Evaluation of A Liver Yin Deficiency Mouse Model Based on Untargeted Metabolomics / 中国实验方剂学杂志

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.

Investigation of Neuroprotective Effect of Geniposide on Chronic Alcoholism Rats Based on Metabonomics / 中国实验方剂学杂志

ObjectiveMetabolomics was used to identify biomarkers of chronic alcoholism， and to evaluate the neuroprotective effect of geniposide， providing reference for the diagnosis and treatment of chronic alcoholism. MethodThe rat model of chronic alcoholism was established by intragastric administration of 50% ethanol with 8 mL·kg-1 for 14 days， and then increased to 12 mL·kg-1 for 21 days. Meanwhile， the intervention was performed by continuous gavage of geniposide （15 mg·kg-1） for 35 days. At the end of the experiment， the biochemical indexes and histopathological morphology of liver and brain tissues of rats were detected. Ultra performance liquid chromatography-quadrupole-time-of-flight tandem mass spectrometry （UPLC-Q-TOF-MS） was used for urine metabonomics. The chromatographic conditions was as follows：ACQUITY UPLC™ HSS T3 column （2.1 mm×100 mm， 1.8 μm）， mobile phase of 0.1% formic acid acetonitrile solution （A）-0.1% formic acid aqueous solution （B） for gradient elution （0-2.5 min， 1%-11%A； 2.5-4.5 min， 11%-21%A； 4.5-7.0 min， 21%-40%A； 7.0-8.5 min， 40%-99%A； 8.5-10.5 min， 99%A； 10.5-10.6 min， 99%-1%A； 10.6-13.0 min， 1%A）， the flow rate of 0.4 mL·min-1. The conditions of mass spectrometry were electrospray ionization （ESI）， positive and negative ion modes， scanning range of m/z 50-1 200. Progenesis QI 2.0 and MassLynx 4.1 were used for data analysis， and biomarkers were identified by matching element composition and secondary fragments with Human Metabolome Database （HMDB）. ResultThe pathological results showed that on the 35th day of model replication， compared with the model group， the cortical neurons in the geniposide group showed a significantly improved state of disorder， nuclear pyknosis， hyperchromatism and cell membrane boundary blurred necrosis. The biochemical results showed that geniposide could significantly increase the activities of glutathione peroxidase （GSH-Px） and superoxide dismutase （SOD）， decrease the activity of acetylcholinesterase （AChE）， decrease the levels of β-endorphin （β-EP） and malondialdehyde （MDA）. A total of 48 biomarkers of chronic alcoholism were identified by metabonomics， involving seven metabolic pathways of tryptophan metabolism， phenylalanine metabolism， pentose and glucuronate interconversions， pyrimidine metabolism， ascorbate and aldarate metabolism， steroid hormone biosynthesis and purine metabolism. The main pathway is 5-hydroxytryptamine pathway of tryptophan metabolism. ConclusionBiomarkers related to nerve injury in chronic alcoholism are mainly derived from the 5-hydroxytryptamine metabolic pathway. Geniposide can regulate this pathway so as to improve oxidative stress in the brain and play a neuroprotective role.

Analysis of Chemical Constituents in Different Parts of Gentiana straminea Based on UPLC-Q-TOF-MS/MS / 中国实验方剂学杂志

ObjectiveUltra-performance liquid chromatography-quadrupole-time-of-flight tandem mass spectrometry （UPLC-Q-TOF-MS/MS） was used to analyze the chemical constituents in the aerial part and roots of Gentiana straminea from different areas of Qinghai province， and the main chromatographic peaks and differential components of different parts were identified. MethodThe chromatographic separation was performed on a Waters ACQUITY UPLC HSS T3 column （2.1 mm×100 mm， 1.8 μm） with 0.1% formic acid aqueous solution （A）-acetonitrile （B） as the mobile phase for gradient elution （0-1 min， 1%-13%B； 1-5 min， 13%-18%B； 5-7 min， 18%-50%B； 7-9.5 min， 50%-60%B； 9.5-11 min， 60%-99%B； 11-14 min； 99%B； 14-15 min， 99%-1%B； 15-16 min， 1%B）， the column temperature at 40 ℃， and the flow rate of 0.3 mL·min-1. Electrospray ionization （ESI） and negative ion full scan mode were selected for the mass spectrometric conditions to analyze the samples， and the detection range was m/z 50-1 200. Chemical constituents of the aerial part were qualitatively analyzed with the reference substances， literature information and ChemSpider. Principal component analysis （PCA） and orthogonal partial least squares discriminant analysis （OPLS-DA） were used to analyze the classification trend， correlation and differential chemical components between aerial part and roots of G. straminea. ResultA total of 68 components， including 24 iridoids， 13 flavonoids， 8 triterpenoids， 6 xanthones， 5 fatty acids， 4 saccharides， 3 phenolic glycosides， 2 alkaloids， 2 sterols and 1 lignan， were preliminarily identified from the aerial part of G. straminea. Among them， 42 components were firstly reported in 4 Gentiana species included in the 2020 edition of Chinese Pharmacopoeia. Eight differential components were screened out， namely sucrose， maltotriose， loganic acid， shanzhiside methyl ester， 6′-O-β-D-glucosylgentiopicroside， swertiamarin， gentiopicrin and isovitexin. ConclusionThe aerial part of G. straminea is rich in chemical constituents and has good medicinal potential. There were significant differences in the chemical components between the aerial part and roots of G. straminea， and the main differential components were iridoids， which could provide a basis for exploring efficacy differences in different parts of G. straminea.

Chemical Composition Analysis and Characterization of Reference Sample of Huangqi Guizhi Wuwutang Based on UPLC-Q-TOF-MS / 中国实验方剂学杂志

ObjectiveTo analyze the chemical composition of the reference sample of Huangqi Guizhi Wuwutang （lyophilized powder）， and to provide quality markers for the formulation of quality standards of this formula. MethodUltra performance liquid chromatography-quadrupole time-of-flight tandem mass spectrometry （UPLC-Q-TOF-MS） was performed on a Waters ACQUITY UPLC™ HSS T3 column （2.1 mm×100 mm， 1.8 μm）， the mobile phase was methanol （A） -0.1% formic acid aqueous solution （B） for gradient elution （0-8 min， 1%-20%A； 8-10 min， 20%-30%A； 10-12 min， 30%-35%A； 12-14 min， 35%-40%A， 14-23 min， 40%-55%A， 23-27 min， 55%-99%A； 27-28 min， 99%A； 28-28.5 min， 99%-1%A； 28.5-30 min， 1%A）， the column temperature was 40 ℃， the injection volume was 2 μL， and the flow rate was 0.3 mL·min-1. The mass spectrometry data of the reference sample of Huangqi Guizhi Wuwutang （lyophilized powder） were collected under positive and negative ion modes. The conditions of mass spectrometry were electrospray ionization （ESI）， scanning range of m/z 50-1 200， and impact energy of 10-30 eV. UNIFI 1.8 and Progenesis QI 2.0 software were used to analyze and characterize the chemical constituents in reference sample of Huangqi Guizhi Wuwutang （lyophilized powder） combined with reference comparison and literature review. ResultA total of 123 chemical constituents were identified， including 33 flavonoids， 26 glycosides， 18 organic acids， 11 terpenoids， 7 phenylpropanoids， 4 gingerol， 3 alkaloids， 3 amino acids， 2 amides and 16 other compounds. ConclusionThe established method can quickly and accurately characterize the chemical components in the reference sample of Huangqi Guizhi Wuwutang （lyophilized powder）， which can provide a basis for the selection of quality evaluation indicators of this formula， and provide a reference for its preparation research.

Material Basis and Mechanism of Epimedii Folium Against Osteoporosis Based on Spectrum-Effect Relationship and Network Pharmacology / 中国实验方剂学杂志

Objective:To study the material basis and potential molecular mechanism of Epimedii Folium against osteoporosis. Method:The chemical components in 14 batches of Epimedii Folium were analyzed by ultra-performance liquid chromatography-quadrupole-time of flight-tandem mass spectrometry （UPLC-Q-TOF-MS/MS）. With the activity of alkaline phosphatase （ALP） as the pharmacodynamic index，the partial least squares regression analysis （PLSR） was conducted to establish a model uncovering the spectrum-effect relationship between UPLC-Q-TOF-MS/MS spectral peak and ALP activity and screen the active components against osteoporosis. Online databases such as the Traditional Chinese Medicine Systems Pharmacology Database and Analysis Platform （TCMSP），Comparative Toxicogenomics Database （CTD），Database for Annotation，Visualization，and Integrated Discovery （DAVID） and Cytoscape 3.6.1 were employed to predict the possible mechanism of action of Epimedii Folium against osteoporosis. Result:A total of 61 peaks and 56 compounds were identified by UPLC-Q-TOF-MS/MS. PLSR showed that icariin，baohuoside Ⅰ，epimedin A，sagittatoside A，and baohuoside Ⅱ might be the active components for Epimedii Folium to inhibit osteoporosis considering their strong correlation with ALP activity. As revealed by the network pharmacological analysis of the five components mentioned above，Epimedii Folium<italic> </italic>mainly regulated seven targets such as tumor necrosis factor （TNF），androgen receptor （AR），and estrogen receptor 1 （ESR1） and eight key pathways like endocrine and other factor-regulated calcium reabsorption，vascular endothelial growth factor （VEGF） signaling pathway，and transient receptor potential （TRP） channels to exert its anti-osteoporosis effect. Conclusion:The exploration of material basis and potential molecular mechanism of Epimedii Folium against osteoporosis based on UPLC-Q-TOF-MS/MS，spectrum-effect relationship，and network pharmacology has provided an experimental basis for the scientific explanation and clinical application of Epimedii Folium in treating osteoporosis.