Metabolomics analysis reveals characteristic metabolites in different levels of oxaliplatin-induced neurotoxicity.

Oxaliplatin (L-OHP), a third-generation platinum-based anti-tumor drug, finds widespread application in the first-line treatment of metastatic colorectal cancer. Despite its efficacy, the drug's usage is curtailed by a litany of side effects, with L-OHP-induced peripheral neuropathy (OIPN) being the most debilitating. This condition can be classified into varying degrees of severity. Employing serum metabolomics, a high-sensitivity, high-throughput technique, holds promise as a method to identify biomarkers for clinical assessment and monitoring of OIPN patients across different severity levels. In our study, we analyzed serum metabolites in patients with different OIPN levels using ultra-performance liquid chromatography-high resolution mass spectrometry. By employing statistical analyses and pathway enrichment studies, we aimed to identify potential biomarkers and metabolic pathways. Our findings characterized the serum metabolic profiles of patients with varying OIPN levels. Notably, pathway analysis revealed a significant correlation with lipid metabolism, amino acid metabolism, and energy metabolism. Multivariate statistical analysis and receiver operator characteristic curve evaluation pointed to anhalamine and glycochenodeoxycholic acid as potential biomarkers for OIPN C and A, which suggest that serum metabolomics may serve as a potent tool for exploring the metabolic status of patients suffering from diverse diseases and for discovering novel biomarkers.

Insights into the metabolite profiles of Rubus chingii Hu at different developmental stages of fruit.

The fruits of Rubus chingii Hu have high medicinal and nutritional values. However, the metabolite profiles of R. chingii, especially the alterations during different development stages of fruit, have not been comprehensively analyzed, hindering the effective utilization of the unique species. In this study, we comprehensively analyzed the metabolites of R. chingii fruit at four developmental stages using systematic untargeted and targeted liquid chromatography-mass spectrometry metabolomics analysis and identified 682 metabolites. Significant changes were observed in metabolite accumulation and composition in fruits during the different developmental stages. The contents of the index components, kaempferol-3-O-rutinoside and ellagic acid, were the highest in immature fruit. The analysis identified 64 differentially expressed flavonoids and 39 differentially expressed phenolic acids; the accumulation of most of these differentially expressed metabolites decreased with the developmental stages of fruit from immaturity to maturity. These results confirmed that the developmental stages of fruit are a critical factor in determining its secondary metabolite compositions. This study elucidated the metabolic profile of R. chingii fruit at different stages of development to understand the dynamic changes in metabolites.

Comprehensive metabolomics analysis of key taste components in different varieties of table grapes.

Grapes are one of the world's largest fruit crops, which are rich in nutrients and taste. Summer Black, Gui Fei, Kyoho Grape, Giant Rose, Shine Muscat, and Rosario Bianco are the six most popular table grapes in Wuxi city, Jiangsu province. Owing to the lack of comprehensive investigations of metabolites in table grapes, the metabolic causes of differences in their taste are unknown. In this study, metabolites of six table grapes were profiled using ultra-high-performance liquid chromatography-Q-Exactive Orbitrap tandem mass spectrometry combined with multivariate analysis. Orthogonal partial least squares discriminant analysis discriminated among the metabolites of these varieties. Metabolic pathway analysis revealed that carbohydrate and amino acid metabolisms were highly conserved among these varieties. Our results suggest that the taste differences in the six table grape varieties can be explained by variations in composition and abundance of carbohydrates, organic acids, amino acids, and polyphenols. This study provides comprehensive insights into the underlying metabolic causes of taste variation in table grapes.

Exploring the contributions of two glutamate decarboxylase isozymes in Lactobacillus brevis to acid resistance and γ-aminobutyric acid production.

BACKGROUND: The glutamate decarboxylase (GAD) system of Lactobacillus brevis involves two isoforms of GAD, GadA and GadB, which catalyze the conversion of L-glutamate to γ-aminobutyric acid (GABA) in a proton-consuming reaction contributing to intracellular pH homeostasis. However, direct experimental evidence for detailed contributions of gad genes to acid tolerance and GABA production is lacking. RESULTS: Molecular analysis revealed that gadB is cotranscribed in tandem with upstream gadC, and that expression of gadCB is greatly upregulated in response to low ambient pH when cells enter the late exponential growth phase. In contrast, gadA is located away from the other gad genes, and its expression was consistently lower and not induced by mild acid treatment. Analysis of deletion mutations in the gad genes of L. brevis demonstrated a decrease in the level of GAD activity and a concomitant decrease in acid resistance in the order of wild-type> ΔgadA> ΔgadB> ΔgadC> ΔgadAB, indicating that the GAD activity mainly endowed by GadB rather than GadA is an indispensable step in the GadCB mediated acid resistance of this organism. Moreover, engineered strains with higher GAD activities were constructed by overexpressing key GAD system genes. With the proposed two-stage pH and temperature control fed-batch fermentation strategy, GABA production by the engineered strain L. brevis 9530: pNZ8148-gadBC continuously increased reaching a high level of 104.38 ± 3.47 g/L at 72 h. CONCLUSIONS: This is the first report of the detailed contribution of gad genes to acid tolerance and GABA production in L. brevis. Enhanced production of GABA by engineered L. brevis was achieved, and the resulting GABA level was one of the highest among lactic acid bacterial species grown in batch or fed-batch culture.

Quality evaluation of Eucommiae Cortex processed by different methods and "sweating" conditions based on simultaneous determination of multiple bioactive constituents combined with gray relational analysis.

Eucommiae Cortex is a classical traditional Chinese medicine, which needs to be processed by "sweating" methods. To select the suitable processing method and "sweating" processing condition for Eucommiae Cortex, in this study, the quality of Eucommiae Cortex was evaluated based on simultaneous determination of multiple bioactive constituents combined with gray relational analysis. The contents of lignans, iridoids, penylpropanoids, flavonoids, and phenols in samples were simultaneously determined using ultra-fast performance liquid chromatography coupled with triple quadrupole-linear ion trap tandem mass spectrometry. The chromatographic separation was performed on a Synergiۛ Hydro-RP 100 Å column (100 mm × 2.0 mm, 2.5 µm) at 30°C with a gradient elution of acetonitrile with 0.1% formic acid/0.1% aqueous formic acid as the mobile phase. Furthermore, gray relational analysis was performed to evaluate and sort the samples according to the contents of 14 constituents by calculating the relative correlation degree of each sample. The results demonstrated that the quality of Eucommiae Cortex "sweating" at source area was better and the better "sweating" condition was to scrape off the cork layer before "sweating" with straw covering and sun drying. The developed method could provide the foundation and support for "sweating" processing method of Eucommiae Cortex in normalization and standardization.

Dynamic changes of metabolite accumulation in Scrophulariae Radix based on liquid chromatography-tandem mass spectrometry combined with multivariate statistical analysis.

Scrophulariae Radix is one of the most popular traditional Chinese medicines. The harvesting time of Scrophulariae Radix is closely related to the quality of products in this traditional Chinese medicine. The goal of the study is to analyze the dynamic changes of metabolite accumulation in Scrophulariae Radix. The difference of constituents in Scrophulariae Radix harvested at different times was analyzed by liquid chromatography with triple quadrupole-time of flight tandem mass spectrometry coupled with principal component analysis and partial least-square discriminant analysis. According to the accurate mass of molecular and product ions provided by triple quadrupole-time of flight tandem mass spectrometry, a total of 30 differential constituents were identified. Furthermore, the contents of ten index differential constituents in Scrophulariae Radix were simultaneously determined by liquid chromatography coupled with triple quadrupole-linear ion trap tandem mass spectrometry. Gray relational analysis was performed to evaluate the samples harvested at different times according to the contents of ten constituents. All of the results demonstrated that the quality of Scrophulariae Radix collected at traditional harvest time was better. This study will provide the basic information for revealing the dynamic change law of metabolite accumulation of Scrophulariae Radix and exploring its quality forming mechanism.

[Difference of chemical constituents in Eucommiae Cortex from different habitats by LC-QTOF MS/MS].

In order to study the influence of ecological environment regarding the synthesis and accumulation of metabolites in Eucommiae Cortex, LC-QTOF MS/MS method combined with multivariate statistical analysis was used to analyze the differences of chemical constituents in Eucommiae Cortex from different habitats. Through the analysis of the multistage tandem mass spectrometry, the characteristic peaks were extracted with mass spectrometry data peak matching, peak alignment, and noise filtering. Principal component analysis (PCA) and partial least-squares discriminant analysis (PLS-DA) were used for data processing. The chemical constituents were identified or tentative presumed according to MS accurate mass and MS/MS spectrometry fragmentation information, combined with the software of database search, comparison with reference standards and literature. The results show the differences among samples of Eucommiae Cortex from different habitats are distinguishable. A total of 23 chemical constituents in Eucommiae Cortex were identified or tentative presumed. Among of them, 14 kinds of common differential chemical constituents (aucubin, geniposidic acid, neochlorogenic acid, syringin, olivil-4',4'-di-O-ß-D-glucopyranoside, chlorogenic acid, cryptochlorogenic acid, 1-hydroxypinoresinol- 4',4'-di-O-ß-D-glucopyranoside, caffeic acid, pinoresinol-di-O-ß-D-glucopyranoside, syringaresionl-di-O-ß-D-glucopyranoside, pinoresinol-4'-O-ß-D-glucopyranoside, eucommiol, isochlorogenic acid C and asiatic acid) presented different changing laws. This study provides basic information for revealing the influence law of ecological environment on the biosynthesis of metabolites in Eucommiae Cortex.

Comparison of Chemical Compositions in Pseudostellariae Radix from Different Cultivated Fields and Germplasms by NMR-Based Metabolomics.

Pseudostellariae Radix (PR) is an important traditional Chinese medicine (TCM), which is consumed commonly for its positive health effects. However, the chemical differences of PR from different cultivated fields and germplasms are still unknown. In order to comprehensively compare the chemical compositions of PR from different cultivated fields, in this study, ¹H-NMR-based metabolomics coupled with high performance liquid chromatography (HPLC) were used to investigate the different metabolites in PR from five germplasms (jr, zs1, zs2, sb, and xc) cultivated in traditional fields (Jurong, Jiangsu, JSJR) and cultivated fields (Zherong, Fujian, FJZR). A total of 34 metabolites were identified based on ¹H-NMR data, and fourteen of them were found to be different in PR from JSJR and FJZR. The relative contents of alanine, lactate, lysine, taurine, sucrose, tyrosine, linolenic acid, γ-aminobutyrate, and hyperoside in PR from JSJR were higher than that in PR from FJZR, while PR from FJZR contained higher levels of glutamine, raffinose, xylose, unsaturated fatty acid, and formic acid. The contents of Heterophyllin A and Heterophyllin B were higher in PR from FJZR. This study will provide the basic information for exploring the influence law of ecological environment and germplasm genetic variation on metabolite biosynthesis of PR and its quality formation mechanism.

Quality Evaluation of Scrophulariae Radix Processed by Different 'Sweating' Methods Based on Simultaneous Determination of Multiple Bioactive Constituents Combined with Grey Relational Analysis.

Scrophulariae Radix is one of the most popular traditional Chinese medicines (TCMs), which needs to be processed by 'sweating' methods. Primary processing of Scrophulariae Radix is an important link which closely relates to the quality of products in this TCM. To facilitate selection of the suitable 'sweating' processing method for Scrophulariae Radix, in this study the quality of Scrophulariae Radix processed by different 'sweating' methods was evaluated based on simultaneous determination of multiple bioactive constituents combined with grey relational analysis. The contents of iridoid glycosides, phenylpropanoid glycosides, and organic acids in Scrophulariae Radix processed by different 'sweating' methods were simultaneously determined using ultra high performance liquid chromatography coupled with triple quadrupole-linear ion trap mass spectrometry (UPLC-QTRAP-MS/MS). Furthermore, grey relational analysis (GRA) was performed to evaluate the 'sweating' processed samples according to the contents of twelve constituents. All of the results demonstrated that the quality of Scrophulariae Radix processed by oven drying at 35 °C and 'sweating' for three days was better. The developed method was useful for the overall assessment on quality of Scrophulariae Radix, and this study may provide the foundation and support for 'sweating' processing of Scrophulariae Radix in normalization and standardization.

Quality Evaluation of Pseudostellariae Radix Based on Simultaneous Determination of Multiple Bioactive Components Combined with Grey Relational Analysis.

Pseudostellariae Radix (PR) is an important traditional Chinese herbal medicine (TCM) with vast clinical consumption because of its positive effects. However, little attention has been devoted to simultaneous analysis of its bioactive components for quality control of PR based on its different harvesting times, different growing habitats, and different processing methods. In this research, the quality of PR was evaluated based on simultaneous determination of multiple bioactive components combined with grey relational analysis (GRA). A reliable method based on ultra-fast liquid chromatography tandem triple quadrupole mass spectrometry (UFLC-QTRAP-MS/MS) was established to simultaneously determine the contents of 30 components in PR, including two cyclopeptides, 12 nucleosides, and 16 amino acids. Furthermore, grey relational analysis was performed to evaluate the quality of PR samples according to the contents of these 30 components. The results showed that the quality of PR harvested in 6 August 2013, cultivated in Jurong, Jiangsu, and treated by oven drying 60 °C was better than that of other PR samples. The proposed method is useful for the overall assessment on the quality of PR, and this study provides valuable information for revealing the dynamic change laws of metabolite accumulation in PR and choosing the most suitable harvesting time and reasonable processing method of PR to obtain the best quality.

[iTRAQ-based quantitative proteomics of cultivated Pseudostellaria heterophylla and its wild type].

This work was designed to investigate proteins differentially expressed in cultivated Pseudostellaria heterophylla and its wild type using i TRAQ proteomics approach. The extracted proteins were digested using FASP method and identified by i TRAQ coupled with LC-MS/MS technology and then analyzed by Protein Pilot 5.0 search engine. Proteins differentially expressed were searched through comparison of relatively quantified proteins. The analysis was conducted using GO(gene ontology), KEGG and STRING. A total of 3 775 proteins were detected, among them, 3 676 proteins can be quantified, of which 127 proteins were up-regulated and 205 were down-regulated in cultivated Pseudostellaria heterophylla. We found 71 significantly differentially expressed proteins for further analysis. These proteins were classified into nine categories: heat shock proteins, transferases, oxidoreductases, lyases, isomerases, ligases, hydrolases, tubulin and translocases. The results indicated that the carbohydrate and cellular amino acids metabolism of cultivated Pseudostellaria heterophylla were weaker than its wild type and its ability of responding to stress was much stronger. GWD1, PHS1, GBE1, PGM, and BAM1 are the important proteins to regulate sucrose; met E and CYS are the key proteins that regulate amino acids in cultivated and wild Pseudostellaria heterophylla. This will provide the basic information for exploring the cause of secondary metabolites differences in different ecotype of Pseudostellaria heterophylla and the protein mechanism of its quality formation.

[Simultaneous determination of thirteen components in Polygoni Multifori Caulis by UPLC-MS/MS].

A comprehensive analytical method based on UPLC-MS/MS was developed for the simultaneous determination of thirteen components including three stilbenes (stilbeneglucoside, polydatin, resveratrol), four anthraquinones (emodin, physcion, emodin-8-ß-D-glucopyranoside, aloe-emodin), five flavonoids (epicatechin, rutin, hyperoside, astragalin,quercetin) and one phenolic acid (gallic acid) in Polygoni Multifori Caulis.The separation was carried out on a Waters BEH C18 column（2.1 mm×100 mm,1.7 µm）with gradient elution of acetonitrile-water (0.1% acetic acid) at a flow rate of 0.25 mL•min⁻¹, and column temperature was 35 ℃. The target compounds were analyzed by multiple reaction monitoring (MRM) mode. TOPSIS analysis ware performed to evaluate the samples from different areas and commercial herbs according to the contents of thirteen components. The correlation coefficients of all the calibration curves were higher than 0.991 5. The average recoveries ranged from 95.24% to 102.3%, and the relative standard deviations were less than 5%. The result of TOPSIS analysis showed that the comprehensive quality of Polygoni Multifori Caulis sample from Guangzhou was better. The developed method with good repeatability and accuracy was suitable for the simultaneous determination of multiple functional substances, which provided a new basis for the comprehensive assessment and overall control of the quality of Polygoni Multifori Caulis.

Integrated analysis of multiple metabolome and transcriptome revealed the accumulation of flavonoids and associated molecular regulation mechanisms in Rubus chingii Hu at different developmental stages.

The traditional Chinese herb Rubus chingii Hu (R. chingii) is widely used in clinical practice due to its beneficial effects. Flavonoids are the important class of pharmacological substances in R. chingii, however, the molecular mechanism underlying the differences in active flavonoid contents in R. chingii at different developmental stages remain poorly understood. In this experiment, we selected four developmental stages (GG, GY, YR, RR) of R. chingii as the research material. We studied the untargeted and targeted metabolic profiles of flavonoids in different periods of R. chingii, combining full-length and comparative transcriptome analyses. Functional analyses were conducted on genes implicated in flavonoid differences. GG and RR displayed relatively higher and lower contents of flavonols, flavones, flavanols, flavanones, and isoflavonoid, respectively. RNA-seq analyses showed structural genes such as RcPAL, RcC4H, Rc4CL, RcCHS, RcCHI, RcF3H, RcF3'H, and RcFLS in flavonoid biosynthesis pathway were upregulated in GG, which were essential for the accumulation flavanones, flavones, and flavonols (effective components). qRT-PCR analyses investigated that six structural genes RcCHI, RcF3H, 2 RcCHS, and 2 Rc4CL, two TFs RcMYB308 and RcMYB123 had a consistent expression pattern with which in transcriptome. Also, an interaction network showed that the RcMYB308 could positively regulate Ka3R, Qu, Qu3G, AS, Hy, Ti through RcF3H. Furthermore, Subcellular localization analysis revealed that RcMYB308 was localization to the nucleus. In tobacco, RcMYB308 was overexpressed, resulting in higher flavonoids, RcF3H, RcF3'H, RcCHI, and RcFLS. RcMYB308 upregulated RcF3H in dual-luciferase assays. These results provide new insights for further understanding the molecular mechanism regulating flavonol biosynthesis in R. chingii fruit, and also provide a potential MYB regulator for molecular breeding of R. chingii.

Comparison of Chemical Constituents in Pseudostellariae Radix with Different Dosage Forms Based on HPLC-Q-Exactive Orbitrap/MS Combined with Multivariate Statistical Analysis.

BACKGROUND: Pseudostellariae Radix (PR) is an important traditional Chinese herbal medicine with vast clinical consumptions, which has two different dosage forms, PR decoction pieces and PR formula granules. However, these two forms are bound to have an impact on the accumulation of the effective components in PR, so the effectiveness of clinical use cannot be guaranteed. OBJECTIVE: To determine the effective composition of PR. METHODS: In this research, PR decoction pieces and formula granules were collected, and their composition was detected by HPLC-Q-Exactive Orbitrap/MS; multivariate statistical analysis was used to distinguish differential metabolites between PR decoction pieces and formula granules. RESULTS: A clear cut difference in the composition of the two groups was observed. 98 differential chemical constituents could be identified in the positive mode, while 52 differential chemical compositions could be identified in the negative mode. The differential chemical compositions were mainly concentrated in flavonoids, organic acids, fatty acids, and amino acids and present different change rules, mainly involved in the isoquinoline alkaloid biosynthesis metabolic pathways. CONCLUSIONS: This study provides basic information to reveal the influence law of different dosage forms on the metabolite synthesis and quality formation mechanism of PR.

Improving the Thermostability of Glutamate Decarboxylase from Lactobacillus brevis by Consensus Mutagenesis.

γ-Aminobutyrate (GABA) is an important bioactive compound synthesized through decarboxylation of L-glutamate by the glutamate decarboxylase (GAD). In this study, stabilized variants of the GAD from Lactobacillus brevis were constructed by consensus mutagenesis. Using Consensus Finder ( http://cbs-kazlab.oit.umn.edu/ ), eight positions with the most prevalent amino acid (over 60% threshold) among the homologous family members were identified. Subsequently, these eight residues were individually mutated to match the consensus sequence using site-directed mutagenesis. Compared to the wild-type, T383K variant displayed the largest shift in thermostability among the single variants, with a 3.0 °C increase in semi-inactivation temperature (T5015), a 1.7-fold improvement of half-life (t1/2) at 55 °C, and a 1.2-fold improvement of t1/2 at 37 °C, respectively, while its catalytic efficiency (kcat/Km) was reduced. To obtain the mutant with improvement in both thermostability and catalytic activity, we performed a site-saturation mutation at T383. Notably, mutants T383V and T383G exhibited an increasement in thermostability and kcat/Km than that of wild-type. This study not only emphasizes the value of consensus mutagenesis for improving the thermostability of GAD but also sheds a powerful guidance to study the thermal stability of other enzymes.

Analysis of the Prunellae Spica transcriptome under salt stress.

Prunella vulgaris L. is a moderately salt tolerant plant commonly found in China and Europe, whose spica (Prunellae Spica) has been used as a traditional medicine. The scant transcriptomic and genomic resources of Prunellae Spica have greatly hindered further exploration of the underlying salt tolerance mechanism of this species. To clarify the genetic basis of its salt tolerance, high-throughput sequencing of mRNAs was employed for de novo transcriptome assembly differential expression analysis of Prunellae Spica under salt stress. 118,664 unigenes were obtained by assembling pooled reads from all libraries with 68,119 sequences annotated. A total of 3857 unigenes were differentially expressed under low, medium and high salt stress, including 2456 up-regulated and 1401 down-regulated DEGs, respectively. Gene ontology analysis revealed that salt stress-related categories involving 'catalytic activity', 'binding', 'metabolic process' and 'cellular process' were highly enriched. KEGG pathway annotation showed that the DEGs from different salt stress treatment groups were mainly enriched in the pathways of translation, signal transduction, carbohydrate metabolism, energy metabolism, lipid metabolism and amino acid metabolism, accounting for over 60% of all DEGs. Finally, it showed that the results of quantitative real-time polymerase chain reaction (qRT-PCR) analysis for 10 unigenes that randomly selected were significantly consistent with RNA-seq data, which further assisted in the selection of salt stress-responsive candidate genes in Prunellae Spica. This study represents a significant step forward in understanding the salt tolerance mechanism of Prunellae Spica, and also provides a significant transcriptomic resource for future work.

[Increasing the thermostability of glutamate decarboxylase from Lactobacillus brevis by introducing proline].

Glutamate decarboxylase, a unique pyridoxal 5'-phosphate-dependent enzyme, catalyzes α-decarboxylation of L-glutamate to γ-aminobutyrate. However, glutamate decarboxylase from different sources has the common problem of poor thermostability that affects its application in industry. In this study, proline was introduced at 13 different positions in glutamate decarboxylase by using the design strategy of homologous sequence alignment between Thermococcus kodakarensis and Lactobacillus brevis CGMCC No.1306. A mutant enzyme G364P with higher thermostability was obtained. Compared to the wild type, thermostability of the mutant G364P was significantly improved, the half-life time (t1/2) at 55 °C and the semi-inactivation temperature (T50 ¹5) of the mutant G364P increased 19.4 min and 5.3 °C, respectively, while kcat/Km of the mutant enzyme remained nearly unchanged. Further analysis of their thermostability by molecular dynamics simulations were performed. The root mean square deviation of G364P and root mean square fluctuation in the loop region including G364 were lower than the wild type at 313 K for 10 ns, and G364P increased one hydrophobic interaction in the loop region. It proves that mutation of flexible 364-Gly to rigid proline endows glutamate decarboxylase with enhanced thermostability.

Comparison of Chemical Constituents in Scrophulariae Radix Processed by Different Methods based on UFLC-MS Combined with Multivariate Statistical Analysis.

Scrophulariae Radix is one of the most popular traditional Chinese medicines (TCMs). Primary processing of Scrophulariae Radix is an important link which closely related to the quality of products in this TCM. The aim of this study is to explore the influence of different processing methods on chemical constituents in Scrophulariae Radix. The difference of chemical constituents in Scrophulariae Radix processed by different methods was analyzed by using ultra fast liquid chromatography-triple quadrupole-time of flight mass spectrometry coupled with principal component analysis and orthogonal partial least squares discriminant analysis. Furthermore, the contents of 12 index differential constituents in Scrophulariae Radix processed by different methods were simultaneously determined by using ultra fast liquid chromatography coupled with triple quadrupole-linear ion trap mass spectrometry. Gray relational analysis was performed to evaluate the different processed samples according to the contents of 12 constituents. All of the results demonstrated that the quality of Scrophulariae Radix processed by "sweating" method was better. This study will provide the basic information for revealing the change law of chemical constituents in Scrophulariae Radix processed by different methods and facilitating selection of the suitable processing method of this TCM.

Transcriptomic analysis of Pseudostellariae Radix from different fields using RNA-seq.

Pseudostellariae Radix is an important traditional Chinese medicine (TCM), which is consumed commonly for its positive health effects. However, a lack of transcriptomic and genomic information hinders research on Pseudostellariae Radix. Here, high-throughput RNA sequencing (RNA-seq) was employed for the de novo assembly to analyze the transcriptome in Pseudostellariae Radix, finding significantly differentially expressed genes in this TCM from different fields based on RNA-seq and bioinformatic analysis. A total of 146,408,539 paired-end reads were generated and assembled into 89,857 unigenes with an average length of 862bp. All of the assembly unigenes were annotated by running BLASTx and BLASTn similarity searches on the Non-redundant nucleotide database (NT), the Non-redundant protein database (NR), Swiss-Prot, Cluster of Orthologous Groups (COG), Kyoto Encyclopedia of Genes and Genomes (KEGG), Gene Ontology (GO), and Interpro. On the basis of bioinformatic analysis and the expression profiles for Pseudostellariae Radix, 29 significantly differentially expressed genes were identified, which provides the basic information for exploring the molecular mechanisms that determine the quality of Pseudostellariae Radix from different fields. The expression levels of 29 genes were validated by real-time quantitative PCR (RT-qPCR). This is the first study to sample Pseudostellariae Radix, which provides an invaluable resource for understanding the genome of this herb.

Chemical Differentiation of Pseudostellariae Radix from Different Cultivated Fields and Germplasms by UPLC-Triple TOF-MS/MS Coupled with Multivariate Statistical Analysis.

To explore rapidly the potential chemical markers for differentiating Pseudostellariae Radix from different cultivated fields :and germplasms, a method is proposed based on ultra-performance liquid chromatographytriple time-of-flight mass/mass spectrometry (UPLC-Triple TOF-MS/MS) coupled with multivariate statistical analysis. Peak matching, peak alignment, and noise filtering were used in analyzing mass spectrometric data. Accurate m/z value analysis of MS data based on software of database search and MS/MS fragment analysis were applied to identify constituents. The obtained. data were statistically analyzed with hierarchical cluster analysis (HCA), principal component analysis (PCA), and partial least squared-discriminant analysis (PLS-DA) to compare the differences among these samples. The PLS-DA loading plot obtained from all mass data showed that 21 compounds were identified as the potential chemical markers to characterize the samples. The results provide experimental data to reveal the influence of ecological environments and germplasms on metabolite biosynthesis in Pseudostellariae Radix.