[Methods for synchronous detection of 14 mycotoxins in Pseudostellariae Radix and investigation on its contamination].

This study aimed to establish a method for synchronous detection of 14 mycotoxins in Pseudostellariae Radix and investigate its contamination with mycotoxins, so as to provide technical guidance for monitoring the quality of Chinese medicinal materials and medication safety. The sample was extracted with 80% acetonitrile in an oscillator for 1 h, purified using the modified QuEChERS purifying agent(0.1 g PSA + 0.3 g C_(18) + 0.3 g MgSO_4), and separated on a Waters HSS T3 chromatographic column(2.1 mm×100 mm, 1.8 µm). The gradient elution was carried out with 0.1% formic acid in water and acetonitrile, followed by the scanning in the multi-reaction monitoring(MRM) mode and the analysis of mycotoxin contamination in 26 Pseudostellariae Radix samples. The recovery rates of the established method were within the range of 82.17%-113.6%, with the RSD values less than 7% and the limits of quantification(LOQ) being 0.019-0.976 µg·kg~(-1). The detection rate of 14 mycotoxins in 26 batches of medicinal materials was 53.85%. The detection rate of sterigmatocystin(ST) was the highest, followed by those of zearalenone(ZEN), aflatoxin G_2(AFG_2), fumonisin B_1(FB_1), HT-2 toxin, and nivalenol(NIV). Their respective detection rates were 38.46%, 26.92%, 23.08%, 11.54%, 11.54%, and 7.69%, with the pollution ranges being 1.48-69.65, 0.11-31.05, 0.11-0.66, 0.28-0.83, 20.86-42.56, and 0.46-1.84 µg·kg~(-1), respectively. The established method for the detection of 14 mycotoxins is accurate, fast and reliable. The research results have very important practical significance for guiding the monitoring and prevention and control of exogenous fungal contamination of Chinese medicinal materials.

[Regional stability analysis of Pseudostellariae Radix new variety "Shitai No.1"].

To provide guidance for certification, popularization and application of Pseudostellariae Radix new variety, the regional adaptation and stabilities of "Shitai No.1" were evaluated. The "Qian taizishen No.1" and "SB-C" varieties (strains) were used as the control varieties. The agronomic, medicinal material traits and medicine quality were used as evaluation index to compare the phenotypic difference of the three varieties (strains) in four planting areas. Compared to the control varieties, 10 agronomic traits of "Shitai No.1" had the smallest coefficient of variation among the 18 agronomic traits, and other 8 agronomic traits placed the middle level. Among 8 medicinal material traits and medicine quality indicators, the coefficient of variation of different regions of the extract content, pseudostellarin B content, the number of 50 g root tuber, the plant medicinal materials weight and weight of single root of "Shitai No.1" were the smallest compare to other varieties (strains). It could be divided into three groups based on the phenotypic difference of the three varieties (strains) in four planting areas. The "Shitai No.1" was classified as one group, while the "Qian taizishen No.1" and "SB-C" had cross clustering. The regional stability of several index about agronomic traits, medicinal material traits and medicine quality of "Shitai No.1" were better than that of the control varieties (strains). "Shitai No.1" was suitable for planting, popularization and application in the appropriate ecological areas of Guizhou province.

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 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.

[Cloning and bioinformatics analysis of abscisic acid 8'-hydroxylase from Pseudostellariae Radix].

Abscisic acid 8'-hydroxylase was one of key enzymes genes in the metabolism of abscisic acid (ABA). Seven menbers of abscisic acid 8'-hydroxylase were identified from Pseudostellaria heterophylla transcriptome sequencing results by using sequence homology. The expression profiles of these genes were analyzed by transcriptome data. The coding sequence of ABA8ox1 was cloned and analyzed by informational technology. The full-length cDNA of ABA8ox1 was 1 401 bp,with 480 encoded amino acids. The predicated isoelectric point (pI) and relative molecular mass (MW) were 8.55 and 53 kDa,respectively. Transmembrane structure analysis showed that there were 21 amino acids in-side and 445 amino acids out-side. High level of transcripts can detect in bark of root and fibrous root. Multi-alignment and phylogenetic analysis both show that ABA8ox1 had a high similarity with the CYP707As from other plants,especially with AtCYP707A1 and AtCYP707A3 in Arabidopsis thaliana. These results lay a foundation for molecular mechanism of tuberous root expanding and response to adversity stress.

[Research model on commodity specification standard of radix Chinese materia medica].

As an important part of the market commodity circulation, the standard grade of Chinese traditional medicine commodity is very important to restrict the market order and guarantee the quality of the medicinal material. The State Council issuing the "protection and development of Chinese herbal medicine (2015-2020)" also make clear that the important task of improving the circulation of Chinese herbal medicine industry norms and the commodity specification standard of common traditional Chinese medicinal materials. However, as a large class of Chinese herbal medicines, the standard grade of the radix is more confused in the market circulation, and lack of a more reasonable study model in the development of the standard. Thus, this paper summarizes the research background, present situation and problems, and several key points of the commodity specification and grade standard in radix herbs. Then, the research model is introduced as an example of Pseudostellariae Radix, so as to provide technical support and reference for formulating commodity specifications and grades standard in other radix traditional Chinese medicinal materials.

Application of HS-SPME-GC-MS combined with electronic nose technology in the odor recognition of pseudostellariae radix.

Correct identification of the substance basis of Pseudostellariae Radix (PR) odor is important not only for the quality control of the products, but also for the safety of the consumers. PR is often described with a special smell, such as strange, moldy or earthy. Electronic nose-based technology coupled with headspace solid phase microextraction gas chromatography-mass spectrometry (HS-SPME-GC-MS) was used to investigate the volatile components in PR from 47 germplasms cultivated in traditional fields. A total of 48 common compounds were identified based on HS- SPME-GC-MS technology, and 25 of them with aroma characteristics were found based on Alpha soft 13.4. The 1-Octen-3-ol, geosim, (E)-2-nonenal and 1- methylnaphthalene as contributing marker compounds of the 'specific smell' of PR were identified. The odor recognition mode, with demonstrated excellent accuracy in recognition abilities, enabled the correct identification of commercial samples including complex mixtures.

Methods for synchronous detection of 14 mycotoxins in Pseudostellariae Radix and investigation on its contamination / 中国中药杂志

This study aimed to establish a method for synchronous detection of 14 mycotoxins in Pseudostellariae Radix and investigate its contamination with mycotoxins, so as to provide technical guidance for monitoring the quality of Chinese medicinal materials and medication safety. The sample was extracted with 80% acetonitrile in an oscillator for 1 h, purified using the modified QuEChERS purifying agent(0.1 g PSA + 0.3 g C_(18) + 0.3 g MgSO_4), and separated on a Waters HSS T3 chromatographic column(2.1 mm×100 mm, 1.8 μm). The gradient elution was carried out with 0.1% formic acid in water and acetonitrile, followed by the scanning in the multi-reaction monitoring(MRM) mode and the analysis of mycotoxin contamination in 26 Pseudostellariae Radix samples. The recovery rates of the established method were within the range of 82.17%-113.6%, with the RSD values less than 7% and the limits of quantification(LOQ) being 0.019-0.976 μg·kg~(-1). The detection rate of 14 mycotoxins in 26 batches of medicinal materials was 53.85%. The detection rate of sterigmatocystin(ST) was the highest, followed by those of zearalenone(ZEN), aflatoxin G_2(AFG_2), fumonisin B_1(FB_1), HT-2 toxin, and nivalenol(NIV). Their respective detection rates were 38.46%, 26.92%, 23.08%, 11.54%, 11.54%, and 7.69%, with the pollution ranges being 1.48-69.65, 0.11-31.05, 0.11-0.66, 0.28-0.83, 20.86-42.56, and 0.46-1.84 μg·kg~(-1), respectively. The established method for the detection of 14 mycotoxins is accurate, fast and reliable. The research results have very important practical significance for guiding the monitoring and prevention and control of exogenous fungal contamination of Chinese medicinal materials.

Effect of Different Drying Methods on Volatile Components in Pseudostellariae Radix Based on GC-IMS / 中国实验方剂学杂志

ObjectiveTo compare the effects of different drying methods on volatile components of Pseudostellariae Radix. MethodThe samples were dried by different methods， including air drying， sun drying， hot air drying （40， 60， 80 ℃） and vacuum freeze drying. Gas chromatography-ion mobility spectrometry （GC-IMS） was used to compare the changes of volatile components in the samples after different treatments. The samples were incubated at 80 ℃ and 500 r·min-1 for 15 min， the injection temperature was 85 ℃， the injection volume was 200 μL， the flow rate of carrier gas was from 2 mL to 150 mL during 20 min， and the temperature of IMS detector was 60 ℃. SE-54 capillary column （0.32 mm×30 m， 0.25 μm） was used， the column temperature was 60 ℃， and the analysis time was 35 min. The differential spectra of volatile components were constructed and analyzed by principal component analysis （PCA）. ResultA total of 37 volatile components were identified from dried Pseudostellariae Radix. The number of compounds in descending order was ketones， aldehydes and alcohols. There were some differences in the volatile components in samples dried by different methods. And the volatile components in samples with sun drying， air drying and hot air drying at 40 ℃ were similar， compared with other drying methods， vacuum freeze drying and hot air drying at 80 ℃ had great effects on the volatile components of Pseudostellariae Radix， and the compounds in the samples with vacuum freeze drying were the least. ConclusionIn this study， GC-IMS for the detection and analysis of volatile components in Pseudostellariae Radix is established， which has the characteristics of high efficiency， nondestructive inspection and simple sample processing. This method can be used for the distinction of Pseudostellariae Radix dried by different methods. And hot air drying at 40 ℃ can effectively retain the volatile components of Pseudostellariae Radix， and achieve similar flavor to samples with sun drying and air drying.

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.

Study on Chinese medicinal materials origin market integration--Taking Lonicerae Japonicae Flos, Lycii Fructus, Isatidis Radix and Pseudostellariae Radix for examples / 中草药

Trading volume in Chinese medicinal materials market has gradually declined, and origin market trade has become a trend. Studying the degree of market integration is helpful to understand the operation of origin market of Chinese medicinal materials, which is of great significance for rational planning of the production of Chinese medicinal materials and improving market efficiency. In this study, four varieties (Lonicerae Japonicae Flos, Lycii Fructus, Isatidis Radix and Pseudostellariae Radix) were selected to study the market integration degree of Chinese medicinal materials. Based on the price index data from 2013 to 2019, this paper makes an empirical analysis by using co-integration test, error correction model and Granger causality test. The results showed that the long-term integration of Chinese medicinal materials market was relatively high, but the short-term integration was far lower than the long-term integration. Finally, the author puts forward four policy suggestions: improving the price information platform conduction of Chinese medicinal materials, building high-quality brand of Chinese medicinal materials in non-authentic producing areas, speeding up the establishment quality traceability system and building modern logistics system of Chinese medicinal materials.

Regional stability analysis of Pseudostellariae Radix new variety "Shitai No.1" / 中国中药杂志

To provide guidance for certification, popularization and application of Pseudostellariae Radix new variety, the regional adaptation and stabilities of "Shitai No.1" were evaluated. The "Qian taizishen No.1" and "SB-C" varieties (strains) were used as the control varieties. The agronomic, medicinal material traits and medicine quality were used as evaluation index to compare the phenotypic difference of the three varieties (strains) in four planting areas. Compared to the control varieties, 10 agronomic traits of "Shitai No.1" had the smallest coefficient of variation among the 18 agronomic traits, and other 8 agronomic traits placed the middle level. Among 8 medicinal material traits and medicine quality indicators, the coefficient of variation of different regions of the extract content, pseudostellarin B content, the number of 50 g root tuber, the plant medicinal materials weight and weight of single root of "Shitai No.1" were the smallest compare to other varieties (strains). It could be divided into three groups based on the phenotypic difference of the three varieties (strains) in four planting areas. The "Shitai No.1" was classified as one group, while the "Qian taizishen No.1" and "SB-C" had cross clustering. The regional stability of several index about agronomic traits, medicinal material traits and medicine quality of "Shitai No.1" were better than that of the control varieties (strains). "Shitai No.1" was suitable for planting, popularization and application in the appropriate ecological areas of Guizhou province.

Molecular cloning and sequence analysis of two GA2ox genes in Pseudostellariae Radix / 中草药

Objective: To clone the gibberellin 2-oxidase (GA2ox) genes from Pseudostellaria heterophylla and perform the bioinformatic and expression mode analysis. Methods: According to P. heterophylla transcriptome annotation, two transcript codings of GA2ox were identified. The full-length cDNA PhGA2ox1 and PhGA2ox8 were determined using RT-PCR. Then the bioinformatic analysis of these genes and encoded proteins were performed. The coding sequences of PhGA2ox1 and PhGA2ox8 were amplified by PCR. And then analyzed the bioinformation of these sequences. The expression levels of PhGA2ox1 and PhGA2ox8 were analyzed using qPCR. Results: Bioinformatic analysis showed that PhGA2ox1 contained 981 bp and encoded a predicted protein of 326 amino acids with molecular weight of 36 871.3 and isoelectric point (PI) of 8.66; PhGA2ox8 contained 1 056 bp and encoded a predicted protein of 351 amino acids with molecular weight of 40 169.9 and PI of 6.91. Both PhGA2ox1 and PhGA2ox8 contained GA2ox conserved domains DIOX_N and 20G-Fell_Oxy, without obvious hydrophobic region, transmembrane domain and signal peptide. Phylogenetic analysis showed that the GA2ox of plant could be divided into three classes, PhGA2ox1 bolongs to Class I and PhGA2ox8 belongs to Class III. The qPCR. showed that the expression of PhGA2ox1 in different tissues and organs of P. heterophylla was constant, but the expression of PhGA2ox8 in root xylem was significantly higher than that in other tissues and organs (P < 0.05). Conclusion: The PhGA2ox1 and PhGA2ox8 genes are cloned for the first time, and provide a foundation for they may play an important role in the growth and development process of P. heterophylla.

1H-NMR based metabolomic analysis of chemical compositions in cultivated and wild pseudostellariae radix / 中国药学杂志

OBJECTIVE: To investigate the differences in the accumulation and biosynthesis of metabolites in different lactic acid of Pseudostellariae Radix. METHODS: 1H-NMR based metabolomic approach combined with multivariate statistical analysis was used to investigate the chemical compositions and find the different metabolites in cultivated Pseudostellariae Radix and its wild-type. RESULTS: Thirty-five metabolites were identified in the 1H-NMR spectra, and 15 of which showed remarkable differences. The multivariate statistical analysis showed that the cultivated Pseudostellariae Radix and its wild-type could be distinguished obviously. The contents of isoleucine, linolenic acid, lactic acid, alanine, lysine, glutamic acid, glutamine, acetoacetic acid, succinic acid, γ-aminobutyric acid, phenylalanine, and sucrose in cultivated Pseudostellaria heterophylla were lower than its wild-type, while the cultivated Pseudostellariae Radix contained more xylose, raffinose, and fumaric acid. CONCLUSION: This study will provide basic information for exploring the quality formation mechanism and revealing the accumulation and biosynthesis of metabolites in different ecotypes of Pseudostellariae Radix.

Cloning and bioinformatics analysis of abscisic acid 8'-hydroxylase from Pseudostellariae Radix / 中国中药杂志

Abscisic acid 8'-hydroxylase was one of key enzymes genes in the metabolism of abscisic acid (ABA). Seven menbers of abscisic acid 8'-hydroxylase were identified from Pseudostellaria heterophylla transcriptome sequencing results by using sequence homology. The expression profiles of these genes were analyzed by transcriptome data. The coding sequence of ABA8ox1 was cloned and analyzed by informational technology. The full-length cDNA of ABA8ox1 was 1 401 bp,with 480 encoded amino acids. The predicated isoelectric point (pI) and relative molecular mass (MW) were 8.55 and 53 kDa,respectively. Transmembrane structure analysis showed that there were 21 amino acids in-side and 445 amino acids out-side. High level of transcripts can detect in bark of root and fibrous root. Multi-alignment and phylogenetic analysis both show that ABA8ox1 had a high similarity with the CYP707As from other plants,especially with AtCYP707A1 and AtCYP707A3 in Arabidopsis thaliana. These results lay a foundation for molecular mechanism of tuberous root expanding and response to adversity stress.

Research model on commodity specification standard of radix Chinese materia medica / 中国中药杂志

As an important part of the market commodity circulation, the standard grade of Chinese traditional medicine commodity is very important to restrict the market order and guarantee the quality of the medicinal material. The State Council issuing the "protection and development of Chinese herbal medicine (2015-2020)" also make clear that the important task of improving the circulation of Chinese herbal medicine industry norms and the commodity specification standard of common traditional Chinese medicinal materials. However, as a large class of Chinese herbal medicines, the standard grade of the radix is more confused in the market circulation, and lack of a more reasonable study model in the development of the standard. Thus, this paper summarizes the research background, present situation and problems, and several key points of the commodity specification and grade standard in radix herbs. Then, the research model is introduced as an example of Pseudostellariae Radix, so as to provide technical support and reference for formulating commodity specifications and grades standard in other radix traditional Chinese medicinal materials.

Q-TRAP-LC-MS/MS analysis on nucleosides in Pseudostellariae Radix with different processing methods / 中草药

Objective: To investigate the influence of different processing methods on the contents of nucleosides in Pseudostellariae Radix (PR). Methods: Q-TRAP-LC-MS/MS method was applied for the analysis on 13 kinds of nucleosides in PR with different processing methods. Results: The contents of cytidine, uridine, inosine, guanosine, and thymidine were higher in PR. The different processing methods of PR had certain influence on the contents of nucleosides. The amount of nucleosides in PR by oven drying was higher than that by drying in the sun. Conclusion: The paper investigates the influence of processing methods on chemical composition in PR, and provides the basis for establishing reasonable processing technology.

Quantitative determination of thirteen nucleosides and nucleobases in pseudostellariae radix in different harvest periods by QTRAP LC-MS/MS / 中国药学杂志

OBJECTIVE: To establish a QTRAP UPLC-MS/MS method for simultaneous determination of thirteen nucleosides and nucleobases in Pseudostellariae Radix, and analyze their dynamic changes at different periods. METHODS: The separation was carried out on a Waters Atlantis T3 column (2.1 mm × 150 mm, 3 μm)eluted by a mobile phase of 5 mmol · mL-1 of ammonium acetate (B)-methanol(A) at a flow rate of 0.4 mL · min-1. The elution program is as follows. 0 -4.5 min, 3% -4% A; 4.5-8 min, 4% -18% A; 8 -10 min, 18%A; 10 -10.1 min, 18% -3% A; 10.1 -13 min, 3%A. The target compounds were analyzed by the positive ion multiple reaction monitoring (MRM) mode. RESULTS: The calibration curves of the 13 nucleosides and nucleobases showed good linearity (r >0.991 0) in the ranges of the tested concentrations, and the average recoveries were between 96.46% - 105.07%. The contents of cytidine, uridine, inosine, guanosine, and thymidine in Pseudostellariae Radix harvested at different periods showed some differences, and the contents were higher in mid-June. CONCLUSION: The method is simple, sensitive, accurate and credible, and provides the basis for exploring the quality forming mechanism of Pseudostellariae Radix medicinal materials and the suitable harvest time.