius L.) has a long history of functional food use for immunological disorder, colitis, cancer, and so on. This study aimed to illustrate the underlying mechanism of American ginseng’s immunomodulatory effect in CTX-induced mice. In this study, all groups of American ginseng (American ginseng polysaccharide [AGP], American ginseng ginsenoside [AGG], co-treated with American ginseng polysaccharide and ginsenoside [AGP_AGG]) have relieve the immune disorder by reversing the lymphocyte subsets ratio in spleen and peripheral blood, as well as stimulating CD4+T cells and IgA-secreting cells in small intestine. These three treatment groups, especially AGP_AGG co-treated group recovered the intestine morphology that up-regulated villus height (VH)/crypt depth (CD) ratio, areas of mucins expression, quantity of goblet cells, and expression of tight junction proteins (ZO-1, occludin). Importantly, the microbiome-metabolomics analysis was applied in this study to illustrate the possible immuno-modulating mechanism. The synergistic effect of polysaccharides and ginsenosides (AGP_AGG group) restored the gut microbiota composition and increased various beneficial mucosa-associated bacterial taxa Clostridiales, Bifidobacterium, and Lachnospiraceae, while decreased harmful bacteria Escherichia-Shigella and Peptococcaceae. Also, AGP_AGG group altered various fecal metabolites such as uric acid, xanthurenic acid, acylcarnitine, 9,10-DHOME, 13-HDoHE, LysoPE15:0, LysoPC 16:0, LysoPI 18:0, and so on, that associated with immunometabolism or protective effect of gut barrier. These results suggest AG, particularly co-treated of polysaccharide and ginsenoside may be used as immunostimulants targeting microbiome-metabolomics axis to prevent CTX-induced side effects in cancer patients.

Panax ginseng C. A. Mey (PGCAM) is a herbaceous perennial belonging to the Araliaceae family, mainly including Mountain-Cultivated Ginseng (MCG) and Garden Ginseng (GG) on the market. We aimed to establish a rapid, accurate and effective method to distinguish 15-year-old MCG and GG using ultra-performance liquid chromatography-quadrupole time-of-flight-tandem mass spectrometry (UPLC-QTOF-MS/MS), and also explored the pharmacological mechanisms of the main components using the Integrative Pharmacology-based Network Computational Research Platform of Traditional Chinese Medicine (TCMIP V2.0; http://www.tcmip.cn/). Altogether, 23 potential quality markers were characterized to distinguish 15-year-old MCG and GG, including ginsenosides Ra2, Rg1, and Ra1, and malonyl-ginsenoside Ra3, etc. The contents of 19 constituents (mainly protopanaxadiol-type) were higher in MCG compared with that in GG, and four constituents (mainly carbohydrate compounds) were higher in GG. The 105 putative targets corresponding to 23 potential quality markers were mainly involved in 30 pathways, which could be divided into 10 models, such as immune regulation, systems (metabolic, nervous, cardiovascular, reproductive), blood-pressure regulation, as well as antitumor, antiaging, antibacterial and anti-inflammatory effects. Furthermore, the potential quality markers of MCG and GG could inhibit the proliferation of breast cancer by regulating the mRNA expression of PSA, S6K, MDM2, and P53 genes by acting on AR, MTOR, PI3K and other targets. The Integrative Pharmacology Strategy may provide an efficient way to identify chemical constituents and explore the pharmacological actions of TCM formulations.

Cohort studies investigating the treatment of chronic non-communicable diseases (NCDs) with traditional Chinese medicine (TCM) have considerably accumulated in recent years. To systematically and for the first time present the achievements and dilemmas of cohort studies, strict inclusion and exclusion criteria were used to search publications from the Web of Science, PubMed, Embase, Cochrane Library, and China National Knowledge Infrastructure databases for cohort studies on NCDs with TCM since the establishment of these databases. Information on the year of publication, exposure factors, diseases, and outcome indicators was obtained, and a literature quality assessment and bibliometric descriptive analysis were conducted. A total of 182 published articles involving 1,615,106 cases were included. There were 110 non-prospective cohort studies and 72 prospective cohort studies. The diseases involved in the cohort studies were, in the order of the number of published articles, malignant tumors (82 articles, 45.05%), cardiovascular diseases (35 articles, 19.23%), neurological diseases (29 articles, 15.93%), chronic kidney diseases (16 articles, 8.79%), liver cirrhosis (8 articles, 4.40%), diabetes mellitus (8 articles, 4.40%), and chronic respiratory diseases (4 articles, 2.20%). The study participants were mainly from China (177 articles, 97.25%). The number of cohort studies increased significantly in the last 5 years (65 articles, 35.71%), and following the Newcastle-Ottawa Scale (NOS) literature quality evaluation, the number of articles that received a score of four to five was high (116 articles, 63.73%), and the overall quality needs to be improved. The application of cohort studies in the field of TCM for the prevention and treatment of NCDs has developed rapidly in the past 5 years, focusing on the prevention and treatment of tumors as well as cardiovascular and cerebrovascular diseases. However, the design and implementation of cohort studies still have considerable limitations. To provide more clinical evidence, researcher should actively cooperate with evidence-based methodologists and standardize the implementation of cohort studies.

(+)-Borneol is a desirable monoterpenoid with effective anti-inflammatory and analgesic effects that is known as soft gold. (+)-bornyl diphosphate synthase is the key enzyme in the (+)-borneol biosynthesis pathway. Despite several reported (+)-bornyl diphosphate synthase genes, relatively low (+)-borneol production hinders the attempts to synthesize it using microbial fermentation. Here, we identified the highly specific (+)-bornyl diphosphate synthase CbTPS1 from Cinnamomum burmanni. An in vitro assay showed that (+)-borneol was the main product of CbTPS1 (88.70% of the total products), and the Km value was 5.11 ± 1.70 μM with a kcat value of 0.01 s–1. Further, we reconstituted the (+)-borneol biosynthetic pathway in Saccharomyces cerevisiae. After tailored truncation and adding Kozak sequences, the (+)-borneol yield was improved by 96.33-fold to 2.89 mg⋅L–1 compared with the initial strain in shake flasks. This work is the first reported attempt to produce (+)-borneol by microbial fermentation. It lays a foundation for further pathway reconstruction and metabolic engineering production of this valuable natural monoterpenoid.

Panax notoginseng, a perennial herb of the genus Panax in the family Araliaceae, has played an important role in clinical treatment in China for thousands of years because of its extensive pharmacological effects. Here, we report a high-quality reference genome of P. notoginseng, with a genome size up to 2.66 Gb and a contig N50 of 1.12 Mb, produced with third-generation PacBio sequencing technology. This is the first chromosome-level genome assembly for the genus Panax. Through genome evolution analysis, we explored phylogenetic and whole-genome duplication events and examined their impact on saponin biosynthesis. We performed a detailed transcriptional analysis of P. notoginseng and explored gene-level mechanisms that regulate the formation of characteristic tubercles. Next, we studied the biosynthesis and regulation of saponins at temporal and spatial levels. We combined multi-omics data to identify genes that encode key enzymes in the P. notoginseng terpenoid biosynthetic pathway. Finally, we identified five glycosyltransferase genes whose products catalyzed the formation of different ginsenosides in P. notoginseng. The genetic information obtained in this study provides a resource for further exploration of the growth characteristics, cultivation, breeding, and saponin biosynthesis of P. notoginseng.

Gastrodia elata, also named Tianma, is a valuable traditional Chinese herbal medicine. It has numerous important pharmacological roles such as in sedation and lowering blood pressure and as anticonvulsant and anti-aging, and it also has effects on the immune and cardiovascular systems. The whole genome sequencing of G. elata has been completed in recent years, which provides a strong support for the construction of the G. elata gene functional analysis platform. Therefore, in our research, we collected and processed 39 transcriptome data of G. elata and constructed the G. elata gene co-expression networks, then we identified functional modules by the weighted correlation network analysis (WGCNA) package. Furthermore, gene families of G. elata were identified by tools including HMMER, iTAK, PfamScan, and InParanoid. Finally, we constructed a gene functional analysis platform for G. elata1. In our platform, we introduced functional analysis tools such as BLAST, gene set enrichment analysis (GSEA), and cis-elements (motif) enrichment analysis tool. In addition, we analyzed the co-expression relationship of genes which might participate in the biosynthesis of gastrodin and predicted 19 mannose-binding lectin antifungal proteins of G. elata. We also introduced the usage of the G. elata gene function analysis platform (GelFAP) by analyzing CYP51G1 and GFAP4 genes. Our platform GelFAP may help researchers to explore the gene function of G. elata and make novel discoveries about key genes involved in the biological processes of gastrodin.

Influenza infection is a highly contagious, acute febrile respiratory disease caused by the influenza virus. Traditional Chinese Medicine (TCM) has dominated plenty of theoretical and practical approaches in the treatment of influenza. It is, therefore, important to highlight the effects of TCM in the clinical treatment of influenza and their impact on inhibiting the growth of this virus in laboratory experiments. We scrutinized existing evidence on whether TCM is effective in clinical applications. Moreover, we described the potential mechanisms of TCM against the influenza virus. Our findings provide analytical evidence that supports the effectiveness of TCM in treating influenza infections as well as their mechanisms against this virus.

The aim of this study was to investigate the precise clinical use of Sinitang decoction (SNT) in ulcerative colitis (UC). Network pharmacology-based analysis of the drug components–targets–diseases–pathways was used to predict the possible clinical applications of SNT. Next, 2,4,6-trinitrobenzenesulfonic acid (TNBS) was used to establish a rat model of UC, and the efficacy of SNT against UC was tested, followed by a proteomic analysis of the specific signatures regulated by SNT against UC. SNT was predicted to be effective in inflammatory bowel disease, UC, and several other diseases. In the rats with UC, SNT decreased the disease activity index and colon mucosal damage index compared to the untreated UC model rats. Additionally, SNT reversed the upregulated levels of serum tumor necrosis factor (TNF)-α, prostaglandin E2 (PGE2), interleukin (IL)-6, and nitric oxide (NO) in UC model rats. The proteomic analysis identified 78 proteins that were differentially regulated by SNT in the rats with UC, which were associated with the Gene Ontology terms sulfur compound binding, calcium ion binding, and Toll-like receptor (TLR)-4 binding. Among these differentially regulated proteins, C-reactive protein (CRP) and collagen alpha-1(XII) chain (COL12A1) were found to be signature proteins associated with the efficacy of SNT against UC. This study represents the first precise investigation of the efficacy and mechanisms of SNT against UC, and shows that SNT is a promising candidate for personalized management of UC.

Mume Fructus (MF) contains a variety of organic acids, free amino acids, and nucleoside components, and studies have not yet analyzed the relationship between the components of free amino acids and nucleosides with the varieties of MF. A rapid and sensitive method was established for simultaneous determination of 21 free amino acids and 9 nucleosides in MF by ultrafast liquid chromatography-mass spectrometry. The analysis was carried out on a Waters XBridge Amide column (100 mm × 2.1 mm, 3.5 μm) with elution by the mobile phase of 0.2% aqueous formic acid (A) and 0.2% formic acid acetonitrile (B) at a flow rate of 0.2 mL/min with 1 μL per injection. The column temperature was maintained at 30°C. The target compounds were analyzed by the positive ion multiple reaction monitoring (MRM) mode. The comprehensive evaluation of the samples was carried out by principal component analysis (PCA) and technique for order preference by similarity to an ideal solution (TOPSIS) analysis. Results showed the method could simultaneously determine 30 components in MF. The content of total analytes in six mainstream varieties was different, exhibited the order Nangao > Daqingmei > Zhaoshuimei > Yanmei > Shishengme > Baimei, and aspartic acid and adenosine were the most abundant amino acid and nucleoside. PCA and OPLS-DA could easily distinguish the samples, and 11 components could be chemical markers of sample classification. TOPSIS implied that the quality of Nangao and Daqingmei was superior to the other varieties. The results could provide a reliable basis for quality evaluation and utilisation of medicinal and edible MF.

Gastrodia elata is an obligate mycoheterotrophic plant with highly reduced leaves and bracts in scape. Here, Yuan et al sequence and analyze its 1.06 Gb genome which provides insights in adaptation to a lifestyle of heterotrophy.

The Chinese yam (Dioscorea polystachya Turcz.) is an underutilized orphan tuber crop. However, in China it has been used in traditional medicine and food for centuries due to the presence of high starch, protein, fiber, and biologically active compounds. Knowledge on the metabolomic profiles of Chinese yam varieties is needed to explore the underutilized metabolites and variety specific uses. Here, the metabolome of eight Chinese yam varieties that are cultivated in different Chinese regions was profiled. A total of 431 metabolites belonging to different biochemical classes was detected. The majority of detected metabolites were classified as amino acids and derivatives. The different yam varieties offer unique uses; e.g., Hebei Ma Yam, Henan Huai Yam, and Henan Wild Yam were the most metabolically enriched and suitable as food and medicine. Yams from Hubei region had comparable nutritional profiles, which is most probably due to their geographical origin. Specifically, Henan Wild Yam had the highest concentrations of diosgenin, vitamins, and polysaccharides. Overall, this study presents a metabolome reference for D. polystachya varieties.

Sterol C24-methyltransferase (SMT) plays multiple important roles in plant growth and development. SMT1, which belongs to the family of transferases and transforms cycloartenol into 24-methylene cycloartenol, is involved in the biosynthesis of 24-methyl sterols. Here, we report the cloning and characterization of a cDNA encoding a sterol C24-methyltransferase from Tripterygium wilfordii (TwSMT1). TwSMT1 (GenBank access number KU885950) is a 1530 bp cDNA with a 1041 bp open reading frame predicted to encode a 346-amino acid, 38.62 kDa protein. The polypeptide encoded by the SMT1 cDNA was expressed and purified as a recombinant protein from Escherichia coli (E. coli) and showed SMT activity. The expression of TwSMT1 was highly up-regulated in T. wilfordii cell suspension cultures treated with methyl jasmonate (MeJA). Tissue expression pattern analysis showed higher expression in the phellem layer compared to the other four organs (leaf, stem, xylem and phloem), which is about ten times that of the lowest expression in leaf. The results are meaningful for the study of sterol biosynthesis of T. wilfordii and will further lay the foundations for the research in regulating both the content of other main compounds and growth and development of T. wilfordii.

Traditional Chinese medicine (TCM) has played a pivotal role in maintaining the health of Chinese people and is now gaining increasing acceptance around the global scope. However, TCM is confronting more and more concerns with respect to its quality. The intrinsic “multicomponent and multitarget” feature of TCM necessitates the establishment of a unique quality and bioactivity evaluation system, which is different from that of the Western medicine. However, TCM is investigated essentially as “herbal medicine” or “natural product”, and the pharmacopoeia quality monographs are actually chemical-markers-based, which can ensure the consistency only in the assigned chemical markers, but, to some extent, have deviated from the basic TCM theory. A concept of “quality marker” (Q-marker), following the “property-effect-component” theory, is proposed. The establishment of Q-marker integrates multidisciplinary technologies like natural products chemistry, analytical chemistry, bionics, chemometrics, pharmacology, systems biology, and pharmacodynamics, etc. Q-marker-based fingerprint and multicomponent determination conduce to the construction of more scientific quality control system of TCM. This review delineates the background, definition, and properties of Q-marker, and the associated technologies applied for its establishment. Strategies and approaches for establishing Q-marker-based TCM quality control system are presented and highlighted with a few TCM examples.

The 4-hydroxy-3-methylbut-2-enyl diphosphate reductase (HDR) is the last step key enzyme of the methylerythritol phosphate (MEP) pathway, synthesizing isopentenyl diphosphate and its allyl isomer dimethylallyl diphosphate, which is important for regulation of isoprenoid biosynthesis. Here the full-length cDNA of HDR, designated TwHDR (GenBank Accession No. KJ933412.1), was isolated from Tripterygium wilfordii for the first time. TwHDR has an open reading frame (ORF) of 1386 bp encoding 461 amino acids. TwHDR exhibits high homology with HDRs of other plants, with an N-terminal conserved domain and three conserved cysteine residues. TwHDR cDNA was cloned into an expression vector and transformed into an Escherichia coli hdr mutant. Since loss-of-function E.coli hdr mutant is lethal, the result showed that transformation of TwHDR cDNA rescued the E.coli hdr mutant. This complementation assay suggests that the TwHDR cDNA encodes a functional HDR enzyme. The expression of TwHDR was induced by methyl-jasmonate (MJ) in T. wilfordii suspension cells. The expression of TwHDR reached the highest level after 1 h of MJ treatment. These results indicate that we have identified a functional TwHDR enzyme, which may play a pivotal role in the biosynthesis of diterpenoid triptolide in T. wilfordii.

Objective: To comprehensively review the literature relating to the use of Artemisiae Argyi Folium (AAF; the dried leaf of Artemisia argyi Lévl. et Vant. from the Asteraceae family), and qi ai (AAF grown in Qichun County and the adjacent areas in Hubei Province, China) in the field of traditional Chinese medicine. Furthermore, this study aimed to give results of the contents determination of three bioactive components in qi ai and AAF. Methods: The literature was reviewed to acquire relevant information regarding the quality and application of AAF and qi ai. In addition, the contents of the bioactive components (essential oil, total flavonoids and tannins) were determined in 29 AAF samples collected from China and Korea using the methods in the Chinese pharmacopoeia (2015) or determined by our experiments. Results: The dried leaf of A. argyi Lévl. et Vant. from the Asteraceae family is a mainstream commodity of AAF. Qi ai is a commercial type of AAF well-known for its good quality, which has been recorded in Chinese ancient literature. Modern research has showed that more intense heat with stronger penetration was generated in moxibustion therapy when qi ai moxa stick was burnt compared with general moxa stick, which led to a better curative effect. Our research also showed that the contents of essential oil, total flavonoids and tannin in qi ai were significantly higher than those of general AAF samples. Conclusion: The superior quality of qi ai compared with general AAF may be due to relatively higher contents of bioactive components. This study provides basic information for further study and utilization of AAF and qi ai.

Crataegus kansuensis Wils. is an important wild eco economical species of the family Rosaceae. The complete chloroplast genome reported here is 159,865 bp in length, including two inverted repeats (IRs) of 26,384 bp, which are separated by a large single-copy (LSC) and a small single-copy (SSC) of 87,815 bp and 19,282 bp, respectively. The whole chloroplast genome of C. kansuensis contains 113 genes, including 79 protein-coding genes, 30 transfer RNA, and 4 ribosome RNA. Phylogenetic analysis indicated that C. kansuensis is closely related to that of C. chungtienensis and C. marshallii, and the genus Crataegus L. was sister to the genus Amelanchier Medik.

The aim of the present study was to investigate the effect of wound location on the fungal communities and volatile distribution of agarwood in Aquilaria sinensis. Two-dimensional gas chromatography with high-resolution time-of-flight mass spectrometry revealed 60 compounds from the NIST library, including 25 sesquiterpenes, seven monoterpenes, two diterpenes, nine aromatics, nine alkanes and eight others. Of five agarwood types, Types IV and II contained the greatest number and concentration of sesquiterpenes, respectively. The fungal communities of the agarwood were dominated by the phylum Ascomycota and were significantly affected by the type of wound tissue. Community richness indices (observed species, Chao1, PD whole tree, ACE indices) indicated that Types I and IV harboured the most and least species-rich fungal communities, and the fungal communities of Types V, I, III and IV/II were dominated by Lasiodiplodia, Hydnellum, Phaeoisaria and Ophiocordyceps species, respectively. Correlations between fungal species and agarwood components revealed that the chemical properties of A. sinensis were associated with fungal diversity. More specifically, the dominant fungal genera of Types V, I and III (Lasiodiplodia, Hydnellum and Phaeoisaria, respectively) were strongly correlated with specific terpenoid compounds. The finding that wound location affects the fungal communities and volatile distribution of agarwood provides insight into the formation of distinct agarwood types.

Salvia miltiorrhiza Bunge, a member of the Lamiaceae family, is valued in traditional Chinese Medicine. Its dried root (named Danshen) has been used for hundreds of years, primarily for the treatment of cardiovascular and cerebrovascular diseases. Tanshinones are the main active ingredients in S. miltiorrhiza and exhibit significant pharmacological activities, such as antioxidant activity, anti-inflammatory activity, cardiovascular effects, and antitumor activity. Danshen dripping pill of Tianshili is an effective drug widely used in the clinical treatment of cardiovascular diseases. With the increasing demand for clinical drugs, the traditional method for extracting and separating tanshinones from medicinal plants is insufficient. Therefore, in combination with synthetic biological methods and strategies, it is necessary to analyze the biosynthetic pathway of tanshinones and construct high-yield functional bacteria to obtain tanshinones. Moreover, the biosynthesis of tanshinones has been studied for more than two decades but remains to be completely elucidated. This review will systematically present the composition, extraction and separation, pharmacological activities and biosynthesis of tanshinones from S. miltiorrhiza, with the intent to provide references for studies on other terpenoid bioactive components of traditional Chinese medicines and to provide new research strategies for the sustainable development of traditional Chinese medicine resources.

Ginseng, the root and rhizome of Panax ginseng C. A. Mey., is a well-known and valuable traditional Chinese medicine. The pharmacological activities of ginseng are mainly attributed to the presence of ginsenosides, which are considered to be critical biomarkers for evaluating ginseng quality. The biosynthesis of triterpenes, which serve defensive functions in plants, is regulated by endogenous phytohormones that play key roles in growth and defense of plant populations. However, the role of major hormones that are closely related to secondary metabolism pathways in P. ginseng is poorly understood. To gain insight into their potential correlation, we performed a spatial synthesis analysis and studied the distribution of endogenous phytohormones and ginsenosides in different tissue regions of the entire P. ginseng plant. Gibberellins are growth hormones that accumulate in the fiber root. In contrast, abscisic acid (ABA), salicylic acid (SA), and jasmonic acid (JA), which are considered stress hormones, were predominantly found in the leaf and leaf peduncle. We observed a tissue-specific distribution of phytohormones consistent with the expression of genes involved in hormone biosynthesis that influenced ginsenoside synthesis and distribution. The aim of this study was to investigate the role of different endogenous phytohormones on triterpene metabolites in ginseng innate immunity.

An amendment to this paper has been published and can be accessed via a link at the top of the paper.

Dioscorea L., the largest genus of the family Dioscoreaceae with over 600 species, is not only an important food but also a medicinal plant. The identification and classification of Dioscorea L. is a rather difficult task. In this study, we sequenced five Dioscorea chloroplast genomes, and analyzed with four other chloroplast genomes of Dioscorea species from GenBank. The Dioscorea chloroplast genomes displayed the typical quadripartite structure of angiosperms, which consisted of a pair of inverted repeats separated by a large single-copy region, and a small single-copy region. The location and distribution of repeat sequences and microsatellites were determined, and the rapidly evolving chloroplast genome regions (trnK-trnQ, trnS-trnG, trnC-petN, trnE-trnT, petG-trnW-trnP, ndhF, trnL-rpl32, and ycf1) were detected. Phylogenetic relationships of Dioscorea inferred from chloroplast genomes obtained high support even in shortest internodes. Thus, chloroplast genome sequences provide potential molecular markers and genomic resources for phylogeny and species identification.

Tripterygium wilfordii is a perennial woody liana medicinal plant with several crucial biological activities. Although studies on tissue culture have previously been conducted, research on genetic transformation is much more challenging and therefore results in slower progress. In the present study, a highly efficient transformation system involving the particle bombardment of T. wilfordii with the reporter egfp gene using the PDS-1000/He system was established. A total of seven parameters affecting the genetic transformation were investigated using an L18 (6 × 36)-type orthogonal array. The result indicated that DNA delivery conditions of 3-cm target distance, 1100 psi helium pressure, 28 mmHg chamber vacuum pressure, three times number of bombardment, CaCl2 as precipitation agent, 2 μg plasmid DNA concentration and 48 h post-bombardment incubation time were optimal for T. wilfordii cell suspensions transformation. The average transformation efficiency was 19.17%. Based on this transformation system, the overexpression of two T. wilfordii farnesyl pyrophosphate synthase genes (TwFPSs) was performed in cell suspensions. Integration of the TwFPSs in the genome was verified by PCR analysis and also by Southern blotting using hygromycin gene as a probe. Real-time quantitative PCR analysis showed that the expression of TwFPS1&2 was highly up regulated in transgenic cell suspensions compared with control cells. The detection of metabolites showed that TwFPS1&2 could highly increase the celastrol content (973.60 μg/g) in transgenic cells. These results indicated that this transformation system is an effective protocol for characterizing the function of genes in the terpenoid biosynthetic pathway.

Bupleurum chinense DC. is a traditional medicinal herb species widely distributed in most provinces of China. In this study, we constructed and annotated a complete circular chloroplast (cp) genome of B. chinense. The cp genome of B. chinense is 155,545 bp in length, including two inverted repeat (IR) regions of 26,305 bp, separated by a large single-copy (LSC) region of 85,430 bp and a small single-copy (SSC) region of 17,505 bp. The GC content of whole cp genome is 37.68%. The genome contains 113 different genes, including 79 protein-coding genes, 30 tRNA genes, and 4 rRNA genes. A maximum-likelihood phylogenomic analysis showed that Bupleurum formed a monophyletic group, and was sister to other groups of Apiaceae.

Gillenia trifoliata (L.) Moench is an ornamental and medicinal species endemic to North America. In this study, a complete circular chloroplast genome of G. trifoliata was assembled and annotated. The chloroplast genome of G. trifoliata is 159,496 bp in size and consists of two IR (26,394 bp) regions separated by LSC (87,647 bp) and SSC (19,061 bp) regions. The GC content of the chloroplast genome is 36.5%. The chloroplast genome comprised of 112 different genes, including 78 protein-coding genes. The phylogenetic analysis revealed that G. trifoliata lies within subfamily Amygdaloideae and forms the progenitor lineage of the pome-bearing Maleae.

Isopentenyl diphosphate isomerase (IPI) catalyzes the isomerization between the common terpene precursor substances isopentenyl diphosphate (IPP) and dimethylallyl diphosphate (DMAPP) during the terpenoid biosynthesis process. In this study, tissue expression analysis revealed that the expression level of the Salvia miltiorrhiza IPI1 gene (SmIPI1) was higher in the leaves than in the roots and stems. Furthermore, color complementation and RNA interference methods were used to verify the function of the SmIPI1 gene from two aspects. A recombinant SmIPI1 plasmid was successfully constructed and transferred into engineered E. coli for validating the function of SmIPI1 through the color difference in comparison to the control group; the observed color difference indicated that SmIPI1 served in promoting the accumulation of lycopene. Transformant hairy root lines with RNA interference of SmIPI1 were successfully constructed mediated by Agrobacterium rhizogenes ACCC 10060. RNA interference hairy roots had a severe phenotype characterized by withering, deformity or even death. The mRNA expression level of SmIPI1 in the RSi3 root line was only 8.4% of that of the wild type. Furthermore the tanshinone content was too low to be detected in the RNA interference lines. These results suggest that SmIPI1 plays a critical role in terpenoid metabolic pathways. Addition of an exogenous SmIPI1 gene promoted metabolic flow toward the biosynthesis of carotenoids in E. coli, and SmIPI1 interference in S. miltiorrhiza hairy roots may cause interruption of the 2-C-methyl-D-erythritol-4-phosphate metabolic pathway.

Tripterygium wilfordii produces not only ent-kaurene, which is an intermediate of gibberellin (GA) biosynthesis in flowering plants, but also 16α-hydroxy-ent-kaurane, whose physiological role has not been characterized. The two compounds are biosynthesized from the universal diterpenoid precursor (E,E,E)-geranylgeranyl diphosphate (GGPP) by diterpene synthases, which have been discovered and functionally characterized in T. wilfordii. Here, we described the functional characterization of four cytochrome P450 reductases (TwCPR) and one ent-kaurene oxidase (TwKO). Four TwCPRs were found to have relatively ubiquitous expression in T. wilfordii root, stem, leaf, and flower tissues. Co-expression of both a TwCPR and TwKO in yeast showed that TwCPR3 has a slightly better activity for providing the electrons required for these reactions, indicating that TwCPR3 is more suitable for use in the functional analysis of other cytochrome P450 monooxygenases. TwKO catalyzed the three-step oxidation of the C4α methyl of the tetracyclic diterpene intermediate ent-kaurene to form ent-kaurenoic acid as an early step in GA biosynthesis. Notably, TwKO could also convert 16α-hydroxy-ent-kaurane to 16α-hydroxy-ent-kaurenoic acid, indicating an important function of 16α-hydroxy-ent-kaurane in the anti-HIV principle tripterifordin biosynthetic pathway in planta. Homology modeling and molecular docking were used to investigate the unknown crucial active amino acid residue involved in the catalytic reaction of TwKO, and one key residue (Leu387) contributed to the formation of 16α-hydroxy-ent-kaurenoic acid, most likely by forming hydrogen bonds with the hydroxyl group (-OH) of 16α-hydroxy-ent-kaurane, which laid the basis for further investigation of the multifunctional nature of KO catalysis. Also, our findings paved the way for the complete biosynthesis of the anti-HIV principle tripterifordin.

The content of active compounds differ in buds and flowers of Lonicera japonica (FLJ) and L. japonica var. chinensis (rFLJ). Chlorogenic acid (CGAs) were major active compounds of L. japonica and regarded as measurements for quality evaluation. However, little is known concerning the formation of active compounds at the molecular level. We quantified the major CGAs in FLJ and rFLJ, and found the concentrations of CGAs were higher in the buds of rFLJ than those of FLJ. Further analysis of CpG methylation of CGAs biosynthesis genes showed differences between FLJ and rFLJ in the 5′-UTR of phenylalanine ammonia-lyase 2 (PAL2). We identified 11 LjbZIP proteins and 24 rLjbZIP proteins with conserved basic leucine zipper domains, subcellular localization, and electrophoretic mobility shift assay showed that the transcription factor LjbZIP8 is a nuclear-localized protein that specifically binds to the G-box element of the LjPAL2 5′-UTR. Additionally, a transactivation assay and LjbZIP8 overexpression in transgenic tobacco indicated that LjbZIP8 could function as a repressor of transcription. Finally, treatment with 5-azacytidine decreased the transcription level of LjPAL2 and CGAs content in FLJ leaves. These results raise the possibility that DNA methylation might influence the recruitment of LjbZIP8, regulating PAL2 expression level and CGAs content in L. japonica.

The poor quality and low productivity of cultivated liquorice (Glycyrrhiza uralensis) continues to put pressure on wild plant populations. As arbuscular mycorrhizal fungi are known to support plant growth and in some cases even to enhance the accumulation of valuable molecules in the plant, the effect of Glomus mosseae on the growth and active ingredient contents was evaluated in liquorice plants grown under nutrient deficiency. We created a nutrient-deficient environment by mixing paddy soil, washed river sand, and pumice at a ratio of 1:5:1. Our results showed that the inoculation of pot-grown liquorice plants with G. mosseae significantly increased the shoot and root biomass (by 25- and 17-folds, respectively) and the contents of glycyrrhizic acid, liquiritin, isoliquiritin, and isoliquiritigenin in the main root (by 1.6-, 4.8-, 6.5-, and 4.4-folds, respectively). Both isoliquiritin and isoliquiritigenin were detectable in the lateral roots of the plants inoculated with G. mosseae, but not in plants without G. mosseae inoculation. G. mosseae inoculation improved the features of the root system and increased photosynthetic efficiency of liquorice. The uptake of P and K by liquorice increased when G. mosseae was inoculated, leading to the depletion of these macronutrients in the soil; G. mosseae also improved the availability of Mg, Cu, Zn, and Mn. Based on these results, we concluded that the inoculation of liquorice plants with G. mosseae is beneficial, particularly for those grown in nutrient-deficient soil, and such positive effect is related to the improvement of the root system and an increased photosynthetic efficiency.

[This corrects the article DOI: 10.1371/journal.pone.0164235.].

Daodi-herb is a part of Chinese culture, which has been naturally selected by traditional Chinese medicine clinical practice for many years. Sweet wormwood herb is a kind of Daodi-herb, and comes from Artemisia annua L. Artemisinin is a kind of effective antimalarial drug being extracted from A. annua. Because of artemisinin, Sweet wormwood herb earns a reputation. Based on the Pharmacopoeia of the People's Republic of China (PPRC), Sweet wormwood herb can be used to resolve summerheat-heat, and prevent malaria. Besides, it also has other medical efficacies. A. annua, a medicinal plant that is widely distributed in the world contains many kinds of chemical composition. Research has shown that compatibility of artemisinin, scopoletin, arteannuin B and arteannuic acid has antimalarial effect. Compatibility of scopoletin, arteannuin B and arteannuic acid is conducive to resolving summerheat-heat. Chemical constituents in A. annua vary significantly according to geographical locations. So, distribution of A. annua may play a key role in the characteristics of efficacy and chemical constituents of Sweet wormwood herb. It is of great significance to study this relationship.We mainly analyzed the relationship between the chemical constituents (arteannuin B, artemisinin, artemisinic acid, and scopoletin) with special efficacy in A. annua that come from different provinces in china, and analyzed the relationship between chemical constituents and spatial distribution, in order to find out the relationship between efficacy, chemical constituents and distribution.A field survey was carried out to collect A. annua plant samples. A global positioning system (GPS) was used for obtaining geographical coordinates of sampling sites. Chemical constituents in A. annua were determined by liquid chromatography tandem an atmospheric pressure ionization-electrospray mass spectrometry. Relationship between chemical constituents including proportions, correlation analysis (CoA), principal component analysis (PCA) and cluster analysis (ClA) was displayed through Excel and R software version2.3.2(R), while the one between efficacy, chemical constituents and spatial distribution was presented through ArcGIS10.0, Excel and R software.According to the results of CoA, arteannuin B content presented a strong positive correlation with artemisinic acid content (p = 0), and a strong negative correlation with artemisinin content (p = 0). Scopoletin content presented a strong positive correlation with artemisinin content (p = 0), and a strong negative correlation with artemisinic acid content (p = 0). According to the results of PCA, the first two principal components accounted for 81.57% of the total accumulation contribution rate. The contribution of the first principal component is about 45.12%, manly including arteannuin B and artemisinic acid. The contribution of the second principal component is 36.45% of the total, manly including artemisinin and scopoletin. According to the ClA by using the principal component scores, 19 provinces could be divided into two groups. In terms of provinces in group one, the proportions of artemisinin are all higher than 80%. Based on the results of PCA, ClA, percentages and scatter plot analysis, chemical types are defined as "QHYS type", "INT type" and "QHS type."As a conclusion, this paper shows the relationship between efficacy, chemical constituents and distribution. Sweet wormwood herb with high arteannuin B and artemisinic acid content, mainly distributes in northern China. Sweet wormwood herb with high artemisinin and scopoletin content has the medical function of preventing malaria, which mainly distributes in southern China. In this paper, it is proved that Sweet wormwood Daodi herb growing in particular geographic regions, has more significant therapeutical effect and higher chemical constituents compared with other same kind of CMM. And also, it has proved the old saying in China that Sweet wormwood Daodi herb which has been used to resolve summerheat-heat and prevent malaria, which distributed in central China. But in modern time, Daodi Sweet wormwood herb mainly has been used to extract artemisinin and prevent malaria, so the Daod-region has transferred to the southern China.

As an edible sclerotia-forming fungus, Poria cocos is widely used as a food supplement and as a tonic in China. High-performance liquid chromatography-quadrupole/time-of-flight mass spectrometry (HPLC-QTOF-MS/MS) was applied to identify triterpene acids in fermented mycelia of P. cocos, as well as the epidermis and inner part of natural sclerotia. A total of 19 triterpene acids were identified in fermented mycelia, whereas 31 were identified in the epidermis and 24 in the inner part. Nine triterpene acids were quantitatively determined, and the concentrations of two valuable triterpenes, dehydropachymic acid and pachymic acid, reached 1.07 mg/g and 0.61 mg/g in the fermented mycelia part, respectively, and were both significantly higher than the concentration in the two natural parts. The fermented mycelia could be a good choice for producing some target triterpene compounds and functional foods through fermentation thanks to the high concentration of some triterpene acids.

A method combining hydrophilic interaction chromatography (HILIC) and electrospray ionization mass spectrometry (ESI-MS) was developed for the characterization and determination of natural Cordyceps. Separation was achieved on a Waters Xbridge Amide column with gradient elution. Identification of 15 target nucleosides and nucleobases was based on retention time, UV spectra and mass measurements of the protonated molecules ([M+H]+) and main fragment ions (ESI-TOF/MS). Eight non-target compounds were tentatively identified by ESI-TOF/MS. The 15 target compounds were quantified by HILIC-ESI-MS/MS using time-programmed selective ion monitoring or multiple reaction monitoring in positive-ion mode under optimized mass conditions. This technique showed good linearity, repeatability and recovery. This approach was also successfully implemented in the analysis of nucleosides and nucleobases in 12 batches of natural Cordyceps samples that were collected from different regions in China. The developed HILIC-ESI-MS method exhibited clear advantages in identifying and determining highly polar bioactive components in Cordyceps, as well as their quality control.

In China, over 40 species of the genus Salvia have been used as medicinal plants for various diseases, some for thousands of years. Recently, research has focused on the biological activities of Salvia medicinal plants used in traditional chinese medicine (TCM). However, to date a scientific survey of the genus Salvia in China has not been carried out. In this paper, we report the results of 10 field surveys of Salvia medicinal plants collected in 17 provinces including detailed information on their local names, growing environment, distribution and therapeutic effects. We also summarize the results of research on the materia medica, phytochemistry and pharmacology of some of the important Salvia medicinal plants. Our study reveals that 35 Salvia plants have been used in TCM in different regions of China, including 20 species used as Danshen to treat heart diseases, and 15 species used to treat a range of other conditions including gynecological diseases, muscular or skeletal problems, hepatitis, urological diseases, and mouth and eye conditions. It is clear that some species of Salvia L. possess significant pharmacological activity in the context of ethnopharmacological knowledge, especially in the treatment of heart disease. Further studies of the phytochemistry and pharmacology of Salvia species will no doubt improve their medical utilization and contribute to the development of new natural drugs.

Tanshinones are abietane-type norditerpenoid quinone natural products found in a well-known traditional Chinese medicinal herb, Salvia miltiorrhiza Bunge. The copalyl diphosphate synthase of S. miltiorrhiza (SmCPS) is the key enzyme in the first step for transformation of geranylgeranyl diphosphate (GGPP) into miltiradiene, which has recently been identified as the precursor of tanshinones. Based on previous gene-to-metabolite network, this study examined the influences of various combined elicitors on the expression of SmCPS and production of tanshinones in S. miltiorrhiza hairy root cultures. Combined elicitors were composed of three classes of elicitors, a heavy metal ion (Ag+), a polysaccharide (yeast extract, YE), and a plant response-signalling compound (methyl jasmonate, MJ). YE + Ag+, Ag+ + MJ, YE + MJ, and YE + Ag+ + MJ were the combinations we tested. The effect of elicitors on the SmCPS expression level was detected by quantitative real-time PCR (qRT-PCR), and the tanshinones accumulation responses to elicitation were analysed by Ultra Performance Liquid Chromatography (UPLC) metabolite profiling. Of these combined elicitors, the expression of SmCPS was significantly enhanced by elicitation, especially at 24 h and 36 h. Of four tanshinones detected, the contents of cryptotanshinone and dihydrotanshinone I were enhanced by treatment with YE + Ag+, Ag+ + MJ, and YE + Ag+ + MJ. Our results indicate that appropriate combined elicitors can enhance tanshinones production in hairy root cultures.

An efficient method for the rapid separation and purification of polyphenols from artichoke by polyamide column chromatography in combination with high-speed counter-current chromatography (HSCCC) was successfully built. The crude ethanol extracts from dry artichoke were first pre-separated by polyamide column chromatography and divided in two parts as sample 1 and sample 2. Then, the samples were further separated by HSCCC and yielded 7.8 mg of chlorogenic acid (compound I), 24.5 mg of luteolin-7-O-β-D-rutinoside (compound II), 18.4 mg of luteolin-7-O-β-D-glucoside (compound III), and 33.4 mg of cynarin (compound IV) with purity levels of 92.0%, 98.2%, 98.5%, and 98.0%, respectively, as determined by high-performance liquid chromatography (HPLC) method. The chemical structures of these compounds were identified by electrospray ionization-mass spectrometry (ESI-MS) and nuclear magnetic resonance (NMR).

The technique of pH-zone-refining counter-current chromatography was successfully applied to preparatively separate three C19-diterpenoid alkaloids from the crude extracts of Aconitum carmichaelii for the first time using a two-phase solvent system of petroleum ether-ethyl acetate-methanol-water (5:5:1:9, v/v/v/v). Mesaconitine (I), hypaconitine (II), and deoxyaconitine (III) were obtained from 2.5 g of the crude alkaloids in a one-step separation; the yields were 4.16%, 16.96%, and 5.05%, respectively. The purities of compounds I, II, and III were 93.0%, 95%, and 96%, respectively, as determined by HPLC. The chemical structures of the three compounds were identified by electrospray ionization mass spectrometry (ESI-MS) and NMR.

Salvia miltiorrhiza Bunge，which is also known as a traditional Chinese herbal medicine，is widely studied for its ability to accumulate the diterpene quinone Tanshinones. In addition to producing a variety of diterpene quinone, S. miltiorrhiza Bunge also accumulates sterol, brassinosteroid and triterpenoids. During their biosynthesis, squalene synthase (SQS, EC 2.5.1.21) converts two molecules of the hydrophilic substrate farnesyl diphosphate into a hydrophobic product, squalene. In the present study, cloning and characterization of S. miltiorrhiza Bunge squalene synthase 2 (SmSQS2, Genbank Accession Number: KM408605) cDNA was investigated subsequently followed by its recombinant expression and preliminary enzyme activity. The full-length cDNA of SmSQS2 was 1 597 bp in length, with an open reading frame (ORF) of 1 245 bp encoding 414 amino acids. The deduced amino acid sequence of SmSQS2 shared high similarity with those of SQSs from other plants. To obtain soluble recombinant enzymes, the truncated SmSQS2 in which 28 amino acids were deleted from the carboxy terminus was expressed as GST-Tag fusion protein in Escherichia coli BL21 (DE3) and confirmed by SDS-PAGE and Western Blot analysis, and the resultant bacterial crude extract was incubated with farnesyl diphosphate and NADPH. GC-MS analysis showed that squalene was detected in the in vitro reaction mixture. The gene expression level was analyzed through Quantitative real-time PCR, and was found to be higher in roots as compared to the leaves, and was up-regulated upon YE+ Ag+ treatment. These results could serve as an important to understand the function of the SQS family. In addition, the identification of SmSQS2 is important for further studies of terpenoid and sterol biosynthesis in S. miltiorrhiza Bunge.

MicroRNAs (miRNAs), which play crucial regulatory roles in plant secondary metabolism and responses to the environment, could be developed as promising biomarkers for different varieties and production areas of herbal medicines. However, limited information is available for miRNAs from Lonicera japonica, which is widely used in East Asian countries owing to various pharmaceutically active secondary metabolites. Selection of suitable reference genes for quantification of target miRNA expression through quantitative real-time (qRT)-PCR is important for elucidating the molecular mechanisms of secondary metabolic regulation in different tissues and varieties of L. japonica. For precise normalization of gene expression data in L. japonica, 16 candidate miRNAs were examined in three tissues, as well as twenty-one cultivated varieties collected from sixteen production areas, using GeNorm, NormFinder, and RefFinder algorithms. Our results revealed combination of u534122 and u3868172 as the best reference genes across all samples. Their specificity was confirmed by detecting the cycling threshold (Ct) value ranges in different varieties of L. japonica collected from diverse production areas, suggesting the use of these two reference miRNAs is sufficient for accurate transcript normalization with different tissues, varieties, and production areas. To our knowledge, this is the first report on validation of reference miRNAs in honeysuckle (Lonicera spp.). Restuls from this study can further facilitate discovery of functional regulatory miRNAs in different varieties of L. japonica.

Pseudostellaria heterophylla (Miq.) Pax is a mild tonic herb widely cultivated in the Southern part of China. The tuberous roots of P. heterophylla accumulate high levels of secondary metabolism products of medicinal value such as saponins, flavonoids, and isoquinoline alkaloids. Despite numerous studies on the pharmacological importance and purification of these compounds in P. heterophylla, their biosynthesis is not well understood. In the present study, we used Illumina HiSeq 4000 sequencing platform to sequence the RNA from flowers, leaves, stem, root cortex and xylem tissues of P. heterophylla. We obtained 616,413,316 clean reads that we assembled into 127, 334 unique sequences with an N50 length of 951 bp. Among these unigenes, 53,184 unigenes (41.76%) were annotated in a public database and 39, 795 unigenes were assigned to 356 KEGG pathways; 23,714 unigenes (8.82%) had high homology with the genes from Beta vulgaris. We discovered 32, 095 DEGs in different tissues and performed GO and KEGG enrichment analysis. The most enriched KEGG pathway of secondary metabolism showed up-regulated expression in tuberous roots as compared with the ground parts of P. heterophylla. Moreover, we identified 72 candidate genes involved in triterpenoids saponins biosynthesis in P. heterophylla. The expression profiles of 11 candidate unigenes were analyzed by quantitative real-time PCR (RT-qPCR). Our study established a global transcriptome database of P. heterophylla for gene identification and regulation. We also identified the candidate unigenes involved in triterpenoids saponins biosynthesis. Our results provide an invaluable resource for the secondary metabolites and physiological processes in different tissues of P. heterophylla.