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Objective: The Huanghuai (HH), which is made from the dried roots of Scutellaria baicalensis (Huangqin in Chinese) and the dried flowers and buds of Sophora japonica (Huaihua in Chinese), is a traditional Chinese formula used to treat dysfunctional uterine bleeding (DUB) (Benglou in Chinese) and proven to treat hemostasis effectively in our previous study. Network pharmacology and molecule docking were performed to study the underlying mechanism of Huanghuai (HH), and pharmacodynamic experiments were conducted to verify its curative effect. Methods: TCMSP, UniProt, GeneCards, STRING, DAVID databases, and Cytoscape 3.7.2 were utilized for the construction of a compound-target-pathway network. Docking the potential effective components with potential targets. The HPLC analysis of the potential effective components was performed. In vivo, the hot plate test model was used to study the analgesic activity, the egg white was used to study the swollen reaction in the sole in mice, and the hemostasis effect was studied by the capillary method, tail-breaking method and abortion uterus test. Results: The results showed that six compounds (acacetin, beta-sitosterol, wogonin, baicalein, kaempferol and quercetin) and four potential targets (PTGS2, AKT1, TP53 and TNF) in the compound-target-pathway network were the potential material basis for HH to treat DUB. It can be seen that the binding energy of the acacetin, wogonin, baicalein, beta-sitosterol, kaempferol and quercetin in HH docked with the receptor proteins PTGS2, AKT1, TP53, and TNF were far less than −5.0 kJ/mol, which means the molecules have low conformational energy, stable structure and high binding activity. And the result of HPLC analysis showed that acacetin, wogonin, baicalein, kaempferol and quercetin were the potential effective components of the hemostasis mechanism of HH, beta-sitosterol was removed due to low content. In vivo testing of the potential effective components, it revealed that the group of potential effective components identified by HPLC could increase the pain threshold, inhibit the swelling hind paws of mice induced by egg white, reduce the bleeding time and clotting time, reduce uterine bleeding, decrease the uterine weight, increase the content of Ca and ET-1, and reduce the content of NO in uterine homogenate tissue, and decrease of E
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Objective:To explore the difference in antibacterial mechanism between <italic>Coptis chinensis</italic> and<italic> </italic>its<italic> </italic>flower stalk based on secondary metabolites and network pharmacology. Method:Based on the ultraperformance liquid chromatography mass spectrometry（UPLC-MS/MS） detection platform，the secondary metabolites database of <italic>C. chinensis</italic> and its flower stalk（MWDB） was built. The common database of metabolites information and the multivariate statistical analysis were used to study the differences of secondary metabolites between <italic>C. chinensis</italic> and its flower stalk and screen out 18 metabolites of<italic> </italic>the<italic> </italic>flower stalk and 11 metabolites of <italic>C. chinensis</italic> with a high content. BATMAN-TCM database was used to obtain the targets of component action，and their corresponding genes were inquired in the UniProt database. GeneCards was retrieved for antimicrobial genes，and the intersection genes of components and antimicrobials were obtained on Venny platform. Through DAVID gene ontology（GO） enrichment analysis and Kyoto encyclopedia of genes and genomes（KEGG） pathway enrichment analysis，the mechanism of its action was predicted，and the results were visualized through histogram and advanced bubble diagram drawn by GraphPad Prism software and OmicShare database. The protein-protein interaction（PPI） network was constructed by STRING， database and the component-target-pathway network was constructed by Cytoscape 3.7.2 software. The antibacterial differences were compared based on the results of network pharmacology analysis. Result:Through network pharmacology，the antibacterial active components of <italic>C. chinensis</italic> were 5 fewer than that of the flower stalk，55 more antibacterial targets than that of the flower stalk； quercetin and berberine were predicted to be the common components of the antagonistic action of <italic>C. chinensis </italic>and the flower stalk. Key genes involved in antimicrobial action were p38 Mitogen-activated protein kinase 14（MAPK14），catalase（CAT）； malaria and Toll-like receptor signaling pathway were different key pathways involved in antimicrobial activity. Conclusion:<italic>C. chinensis </italic>and the flower stalk mainly exert the antibacterial effect in a multi-target and multi-pathway manner，which can offer new ideas and clues for the study of antibacterial mechanism of<italic> C. chinensis</italic> and the flower stalk，and provide a new development direction for the comprehensive development and rational application of the flower stalk resources.
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By referring to Chinese Pharmacopoeia 2015 edition and relevant literatures, combining the Technical Requirements for Quality Control and Standard Formulation of Traditional Chinese Medicine Formula Granules (Draft for Comments) issued by China Pharmacopoeia Committee in 2016, and considering the actual production situation of formula granules, the specificity of quality standards, the selection of quantitative detection indexes, index component transfer rate and other aspects in the Publicity of Uniform Standards for Trial Use of Traditional Chinese Medicine Formula Granules were comprehensively discussed. According to the results of the discussion, relevant suggestions are put forward for the development and improvement of the quality standard of traditional Chinese medicine formula granules, which provides reference for promoting the healthy development of formula granule industry.
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Objective:To establish the fingerprint of standard decoction of Citri Reticulatae Pericarpium and evaluate its quality. Method:According to the preparation conditions of the standard decoction,15 batches of standard decoction of Citri Reticulatae Pericarpium were prepared.HPLC was employed to determine the content of hesperidin in this standard decoction.Ultraviolet spectroscopy(UV) and infrared spectroscopy(IR) were used to establish the fingerprint of standard decoction of Citri Reticulatae Pericarpium.The correlation coefficient method and double index sequence analysis method were used to compare and analyze the spectra of different batches of this standard decoction. Result:The content of hesperidin in 15 batches of this standard decoction were 0.82%-2.60%,and the measured value of dry extract rate was 32.02%-46.11%.Compared with ultraviolet and infrared control fingerprint,the fingerprint similarities of the standard decoction of each batch were > 0.897 and > 0.942,respectively.The double index analysis results showed that the common peak ratio was more than 62.50%,variation peak ratio was less than 46.67%. Conclusion:The quality evaluation method established in this study can be used for systematic evaluation of standard decoction of Citri Reticulatae Pericarpium,and it can provide theoretical reference for the formulation of quality standard of Citri Reticulatae Pericarpium dispensing granules and other related preparations.
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Objective To establish the quality evaluation system of Berberidis Radix (BR) standard decoction. Methods According to the preparation requirements of BR standard decoction, 15 batches of BR standard decoction powder was prepared, and, the index components in BR standard decoction was identified by using TLC chromatography. The content of index components was then detected by using HPLC, and the transfer rate of the index composition and the yield of the paste were calculated. Using infrared spectrometer to scan 15 batches of three standard decoctions. The infrared fingerprint spectrum was established, and the correlation between each batch was analyzed by using the two-index sequence analysis method. UV-vis spectrophotometer was used to respectively scan 15 batches of three needle standard medicinal broth for, establishing ultraviolet control map. The similarity between the fingerprint of sample and control was analyzed by SPSS software. Results The yield of 15 batches of standard decoction was 3.42%-6.36%. The content of berberine hydrochloride was 7.53%-13.98%, and the total transfer rate was 39.64%-73.62%. The same colored fluorescent spots were appeared at the corresponding positions of BR standard decoction, the berberine hydrochloride control, and BR control solution and the spots were in the same position of the same silica gel G thin layer. The lower limit of total peak rate of IR fingerprint was 50.0%, and the variation peak rate was 0.0%-62.5%. The similarity of UV fingerprints was all greater than 0.9, and the variation acceptable range was 0.768-1.000. Conclusion A stable and reliable method for the comprehensive quality evaluation of BR standard decoction was established, which can provide reference for the quality control of BR formula granules.
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Objective: The quality standard of Compound Lumbrical Capsule (CLC) was re-evaluated using blood and anti-coagulation intensity as indexes. Methods: Fibrin plate method was used to determine the strength of promoting blood circulation and anti-coagulation for CLC by selecting urokinase and thrombin as reference substances. Results: The results of urokinase concentration and transparent circle area showed a good linear relationship in 200-1 000 U/mL, r = 0.999; The results of thrombin concentration and precipitation circle area showed a good linear relationship in 8-40 U/mL, r = 0.997. The blood anti-coagulant activity of compound earthworm capsule of temporary regulations were not less than 12 840 U/g and 113 822 U/g. Conclusion: The method is rapid, simple, and accurate for determining the activity of promoting blood circulation and anticoagulation for compound lumbrical capsule, thus it can be used for the quality control of CLC.
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Objective: To evaluate the quality of Citri Reticulatae PericarpiumViride(CRPV) through the clinically utilizedquantity of hesperidin. Methods: On the basis of single-factor experiments, extraction technology of CRPV was optimized by solvent volume and extraction time as independent variables, overalldesirability value of extract yield and the extraction transfer rate of hesperidin as evaluation indexes. Centralcomposite design was adopted to design a two factors-five levels table, and a quadratic-multinomial fitting wasapplied. Extraction process was selected by response surface methodology. According to the optimum extractionprocess, the clinically utilized quantity of 11 batches of CRPV was determined basedon the real content and extraction transfer rate. Results: Optimal extraction technology was as follows: extractedfor three times with 24-folds of water, 2.5h each time. The model-predicted and experimentally measured values ofoverall desirability were 0.822 0 and 0.808 8, respectively, revealing a relative error of only 1.61% between them.The clinically utilized quantity and the contents of hesperidin of 11 batches of CRPVwas not positive correlation. Conclusion: The method established in this paper is related to the effectivecomponents of the medicinal materials and the clinical practice, which provides a new way of thinking for thequality evaluation of Chinese medicinal materials.
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Objective: To study the effect of compatibility of Notoginseng Radix et Rhizoma (NRR) with Salviae Miltiorrhizae Radix et Rhizoma (SMRR) on the extraction of SMRR compositions. Methods: The optimum technology conditions of aqueous and alcohol extraction of SMRR were elected by single factor and orthogonal experiments with the contents of cryptotanshinone, tanshinone IIA, rosmarinic acid, salvianolic acid B and solid content as the investigation index. Under the optimum extraction conditions, the dissolution compositions of SMRR were detected as it was compatible with NRR by HPLC, and data were analyzed. Results: The optimum technology conditions of the SMRR's alcohol extraction were 10 times amount of 75% alcohol refluxing and extracting twice, each time 1.5 h, and soaking for 0 h. The optimum technology conditions of the SMRR's aqueous extraction were 8 times amount of water refluxing and extracting 3 times, each time 2 h. NRR had no significant effect on the extraction of tanshinone IIA and salvianolic acid B as principal components from SMRR, and the effect of NRR on other alcohol-soluble components and water-soluble extraction of SMRR also had no significant improvement. Conclusion: NRR had no obvious effect on the dissolution of SMRR compositions.
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The aim of this study was to understand the enterovirus types and biological features of pediatric cases of HFMD in Sanmenxia City during 2011, and compare the latter to a cohort of healthy children. Stool samples of 55 cases of HFMD and 60 healthy children were collected for the isolation and identification of enteroviruses using RNA extraction and real-time RT-PCR assays. EV71 and CA16 were identified by nucleotide sequencing using virus-specific VP1 primers; for the other enteroviruses, 012/011 and 008/013 primers were used for amplification and sequencing. The results were analysed by sequence alignment with known sequences, and the characteristics of the EV71 VP1 gene were also analyzed. The detection rates for enteroviruses in cases of HFMD and healthy children were 52.73% (29/55) and 18.33% (11/60), respectively. Among these, there were 22 cases of EV71, four cases of CA16 and three cases of other enteroviruses in the cases with HFMD. Eleven healthy children had intestinal viruses, of which nine were Coxsackie B virus strains (81.82%, 9/11). Gene sequencing of the 19 EV71 strains illustrated that they were all subgenotype C4a, but the evolutionary tree showed an obvious clustering between cases from Lingbao City and Lushi County. This study demonstrates that the EV71 subgenotype C4a and CA16 strains were the most common cause of HFMD in Sanmenxia City in 2011, and that Coxsackie B strains were prevalent in healthy children. This finding may indicate that there is a widespread source of recessive infection in the community.
Subject(s)
Child , Child, Preschool , Female , Humans , Infant , Male , China , Epidemiology , Cities , Epidemiology , Enterovirus A, Human , Classification , Genetics , Hand, Foot and Mouth Disease , Epidemiology , Virology , Phylogeny , Viral Proteins , GeneticsABSTRACT
Molecular detection of enterovirus (EV)71 RNA based on PCR methods is a quick and sensitive approach. At present, different PCR-based methods for EV71 RNA detection are available, but comparisons of results obtained using different approaches are limited. This study is to compare the analytical sensitivity and specificity of different real-time reverse transcription-polymerase chain reaction (rRT-PCR) and conventional reverse transcription-polymerase chain reaction (cRT-PCR) assays for enterovirus and EV71 detection, Altogether, three rRT-PCR assays and one cRT-PCR assay targeting the 5'UTR gene for universal detection of enterovirus; two rRT-PCR assays andone cRT-PCR assay targeting the VP1 gene for specific detection of EV 71 were examined. All assays showed good specificity. The detection sensitivity ranged from 8.19 x 10 to 8.19 x 10(5) copy equivalents. In general, rRT-PCR assays were more sensitive than cRT-PCR assays. All rRT-PCR assays showed 100% sensitivity for clinical specimens.
Subject(s)
Humans , Enterovirus A, Human , Classification , Genetics , Enterovirus Infections , Diagnosis , Virology , Real-Time Polymerase Chain Reaction , Methods , Reverse Transcriptase Polymerase Chain Reaction , Methods , Sensitivity and SpecificityABSTRACT
<p><b>OBJECTIVE</b>To understand etiological types and distribution features of hand-foot-mouth disease (HFMD) in Henan province between 2008 and 2011.</p><p><b>METHODS</b>A total of 30 486 specimens of feces, rectal swabs or throat swabs from HFMD patients were collected by each Municipal CDC in Henan from 2008 to 2011. The enterovirus 71 (EV71), coxsackie virus A16 (CA16) and other enterovirus (EV) were detected by RT-PCR or real time RT-PCR. The VP1 gene of EV71 was amplified and the sequences were analyzed by bioinformatics software. A genetic evolution tree of the sequence was constructed as well.</p><p><b>RESULTS</b>The positive rates of EV71, CA16 and other EV were 62.70% (11 209/17 876), 12.03% (2150/17 876), 25.27% (4517/17 876) in 17 876 laboratory diagnosed cases, respectively. The differences were statistically significant (χ(2) = 157.17, P < 0.05). The positive rates of EV71, CA16 and other EV were 63.40% (7370/11 624), 11.58% (1346/11 624) and 25.02% (2908/11 624) in male patients and 61.40% (3839/6252), 12.86% (804/6252) and 25.74% (1609/6252) in female patients, respectively. The differences were statistically significant (χ(2) = 4.06, P < 0.05). The children under 5 years old were high-risk population of HFMD, accounting to 97.67% (17 459/17 876) of the laboratory-diagnosed patients.86.92% (15 537/17 876) cases were children between 1 to 3 years old. Constituent ratio of EV71 changed seasonally during a year, there was a high infection ratio of EV71 between April and June, especially in May, the infection ratio reached 69.34% (2384/3438). The positive rates of EV71, CA16 and other EV were 82.48% (5715/6929), 1.76% (122/6929) and 15.76% (1092/6929) among the 6929 laboratory-diagnosed severe cases, respectively. The positive rates of EV71 was higher than CA16 and other EV (χ(2) = 9259.17, 6170.81, P < 0.05, respectively). There were 117 deaths because of severe HFMD, 55 (47.01%) of which were laboratory confirmed. 50 death cases were infected by EV71, and according to the genetic evolution analysis, the VP1 gene of EV71 strain was belonged to subtype C4 of gene C.</p><p><b>CONCLUSION</b>The EV71 and CA16 were the main pathogens which caused HFMD in Henan province, and EV71 virus was the dominant strain, belonging to C4 subtype of gene C.</p>
Subject(s)
Child , Child, Preschool , Female , Humans , Infant , Male , China , Epidemiology , Enterovirus A, Human , Classification , Genetics , Evolution, Molecular , Hand, Foot and Mouth Disease , Epidemiology , Virology , PhylogenyABSTRACT
This report presents an overview of human enterovirus B species in Henan Province. A total of 14 isolates of HEV-B species isolated under HFMD surveillance network during the six months in 2010 were examined. Based on molecular typing results targeting VP1 region, 14 isolates were classified into 6 serotypes of HEV-B. Furthermore, comparison of these 14 isolates with reference strains and strains in mainland China was conducted. The phylogenetic analysis revealed that E25, E11 and E6 showed homology with those from Shandong Province which adjoins Henan Province. E1 and E13 showed homology with those from Yunnan Province, and E30 showed homology with Henan strain isolated in 2008. Cocirculation of two lineages of echovirus 6 was observed.
Subject(s)
Humans , China , Epidemiology , Enterovirus B, Human , Classification , Genetics , Enterovirus Infections , Epidemiology , Virology , Feces , Virology , Molecular Sequence Data , PhylogenyABSTRACT
To reveal the genomic sequence characteristics of coxsackievirus A16 (CoxA16) strain isolated from patients with hand-foot-mouth disease (HFMD) in Henan province. A total of 406 samples were detected by reverse-transcription polymerase chain reaction (RT-PCR) and cell-culture-based isolation of coxsackievirus A16. The whole genome of CoxA16 isolate was amplified using 10 pairs of primers, the sequences were analyzed and phylogenetic tree was generated by bioinformatics software. The full length of HN1162/HN/CHN/2010 genome was 7411bp. Compared with the other CoxA16 strains released in GenBank, the nucleotide similarities were 87.0-97.9%, 77.0%-95.4%, 80.3%-96.9%, 77.9% 96.2%, 80.5-100% in 5'UTR, P1, P2, P3, 3'UTR region, respectively; The similarities of nucleotide and amino acid sequences in VP1 region were 91.4%-96.4% and 99.3%-99.7%, respectively. Phylogenetic tree analysis showed that CoxA16 strains isolated from Henan, Shenzhen, Guangzhou and Fujian belonged to the same cluster. The newly isolated CoxA16 from Henan province belonged to subgenotype C2/B-2. These results will have great significance in monitoring CoxA16 and for prevention and control of hand-foot-mouth disease.
Subject(s)
Child , Child, Preschool , Female , Humans , Infant , Male , China , Epidemiology , Enterovirus A, Human , Classification , Genetics , Genomics , Hand, Foot and Mouth Disease , Epidemiology , Virology , Molecular Sequence Data , PhylogenyABSTRACT
To reveal the genetic features and recombination of enterovirus 71 isolates between 2008 and 2010. A total of 5 enterovirus 71 isolates were sequenced completely and phylogenetic analysis and recombination were performed. Phylogenetic analysis based on VP1 regions revealed that the Henan enterovirus 71 between 2008 and 2010 belonged to C4a in subgenotype C4. Bootscan analyses and phylogenetic analysis based on the 5'UTR, P1, P2, P3 genomic regions revealed the recombinations between EV71 genotypes B and C at the 2A-2B junction, and between EV71 genotype B and CA16 strain G-10 at the 3B-3C junction. Henan enterovirus 71 isolates between 2008 and 2010 belonged to C4a in subgenotype C4 which was the predominant virus genotype circulating in mainland China since 2004, a combination of intratypic and intertypic recombination were found in EV71 subgenotype C4.