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Objective To establish method for simultaneous determination of hesperidin, cinnamaldehyde and eugenol in Chunyang Zhengqi capsules by high performance liquid chromatography. Methods The column was Agilent PorosheⅡ 120 EC-C18 (4.6 mm×150 mm, 4 μm). The mobile phase was acetonitrile-water with gradient elution. The column temperature was 35℃. The flow rate was 1.0 ml/min, and the detection wavelength was 284 nm. Results The methodological verification showed that hesperidin, cinnamaldehyde and eugenol had a good linearity (r≥0.999 9). The precisions were less than 2.0%. The average recovery was between 98.0% and 101.9%. The stability and repeatability of RSD were also less than 3.0%, which met the requirements of method validation. Conclusion The method is simple, stable, reproducible and accurate, which could be used to the quality control of Chunyang Zhengqi capsules.
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ObjectiveTaking Achyranthis Bidentatae Radix（ABR） from different origins as samples， to quantitatively analyze the chemical composition and chromaticity of ABR with different processing degrees， and clarify the correlation and change law between color and composition in the processing process of ABR， so as to provide reference for the quality evaluation of processed products of ABR. MethodThe colorimeter is used to measure the chromaticity values of three kinds of processing degrees of ABR in different origins to show the color value change trend during the processing process， and the color parameters of wine-processed and salt-processed products of ABR with different processing degrees were analyzed by principal component analysis（PCA）， orthogonal partial least squares-discriminant analysis（OPLS-DA） and other analysis methods. The contents of eight representative components of ABR were measured by high performance liquid chromatography（HPLC）， the correlation between chromaticity and each representative component was analyzed by Pearson correlation analysis， and the applicability of the selected eight representative components was further verified by Fisher linear discriminant analysis， and the wine-processed and salt-processed products of ABR with different processing degrees were grouped according to the degree of processing， and 48 samples of wine-processed and salt-processed products with different processing degrees were used as training samples. Taking the contents of 5-hydroxymethylfurfural， polypodine B， β-ecdysterone， 25R-inokosterone， 25S-inokosterone， ginsenoside Ro， chikusetsusaponin Ⅳa and polysaccharides as variables， the discriminant function was established respectively， and 12 samples of wine-processed and salt-processed products of ABR with different processing degrees were back-tested to verify the discriminant function and test the reliability of the function. ResultPCA and OPLS-DA results showed that ABR samples with different processing degrees were classified into clusters， and the results could significantly distinguish different processed products. During the process of wine and salt processing， the contents of 5-hydroxymethylfurfural， ginsenoside Ro， and chikusetsusaponin Ⅳa gradually increased with the deepening of the processing degree， while the contents of polypodine B， β-ecdysterone， 25R-inokosterone， 25S-inokosterone and polysaccharides showed a gradual decreasing trend， indicating these 8 components increased and decreased to different degrees in the process of wine and salt processing. The results of Pearson correlation analysis showed that the 5-hydroxymethylfurfural content of the samples with different processing degrees of wine-processed and salt-processed products were negatively correlated with the brightness value（L*） and the total color difference value（E*ab）（P<0.01）， and positively correlated with the red-green value（a*） and the yellow-blue value（b*）（P<0.01）， and that the content of polypodine B and polysaccharides were positively correlated with L* and E*ab（P<0.01）. The discriminant functions of wine-processed and salt-processed products of ABR were established by Fisher linear discriminant analysis， and their accuracy rates in the training samples were 93.75% and 95.83%， respectively. Twelve test samples of wine-processed and salt-processed products with different processing degree were back substitution， and the correct rate was 100%. ConclusionThe trend of composition and color changes of ABR with different processing degrees in different production areas is relatively consistent， and the color value can better distinguish ABR with different processing degrees， and the color of ABR is related to some representative components in the processing process， indicating that the color can provide reference for the identification of the processing degree of ABR and the prediction of component content.
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OBJECTIVE To establish a method for the content determination of 11 components such as protodioscin in Guge fengtong tablets， and to evaluate the comprehensive quality of Guge fengtong tablets by combining with chemometric analysis and entropy weight-technique for order preference by similarity to ideal solution （EW-TOPSIS） method. METHODS HPLC method was adopted. The determination was performed on Agilent Eclipse Plus C18 column with a mobile phase consisted of acetonitrile- 0.2% phosphoric acid solution at the flow rate of 1.0 mL/min by gradient elution. The column temperature was set at 30 ℃ . The detection wavelengths were set at 203 nm （0-28 min， protodioscin， methyl protodioscin， pseudoprotodioscin， dioscin） and 280 nm （28-60 min， catechin， epicatechin， liquiritigenin， medicarpin， 6-gingerol， 8-gingerol， 10-gingerol）； the sample size was 10 μL. Using epicatechin as the internal reference， quantitative analysis of multi-components by single marker （QAMS） method was used to determine the contents of protodioscin， methyl protodioscin， pseudoprotodioscin， dioscin， catechin， liquiritigenin， medicarpin， 6-gingerol， 8-gingerol and 10-gingerol， which were compared with the results of the external standard method. SPSS 26.0 software and SIMCA 14.1 software were used for principal component analysis and orthogonal partial least squares-discriminant analysis， with variable importance in projection （VIP） value greater than 1 as the standard， to screen for differential markers that affect the quality； the EW-TOPSIS method was adopted to evaluate the quality of 15 batches of samples comprehensively.RESULTS The contents of protodioscin， methyl protodioscin， pseudoprotodioscin， dioscin， catechin， liquiritigenin， medi-carpin， 6-gingerol， 8-gingerol and 10-gingerol determined by HPLC combined with QAMS were 6.330-10.863， 1.150-2.274， 0.431- 0.740， 2.818-4.823， 0.826-1.510， 0.043-0.094， 0.079-0.231， 0.479-1.020， 0.146-0.288， 0.118-0.318 mg/g， respectively； there were no statistical significances， compared with the external standard method （P＞0.05）. A total of 15 batches of samples were clustered into 3 groups， with S1-S6， S7-S10， and S11-S15 clustered into one group， respectively. The VIP values of protodioscin， epicatechin， dioscin and 6-gingerol were greater than 1. Euclidean closeness values of the optimal solution （C）i for 15 batches of samples were 0.163 5 to 0.703 7， and Ci values of S11-S15 were all higher than 0.6. CONCLUSIONS The established QAMS method is accurate and simple， and can be used for comprehensive quality evaluation of Guge fengtong tablets， by combining with chemometric analysis and EW-TOPSIS method. Protodioscin， epicatechin， dioscin and 6-gingerol are the differential markers that affect the quality of Guge fengtong tablets. Samples S11-S15 have better quality.
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OBJECTIVE To establish an HPLC fingerprint of Xiao’er resuqing oral liquid， and to determine the contents of twelve index components. METHODS HPLC method was adopted. The determination was performed on Venusil MP C18 column with mobile phase consisting of acetonitrile-0.1% phosphate aqueous solution （gradient elution） at a flow rate of 1.0 mL/min. The detection wavelength was set at 210 nm， the column temperature was 30 ℃， the injection volume was 10 μL. HPLC fingerprint of Xiao’er resuqing oral liquid was established by using the Similarity Evaluation System of Chromatographic Fingerprint of TCM （2012 edition） to evaluate the similarity. The contents of 12 components were determined， including （R， S）-goitrin， 3，5-O-dicaffeoyl quinic acid， puerarin， forsythin， forsythoside A， chlorogenic acid， baicalin， saikosaponins d， wogonoside， baicalein， emodin and chrysophanol. RESULTS The similarity of HPLC fingerprints of 13 batches of Xiao’er resuqing oral liquid was greater than 0.97， and 14 common peaks were confirmed. The contents of the above 12 index components in 13 batches of Xiao’er resuqing oral liquid were as follows： 0.078-0.172， 1.564-2.736， 1.338-2.578， 0.426-0.872， 1.477-2.628， 1.396-2.447， 4.052-9.146， 0.367- 0.692， 1.974-4.674， 1.274-2.969， 0.085-0.167 and 0.155-0.307 mg/mL. CONCLUSIONS The established HPLC fingerprint and content determination methods have high accuracy and high specificity， which can be used for the quality evaluation of Xiao’er resuqing oral liquid.
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ObjectiveTo improve the quality standard of Yuanhu Zhitong oral liquid in order to strengthen the quality control of this oral liquid. MethodThin layer chromatography（TLC） was used for the qualitative identification of Corydalis Rhizoma and Angelicae Dahuricae Radix in Yuanhu Zhitong oral liquid by taking tetrahydropalmatine， corydaline reference substances and Corydalis Rhizoma reference medicinal materials as reference， and cyclohexane-trichloromethane-methanol（5∶3∶0.5） as developing solvent， Corydalis Rhizoma was identified using GF254 glass thin layer plate under ultraviolet light（365 nm）. And taking petroleum ether（60-90 ℃） -ether-formic acid（10∶10∶1） as developing solvent， Angelicae Dahuricae Radix was identified using a silica gel G TLC plate under ultraviolet light（305 nm）. High performance liquid chromatography（HPLC） was performed on a Waters XSelect HSS T3 column（4.6 mm×250 mm， 5 μm） with acetonitrile（A）-0.1% glacial acetic acid solution（adjusted pH to 6.1 by triethylamine）（B） as the mobile phase for gradient elution（0-10 min， 20%-30%A； 10-25 min， 30%-40%A； 25-40 min， 40%-50%A； 40-60 min， 50%-60%A）， the detection wavelength was set at 280 nm， then the fingerprint of Yuanhu Zhitong oral liquid was established， and the contents of tetrahydropalmatine and corydaline were determined. ResultIn the thin layer chromatograms， the corresponding spots of Yuanhu Zhitong oral liquid， the reference substances and reference medicinal materials were clear， with good separation and strong specificity. A total of 12 common peaks were identified in 10 batches of Yuanhu Zhitong oral liquid samples， and the peaks of berberine hydrochloride， dehydrocorydaline， glaucine， tetrahydropalmatine and corydaline. The similarities between the 10 batches of samples and the control fingerprint were all >0.90. The results of determination showed that the concentrations of corydaline and tetrahydropalmatine had good linearity with paek area in the range of 0.038 6-0.193 0， 0.034 0-0.170 0 g·L-1， respectively. The methodological investigation was qualified， and the contents of corydaline and tetrahydropalmatine in 10 batches of Yuanhu Zhitong oral liquid samples were 0.077 5-0.142 9、0.126 1-0.178 2 g·L-1， respectively. ConclusionThe established TLC， fingerprint and determination are simple， specific and reproducible， which can be used to improve the quality control standard of Yuanhu Zhitong oral liquid.
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OBJECTIVE To establish the method for content determination of related substances in Oxcarbazepine tablets. METHODS Ultra-high performance liquid chromatography （UPLC） method was adopted and the separation was performed on ZORBAX Eclipse Plus C18 column with mobile phase consisted of acetonitrile-0.01 mol/L ammonium acetate solution （pH6.0） （gradient elution） at the flow rate of 0.5 mL/min. The detection wavelength was 230 nm and column temperature was set at 35 ℃. The sample size was 10 μL. RESULTS The linear ranges of oxcarbazepine and impurity A， B， C， D， E， I， K， L and N were 0.192-1.440， 1.019-7.639， 0.208-1.559， 0.230-1.727， 0.389-2.915， 0.182-1.364， 0.393-2.945， 0.199-1.493， 0.199-1.490 and 0.200- 1.503 μg/mL， respectively （all r＞0.999）. The detection limits were 0.046， 0.037， 0.049， 0.027， 0.077， 0.040， 0.114， 0.054， 0.055 and 0.039 μg/mL. The quantitation limits were 0.152， 0.122， 0.162， 0.090， 0.258， 0.132， 0.380， 0.181， 0.185 and 0.130 μg/mL. RSDs of precision， repeatability， stability （24 h） and durability tests were all lower than 5.0%. The average recoveries were 92.8%-105.6% （RSD≤3.0%， n＝9）. Only impurity K and unknown impurity were detected in the original preparation sample， with a total content of 0.078% to 0.083%； impurities A， B， D， I and unknown impurity were detected in the generic preparations produced by domestic enterprise Ⅰ， with a total content of 0.147% to 0.163%； impurities A， B， I and unknown impurity were detected in the generic preparations produced by domestic enterprise Ⅱ， with a total content of 0.085% to 0.161%. CONCLUSIONS The established method is rapid， sensitive， accurate， stable and durable. It can be used for the content determination of 9 known impurities in Oxcarbazepine tablets.
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Objective To determine the content of five alkaloids from extracts of Piper longum and test the pharmacodynamic effect of them on rats with experimental myocardial ischemia induced by injection of pituitrin. Methods The content of five alkaloids was determined simultaneously by HPLC. The experimental myocardial ischemia in rats was induced by injection of pituitrin, and the absolute value of T wave change and change of heart rate before and after model establishment were chosen to be the observation index. The effects of large, medium and small dose groups were evaluated. Results Three batches of samples were analyzed, with the contents of piperine for 56.1%, 49.7%, 51.6%; N-isobutyl-(2E,4E)-octadecatrienamide for 4.5%, 4.2%, 4.3%; guineensine for 0.46%, 0.38%, 0.40%; piplartine for 1.73%, 1.67%, 1.70% and piperamide for 0.55%, 0.46%, 0.49%, respectively. All dose groups from extracts of piper longum had significantly reduced the absolute value of T wave and almost have no effect on the change of heart rate, except the high dose group showed the effect of reducing heart rate at some time . Conclusion The HPLC method was suitable for the simultaneous determination of five alkaloids from extracts of Piper longum. It was shown that extracts of Piper longum had good bioactivity in anti-myocardial ischemia.
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OBJECTIVE To establish the method for the content determination of 5-hydroxymethylfurfural （5-HMF） in glucosamine hydrochloride tablets， and to analyze its regularity and influential factors. METHODS Quantitative analysis of 5-HMF was performed using high-performance liquid chromatography. The analysis was conducted on Shim-pack GIST C18-AQ column with mobile phase consisted of 0.1% phosphoric acid solution-methanol （90∶10， V/V） at the flow rate of 1.0 mL/min. The column temperature was 30 ℃， and detection wavelength was 284 nm. The injection volume was 20 μL. Reaction kinetics test of different temperatures was adopted to analyze the relationship of 5-HMF content with reaction temperature and reaction time， and utilized to build its formation kinetic model. RESULTS The linger range of 5-HMF was 0.057-5.698 μg/mL （r＝0.999 9）. The limits of detection and quantitation were 5.70 and 17.09 ng/mL； RSDs of precision， repeatability and stability （24 h） tests were all lower than 1.0% （n＝6）. The average recoveries ranged from 99.38% to 99.73%（RSD＝0.53%， n＝9）. The contents of the 5-HMF in 8 batches of samples ranged 4.10-35.13 μg/g. Results of data fitting in reaction kinetics test showed that the higher reaction temperature and the longer reaction time， the higher 5-HMF content in the sample. At 50， 60， 70 and 80 ℃ ， the relationship between the content of 5-HMF and the reaction time was linear， in accordance with a zero-order kinetic model. The reaction rate constants were 6.789， 7.715， 8.815 and 11.430， respectively. CONCLUSIONS The established method has strong specificity， high sensitivity， and good accuracy； the reaction temperature and reaction time are important influential factors for the formation of 5-HMF in glucosamine hydrochloride tables. The change rule of its content conforms to the zero-order kinetic model.
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OBJECTIVE To evaluate the quality of Indigo Naturalis， and to provide reference for the quality control of Indigo Naturalis. METHODS UPLC-MS/MS method was used to determine the contents of 6 indole alkaloids （indigo， indirubin， isatin， tryptanthrin， indole and indole-3-carboxaldehyde） in Indigo Naturalis from different origins. Cluster analysis， principal component analysis and partial least squares-discriminant analysis （PLS-DA） were used to evaluate the quality of Indigo Naturalis from different origins. RESULTS The contents of indigo， indirubin， isatin， tryptanthrin， indole and indole-3-carboxaldehyde in Indigo Naturalis from different origins were 20 320.83-26 585.01， 1 327.69-3 102.25， 141.69-894.50， 2.17-5.27， 2.14-5.93 and 1.69-4.34 μg/g， respectively. The Indigo Naturalis from different areas were clustered into two categories by cluster analysis. Samples S1， S2， S4， S6， S7， S9 and S10 were clustered into category Ⅰ， and samples S3， S5， S8， S11 and S12 were clustered into category Ⅱ. Indigo Naturalis from different origins was evaluated with 3 principal components. The results showed that category Ⅰ sample scored higher and had better quality， while category Ⅱ sample scored lower and had worse quality. PLS-DA showed that indigo， indirubin， tryptanthrin and isatin were the main substances that reflected the quality difference of Indigo Naturalis. CONCLUSIONS The quality of Indigo Naturalis from different origins is different， and the quality of Indigo Naturalis of different batches from the same area is not stable. The quality evaluation method of Indigo Naturalis established in this paper is stable and reliable， which can provide a basis for the quality control of Indigo Naturalis.
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OBJECTIVE To establish the fingerprint of total saponins from Mussaenda pubescens， and to study the spectrum- effect relationship of its hepatoprotective activity. METHODS Ten batches of total saponins from M. pubescens from different origins were prepared using 75% ethanol as solvent. High-performance liquid chromatography （HPLC） and the Similarity Evaluation System for Traditional Chinese Medicine Chromatographic Fingerprints （2012 edition） were used to draw the fingerprints of 10 batches of total saponins from M. pubescens. The similarity evaluation and identification of common peaks were conducted. The same HPLC method was adopted to determine the contents of five triterpenoid saponins （mussaendoside H， mussaendoside U， mussaglaoside C， mussaendoside G and mussaendoside O）. The hepatoprotective effect of total saponins from M. pubescens was investigated by establishing carbon tetrachloride-induced acute liver injury model mice， and the spectrum-effect relationship was studied by using grey correlation analysis. RESULTS There were 11 common peaks in 10 batches of total saponins from M. pubescens， 5 of which were identified， i.e. mussaendoside H （peak 3）， mussaendoside U （peak 7）， mussaglaoside C （peak 8）， mussaendoside G （peak 9） and mussaendoside O （peak 11）； the similarities of 10 batches of samples ranged 0.940- 0.991. Average contents of mussaendoside H， mussaendoside U， mussaglaoside C， mussaendoside G， mussaendoside O were 0.01- 0.05， 0.10-0.21， 0.10-0.18， 0.03-0.08， 0.20-0.40 mg/g， respectively. Ten batches of total saponins from M. pubescens could generally reduce the contents of alanine aminotransferase （ALT） and aspartate aminotransferase （AST） in serum， and tumor necrosis factor-α （TNF-α）， interleukin-6 （IL-6） and IL-1β in liver tissue of model mice （P＜0.05 or P＜0.01）. The E-mail：13878195336@139.com correlation between the common peak areas and the contents of ALT， AST， TNF-α， IL-6 and IL-1β were 0.602-0.757， 0.585-0.833， 0.593-0.795， 0.618-0.820， 0.607-0.804， respectively； the peaks with high correlation were peaks 11， 9 and 8 in order. CONCLUSIONS Ten batches of total saponins from M. pubescens have similar components， and the average contents of mussaendoside H， mussaendoside U， mussaglaoside C， mussaendoside G and mussaendoside O are different. The batches of samples have a certain degree of hepatoprotective effect； mussaendoside O， mussaendoside G and mussaglaoside C may be its main active components.
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OBJECTIVE To provide a reference for comprehensive quality evaluation and control of the effective parts of Dracocephalum moldavica （EPDM）. METHODS A total of 10 batches of EPDM were prepared， and chemical information of EPDM was collected by HPLC-Q-Exactive-MS. EPDM components were identified by literature search， database comparison and manual analysis. HPLC fingerprints of 10 batches of EPDM were established by using the Similarity Evaluation System for Traditional Chinese Medicine Chromatographic Fingerprint （2004 A edition）； the similarity evaluation and common peak identification were carried out， and the contents of 5 index components were determined by HPLC. RESULTS A total of 11 compounds in EPDM were identified. The fingerprint similarities of EPDM samples from 10 batches were all above 0.96. Among 11 chromatographic peaks， 5 peaks were identified， such as luteolin-7-O-β-D-glucuronide（LG）， apigenin-7-O-glucuronide（APG）， rosmarinic acid（RA）， diosmetin-7-O-β-D-glucuronide（DG）， tilianin（TL） . The results of the quantitative analysis showed that all above 5 components had good linearity （R2≥0.999）， and the average recoveries were in the range of 95.12%-98.37%. The contents of LG， APG， RA， DG， TL were 21.268 3-29.243 9， 6.365 4-7.771 7， 37.327 4-45.671 2， 17.169 9-21.985 9， 66.940 4-91.206 3 mg/g， respectively. CONCLUSIONS The established method of component identification， fingerprint and content determination is stable， feasible and reliable， which is suitable for the comprehensive quality evaluation and control of EPDM.
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OBJECTIVE To establish methods to identify the chemical components of Gantaishu capsule， and determine the contents of 6 index components including glycyrrhizic acid. METHODS The chemical components of Gantaishu capsule were determined by HPLC-TOF/MS； the contents of 6 index components including glycyrrhizic acid were determined by UPLC-MS/MS. RESULTS A total of 41 chemical components were identified in Gantaishu capsules. The linear ranges of glycyrrhizic acid， mangiferin， luteolin， costunolide， oleanolic acid and berberine were 200-10 000 ng/mL（r were all greater than 0.999）. The limits of quantification were 200， 20， 10， 1， 10， 0.5 ng/mL， and the limits of detection were 100， 10， 5， 0.5， 5， 0.25 ng/mL， respectively； RSDs of precision， stability （24 h） and reproducibility tests were all less than 5.0% （n＝6 or n＝3）； the recoveries were 99.05%-101.08% （RSD were all less than 2.0%， n＝6）. The contents of them were 2.42-2.66， 0.85-1.16， 0.35-0.46， 6.18- 6.46， 0.99-1.29， 5.22-5.56 mg/g. CONCLUSIONS The established methods for identification and content determination are rapid and simple， and can be used for the identification of chemical components and the content determination of index components in Gantaishu capsule.
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Objective To establish a gas chromatography for simultaneous determination of camphor residue and borneolum content in Qingchang Suppository. Methods Gas chromatograph method was used. The chromatographic column was Agilent capillary column(30 m×0.25 mm×0.25 µm). The column temperature was 140 ℃. The sample injection temperature was 250 ℃. The FID detector temperature was 250 ℃. Results Camphor，borneol and isoborneol content showed good linear in the extent of 0.0299~1.497(r=1.000), 0.0205~1.025(r=1.000), 0.0097~0.4830 µg (r=1.000). RSDs of precision，stability and repeatability test results were less than 2%. The recovery was 99.7%, 101.0%, 102.5%. Conclusion This method is simple and quick with accurate result, which could be used for the content determination of Borneol in Qingchang Suppository.
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ObjectiveTo investigate the effect of microemulsion on the distribution of index components in different phases of Zexietang extract based on high performance liquid chromatography（HPLC） and phase separation process. MethodParticle size meter and transmission electron microscope were used to characterize the colloidal particles in blank microemulsion， aqueous extract of Zexietang and microemulsion extract of Zexietang. The phase separation process was established by high-speed centrifugation and dialysis， and based on this process， the aqueous extract and microemulsion extract of Zexietang were separated into the true solution phase， the colloidal phase and the precipitation phase， respectively. The contents of six components， including atractylenolide Ⅲ， atractylenolide Ⅱ， 23-acetyl alisol C， alisol A， alisol B and alisol B 23-acetate， were determined by HPLC with the mobile phase of water（A）-acetonitrile（B） for gradient elution（0-5 min， 40%-43%B； 5-20 min， 43%-45%B； 20-45 min. 45%-60%B； 45-75 min， 60%-80%B）. The solubility of the index components in water and microemulsion was determined by saturation solubility method. ResultThe colloidal particles in the aqueous extract， microemulsion extract and blank microemulsion were all spherical， and the particle size， polydispersity index（PDI） and Zeta potential of the colloidal particles were in the order of aqueous extract >microemulsion extract >blank microemulsion. The results of phase separation showed that the colloidal phase and the true solution phase could be completely separated by dialysis for 2.5 h， and the phase separation process was tested to be stable and feasible. Compared with the aqueous extract of Zexietang， the use of microemulsion as an extraction solvent could increase the contents of atractylenolide Ⅲ， 23-acetyl alisol C， atractylenolide Ⅱ ， alisol A， alisol B and alisol B 23-acetate by 3.75， 6.82， 35.47， 10.66， 35.41， 27.75-fold， and could increase the extraction efficiencies of the latter five constituents by 2.03， 1.15， 1.70， 6.43， 5.53 times. The solubility test showed that the microemulsion could significantly improve the solubility of atractylenolide Ⅱ， alisol A， alisol B and alisol B 23-acetate， but it had less effect on the solubility of atractylenolide Ⅲ and 23-acetyl alisol C. ConclusionMicroemulsion can improve the extraction efficiency and increase the distribution of the index components in the colloidal phase state of Zexietang to different degrees， providing a reference for the feasibility of microemulsion as an extraction solvent for traditional Chinese medicine.
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ObjectiveTo clone coumarate-3-hydroxylase gene （C3H） from Angelica sinensis， and analyze the correlation between its bioinformatics， expression patterns and content of ferulic acid， and to explore the functions of ASC3H. MethodReal-time polymerase chain reaction （Real-time PCR） was used to clone the full-length cDNA of ASC3H based on the transcriptome dataset of A. sinensis， and the bioinformatics analysis of the gene sequence was carried out. Real-time PCR and high performance liquid chromatography （HPLC） were used to determine relative expression of ASC3H and content of ferulic acid in different root tissues of A. sinensis （periderm， cortex and stele）. ResultThe open reading frame （ORF） of ASC3H （GenBank accession number： MN2550298） was 1 530 bp， encoding 509 amino acids， with a theoretical molecular weight of 57.86 kDa and an isoelectric point of 8.36. It was a hydrophilic protein that was located in the chloroplast with multiple phosphorylation sites and a transmembrane region， and contained a conserved domain CGYDWPKGYGPIINVW_P450 （383-399 aa） in cytochrome P450. Multiple amino acid sequence alignment analysis showed that ASC3H had high similarity with C3H from other plants， especially Ammi majus in Umbelliferae. The Real-time PCR revealed that ASC3H had different expressions in periderm， cortex and stele tissues of A. sinensis roots. It was found from HPLC that the cortex tissues had the highest content of ferulic acid， and the stele tissues had the lowest. ConclusionASC3H was successfully cloned from A. sinensis， and its sequence characteristics were understood more clearly， suggesting that ASC3H might be involved in the ferulic acid biosynthesis pathway of A. sinensis. This paper provided a basis for further studying the functions of the gene and exploring the biosynthesis and regulation mechanism of ferulic acid in A. sinensis， while laying the foundation for the genetic improvement of A. sinensis．
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Objective To establish a method for the determination of cisatracurium besylate in human plasma by UPLC-MS/MS which could be used in the monitoring of drug residual in plasma of elderly patients after operation. Methods The samples were precipitated with 0.1% formic acid-acetonitrile solution and separated by an SHISEIDO ADME column(3.0 mm×100 mm, 2.7 μm) for isocratic elution with the mobile phase of water containing 0.1% formic acid with 2 mmol/L ammonium acetate and acetonitrile (30:70, V/V). MS condition was optimized in the positive ion detection mode by multiple reaction monitoring (MRM), along with the Agilent jet stream electrospray source interface (AJS-ESI). The precursors to the product ion transitions were m/z 464.3→358.2 for cisatracurium besylate and m/z 557.4→356.3 for vecuronium bromide (the internal standard, IS). Plasma samples of elderly patients undergoing spinal surgery were collected after anesthesia induction, at the end of surgery, 0.5 h and 1 h after surgery, and from the blood bags while autologous blood transfusion, and stored in cryopreservation tubes with 2% formic acid solution. Then the contents of cisatracurium besylate were determined. The effects of autogenous blood transfusion on plasma concentration of cisatracurium besylate in elderly patients after surgery evaluated. Results The calibration curve was linear in the range of 20-5 000 ng/ml for cisatracurium besylate in human plasma, r=0.999 7. The intra-day and inter-day precision and accuracy were good (RSD<10%, RE<±10%). The matrix effect of different concentrations was 71.88%~80.64%. The recovery of different concentrations was 83.62%~88.87%. The recovery of vecuronium bromide (IS) was 125.91%, which conformed with the requirement of methodological validation. There was a certain degree of residual cisatracurium besylate in the plasma of elderly patients, so the extubation time should be strictly controlled and the stay time of patients in the anesthesia recovery room should be appropriately extended. Conclusion The method is sensitive, accurate, and efficient, which could be used for the determination of cisatracurium besylate in human plasma of elderly patients after operation.
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OBJECTIVE To establish the quality standard of Clinopodium gracile. METHODS Ten batches of C. gracile were collected to perform appearance and property identification， microscopic identification and thin layer chromatography （TLC） identification. Moisture， total ash， acid-insoluble ash and dilute ethanol extract were detected， and the content of rosmarinic acid was determined by HPLC. RESULTS The stem of C. gracile was slender， square columnar， covered by white fluff， the surface was grayish green or greenish brown； epidermal cells， non-glandular hairs， cortical cells and so on were seen in the cross section of the stem. Non-glandular hairs， ducts， wood fibers， mesophyll cells and so on could be seen in the powder. Results of TLC identification showed that there were spots of the same color in the chromatographic position corresponding to the chromatographic position of buddlejasaponin Ⅳb control. The contents of water， total ash， acid-insoluble ash， dilute ethanol extract and rosmarinic acid in 10 batches of samples were 8.69%-12.33%， 5.96%-13.33%， 0.14%-3.29%， 18.57%-32.61%， 0.35%-0.82%， respectively. The average values were 10.10%， 9.73%， 1.06%， 23.54% and 0.56%， respectively. CONCLUSIONS The established method can be used for quality control of C. gracile. It is preliminarily proposed that the ash content in the herb should not exceed 12.0%， the total ash content should not exceed 12.0%， the acid-insoluble ash content should not exceed 1.5%， the dilute ethanol extract should not be less than 18.0%， and the rosmarinic acid content should not be less than 0.45%.
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Based on ultra high-performance liquid chromatography-tandem mass spectrometry (UHPLC-MS/MS), a rapid and simultaneous quantitative method for the measurement of seven components (kinsenoside; rutin; kaempferol-3-O-rutinoside; quercimeritrin; narcissin; isorhamnetin-3-O-glucoside; quercetin) of A. roxburghii was established. The separation was performed over 8.0 minutes on a Waters Acquity UPLC BEH Shield RP18 (2.1 mm × 100 mm; 1.7 μm) with a mobile phase consisting of acetonitrile (A) and 0.1% formic acid water solution (B) with gradient elution at a flow rate of 0.2 mL·min-1; the column temperature was 30 ℃ and the injection volume was 2 μL. Electrospray spray ionization source (ESI source) was used for mass spectrometry, and positive and negative ion modes were detected at the same time. The results showed good linearity (R2 ≥ 0.998 0), with good precision, repeatability and stability, and the average recovery was 97.71%-103.33%. Through cluster heat map and redundancy analysis, we found that kinsenoside was mainly distributed in stems, followed by leaves, and the lowest content in roots. The content of kinsenoside increased significantly in the stems of plants 6 months, but less change was evident in the roots and leaves. Flavonoids and flavonoid glycosides were mainly distributed in leaves. The UHPLC-MS/MS method established in this paper can be used for the quality control of A. roxburghii and provides a reference for establishing a more comprehensive quality detection method for this medicinal.
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Objective To optimize the microwave-assisted extraction process of green tea polyphenols. Methods The extraction yield of tea polyphenols was figured up by building the standard curve of gallic acid and examining the concentration of tea polyphenols in green tea extract with the introduction of a correction factor. The effects of four single factor levels of microwave extraction time, microwave output power, liquid-to-material yield, and ethanol volume fraction on the extraction yield of tea polyphenols were primarily studied in this experiment. The response surface was applied to further optimize the extraction process of green tea polyphenols after exploring the appropriate range of four single factor levels. Results The optimal extraction process was as follows: extraction time 37 s, microwave output power 350 w, material - liquid yield 1∶45 (g/ml), ethanol volume fraction 55%, and the actual extraction yield of tea polyphenols was 25.65%, which was not much different from the theoretical value. Conclusion The microwave-assisted green tea polyphenol extraction process optimized by response surface methodology is time-saving and practicable, and the extraction yield is high.
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@#The UPLC fingerprint of colistimethate sodium was established for the study of quality consistency.The chromatographic column was Acquity UPLC? Peptide CSH C18 (2.1 mm × 150 mm, 1.7 μm).The mobile phase A was phosphate buffer-acetonitrile (19∶1), and the mobile phase B was phosphate buffer-acetonitrile (1∶1).The mobile phase was in gradient elution at a flow rate of 0.3 mL/min.The column temperature was set at 30 °C and the detection wavelength was 210 nm.The similarity of the fingerprints was analyzed with the Similarity Evaluation System for Chromatographic Fingerprint of Tradition Chinese Medicine (Version 2012) in combination with content determination of multiple index components to evaluate the quality consistency of imported and domestic bulk drugs.The result showed that both the original and generic bulk drugs met the specified limit requirements in the European Pharmacopoeia standards, and that their UPLC fingerprints were highly similar, indicating that the quality of the two substances was consistent.Establishing a fingerprint for similarity evaluation and combining it with the results of indicator component content determination as a comprehensive evaluation method for the study of drug quality consistency of complex components has the characteristics of fast, accurate, and comprehensive, which is helpful for drug quality evaluation and provides ideas for the evaluation of antibiotic quality consistency of complex components.