微滴式数字聚合酶链式反应(droplet digital polymerase chain reaction,ddPCR)是一种新型核酸扩增技术,可对DNA或RNA分子采用绝对定量的方式进行分析。其结果具有更高的精准度、准确性和灵敏度,大大提升了数字PCR技术的可扩展性与实用...微滴式数字聚合酶链式反应(droplet digital polymerase chain reaction,ddPCR)是一种新型核酸扩增技术,可对DNA或RNA分子采用绝对定量的方式进行分析。其结果具有更高的精准度、准确性和灵敏度,大大提升了数字PCR技术的可扩展性与实用性,促进了现代分子生物学在精准定量检测方面的发展和应用。本文重点论述了ddPCR法的技术原理、优势以及在食源性致病微生物定量检测、转基因成分分析、食品源性成分检测等食品安全检测领域的应用研究进展情况。展开更多
目的：采用UPLC-Q-TOF/MS对大鼠血液中痰热清注射液成分进行定性鉴别分析,为该制剂的临床应用提供参考。方法：采用Agilent Eclipse Plus C_（18）色谱柱进行色谱分离,流动相0.1%甲酸水溶液-乙腈梯度洗脱。在正、负离子模式下使用电喷...目的：采用UPLC-Q-TOF/MS对大鼠血液中痰热清注射液成分进行定性鉴别分析,为该制剂的临床应用提供参考。方法：采用Agilent Eclipse Plus C_（18）色谱柱进行色谱分离,流动相0.1%甲酸水溶液-乙腈梯度洗脱。在正、负离子模式下使用电喷雾离子源（ESI）模式获取数据,数据采集范围m/z 100-1 100,通过同对照品对比、质谱分析、检索文献等方法进行物质鉴别。结果：共鉴别分析出27种入血成分,获得其中5种主要指标成分绿原酸、黄芩苷、咖啡酸、熊去氧胆酸与鹅去氧胆酸的主要代谢产物及代谢途径。咖啡酸、绿原酸可生成多种Ⅰ相和Ⅱ相消除产物,代谢途径多样;而熊去氧胆酸、鹅去氧胆酸与黄芩苷则主要以Ⅱ相消除为主。结论：痰热清注射液中仅有部分次要成分不会被吸收入血。建立的UPLC-Q-TOF/MS可对痰热清注射剂入血成分进行快速有效的定性鉴别,并可确定主要成分的代谢状态。展开更多
AIM: To develop and validate a high performance liquid chromatography(HPLC) coupled with diode array and evaporative light scattering detectors(DAD-ELSD) method for the quantitative determination and fingerprint analy...AIM: To develop and validate a high performance liquid chromatography(HPLC) coupled with diode array and evaporative light scattering detectors(DAD-ELSD) method for the quantitative determination and fingerprint analysis of ten active constituents in three chemical classes(namely, xanthone glycosides, steroidal saponins, and alkaloids) in Zhimu-Huangbai herb pair(ZB). METHOD: Chromatographic separation was performed on a Diamonsil C18 column(4.6 mm × 250 mm, 5 μm, Dikma) by gradient elution using acetic acid in acetonitrile solution at a flow rate of 1.0 mL·min–1 at 260 nm. The drift tube temperature of ELSD was set to 60 ℃ and nebulizer gas pressure was 4.0 Bar. Method validation was performed to assure its linearity, limits of detection and quantification, precision, repeatability, stability, and accuracy. RESULTS: The HPLC-DAD-ELSD method allowed the quantification of ten compounds(phellodendrine, jatrorrhizine, palmatine, berberine, neomangiferin, mangiferin, timosaponin E-I, timosaponin B-II, timosaponin B, and timosaponin A-III), and was successfully applied to fingerprint analysis for ten batches of ZB samples. CONCLUSION: This was the first time to apply the combination of DAD and ELSD for the simultaneous determination of ten active ingredients in ZB. The results showed that the combination of quantitative analysis for marker ingredients and chemical fingerprint for the TCM herb pair provides a potentially powerful, widely introduced, and internationally accepted strategy for assessment of complex TCM formulas.展开更多
Phenolic compounds, metabolites of the phenylpropanoid pathway, play an important role in the growth and environmental adaptation of many plants. Phenylalanine ammonia-lyase(PAL) is the first key enzyme of the phenylp...Phenolic compounds, metabolites of the phenylpropanoid pathway, play an important role in the growth and environmental adaptation of many plants. Phenylalanine ammonia-lyase(PAL) is the first key enzyme of the phenylpropanoid pathway. The present study was designed to investigate whether there is a multi-gene family in I. Indigotic and, if so, to characterize their properties. We conducted a comprehensive survey on the transcription profiling database by using tBLASTn analysis. Several bioinformatics methods were employed to perform the prediction of composition and physicochemical characters. The expression levels of IiPAL genes in various tissues of I. indigotica with stress treatment were examined by quantitative real-time PCR. Protoplast transient transformation was used to observe the locations of IiPALs. IiPALs were functionally characterized by expression with pET-32a vector in Escherichia colis strain BL21(DE3). Integration of transcripts and metabolite accumulations was used to reveal the relation between IiPALs and target compounds. An new gene(IiPAL2) was identified and both IiPALs had the conserved enzymatic active site Ala-Ser-Gly and were classified as members of dicotyledon. IiPAL1 and IiPAL2 were expressed in roots, stems, leaves, and flowers, with the highest expression levels of IiPAL1 and IiPAL2 being observed in stems and roots, respectively. The two genes responded to the exogenous elicitor in different manners. Subcellular localization experiment showed that both IiPALs were localized in the cytosol. The recombinant proteins were shown to catalyze the conversion of L-Phe to trans-cinnamic acid. Correlation analysis indicated that Ii PAL1 was more close to the biosynthesis of secondary metabolites than IiPAL2. In conclusion, the present study provides a basis for the elucidation of the role of Ii PALs genes in the biosynthesis of phenolic compounds, which will help further metabolic engineering to improve the accumulation of bioactive components in I.indigotica.展开更多
Tanreqing injection(TRQ), a well-known traditional Chinese medicine formula, is commonly used to treat respiratory diseases. In the present study, a rapid, selective, and sensitive liquid chromatography-tandem mass sp...Tanreqing injection(TRQ), a well-known traditional Chinese medicine formula, is commonly used to treat respiratory diseases. In the present study, a rapid, selective, and sensitive liquid chromatography-tandem mass spectrometry(LC-MS/MS) method was developed and validated to simultaneously determinate the plasma contents of 5 major constituents of TRQ, including chlorogenic acid(CHA), caffeic acid(CFA), baicalin(BA), ursodeoxycholic acid(UDCA) and chenodeoxycholic acid(CDCA) in rats after intravenous administration of TRQ. Chromatographic separation was performed on an Agilent Zorbax SB-C_(18) column(3.5 μm, 100 mm × 2.1 mm), with acetonitrile and 0.1% aqueous formic acid as mobile phase at a flow rate of 0.3 m L·min^(^(-1)). The calibration curves were linear over the ranges of 27.0–13 333.0 ng·m L^(-1) for CFA, 30.0–14 933.0 ng·m L^(-1) for CHA, 50.0–50 333.0 ng·m L^(-1) for BA, 550.0–55 000.0 ng·m L^(-1) for UDCA, and 480.0–48 000.0 ng·m L^(-1) for CDCA, respectively. Intra- and inter-day precisions(relative standard deviations, RSDs) were from 3.11% to 14.08%. The extraction recoveries were greater than 71% and accuracy(relative recovery) was from 89% to 137% for all analytes, except endogenous bile acids. This validated method was successfully applied to the first pharmacokinetic study of CFA, CHA, BA, UDCA and CDCA in rat plasma after intravenous administration of TRQ.展开更多
AIMS: To develop an HPLC-MS/MS method for the quantification of platycodin D(PD) in rat plasma, and to acquire the main pharmacokinetic parameters of PD after oral administration of pure PD or of Platycodi Radix extra...AIMS: To develop an HPLC-MS/MS method for the quantification of platycodin D(PD) in rat plasma, and to acquire the main pharmacokinetic parameters of PD after oral administration of pure PD or of Platycodi Radix extract(PRE) containing PD. METHOD: Plasma samples were pretreated with solid-phase extraction using an Oasis HLB SPE cartridge. Madecassoside was used as the internal standard(IS). Chromatographic separation was achieved on an ODS column(100 mm × 2.1 mm i.d., 3.5 μm) with a mobile phase consisting of acetonitrile/water(30 : 70, V/V) containing 0.1 mmol L 1ammonium acetate at a flow rate of 0.25 mL min 1. The detection was performed on a triple quadruple tandem mass spectrometer using an electrospray ionization(ESI) source with a chromatographic run time of 3.0 min. The detection was operated by multiple reaction monitoring(MRM) of the transitions of m/z 1 223.6→469.2 for PD and of m/z 973.6→469.2 for madecassoside(IS), respectively. RESULTS: The calibration curve was linear from 5 to 2 000 ng mL 1(r2>0.99) with a lower limit of quantification(LLOQ) of 5 ng mL 1. The intra- and inter-day precision(relative standard deviation, RSD) values were below 15% and the accuracy(relative error, RE) was from 15% to +15% at three quality control(QC) levels. Plasma concentrations of PD were determined for 24 h after i.v. administration of PD, and oral administration of PD and PRE, respectively. The absolute oral bioavailability of PD in rats was found to be(0.48 ± 0.19)% when administered PD, and to be(1.81 ± 0.89)% when administered PRE. CONCLUSION: The developed HPLC-MS/MS method was successfully applied to assess the pharmacokinetic parameters and oral bioavailability of PD in rats after administration of PD and Platycodi Radix extract.展开更多
文摘微滴式数字聚合酶链式反应(droplet digital polymerase chain reaction,ddPCR)是一种新型核酸扩增技术,可对DNA或RNA分子采用绝对定量的方式进行分析。其结果具有更高的精准度、准确性和灵敏度,大大提升了数字PCR技术的可扩展性与实用性,促进了现代分子生物学在精准定量检测方面的发展和应用。本文重点论述了ddPCR法的技术原理、优势以及在食源性致病微生物定量检测、转基因成分分析、食品源性成分检测等食品安全检测领域的应用研究进展情况。
基金supported by the Natural Science Foundation of Shanghai City,China(No.10411969800)the National Nature Science Foundation of China(Nos.81202866 and 81302856)
文摘AIM: To develop and validate a high performance liquid chromatography(HPLC) coupled with diode array and evaporative light scattering detectors(DAD-ELSD) method for the quantitative determination and fingerprint analysis of ten active constituents in three chemical classes(namely, xanthone glycosides, steroidal saponins, and alkaloids) in Zhimu-Huangbai herb pair(ZB). METHOD: Chromatographic separation was performed on a Diamonsil C18 column(4.6 mm × 250 mm, 5 μm, Dikma) by gradient elution using acetic acid in acetonitrile solution at a flow rate of 1.0 mL·min–1 at 260 nm. The drift tube temperature of ELSD was set to 60 ℃ and nebulizer gas pressure was 4.0 Bar. Method validation was performed to assure its linearity, limits of detection and quantification, precision, repeatability, stability, and accuracy. RESULTS: The HPLC-DAD-ELSD method allowed the quantification of ten compounds(phellodendrine, jatrorrhizine, palmatine, berberine, neomangiferin, mangiferin, timosaponin E-I, timosaponin B-II, timosaponin B, and timosaponin A-III), and was successfully applied to fingerprint analysis for ten batches of ZB samples. CONCLUSION: This was the first time to apply the combination of DAD and ELSD for the simultaneous determination of ten active ingredients in ZB. The results showed that the combination of quantitative analysis for marker ingredients and chemical fingerprint for the TCM herb pair provides a potentially powerful, widely introduced, and internationally accepted strategy for assessment of complex TCM formulas.
基金supported by the Natural Science Foundation of China(Nos.31100221 and 81325024)
文摘Phenolic compounds, metabolites of the phenylpropanoid pathway, play an important role in the growth and environmental adaptation of many plants. Phenylalanine ammonia-lyase(PAL) is the first key enzyme of the phenylpropanoid pathway. The present study was designed to investigate whether there is a multi-gene family in I. Indigotic and, if so, to characterize their properties. We conducted a comprehensive survey on the transcription profiling database by using tBLASTn analysis. Several bioinformatics methods were employed to perform the prediction of composition and physicochemical characters. The expression levels of IiPAL genes in various tissues of I. indigotica with stress treatment were examined by quantitative real-time PCR. Protoplast transient transformation was used to observe the locations of IiPALs. IiPALs were functionally characterized by expression with pET-32a vector in Escherichia colis strain BL21(DE3). Integration of transcripts and metabolite accumulations was used to reveal the relation between IiPALs and target compounds. An new gene(IiPAL2) was identified and both IiPALs had the conserved enzymatic active site Ala-Ser-Gly and were classified as members of dicotyledon. IiPAL1 and IiPAL2 were expressed in roots, stems, leaves, and flowers, with the highest expression levels of IiPAL1 and IiPAL2 being observed in stems and roots, respectively. The two genes responded to the exogenous elicitor in different manners. Subcellular localization experiment showed that both IiPALs were localized in the cytosol. The recombinant proteins were shown to catalyze the conversion of L-Phe to trans-cinnamic acid. Correlation analysis indicated that Ii PAL1 was more close to the biosynthesis of secondary metabolites than IiPAL2. In conclusion, the present study provides a basis for the elucidation of the role of Ii PALs genes in the biosynthesis of phenolic compounds, which will help further metabolic engineering to improve the accumulation of bioactive components in I.indigotica.
基金supported by National Natural Science Foundation of China(No.81325024)National Significant Projects of New Drugs Creation(No.2013ZX09507005)
文摘Tanreqing injection(TRQ), a well-known traditional Chinese medicine formula, is commonly used to treat respiratory diseases. In the present study, a rapid, selective, and sensitive liquid chromatography-tandem mass spectrometry(LC-MS/MS) method was developed and validated to simultaneously determinate the plasma contents of 5 major constituents of TRQ, including chlorogenic acid(CHA), caffeic acid(CFA), baicalin(BA), ursodeoxycholic acid(UDCA) and chenodeoxycholic acid(CDCA) in rats after intravenous administration of TRQ. Chromatographic separation was performed on an Agilent Zorbax SB-C_(18) column(3.5 μm, 100 mm × 2.1 mm), with acetonitrile and 0.1% aqueous formic acid as mobile phase at a flow rate of 0.3 m L·min^(^(-1)). The calibration curves were linear over the ranges of 27.0–13 333.0 ng·m L^(-1) for CFA, 30.0–14 933.0 ng·m L^(-1) for CHA, 50.0–50 333.0 ng·m L^(-1) for BA, 550.0–55 000.0 ng·m L^(-1) for UDCA, and 480.0–48 000.0 ng·m L^(-1) for CDCA, respectively. Intra- and inter-day precisions(relative standard deviations, RSDs) were from 3.11% to 14.08%. The extraction recoveries were greater than 71% and accuracy(relative recovery) was from 89% to 137% for all analytes, except endogenous bile acids. This validated method was successfully applied to the first pharmacokinetic study of CFA, CHA, BA, UDCA and CDCA in rat plasma after intravenous administration of TRQ.
基金supported by the National Natural Science Foundation of China(No.81073030)the National Key Technology R&D Program in the 11th Five Year Plan of China(No.2008BA151B00-2)
文摘AIMS: To develop an HPLC-MS/MS method for the quantification of platycodin D(PD) in rat plasma, and to acquire the main pharmacokinetic parameters of PD after oral administration of pure PD or of Platycodi Radix extract(PRE) containing PD. METHOD: Plasma samples were pretreated with solid-phase extraction using an Oasis HLB SPE cartridge. Madecassoside was used as the internal standard(IS). Chromatographic separation was achieved on an ODS column(100 mm × 2.1 mm i.d., 3.5 μm) with a mobile phase consisting of acetonitrile/water(30 : 70, V/V) containing 0.1 mmol L 1ammonium acetate at a flow rate of 0.25 mL min 1. The detection was performed on a triple quadruple tandem mass spectrometer using an electrospray ionization(ESI) source with a chromatographic run time of 3.0 min. The detection was operated by multiple reaction monitoring(MRM) of the transitions of m/z 1 223.6→469.2 for PD and of m/z 973.6→469.2 for madecassoside(IS), respectively. RESULTS: The calibration curve was linear from 5 to 2 000 ng mL 1(r2>0.99) with a lower limit of quantification(LLOQ) of 5 ng mL 1. The intra- and inter-day precision(relative standard deviation, RSD) values were below 15% and the accuracy(relative error, RE) was from 15% to +15% at three quality control(QC) levels. Plasma concentrations of PD were determined for 24 h after i.v. administration of PD, and oral administration of PD and PRE, respectively. The absolute oral bioavailability of PD in rats was found to be(0.48 ± 0.19)% when administered PD, and to be(1.81 ± 0.89)% when administered PRE. CONCLUSION: The developed HPLC-MS/MS method was successfully applied to assess the pharmacokinetic parameters and oral bioavailability of PD in rats after administration of PD and Platycodi Radix extract.
