Research progress relating to the role of cytochrome P450 in the biosynthesis of terpenoids in medicinal plants.

Terpenoids are an extensive and diverse group of plant secondary metabolites. To date, they have been applied in many fields including industry, medicine and health. The wide variety of terpenoid compounds cannot arise solely from simple cyclisations of a precursor molecule or from a single-step reaction; their structural diversity depends on the modification of many specific chemical groups, rearrangements of their skeletal structures and on the post-modification reactions. Most of the post-modification enzymes that catalyse these reactions are cytochrome P450 monooxygenases. Therefore, the discovery and identification of plant P450 genes plays a vital role in the exploration of terpenoid biosynthesis pathways. This review summarises recent research progress relating to the function of plant cytochrome P450 enzymes, describes P450 genes that have been cloned from full-length cDNA and identifies the function of P450 enzymes in the terpenoid biosynthesis pathways of several medicinal plants.

[Cloning and bioinformatics analysis of ent-kaurene oxidase synthase gene in Salvia miltiorrhiza].

Based on the transcriptome database of Salvia miltiorrhiza, specific primers were designed to clone a full-length cDNA of ent-kaurene oxidase synthase (SmKOL) using the RACE strategy. ORF Finder was used to find the open reading frame of SmKOL cDNA, and ClustalW has been performed to analysis the multiple amino acid sequence alignment. Phylogenetic tree has been constructed using MEGA 5.1. The transcription level of SmKOL from the hairy roots induced by elicitor methyl jasmonate (MeJA) was qualifiedby real-time quantitative PCR. The full length of SmKOL cDNA was of 1 884 bp nucleotides encoding 519 amino acids. The molecular weight of the SmKOL protein was about 58.88 kDa with isoelectric point (pI) of 7.62. Results of real-time quantitative PCR analyses indicated that the level of SmKOL mRNA expression in hairy roots was increased by elicitor oMeJA, and reached maximum in 36 h. The full-length cDNA of SmKOL was cloned from S. miltiorrhiza hairy root, which provides a target gene for further studies of its function, gibberellin biosynthesis and regulation of secondary metabolites.

[Cloning and induced expression analysis of 4-hydroxy-3-methyl-but-2-enyl diphosphate reductase gene (smHDR) of Salvia miltiorrhiza].

This study reported the obtainment of the full-length cDNA of Salvia miltiorrhiza hairy roots (Abbr: SmHDR, GenBank number: JX233817), via extracting Salvia miltiorrhiza hairy roots total RNA, designing specific primers according to the transcriptome data and using the RACE strategy, and then analyzed it with bioinformatics approaches. On this basis, using the real-time PCR to detect SmHDR gene expression after Ag+ induction, and testing tanshinones contents of corresponding samples by UPLC. SmHDR has 1 647 nucleotides, and an open reading frame (ORF) encoding a protein of 463 amino acid residues. The deduced protein has isoelectric point (pI) of 5.72 and a calculated molecular weight about 51.88 kD. In the secondary structure, the percentage of alpha helix, beta turn and random coil were 35.64%, 20.30% and 44.06%, respectively. Sequence alignment and phylogenetic analysis demonstrated that SmHDR had relative close relationship to the HDR of Picrorhiza kurrooa, similar to HDR from other species of plants. Real time PCR results indicated that elicitor of Ag+ stimulated the increase of mRNA expression of SmHDR. At the same time, results of ultra performance liquid chromatography (UPLC), used to examine the accumulation of diterpenoid tanshinones in hairy roots, showed that the contents of diterpenoid tanshinones in hairy roots of Salvia miltiorrhiza were increased dramatically at 12 h after treated with Ag+, and then decreased significantly. This result showed a positive correlation between the levels of mRNA expression and tanshinones accumulation in Salvia miltiorrhiza stimulated by Ag+. The content of tanshinones was gradually raised, and it had an obvious increase at 120 h. The bioinformatics analysis and gene expression indicated that SmHDR might be involved in tanshinones biosynthesis, which laid the foundation for further study of secondary metabolic regulation mechanism of tanshinones.

[Advances in studies on 3-hydroxy-3-metllylglutaryl coenzyme A reductase in terpenoids biosynthesis of medicinal plants].

There exists many kinds and a huge number of terpenoid in medicinal plants, which show a wide range of pharmacological activities. 3-Hydroxy-3-metllylglutaryl coenzyme A reductase(HMGR) is a key rate-limiting enzyme in terpenoid biosynthetic pathway . HMGR plays an important role in the regulation of secondary metabolism of the terpenoid. The paper summarized the biological function and the catalytic mechanism of HMGR, the cloning and the structure of the gene as well as its research progress in some medicinal plants.

[Cloning and bioinformatics analysis of 2-c-methyl-D-erythritol 2,4-cyclodiphosphate synthase gene in Salvia miltiorrhiza].

OBJECTIVE: To clone and analysis a 2-C-methyl-D-erythritol 2,4-cyclodiphosphate synthase (SmMCS) full-length eDNA from Salvia miltiorrhiza hairy roots. METHOD: A full-length eDNA of SmMCS has been cloned by designing specific primers according to the transcriptome database and using the RACE strategy. ORF Finder was used to find the open reading frame of SmMCS cDNA and ClustalW has been performed to analysis the multiple amino acid sequence alignment. Phylogenetic tree has been constructed using MEGA 5. 1. Real-time quantitative PCR have been applied to detect the transcription level of SmMCS from hairy roots after elicitor Ag+ supplied. RESULT: The SmMCS cDNA sequence was obtained. The full length of SmMCS (DNA was 988 bp encoding 234 amino acids. The deduced protein had isoelectric point (pI) of 8.53 and a calculated molecular weight about 24. 6 kDa. Results of real time PCR indicated that elicitor of Ag+ stimulated the increase of mRNA expression of SmMCS in hairy roots, and were increased dramatically at 12 h. CONCLUSION: The full-length cDNA of SmMCS was cloned from S. miltiorrhiza hairy root,which can provide a gene target for further studies of tanshinones biosynthesis and terpenoid secondary metabolites.

Therapeutic potential of traditional Chinese medicine against atherosclerosis: Targeting trimethylamine N-oxide.

BACKGROUND: Recent studies have shown that plasma trimethylamine-N-oxide (TMAO) level is highly correlated with the risk of atherosclerosis (AS), and the elevated level is significantly positively correlated with the incidence of AS. PURPOSE: The purpose of this article is to offer a useful summary of the correlation between TMAO and AS, and the effect of herbal monomers, herbal extracts, and formulas on anti-atherosclerosis mediated by TMAO. METHOD: The data contained in this article comes from PubMed, Web of Science, and China National Knowledge Infrastructure. RESULTS: This review discusses the main mechanism of AS induced by TMAO, including endothelial dysfunction, macrophage foaming, platelet reactivity, and cholesterol metabolism, and summarizes 6 herb monomers, 5 herb extracts, and 2 formulas that have been tested for their anti-TMAO activity. CONCLUSION: The current understanding of possible ways to reduce TMAO generation is discussed, with the effect and potential of herb monomers, herb extracts, and formulas highlighted.

Establishment of a High Content Image Platform to Measure NF-κB Nuclear Translocation in LPS-induced RAW264.7 Macrophages for Screening Anti-inflammatory Drug Candidates.

BACKGROUND: High Content Image (HCI), an automatic imaging and analysis system, provides a fast drug screening method by detecting the subcellular distribution of protein in intact cells. OBJECTIVE: This study established the first standardized HCI platform for lipopolysaccharide (LPS)-induced RAW264.7 macrophages to screen anti-inflammatory compounds by measuring nuclear factor-κB (NF-κB) nuclear translocation. METHODS: The influence of the cell passages, cell density, LPS induction time and concentration, antibody dilution, serum, dimethyl sulfoxide, and analysis parameters on NF-κB nuclear translocation and HCI data quality was optimized. The BAY-11-7085, the positive control for inhibiting NF-κB, and the Western blot assay were separately employed to verify the stability and reliability of the platform. Lastly, the effect of BHA on NO release, iNOS expression, IL-1ß, IL-6, and TNF-α mRNA in LPS-induced RAW264.7 cells was detected. RESULTS: The optimal conditions for measuring NF-κB translocation in LPS-induced RAW264.7 cells by HCI were established. Cells that do not exceed 22 passages were seeded at a density of 10 k cells/well and pretreated with compounds following 200 ng/mL LPS for 40 min. Parameters including the nuclear area of 65 µm2, cell area of 80 µm2, collar of 0.9 µm, and sensitivity of 25% were recommended for image segmentation algorithms in the analysis workstation. Benzoylhypaconine from aconite was screened for the first time as an anti-inflammatory candidate by the established HCI platform. The inhibitory effect of benzoylhypaconine on NF-κB translocation was verified by Western blot. Furthermore, benzoylhypaconine reduced the release of NO, inhibited the expression of iNOS, and decreased the mRNA levels of IL-1ß, IL-6, and TNF-α. CONCLUSION: The established HCI platform could be applied to screen anti-inflammatory compounds by measuring the NF-κB nuclear translocation in LPS-induced RAW264.7 cells.

Identification of blood-red color formation in edible bird's nests provides a new strategy for safety control.

In order to reveal the color formation mechanism of blood-red edible bird's nests (EBNs) and develop a quick and specific strategy to distinguish the artificial fake one, multiple methods of UPLC-TOF/MS, UV, NMR, FT-IR and 2D IR were used to detect the chemical markers of the reddening reaction, the results showed that the reddening substances were C9H10N2O5 and C9H9NO6, which were verified as products of a phenol-keto tautomerism evolved from l-tyrosine. Moreover, natural and artificial red EBNs with varying degrees of chemical fumigation also can be successfully distinguished using the chemical markers, and the protein variation in SDS-PAGE gel could also support the distinction. This work established a systematic method of chemical identification for both natural and artificial blood-red EBNs, and provided a new identification strategy for food safety control that can promote the development of a healthier market of EBNs.

Review on the Development and Applications of Medicinal Plant Genomes.

With the development of sequencing technology, the research on medicinal plants is no longer limited to the aspects of chemistry, pharmacology, and pharmacodynamics, but reveals them from the genetic level. As the price of next-generation sequencing technology becomes affordable, and the long-read sequencing technology is established, the medicinal plant genomes with large sizes have been sequenced and assembled more easily. Although the review of plant genomes has been reported several times, there is no review giving a systematic and comprehensive introduction about the development and application of medicinal plant genomes that have been reported until now. Here, we provide a historical perspective on the current situation of genomes in medicinal plant biology, highlight the use of the rapidly developing sequencing technologies, and conduct a comprehensive summary on how the genomes apply to solve the practical problems in medicinal plants, like genomics-assisted herb breeding, evolution history revelation, herbal synthetic biology study, and geoherbal research, which are important for effective utilization, rational use and sustainable protection of medicinal plants.

Discovery of Quality Markers in Hugan Qingzhi Formula by Integrating a Lipid-Lowering Bioassay with UHPLC-QQQ-MS/MS.

Nonalcoholic fatty liver disease (NAFLD) is a prevalent chronic liver disease. The Hugan Qingzhi formula (HGQZ) has been proven effective in treating NAFLD through clinical and pharmacological mechanism studies. A screening study of the chemical components was carried out to better control the quality of this formula. Current research has combined biological activity assessment with chemical analysis to screen and identify the bioactive compounds in HGQZ for use as potential quality markers (Q-markers) to control the quality of this herbal product. The HGQZ extracted by three different solvents was evaluated in a free fatty acid-induced hepatic steatosis LO2 cell model. Simultaneously, the twelve major chemical constituents of these extracts were quantitatively measured by ultrahigh-performance liquid chromatography coupled with triple quadrupole mass spectrometry (UHPLC-QQQ-MS/MS). Extraction with 50% ethanol showed the most potent lipid-lowering effect in steatosis LO2 cells and the highest extraction rate of major chemical constituents. Correlation analysis was used to establish the relationship between the biological activities and chemical characteristics of these extracts. The results showed that the contents of typhaneoside, hyperoside, isoquercitrin, isorhamnetin-3-O-neohesperidoside, notoginsenoside R1, and alisol B 23-acetate were positively correlated to the lipid-lowering effect. The subsequent bioassay confirmed that typhaneoside, isoquercitrin, and alisol B 23-acetate played the role of reducing the lipid effect. In conclusion, 50% of ethanol extraction produced the most active extract of HGQZ. Typhaneoside, isoquercitrin, and alisol B 23-acetate could be considered potential Q-markers for the quality control of HGQZ.

Novel SNP markers on ginsenosides biosynthesis functional gene for authentication of ginseng herbs and commercial products.

Panax ginseng and Panax quinquefolius have similar bioactive components and morphological characteristics, but they are known to have different medicinal values, high-sensitive and accurate method is expected to identify the sources of ginseng products and evaluate the quality, but with a huge challenge. Our established UHPLC-TOF/MS method coupled with orthogonal partial least squares discriminant analysis (OPLS-DA) model based on 18 ginsenosides was applied to discriminate the sources of raw medicinal materials in ginseng products, and nested PCR strategy was used to discover 6 novel single nucleotide polymorphism (SNP) sites in functional dammarenediol synthase (DS) gene for genetic authentication of P. ginseng and P. quinquefolius for the first time. OPLS-DA model could identify the sources of raw ginseng materials are real or not. SNP markers were applied to identify ginseng fresh samples as well as commercial products, and proved to be successful. This established molecular method can tell exact source information of adulterants, and it was highly sensitive and specific even when total DNA amount was only 0.1 ng and the adulteration was as low as 1%. Therefore, this study made an attempt at the exploration of new type SNP marker for variety authentication and function regulation at the same time, and the combination of chemical and molecular discrimination methods provided the comprehensive evaluation and authentication for the sources of ginseng herbs and products.

Discovery of differential sequences for improving breeding and yield of cultivated Ophiocordyceps sinensis through ITS sequencing and phylogenetic analysis.

To accelerate the breeding process of cultivated Ophiocordyceps sinensis and increase its yield, it is important to identify molecular fingerprint of dominant O. sinensis. In the present study, we collected 3 batches of industrially cultivated O. sinensis product with higher yield than the others and compared their internal transcribed spacer (ITS) sequences with the wild and the reported. The ITS sequence was obtained by bidirectional sequencing and analyzed with molecular systematics as a DNA barcode for rapid and accurate identification of wild and cultivated O. sinensis collected. The ITS sequences of O. sinensis with detailed collection loci on NCBI were downloaded to construct a phylogenetic tree together with the sequences obtained from the present study by using neighbor-joining method based on their evolution relationship. The information on collection loci was analyzed with ArcGIS 10.2 to demonstrate the geographic distribution of these samples and thus to determine the origin of the dominant samples. The results showed that all wild and cultivated samples were identified as O. sinensis and all sequences were divided into seven phylogenetic groups in the tree. Those groups were precisely distributed on the map and the process of their system evolution was clearly presented. The three cultivated samples were clustered into two dominant groups, showing the correlation between the industrially cultivated samples and the dominant wild samples, which can provide references for its optimized breeding in the future.