Online discovery of the molecular mechanism for directionally detoxification of Fuzi using real-time extractive electrospray ionization mass spectrometry.

ETHNOPHARMACOLOGICAL RELEVANCE: Aconitum carmichaelii Debeaux, a famous traditional medicinal herb for collapse, rheumatic fever, and painful joints, always raises global concerns about its fatal toxicity from toxic alkaloids when improperly processed. Therefore, it is urgent to clarify the internal molecular mechanism of processing detoxification on Aconitum and develop simple and reliable approaches for clinical application, which is also of great significance to the rational medicinal use of Aconitum. AIM OF THE STUDY: The study aimed at developing a complete molecular mechanism exploration strategy in complex medicinal herb decocting system, clarifying the internal molecular mechanism of processing detoxification on Aconitum, and exploring valid approaches for detoxification. MATERIALS AND METHODS: Aconiti Lateralis Radix Praeparata (Fuzi) was selected as the model for exploring the complex Aconitum detoxification mechanism using an advanced online real-time platform based on extractive electrospray ionization mass spectrometry. The methods realized the sensitive capture of dynamic trace intermediates, accurate qualitative and quantitative analysis, and real-time and long-term monitoring of multi-components with satisfactory accuracy and resistance to complex matrices. RESULTS: Components in the complex Aconitum decocting system were real-timely characterized and fat meat was discovered and verified to directionally detoxify Aconitum while reserving the therapy effect. More importantly, the dynamic detoxification mechanism in the chemically complex Aconitum decoction was molecularly profiled. A novel reaction pathway based on nucleophilic substitution reaction mechanism was proposed. As confirmed by the theoretic calculations at DFT B3LYP/6-31G (d) levels, fatty acids (e.g., palmitic acid) acted as a green, cheap, and high-performance catalyst and promote the decomposition of toxic diester alkaloids to non-toxic and active benzoyl-monoester alkaloids through the discovered mechanism. CONCLUSION: The study exposed a novel detoxification molecular mechanism of Aconitum and provided an effective method for the safe use of Aconitum, which could effectively guide the development of traditional processing technology and compatibility regulation of the toxic herb and had great value to the modernization and standardization development of traditional medicine.

Exploring the resources of the genus Viscum for potential therapeutic applications.

ETHNOPHARMACOLOGICAL RELEVANCE: The genus Viscum comprises approximately 100 species that are mainly distributed across Africa, Asia and Europe. The extracts and preparations of Viscum species are widely used as common complementary and alternative medicines in the treatment of rheumatism and cancer. AIM OF THE REVIEW: This review aims to explore the medicinal properties of twelve species belonging to the genus Viscum for potential therapeutic applications. MATERIALS AND METHODS: We collected online information (including PubMed, CNKI, Google Scholar, and Web of Science) from January 1915 to April 2021 and knowledge from classical books on Chinese herbal medicines available for 12 species of the genus Viscum, including Viscum coloratum (Kom.) Nakai, Viscum album L., Viscum articulatum Burm. f., Viscum liquidambaricola Hayata, Viscum ovalifolium DC., Viscum capitellatum Sm., Viscum cruciatum Sieber ex Boiss., Viscum nudum Danser, Viscum angulatum B.Heyne ex DC., Viscum tuberculatum A.Rich., Viscum multinerve Hayata, and Viscum diospyrosicola Hayata. RESULTS: At least 250 different compounds have been reported across twelve Viscum species, including amino acid and peptides, alkaloids, phenolic acids, flavonoids, terpenoids, carbohydrates, fatty acids, lipids, and other types of compounds. In particular, for Viscum coloratum (Kom.) Nakai and Viscum album L., the plants, preparations, and bioactive components have been thoroughly reviewed. This has allowed to elucidate the role of active components, including lectins, viscotoxins, flavonoids, terpenoids, phenolic acids, and polysaccharides, in multiple bioactivities, such as anti-cancer, anti-rheumatism arthralgia, anti-inflammation, anti-cardiovascular diseases, enhancing immunity, and anti-chemotherapy side effects. We also evaluated quality control methods based on active compounds, in vivo exposure compounds, and discriminated chemical markers. CONCLUSIONS: This is the first report to systematically review the pharmaceutical development history, chemical composition, clinical evidence, pharmacological activity, discriminated chemical markers, in vivo exposure, and quality control on twelve distinct species of Viscum plants with medicinal properties. The significant safety and efficacy, along with the minor side effects are constantly confirmed in clinics. The genus Viscum is thus an important medicinal resource that is worth exploring and developing in future pharmacological and chemical studies.

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.

[Establishment of a genetic transformation system for hairy root culture of Erigeron breviscapus].

The electroporation method was performed to transfer plasmid DNA of PBI-1300 carrying GFP gene into Agrobacterium rhizogenes C58C1 strains. Mediated by A. rhizogenes C58C1, the GFP gene were transformed into Erigeron breviscapus aseptic leaves by leaf disc method, then the hairy roots were induced and the infected hairy roots were screened by hygromycin resistance. The chromosomal DNA of the hairy root was used as the templates for the PCR amplification with the GFP-specific primers and then the expected amplified DNA bands appeared, the green fluorescent of GFP in the cut hairy roots was observed by two-photon microscope. These results indicated that GFP gene was integrated into the genome of E. breviscapus and was expressed stably. This study laid the groundwork for foreign gene high-efficiency expression inthe genetic transformation system for hairy root culture of E. breviscapus.

Cloning and functional analysis of two sterol-C24-methyltransferase 1 (SMT1) genes from Paris polyphylla.

The biosynthetic pathways of phytosterols and steroidal saponins are located in two adjacent branches which share cycloartenol as substrate. The rate-limiting enzyme S-adenosyl-L-methionine-sterol-C24-methyltransferase 1 (SMT1) facilitates the metabolic flux toward phytosterols. It catalyzes the methylation of the cycloartenol in the side chain of the C24-alkyl group, to generate 24(28)-methylene cycloartenol. In this study, we obtained two full-length sequences of SMT1 genes from Pari polyphylla, designated PpSMT1-1 and PpSMT1-2. The full-length cDNA of PpSMT1-1 was 1369 bp long with an open reading frame (ORF) of 1038 bp, while the PpSMT1-2 had a length of 1222 bp, with a 1005 bp ORF. Bioinformatics analysis confirmed that the two cloned SMTs belong to the SMT1 family. The predicted function was further validated by performing in vitro enzymatic reactions, and the results showed that PpSMT1-1 encodes a cycloartenol-C24-methyltransferase, which catalyzes the conversion of cycloartenol to 24-methylene cycloartenol, whereas PpSMT1-2 lacked this catalytic activity. The tissue expression patterns of the two SMTs revealed differential expression in different organs of Paris polyphylla plants of different developmental stage and age. These results lay the foundation for detailed genetic studies of the biosynthetic pathways of steroid compounds, which constitute the main class of active substances found in P. polyphylla.

[Cloning and protein expression analysis of geranyl diphosphate synthase genes in Tripterygium wilfordii].

Based on the transcriptome data, the study cloned full-length cDNA of TwGPPS1 and TwGPPS2 genes from Tripterygium wilfordii suspension cells and then analyzed the bioinformation of the sequence and protein expression. The cloned TwGPPS1 has a 1 278 bp open reading frame (ORF) encoding a polypeptide of 425 amino acids. The deduced isoelectric point (pI) was 6.68, a calculated molecular weight was about 47.189 kDa. The full-length cDNA of the TwGPPS2 contains a 1 269 bp open reading frame (ORF) encoding a polypeptide of 422 amino acids. The deduced isoelectric point (pI) was 6.71, a calculated molecular weight was about 46.774 kDa.The entire reading frame of TwGPPS1,2 was cloned into the pET-32a(+) vector and expressed in E. coli BL21 (DE3) cells to obtain the TwGPPS protein, which laid a basis for further study on the regulation of terpenoid secondary metabolism and biological synthesis.

The MVA pathway genes expressions and accumulation of celastrol in Tripterygium wilfordii suspension cells in response to methyl jasmonate treatment.

Celastrol is an important bioactive triterpenoid in traditional Chinese medicinal plant, Tripterygium wilfordii. Methyl Jasmonate (MJ) is a common plant hormone which can regulate the secondary metabolism in higher plants. In this study, the mevalonate (MVA) pathway genes in T. wilfordii were firstly cloned. The suspension cells of T. wilfordii were elicited by MJ, and the expressions of MVA pathway genes were all enhanced in different levels ranging from 2.13 to 22.33 times of that at 0 h. The expressions were also enhanced compared with the CK group separately. The accumulation of celastrol in the suspension cells after the treatment was quantified and co-analyzed with the genes expression levels. The production of celastrol was significantly increased to 0.742 mg g(-1) after MJ treatment in 288 h which is consistent with the genes expressions. The results provide plenty of gene information for the biosynthesis of terpenoids in T. wilfordii and a viable way to improve the accumulation of celastrol in T. wilfordii suspension cells.

[Cloning and bioinformatics analysis of geranylgeranyl diphosphate synthase gene of Tripterygium wilfordii].

A full-length cDNA of GGPPS gene from Tripterygium wilfordii suspension cells was obtained by use of RACE strategy (GeneBank: KM978333), and then analyzed by bioinformatics approaches. TwGGPPS cDNA has 1857 nucleotides and an open reading frame (ORF) encoding a protein of 514 amino acid residues. The deduced protein has isoelectric point (pI) of 7.85, a calculated molecular weight about 57.13 kD, 5 conserved domains and 2 functional domains. PSORT Prediction showed it was located at plasma membrane. Phylogenetic analysis demonstrated that TwGGPPS1 was similar to GGPPS from other species of plants. For the first time the cloning of geranylgeranyl diphosphate synthase gene from T. wilfordii was reported, it lays the foundation for further research of diterpenoids biosynthetic pathway.

[Cloning and expression analysis of a acetyl-CoA U-acetyltransferase gene (TwAACT) from Tripterygium wilfordii].

In this study, based on the transcriptome data, we cloned the full-length cDNAs of TwAACT gene from Tripterygium wilfordii suspension cells, and then analyzed the bioinformation of the sequence, detected the genetic differential expression after being induced by methyl jasmonate (MeJA) by RT-PCR. The full-length cDNA of the TwAACT was 1 704 bp containing a 1 218 bp open reading frame (ORF) encoding a polypeptide of 405 amino acids (GeneBank accession No. KP297934). The deduced isoelectric point (pI) was 6.10, a calculated molecular weight was about 41.20 kDa, and online prediction showed that TwAACT had two catalytic active sites. After the induction of MeJA, the relative expression level of TwAACT increased rapidly. The expression level of TwAACT was highest at 24 h. TwAACT was cloned firstly, that laid the foundation for identifying thegene and illustrating thebiosynthesis mechanism and its synthetic biology.

[Cloning and expression analysis of 4- (cytidine-5-diphospho) -2-C-methyl-D-erythritol kinase gene in Tripterygium wilfordii].

4-(Cytidine-5-diphospho) -2-C-methyl-D-erythritol kinase is a key enzyme in the biosynthesis pathway of terpenoids. According to the transcriptome database, the specific primers were designed and used in PCR. The bioinformatic analysis of the sequenced TwCMK gene was performed in several bioinformatics software. The Real-time fluorescence quantification polymerase chain reaction (RT-qPCR) were used to detect the expression levels of TwCMK from T. wilfordii after elicitor MeJA supplied. The results showed that the full length of TwCMK cDNA was 1 732 bp encoding 387 amino acids. The theoretical isoelectric point of the putative TwCMK protein was 5.79 and the molecular weight was about 42.85 kDa. MeJA stimulated the rising of TwCMK expression in suspension cell and signally impacted at 24 h. The research provides a basis for further study on the regulation of terpenoid secondary metabolism and biological synthesis.

[Cloning and expression analysis of 2-C-methyl-D-erythritol 4-phosphate cytidylyltransferase gene in Tripterygium wilfordii].

To clone the 2-C-methyl-D-erythritol 4-phosphate cytidylyltransferase (TwMCT) full length cDNA from Tripterygium wilfordii, the specific primers were designed according to the transcriptome data and the LCPCR were carried out. After a series of bioinformatics analysis on the TwMCT, the MeJA induced expression content were investigated by real-time fluorescence quantification polymerase chain reaction (RT-qPCR). The result showed that the full of TwMCTcDNA was 1 318 bp nucleotides encoding 311 amino acids. The molecular weight of the deduced TwMCT protein was about 34.14 kDa and the theoretical isoelectric point was 8.65. Result of the RT-qPCR analysis indicated that the content of TwMCT mRNA expression in T. wilfordii suspension cell was rising after treating with MeJA and reached the maximum in 24 h. Cloning and analyzing TwMCT gene from T. wilfordii provided gene element for studying the function and expression regulation of secondary metabolites.

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.

Safety and efficacy of anisodamine on prevention of contrast induced nephropathy in patients with acute coronary syndrome.

BACKGROUND: Previous studies have proved the renal protective effects of anisodamine in patients with septic shock. The aim of this study was to investigate anisodamine for the prevention of contrast induced nephropathy (CIN) in patients with acute coronary syndrome (ACS). METHODS: Consecutive ACS patients undergoing elective percutaneous coronary intervention (PCI) were randomly assigned to one of two groups: patients in the anisodamine group (ANI group) were assigned to receive intravenous infusions of anisodamine by an adjusted-dose (0.1 - 0.2 µg × kg(-1)× min(-1)) from the PCI procedure to 24 hours after PCI, and the control group (CON group) received 0.9% isotonic saline of the same volume. All patients were hydrated for 6 to 12 hours before and 12 hours after PCI. Blood samples were taken on the day of PCI and at 24, 48 and 72 hours after PCI to measure the serum creatinine (SCr). RESULTS: A total of 177 patients were involved in the study, 88 in the ANI group and 89 in the CON group. In both groups, the SCr concentrations significantly increased after PCI, with the peak value occurring at 48 hours. At 72 hours, the SCr concentration in the ANI group retuned to the baseline level (P > 0.05), but the SCr concentration in CON group was still higher than baseline level (P < 0.01). The SCr concentrations at 48 and 72 hours after PCI were much lower in the ANI group than those in the CON group (both P < 0.01). The estimated glomerular filtration rate (eGFR) significantly decreased after PCI, the lowest value occurred at 48 hours. In the ANI group, the eGFR at 72 hours was similar to the baseline level. In the CON group, the eGFR failed to return to baseline at 72 hours (P < 0.01). The eGFR at 24, 48 and 72 hours after PCI were higher in the ANI group (all P < 0.05). The incidence of CIN in the ANI group was lower than that in the CON group within 72 hours after PCI (P < 0.05). The results of multiple Logistic regression proved that both diabetes and left ventricular ejection fraction (LVEF) were independent predictors of CIN, and treatment with anisodamine was an independent preventive factor of CIN (OR 0.369 and 95%CI 0.171 to 0.794, P = 0.011). No serious side effects were found in the ANI group. CONCLUSION: Intravenous infusion of anisodamine during and after elective PCI may safely prevent the occurrence of CIN in ACS patients.