Qualitative and Quantitative Analysis of Chemical Constituents in Liu Junzitang by UPLC-Q-TOF-MS/MS and UPLC / 中国实验方剂学杂志

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.

Toxicity Attenuation Mechanism on Processing Method for Aconiti Lateralis Radix Praeparata in Guilingji Based on Urine Metabolomics / 中国实验方剂学杂志

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.

Analysis of Antidepressant Quality Markers of Bupleuri Radix Before and After Vinegar-processing Based on UPLC-Q-TOF-MS and Network Pharmacology / 中国实验方剂学杂志

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.

[Determination of seven mycotoxins in cereals by ultra performance liquid chromatography-quadrupole-time of flight mass spectrometry based on the self-built database].

A method for the determination of seven mycotoxins in rice and wheat by ultra performance liquid chromatography-quadrupole-time of flight mass spectrometry (UPLC-Q-TOF/MS) based on self-built database was established. Samples were extracted with 0.2% formic acid aqueous solution-acetonitrile (50∶50, v/v), dehydrated and salted according to the QuEChERS method (4 g of magnesium sulfate, 1 g of sodium chloride, 1 g of sodium citrate, 0.5 g of citrate disodium salt), and separated on an HSS T3 column (100 mm×2.1 mm, 1.8 µm). UPLC-Q-TOF/MS with MSE screening was performed, and the positive and negative ions of the screened mycotoxins were calibrated and quantified using matrix-matched standard curves with time of flight multiple reaction monitoring (TOF-MRM). The results showed that aflatoxin G1 (AFG1), aflatoxin G2 (AFG2), aflatoxin B1 (AFB1), aflatoxin B2 (AFB2), and ochratoxin A (OTA) exhibited moderate matrix effects in rice, while OTA and zearalenone (ZEN) exhibited moderate matrix effects in wheat. The seven mycotoxins showed good linearities in their respective concentration ranges, with correlation coefficients (r) of 0.9900-0.9998. The limits of detection (LODs) for rice and wheat were 0.50-400 and 0.50-200 µg/kg, respectively, and the limits of quantification (LOQs) for both cereals were 1.00-800 µg/kg. In rice, the average recoveries at three spiked levels of low, medium, and high were 88.1%-123.9%, with relative standard deviations (RSDs) of 0.2%-13.6%. In wheat, the average recoveries at three spiked levels of low, medium, and high were 102.0%-123.4%, with RSDs of 0.8%-14.8%. As a result, one batch of 46 batches of rice was screened out for AFB1 and AFB2, with a screening rate of 2.2%, of which the measured values were 10.8 µg/kg and 1.2 µg/kg, respectively. According to GB 2761-2017, the maximum allowable level of AFB1 in rice is 10 µg/kg; thus, the exceeding rate for AFB1 is 2.2%. Deoxynivalenol (DON) was screened out in 9 out of 24 batches of wheat (screening rate, 37.5%), while ZEN was screened out in 19 batches (screening rate, 79.2%). According to GB 2761-2017, the maximum allowable levels of DON and ZEN in wheat are 1000 and 60 µg/kg, respectively. The levels of DON and ZEN detected in the wheat samples did not exceed these limits. The proposed method uses MSE for qualitative screening to avoid the occurrence of false positives caused by interfering compounds with mass numbers and retention times similar to those of the analytes. TOF-MRM mode is then used to quantify the positively screened mycotoxins. The method is fast, accurate, sensitive, and suitable for the isolation and quantitative detection of mycotoxin residues in rice and wheat samples. The findings provide powerful technical support for mycotoxin contamination monitoring in rice and wheat and early risk-warning efforts.

[Establishment of non-targeted screening database and confirmation method for 18 mycotoxins in grains using ultra performance liquid chromatography-quadrupole-time of flight mass spectrometry].

A mass spectral library of 18 mycotoxins was developed based on ultra performance liquid chromatography-quadrupole-time of flight mass spectrometry (UPLC-Q-TOF/MS), which was used to establish a non-targeted screening method for mycotoxins in rice and wheat matrices. Eighteen mycotoxin standards were separated on an HSS T3 column, and data were collected for both positive and negative ionization under the MSE mode of the UPLC-Q-TOF/MS. Details including formulas, retention times, theoretical exact masses, measured exact masses of the adduct and fragment ions, and ion abundance ratios were recorded to establish the mass spectral library of the 18 mycotoxins in UNIFI Software. Analyte detection was based on a retention time deviation of 0.3 min, and the exact mass deviation of the adduct ions and fragment ions was set to 5×10-6. The screening detection limit (SDL) was used as the main threshold for verifying the screening method. In the validation process, 18 mycotoxins were classified into two types: with maximum levels (MLs) and without MLs. The results showed that the mycotoxins with MLs could be accurately screened at their limited level, and the mycotoxins without MLs had a range of SDL concentration from 2 to 800 µg/kg. The matrix effect results showed that 14 mycotoxins in rice and 11 in wheat had moderate matrix effects. Finally, 25 batches of rice and wheat were purified using QuEChERS and HLB columns after acetonitrile extraction and screening were performed by employing the established method. The results revealed that aflatoxin G1 (AFG1), aflatoxin G2 (AFG2), fumonisins B1 (FB1), and sterigmatocystin (ST) were detected in one batch of rice, FB1 and ST were detected in another batch of rice, FB1 and ochratoxin A (OTA) were detected in two batches of wheat, and no other mycotoxins were detected. This method is characterized by high throughput, simplicity, rapidity, accuracy, and can be applied to accurately screen mycotoxins with concentrations higher than the SDLs and qualitatively screen various mycotoxins in rice and wheat without standards.

Analysis of Chemical Constituents in Puerariae Lobatae Radix Dispensing Granules by UPLC-Q-TOF-MS/MS / 中国实验方剂学杂志

ObjectiveA rapid method for identification of chemical constituents in Puerariae Lobatae Radix dispensing granules was established in order to clarify the material basis. MethodThe chemical constituents of Puerariae Lobatae Radix dispensing granules was qualitatively analyzed by ultra-performance liquid chromatography-quadrupole-time-of-flight mass spectrometry（UPLC-Q-TOF-MS/MS） under positive and negative ion modes， and the chromatographic conditions were on an ACQUITY UPLC HSS T3 column（2.1 mm×100 mm， 1.8 μm） with 0.1% formic acid aqueous solution（A）-0.1% formic acid acetonitrile solution（B） as mobile phase for gradient elution（0-4 min， 5%-10%B； 4-10 min， 10%-15%B； 10-20 min， 15%-16%B； 20-27 min， 16%-31%B； 27-33 min， 31%-59%B； 33-42 min， 59%-95%B； 42-42.1 min， 95%-5%B； 42.1-45 min， 5%B）， the flow rate was 0.35 mL·min-1， the column temperature was 40 ℃， the injection volume was 5 μL， and electrospray ionization（ESI） was selected. Then these chemical constituents were comprehensively identified based on PeakView 1.2， PubChem， ChemicalBook， ChemSpider， comparative control profiles and literature information. ResultA total of 128 chemical constituents were identified from the dispensing granules， including 60 flavonoids， 26 organic acids， 7 glycosides， 6 coumarins， 3 nucleosides and 26 other compounds. By focusing on the cleavage patterns of flavonoids， organic acids， glycosides， coumarins， nucleosides and other compounds， 12 compounds that have not been reported in Puerariae Lobatae Radix species were identified from the dispensing granules. ConclusionThe established method can systematically and rapidly identify the chemical constituents in Puerariae Lobatae Radix dispensing granules， and cleared it composition is mainly flavonoids and organic acids. Laying a foundation for the study of the material basis， mechanism of action and clinical application of the dispensing granules.

Quality Marker （Q-marker） of Tinosporae Radix Associated with Efficacy of "Relieving Sore Throat" / 中国实验方剂学杂志

ObjectiveTo study the potential quality marker （Q-marker） of Tinosporae Radix associated with efficacy of "relieving sore throat" based on ultra-performance liquid chromatography-quadrupole time-of-flight mass spectrometry （UPLC-Q-TOF-MS）， multivariate statistical analysis （MSA）， and network pharmacology. MethodUPLC-Q-TOF-MS was used to identify the main chemical components in 18 batches of Tinosporae Radix. On this basis， principal component analysis （PCA） and orthogonal partial least squares-discriminant analysis （OPLS-DA） were employed to screen out the main marker components that caused differences between groups. Moreover， network pharmacology technology was applied to predict the potential "sore throat-relieving" components， and the molecular docking between the common components resulting from MSA and network pharmacology and the core targets was carried out to verify the marker components. ResultA total of 17 compounds， including alkaloids， diterpenoid lactones， and sterols， were identified by UPLC-Q-TOF-MS. Five main differential components were found by MSA： Columbamine， jatrorrhizine， palmatine， menisperine， and columbin. Network pharmacology analysis yielded six compounds： tetrahydropalmatine， palmatine， menisperine， fibleucin， neoechinulin A， and columbin which were selected as potential "sore throat-relieving" components of Tinosporae Radix. They may relieve sore throat by acting on interleukin-6， epidermal growth factor receptor， prostaglandin G/H synthase 2， matrix metalloproteinase-9， proto-oncogene tyrosine-protein kinase Src and other targets， and regulating Hepatitis B， influenza A， human T-cell virus infection， human cytomegalovirus infection， coronavirus disease-2019， and other signaling pathways. The common active components in Tinosporae Radix resulting from MSA and network pharmacology analysis were palmatine， menisperine， and columbin， which had high binding affinity with six core targets and can be used as the Q-marker components of Tinosporae Radix in "relieving sore throat". ConclusionThis study predicts the "sore throat-relieving" Q-marker of Tinosporae Radix， which lays a basis for developing the quality standard of Tinosporae Radix based on the efficacy and improving the quality evaluation system of the medicinal.

Exploration of Material Basis of "Clearing Excessive Fire of Liver-gallbladder" of Bile-processed Coptidis Rhizoma Based on UPLC-Q-TOF/MS Metabolomics and Molecular Docking Technology / 中国实验方剂学杂志

ObjectiveTo explore the material basis of bile-processed Coptidis Rhizoma clearing excessive fire of liver-gallbladder based on ultra performance liquid chromatography-quadrupole-time-of-flight mass spectrometry（UPLC-Q-TOF/MS） metabolomics and molecular docking. MethodUPLC-Q-TOF/MS metabolomics was used to analyze the chemical constituents of Coptidis Rhizoma， water-processed Coptidis Rhizoma and bile-processed Coptidis Rhizoma. Chromatographic separation was achieved with 0.1% formic acid aqueous solution（A）-acetonitrile（B） as the mobile phase in gradient elution（0-2 min， 5%B； 2-20 min， 5%-65%B； 20-40 min， 65%-10%B； 40-45 min， 10%B； 45-46 min， 10%-95%B； 46-49 min， 95%B）， and electrospray ionization（ESI） was applied and operated in positive and negative ion modes， the acquisition range was m/z 80-1 200. Based on this， partial least squares-discriminant analysis（PLS-DA） and variance analysis were used to screen the differential compounds among the three products of Coptidis Rhizoma. Network pharmacology and molecular docking were used to verify the degree of association between differential compounds and excessive fire of liver-gallbladder syndrome. ResultA total of 33 chemical constituents were identified， including 2 phenolic acids， 5 binding bile acids and 26 alkaloids. And 16 differential compounds were identified by multivariate statistical analysis， including 11 alkaloids and 5 binding bile acids. Pathway enrichment analysis in the Kyoto Encyclopedia of Genes and Genomes（KEGG） database yielded 8 pathways related to excessive fire of liver-gallbladder， and the key protein phosphatidylinositol 4，5-bisphosphate 3-kinase catalytic subunit alpha isoform（PIK3CA） was obtained according to the "component-target-pathway" network analysis. Molecular docking results showed that 11 alkaloids had good binding ability with PIK3CA. ConclusionPorcine bile is unique in the processing of bile-processed Coptidis Rhizoma， which can promote the production and dissolution of 11 alkaloids， including berberine and dihydrochelerythrine. Based on the results of molecular docking and reported pharmacological experiments， it can be concluded that 16 different compounds such as berberine， dihydrochelerythrine and taurohyodeoxycholic acid are the material basis of bile-processed Coptidis Rhizoma.

Analysis on Material Basis of Anti-COPD Effect of Euphorbia helioscopia Based on Serum Pharmacochemistry and Network Pharmacology / 中国实验方剂学杂志

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.

Establishment of A Quality Control Method for Simultaneous Determination of Multiple Active Components in Uygur Medicine of Nymphaeae Flos / 中国实验方剂学杂志

ObjectiveTo develop a quality control method for the simultaneous determination of multiple active components in Nymphaeae Flos aiming at the problems of the single index for quality control and the relatively low overall quality control level. MethodUltra performance liquid chromatography-quadrupole-time-of-flight mass spectrometry（UPLC-Q-TOF-MS/MS）was used to identify and select the index components for quality control with the mobile phase of 0.1% formic acid aqueous solution（A）-acetonitrile（B）for gradient elution （0-2 min， 3%-8%B； 2-4 min， 8%-10%B； 4-13 min， 10%-15%B； 13-19 min， 15%-20%B； 19-26 min， 20%-45%B） at a flow rate of 0.4 mL·min-1， detection wavelength of 350 nm， electrospray ionization（ESI）， negative ion scanning mode， ion source temperature of 120 ℃， scanning range of m/z 100-1 200， transmit collision energy of 6 eV for low-energy scanning and 25-50 eV for high-energy scanning. High performance liquid chromatography（HPLC）was used to establish the quality control method for the simultaneous determination of multi-index components with the mobile phase of 0.2% phosphoric acid aqueous solution（A）-acetonitrile（B） for gradient elution（0-30 min， 12%-15%B； 30-60 min， 15%-22%B； 60-90 min， 22%-40%B）and detection wavelength of 350 nm. The preparation method of the test solution for content determination was refluxing extraction for 60 min with 80 times the amount of 70% methanol. ResultBy comparing the retention time， ultraviolet absorption characteristics， MS and MS/MS spectrometric signals in the samples with the reference substances， 8 active components with high contents， including brevifolincarboxylic acid， ellagic acid， rutin， nicotiflorin， astragalin， quercetin， quercetin-3-methylether and kaempferol， were identified qualitatively from Nymphaeae Flos， which were selected as the index components for quality control. Under the established HPLC conditions， the above 8 components could be well separated（resolution>1.5）， and showed good linearity（r=0.999 9）between the concentration ranges of 1.99-99.6， 1.76-176， 1.52-75.8， 3.60-180， 0.964-96.4， 1.18-118， 1.94-96.8， 1.04-104 mg·L-1 and the peak areas， respectively. The detection limits of them were 10-49 μg·L-1， and the limits of quantitation were 34-164 μg·L-1. The average recoveries were 97.12%-103.1% with the relative standard deviations （RSDs） were 1.1%-2.2%. ConclusionA quality control method for simultaneous determination of the multiple active components in Nymphaeae Flos have been developed， which is simple， accurate and reproducible， and it can provide a scientific basis for the formulation of quality standard of this herb and lay a research foundation for the transformation of Uygur hospital preparations containing Nymphaeae Flos into new drugs.

[Rapid screening and confirmation of 86 illegally added chemicals in fishery drugs by ultra performance liquid chromatography-quadrupole-time-of-flight mass spectrometry].

With the rapid expansion of fisheries, one of the most significant limitations to the sustainable development of fisheries in China is the quality and safety of fishery products owing to the abuse of fishery drugs and the use of illegal and/or restricted chemicals in fishery drugs. A range of chemicals that are potential hazards to fishery drugs were selected for screening in this study. A comprehensive analytical method was developed, based on ultra performance liquid chromatography-quadrupole-time-of-flight mass spectrometry (UPLC-Q-TOF-MS), for the rapid screening of 86 types of illegally added chemicals in fishery drugs. The fishery drug samples were extracted with 80% (v/v) acetonitrile aqueous solution and diluted to reduce matrix effects. The 86 target compounds were separated on an ACQUITY PREMIER HSS T3 column (100 mm×2.1 mm, 1.8 µm), with methanol and 0.1% formic acid as mobile phases, via gradient elution. The extract was directly analyzed by UPLC-Q-TOF-MS using electrospray ionization in the positive mode. The external standard method was used for quantification. In this study, the extraction reagent and purification procedure were selected to develop a simple and effective pre-treatment protocol. The effects of the chromatographic column, mobile phase, and fragmentation voltage on the separation and sensitivity of the 86 substances were evaluated to determine the optimum instrument conditions. An accurate mass database and fragment ion library were created for the rapid qualitative and quantitative analysis of the 86 illegally added chemicals in fishery drugs. The retention time, isotopic abundance and spacing, and precise mass of the principal diagnostic ion for each analyte were used for identification. The information on the fragment ions obtained from the target MS/MS profiles was compared with that from a database to ensure the accuracy of the qualitative results. The chromatographic peak area of each target analyte was used for quantification. The analytical detection was based on the retention time deviation of ±0.35 min, accurate mass deviation of ±10×10-6, and major adduct forms, including [M+H]+, [M+Na]+, and [M+NH4]+. To evaluate the matrix effects of the 86 target chemicals at varied dilution ratios, two types of antibiotics and four types of Chinese herbal medicines were selected as typical samples. Considering the instrument tolerance as well as sensitivity and accuracy of the procedure, the recommended dilution ratios for antibiotics and Chinese herbal medicines were 50 times and 10 times, respectively. Two different types of calibration curves were prepared; one was the solvent calibration curve for antibiotics and the other was the matrix calibration curve for Chinese herbal medicines. For a given concentration, the calibration curves of the 86 target chemicals were linear with correlation coefficients of at least 0.99. The recoveries ranged from 76.8% to 112.1% with relative standard deviations (RSDs) (n=3) of less than 11.7%. The limit of quantification (LOQ) ranges of the compounds in Chinese herbal medicines and antibiotics were 1-15 mg/kg and 5-75 mg/kg, respectively. To evaluate the screening detection limits (SDLs) of each compound, a mixed standard solution was added to a fishery drug sample at varied concentrations. The SDL ranges of the compounds in Chinese herbal medicines and antibiotics were 1-15 and 5-50 mg/kg, respectively. This approach resulted in SDLs that satisfy the actual screening requirements. Because of its rapid nature, simplicity, accuracy, and sensitivity, the method may be used in the high-throughput screening and identification of illegally added chemicals in many types of fishery drugs. This method was applied to a monitoring project for the quality and safety of fishery inputs in Zhejiang Province. Sixty fishery drug samples were evaluated, among which eight Chinese herbal medicine samples were found to contain unspecified ingredients and one antibiotic sample was found to be free of any active substances. Thus, an effective technical method to monitor the quality and safety of fishery drugs was developed.

Metabolomics Analysis of Plasma at Different Stages Based on UPLC-Q-TOF/MS： Modeling of Spleen Deficiency with Dampness Retention-ulcerative Colitis Rats and Administration of Shenling Baizhusan / 中国实验方剂学杂志

ObjectiveBased on ultra performance liquid chromatography-quadrupole time-of-flight mass spectrometry （UPLC-Q-TOF/MS）， the changes of endogenous markers in rat plasma at the different stage， namely modeling and administration of Shenling Baizhusan （SLBZS）， and the mechanism of SLBZS in the treatment of ulcerative colitis （UC） were studied. MethodIn the modeling stage， rats were randomly divided into normal group， spleen deficiency with dampness retention-UC （SDDR-UC） and pure-UC （P-UC） model group. In the administration stage， SLBZS was given to the above two different model groups. After modeling and administration， rat plasma was collected and determined by UPLC-Q-TOF/MS. The mobile phase was 0.1% formic acid aqueous solution （A）-acetonitrile （B） for gradient elution （in positive ion mode：0-2 min， 99%A； 2-9 min， 99%-73%A； 9-10 min， 73%-44%A； 10-13 min， 44%-38%A； 13-19 min， 38%-28%A； 19-21 min， 28%-2%A； 21-23 min， 2%A； 23-25 min， 2%-10%A； 25-27 min， 10%-99%A； in negative ion mode：0-2 min， 85%A； 2-3 min， 85%-65%A； 3-5.5 min， 65%-44%A； 5.5-8 min， 44%-25%A； 8-10 min， 25%-2%A； 10-16 min， 2%-85%A）. The electrospray ionization （ESI） temperature was 120 ℃ under the positive and negative ion modes， and the acquisition range was 50-1 000. Partial least squares-discriminant analysis （PLS-DA） was used to analyze the changes of endogenous metabolites in the above two different model rats from the different stage. MetaboAnalyst 5.0 was used to analyze the metabolic pathways of these identified metabolites. ResultSixteen potential biomarkers were screened and identified in the modeling stage， among which 11 potential biomarkers were common in the two model rats， which mainly affected the primary bile acid biosynthesis pathway. Twenty-three potential biomarkers were screened and identified during the administration stage， among which 3 potential biomarkers were shared by the two model rats， and SDDR-UC and P-UC model rats had 11 and 9 potential biomarkers， respectively. It mainly affected 6 pathways such as purine metabolism， pentose phosphate pathway， pyrimidine metabolism， retinol metabolism， primary bile acid biosynthesis and steroid hormone synthesis. ConclusionThe primary bile acid biosynthesis pathway appears in the different stage of modeling and administration of UC， showing a dynamic change process. The therapeutic effect of SLBZS on SDDR-UC rats may be related to inhibiting the expression of nuclear transcription factor -κB （NF-κB） signaling pathway， activating farnesoid X receptor （FXR） and enhancing the expression of cytochrome P450.

Explanation of Scientific Connotation of Euodiae Fructus Stir-fried with Coptidis Rhizoma Based on UPLC-Q-TOF/MS and Pattern Recognition Technology / 中国实验方剂学杂志

ObjectiveTo analyze changes of the chemical composition in Euodiae Fructus before and after processing with Coptidis Rhizoma decoction， so as to provide scientific basis for elucidating the processing mechanism of this decoction pieces. MethodUltra-performance liquid chromatography-quadrupole-time-of-flight mass spectrometry （UPLC-Q-TOF/MS） was performed on a Titank C18 column （2.1 mm×100 mm， 1.8 μm）， the mobile phase was 0.1% formic acid aqueous solution-acetonitrile for gradient elution， the column temperature was set at 40 ℃， the flow rate was 0.25 mL·min-1. Electrospray ionization （ESI） was used to scan in positive and negative ion modes， and the scanning range was m/z 50-1 250. The chemical constituents in Euodiae Fructus were identified before and after processing by reference substance comparison， database matching and literature reference， and MarkerView™ 1.2.1 software was used to normalize the obtained data， SIMCA-P 14.1 software was employed to perform principal component analysis （PCA） and orthogonal partial least squares-discriminant analysis （OPLS-DA） on MS data of raw and processed products to screen the differential components before and after processing. ResultA total of 50 compounds were identified， including 48 kinds of stir-fried products with Coptidis Rhizoma decoction and 44 kinds of raw products. After processing， six compounds were added， including danshensu， noroxyhydrastinine， oxyberberine， 13-methylberberrubine， protopine and canadine. However， two kinds of compounds， including （S）-7-hydroxysecorutaecarpine and wuchuyuamide Ⅱ， were not detected after processing. In general， after processing， the overall contents of phenolic acids and flavonoids decreased significantly， the overall content of limonoids increased， and the overall content of alkaloids did not decrease insignificantly. The results of PCA and OPLS-DA showed that there were significant differences in the composition and content of the chemical components of Euodiae Fructus before and after processing， and a total of 12 variables such as quercetin， dihydrorutaecarpine and dehydroevodiamine were obtained by screening. ConclusionEuodiae Fructus stir-fried with Coptidis Rhizoma decoction mainly contains phenolic acids， flavonoids， limonoids and alkaloids. The composition and content of the chemical components have some changes before and after processing. The addition of processing excipients and hot water immersion are the main reasons for the difference， which can provide experimental basis for interpretation of the processing mechanism of this characteristic processed products of Euodiae Fructus.

Analysis of Effective Terpenoids in Xinjiang Pleurotus ferulae Against Esophageal Carcinoma by UPLC-Q/TOF-MS Technique / 中国实验方剂学杂志

Objective:To discover the effective terpenoids in Xinjiang <italic>Pleurotus ferulae</italic> with the activity of anti-esophageal carcinoma. Method:By screening the activity of human esophageal cancer Eca109 cells， the ethyl acetate extract phase of <italic>P. ferulae </italic>ethanol extract （PFEP-E） was separated and purified by silica gel chromatography and preparative thin layer chromatography. Ultra-performance liquid chromatography-quadrupole-time-of-flight mass spectrometry （UPLC-Q/TOF-MS） combined with online network databases such as Metlin， MassBank， PubChem and related literature were used to identify the effective elution sites and analyze their contents. Result:The elution fraction （Fr_2-3∶1） of petroleum ether-ethyl acetate （3∶1） on the silica gel column had the strongest inhibitory activity on the proliferation of human esophageal cancer Eca109 cells. The component analysis showed that 24 effective terpenoids were identified under positive ion mode， and 28 effective terpenoids were identified under negative ion mode， a total of 52 terpenoids were identified， which were isolated from this edible fungus for the first time. Content of total terpenoids in Fr_2-3∶1 were 62.88 mg·g^-1， including 2 monoterpenoids， 5 sesquiterpenoids， 15 diterpenoids and 30 triterpenoids， accounting for 1.32%， 12.04%， 47.55%， 39.09% of the total terpenoids， respectively. Diterpenoids and triterpenoids were the main components of the effective terpenoids in <italic>P. ferulae</italic>， accounting for 86.64% of the total terpenoids. Gibberellins were the main diterpenoids， accounting for 79.70% of the total diterpenoids， triterpenoids were mainly ganoderic acids， accounting for 29.25% of the total triterpenoids. The results of methyl thiazolyl tetrazolium （MTT） test showed that gibberellin A₃ and gibberellin A₅ had weak anti-esophageal cancer activity， while gypsogenin and oleanolic acid had strong anti-esophageal cancer activity. Conclusion:The effective terpenoids of <italic>P. ferulae</italic> against esophageal cancer are triterpenoids mainly composed of ganoderic acids， which can provide theoretical basis for the development of terpenoids of <italic>P. ferulae</italic> as anti-tumor drugs and the development of functional foods， and help to effectively improve the additional output value of <italic>P. ferulae</italic>.

Identification of Chemical Constituents in Suanzaoren Tang Granules by UPLC-Q-TOF-MS/MS / 中国实验方剂学杂志

Objective:A comprehensive and in-depth analysis method for identification of chemical constituents in Suanzaoren Tang granules was established. Method:Ultra-performance liquid chromatography-quadrupole-time of flight mass spectrometry （UPLC-Q-TOF-MS/MS） was employed with the mobile phase of 0.1% formic acid aqueous solution （A）-acetonitrile （B） for gradient elution （0-8 min， 5%-17%B； 8-10 min， 17%B； 10-11 min， 17%-18%B； 11-12 min， 18%-20%B； 12-17 min， 20%-23%B； 17-22 min， 23%-33%B； 22-30 min， 33%-60%B； 30-32 min， 60%-100%B； 32-36 min， 100%B）， the flow rate of 0.3 mL·min^-1 and electrospray ionization （ESI）. High quality MS/MS data were scanned in positive and negative ion modes with scanning range of <italic>m</italic>/<italic>z</italic> 50-1 500. The local database of the chemical components from different Chinese medicines in Suanzaoren Tang granules was established by SCIEX OS software. Then the chemical components in Suanzaoren Tang granules were characterized by matching with the local database and comparing with the reference substance and literature information. Result:A total of 134 compounds were characterized and identified under positive and negative ion modes， mainly including flavonoids， triterpenoids， phthalides， steroidal saponins， alkaloids and organic phenolic acids. In addition， the sources of Chinese medicines for all compounds identified in Suanzaoren Tang granules were assigned. Among them， 41 were from Ziziphi Spinosae Semen， 11 were from Poria， 22 were from Anemarrhenae Rhizoma， 28 were from Chuanxiong Rhizoma and 35 were from Glycyrrhizae Radix et Rhizoma. Conclusion:The method can be used to identify the chemical constituents in Suanzaoren Tang granules systematically， quickly and accurately， which can provide a new strategy for the rapid and accurate identification of other Chinese patent medicines.

Analysis of Chemical Constituents in Paullinia cupana Dried Seeds by UPLC-Q-TOF-MS / 中国实验方剂学杂志

Objective:The chemical constituents in guarana （<italic>Paullinia cupana</italic> dried seeds） were systematically analyzed to provide a basis for further research， development and utilization of this plant. Method:The contents of crude protein， crude fat， crude polysaccharide and crude fiber in guarana were determined according to national standards and related documents， and the chemical constituents of guarana was qualitatively analyzed by ultra performance liquid chromatography-quadrupole-time-of-flight mass spectrometry （UPLC-Q-TOF-MS）， ACQUITY UPLC-HSS-T3 column （2.1 mm×100 mm， 1.8 μm） was used with 0.1% formic acid aqueous solution （A）-0.1% formic acid acetonitrile solution （B） as mobile phase for gradient elution （0-5 min， 2%-10%B； 5-6 min， 10%-20%B； 6-9 min， 20%-30%B； 9-9.5 min， 30%-35%B； 9.5-10.5 min， 35%-45%B； 10.5~13 min， 45%-55%B； 13-15 min， 55%-80%B； 15-19 min， 80%-98%B； 19-20 min， 98%B； 20-20.3 min， 98%-2%B； 20.3-23 min， 2%B）， the electrospray ionization （ESI） was used for detection in positive and negative ion modes， the scanning range was <italic>m</italic>/<italic>z</italic> 50-1 500， and the structure was identified according to the relative molecular weight and fragment information combined with database matching and comparison of reference substances. Result:The contents crude protein， crude fat， crude polysaccharide and crude fiber in guarana were （0.63±0.03）%， （2.73±0.09）%， （3.23±0.12）% and （8.89±0.59）%， respectively. A total of 42 chemical constituents in guarana were identified by UPLC-Q-TOF-MS， including 3 methylxanthines， 2 nucleosides， 1 amino acid， 3 organic acids， 33 flavonoids， 3 （<italic>L</italic>-tryptophan， epigallocatechin gallate， daidzein） of which were first discovered in guarana. Conclusion:Guarana is rich in nutrients and has good potential to be developed as a functional food. UPLC-Q-TOF-MS technique provides a simple， rapid and accurate method for the identification of chemical constituents in guarana. Methylxanthines and proanthocyanidins are the main chemical constituents of guarana， which is meaningful for quality evaluation and material basis of guarana.

Investigation of Transdermal Constituents and Molecular Mechanism of Euodiae Fructus in Treatment of Diarrhea by Transdermal Drug Delivery Based on Integrated Pharmacology and UPLC-Q-TOF-MS / 中国实验方剂学杂志

Objective:To identify the transdermal constituents of Euodiae Fructus and predict its molecular mechanism in treating diarrhea by transdermal drug delivery. Method:Ultra performance liquid chromatography-quadrupole time-of-flight mass spectrometry （UPLC-Q-TOF-MS） and integrated pharmacology methods were used. The rapid identification of transdermal constituents of Euodiae Fructus was realized by the means of comparison of reference substances， analysis of UNIFI system and mass spectrometry. On this basis， Integrative Pharmacology-based Research Platform of Traditional Chinese Medicine （TCMIP） v2.0， SymMap， DisGeNET databases and literature were used to collected potential targets of transdermal constituents of Euodiae Fructus and targets for diarrhea-related diseases. The disease targets and drug targets were topologically analyzed to obtain the core targets， which were used for the Gene Ontology （GO） function and Kyoto Encyclopedia of Genes and Genomes （KEGG） pathway enrichment analysis. Finally， Cytoscape 3.6.0 was used to build up a network of transdermal constituents-core targets-key pathways. Result:A total of 19 chemical constituents were speculatively identified from Euodiae Fructus extract， including quinolone alkaloids， limonins， indole alkaloids， organic acids and sterols. A total of 174 core targets of Euodiae Fructus for treating diarrhea were obtained by a topology analysis， signaling pathways of inflammatory response， cell proliferation， nutrient regulation and energy metabolism， signal transduction， bacterial infection were obtained through the analysis of KEGG enrichment. Conclusion:In this study， the transdermal constituents of Euodiae Fructus are identified for the first time， they can participate in the regulation of intestinal inflammation， maintain the integrity of intestinal mucosa， repaire and adjust the metabolism of the body by acting on Rac protein family， phosphatidylinositol 3-kinase， cytochrome P450 enzymes and aldo-keto reductase， respectively. In general， the molecular mechanism of Euodiae Fructus in the treatment of diarrhea is preliminarily elucidated.

Anti-acetylcholinesterase Active Ingredients from Aconitum tanguticum by UPLC-Q-TOF-MS/MS / 中国实验方剂学杂志

Objective:To identify the anti-acetylcholinesterase active ingredients in <italic>Aconitum tanguticum</italic>， so as to lay the foundation for finding new anti-Alzheimer's disease （AD） drugs. Method:The anti-acetylcholinesterase active fractions of <italic>A. tanguticum</italic> were screened by the modified Ellman's method， and the chemical composition of the active fraction was analyzed by ultra-performance liquid chromatography-quadrupole time-of-flight mass spectrometry （UPLC-Q-TOF-MS/MS）. The chromatographic separation was performed on an ACQUITY UPLC BEH C₁₈ column （2.1 mm×50 mm， 1.7 μm） with acetonitrile （A）-0.4% ammonia aqueous solution （B） as mobile phase for gradient elution， and the column temperature was set at 30 ℃ with the flow rate of 0.4 mL·min^-1. Phase A of the dichloromethane fraction changed with time as follows：0-3 min， 5%A； 3-7 min， 5%-20%A； 7-11.5 min， 20%-33%A； 11.5-15.5 min， 33%-50%A； 15.5-20.5 min， 50%-80%A； 20.5-23 min， 80%-85%A； 23-25 min， 85%-95%A. Phase A of the <italic>n</italic>-butanol fraction changed with time as follows：0-2 min， 5%A； 2-8 min， 5%-20%A； 8-11 min， 20%-33%A； 11-15 min， 33%-95%A. Mass spectrometry was performed on electrospray ionization， data were collected in positive ion mode， and the detection range was <italic>m</italic>/<italic>z</italic> 100-1 500. Result:Both the dichloromethane and <italic>n</italic>-butanol fractions had a certain inhibitory effect on acetylcholinesterase， their half inhibitory concentration （IC₅₀） values were （64±4.4） mg·L^-1 and （85.7±3.8） mg·L^-1， respectively. By UPLC-Q-TOF-MS/MS analysis， a total of 21 alkaloids were identified from the dichloromethane fraction， and 11 alkaloids were identified from <italic>n</italic>-butanol fraction. Guan-fu base Ⅰ， found in both fractions， was first discovered in <italic>A. tanguticum</italic>. Conclusion:Diterpene alkaloids are the main anti-acetylcholinesterase substances of <italic>A. tanguticum</italic>， which is worth further exploration.

Difference Analysis of Chemical Constituents in Different Varieties of Puerariae Flos by UPLC-Q-TOF-MS / 中国实验方剂学杂志

Objective:To compare the chemical constituents of Puerariae Flos from three different varieties of <italic>Pueraria montana</italic> var. <italic>lobata</italic>， <italic>P. montana</italic> var. <italic>thomsonii</italic> and <italic>P</italic>. <italic>montana</italic> var<italic>. montana</italic>. Method:Ultra-performance liquid chromatography-quadrupole-time-of-flight mass spectrometry （UPLC-Q-TOF-MS） was used with the mobile phase of 0.1% formic acid aqueous solution （A）-acetonitrile （B） for gradient elution （0-20 min， 10%-30%B； 20-30 min， 30%-55%B； 30-35 min， 55%-95%B； 35-37 min， 95%B； 37-40 min， 95%-10%B）， the flow rate was 0.25 mL·min^-1. Electrospray ionization （ESI） was used to scan and collect MS data in positive and negative ion modes with scanning range of <italic>m</italic>/<italic>z</italic> 50-1 500. The chemical components from different sources of Puerariae Flos were identified in combination with the chemical composition database and literature information. After the obtained data were normalized by MarkerView^TM 1.2.1， they were imported into SICMA-P 14.1 software for principal component analysis （PCA） and orthogonal partial least squares discriminant analysis （OPLS-DA） to select the main differentiated components among the three different varieties. Result:A total of 35 compounds were identified from three different varieties of Puerariae Flos， including 22 isoflavones， 6 flavonoids and 7 saponins. The flowers of <italic>P</italic>. <italic>lobata</italic>， <italic>P. montana</italic> var. <italic>thomsonii</italic> and <italic>P</italic>. <italic>montana</italic> var<italic>. montana</italic> contained 32， 35， 33 compounds， respectively. And 18 differential compounds were screened under the positive and negative ion modes， including kakkalide， tectoridin， 6″-<italic>O</italic>-xylosyl-tectoridin， 4'-methyltectorigenin-7-glucoside， glycitin， 6″-<italic>O</italic>-xylosyl-glycitin， irisolidone， kaikasaponin Ⅲ， 6″-<italic>O</italic>-malonylglycitin， kakkalidone， tectorigenin， rutin， soyasaponin BB， vitexin， biochanin A， genistin， kakkatin， azukisaponin Ⅱ. Conclusion:This research is the first to systematically study the chemical constituents of the flower of <italic>P</italic>. <italic>montana</italic> var<italic>. montana</italic>， although the flower of <italic>P</italic>. <italic>montana</italic> var<italic>. montana</italic> is used as adulterants， it has high contents of tectoridin and 6″-<italic>O</italic>-xylosyl-tectoridin， which has great potential for development. The efficacy components such as kakkalide and tectoridin in Puerariae Flos from the three sources of varieties are obviously different， and it is necessary to carefully consider the application of these three varieties as Puerariae Flos.

The study of neuroprotective effect of ferulic acid based on cell metabolomics.

Ferulic acid (FA), a naturally derived phenolic compound, has antioxidant and antidepressant-like effects. It is still a challenge to study its mechanism due to the complexity of the pathophysiology of depression. In this study, ultra-high performance liquid chromatography coupled with quadrupole time-of-flight mass spectrometry (UPLC-QTOF-MS) was used to perform metabolomics studies based on biochemical changes in differentiated rat pheochromocytoma (PC12) cells treated with corticosterone-induced neurological damage after FA treatment. A total of 31 metabolites were identified as potential biomarkers for corticosterone-induced PC12 cells injury. Among them, 24 metabolites were regulated after FA treatment. Pathway analysis revealed that these metabolites were mainly involved in the amino acid metabolism, energy metabolism and glycerophospholipid metabolism. In addition, based on the results of metabolomics, three cell signaling pathways related to glutamate were discovered. To further study the interactions between FA and major targets in three signaling pathways, a molecular docking method was employed. The results showed that FA had the strongest binding power with protein kinase B (AKT). Furthermore, the result of mRNA changes analyzed by quantitative real time RT-PCR indicated that AKT and protein kinase A (PKA) in the signaling pathway were up regulated after treatment with FA compared with model group. This study shows that strategies based on cell metabolomics associated with molecular docking and molecular biology is a helpful tool to elucidate the neuroprotective mechanism of FA.