Exploring the mechanism of Xihuang Pill's anti-hyperplasia of mammary glands effect based on UPLC-Q-orbitrap-MS technology / 药学学报

Based on UPLC-Q-orbitrap-MS and biological network analysis tools, the mechanism of Xihuang Pill in improving hyperplasia of mammary glands was systematically analyzed. The rat model of hyperplasia of mammary glands was established by intramuscular injection of estradiol benzoate and progesterone. LC-MS tissue metabolomics was used to explore the key metabolites and metabolic pathways of Xihuang Pill in improving hyperplasia of mammary glands in rat. The network analysis of the key metabolites regulated by Xihuang Pill was carried out by integrating biological network analysis tools, focusing on the key metabolic pathways, and exploring the potential targets of Xihuang Pill to improve hyperplasia of mammary glands. Compared with the control group, there were significant differences in the content of 49 differential metabolites in the tissues of the model group (P < 0.05). Xihuang Pills could significantly call back 17 metabolites such as L-alanine, threonine, indole-3-carboxylic aldehyde, lysine, arginine, alanylleucine, glycyltyrosine, γ-glutamyl leucine, vitamin B3, serine leucine, threonine leucine, isoleucine glutamic acid, γ-glutamyl tyrosine, decanoyl-L-carnitine, uric acid, leucylleucine, S-adenosyl-methionine. Further network analysis and literature research on the key metabolites regulated by Xihuang Pills showed that the AGE-RAGE signaling pathway may be one of the important pathways for Xihuang Pills to improve hyperplasia of mammary glands. STAT3, MAPK1, EGFR, CASP3, CASP8, PRKCA and JUN in the AGE-RAGE signaling pathway may be potential targets for Xihuang Pills to improve hyperplasia of mammary glands. The animal experiment operations involved in this paper follow the provisions of the Animal Ethics Committee of Gansu University of Traditional Chinese Medicine and pass the ethical review of animal experiments (approval number: 2022-705).

Urinary Metabolomics Aanlysis of Differences in Effect of Aconiti Coreani Radix and Typhonii Rhizoma on Gerbils with Stroke / 中国实验方剂学杂志

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.

Analysis of Chemical Components and Tissue Distribution of Lujiao Formula Based on UPLC-Q-Orbitrap-MS / 中国实验方剂学杂志

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.

UPLC-Q-Orbitrap-MS based metabolomics and analysis of the effect of Suanzaoren Decoction on serum of chronic unpredictable mild stress depression rats / 药学学报

A UPLC-Q-Orbitrap-MS based metabolomic approach combined with biochemical assay and histopathological inspection were employed to study the intervention effects of Suanzaoren Decoction (SZRD) on chronic unpredictable mild stress (CUMS) depression rats, and to clarify the metabolic regulation pathway of SZRD. The rats were randomly divided into normal control group, CUMS model group, positive drug venlafaxine group, SZRD high (24 g·kg-1) and low (12 g·kg-1) dose groups, respectively. The CUMS model was replicated by subjecting to a variety of stimulus, such as thermal stimulation, ice water swimming, ultrasonic stimulation, tail clamping, day and night reversal, plantar electric shock and so on for rats. After oral administration of drugs for 28 days, the behavioral indexes of rats in each group were observed and the hippocampus and serum samples of rats were collected for biochemical assay and histopathological inspection. Compared with the CUMS model group, low dose and high dose SZRD groups can significantly reduce the immobility time of forced swimming (P < 0.001, P < 0.001), increase the sucrose preference rate (P < 0.01, P < 0.05), the number of crossings (P < 0.05, P < 0.01) and the number of uprights (P < 0.05, P < 0.01) in the open field test, suggesting that SZRD can significantly improve the depression-like behavior of CUMS model rats. In addition, SZRD could significantly reduce the levels of serum IL-6, IL-1β and TNF-α of CUMS model rats. A total of 21 differential metabolites in serum were identified by comparison with the data from the literature and databases. In addition, low-dose SZRD and high-dose SZRD improved the 8 and 11 perturbed potential serum biomarkers that were induced by CUMS, respectively, which related to alanine, aspartic acid and glutamic acid, tryptophan and arachidonic acid metabolism. This study provides a scientific basis for expanding the clinical indications of SZRD. This experiment was approved by the Animal Ethics Committee of Shanxi University (Approval No. SXULL2020028).

Urine metabolomics study of intervention of Xihuang Pills in rats with hyperplasia of mammary gland / 中国中药杂志

This study aimed to investigate the mechanism of Xihuang Pills in improving hyperplasia of mammary gland(HMG) in rats based on urine metabolomics using ultra-performance liquid chromatography-quadrupole-Orbitrap mass spectrometry(UPLC-Q-Orbitrap-MS). The HMG rat model was established by intramuscular injection of estradiol benzoate solution(0.5 mg·kg~(-1), 25 days) followed by progesterone injection(5 mg·kg~(-1), 5 days). UPLC-Q-Orbitrap-MS technology was used to establish the endogenous small-molecule metabolic profiles in urine samples of rats in the blank group, the HMG model group, and Xihuang Pills group. Multivariate statistical analysis was performed for pattern recognition, t test and variable importance in the projection(VIP) were used to screen potential biomarkers. The significantly changed differential metabolites were identified using the online database Human Metabolome Database(HMDB). Metabolic pathway enrichment analysis was conducted using the MetaboAnalyst 5.0 database. The results showed that 90 differential metabolites with significant changes(P<0.05) were identified between the blank group and the HMG model group using the HMDB. Among them, 48 metabolites significantly reverted(P<0.05) after administration of Xihuang Pills, which may be related to the regulatory effect of Xihuang Pills. Thirteen metabolic pathways significantly associated with HMG were identified when the differential metabolites were imported into the MetaboAnalyst 5.0 database, and Xihuang Pills could modulate seven of these pathways. These metabolic pathways mainly involved histidine metabolism, arginine and proline metabolism, β-alanine metabolism, glycine, serine and threonine metabolism, tryptophan metabolism, pyrimidine metabolism, and amino sugar and nucleotide sugar metabolism. This study utilized UPLC-Q-Orbitrap-MS and urine metabolomics technology to analyze the mechanism of Xihuang Pills in improving HMG, laying the foundation for further in-depth research.

Analysis on Chemical Constituents in Xiao Chengqitang by UPLC-Q-Orbitrap-MS / 中国实验方剂学杂志

Objective:To rapidly identify the chemical constituents in Xiao Chengqitang by ultra-performance liquid chromatography-quadrupole-electrostatic field orbitrap high resolution mass spectrometry （UPLC-Q-Orbitrap-MS）. Method:The method was established by the Waters CORTECS T3 column （2.1 mm×150 mm， 1.6 μm）， mobile phase was methanol （A）-0.1% formic acid aqueous solution （B） for gradient elution （0-5 min， 3%-21%A； 5-20 min， 21%-36%A； 20-32 min， 36%-50%A； 32-42 min， 50%-62%A； 42-50 min， 62%-85%A； 50-60 min， 85%-95%A）， the flow rate was 0.2 mL·min^-1， and the column temperature was 30 ℃. UPLC-Q-Orbitrap-MS was operated in positive and negative ion modes， the scanning range was 100-1 200 with mode of Full MS/dd-MS²， and the collision energies were 20， 40 eV. The compounds were identified by comparing with reference substances and combining with literature reports and MS database information. Result:A total of 123 components were identified in Xiao Chengqitang， including 33 flavonoids， 25 anthraquinones and anthrones， 23 phenylpropanoids， 15 tannins， 10 nitrogen-containing components and 17 other components. Among them， 32 components were determined by reference substances. Conclusion:The material basis of Xiao Chengqitang is flavonoids， anthraquinones and anthrones， phenylpropanoids， which is derived from Aurantii Fructus Immaturus，<italic> </italic>Rhei Radix et Rhizoma and Magnoliae Officinalis Cortex， respectively.

Study on difference of chemical compositions of Guilingji before and after alchemy based on mass spectrometry and multivariate statistical analysis / 中国中药杂志

UPLC-Q-Orbitrap MS/MS and ICP-MS coupled with multivariate statistical analysis was employed to explore the differences in chemical compositions of Guilingji(GLJ) before and after alchemy.The changes in organic chemical compositions and inorganic elements were observed and 39 differential organic compositions were found in GLJ after alchemy, 24 compounds of which were identified. The differential compositions of GLJ included violet ketones, chalcones, amides, and fatty acids whose contents were increased after alchemy, as well as flavones, isoflavones, dihydroflavones, flavonoid glycosides, and coumarins whose content were decreased after alchemy. This study showed 6 inorganic elements filtered out as markers for distinguishing GLJ before and after alchemy, including B, Si, Mg, K, Cr, and Ni.The contents of Mg, K, Cr and Ni were increased while the contents of B and Si were decreased after alchemy.The difference of the contents after alchemy changed the cold and hot properties of the compound, showing the decrease of dryness, and the hot property was changed to warm and neutral properties; in addition, the membrane permeability and absorption of the compound compositions were improved. In this study, we preliminarily investigated the changes of chemical compositions in GLJ before and after alchemy as well as the effects of alchemy on physical and chemical properties and cold-heat nature of GLJ, laying a foundation for further clarifying the scientific connotation of alchemy process.