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This study aimed to identify the related substances of phloroglucinol injection by two-dimensional liquid chromatography quadrupole time-of-flight mass spectrometry (2D-LC-Q-TOF/MS). The first-dimensional separation was carried out on an HSS T3 (250 mm × 4.6 mm, 5 μm) column by gradient elution using 1.36 g·L-1 potassium dihydrogen phosphate buffer solution (pH adjusted to 3.0 with diluted phosphoric acid) and acetonitrile as the mobile phases. The separated components were then trapped in switch valve tube lines respectively and delivered to the second-dimensional desalting gradient elution which was performed with a BDS C18 (100 mm × 4.6 mm, 2.4 μm) column using 0.1% formic acid and methanol as the mobile phases. After rapid desalting, electrospray-ionization quadrupole time-of-flight high resolution mass spectrometry was used for determining the accurate masses and elemental compositions of the parents and their product ions for both phloroglucinol and its related substance. Structures of the related substances were then figured out by mass spectrometry elucidation, organic reaction mechanism analysis, and/or comparison with reference substances. Under the established analytical conditions, phloroglucinol and its related substances were adequately separated, 17 main related substances were detected and identified in the injection and its stressed samples for the first time. The identification results can provide reference for the quality control of phloroglucinol injection.
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Abstract Changes in metabolite levels of patients using the long-term drug can be comprehensively demonstrated by pharmacometabolomic studies. In this study, biological alterations induced by the administration of solifenacin succinate were investigated with a pharmacometabolomics approach on rat metabolism. Plasma samples obtained from rats were analyzed by LC-Q- TOF/MS/MS. METLIN and HMDB databases were used to identify metabolites. Data were processed and classified with MATLAB 2017b. 53 m/z values were found to be significantly different between the drug and control groups (p ≤ 0.01 and fold analysis > 1.5) and identified by comparing METLIN and HMDB databases. According to multivariate data analysis, changes in arachidonic acid, thromboxane A2, palmitic acid, choline, calcitriol, histamine phosphate, retinyl ester, l-cysteine, l-leucine, beta-alanine, l-histidine levels were found to be statistically significant compare to the control group. Differences in the biosynthesis of phenylalanine, aminoacyl-tRNA, tyrosine, tryptophan, metabolism of glycerophospholipid, cysteine, methionine, histidine, arachidonic metabolism have been successfully demonstrated by the metabolomics approach. Our study provides important information to explain the efficacy and toxicity of chronic administration of solifenacin succinate
Subject(s)
Animals , Rats , Metabolome/drug effects , Metabolomics/methods , Solifenacin Succinate/pharmacology , Metabolism/drug effects , Rats, WistarABSTRACT
Objective:To systematically study the chemical components of Qianyang Yuyin granules and explore its main pharmacodynamic substances and mechanism in the prevention and treatment of hypertensive renal damage. Method:Liquid chromatography coupled with quadrupole time-of-flight mass spectrometry （LC/Q-TOF-MS） was employed to comprehensively analyze the chemical components of Qianyang Yuyin granules. Agilent Poroshell 120 SB-C<sub>18</sub> column （3.0 mm×100 mm， 2.7 μm） was used， flow rate was 0.4 mL·min<sup>-1</sup>， electrospray ionization （ESI） was applied and operated in positive and negative ion modes， the acquisition range was <italic>m</italic>/<italic>z</italic> 25-1 000. Mobile phase in positive ion mode consisted of water+10 mmol·L<sup>-1</sup> ammonium formate+0.125% formic acid+0.1% methanol （A）-［acetonitrile-water （9∶1）+10 mmol·L<sup>-1</sup> ammonium formate+0.125% formic acid］ （B）， and in negative ion mode consisted of water+10 mmol·L<sup>-1</sup> ammonium formate+0.1% methanol （A）-［acetonitrile-water （9∶1）+10 mmol·L<sup>-1</sup> ammonium formate］ （B） with the gradient elution （0-3.5 min， 5%B； 3.5-4 min， 5%-10%B； 4-9 min， 10%-25%B； 9-18 min， 25%-30%B； 18-25 min， 30%-50%B； 25-27 min， 50%-90%B； 27-32 min， 90%B； 32-33 min， 90%-5%B； 33-39 min， 5%B）. According to the information of the accurate mass， the multistage fragment ions， the mass spectrometric data of the standard substances and the relative reference literature， the structures of the chemical components in Qianyang Yuyin granules were identified. Based on the identified components， network pharmacology study， including target prediction and functional enrichment was applied to screen out the main active substances against hypertensive renal damage， and explore the potential mechanism. Result:A total of 99 chemical components were identified， from which 43 active substances and 48 key targets were screened out. The key components contained kaempferol， quercetin， ferulic acid， luteolin， caffeic acid methyl ester， cinnamic acid， aloe-emodin， emodin， gallic acid， <italic>N</italic>-<italic>trans</italic>-feruloyltyramine， isoorientin， 8-<italic>O</italic>-feruloylharpagide， ethyl caffeate， isookanin， cyasterone， 2，3，5，4'-tetrahydroxystilbene-2-<italic>O</italic>-<italic>β</italic>-<italic>D</italic>-glucopyranoside， loganin， alisol B-23-acetate and harpagide. The key targets included vascular endothelial growth factor A （VEGFA）， serine/threonine protein kinase 1 （AKT1）， Jun proto-oncogene （JUN）， etc. Conclusion:Qianyang Yuyin granules mainly exert the effects of removing heat from the liver， tonifying the kidney and removing blood stasis via modulation of vascular endothelium， angiogenesis， inflammatory reaction， oxidative stress， immune response and so on.
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Jie-Geng-Tang (JGT), a traditional formula, is employed in the treatment of sore throat and cough and comprises Platycodonis Radix and Glycyrrhizae Radix et Rhizoma in the ratio 1 : 2. Our previous study demonstrated that JGT protected mice from S. aureus-induced acute lung injury (ALI). Five constituents of JGT showed antibacterial activities against S. aureus in vitro. However, the potential effective constituents of JGT in vivo were still unclear. In this study, the chemical constituents of JGT were identified by liquid chromatography with quadrupole time-of-flight spectrometry (LC-Q-TOF-MS). A total of 96 constituents were identified or assumed, including seven organic acids, 45 flavonoids, 36 triterpene saponins, and eight compounds of other types. The structures of 31 of the constituents were confirmed by comparing them with corresponding authentic standards. Moreover, 15 prototypes and 49 metabolites were deduced in the serums of mice, 24 prototypes and 47 metabolites were deduced in the lungs of mice after the oral administration of JGT. Three types of constituents, namely organic acids, flavonoids, and triterpene saponins, could be absorbed into the blood. Moreover, flavonoids and triterpene saponins were more likely distributed in the lung than in the blood. To the best of our knowledge, this is the first report on the systematic metabolites profile of JGT in vivo. The results reported were beneficial to the elucidation of the effective material basis of JGT.
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Efonidipine HCl Ethanolate is an antihypertensive drug with 1,4 dihydropyridine and phosphinane derivative. Forceddegradation study was performed in Efonidipine as per the guidelines by International Conference on Harmonization(ICH) Q1A (R2). Extensive degradation and slight degradation were observed in alkaline and photolytic conditions,respectively, whereas acidic, oxidative, and thermal conditions did not show any degradation. Degradation productswere separated on Thermo Hypersil BDS C18 column (250 × 4.6 mm, 5 µ), mobile phase in gradient mode usingammonium acetate buffer and acetonitrile with detection at a wavelength of 254 nm. Six degradation products in alkalinecondition and four degradation products in photolytic condition were identified by HPLC and characterized by massspectrometry using LC-Q-TOF-MS, and degradation pathway was proposed. This is the typical case of degradation,where co-solvent methanol reacts with Efonidipine to form pseudo degradation products such as DP1, DP4, DP5, andDP6. Three degradation products DP1, DP3, and DP4 in alkaline condition were isolated by preparative HPLC andwere characterized by LC-Q-TOF-MS, 1H/13C NMR, and IR techniques. By characterization with these techniques,DP1 is characterized as 3-2-(N-benzylanilino)ethyl 3-oxo-2,2-dimethylpropyl hydrogen 1,4-dihydro-2,6-dimethyl-4-(3-nitrophenyl) pyridin-3-yl-3-phosphonate, DP3 is characterized as 2-(N-benzyl-N-phenylamino) ethanol, and DP4is characterized as 3-methoxy-2,2-dimethylpropyl hydrogen 1,4-dihydro-2,6-dimethyl-5-methyloxycarbonyl-4-(3-nitro)phenylpyridin-3-yl-3-phosphonate. The developed method was validated as per guidelines by ICH with respectto linearity, accuracy, precision, limit of detection, and robustness.
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Identification of components and metabolites of traditional Chinese medicines (TCMs) employing liquid chromatography-quadrupole time-of-flight mass spectrometry (LC-Q-TOF MS) techniques with information-dependent acquisition (IDA) approaches is increasingly frequent. A current drawback of IDA-MS is that the complexity of a sample might prevent important compounds from being triggered in IDA settings. Sequential window acquisition of all theoretical fragment-ion spectra (SWATH) is a data-independent acquisition (DIA) method where the instrument deterministically fragments all precursor ions within the predefined m/z range in a systematic and unbiased fashion. Herein, the superiority of SWATH on the detection of TCMs' components was firstly investigated by comparing the detection ef-ficiency of SWATH-MS and IDA-MS data acquisition modes, and sanguisorbin extract was used as a mode TCM. After optimizing the setting parameters of SWATH, rolling collision energy (CE) and variable Q1 isolation windows were found to be more efficient for sanguisorbin identification than the fixed CE and fixed Q1 isolation window. More importantly, the qualitative efficiency of SWATH-MS on sanguisorbins was found significantly higher than that of IDA-MS data acquisition. In IDA mode, 18 kinds of sangui-sorbins were detected in sanguisorbin extract. A total of 47 sanguisorbins were detected when SWATH-MS was used under rolling CE and flexible Q1 isolation window modes. Besides, 26 metabolites of sangui-sorbins were identified in rat plasma, and their metabolic pathways could be deduced as decarbonylation, oxidization, reduction, methylation, and glucuronidation according to their fragmental ions acquired in SWATH-MS mode. Thus, SWATH-MS data acquisition could provide more comprehensive information for the component and metabolite identification for TCMs than IDA-MS.
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@#To identify the amino alcohols related substances in atenolol. The related substances in atenolol and its stressed samples were pre-column derivatized with 9-fluorenylmethyl chloroformate. The separation was carried out on an Inertsil ODS-SP column (250 mm×4.6 mm, 5 μm) with linear gradient elution by methanol-ammonium acetate solution as the mobile phases. Electrospray positive ionization high-resolution Q-TOF/MS was used for the determination of the accurate masses and elemental compositions of the parent and fragment ions of these related substance derivatives. The structures of all the detected substances were identified by spectral analysis and synthetic analysis. Under the established conditions, atenolol and its amino alcohols related substances were well separated, and a total of 14 impurity peaks were detected and identified, of which 12 were related substances and 2 were derivatization reaction by-products. The established LC-MS method provides a reference for the examination and quality control of atenolol related substances.
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Objective: The present work deals with the qualitative study of the phytoconstituents present in Aegialitis rotundifolia Roxb., ethanolic leaves extract and evaluate its antioxidant properties in vitro. Methods: The qualitative phytochemical analysis of the extract was performed first using preliminary phytochemical tests and then by liquid chromatography quadrupole-time-of-flight mass spectrometry (LC-Q-TOF-MS). The antioxidant properties were investigated comprehensively using seven in vitro models viz., 2, 2-diphenyl-1-picrylhydrazyl (DPPH) radical scavenging, nitric oxide (NO) scavenging, hydrogen peroxide (H2O2) scavenging, superoxide (SOD) radical scavenging, lipid peroxidation (LPO) assay, reducing power (RP), and total antioxidant activity. Results: The preliminary phytochemical analysis revealed the presence of several important phytochemical groups whereas the LC-Q-TOF-MS analysis detected 25 phytoconstituents in the extract mostly belonging to flavonoids and alkaloids. The test extract showed strong dose-dependent antioxidant activity in all the seven in vitro models, however, the activity of the extracts was slightly lower compared to the reference standard ascorbic acid. Conclusion: The test extract showed strong antioxidant properties which could be possibly due to the phytoconstituents detected in the extract.
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Objective: To identify the common compounds and variance compounds of Sophorae Fructus from different regions by ultra performance liquid chromatography coupled with time-of-fight mass spectrometry (UFLC-Q-TOF-MS). Methods: The separation was performed on Waters Acquity UPLC BEH C18 column (100 mm × 2.1 mm, 1.7 μm), with a mobile phase using water with 0.1% formic acid (A) and acetonitrile with 0.1% formic acid (B) for gradient elution; Q-TOF/MS and electrospray ion (ESI) source were applied for the analysis under the positive ion mode and the negative ion mode; 1 000 ions were extracted through Markerview 1.2.1 software from Sophorae Fructus of 11 different regions. And common ions (compounds) were selected according to the following principles: One ion can be detected in all samples, and most of the peak area was greater than the 1 × 104; The variance ions (compounds) were selected according to principal components analysis. The formula of common ions was then determined by accurate mass and isotopic abundance ratio from target screening function of Peakview 2.0/masterview 1.0 software, and its structure were determined by analysis of MS/MS fragment or comparison with standard substances and references. Results: A total of 24 common compounds and 21 variance compounds were identified and inferred in Sophorae Fructus from different producing areas. Conclusion: UPLC-Q-TOF/MS method which develops a new strategy can identify the main chemical constituents from Sophorae Fructus rapidly and accurately. The determination of the main common components lays a foundation for the selection of quality evaluation indexes and the in-depth study of pharmacodynamic substances. The variance components can also be used as one of the bases for the identification of origin and authentic evaluation of Sophorae Fructus.
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Human intestinal bacteria play an important role in the metabolism of herbal medicines, leading to the variations in their pharmacological profile. The present study aimed to investigate the metabolism of Xiao-Cheng-Qi decoction (XCQD) by human intestinal bacteria and to discover active component combination (ACC) contributing to the anti-inflammatory activity of XCQD. The water extract of XCQD was anaerobically incubated with human intestinal bacteria suspensions for 48 h at 37 °C. A liquid chromatography-hybrid quadrupole time-of-flight mass spectrometry (LC-Q-TOF/MS) method was performed for identification of the metabolites. In addition, the anti-inflammatory effects of XCQD and biotransformed XCQD (XCQD-BT) were evaluated in vitro with cytokines in RAW264.7 cells induced by lipopolysaccharide (LPS). A total of 51 compounds were identified in XCQD and XCQD-BT. Among them, 20 metabolites were proven to be transformed by human intestinal bacteria. Significantly, a combination of 14 compounds was identified as ACC from XCQD-BT, which was as effective as XCQD in cell models of inflammation. In conclusion, this study provided an applicable method, based on intestinal bacterial metabolism, for identifying combinatory compounds responsible for a certain pharmacological activity of herbal medicines.
Subject(s)
Animals , Humans , Mice , Anti-Inflammatory Agents , Pharmacology , Therapeutic Uses , Bacteria , Metabolism , Biotransformation , Cytokines , Metabolism , Drugs, Chinese Herbal , Chemistry , Metabolism , Feces , Microbiology , Gastrointestinal Microbiome , Inflammation , Drug Therapy , Lipopolysaccharides , Pharmacology , Macrophages , Metabolism , Models, Biological , Molecular StructureABSTRACT
Human intestinal bacteria play an important role in the metabolism of herbal medicines, leading to the variations in their pharmacological profile. The present study aimed to investigate the metabolism of Xiao-Cheng-Qi decoction (XCQD) by human intestinal bacteria and to discover active component combination (ACC) contributing to the anti-inflammatory activity of XCQD. The water extract of XCQD was anaerobically incubated with human intestinal bacteria suspensions for 48 h at 37 °C. A liquid chromatography-hybrid quadrupole time-of-flight mass spectrometry (LC-Q-TOF/MS) method was performed for identification of the metabolites. In addition, the anti-inflammatory effects of XCQD and biotransformed XCQD (XCQD-BT) were evaluated in vitro with cytokines in RAW264.7 cells induced by lipopolysaccharide (LPS). A total of 51 compounds were identified in XCQD and XCQD-BT. Among them, 20 metabolites were proven to be transformed by human intestinal bacteria. Significantly, a combination of 14 compounds was identified as ACC from XCQD-BT, which was as effective as XCQD in cell models of inflammation. In conclusion, this study provided an applicable method, based on intestinal bacterial metabolism, for identifying combinatory compounds responsible for a certain pharmacological activity of herbal medicines.
Subject(s)
Animals , Humans , Mice , Anti-Inflammatory Agents , Pharmacology , Therapeutic Uses , Bacteria , Metabolism , Biotransformation , Cytokines , Metabolism , Drugs, Chinese Herbal , Chemistry , Metabolism , Feces , Microbiology , Gastrointestinal Microbiome , Inflammation , Drug Therapy , Lipopolysaccharides , Pharmacology , Macrophages , Metabolism , Models, Biological , Molecular StructureABSTRACT
Objective: To identify the major chemical constituents in Cortex Juglandis Mandshuricae by ultra performance liquid chromatography coupled with time-of-flight mass spectrometry (UPLC-Q-TOF/MS). Methods: The separation was performed on Waters Acquity UPLC BEH C18 column (100 mm×2.1 mm, 1.7 μm), with a mobile phase using water with 0.1% formic acid (A) and acetonitrile with 0.1% formic acid (B) for gradient elution; Q-TOF/MS and electrospray ion (ESI) source was applied for the analysis under the positive and negative ion modes; Target search and non-target search were performed by Peakview 2.0/masterview1.0 or Markerview 1.2.1 software. Then the formulas of compounds were determined by accurate mass and isotopic abundance ratio from target screening function of software. Their structures were determined by analysis of MS/MS fragment or comparison with standard substances and references. Results: Forty-four major chemical compounds including 13 naphthalene quinones, 3 diarylheptanoids, 15 flavonoids, and 13 other compounds were identified or inferred in Cortex Juglandis Mandshuricae. Conclusion: UPLC-Q-TOF/MS method which develops a new strategy can identify the main chemical constituents from Cortex Juglandis Mandshuricae rapidly and accurately, main chemical constituents can be used for quality evaluation and pharmacological research.
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A suitable liquid chromatography quadrupole time-of-flight mass spectrometric (LC–Q-TOF–MS) method was developed for separation and characterization of related substances in bacitracin test drug. The separation was performed on LiChrospher RP-18 column using methanol as mobile phase A and 0.2% ammonium acetate buffer solution as mobile phase B in gradient elution. A total of 12 related substances were detected through high resolution mass spectrometric determination in a positive electrospray ionization mode. They were identified as co-existing active components and degradation products of bacitracin through the analysis and elucidation of both the protonated parents and the product ions of all the related substances and their fragmentation pathways were also proposed.
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To analyze the dynamic changes in components in exocarp of Juglans mandshurica at different browning periods. Twenty-six batches of exocarp of J. mandshurica samples from thirteen browning periods were assessed by UPLC-Q-TOF-MS/MS. The formula of different compounds were determined by accurate mass and isotopic abundance ratio from target screening function of Peakview 2.0/masterview1.0 software. Then their structures were determined by analysis of MS/MS fragment or comparison with standard substances and references. The contents of chemical components were changed significantly in different browning periods and twenty five compounds were identified or inferred. Of the 13 naphthoquinone compounds, the contents of 6 compounds with similar parent nucleus as juglone and 3 naphthoquinone glycosides compounds were decreased significantly, and 4 naphthoquinone derivatives such as regiolone were produced; the contents of four flavones and two phenolic acids compounds were decreased significantly; and the contents of 6 diarylheptanoids compounds were increased significantly. UPLC-Q-TOF/MS method can be used to identify and analyze the chemical constituents from exocarp of J. mandshurica rapidly and accurately, and analyze the rules of dynamic changes, to reveal the browning of Chinese medicinal materials and its effects on compositions of fruits and vegetables.
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<p><b>OBJECTIVE</b>To compare the effects of different acupuncture methods on urine metabolites in rheumatoid arthritis (RA) rabbits, and to explore the specificity mechanism of heat-reinforcing acupuncture for RA.</p><p><b>METHODS</b>A total of 40 clean purple-blue rabbits were randomly allocated to a normal group, a model group, a mild reinforcing-reducing needling (MRRN) group, a twirling-reinforcing needling (TRN) group and a heat-reinforcing needling (HRN) group, 8 rabbits in each one. Except the normal group, the rabbits in the remaining groups were treated with ovalbumin and freezing to establish RA model. The rabbits in the MRRN group, TRN group and HRN group were treated with MRRN, TRN and HRN at "Zusanli" (ST 36), respectively, 30 min per treatment, once a day for seven days. After treatment, 24-h urine was collected. The rabbits were sacrificed to collect synovial tissues of knee to perform morphology observation; the liquid chromatography quadrupole time-of-flight mass spectrometry (LC-Q/TOF-MS) was applied to measure urine metabolites. All the data were analyzed by principal component analysis (PCA) and partial least squares discriminant analysis (PLS-DA).</p><p><b>RESULTS</b>Compared with the normal group, the leucine-related metabolites, as main urine metabolites, were decreased in the model group (<0.05), while the purine-related metabolites and tryptophane-related metabolites were increased (<0.05). Compared with the model group, the leucine-related metabolites, as main urine metabolites, were increased in the three needling groups after treatment (<0.05), while the tryptophan-related metabolites andpurine-related metabolites were decreased (<0.05), moreover, the leucine-related metabolites in the HRN group were obviously higher than those in the MRRN group and TRN gruop (<0.05).</p><p><b>CONCLUSIONS</b>MRRN, TRN and HRN can regulate the pathway of leucine metabolism (energy metabolism), purine metabolism (oxidative damage) and tryptophane metabolism (immune regulation) for RA, The specificity of HRN for RA focuses on regulation of leucine metabolism (energy metabolism).</p>
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Si-Miao-Wan (SMW), a tradiational Chinese medicinal formula consisting of Atractylodis Rhizoma, Phellodendri Chinensis Cortex, Coicis Semen, and Achyranthis Bidentatae Radix, has been used for the treatment of gout and gouty arthritis for many years. In the present study, a liquid chromatography quadrupole-time-of-flight mass spectrometry (LC-Q-TOF/MS) method was established to identify the multiple constituents of SMW and its metabolites in rat biological samples after oral administration. A total of 48 compounds in SMW, including 21 alkaloids, 12 organic acids, 2 terpenes, 3 lactones, 2 phytosterols, and 8 other compounds, were tentatively characterized with the diagnostic-ion filtering strategy. Based on the diagnostic ions applied to identify compounds in SMW, 28 prototype compounds and 10 metabolic compounds were detected in the biological samples. This was the first comprehensive drug metabolism investigation of SMW in rats. The developed method could be a useful means for identifying the multi-components in SMW and the metabolic components. The results may help explore the possible metabolic processes and mechanism of action for SMW in vivo.
Subject(s)
Animals , Male , Rats , Alkaloids , Chemistry , Metabolism , Chromatography, High Pressure Liquid , Methods , Drugs, Chinese Herbal , Chemistry , Metabolism , Rats, Sprague-Dawley , Spectrometry, Mass, Electrospray Ionization , Methods , Tandem Mass Spectrometry , MethodsABSTRACT
Objective: To identify the major chemical compounds in 78 batches of the exocarp of Juglans mandshurica from different origins by ultra performance liquid chromatography coupled with time-of-fight mass spectrometry (UFLC-Q-TOF/MS) and determine the major active chemical components. Methods: The separation was performed on Waters Acquity UPLC BEH C18 column (100 mm × 2.1 mm, 1.7 μm), with a mobile phase using water with 0.1% formic acid (A) and acetonitrile with 0.1% formic acid (B) for gradient elution; Q-TOF/MS and electrospray ion (ESI) source were applied for the analysis under the positive ion mode; One thousand ions were extracted through Markerview 1.2.1 software from 78 batches. And common ions (compounds) were selected according to the following principles: One ion can be detected in all samples, and the relative strength is greater than the e4. Then the formula of common ions were determined by accurate mass and isotopic abundance ratio from target screening function of Peakview 2.0/masterview1.0 software. Their structures were determined by analysis of MS/MS fragment or comparison with standard substances and references. Results: Thirty-one major chemical compounds including eleven naphthalene quinones, three diarylheptanoids, three flavonoids, eight triterpenes, and six other compounds were identified or inferred in the exocarp of J. mandshurica. Conclusion: UPLC-Q-TOF/MS method which develops a new strategy can identify the main chemical constituents from the exocarp of J. mandshurica rapidly and accurately, main chemical constituents can be used for the quality evaluation and efficacy material research.
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The changes in effective components of Juglans mandshurica at different harvest periods were analyzed by UPLC-Q-TOF-MS/MS. Eighteen batch samples of J. mandshurica from six harvest periods were assessed by multivariate statistical analysis with Markerview software. The formula of different compounds were determined by accurate mass and isotopic abundance ratio from target screening function of Peakview 2.0/Masterview1.0 software. Then their structure were determined by analysis of MS/MS fragment or comparison with standard substances and references. Naphthoquinone are the major markers in samples of Juglans mandshurica from different harvest periods. Thirty-eight of naphthalenequinones were identified or inferred in J. mandshurica and contents decline gradually. UPLC-Q-TOF-MS method which develops a new strategy can identify and analyze chemical constituents from J. mandshurica rapidly and accurately, main chemical constituents can be used for quality evaluation and efficacy material research. The dynamic changes in the metabolite accumulation of J. mandshurica the basic data for harvesting medicinal plants at different times.
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To investigate the metabolism of six saponins by rat intestinal bacteria in vitro.Six saponins, including notoginsenoside R₁, ginsenoside Rg₁, ginsenoside Rg₂, ginsenoside Re, ginsenoside Rd and ginsenoside Rb₁, were incubated for 8 and 24 h with rat intestinal bacteria under anaerobic environment, respectively. After the samples were precipitated by acetonitrile and extracted with ethyl acetate, LC-Q-TOF-MS/MS was applied for the qualitative analysis of the metabolites. The potential metabolites in rat feces were analyzed by comparing the total ion current of the test samples and blank samples and analyzing the quasi-molecular ion and fragment ion of all chromatograms. The results showed that six saponins could be easily metabolized by rat intestinal bacteria. Notoginsenoside R₁ was mainly metabolized into five metabolites, and it's metabolic pathway was notoginsenoside R₁→ginsenoside Rg₁→ginsenoside Rh₁ and ginsenoside F₁→protopanaxatriol→dehydrogenated protopanaxatriol. Ginsenoside Rg₁ was mainly metabolized into four metabolites, and it's metabolic pathway was ginsenoside Rg₁→ginsenoside Rh₁ and ginsenoside F₁→protopanaxatriol→dehydrogenated protopanaxatriol. Ginsenoside Rg₂ was mainly metabolized into two metabolites, and it's metabolic pathway was ginsenoside Rg₂→ protopanaxatriol→dehydrogenated protopanaxatriol. Ginsenoside Re was mainly metabolized into four metabolites, and it's metabolic pathway was ginsenoside Re→ginsenoside Rg₂→ginsenoside F₁→protopanaxatriol→dehydrogenated protopanaxatriol. Ginsenoside Rd was mainly metabolized into four metabolites, and it's metabolic pathway was ginsenoside Rd→ginsenoside Rg₃ and ginsenoside F₂→ginsenoside Rh₂→protopanaxadiol. Ginsenoside Rb1 was mainly metabolized into five metabolites, and it's metabolic pathway was ginsenoside Rb₁→ginsenoside Rd→ginsenoside Rg₃ and ginsenoside F₂→ginsenoside Rh₂→protopanaxadiol. In summary, six saponins could be quickly metabolized by rat intestinal bacteria in vitro. Their major metabolic pathways were deglycosylation and dehydrogenation.
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The present study aimed at identifying potential lead compounds for diabetes mellitus drug discovery. We developed a novel method involving centrifugal ultrafiltration separation subsequent liquid chromatography with quadrupole time of flight tandem mass spectrometry (LC-Q/TOF-MS/MS) determination to screen α-glucosidase inhibitors in complex Scutellaria baicalensis Georgi (SBG) extract. By adding a second filter to the screening process, the level of non-specific binding of Compounds 1, 3, 10 and 11 was significantly decreased, and the level of non-specific binding of Compounds 5 and 15 also was reduced. As a result, five flavonoids identified as baicalein, baicalein, wogonin, chrysin, and oroxylin A, were rapidly found to interact with α-glucosidase and possess potent anti-α-glucosidase activity in vitro. Specific binding of ligands to α-glucosidase was demonstrated though the proposed method and the ligands could be ranked in order of affinity for α-glucosidase, which were corresponded to the order of inhibitory activity in vitro. In conclusion, our results indicated that the developed method is a rapid and effective screening method for rat intestinal α-glucosidase inhibitors from complex herbal medicines such as SBG.