RESUMO
ObjectiveBased on ultra performance liquid chromatography-quadrupole-time-of-flight tandem mass spectrometry（UPLC-Q-TOF-MS）， to evaluate the establishment of a mouse model of liver Yin deficiency by thyroid tablet suspension combined with 10% carbon tetrachloride（CCl4） from the perspective of non-targeted metabolomics， in order to lay the foundation for the establishment of a traditional Chinese medicine（TCM） syndrome model. MethodA total of 24 mice were randomly divided into blank group and model group. The model group was given thyroid tablet suspension（0.003 2 g·kg-1） by gavage for 14 consecutive days， and 10% CCl4（5 mL·kg-1） was intraperitoneally injected once a week to establish a liver Yin deficiency model， while the blank group was injected with an equal amount of olive oil intraperitoneally and gavaged with an equal amount of distilled water， and was fed with normal feed. After the modeling was completed， 6 mice in each group were randomly selected， the levels of alanine aminotransferase（ALT）， aspartate aminotransferase（AST）， cyclic adenosine monophosphate（cAMP）， cyclic guanosine monophosphate（cGMP）， interleukin（IL）-6， IL-10， tumor necrosis factor-α（TNF-α）were measured in the mice serum， and malondialdehyde（MDA）， superoxide dismutase（SOD）， total protein（TP）， hydroxyproline（HYP） and other indicators were measured in the mice liver. Liver tissue sections were taken for hematoxylin-eosin（HE） staining and observing pathological changes. The remaining 6 mice in each group were subjected to UPLC-Q-TOF-MS combined with principal component analysis（PCA） and orthogonal partial least squares-discriminant analysis（OPLS-DA） were used to screen differential metabolites in the liver Yin deficiency mouse model， Kyoto Encyclopedia of Genes and Genomes（KEGG） database was used to analyze the corresponding metabolic pathways of differential metabolites. ResultCompared with the blank group， mice in the model group showed liver Yin deficiency manifestations such as reduced body weight， fatigue and sleepiness， disheveled and lusterless hair， irritability. The levels of ALT， cAMP/cGMP， IL-6， AST， MDA， cAMP， TNF-α significantly increased（P<0.05， P<0.01）， while the levels of SOD， IL-10 and cGMP significantly decreased（P<0.05， P<0.01）， and the changes of HYP and TP were not statistically significant. Hepatic steatosis and distortion of the radial arrangement of the liver plate cells were seen in the section images of the model group， endogenous substances were clearly separated， and 252 differential metabolites were identified in the serum samples， which were mainly involved in the metabolic pathways of purine metabolism， steroid hormone biosynthesis and pyrimidine metabolism. A total of 229 differential metabolites were identified in the liver samples， mainly involving nucleotide metabolism， purine metabolism， steroid hormone biosynthesis， pyrimidine metabolism， antifolate resistance， insulin resistance， primary bile acid biosynthesis， prostate cancer， sulfur relay system， arachidonic acid metabolism and other metabolic pathways. ConclusionThe successful establishment of liver Yin deficiency model in mice by CCl4 combined with thyroid hormone is evaluated through the investigation of serum and liver metabolomics， combined with biochemical indicators， which provides a biological basis and experimental foundation for the Yin deficiency syndrome model of TCM.
RESUMO
ObjectiveTo establish a qualitative and quantitative analysis method for chemical constituents in Liu Junzitang（LJZT）， and to clarify its material basis. MethodThe chemical constituents in LJZT were analyzed by ultra performance liquid chromatography-quadrupole-time-of-flight mass spectrometry（UPLC-Q-TOF-MS/MS）， and the resulting compounds were identified by using databases， such as MassBank， PubChem， ChemSpider， Traditional Chinese Medicine Systems Pharmacology Database and Analytical Platform（TCMSP）， and by combining with relevant literature. UPLC was used to establish a quantitative method for analysis of 9 compounds in LJZT， including liquiritin， hesperidin， lobetyolin， liquiritigenin， glycyrrhizic acid， nobiletin， tangeretin， atractylenolide Ⅱ and Ⅰ. ResultBy combining the relevant literature， database and MS information， a total of 79 compounds were identified from LJZT， including 31 flavonoids， 15 terpenoids， 14 nitrogen-containing compounds， 6 phenylpropanoids， 6 organic acids and 7 other compounds. The established quantitative analytical method for the nine representative components showed good linearity within their respective linear ranges， and the precision， stability， reproducibility and recovery were in accordance with the requirements. The quantitative results showed that the contents of liquiritin， hesperidin， lobetyolin， liquiritigenin， glycyrrhizic acid， nobiletin， tangeretin， atractylenolide Ⅱ and Ⅰ in LJZT were 0.376 5， 2.602 1， 0.082 6， 0.128 1， 1.778 6， 0.015 7， 0.006 7， 0.030 4， 0.003 2 mg·g-1， respectively. ConclusionThe established method can quickly， sensitively and accurately analyze the chemical constituents in LJZT， clarify that the material basis of LJZT is mainly flavonoids， terpenoids and nitrogen-containing compounds， and simultaneously determine the contents of the 9 components， which can lay a foundation for the research on quality control， mechanism and clinical application of LJZT.
RESUMO
ObjectiveUltra-performance liquid chromatography-quadrupole-time-of-flight mass spectrometry（UPLC-Q-TOF-MS） was used to identify the metabolites of harmine in rats， in order to explore the differences in distribution of metabolites in rats after single dose（40 mg·kg-1） intragastric administration of harmine， as well to speculate the metabolic pathways. MethodSD rats were given a single dose of harmine by intragastric administration. Plasma， bile， urine and feces samples were collected after administration， and the samples were processed for determination by UPLC-Q-TOF-MS. The separation was performed on an ACQUITY UPLC™ HSS T3 columu（2.1 mm×100 mm， 1.8 μm） with acetonitrile（A）-0.1% formic acid aqueous solution（B） as mobile phase for gradient elution（0-2 min， 5%A； 2-9 min， 5%-35%A； 9-9.5 min， 35%-100%A； 9.5-12 min， 100%A； 12-12.5 min， 100%-5%A； 12.5-14 min， 5%A）， the mass spectra were obtained in positive ion mode with electrospray ionization（ESI）， the scanning range was m/z 50-1 200. The metabolites of harmine were identified based on the information of the obtained compounds and the literature data， and the metabolic pathways were hypothesized. ResultA total of 42 compounds（harmine and its metabolites） were identified in rats， including 27 in plasma， 17 in bile， 26 in urine and 13 in feces. The metabolic pathways involved in these 42 metabolites included monohydroxylation， dihydroxylation， demethylation， glucuronidation and sulfation. ConclusionHarmine can undergo phase Ⅰ and phase Ⅱ metabolic reactions in rats， and the prototype drug is metabolized rapidly in vivo， and the metabolites are mainly excreted by the kidneys， which can provide a reference basis for the pharmacodynamics and material basis of harmine.
RESUMO
A new method for simultaneous determination of 23 kinds of per-and polyfluoroalkyl substances(PFASs)(13 kinds of perfluoro carboxylic acids,4 kinds of perfluoro sulfonic acids,and 6 kinds of new substitutes)in plant leaf tissue by ultra-high performance liquid chromatography-tandem mass spectrometry(UHPLC-MS/MS)using automatic online solid phase extraction(SPE)to remove the matrix interference components in plant crude extracts was developed.The plant leaf samples were extracted twice with 1%formic acid-methanol solution,then evaporated to dry,redissolved with 70%methanol solution,and directly injected for analysis.After 23 kinds of target PFASs were purified automatically by online SPE with a WAX column,the six-way valve was switched to rinse PFASs onto an alkaline mobile phase system-compatible C18 analytical column.Then,the 23 kinds of target PFASs were separated within 16 min by gradient elution using a binary mobile phase system of methanol/water(Containing 0.4%ammonium hydroxide).Tandem mass spectrometry was performed in multiple reaction monitoring(MRM)mode for online detection of various PFASs,and quantification was carried out by internal standard method.The results of the method validation showed that satisfactory average recoveries of 23 kinds of PFASs in plant leaf samples(64.2%-125.5%),precision(relative standard deviations(RSDs)of 0.7%-12.8%),linearity(R2＞0.990),and sensitivity(the detection limits(S/N=3)were in the range of 0.02-0.50 μg/kg)were achieved.Finally,this method was used to detect PFASs in the marine green tide algae(Enteromorpha prolifera)and several tree leaves,and a total of 6 kinds of PFASs were detected,in which PFBA was the main contaminant.Compared with the reported offline SPE methods,the proposed online SPE technique significantly simplified the sample pretreatment process and provided an automatic,simple,and environment-friendly method for the routine monitoring of legacy and emerging PFASs in plant tissues.
RESUMO
BACKGROUND:Traumatic spinal cord injury primarily relies on scale assessment and imaging examinations in clinical practice.However,there are limitations in predicting the prognosis of the injury.Therefore,the use of metabolomics technology for biomarker screening is significant for estimating the extent of damage,injury and recovery,as well as developing new therapies. OBJECTIVE:To characterize the metabolic features of patients with traumatic spinal cord injury using metabolomics technology and explore potential biomarkers and disrupted metabolic pathways. METHODS:Serum and urine samples were collected from 20 patients with traumatic spinal cord injury(observation group)and 10 healthy subjects(control group).Metabolites were analyzed and multivariate statistical analysis was then performed for data processing to screen differential metabolites.Metabolic pathway enrichment was performed using MetaboAnalyst software.Logistic regression was applied to construct a biomarker combination model,and its relationship with the American Spinal Injury Association grading was analyzed. RESULTS AND CONCLUSION:Significant differences in 160 and 73 metabolites were detected in the serum and urine samples of the two groups,respectively.Pathway enrichment analysis showed evident disturbances in lipid metabolism after traumatic spinal cord injury,including sphingolipid,arachidonic acid,α-linolenic acid,and arachidonic acid metabolism,as well as glycerophospholipid and inositol phosphate biosynthesis.The combination of two identified biomarkers,telmisartan and quercetin glycoside,showed a correlation with the American Spinal Injury Association grading in both serum and urine levels.Thus,metabolomics technology provides assistance in further understanding the pathological mechanisms of traumatic spinal cord injury and screening therapeutic targets.The identified metabolic biomarker combination may serve as a reference for assessing the severity of traumatic spinal cord injury.
RESUMO
ObjectiveTo investigate the potential active ingredients and targets of Baihu Jia Renshentang（BHJRST） for the treatment of obesity combined with type 2 diabetes mellitus（T2DM） by network pharmacology and in vivo experiments. MethodUltra performance liquid chromatography-quadrupole/electrostatic field orbitrap high-resolution mass spectrometry（UPLC-Q-Exactive Orbitrap MS） was used to analyze and identify the material basis of BHJRST. Subsequently， potential targets for the action of the active ingredients were queried in databases such as ChEMBL， Therapeutic Target Database（TTD）， YaTCM， DisGeNET and Traditional Chinese Medicine on Immuno-Oncology（TCMIO）， and the shared targets were identified by taking the intersection of these targets with disease targets. The shared targets were imported into the STRING database to construct a protein-protein interaction（PPI） network， the hub genes were identified by cytoHubba plug-in， and molecular docking was used to validate the binding energy of the hub genes to the bioactive ingredients in BHJRST. Meanwhile， the shared targets were imported into the DAVID platform for gene ontology（GO） functional annotation and Kyoto Encyclopedia of Genes and Genomes（KEGG） pathway enrichment analysis. The predicted results were subsequently verified by animal experiments. Eighteen 8-week-old male skeletal muscle insulin-like growth factor-1 receptor dysfunction（MKR） mice were induced by a high-fat diet for 12 weeks in order to prepare a mouse model of obesity combined with T2DM. The mice were randomly divided into the model group， metformin group（0.2 g·kg-1） and BHJRST group（27 g·kg-1 in raw material）， and another 6 male FVB mice of the same age as the normal group. The mice in each group were were given the corresponding drugs by gavage， and the normal and model groups were given the same amount of distilled water by gavage， 1 time/d for 6 consecutive weeks. At the end of administration， the body mass， Lee's index， fasting blood glucose（FBG）， oral glucose tolerance test（OGTT） of mice in each group were examined， and the pathological morphology of the white adipose tissue of the epididymis was observed， and the expression of the mRNA of the hub genes in the white adipose tissue of the epididymis was detected by real-time fluorescence quantitative polymerase chain reaction（Real-time PCR）. ResultA total of 200 bioactive components of BHJRST were identified， of which 64 bioactive components were reverse-matched to 384 targets， and a total of 308 targets were associated with obesity combined with T2DM. Hub genes included mitogen-activated protein kinase 1（MAPK1）， signal transducer and activator of transcription 3（STAT3）， MAPK3， interleukin（IL）-2， Janus kinase 1（JAK1）， nuclear transcription factor-κB p65（RELA）， estrogen receptor 1（ESR1）， transcription factor AP-1（JUN）， MAPK14 and lymphocyte-specific protein tyrosine kinase（LCK）. GO functional annotation showed that it was mainly enriched in cytoplasm， cell membrane and nucleus， and was closely related to important biological processes such as peptide serine phosphorylation， protein phosphorylation and inflammation. In KEGG enrichment analysis， metabolic pathway， lipid and atherosclerosis， phosphatidylinositol 3-kinase（PI3K）/protein kinase B（Akt） and MAPK signal pathways were significantly enriched. The molecular docking results showed that the hub genes had a stable binding relationship with 10 bioactive components， including quercetin， isoliquiritigenin， and morin， in BHJRST. The results of animal experiments showed that BHJRST could significantly reduce body mass， Lee's index and FBG levels（P<0.01） in mice with obesity combined with T2DM， improve the pathological changes of white adipose tissue， and down-regulate the the mRNA expression of the hub genes in white adipose tissue of the epididymis（P<0.01）. ConclusionIn this study， 10 potentially active components such as quercetin， isoliquiritigenin， and morin in BHJRST are identified through network pharmacology and animal experiments， and it is possible to treat obesity combined with T2DM by regulating lipid and atherosclerosis， phosphatidylinositol PI3K/Akt and MAPK signal pathways， which provides important clues and theoretical basis for the study of its mechanism and clinical application.
RESUMO
ObjectiveTo investigate the influence of concentration ratio（CR） between the internal reference and target components on the quantitative accuracy of quantitative analysis of multi-components by single marker（QAMS） by taking ginsenosides as an example. MethodUltra performance liquid chromatography（UPLC） was employed， the contents of nine components in Ginseng Radix et Rhizoma（ginsenosides Rg1， Re， Rf， Rh1， Rb1， Rc， Rb2， Rb3， Rd） were determined by external standard method（ES）. Using ginsenoside Rg1 as the internal reference， the contents of the remaining 8 ginsenosides were determined by QAMS， and the quantitative results were compared with those of ES to evaluate the quantitative accuracy of the established QAMS. According to the contents of these 9 ginsenosides， the simulated sample solutions with different CRs of ginsenoside Rg1 to ginsenosides Rf， Rb2， Rd were prepared with the reference substance（CR=100∶1， 10∶1， 5∶1， 2∶1， 1∶1， 0.5∶1， 0.25∶1）， in order to validate the effect of the CRs between the internal reference and other components on the quantitative accuracy of the QAMS. ResultThe results of ginsenosides Re， Rf， Rb1， Rc， Rb2 calculated by the two methods were the same with the relative standard deviation（RSD）<3%， however， the results of ginsenosides Rh1， Rb3 and Rd calculated by the two methods were different with RSDs of 7.06%-9.61%. According to the result of the simulated sample solution， the difference between the calculated results of ES and QAMS was large when the CR between the internal reference（ginsenoside Rg1） and other components was 5 or 10 or even higher. ConclusionThe quantitative error of QAMS will increase when the CR between the quantitative component and the internal reference is too large， so it is suggested that when establishing the QAMS， the components with close concentration to the internal reference should be selected for quantification.
RESUMO
ObjectiveTo explore the potential mechanism of different processed products of Baiyaojian and its compound Xiangmei pills in rats with ulcerative colitis（UC） by comparing the pharmacodynamic and metabolomic differences. MethodEighty SD rats were acclimatized and kept for 3 d， and randomly divided into 8 groups［blank group， model group， mesalazine group（0.4 g·kg-1）， Baiyaojian group（1.89 g·kg-1）， stir-fried Baiyaojian group（1.89 g·kg-1）， carbonized Baiyaojian group（1.89 g·kg-1）， and Xiangmei pills low and high dose groups（1.89， 5.67 g·kg-1）］， with 10 rats in each group. Rats in the blank group were administered physiological saline by gavage， and rats in the remaining 7 groups were orally administered 5% dextran sodium sulfate（DSS） solution daily for 8 consecutive days to induce UC model. After successful modeling， each dosing group was given the corresponding dose of drug solution by gavage， and the blank and model groups were given equal amounts of saline by gavage， and the drug was administered continuously for 8 d. Then serum levels of tumor necrosis factor-α（TNF-α）， interleukin（IL）-6， IL-10 and IL-1β were measured by enzyme-linked immunosorbent assay（ELISA）， hematoxylin-eosin（HE） staining was used to observe the histopathological changes of colon tissue， the proportion of T helper 17 cells（Th17） and regulatory T cells（Treg） in the peripheral blood of rats in each group was detected by flow cytometry. The endogenous metabolites in serum of rats were detected by ultra performance liquid chromatography-quadrupole/electrostatic field orbitrap high-resolution mass spectrometry（UPLC-Q-Exactive Orbitrap MS）， and the differential metabolites were characterized by combining principal component analysis（PCA） and orthogonal partial least squares-discriminant analysis（OPLS-DA）， and were analyzed according to the variable importance in the projection（VIP） value>1.0 and P<0.05， and potential metabolic pathways were analyzed according to Human Metabolome Database（HMDB）. ResultCompared with the blank group， the colon tissue of the model group was congested and the mucosa was ulcerated， the colon length was significantly reduced（P<0.01） and the quality was significantly increased（P<0.05）， the proportion of Th17/Treg in the peripheral blood and the serum levels of TNF-α， IL-6 and IL-1β were significantly increased， while the IL-10 expression wassignificantly reduced（P<0.05， P<0.01）. Compared with the model group， the colon tissue of UC rats in each treatment group was improved with scattered ulcers， reduced inflammatory cell infiltration， significantly increased colon length， and significantly decreased mass（P<0.05）， the proportion of Th17/Treg in the peripheral blood decreased， the expression of TNF-α，IL-6 and IL-1β was significantly reduced（P<0.05， P<0.01）， while the IL-10 expression was significantly increased（P<0.01）. The therapeutic effect of different administration groups on UC was in the order of high dose group of Xiangmei pills>low dose group of Xiangmei pills>carbonized Baiyaojian group>stir-fried Baiyaojian group>Baiyaojian group. And a total of 26 differential metabolites were screened in the metabolomics results. Compared with the blank group， 14 differential metabolites were up-regulated and 5 metabolites were down-regulated in the model group， and 14， 9， 14， 12 and 17 metabolites could be recalled in the Baiyaojian group， stir-fried Baiyaojian group， carbonized Baiyaojian group， Xiangmei pills low and high dose groups. The main metabolic pathways involved were citrate cycle pathway， pantothenic acid and coenzyme A biosynthesis pathway， aromatic hydrocarbon receptor（AhR） signaling pathway， glycolysis/gluconeogenesis pathway. ConclusionThe therapeutic effect of Baiyaojian on UC is significantly improved after charcoal stir-frying， and the efficacy is more prominent when combined with Angelicae Dahuricae Radix and Mume Fructus Carbonisata， which can provide a basis for the development of Baiyaojian compound preparations.
RESUMO
Backgroud At present, there is no unified standard for the detection of glufosinate ammonium and three metabolites in urine, which affects the accurate assessment of occupational exposure risk to a certain extent. It is of great significance to establish a rapid and effective inspection method to ensure occupational safety and public health. Objective To establish an ultra performance liquid chromatography-tandem mass spectrometry for simultaneous determination of glufosinate ammonium and three metabolites in urine. Methods The effects of dilution solvents and dilution ratios on the response values of glufosinate ammonium and three metabolites were compared, and the retention capacities of solid phase extraction columns for targets as well as the effects of chromatographic columns and mobile phase systems on chromatographic peaks were analyzed. Samples were quantified by matrix effect matching external standard method. Accuracy of the method was evaluated by recovery rate of standard addition, and precision of the method was evaluated by relative standard deviation of intra-day and inter-day measurements. Urine samples of 30 health individuals were collected to evaluate the application of the method. Results The urine samples were diluted with 0.2 mL water and 0.6 mL acetonitrile, purified by HLB solid phase extraction columns, and separated by Dikma Polyamino HILIC columns, and gradient elution was carried out with 0.5 mmol·L−1 ammonium acetate and 0.1% ammonia water as mobile phase, which achieved a good peak shape and mass spectrum response. The linearities of the four target compounds were good in the range of 0.5-50 ng·mL−1, and the correlation coefficients (r) were all greater than 0.998. The detection limits were 0.56-2.86 μg·L−1, the quantification limits were 1.87-29.54 μg·L−1, and the recovery rates of standard addition ranged from 75.0% to 103.6%, The relative standard deviations of intra-batch and inter-batch were from 2.5% to 8.1% and from 4.3% to 9.3% respectively. The method was applied to detect 30 urine samples of subjects, and no target was detected. Conclusion The method is simple, rapid, sensitive, and accurate. It is suitable for the determination of glufosinate ammonium and its metabolites in human urine without derivatization.
RESUMO
ObjectiveBased on ultra performance liquid chromatography-mass spectrometry（UPLC-MS） and non-targeted metabolomics technology to discuss the central regulatory effect of Chaishao Liujuntang on chronic atrophic gastritis（CAG） rats with liver-depression and spleen-deficiency， and to look for the correlation between cerebral cortex， hypothalamus and metabolic status of gastric tissues. MethodA CAG rat model with liver-depression and spleen-deficiency was established by chemical induction， hunger and satiety disorders， chronic restraint and tail clamping stimulation， lasting for 16 weeks. Twenty-eight Wistar rats were randomly divided into a blank group of 8 rats and a model group of 20 rats. After the completion of modeling， 4 rats in the model group were taken to observe the pathological changes of gastric mucosa. The remaining model rats were randomly divided into a model group of 8 rats and a Chaishao Liujuntang group of 8 rats. Chaishao Liujuntang group rats were given 5.1 g·kg-1 by gavage， and the remaining rats were given equal volume sterilized water by gavage for 4 weeks. Macroscopic characteristics， behavioral indicators and histopathological changes of the gastric mucosa of rats in each group were observed and compared. UPLC-MS non-targeted metabolomics was used to explore the metabolic regulation effect of Chaishao Liujuntang on the cerebral cortex， hypothalamus and stomach tissues of CAG rats with liver-depression and spleen-deficiency. Pearson correlation coefficient method was used to analyze the correlation between different tissue metabolites. ResultCompared with the model group， the macroscopic characteristics of rats in Chaishao Liujuntang group were improved， such as hair color， mental state and stool properties， and the number of times of crossing and standing in the open field experiment was significantly increased， and the static time of forced swimming was significantly reduced（P<0.01）， and the gastric mucosa atrophy was reduced. The metabolic data from the cerebral cortex of rats in each group identified a total of 3 common potential biomarkers， but not enriched in pathways， 26 common potential biomarkers were identified in the hypothalamus， and the key metabolic pathways involved were mainly enriched in purine metabolism， glycerol phospholipid metabolism， D-glutamine and D-glutamic acid metabolism. Seventeen common potential biomarkers were identified in the stomach， and the key metabolic pathways involved were mainly enriched in thiamine metabolism， valine， leucine and isoleucine biosynthesis， and taurine and taurine metabolism. Correlation analysis of metabolites in different tissues revealed that multiple amino acids and their derivatives mediated metabolic connections between the cerebral cortex， hypothalamus and stomach of rats. ConclusionThe metabolic disorders in the cerebral cortex， hypothalamus and stomach of CAG rats with liver-depression and spleen-deficiency have their own characteristics， mainly manifested by changes in the content of glycerol phospholipids， fatty acids and bile acid metabolites. Moreover， Chaishao Liujuntang may play a central regulatory role in CAG rats with liver-depression and spleen-deficiency by correcting the metabolic disorders of amino acids.
RESUMO
ObjectiveTo identify the prototypical components and metabolites absorbed into blood and cerebrospinal fluid of Schisandrae Chinensis Fructus（SCF） based on sequential metabolism combined with liquid chromatography-mass spectrometry. MethodBlood and cerebrospinal fluid samples of integrated metabolism， intestinal metabolism and hepatic metabolism were collected from male SD rats after gavage and in situ intestinal perfusion administration， and ultra-performance liquid chromatography-quadrupole/electrostatic field orbitrap high-resolution mass spectrometry（UPLC Q-Exactive Orbitrap MS） was used to analyze and compare the differences in the spectra of SCF extract， blank plasma， administered plasma， blank cerebrospinal fluid and administered cerebrospinal fluid with ACQUITY UPLC BEH Shield RP18 column（2.1 mm×100 mm， 1.7 µm）， the mobile phase was acetonitrile（A）-0.1% formic acid aqueous solution（B） for gradient elution（0-7 min， 95%B； 7-12 min， 95%-35%B； 12-17 min， 35%-15%B； 17-20 min， 15%-12%B； 20-22 min， 12%-5%B； 22-23 min， 5%B； 23-25 min， 5%-95%B； 25-28 min， 95%B）. And heated electrospray ionization（HESI） was used with positive and negative ion modes， the scanning range was m/z 100-1 500. The prototypical constituents and their metabolites absorbed into blood and cerebrospinal fluid of SCF were identified according to the retention time， characteristic fragments， molecular formulae and the information of reference substances. ResultA total of 42 chemical components were identified in the extract of SCF， including lignans， flavonoids， amino acids， tannins， and others， of which lignans were the main ones. A total of 27 prototypical components and 14 metabolites were identified in plasma samples from different sites. A total of 15 prototypical components and 9 metabolites were identified in cerebrospinal fluid. The main metabolic reactions involved in the formation of metabolites were mainly demethylation， methylation， demethoxylation and hydroxylation. ConclusionThrough the systematic identification of the prototypical components and metabolites of SCF in rats， it provides data support for further better exploring the material basis of SCF in the treatment of central nervous system diseases.
RESUMO
ObjectiveTo analyze the antidepressant quality markers（Q-Marker） of Bupleuri Radix（BP） before and after vinegar-processing by ultra-performance liquid chromatography-quadrupole-time-of-flight mass spectrometry（UPLC-Q-TOF-MS）， multivariate statistical analysis and network pharmacology. MethodUPLC-Q-TOF-MS was used to analyze the chemical basis of raw and vinegar-processed products of BP， and principal component analysis（PCA） orthogonal partial least squares-discriminant analysis（OPLS-DA） were used to identify the differential components in BP that changed significantly before and after vinegar-processing， which were regarded as candidate quality markers（Q-Marker）. Then the disease-drug-component-target network related to antidepressant effect of BP was constructed by network pharmacology， and the antidepressant Q-Marker of raw and vinegar-processed products of BP was determined. Rats were randomly divided into blank group， model group， fluoxetine group（2.67 mg·kg-1） and total saponin group（0.72 mg·kg-1）， except the blank group， rats in the other groups were subjected to chronic unpredictable mild stress（CUMS）. Three weeks after the start of modeling， rats in each administration group were given the corresponding dose of drugs once a day for 4 weeks， and rats in the blank and model groups were given normal saline with dose of 10 mL·kg-1. At 1 day before modeling， 21 days and 28 days after administration， body mass weighing， sucrose preference test and open field test were performed on each group . After 28 days of administration， real-time fluorescence quantitative polymerase chain reaction（Real-time PCR） was used to detect the mRNA expression levels of phosphatidylinositol 3-kinase（PI3K）， protein kinase B（Akt）， mammalian target of rapamycin（mTOR）， glycogen synthase kinase-3β（GSK-3β）， forkhead box transcription factor O3a（FoxO3a） and β-catenin in hippocampal tissues of rats in each group， while protein expression levels of PI3K， Akt， mTOR and FoxO3a in hippocampal tissues of rats in each group were detected by Western blot. ResultThere were 19 components in BP showed significant changes before and after vinegar-processing， and 9 components such as saikosaponin A， saikosaponin B1， saikosaponin B2， saikosaponin C and saikosaponin D were identified as potential Q-Marker through S-plot differential marker screening. Combined with the disease-drug-component-target network， saikosaponin A， saikosaponin B1， saikosaponin B2 and saikosaponin D were identified as antidepressant Q-Marker of raw and vinegar-processed products of BP. According to the results of pharmacodynamic tests， after 28 d of administration， compared with the blank group， the body mass， sucrose preference index and open field total score of rats in model group， fluoxetine group and total saponin group decreased significantly（P<0.01）. Compared with the model group， the body mass， sucrose preference index and open field total score in total saponin group increased significantly（P<0.01）. Compared with the blank group， mRNA expression levels of PI3K， Akt， mTOR and β-catenin in hippocampus of rats in the model group decreased significantly（P<0.05）， while mRNA expression levels of GSK-3β and FoxO3a increased significantly（P<0.05）. Compared with the model group， mRNA expression levels of PI3K， Akt， mTOR and β-catenin in hippocampus of rats in the total saponin group were increased significantly（P<0.05）， while mRNA expression levels of GSK-3β and FoxO3a decreased significantly（P<0.05）. Compared with the blank group， the protein expression levels of Akt and mTOR in hippocampus of the model group decreased significantly（P<0.01）， while the protein expression levels of PI3K and FoxO3a increased significantly（P<0.01）. Compared with the model group， the expression level of Akt in hippocampus of the total saponin group increased significantly（P<0.01）， the mTOR expression level was increased but not statistically significant， while the protein expression levels of PI3K and FoxO3a decreased significantly（P<0.01）. ConclusionThe chemical constituents of BP changed greatly after vinegar-processing， and the antidepressant Q-Marker of raw and vinegar-processed products of BP was determined by chemical basis， pharmacodynamics， network pharmacology and signaling pathway， which provided a reference for further research on quality control， pharmacodynamic substance basis and processing mechanism of BP.
RESUMO
ObjectiveMetabolomics was used to reveal the mechanism of Aconiti Lateralis Radix Praeparata（ALRP） in attenuating toxicity by processing from the aspects of amino acid metabolism， oxidative stress and energy metabolism by analyzing multiple metabolic pathways. MethodTwenty-four rats were randomly divided into control group， raw group and processed group， 8 rats in each group. The raw and processed group were given with 0.64 g·kg-1 of raw ALRP and processed ALRP respectively every day， the control group was given with an equal amount of normal saline once a day. After continuous administration for 7 days， the urine， serum and heart tissue of rats were collected. Pathological examination of the heart was carried out using hematoxylin-eosin（HE） staining， and the activities of lactate dehydrogenase（LDH） and creatine kinase-MB（CK-MB） in serum and cardiac tissues were detected by microplate assay and immunoinhibition assay. The effects of ALRP on rat heart before and after processing were compared and analyzed. Ultra performance liquid chromatography-quadrupole-time-of-flight mass spectrometry（UPLC-Q-TOF-MS） was used to perform urine metabolomics analysis， and multivariate statistical analysis was used to screen for differential metabolites related to ALRP in attenuating toxicity by processing， and pathway enrichment analysis was carried out to explore the processing mechanism. ResultHE staining showed that no obvious pathological changes were observed in the heart tissue of the control group， while obvious infiltration of inflammatory cells such as plasma cells and granulocytes was observed in the heart tissue of the raw group， indicating that the raw ALRP had strong cardiotoxicity. There was no significant difference in HE staining of heart tissue between the processed group and the control group， indicating that the toxicity of ALRP was significantly reduced after processing. Compared with the control group， the activities of LDH and CK-MB were significantly increased in serum and heart tissue of the raw group， and those were significantly decreased in serum and heart tissue of the processed group， suggesting that the myocardial toxicity of processed ALRP was reduced. A total of 108 endogenous differential metabolites associated with the raw ALRP were screened using multivariate statistical analysis in positive and negative modes， of which 51 differential metabolites were back-regulated by the processed ALRP. Biological analysis of the key regulatory pathways and associated network changes showed that the pathways related to toxicity of ALRP mainly included tryptophan metabolism， arginine and proline metabolism， phenylalanine metabolism， aminoacyl-tRNA biosynthesis， alanine， aspartate and glutamate metabolism， etc. The metabolic pathways related to the attenuation of processed ALRP mainly included aminoacyl-tRNA biosynthesis， tryptophan metabolism， phenylalanine， tyrosine and tryptophan biosynthesis， phenylalanine metabolism and caffeine metabolism. ConclusionThe processing technology of ALRP in Guilingji can significantly attenuate the cardiotoxicity of raw products， the mechanism mainly involves amino acid metabolism， oxidative stress and energy metabolism， which can provide experimental bases for the research related to the mechanism of toxicity reduction of ALRP by processing and its clinical safety applications.
RESUMO
ObjectiveTo qualitatively analyze the chemical constituents and their tissue distribution in Lujiao formula based on ultra performance liquid chromatography-quadrupole-electrostatic field orbitrap high resolution mass spectrometry（UPLC-Q-Orbitrap-MS）. MethodThe separation was performed on an ACQUITY UPLC® BEH C18 column（2.1 mm×100 mm， 1.7 μm） with the mobile phase of 0.1% formic acid aqueous solution（A）-methanol（B） in a gradient elution（0-2 min， 4%B； 2-6 min 4%-12%B； 6-38 min， 12%-70%B； 38-38.5 min， 70%B； 38.5-39 min， 70%-95%B； 39-43 min， 95%B； 43-43.1 min， 95%-4%B； 43.1-45 min， 4%B）， the flow rate was 0.3 mL·min-1 with the column temperature of 40 ℃ and the injection volume of 5 µL. The data were acquired by an electrospray ionization（ESI） in the full scanning mode of positive and negative ions， the scanning rang was set at m/z 100-1 500， the collision energies were 10， 20， 40 eV. Retention time， precise relative molecular mass and secondary mass spectrometry fragment ions were used to identify the compounds in Lujiao formula and analyze their tissue distribution， combing with self-established database and comparing with standard substances and published literature data. ResultA total of 260 compounds， including 156 flavonoids， 43 terpenoids， 18 coumarins， 13 organic acids， 7 phenylethanoids， 7 alkaloids and 16 others， were identified or hypothesized from Lujiao formula， 68 of which were identified by comparison with standard substances. And the results of tissue distribution showed that 100， 143， 129 and 126 prototype components were detected in blood， heart， liver and kidney， respectively. ConclusionThe chemical composition of Lujiao formula and their tissue distribution were systematic analyzed， which can provide reference for the quality control， clinical application， pharmacokinetics and pharmacodynamic material basis of Lujiao formula and its medicinal materials.
RESUMO
ObjectiveTo investigate the effects of Aconiti Coreani Radix and Typhonii Rhizoma on the urinary metabolites of gerbils with stroke by non-targeted metabolomics technique， and then to clarify the mechanism of the two， as well as their similarities and differences. MethodTwenty-four gerbils were randomly divided into control group（CG）， model group（MG）， Aconiti Coreani Radix group（RA） and Typhonii Rhizoma group（RT）. Except for the CG， ischemic stroke model was constructed using right unilateral ligation of gerbil carotid artery in the remaining groups. Except for the CG and MG， rats in the other groups received whole powder suspension（0.586 mg·g-1） was administered for 14 days. The neurological deficit in each group was scored by Longa scoring on days 0， 3， 7 and 14. After the end of administration， the serum， brain tissue and urine of gerbils in each group were collected， and the rate of cerebral infarction was detected by 2，3，5-triphenyltetrazolium chloride（TTC）， and the levels of interleukin（IL）-6， tumor necrosis factor（TNF）-α， malondialdehyde（MDA）， superoxide dismutase（SOD）， glutathione（GSH）， and nitric oxide（NO） in serum and brain tissue were determined by enzyme-linked immunosorbent assay（ELISA）. The urine metabolomics of gerbils in each group was studied by ultra performance liquid chromatography-quadrupole-electrostatic field orbitrap high resolution mass spectrometry（UPLC-Q-Orbitrap-MS）， and the data were processed by multivariate statistical analysis， and differential metabolites were screened based on value of variable importance in the projection（VIP） of the first principal component>1 and t-test P<0.05. Metabolic pathway analysis of the screened differential metabolites was performed using Kyoto Encyclopedia of Genes and Genomes（KEGG） database and Metaboanalyst 5.0. ResultCompared with the CG， the neurological deficit score was significantly increased in the MG（P<0.05）， compared with the MG， the neurological deficit scores in the RA and RT were significantly reduced after 7 d and 14 d（P<0.05）. Compared with the CG， the rate of cerebral infarction was significantly increased in the MG（P<0.05）， compared with the MG， the rates of cerebral infarction in the RA and RT were significantly reduced（P<0.05）. Compared with the CG， the levels of IL-6， TNF-α， and MDA in the serum and brain tissue of gerbils from the MG were significantly increased（P<0.05）， and the levels of SOD， GSH and NO were significantly reduced（P<0.05）. Compared with the MG， Aconiti Coreani Radix and Typhonii Rhizoma could down-regulate the levels of IL-6， TNF-α and MDA， and up-regulated the levels of SOD， GSH and NO. A total of 112 endogenous differential metabolites were screened by urine metabolomics， of which 16 and 26 metabolites were called back by Aconiti Coreani Radix and Typhonii Rhizoma， and could be used as potential biomarkers for both treatments in stroke gerbils， respectively. The results of the pathway analysis showed that both Aconiti Coreani Radix and Typhonii Rhizoma had regulatory effects on arginine and proline metabolism， pyrimidine metabolism， and aminoacyl-tRNA biosynthesis. In addition， Aconiti Coreani Radix could also regulate riboflavin metabolism， Typhonii Rhizoma could also regulate purine metabolism， glycine， serine and threonine metabolism， arachidonic acid metabolism， biosynthesis of pantothenate and coenzyme A， and β-alanine metabolism. ConclusionBoth Aconiti Coreani Radix and Typhonii Rhizoma have better therapeutic effects on stroke， with Aconiti Coreani Radix having stronger effects. From the metabolomics results， the main metabolic pathways regulated by Aconiti Coreani Radix involve amino acid metabolism， oxidative stress and so on， while Typhonii Rhizoma mainly involve amino acid metabolism， lipid metabolism， energy metabolism， etc.
RESUMO
ObjectiveTo establish a rapid and stable liquid chromatography-mass spectrometry（LC-MS） for simultaneous analysis of 17 chemical components in Gnaphalium affine aboveground parts with flowers， so as to provide experimental basis for improving the quality standard of this herb. MethodUltra performance liquid chromatography-quadrupole/electrostatic field orbitrap mass spectrometry（UPLC-Q-Exactive Orbitrap MS） was used for the quantitative analysis of 17 constituents in 15 batches of G. affine from different origins， the separation was performed on an ACQUITY UPLC® BEH C18 column（2.1 mm×100 mm， 1.7 μm） with the mobile phase of methanol（A）-0.1% formic acid aqueous solution（B） for gradient elution（0-1.0 min， 8%A； 1.0-4.0 min， 8%-26%A； 4.0-9.0 min， 26%A； 9.0-14.0 min， 26%-34%A； 14.0-14.5 min， 34%-45%A； 14.5-15.0 min， 45%-60%A； 15.0-18.0 min， 60%-90%A； 18.0-19.0 min， 90%A； 19.0-19.01 min， 90%-8%A； 19.01-20.0 min， 8%A）， the flow rate was 0.3 mL·min-1， the column temperature was 40 ℃ and the injection volume was 2 μL. And the electrospray ionization was used with full scanning in both positive and negative ion modes， and the scanning range was m/z 100-1 000. ResultThe established method has been verified by the methodology and could be used for the simultaneous quantification of 17 components in G. affine. The content ranges of the 17 components（quinic acid， gallic acid， protocatechuic acid， neochlorogenic acid， chlorogenic acid， cryptochlorogenic acid， caffeic acid， 1，3-O-dicaffeoylquinic acid， isochlorogenic acid A， isoquercitrin， 1，5-O-dicaffeoylquinic acid， apigenin-7-O-glucoside， astragalin， isochlorogenic acid C， luteolin， apigenin and hispidulin） in 15 batches of G. affine samples was 39.60-179.12， 0.17-0.84， 2.41-8.38， 4.33-31.50， 13.63-180.38， 2.43-14.75， 1.16-19.68， 0.49-5.63， 55.77-445.16， 0.23-10.26， 62.04-530.10， 1.11-18.01， 11.36-90.61， 12.22-65.98， 7.22-69.84， 3.37-45.65， 0.30-2.59 μg·g-1， respectively. The content of organic acids was higher than that of flavonoids in G. affine， and the contents of 1，5-O-dicaffeoylquinic acid， isochlorogenic acid A， quinic acid and chlorogenic acid were higher. Meanwhile， the content of flavonoids in the samples from Guizhou was higher than that from Jiangsu， while the content of organic acids in the samples from Jiangsu was higher than that from Guizhou. ConclusionThe established method can be used for the rapid and accurate determination of 17 components in G. affine， which clarifies the content range of the main components in this herb， and can provide a reference for the selection of quality control markers of G. affine.
RESUMO
ObjectiveThrough the correlation analysis between intestinal absorption profile and inhibition of macrophage foaming， the pharmacodynamic components of Zhuriheng dripping pills（ZRH） were explored to provide a basis for establishing its quality standard. MethodIntestinal absorption fluids with 0， 5， 10， 15， 20 times clinical equivalent doses were prepared by a rat everted gut sac（EGS）， and the oxidized low density lipoprotein（ox-LDL）-induced RAW264.7 macrophage foaming model was used to investigate the effect of intestinal absorption fluid with different doses on the accumulation of lipids in RAW264.7 cells by oil red O staining and cholesterol content determination， and to screen for the optimal dose. Ultra performance liquid chromatography-quadrupole-electrostatic field orbitrap high-resolution mass spectrometry（UPLC-Q-Exactive Orbitrap MS） was used to analyze and identify intestinal absorption fractions of ZRH intestinal absorption fluids， and partial least squares-discriminant analysis（PLS-DA） and orthogonal partial least squares-discriminant analysis（OPLS-DA） were performed on different doses of ZRH intestinal absorption fluids using SIMCA 13.0 with peak area as the independent variable and the pharmacodynamic indicators as the dependent variables to screen the compounds with variable importance in the projection（VIP） value>1.0 as contributing components， and Pearson correlation analysis was used to determine the spectral effect relationship， determined the compounds and positive correlation with pharmacodynamic were as active ingredients. Molecular docking was used to verify the binding energy of peroxisome proliferator-activated receptor α（PPARα）， PPARγ， PPARβ， human retinoid X receptor α（RXRA） and nuclear transcription factor-κB（NF-κB） with the active ingredients in ZRH intestinal absorption fluids. Real-time fluorescence quantitative polymerase chain reaction（Real-time PCR） was performed to detect the mRNA levels of PPARγ， scavenger receptor A1（SRA1） and adenosine triphosphate-binding cassette transporter A1（ABCA1） in RAW264.7 cells， Westen blot was used to detect the expression level of PPARγ protein in RAW264.7 cells， and enzyme-linked immunosorbent assay（ELISA） was used to detect the levels of interleukin（IL）-1β and NF-κB in RAW264.7 cells. ResultAccording to the results of oil red O staining and cholesterol content determination， the ZRH intestinal absorption fluids could significantly reduce macrophage foaming， and intestinal absorption fluids with 15， 20 times clinical equivalent doses had the best effect， the 15-fold ZRH intestinal absorption fluid was finally determined as the study subject. Spectral effect relationship showed that 52 corresponding peaks in the ZRH-containing intestinal fluid were positively correlated with the efficacy， including organic acids， phenylpropanoids， iridoids， flavonoids， bile acids， coumarins and chromones. Target validation results showed that 86.9%-96.2% of the total components processed good binding activities with the key targets of PPARα， PPARγ， PPARβ， RXRA and NF-κB， and the docking energy values were all less than -6.0 kcal·mol-1（1 cal≈4.19 J）. The results of validation showed that， compared with the normal group， the model group showed a significant increase in the levels of SRA1 and PPARγ mRNA expression， a significant decrease in ABCA1 mRNA expression， a significant increase in the level of PPARγ protein expression， and a significant increase in the levels of IL-1β and NF-κB（P<0.01）， compared with the model group， the 15-fold intestinal absorption fluid group showed a significant decrease in the levels of SRA1 and PPARγ mRNA expression（P<0.05， P<0.01）， ABCA1 mRNA expression level was significantly up-regulated， the levels of IL-1β and NF-κB were significantly reduced（P<0.01）， and PPARγ protein expression level was significantly reduced（P<0.05）. ConclusionThis study identifies 52 components and their metabolites in ZRH intestinal absorption fluid that are positively correlated with the inhibition of macrophage foaming， which may be related to the regulation of the PPARs pathway in cells and the reduction of the levels of inflammatory factors， and can provide a reference for the quality control and clinical application of ZRH.
RESUMO
ObjectiveTo determine the pharmacodynamic substance basis of Epimedii Folium（EF） and Epimedii Wushanensis Folium（EWF） in promoting osteogenic differentiation， and to establish a method to analyze the material basis of Chinese materia medica based on the correlation between chemical fingerprint and cellular metabolomics. MethodThe chemical fingerprints of 15 batches of EF with 4 species and 3 batches of EWF were analyzed by ultra performance liquid chromatography-quadrupole-electrostatic field orbitrap high-resolution mass spectrometry（UPLC-Q-Exactive Orbitrap-MS）， and partial least squares-discriminant analysis（PLS-DA） was used to analyze the peak areas of chemical fingerprints of samples. The effects of different samples on proliferative activity of MC3T3-E1 osteoblast precursors， as well as the activity of alkaline phosphatase（ALP） in osteoblasts were detected by cell counting kit-8（CCK-8） and enzyme-linked immunosorbent assay（ELISA）. At the same time， UPLC-quadrupole-time-of-flight mass spectrometry（UPLC-Q-TOF-MS/MS） was used to analyze the effects of different samples on the metabolomics of MC3T3-E1 cells， then metabolic peak table of osteogenic differentiation cells was constructed， and pharmacodynamic index mean Y0 was introduced into the peak table. PLS was used to calculate mean Y0 of each group， and the mean Y0 was added to the peak table of chemical fingerprint to construct the correlation between chemical fingerprint and cell metabolome， the pharmacodynamic components of EF and EWF that promote bone differentiation were screened according to variable importance in the projection（VIP） value>1. The pharmacodynamic effects of EF and EWF were evaluated according to the mean Y0 of each group. ResultThe chemical fingerprints of EF with different origins and EWF were completely separated. Compared with the blank group， the activity of MC3T3-E1 cells in EF and EWF groups was significantly increased， the activity of ALP in the Epimedium brevicornu（Gansu province）， E. koreanum and E. pubescens groups was significantly increased（P<0.05）. The results of cell metabolomics showed that the blank group and the model group had an obvious trend of separation. EF with different origins and EWF had different distance from the model group， indicating that EF with different origins and EWF had different effect on promoting osteogenic differentiation. Chemical fingerprint-cell metabolomics integration analysis screened 9 components closely related to the efficacy of EF and EWF， including diphylloside B， epimedin C， icariin， baohuoside Ⅰ， yinyanghuo B， β-anhydroicaritin， magnoflorine， cryptochlorogenic acid and quercetin. E. koreanum had the strongest effect on promoting osteogenic differentiation. ConclusionThis study determined that the material basis of EF and EWF promoting osteogenic differentiation were mostly flavonoids， alkaloids and organic acids， which provided ideas and methods for the screening of pharmacodynamic components and the prediction of therapeutic effect of Chinese materia medica.
RESUMO
ObjectiveTo establish a qualitative analysis method for the chemical constituents of the reference sample of Xiao Xumingtang， and to establish the fingerprint of 15 batches of Xiao Xumingtang， so as to evaluate the quality consistency among batches. MethodAccording to the key information of Xiao Xumingtang in the Key Information Table of Ancient Famous Classical Formulas（25 Formulas）， the reference sample of this formula was prepared， and it was detected by ultra performance liquid chromatography-quadrupole-time-of-flight mass spectrometry（UPLC-Q-TOF-MS/MS）. The chemical components were identified by self-constructed database， consulting relevant literature， and comparing with the reference substances， and the components were assigned by comparing with single drug samples and negative samples lacking single drug. The fingerprint of the reference sample of Xiao Xumingtang was established using high performance liquid chromatography（HPLC）， and the common peaks were assigned and identified through single drug samples and negative samples lacking single drug. ResultBased on the information of MS fragments， relevant literature， and database retrieval， a total of 64 compounds were identified and inferred from the reference sample of Xiao Xumingtang， including 31 flavonoids， 8 terpenoids， 12 triterpenoid saponins， 2 phthalides， 3 phenylpropanoids， 2 gingerols， 5 alkaloids， and 1 cyanoside. Among them， 21 were derived from Scutellariae Radix， 10 from stir-fried Glycyrrhizae Radix et Rhizoma， 9 from Ginseng Radix et Rhizoma， 8 from Paeoniae Radix Alba， 4 from Saposhnikoviae Radix， 3 from Stephaniae Tetrandrae Radix， 3 from Chuanxiong Rhizoma， 2 from Aconiti Lateralis Radix Praeparata， 2 from Zingiberis Rhizoma Recens， 1 from Ephedrae Herba， and 1 from Armeniacae Semen Amarum. The established HPLC fingerprint of the reference sample of Xiao Xumingtang had 23 common peaks， among which， peaks 1 and 2 were derived from Paeoniae Radix Alba， peaks 3 and 7 from Saposhnikoviae Radix， peaks 4， 8 and 9 from Stephaniae Tetrandrae Radix， peaks 10， 17， 18， 20 and 21 from stir-fried Glycyrrhizae Radix et Rhizoma， peaks 11-16， 19 and 22 from Scutellariae Radix， peak 5 from Chuanxiong Rhizoma， peak 23 from Zingiberis Rhizoma Recens， peak 6 was the common component of stir-fried Glycyrrhizae Radix et Rhizoma and Scutellariae Radix. A total of 10 compounds including albiflorin（peak 1）， paeoniflorin（peak 2）， cimicifugoside（peak 3）， 5-O-methylvisammioside（peak 7）， baicalin（peak 11）， sec-O-glucosylhamaudol（peak 13）， oroxylin A-7-O-β-D-glucuronide（peak 15）， wogonoside（peak 16）， glycyrrhizic acid（peak 21） and 6-gingerol（peak 23） were identified. The similarities of 15 batches of reference samples were>0.999， indicating that the reference samples had good consistency. ConclusionThrough the identification of the chemical constituents in the reference sample of Xiao Xumingtang， it is clear that the composition of the samples is mainly composed of flavonoids and triterpenoid saponins. The established fingerprint can basically reflect the overall chemical characteristics of the reference sample of Xiao Xumingtang， which can provide a basis for the quality research of its compound preparations.
RESUMO
ObjectiveTo analyze the migrating components absorbed into blood of the aqueous extract of Euphorbia helioscopia， and to explore the pharmacodynamic material basis of the aqueous extract of E. helioscopia against chronic obstructive pulmonary disease（COPD）. MethodUltra-performance liquid chromatography-quadrupole-time-of-flight mass spectrometry（UPLC-Q-TOF-MS/MS） was used to detecte the migrating components absorbed into blood of rats after intragastric administration of aqueous extract of E. helioscopia. An Agilent RRHD SB-C18 column（3 mm×100 mm， 1.8 μm） was used with 0.1% formic acid aqueous solution（A）-acetonitrile（B） as the mobile phase for gradient elution（0-15 min， 5%-30%B； 15-20 min， 30%-50%B； 20-30 min， 50%-95%B； 30-35 min， 95%-5%B）， and the detection wavelength of 190-800 nm， column temperature of 40 ℃， flow rate of 0.3 mL∙min-1 and injection volume of 4 μL. The electrospray ionization（ESI） was used in positive and negative ion modes， and the detection range was m/z 50-1 250. Network pharmacology was used to screen out the key components and the key targets of COPD through the interaction analysis. Metascape database was used to predict the molecular function， biological process， cellular composition and signal pathways mainly involved in the anti-COPD effect of E. helioscopia. Molecular docking technique was used to determine the affinity of key targets with key components. ResultA total of 29 migrating components absorbed into blood of rats were identified after intragastric administration of aqueous extract of E. helioscopia， 9 of which were prototype components and 20 were metabolites. Network pharmacological analysis showed that luteolin， quercetin， apigenin， naringenin and helioscopinolide C were the key components of E. helioscopia against COPD， and vascular endothelial growth factor A（VEGFA）， albumin（ALB）， protein kinase B1（Akt1）， tumor necrosis factor（TNF） and interleukin-6（IL-6） were the key targets. Molecular docking results showed that one diterpene lactone（helioscopinolide C） and three flavonoids（naringenin， luteolin， apigenin） in the migrating components absorbed into blood all had strong binding activity to the key targets of E. helioscopia against COPD. ConclusionNaringenin， helioscopinolide C， luteolin and apigenin may be the main anti-COPD active substances of E. helioscopia.