Nudix hydrolase WvNUDX24 is involved in borneol biosynthesis in Wurfbainia villosa.

Borneol, camphor, and bornyl acetate are highly promising monoterpenoids widely used in medicine, flavor, food, and chemical applications. Bornyl diphosphate (BPP) serves as a common precursor for the biosynthesis of these monoterpenoids. Although bornyl diphosphate synthase (BPPS) that catalyzes the cyclization of geranyl diphosphate (GPP) to BPP has been identified in multiple plants, the enzyme responsible for the hydrolysis of BPP to produce borneol has not been reported. Here, we conducted in vitro and in vivo functional characterization to identify the Nudix hydrolase WvNUDX24 from W. villosa, which specifically catalyzes the hydrolysis of BPP to generate bornyl phosphate (BP), and then BP forms borneol under the action of phosphatase. Subcellular localization experiments indicated that the hydrolysis of BPP likely occurs in the cytoplasm. Furthermore, site-directed mutagenesis experiments revealed that four critical residues (R84, S96, P98, and G99) for the hydrolysis activity of WvNUDX24. Additionally, the functional identification of phosphatidic acid phosphatase (PAP) demonstrated that WvPAP5 and WvPAP10 were able to hydrolyze geranylgeranyl diphosphate (GGPP) and farnesyl diphosphate (FPP) to generate geranylgeranyl phosphate (GGP) and farnesyl phosphate (FP), respectively, but could not hydrolyze BPP, GPP, and neryl diphosphate (NPP) to produce corresponding monophosphate products. These findings highlight the essential role of WvNUDX24 in the first step of BPP hydrolysis to produce borneol and provide genetic elements for the production of BPP-related terpenoids through plant metabolic engineering and synthetic biology.

Comparing genomes of Fructus Amomi-producing species reveals genetic basis of volatile terpenoid divergence.

Wurfbainia longiligularis and Wurfbainia villosa are both rich in volatile terpenoids and are 2 primary plant sources of Fructus Amomi used for curing gastrointestinal diseases. Metabolomic profiling has demonstrated that bornyl diphosphate (BPP)-related terpenoids are more abundant in the W. villosa seeds and have a wider tissue distribution in W. longiligularis. To explore the genetic mechanisms underlying the volatile terpenoid divergence, a high-quality chromosome-level genome of W. longiligularis (2.29 Gb, contig N50 of 80.39 Mb) was assembled. Functional characterization of 17 terpene synthases (WlTPSs) revealed that WlBPPS, along with WlTPS 24/26/28 with bornyl diphosphate synthase (BPPS) activity, contributes to the wider tissue distribution of BPP-related terpenoids in W. longiligularis compared to W. villosa. Furthermore, transgenic Nicotiana tabacum showed that the GCN4-motif element positively regulates seed expression of WvBPPS and thus promotes the enrichment of BPP-related terpenoids in W. villosa seeds. Systematic identification and analysis of candidate TPS in 29 monocot plants from 16 families indicated that substantial expansion of TPS-a and TPS-b subfamily genes in Zingiberaceae may have driven increased diversity and production of volatile terpenoids. Evolutionary analysis and functional identification of BPPS genes showed that BPP-related terpenoids may be distributed only in the Zingiberaceae of monocot plants. This research provides valuable genomic resources for breeding and improving Fructus Amomi with medicinal and edible value and sheds light on the evolution of terpenoid biosynthesis in Zingiberaceae.

Chromosome-level genome assembly and functional characterization of terpene synthases provide insights into the volatile terpenoid biosynthesis of Wurfbainia villosa.

Wurfbainia villosa is a well-known medicinal and edible plant that is widely cultivated in the Lingnan region of China. Its dried fruits (called Fructus Amomi) are broadly used in traditional Chinese medicine for curing gastrointestinal diseases and are rich in volatile terpenoids. Here, we report a high-quality chromosome-level genome assembly of W. villosa with a total size of approximately 2.80 Gb, 42 588 protein-coding genes, and a very high percentage of repetitive sequences (87.23%). Genome analysis showed that W. villosa likely experienced a recent whole-genome duplication event prior to the W. villosa-Zingiber officinale divergence (approximately 11 million years ago), and a recent burst of long terminal repeat insertions afterward. The W. villosa genome enabled the identification of 17 genes involved in the terpenoid skeleton biosynthesis pathway and 66 terpene synthase (TPS) genes. We found that tandem duplication events have an important contribution to the expansion of WvTPSs, which likely drove the production of volatile terpenoids. In addition, functional characterization of 18 WvTPSs, focusing on the TPS-a and TPS-b subfamilies, showed that most of these WvTPSs are multi-product TPS and are predominantly expressed in seeds. The present study provides insights into the genome evolution and the molecular basis of the volatile terpenoids diversity in W. villosa. The genome sequence also represents valuable resources for the functional gene research and molecular breeding of W. villosa.

Rhamnosyltransferases involved in the biosynthesis of flavone rutinosides in Chrysanthemum species.

Linarin (acacetin-7-O-rutinoside), isorhoifolin (apigenin-7-O-rutinoside), and diosmin (diosmetin-7-O-rutinoside) are chemically and structurally similar flavone rutinoside (FR) compounds found in Chrysanthemum L. (Anthemideae, Asteraceae) plants. However, their biosynthetic pathways remain largely unknown. In this study, we cloned and compared FRs and genes encoding rhamnosyltransferases (RhaTs) among eight accessions of Chrysanthemum polyploids. We also biochemically characterized RhaTs of Chrysanthemum plants and Citrus (Citrus sinensis and Citrus maxima). RhaTs from these two genera are substrate-promiscuous enzymes catalyzing the rhamnosylation of flavones, flavanones, and flavonols. Substrate specificity analysis revealed that Chrysanthemum 1,6RhaTs preferred flavone glucosides (e.g. acacetin-7-O-glucoside), whereas Cs1,6RhaT preferred flavanone glucosides. The nonsynonymous substitutions of RhaTs found in some cytotypes of diploids resulted in the loss of catalytic function. Phylogenetic analysis and specialized pathways responsible for the biosynthesis of major flavonoids in Chrysanthemum and Citrus revealed that rhamnosylation activity might share a common evolutionary origin. Overexpression of RhaT in hairy roots resulted in 13-, 2-, and 5-fold increases in linarin, isorhoifolin, and diosmin contents, respectively, indicating that RhaT is mainly involved in the biosynthesis of linarin. Our findings not only suggest that the substrate promiscuity of RhaTs contributes to the diversity of FRs in Chrysanthemum species but also shed light on the evolution of flavone and flavanone rutinosides in distant taxa.

[Main components in different varieties of Xihuangcao by UPLC-Q-TOF-MS and UPLC-DAD].

This study established an ultra-high performance liquid chromatography coupled with quadrupole-time-of-flight mass spectrometry(UPLC-Q-TOF-MS) method to analyze the main components in different varieties of Xihuangcao and established a UPLC-DAD method to simultaneously determine the five active components(caffeic acid, rosmarinic acid, schaftoside, isoschaftoside, and oridonin).The chromatographic separation was performed on a Waters ACQUITY UPLC BEH C_(18) column(2.1 mm×100 mm, 1.7 µm) with a gradient elution of methanol(B)-water containing 0.1% formic acid(A) at a flow rate of 0.3 mL·min~(-1).The column temperature was 30 ℃.The Q-TOF-MS discriminant analysis was performed under positive electrospray ion mode and the split ratio was 1∶1. Quantitative analysis was carried out by UPLC-DAD.The determination wavelength was set at 245 nm.Thirty-two main components of Xihuangcao were separated and identified by UPLC-Q-TOF-MS, where 19 were identified in Rabdosia serra, nine in R.nervosa, 10 in R.lophanthoides, 15 in R.lophanthoides var.graciliflora, 10 in R.lophanthoides var.gerardianus, and seven in R.stracheyi.The UPLC-DVD method was developed for simultaneously determining five active components in different varieties of Xihuangcao.The standard curves for five compounds showed good linearity with correlation coefficients higher than 0.999 0.The precision, repeatability, and stability were good.The average recoveries(n=6) were between 97.01% and 102.7% with RSD<3.0%.The results of UPLC-Q-TOF-MS analysis provided a scientific basis for the use of R.stracheyi as a medicinal material of Xihuangcao and the equivalent use of R.lophanthoides var.gerardianus with R.lophanthoides var.graciliflora to some extent.The UPLC-DAD method for simultaneously determining five active components is simple, rapid, and accurate.This study can provide the basis for the quality control of different varieties of Xihuangcao.

Isolation and characterization of AaZFP1, a C2H2 zinc finger protein that regulates the AaIPPI1 gene involved in artemisinin biosynthesis in Artemisia annua.

MAIN CONCLUSION: AaZFP1, a C2H2-type transcription factor, was found to bind the AGT-N1-10-AGT box of AaIPPI1pro and activate the expression of AaIPPI1 involved in artemisinin biosynthesis. Artemisinin, an endoperoxide sesquiterpene lactone, is a widely used antimalarial drug isolated from Artemisia annua L. Isopentenyl pyrophosphate isomerase (AaIPPI1) catalyzes the interconversion of isopentenyl diphosphate and dimethylallyl diphosphate and is the key gene involved in the biosynthesis of artemisinin. However, the AaIPPI1 gene regulation network remains largely unknown. Here, we isolated the AaIPPI1 promoter (AaIPPI1pro) and predicted that it contains cis-elements involved in stress responses, including the TGACG motif (a methyl jasmonate-responsive element), GARE motif (a gibberellin-responsive element), ABRE (an abscisic acid-responsive element), TC-rich repeats (a stress-responsive element), and the AGT-N1-10-AGT box, which is the binding site of Cys/His2 zinc finger protein (C2H2 ZFP). The C2H2 ZFP gene AaZFP1 was discovered by screening a cDNA library using AaIPPI1pro as bait in yeast. AaZFP1 contains two conserved C2H2 regions, a nuclear localization domain (B box), a Leu-rich domain (L box), and a conserved DLN sequence (DLN box) close to its C terminus. A subcellular localization assay indicated that AaZFP1 protein is localized in the nucleus and cytoplasm. An electrophoretic mobility shift assay demonstrated that AaZFP1 binds to the AGT-N1-10-AGT box of AaIPPI1pro. A dual-luciferase assay indicated that AaZFP1 enhanced the promoter activity of AaIPPI1 in vivo. Transient overexpression of AaZFP1 in A. annua increased the expression of AaIPPI1 and the content of artemisinin. Our data demonstrated that AaZFP1 functions as a transcriptional activator that regulates the expression of AaIPPI1 by directly binding to its promoter. The present study provides insights into the transcriptional regulation of genes involved in artemisinin biosynthesis in A. annua.

[Cloning of transcription factor PcFBA-1 in Pogostemon cabin and its interaction with FPPS promoter].

Farnesyl diphosphate synthase(FPPS) is a key enzyme at the branch point of the sesquiterpene biosynthetic pathway, but there are no reports on the transcriptional regulation of FPPS promoter in Pogostemon cabin. In the early stage of this study, we obtained the binding protein PcFBA-1 of FPPS gene promoter in P. cabin. In order to explore the possible mechanism of PcFBA-1 involved in the regulation of patchouli alcohol biosynthesis, this study performed PCR-based cloning and sequencing analysis of PcFBA-1, analyzed the expression patterns of PcFBA-1 in different tissues by fluorescence quantitative PCR and its subcellular localization using the protoplast transformation system, detected the binding of PcFBA-1 protein to the FPPS promoter in vitro with the yeast one-hybrid system, and verified its transcriptional regulatory function by dual-luciferase reporter gene assay. The findings demonstrated that the cloned PcFBA-1 had an open reading frame(ORF) of 1 131 bp, encoding a protein of 376 amino acids, containing two conserved domains named F-box-like superfamily and FBA-1 superfamily, and belonging to the F-box family. Moreover, neither signal peptide nor transmembrane domain was contained, implying that it was an unstable hydrophilic protein. In addition, as revealed by fluorescence quantitative PCR results, PcFBA-1 had the highest expression in leaves, and there was no significant difference in expression in roots or stems. PcFBA-1 protein was proved mainly located in the cytoplasm. Furthermore, yeast one-hybrid screening and dual-luciferase reporter gene assay showed that PcFBA-1 was able to bind to FPPS promoter both in vitro and in vivo to enhance the activity of FPPS promoter. In summary, this study identifies a new transcription factor PcFBA-1 in P. cabin, which directly binds to the FPPS gene promoter to enhance the promoter activity. This had laid a foundation for the biosynthesis of patchouli alcohol and other active ingre-dients and provided a basis for metabolic engineering and genetic improvement of P. cabin.

Effect of Pretreatment on Detection of 37 Pesticide Residues in Chrysanthemum indicum.

This study aimed to develop a method, followed by gas chromatography-mass spectrometry, for detecting 37 pesticides in Chrysanthemum indicum (C. indicum) and investigating the decrease in the matrix-induced enhancement effect. The influence of QuEChERS extraction and matrix solid-phase dispersion (MSPD) on the recovery and matrix effect (ME) was compared. extraction and matrix solid-phase dispersion (MSPD) on the recovery and matrix effect (ME) was compared to decrease the ME. The cleanup sorbents, volume and type of solvent, and treatment time were optimized. The accuracy (as recovery), precision (as relative standard deviation, RSD), linearity, limit of quantitation, and limit of detection were determined. The recoveries at the three levels using mixed standard solution ranged between 76% and 120% with RSD ≤15%, and 76% and 120% with RSD ≤11% for MSPD and QuEChERS extraction, respectively. The results suggested that the ME for 21 pesticides was in the range of 80%-120% after MSPD and 15% after QuEChERS extraction. QuEChERS extraction was simpler and faster than MSPD. This methodology was applied in the analysis of 27 C. indicum samples; phorate was most frequently detected (63.0% of the sample).

[SSR loci information analysis in transcriptome of Andrographis paniculata].

To study the SSR loci information and develop molecular markers, a total of 43 683 Unigenes in transcriptome of Andrographis paniculata were used to explore SSR. The distribution frequency of SSR and the basic characteristics of repeat motifs were analyzed using MicroSAtellite software, SSR primers were designed by Primer 3.0 software and then validated by PCR. Moreover, the gene function analysis of SSR Unigene was obtained by Blast. The results showed that 14 135 SSR loci were found in the transcriptome of A. paniculata, which distributed in 9 973 Unigenes with a distribution frequency of 32.36%. Di-nucleotide and Tri-nucleotide repeat were the main types, accounted for 75.54% of all SSRs. The repeat motifs of AT/AT and CCG/CGG were the predominant repeat types of Di-nucleotide and Tri-nucleotide, respectively. A total of 4 740 pairs of SSR primers with the potential to produce polymorphism were designed for maker development. Ten pairs of primers in 20 pairs of randomly picked primers produced fragments with expected molecular size. The gene function of Unigenes containing SSR were mostly related to the basic metabolism function of A. paniculata. The SSR markers in transcriptome of A. paniculata show rich type, strong specificity and high potential of polymorphism, which will benefit the candidate gene mining and marker-assisted breeding.

Evaluation of analgesic and anti-inflammatory activities of Rubia cordifolia L. by spectrum-effect relationships.

The objective of the current work was to evaluate the spectrum-effect relationships between high-performance liquid chromatography fingerprints and analgesic and anti-inflammatory effects of Rubia cordifolia L. extract (RCE), and to identify active components of RCE. Chemical fingerprints of ten batches of RC from various sources were obtained by HPLC, and similarity and hierarchical clustering analyses were carried out. Pharmacodynamic assays were performed in adjuvant-induced arthritis rat model to assess the analgesic and anti-inflammatory properties of RCE. The spectrum-effect relationships between chemical fingerprints and the analgesic and anti-inflammatory effects of RCE were established by gray correlation analysis. UPLC-ESI-MS was used to identify the structures of potential active components, by reference standards comparison. The results showed that a close correlation existed between chemical fingerprints with analgesic and anti-inflammatory activities, and alizarin, 6-hydroxyrubiadin, purpurin and rubiadin might be the active constituents of RCE. In addition, RCE attenuated pathological changes in adjuvant-induced arthritis. The current findings provide a strong basis for combining chemical fingerprints with analgesic and anti-inflammatory activities in assessing the spectrum-effect relationships of RCE.

An investigation of fungal contamination on the surface of medicinal herbs in China.

BACKGROUND: The dried parts of medicinal herbs are susceptible to the infection of fungi during pre- or post-harvest procedure. This study aimed to investigate the presence of fungi and their metabolites mycotoxins on the surface of medicinal herbs collected from China. METHODS: Forty-five retail samples of 15 different medicinal herbs were collected from 3 different regions in China. Then the potential fungi were immediately washed off from the surface of each sample with 0.1% Tween-20 followed by incubation of the rinse on petri-dish with potato dextrose agar containing chloramphenicol at 28 °C. The obtained fungi were isolated as single colonies and then characterized by morphology and molecular identification using internal transcribed spacer (ITS) sequencing with extracted DNA. Meanwhile, the mycotoxin-producing potential of the isolates was studied by liquid chromatography-tandem mass spectrometry (LC-MS/MS). RESULTS: A total of 126 fungi were identified from the surface of samples by morphology and ITS sequencing, with Aspergillus and Penicillium genera as the predominant contaminants. The mycotoxin-producing potential analysis showed that 6 of 8 A. versicolor isolates could produce sterigmatocystin. All 3 A. aculeatus isolates produced ochratoxin A, but only 1 of 3 A. flavus strains produced aflatoxins B1 and B2 without G1 and G2. Although the sample contamination ratios were high (≥95.6%), there was no significant difference (χ2 = 1.05, P = 1.0) among the samples from 3 regions, which demonstrates the prevalent fungal contamination in the herbal medicines. CONCLUSION: The prevalent contamination phenomenon of fungi and high potential risk of sterigmatocystin and ochratoxin A were observed in 45 medicinal herbs collected from China.

Qualitative and Quantitative Analysis of Volatile Components of Zhengtian Pills Using Gas Chromatography Mass Spectrometry and Ultra-High Performance Liquid Chromatography.

Zhengtian pills (ZTPs) are traditional Chinese medicine (TCM) which have been commonly used to treat headaches. Volatile components of ZTPs extracted by ethyl acetate with an ultrasonic method were analyzed by gas chromatography mass spectrometry (GC-MS). Twenty-two components were identified, accounting for 78.884% of the total components of volatile oil. The three main volatile components including protocatechuic acid, ferulic acid, and ligustilide were simultaneously determined using ultra-high performance liquid chromatography coupled with diode array detection (UHPLC-DAD). Baseline separation was achieved on an XB-C18 column with linear gradient elution of methanol-0.2% acetic acid aqueous solution. The UHPLC-DAD method provided good linearity (R (2) ≥ 0.9992), precision (RSD < 3%), accuracy (100.68-102.69%), and robustness. The UHPLC-DAD/GC-MS method was successfully utilized to analyze volatile components, protocatechuic acid, ferulic acid, and ligustilide, in 13 batches of ZTPs, which is suitable for discrimination and quality assessment of ZTPs.

[Comparative Study on Effects of Anti-Gastritis,Gastric Mucosal Protection and Gastrointestinal Movement Promotion of Root and Stem of Zanthoxylum nitidum].

Objective: To provide the experimental evidence for expansion of medicinal parts of Zanthoxylum nitidum by comparing the effects of anti-gastritis,gastric mucosal protection and gastrointestinal movement promotion of its root and stem. Methods: The pharmacological effects between root and stem of Zanthoxylum nitidum were compared by observing the anti-gastritis effect on rats with chronic superficial gastritis induced by iodoacetamide, evaluating the gastric mucosal protective effect on rats' gastric ulcer induced by stress, indometacin and pylorus ligation test, and investigating gastrointestinal movement promotion effect on mice gastric evacuation and intestinal propelling. Results: Both root and stem of Zanthoxylum nitidum showed effects of relieving the inflammation symptoms of rats' gastric mucosa induced by iodoacetamide, gastric ulcer respectively induced by stress, and presenting a strong inhibition of free acid and pepsin activity in gastric juice. Furthermore stem parts of Zanthoxylum nitidum in promoting gastrointestinal motility even showed better efficacy than root. Conclusion: Stem of Zanthoxylum nitidum has similar effects of anti-gastritis, gastric mucosal protection and gastrointestinal movement promotion with root of Zanthoxylum nitidum.

[Preliminary study on effect of Rhodiolae Crenulatae Radix et Rhizoma cell wall-broken decoction pieces on intestinal flora of mice].

This study aims to analyze and compare the effect of cell wall-broken decoction pieces, conventional decoction pieces and conventional powder of Rhodiolae Crenulatae Radix et Rhizoma on the intestinal flora of normal mice. The conventional bacterial culture and PCR-DGGE (polymerase chain reaction-denaturing gradient gel electrophoresis) were adopted for the mice after the oral administration for 14 days. According to the bacterial culture results, the 1/8 dose cell wall-broken decoction pieces group showed fewer Enterococcus and Escherichia coli bacillus but more Lactobacillus and Bifidobacterium than the conventional decoction pieces group and the traditional powder group (P <0.05). Meanwhile, on the basis of the PCR-DGGE results, the 1/8 dose cell wall-broken decoction pieces group revealed the highest Shannon-Wiener index (H) and species richness (S) among the seven groups, with extremely significant differences compared with the normal group (P <0.01), significant differences compared with the conventional decoction pieces group and the conventional powder group (P <0.05) and a high intra-group similarity. In conclusion, the long-term intake of 1/8 dose Rhodiolae Crenulatae Radix et Rhizoma cell wall-broken decoction pieces showed a certain effect in regulating intestinal tract by promoting the growth of Lactobacillus and Bifidobacterium. Furthermore, the intestinal flora community will become more stable.

[Comparative Study on Effects of Anti-contusion Injury, Analgesia and Anti-inflammation of Root and Stem of Zanthoxylum nitidum].

OBJECTIVE: To provide the scientific evidence for expansion of medicinal parts of Zanthoxylum nitidum by comparing the effects of anti-contusion injury, analgesia and anti-inflammation of its root and stem. METHODS: The pharmacological effects between root and stem of Zanthoxylum nitidum were compared by observing the anti-injury effect in rats with injury struck by hammer. The analgesic effect in mice was evaluated by writhing test and hot plate test, and the anti-inflammatory effect on paw edema induced by carrageenan and granuloma induced by cotton pellet were investigated in rats. RESULTS: Both root and stem of Zanthoxylum nitidum relieved the exterior and histological symptoms of rats' injury legs struck by hammer, decreased the numbers of mice's writhing, enhanced pain threshold of mice on heat plate, inhibited the edema of rats induced by carrageenan, and suppressed the granuloma of rats induced by cotton pellet. CONCLUSION: Stem of Zanthoxylum nitidum has similar effects of anti-contusion injury, analgesia and anti-inflammation with root of Zanthoxylum nitidum.

[A high throughput coupled with high performance liquid chromatography-tandem mass spectrometry method for determination of aflatoxin B1, B2, G1, G2 in 10 traditional Chinese medicines].

As the dilution procedure was applied, a simple, rapid and cost-effective high-performance liquid chromatography-tandem mass spectrometry (LC-MS/MS) method for determination of aflatoxin B1, B2, G1, and G2 was successfully by performed in a total 83 samples of 10 traditional Chinese medicines (TCMs), which were collected from 5 different hospital pharmacies and 5 different medical stores in Guangzhou city. Matrix effects of these 10 TCMs were ranged from 80.23% to 115.5% in low, intermediate and high concentration levels, indicating that the negative effect was overcome in this study. Meanwhile, the analysis method was proved to be stable and reliable during the whole analysis using Semen Armeniacae Amarum spiked 3 concentration levels of standard solution as quality control samples and the RSD < 6.6% was obtained. The contamination levels of 83 investigated samples were 13.89% and 17.02% in hospital pharmacies and medical stores, respectively. The result was presented to provide relevant reference and supplement to those researchers in TCMs analysis and screening.

[Separation and molecular identification of fungal contamination on surface of 15 Chinese herbal medicines].

OBJECTIVE: To evaluate fungal contamination on the surface of Chinese herbal medicines and explore an appropriate method for fast and efficient identification of contaminant fungi. METHOD: Chinese herbal medicines were first washed and the washing solution was plated onto potato dextrose agar (PDA) to obtain the pure isolates. For molecular identification, two new pairs of specific primers were designed according to ITS region of fungi genome sequences. The strains were identified through polymerase chain reaction (PCR) and sequence analysis. RESULT: Fifty fungal strains were obtained from the surface of 15 Chinese herbal medicines with the percent of contaminated samples of 93.3%. Twenty-seven strains among them were successfully identified. CONCLUSION: Fungal contamination on the surface of Chinese herbal medicines is quite common. Although different fungal species were isolated, the genus Aspergillus was the predominant. The primer pairs developed in this study are compatible and can be used to identify fungal species from the surface of Chinese herbal medicines.

[Determination of aflatoxin B1, B2, G1, G2 in armeniacae semen amarum by high-performance liquid chromatography-tandem mass spectrometry].

A simple, rapid and cost-effective high-performance liquid chromatography-tandem mass spectrometry (LC-MS/ MS) method was established for simultaneous determination of aflatoxins (AFB1, AFB2, AFG1, AFG2) in Armeniacae Semen Amarum and the application was performance in 11 samples collected from different markets, medical stores and hospitals. The sample was extracted with 84% acetonitrile/water and 250 microL extraction was directly injected into a LC-MS/MS system without further purification procedure after being redissolved with methanol. The LC separation was performed on a C18 column with a linear gradient elution program of 4 mmol x L(-1) NH4 Ac-0.1% formic acid solution and menthol as the mobile phase. Selected reaction monitoring (SRM) was used for selective determination of the four aflatoxins on a triple quadruple mass spectrometer, which was operated in positive ionization modes. All the four aflatoxins showed a good linear relationship with r > 0.999 0, the average recoveries were between 87.88% and 102.9% and the matrix effect was ranged from 90.71% to 99.30% in low, intermediate and high levels. Furthermore, the higher recovery was obtained by the method reported in this study, comparing to the cleanup procedure with the Mycosep 226 purification column. Eleven samples collected were detected and the contamination levels of the AFB1 were between 1.590-2.340 microg x kg(-1) and the AF (B1 + B2 + G1 + G2) was ranged from 2.340 to 3.340 microg x kg(-1). In summary, the developed method was suitable to detect and screen AFB1, AFB2, AFG1, AFG2 in Armeniacae Semen Amarum.

[Study on HPLC fingerprint chromatograms of cell wall-broken decoction pieces of Notoginseng].

OBJECTIVE: To establish the HPLC fingerprint chromatograms of crude Notoginseng, cell wall-broken powder and cell wall-broken decoction pieces of Notoginseng and provide evidence for quality control of cell wall-broken decoction pieces of Notoginseng. METHODS: The HPLC procedure was performed on the chromatographic column of Hypersil ODS2, and the mobile phase was acetonitrile and water in gradient elution with the flow velocity of 1.0 mL/min. The detection wavelength was 203 nm and the column temperature was 25 degrees C. The chromatograms was analyzed with the software of "similarity evaluation system for chromatographic fingerprint of TCM". RESULTS: Eight common peaks were pinpointed from the chromatograms of different batches of crude Notoginseng, cell-broken powder and cell-broken decoction pieces, the similarities of the chromatograms were all larger than 0.9. CONCLUSION: The method of the HPLC fingerprint chromatogram is of good precision, reproducibility and stability,which is suitable for quality control of cell wall-broken decoction pieces of Notoginseng.

[HPLC fingerprint of flavonoids and phenols of Dendrobium nobile].

OBJECTIVE: To establish HPLC fingerprint of flavonoids and phenols of Dendrobium nobile. METHODS: Phenomenex prodigy ODS(3) C18 column (250 mm x 4.6 mm, 5 microm) was used with a mixture of acetonitrile-0.1% acetic acid as the mobile phase in a gradient mode, the column temperature was 25 degrees C, the flow rate was 1.0 mL/min, and the detection wavelength was 254 nm. RESULTS: The flavonoids and phenols of Dendrobium nobile were well separated, and 10 fingerprint peaks in common were confirmed. CONCLUSION: This method is simple, accurate with good reproducibility, and can be used specifically for the quality control of Dendrobium nobile.