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).

Analysis on Components Absorbed into Blood and Cerebrospinal Fluid of Schisandrae Chinensis Fructus Based on Sequential Metabolism / 中国实验方剂学杂志

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.

Exploration of Spectrum-effect Relationship of Zhuriheng Dropping Pills Against Macrophage Foaming Based on UPLC-Q-Exactive Orbitrap MS / 中国实验方剂学杂志

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.

Quantitative Analysis of Multi-components in Gnaphalium affine Based on UPLC-Q-Exactive Orbitrap MS / 中国实验方剂学杂志

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.

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.

Simultaneous determination of fifteen components in Niuhuang Jiangya Pills by UPLC-Q Exactive-Orbitrap MS / 药学学报

An ultra performance liquid chromatography-quadrupole/electrostatic field orbitrap high resolution mass spectrometry (UPLC-Q Exactive-Orbitrap MS) method for the simultaneous determination of 15 compounds (taurocholic acid, 7-keto-3α,12-α-dihydroxycholanic acid, glycocholic acid, 3-oxo-7α,12α-hydroxy-5β-cholanoic acid, taurochenodeoxycholic acid, 3α-hydroxy-7-oxo-5β-cholanic acid, hyocholic acid, sodium taurodeoxycholate, hyodeoxycholic acid, cholic acid, glycochenodeoxycholic acid, glycodeoxycholic acid, taurolithocholic acid sodium salt, chenodeoxycholic acid, deoxycholic acid) in Niuhuang Jiangya Pills was established. The separation was performed on a Thermo Fisher Scientfic Bremen HYPERSIL GOLD C18 column (100 mm × 2.1 mm, 1.9 μm). Methanol and water (containing 0.1% formic acid)were adopted as the mobile phase by gradient elution．MS detection was performed with multiple reaction monitoring mode．The results showed that fifteen compounds had a good linearity within their respective concentration ranges (r > 0.999 0). The average recovery rates were 93.7%- 105.2% (n = 9). The established method was used to determine the content of 15 batches of samples, and the results showed that the content of cholic acid was quite different. The present study provides an important reference for the overall quality control of Niuhuang Jiangya Pills.

Quality evaluation of Qilong Zhuang'er oral liquid based on UHPLC-Q-exactive orbitrap MS and chemometrics analysis / 药学学报

An UHPLC-Q-exactive orbitrap MS method for the simultaneous determination of 19 chemical components in Qilong Zhuang'er oral liquid was established and the quality differences between different batches of samples was compared by chemometric analysis to provide a basis for the quality evaluation of the preparation. The contents of allantoin, L-proline, pyroglutamic acid, hordenine, adenosine, L-phenylalanine, guanosine, L-tryptophan, caffeic acid, calycosin-7-glucoside, verbascoside, isoacteoside, ononin, calycosin, 3-hydroxy-9,10-dimethoxyptercarpan, formononetin, atractylenolide III, atractylenolide II and astragaloside A were analyzed by cluster heat map, principal component analysis (PCA) and partial least squares discriminant analysis (PLS-DA) using Hiplot platform and MarkerlynxXS software to comprehensively evaluate the quality difference of different batches of Qilong Zhuang'er oral liquid. The 19 chemical compounds showed good linearity in their respective concentration ranges (r ≥ 0.999). The RSD of precision, repeatability and stability (24 h) tests were all less than 1.94%. The average recovery was 97.24%-102.75% (RSD < 2.74%, n = 6). The 10 batches of samples were divided into two categories by cluster heat map and PCA analysis. 3-Hydroxy-9,10-dimethoxyptercarpan, atractylenolide III, calycosin, atractylenolide II, formononetin, allantoin and caffeic acid were identified as differential markers by PLS-DA. The established multi component quantitative method of Qilong Zhuang'er oral liquid combined with chemometric analysis can provide reference for the quality evaluation of the preparation.

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).

Comparison of alkaloids in Aconiti Kusnezoffii Radix, Aconiti Radix, and Aconiti Lateralis Radix Praeparata based on UHPLC-Q-Exactive Orbitrap MS/MS / 中国中药杂志

UHPLC-Q-Exactive Orbitrap MS/MS was used to systematically analyze and compare the alkaloids in Aconiti Kusnezoffii Radix, Aconiti Radix, and Aconiti Lateralis Radix Praeparata. After the samples were pretreated in the solid-phase extraction cartridges, 0.1% ammonium hydroxide(A)-acetonitrile(B) was used for gradient elution. The LC-MS method for characterization of alkaloids in the three herbal medicines was established in ESI positive ion mode to collect high resolution MS data of reference substances and samples. On the basis of the information of reference substance cracking behavior, retention time, accurate molecular mass, and related literature, a total of 155 alkaloids were identified in Aconiti Kusnezoffii Radix, Aconiti Radix, and Aconiti Lateralis Radix Prae-parata. Specifically, 130, 127, and 92 alkaloids were identified in Aconiti Kusnezoffii Radix, Aconiti Radix, and Aconiti Lateralis Radix Praeparata, respectively. Monoester alkaloids and amino-alcohol alkaloids were dominant in the three herbal medicines, and the alkaloids in Aconiti Kusnezoffii Radix and Aconiti Radix were similar. This paper can provide a reference for elucidating the pharmacological effects and clinical application differences of the three herbal medicines produced from plants of Aconitum.

Rapid identification of components in Wuzhuyu Decoction using UHPLC Q-Exactive Orbitrap MS~n and molecular network technology / 中国中药杂志

Wuzhuyu Decoction, the classical formula recorded in the Treatise on Febrile Diseases(Shang Han Lun), has been included in the Catalogue of Ancient Classic Prescriptions(the First Batch). Consisting of Euodiae Fructus, Ginseng Radix et Rhizoma, Zingiberis Rhizoma Recens, and Jujubae Fructus, it is effective in warming the middle, tonifying deficiency, dispelling cold, and descending adverse Qi, and is widely applied clinically with remarkable efficacies. For a classical formula, the chemical composition is the material basis and an important premise for quantity value transfer. This study aimed to establish a rapid identification method of chemical components in Wuzhuyu Decoction by high-resolution mass spectrometry(HR-MS) and molecular network. AQUITY UPLC BEH C_(18) column(2.1 mm×100 mm, 1.7 μm) was used for sample separation, and acetonitrile-0.1% formic acid in water was used as mobile phases for gradient elution. Q-Exactive Orbitrap MS data were collected in positive and negative ion modes, and GNPS molecular network was plotted according to the similarity of MS/MS fragmentation modes. Cytoscape 3.6.1 was used to screen molecular clusters with similar structures. Finally, the chemical components of Wuzhuyu Decoction were rapidly identified according to the controls, as well as the information of retention time, accurate relative molecular weight of HR-MS, and MS/MS multistage fragments. A total of 105 chemical components were identified in Wuzhuyu Decoction. This study can provide data for the follow-up quality control, standard substance research, and pharmacodynamic material research on Wuzhuyu Decoction, as well as references for the rapid qualitative analysis of the chemical components of Chinese medicine.

Chemical constituents in different parts of Ixeris sonchifolia based on UPLC-LTQ-Orbitrap-MS~n / 中国中药杂志

The chemical constituents in stem leaf, root, and flower of Ixeris sonchifolia were identified by the ultra performance li-quid chromatography coupled with linear ion trap quadrupole-orbitrap mass spectrometry(UPLC-LTQ-Orbitrap-MS~n). The separation was performed on an Acquity UPLC BEH C_(18) column(2.1 mm×100 mm, 1.7 μm) with a mobile phase of water(containing 0.1% formic acid, A)-acetonitrile(B) with gradient elution. With electrospray ionization source, the data of 70% methanol extract from stem leaf, root and flower of I. sonchifolia were collected by high-resolution full-scan Fourier transform spectroscopy, data dependent acquisition, precursor ion scan, and selected ion monitoring in the negative and positive ion modes. The compounds were identified based on accurate molecular weight, retention time, fragment ions, comparison with reference standard, Clog P and references. A total of 131 compounds were identified from the 70% methanol extract of I. sonchifolia, including nucleosides, flavonoids, organic acids, terpenoids, and phenylpropanoids, and 119, 110, and 126 compounds were identified from the stem leaf, root and flower of I. sonchifolia, respectively. In addition, isorhamnetin, isorhamnetin-7-O-sambubioside and caffeylshikimic acid were discovered from I. sonchifolia for the first time. This study comprehensively analyzed and compared the chemical constituents in different parts of I. sonchifolia, which facilitated the discovery of effective substances and the development and application of medicinal material resources of I. sonchifolia.

Effect of processing method on chemical constituents of Rehmanniae Radix: based on UHPLC-LTQ-Orbitrap MS / 中国中药杂志

This study aims to explore the chemical composition of Rehmanniae Radix braised with mild fire and compare the effect of processing method on the chemical composition of Rehmanniae Radix. To be specific, ultra-high performance liquid chromatography with linear ion trap-orbitrap mass spectrometry(UHPLC-LTQ-Orbitrap MS) was used to screen the chemical constituents of Rehmanniae Radix. The chemical constituents were identified based on the relative molecular weight and fragment ions, literature information, and Human Metabolome Database(HMDB). The ion peak area ratio of each component before and after processing was used as the index for the variation. SIMCA was employed to establish principal component analysis(PCA) and orthogonal partial least squares discriminant analysis(OPLS-DA) models of different processed products. According to the PCA plot, OPLS-DA plot, and VIP value, the differential components before and after the processing were screened out. The changes of the content of differential components with the processing method were analyzed. A total of 66 chemical components were identified: 57 of raw Rehmanniae Radix, 55 of steamed Rehmanniae Radix, 55 of wine-stewed Rehmanniae Radix, 51 of repeatedly steamed and sundried Rehmanniae Radix Praeparata, 62 of traditional bran-braised Rehmanniae Radix, and 63 of electric pot-braised Rehmanniae Radix. Among them, the 9 flavonoids of braised Rehmanniae Radix were from Citri Reticulatae Pericarpium. PCA suggested significant differences in the chemical composition of Rehmanniae Radix Praeparata prepared with different processing methods. OPLS-DA screened out 32 chemical components with VIP value >1 as the main differential components. Among the differential components, 9 were unique to braised Rehmanniae Radix(traditional bran-braised, electric pot-braised) and the degradation rate of the rest in braised(traditional bran-braised, electric pot-braised) or repeatedly steamed and sundried Rehmanniae Radix was higher than that in the steamed or wine-stewed products. The results indicated the chemical species and component content of Rehmanniae Radix changed significantly after the processing. The 32 components, such as rehmapicrogenin, martynoside, jionoside D, aeginetic acid, hesperidin, and naringin, were the most important compounds to distinguish different processed products of Rehmanniae Radix. The flavonoids introduced by Citri Reticulatae Pericarpium as excipient may be the important material basis for the effectiveness of braised Rehmanniae Radix compared with other processed products.

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

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

Characterization of Effective Components in Guizhi Jia Gegentang for Intervention of Influenza Virus Pneumonia Based on Serum Pharmacochemistry of Traditional Chinese Medicine / 中国实验方剂学杂志

ObjectiveTo characterize the efficacy components of Guizhi Jia Gegentang（GGT） in intervening influenza virus pneumonia by ultra-performance liquid chromatography-quadrupole-electrostatic field orbitrap high resolution mass spectrometry（UPLC-Q-Exactive Orbitrap MS）. MethodBALB/c mice were randomly divided into normal group and GGT group（36 g·kg-1·d-1） with six mice in each group. GGT group was continuously administered GGT extract for 5 d， while the normal group was administered an equal amount of ultrapure water. Serum and lung tissue were collected after administration， and UPLC-Q-Exactive Orbitrap MS was used to characterize the prototypical and metabolic components of GGT in serum and lung tissue of mice. The components existed simultaneously in the serum and lung tissue of mice from the GGT group were defined as its functional components， and the targets of efficacy components were searched by SwissTargetPrediction database， and GeneCards database was used to query the target of influenza virus pneumonia， and then the intersection was taken to obtain potential targets of GGT for intervening in the disease. Protein-protein interaction（PPI） network analysis of potential targets was performed by STRING database， and Kyoto Encyclopedia of Genes and Genomes（KEGG） pathway enrichment analysis on potential targets was performed by Metascape. ResultA total of 29 prototypical components and 28 metabolic components of GGT were detected in the drug-containing serum of mice， of which 11 prototypical components and 4 metabolic components were detected in the lung tissue of mice. The main metabolic pathways included reduction， hydroxylation， methylation， glucuronidation and sulfation. The results of PPI network and KEGG analysis showed that these functional components may act through their effects on targets such as albumin（ALB）， epidermal growth factor receptor（EGFR）， steroid receptor coactivator（SRC）， Toll-like receptor 4（TLR4）， nuclear transcription factor（NF）-κB and adhesion junction. ConclusionThe 11 prototypical components and 4 metabolites present simultaneously in the drug-containing serum and lung tissue of mice may be the potential therapeutic components of GGT in interfering with influenza viral pneumonia， and act through interfering with inflammatory metabolic pathways. This study can provide a reference for the mechanism study of GGT in the treatment of influenza viral pneumonia.

Component profile analysis of Sanhan Huashi Formula based on UHPLC-LTQ-Orbitrap-MS, GC-MS, and UHPLC-QQQ-MS/MS / 中国中药杂志

This study comprehensively analyzed the active components of Sanhan Huashi Formula using qualitative and quantitative mass spectrometry techniques, laying the foundation for understanding its pharmacological substance basis. UHPLC-LTQ-Orbitrap-MS and GC-MS technologies were used to analyze and identify the volatile and non-volatile components in Sanhan Huashi Formula. UHPLC-QQQ-MS/MS technology was used to simultaneously determine the content of 27 major active components in the formula. The results showed that 308 major chemical components were identified in Sanhan Huashi Formula, among which 60 compounds were identified by comparing with reference standards, mainly including alkaloids, flavonoids, coumarins, triterpenoid saponins, amino acids, and nucleosides. GC-MS technology preliminarily identified 52 volatile compounds, with γ-eudesmol and β-eudesmol as the main components. The quantitative results demonstrated good linearity(r>0.99) for the 27 active components, indicating the stability, simplicity, and reliability of the established method. Among them, amygdalin, nodakenin, arecoline, ephedrine, and pseudoephedrine had relatively high content and were presumably the main pharmacologically active substances. In conclusion, this study systematically and comprehensively characterized the major chemical components and patterns in Sanhan Huashi Formula, providing a basis for understanding its pharmacological mechanisms and clinical applications.

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.

Chemical constituents and mechanism of Chuanzhi Tongluo Capsules based on UPLC-Q-Exactive Orbitrap-MS and network pharmacology / 中国中药杂志

The chemical constituents of Chuanzhi Tongluo Capsules were analyzed and identified using ultra-high performance liquid chromatography-quadrupole/electrostatic field orbitrap high-resolution mass spectrometry(UPLC-Q-Exactive Orbitrap-MS) to clarify the pharmacological substance basis. In addition, network pharmacology was employed to explore the mechanism of Chuanzhi Tongluo Capsules in the treatment of cerebral infarction. Gradient elution was performed using acetonitrile and 1% acetic acid in water as the mobile phase. Mass spectrometry was performed in positive and negative ion modes. Xcalibur 4.2 software was used for compound analysis, including accurate mass-to-charge ratio and MS/MS fragment information, combined with the comparison of reference standards and literature data. A total of 152 compounds were identified, including 32 organic acids, 35 flavonoids and their glycosides, 33 diterpenes, 13 phthalides, 12 triterpenes and triterpene saponins, 23 nitrogen-containing compounds, and 4 other compounds, and their fragmentation patterns were analyzed. SwissTargetPrediction, GeneCards, DAVID, and other databases were used to predict and analyze the core targets and mechanism of Chuanzhi Tongluo Capsules. Protein-protein interaction(PPI) network topology analysis identified 10 core targets, including TNF, VEGFA, EGFR, IL1B, and CTNNB1. KEGG enrichment analysis showed that Chuanzhi Tongluo Capsules mainly exerted their effects through the regulation of lipid and atherosclerosis, glycoproteins in cancer, MicroRNAs in cancer, fluid shear stress, and atherosclerosis-related pathways. Molecular docking was performed between the key constituents and core targets, and the results demonstrated a strong binding affinity between the key constituents of Chuanzhi Tongluo Capsules and the core targets. This study comprehensively elucidated the chemical constituents of Chuanzhi Tongluo Capsules and explored the core targets and mechanism in the treatment of cerebral infarction based on network pharmacology, providing a scientific reference for the study of the pharmacological substance basis and formulation quality standards of Chuanzhi Tongluo Capsules.

Exploring of Transformation Rule of Saponins in Polygalae Radix Before and After Processing Based on Simulated Processing Technology / 中国实验方剂学杂志

ObjectiveTo investigate the transformation mechanism and content variation of saponins from Polygalae Radix before and after being boiled with licorice juice and water. MethodSimulated licorice juice boiled products and simulated water boiled products of onjisaponin B， onjisaponin Z， onjisaponin F， polygalasaponin ⅩⅩⅧ were prepared by simulated processing technology， and analyzed by ultra-performance liquid chromatography-quadrupole-electrostatic field orbitrap high resolution mass spectrometry（UPLC-Q-Exactive Orbitrap/MS）. Then the contents of onjisaponin B， onjisaponin Z， onjisaponin F， polygalasaponin ⅩⅩⅧ and tenuifolin in Polygalae Radix， licorice-boiled Polygalae Radix and water-boiled Polygalae Radix were determined by UPLC-triple quadrupole tandem mass spectrometry（UPLC-QQQ-MS/MS）. ResultDuring the boiling process with licorice juice and water， onjisaponin B could be hydrolyzed to produce 4-methoxycinnamic acid， desacylsenegin Ⅲ， polygalasaponin ⅩⅩⅧ and tenuifolin， onjisaponin Z could be hydrolyzed to produce 3，4，5-trimethoxycinnamic acid， onjisaponin TF， polygalasaponin ⅩⅩⅧ and tenuifolin， onjisaponin F could be hydrolyzed to produce 3，4，5-trimethoxycinnamic acid， onjisaponin G， polygalasaponin ⅩⅩⅧ and tenuifolin， and polygalasaponin ⅩⅩⅧ was hydrolyzed to produce tenuifolin. After being boiled with licorice juice or water， the content of onjisaponin B decreased significantly（P<0.05， P<0.01）， but the contents of onjisaponin Z， onjisaponin F， polygalasaponin ⅩⅩⅧ and tenuifolin increased significantly（P<0.05， P<0.01） in Polygalae Radix. Compared with the water-boiled products， the contents of onjisaponin Z and tenuifolin increased significantly（P<0.05， P<0.01）， and the change of tenuifolin content was the most significant in the licorice-boiled products.However， there was no significant difference in the content of onjisaponin B， onjisaponin F and polygalasaponin ⅩⅩⅧ between the water-boiled products and the licorice-boiled products. ConclusionBeing boiled with licorice juice or water can hydrolyze onjisaponin B， onjisaponin Z， onjisaponin F and polygalasaponin ⅩⅩⅧ， and generate secondary glycosides and aglycones（organic acids） through deglycosylation， which leads to obvious changes in the contents of onjisaponins after Polygalae Radix being processed.It is inferred that licorice juice can promote the hydrolysis of some onjisaponins in Polygalae Radix to onjisaponin Z and tenuifolin.This study provides an experimental basis for revealing processing mechanism of Polygalae Radix.

Establishment of Specific Chromatogram and TLC Identification for Qingxin Lianziyin / 中国实验方剂学杂志

ObjectiveTo establish the specific chromatogram and thin layer chromatography（TLC） of Qingxin Lianziyin（QXLZY） benchmark samples， in order to clarify the key quality attributes and provide a reference for the quality evaluation of QXLZY. MethodHigh performance liquid chromatography（HPLC） specific chromatogram of QXLZY benchmark samples was developed by using a YMC Hydrosphere C18 column（4.6 mm×250 mm， 5 μm） with the mobile phase of acetonitrile（A）-0.2% formic acid aqueous solution（B） for gradient elution（0-10 min， 5%-20%A； 10-20 min， 20%A； 20-25 min， 20%-24%A； 25-40 min， 24%-30%A； 40-55 min， 30%-50%A； 55-65 min， 50%-100%A； 65-75 min， 100%A； 75-75.1 min， 100%-5%A； 75.1-90 min， 5%A）， and the detection wavelength was 360 nm. Ultra-high performance liquid chromatography-linear ion trap/orbitrap mass spectrometry（UHPLC-LTQ-Orbitrap MS） with electrospray ionization（ESI） was used to identify the components of QXLZY benchmark samples by accurate relative molecular weight and multilevel MS fragment ion information， the detection conditions were positive and negative ion modes and data dependency scanning mode. TLC identification methods for Ophiopogonis Radix， Lycii Cortex， Nelumbinis Semen， Poria， Astragali Radix and Ginseng Radix et Rhizoma in QXLZY were established. ResultA total of 15 characteristic peaks were identified from Glycyrrhizae Radix et Rhizoma， Plantaginis Semen and Scutellariae Radix， and the relative standard deviations of the retention times of 15 characteristic peaks in 15 batches of QXLZY benchmark samples were≤3% with peak 8（baicalin） as the reference peak. A total of 100 compounds， including flavonoids， organic acids， saponins， amino acids and others， were identified in the benchmark samples by UHPLC-LTQ-Orbitrap MS. The established TLC had good separation and was suitable for the identification of Ophiopogonis Radix， Lycii Cortex， Nelumbinis Semen， Poria， Astragali Radix and Ginseng Radix et Rhizoma in QXLZY. ConclusionThe material basis of QXLZY benchmark samples is basically determined by MS designation and source attribution. The established specific chromatogram and TLC of QXLZY are simple， stable and reproducible， which can provide a reference for the development and quality control of QXLZY.