Common diseases and drug use of Pseudostellaria heterophylla / 中国中药杂志

Pseudostellaria heterophylla in large-scale cultivation needs to apply pesticides to control diseases, and non-standard use of pesticide may cause excessive pesticide residues in medicinal materials, increasing the risk of clinical medication. To accurately monitor the residual pesticides, this paper investigated the drug use during the process of P. heterophylla disease prevention in 25 P. he-terophylla planting enterprises or individual households in Guizhou province. It was found that there were 8 common diseases in P. he-terophylla planting, including leaf spot, downy mildew, virus disease, root rot, dropping disease, purple feather disease, white silk disease, and damping-off disease. Twenty-three kinds of pesticides were used in disease control, mainly chemical synthetic pesticides, accounting for 78.3%, followed by biological pesticides and mineral pesticides, accounting for 13.0% and 8.7%, respectively. The disease prevention and control drugs were all low-toxic pesticides, and there were no varieties banned in the Chinese Pharmacopoeia(2020 edition). However, the pesticides used have not been registered on P. heterophylla, and the excessive use of drugs was serious. The present monitoring of pesticide residues in P. heterophylla is mainly based on traditional pesticides such as organochlorine, organophosphorus, and carbamate, which does not effectively cover the production of drugs and had certain safety risks. It is suggested to speed up the research and registration of drug use in the production of P. heterophylla, increase the use of biological pesticides, and further improve the monitoring indicators of pesticide residues in combination with the actual production of drugs, so as to promote the high-quality development of P. heterophylla industry.

Cloning and functional verification of PhAEP gene, a key enzyme for biosynthesis of heterophyllin A in Pseudostellaria heterophylla / 中国中药杂志

This paper aimed to study the role of asparagine endopeptidase(AEP) gene in the biosynthesis mechanism of cyclic peptide compounds in Pseudostellaria heterophylla. The transcriptome database of P. heterophylla was systematically mined and screened, and an AEP gene, tentatively named PhAEP, was successfully cloned. The heterologous function verification by Nicotiana benthamiana showed that the expression of the gene played a role in the biosynthesis of heterophyllin A in P. heterophylla. Bioinformatics analysis showed that the cDNA of PhAEP was 1 488 bp in length, encoding 495 amino acids with a molecular weight of 54.72 kDa. The phylogenetic tree showed that the amino acid sequence encoded by PhAEP was highly similar to that of Butelase-1 in Clitoria ternatea, reaching 80%. The sequence homology and cyclase active site analysis revealed that the PhAEP enzyme may specifically hydrolyse the C-terminal Asn/Asp(Asx) site of the core peptide in the HA linear precursor peptide of P. heterophylla, thereby participating in the ring formation of the linear precursor peptide. The results of real-time quantitative polymerase chain reaction(RT-qPCR) showed that the expression level of PhAEP was the highest in fruits, followed by in roots, and the lowest in leaves. The heterophyllin A of P. heterophylla was detected in N. benthamiana that co-expressed PrePhHA and PhAEP genes instantaneously. In this study, the PhAEP gene, a key enzyme in the biosynthesis of heterophyllin A in P. heterophylla, has been successfully cloned, which lays a foundation for further analysis of the molecular mechanism of PhAEP enzyme in the biosynthesis of heterophyllin A in P. heterophylla and has important significance for the study of synthetic biology of cyclic peptide compounds in P. heterophylla.

Identification and biological characterization of pathogen causing black spot of Pseudostellaria heterophylla in Fujian province / 中国中药杂志

In Zherong county, Fujian province, the black spot of Pseudostellaria heterophylla often breaks out in the rainy season from April to June every year. As one of the main leaf diseases of P. heterophylla, black spot seriously affects the yield and quality of the medicinal material. To identify and characterize the pathogens causing black spot, we isolated the pathogens, identified them as a species of Alternaria according to Koch's postulates, and then tested their pathogenicity and biological characteristics. The results showed that the pathogens causing P. heterophylla black spot were A. gaisen, as evidenced by the similar colony morphology, spore characteristics, sporulation phenotype, and the same clade with A. gaisen on the phylogenetic tree(the maximum likelihood support rate of 100% and the Bayesian posterior probability of 1.00) built based on the tandem sequences of ITS, tef1, gapdh, endoPG, Alta1, OPA10-2, and KOG1077. The optimum conditions for mycelial growth of the pathogen were 25 ℃, pH 5-8, and 24 h dark culture. The lethal conditions for mycelia and spores were both treatment at 50 ℃ for 10 min. We reported for the first time the A. gaisen-caused black spot of P. heterophylla. The results could provide a theoretical basis for the diagnosis and control of P. heterophylla leaf spot diseases.

Comparative chemical characters of Pseudostellaria heterophylla from geographical origins of China / 中草药·英文版

OBJECTIVE@#Pseudostellaria heterophylla has been paid more attention in recent years, mainly as a medicine food homology plant. The content determination of P. heterophylla is not specified in the Chinese Pharmacopoeia (version 2020). The environmental conditions in different production areas could exert an influence on the quality of P. heterophylla. The purpose of this study is to discriminate P. heterophylla collected from different geographical origins of China.@*METHODS@#In this study, the content of polysaccharide in 28 batches of P. heterophylla was determined using phenol-sulfuric acid. HPLC fingerprints were established under optimised HPLC-PDA methods. Subsequently, the similarity analysis (SA) and the quantification of heterophyllin B were analyzed. The metabolites of P. heterophylla were identified and evaluated using UHPLC-Q Exactive HF orbitrap MS system. Principal component analysis (PCA), partial least squares discriminant analysis (PLS-DA), hierarchical cluster analysis (HCA) and orthogonal PLS-DA (OPLS-DA) were performed based on all peak areas.@*RESULTS@#The polysaccharide content in Guizhou and Jiangsu was higher than that of other production areas, which varied significant from different origins. While the content of heterophyllin B in Anhui and Jiangsu was high. The correlation coefficients of HPLC fingerprints for 28 batches samples ranged from 0.877 to 0.990, and the characteristic map can be used to identify and evaluate the quality of P. heterophylla. The samples from Fujian, Guizhou, Jiangsu provinces can be relatively separated using multivariate statistical analysis including PCA, PLS-DA, HCA, OPLS-DA, indicating that their metabolic compositions were significantly different. Ultimately, a total of 15 metabolites which were filtrated by a VIP-value > 1 and a P-value < 0.05 associated with the separation of different origins were identified.@*CONCLUSION@#HPLC fingerprint was established to evaluate the quality and authenticity of P. heterophylla. The present work showed that the difference of geographic distributions had an influence on the internal chemical compositions. A sensitive and rapid untargeted metabolomics approach by UHPLC-Q Exactive HF orbitrap MS was utilized to evaluate P. heterophylla from different origins in China for the first time. Overall, this study provides insights to metabolomics of P. heterophylla and supplies important reference values for the development of functional foods.

Identification and biological characteristics of violet root rot pathogen of Pseudostellaria heterophylla in Fujian province / 中国中药杂志

Violet root rot is one of the main root diseases in the production process of Pseudostellaria heterophylla. To clarify the pathogenic species that cause the violet root rot of P. heterophylla in Fujian province, the roots and the sclerotia with violet root rot symptoms were collected from the main producing areas of P. heterophylla(Fujian province) from 2017 to 2021, and the pathogens were isolated by tissue separation method and identified by morphology and multi-gene phylogenetic analysis. Additionally, the biological characteristics of the pathogens were studied and the fungicides were determined. The results showed that 78 strains of violet root rot were isolated from the collected root samples, which belonged to one type after preliminary morphological identification. Two represen-tative strains were selected from the pathogens for multi-gene phylogenetic analysis, and they were clustered with Helicobasidium mompa together. The suitable culture conditions for the mycelium were OA medium, 25 ℃, pH 6, and ammonium oxalate as the nitrogen source. The lethal temperature of the mycelium was 50 ℃ for 10 minutes. Moreover, 99.1% propiconazole and 98.7% azoxystrobin had the optimal bacteriostatic effect, and the concentrations with the 50% bacteriostatic rate were 16.85 and 12.24 μg·mL~(-1), respectively. On the basis of the above results, the pathogen causing violet root rot of P. heterophylla in Fujian province was H. mompa. The medium type, growth temperature, pH value, nitrogen source, etc. had significant effect on the growth of mycelium.

Chemical constituents of cyclic peptides from fibrous roots of Pseudostellaria heterophylla / 中国中药杂志

Four cyclic peptides were isolated from the 75% ethanol extract of the fibrous roots of Pseudostellaria heterophylla by silica gel, Sephadex LH-20 column chromatography, and semi-preparative HPLC. Through mass spectrometry, NMR and other methods, they were identified as pseudostellarin L(1), heterophyllin B(2), pseudostellarin B(3), and pseudostellarin C(4). Among them, compound 1 was a new cyclic peptide, and compounds 2-4 were isolated from the fibrous roots of P. heterophylla for the first time. None of these compounds displayed cytotoxic activities against MCF-7, A549, HCT-116, and SGC-7901 cells.

Isolation of Fusarium and identification of its toxins from tuberous root of Pseudostellaria heterophylla / 中国中药杂志

Fusarium is the major pathogen of root rot of Pseudostellaria heterophylla. This study aims to explain the possible distribution of Fusarium species and the contamination of its toxin-chemotypes in tuberous root of P. heterophylla. A total of 89 strains of fungi were isolated from the tuberous root of P. heterophylla. Among them, 29 strains were identified as Fusarium by ITS2 sequence, accounting for 32.5%. They were identified as five species of F. avenaceum, F. tricinctum, F. fujikuroi, F. oxysporum, and F. graminearum based on β-Tubulin and EF-1α genes. LC-MS/MS detected 18, 1, and 5 strains able to produce ZEN, DON, and T2, which accounted for 62.1%, 3.4%, and 17.2%, respectively. Strain JK3-3 can produce ZEN, DON, and T2, while strains BH1-4-1, BH6-5, and BH16-2 can produce ZEN and T2. PCR detected six key synthase genes of Tri1, Tri7, Tri8, Tri13, PKS14, and PKS13 in strain JK3-3, which synthesized three toxins of ZEN, DON, and T2. Four key synthase genes of Tri8, Tri13, PKS14, and PKS13 were detected in strains BH1-4-1, BH6-5, and BH16-2, which were responsible for the synthesis of ZEN and T2. The results showed that the key genes of toxin biosynthesis were highly correlated with the toxins produced by Fusarium, and the biosynthesis of toxin was strictly controlled by the genetic information of the strain. This study provides a data basis for the targeted prevention and control of exo-genous mycotoxins in P. heterophylla and a possibility for the development of PCR for rapid detection of toxin contamination.

Cloning and ectopic expression analysis of squalene epoxidase 1 gene from Pseudostellaria heterophylla / 中草药

Objective: To clone the full-length cDNA encoding squalene epoxidase 1 (SQE1), a key enzyme of triterpenes biosynthesis, from Pseudostellaria heterophylla and to perform functional analysis. Methods: With the total RNA as template, the full-length cDNA of SQE1 in P. heterophylla was cloned via RT-PCR and rapid amplification of cDNA ends (RACE) techniques. The bioinformatics of the cloned SQE1 gene was performed. The target gene was transfered into tobacco by Agrobacterium-mediated transformation. Results: The full-length cDNA (2 038 bp) of SQE1 gene was obtained with an open reading frame of 1 554 bp, encoding 517 amino acid polypeptides, which had higher homology with the known SQEs in other medicinal species. The calculated relative molecular mass was 5.67 × 104, the isoelectric point was 8.8. The deduced protein sequence exhibited FAD-binding domains and four transmembrane regions. The content of total triterpenes was increased in transgenic tobacco plants. Conclusion: This is the first report that the full-length cDNA encoding SQE1 from P. heterophylla is cloned. The ectopic expression of SQE1 could promote to increase the content of total triterpenes in transgenic plant. This work provides a foundation for exploring the biosynthetic pathway of triterpenes in P.heterophylla and their applications in bioengineering.

Cloning and expression analysis on full length CDS of zeaxanthin epoxidase gene in Pseudostellaria heterophylla / 中国中药杂志

Zeaxanthin epoxidase plays an important role in indirect pathway of plant abscisic acid biosynthesis. According to the data of Pseudostellaria heterophylla transcriptome, zeaxanthin epoxidase gene was isolated and named as PhZEP. The results of bioinformatics analysis showed that the coding sequence of PhZEP was 1 263 bp long and encoded 420 amino acids. The putative protein molecular weight was 47.34 kDa and its theoretical isoelectric point was 6.64. The characteristic structure domains were predicted, including binding site of lipoprotein and flavoprotein monooxyenase. A signal peptide was discovered at the N-terminal of amino acids. The Real-time PCR revealed that PhZEP had a higher expression level in leaves than other tissues of P.heterophylla. Highly expressed PhZEP was also observed at 10 d and 40 d tuberous root after flowering. PhZEP presented a different expression after treatment with ABA, fluridone and ABA +fluridone compared to the control. The expression of PhZEP in tuberous root after ABA treatment was close to that in control group, while PhZEP showed significant up-regulation in the fluridone treatment group. In this study, the PhZEP gene from P. heterophylla was cloned and this result has important significance for its functional identification. This research provides a basis for the further analysis on functional mechanism of ABA during development of P. heterophylla.

iTRAQ-based quantitative proteomics of cultivated Pseudostellaria heterophylla and its wild type / 药学学报

This work was designed to investigate proteins differentially expressed in cultivated Pseudostellaria heterophylla and its wild type using iTRAQ proteomics approach. The extracted proteins were digested using FASP method and identified by iTRAQ 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; metE 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.

Cloning and sequence analysis of three Actin gene fragments from Pseudostellaria heterophylla / 中草药

Objective: Cloning and sequence analysis of the cDNA fragments encoding Actin gene from Pseudostellaria heterophylla. Methods: Degenerate primers were designed based on the conservated sequences of the cloned Actin gene from other plant species. The core fragments of Actin gene were obtained by reverse transcription polymerase chain reaction (RT-PCR) and the flanking fragments were cloned by using suppression PCR. The expression profiles of Actin gene in different cultivated provenances, organs, and development stages were analyzed by semiquantitative PCR. Results: The three Actin genes obtained were designated PhACT1, PhACT2, and PhACT3, which were extended to 1008, 1008, and 975 bp through suppression PCR, encoding 336, 336, and 325 amino acids, respecifively. The semiquantitative PCR analysis showed that PhACT2 and PhACT3 had a stable expression except PhACT1.Conclusion: Three Actin genes are cloned and the PhACT2 could be used as an internal standard gene for the expression analysis of the functional genes in P. heterophylla.

Research on quality regionalization of cultivated Pseudostellaria heterophylla based on climate factors / 中国中药杂志

Maxent model was applied in the study to filtering the climate factors layer by layer. Polysaccharides and pseudostellarin B the two internal quality evaluation index were combined to analyse the interlinkages between climate factors and chemical constituents in order to search for the critical climate factors of Pseudostellaria heterophylla. Then based on the key climate factors to explicit the quality spatial distribution of P. heterophylla. The results showed that polysaccharides and climatic factors had no significant correlation, suggesting that the indicator was not climate-driven metabolites. Pseudostellarin B could construct regression model with the precipitation. And quality regionalization results showed that pseudostellarin B content presented firstly increased and then decreased trend from southeast to northwest, which was the consistent change with precipitation. It clearly proposed that precipitation was the key climate factor, which affected the accumulation of cyclopeptide compound for Pseudostellariae Radix.

Quality evaluation and growing regionalization of Pseudostellaria heterophylla in Guizhou / 中国中药杂志

This paper is aimed to study the potential ecological suitability regionalization of Pseudostellaria heterophylla for selecting GAP planting base location and designing rational production layout. The ecological factors and contribution rate were determined by using maximum entropy (Maxent) model. Then, the information entropy theory was used to determine the relative importance of each environmental factor, and thus to determine the most limiting habitat criteria. Finally, the probable spatial distribution of P. heterophylla was determined based on GIS spatial analysis of habitat conditions. Meanwhile, the optimal index range of ecological factors was quantified. The moderately and highly suitable habitats were mainly located in Shibing, Huangping, Cengong, the middle and east of Kaili, the south of Yuqing, the west of Tongren. The percentage of moderately and highly suitable habitats for P. heterophylla in the study area was 3.64%, and its area was 6 405.39 km². The results also showed that seven dominant ecological factors controlled the distribution of P. heterophylla. These factors included agrotype, the warmest rain, aspect, slope, the warmest and highest temperature, contents of soil organic carbon, and the driest month precipitation. The habitat suitability assessment model based on GIS and Maxent model theory could accurately evaluate the habitat suitability distribution of P. heterophylla in Guizhou. In addition, we recommended Cengong and Zhenyuan county in Guizhou province as the worthy developing potential planting areas.

Cloning and expression analysis of four carotenoid cleavage dioxygenases genes from Pseudostellaria heterophylla / 中国中药杂志

To investigate the molecular mechanism of quality formation of Pseudostellaria heterophylla, the carotenoid cleavage dioxygenases (CCDs) genes were cloned from the transcriptome database of P. heterophylla, and analyzed them with bioinformatics analysis and expression analysis. The sequence length of four new gene were 1 617, 1 461, 1 746, 1 875 bp, and subsequently, named as PhCCD1,PhNCED2,PhNCED3 and PhCCD4 according to its genetic relationship with Arabidopsis thaliana. The sequence analysis showed that four new gene were all containing REP65 domains and binding sites of ferrous ion, such as histidine, glutamates and aspartates. Analysis phylogeny showed that PhNCED2 and PhNCED3 were the cluster of NCEDs, PhCCD1 and PhCCD4 were the cluster of CCDs. In addition, PhCCD1 and AtCDD1 of Arabidopsis thaliana, PhCCD4 and AtCCD4 of A. thaliana,PhNCED2, PhNCED3 and AtNCED3 of A. thaliana have high similarities. Analysis of real-time fluorescence quantitative showed that PhNCED2 and PhNCED3 were expressed mainly in underground part, the expression quantity of PhNCED2 reached the highest in fibrous root, PhNCED3 keeps higher in phloem and xylem, it may be the key enzymes of ABA biosynthesis genes. Moreover,PhCCD1 and PhCCD4 were expressed mainly in aerial part,the expression quantity of PhCCD1 reached the highest in leaf,PhCCD4 keeps higher in stem and leaf.It may be involved in the biosynthesis of carotenoids for P. heterophylla. The study obtained CDDs gene of P. heterophylla for the first time,this would lay the foundation of developing the response mechanism of P. heterophylla about external stress further,and then exploring the biological approach of quality formation in P. heterophylla.

Identification of Pseudostellaria heterophylla from different idioplasms by analysis of rDNA ITS sequences / 中草药

Objective: To provide the DNA molecular marker for the identification of Pseudostellaria heterophylla from the different idioplasms by analysis of rDNA ITS sequences. Methods: PCR amplification, cloning, and sequencing were carried out using specified primer, and the rDNA ITS base sequences were compared. Results: The ITS mutation extension was 623-624 pb among nine idioplasms of P. heterophylla. Thereinto, the ITS-1 was 224 pb and its G + C content was 52.91%-54.26%, the 5.8S rDNA was 155 bp and its G + C content was 54.49%-55.13%, the ITS-2 was 244-245 bp and its G + C content was 55.55%-56.41%. There were 17 mutation sites (2.72%) in the whole ITS sequences. There were 7, 7, and 3 mutation sites in ITS1, ITS2, and 5.8S, respectively. The different idioplasms had a number of specific single nucleotide mutation sites. Their homologies with each other were upwards 99.9% and their sequence genetic distances were 0.003-0.013. These results showed that the mutation in species from different producing areas and idioplasms was within no more than one species. Conclusion: The mutation of ITS sequences could be used to authenticate P. heterophylla from different idioplasms.

Comparison of the Content of Polysaccharide in the Body and in the Root of Pseudostellaria Heterophylla / 中国中医药信息杂志

Objective To compare the polysaccharide content of pseudostellaria heterophylla which produced in various areas and in different parts of them. Methods The polysaccharide content of pseudostellaria heterophylla was determined by phenol-sulfuric acid method. Results The polysaccharide content of pseudostellaria heterophylla produced in Fujian is the highest, Jiangsu and Guizhou are second-class. The polysaccharide content has some disparity in the bodies and in the roots of pseudostellaria heterophylla. Conclusions The polysaccharide content in the bodies differs 1.96% with that in the roots. This experiment provides theory foundation for scientific preparation of pseudostellaria heterophylla.

Study on Quality Standards of Pseudostellaria heterophylla / 中国药房

OBJECTIVE:To establish the quality standards of Pseudostellaria heterophylla.METHODS:P.cyclopeptides was identified by thin layer chromatography protosite reaction with ninhydrin reagent.The contents of moisture,total ash,acid-insoluble ash,water-soluble extract and alcohol-soluble extract were detected according to the standards stated in Chinese Pharmacopoeia.The content of pseudostellarin B was determined by HPLC.RESULTS:TLC identification of P.cyclopeptides and HPLC determination of pseudostellarin B were established preliminarily.Limitation of moisture content,ash content,extract and pseudostellarin B were defined.CONCLUSION:The qualitative method and quantitative method and physicochemical index can be used as standards for the quality control of P.heterophylla.

Sequence analysis of rDNA ITS of de-virus seedlings of Pseudostellaria heterophylla / 中草药

Objective To study rDNA ITS sequence similarities and differences between the de-virus seedlings and outside seedlings of Pseudostellaria heterophylla. Methods The sequences of ITS region (including ITS1, ITS2, and 5.8S) from nine de-virus seedlings and four outside seedlings were studied by the method of DNA sequence analysis. Results There were the same sequences in 5.8S region between the de-virus seedlings and outside seedlings of P. heterophylla. Variable sites lay in the initial position of ITS1 and termination of ITS2 basically. The de-virus seedlings from Liyang (Jiangsu Province) and Xuancheng (Anhui Province) had more variable sites. Phylogenetic dendrogram based on ITS was constructed by using UPGMA methods, which showed the inherent relation at the level of molecular biology. Conclusion The analysis of rDNA ITS of P. heterophylla has further clarified the inherit stability of de-virus seedlings at the molecular level.