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Objective: Using LC-MS to explore the pharmacokinetic process in rats of Shenling Baizhu Pulvis (SBP), which was modified by particle design technology. Methods: Particle design powder of SBP was prepared by particle design technology. A scientific and feasible LC-MS analysis method was established to determine the blood concentration of index compounds such as ginsenoside Re (GI-Re), ginsenoside Rb1 (GI-Rb1), ginsenoside Rg1 (GI-Rg1), atractylenolide I (AT-I), atractylenolide II (AT-II) and pachymic acid (PA) in rats at different time points after administration. DAS 3.2.8 pharmacokinetic software was adopted to analyze the data, which related to blood concentration of index compounds, and the pharmacokinetics parameters were calculated by the non-compartmental model. Results: LC-MS analysis method was established, which has a good linear relationship and specificity for the index compounds in rats, and the RSD of precision, accuracy, extraction recovery and stability were all less than 5% or 10%. Compared with ordinary powder, the particle design powder displayed increased Cmax and AUC0-∞ after administration, and the AUC0-∞ of GI-Re, GI-Rb1, GI-Rg1, AT-I, AT-II and PA were increased to 1.52, 2.02, 1.22, 1.41, 1.13 and 1.43 times, respectively. Conclusion: The LC-MS analysis method meet the requirements of biological sample analysis in Pharmacopoeia of the People's Republic of China. After particle design and modification, the absorption speed of SBP in vivo become faster and the bioavailability is improved significantly.
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Objective: To establish a quality evaluation method of Shirebi Tablet based on HPLC fingerprints, quantitative analysis of multi-components and chemometrics analysis. Methods: The chromatographic column was Waters Symmetry C18 column (250 mm × 4.6 mm, 5 μm), and the column temperature was set at 30 ℃. The detection wavelength was set at 303 nm (for mulberroside A, mulberroside F and moracin M) and 270 nm (for forsythoside B, forsythoside A, forsythin, atractylodinol, atractylenolide II, and atractylodin). The mobile phase was composed of acetonitrile-0.2% phosphate acid solution in gradient elution manner at a flow rate of 1.0 mL/min. The HPLC fingerprint of Shirebi Tablet was established, the common peaks were determined by similarity evaluation system for chromatographic fingerprint of TCM (Version 2012.130723), and the similarity was calculated. The content determination methods for mulberroside A, mulberroside F, moracin M, forsythoside B, forsythoside A, forsythin, atractylodinol, atractylenolide II, and atractylodin were validated. The chemometrics methods such as cluster analysis and principal component analysis were used to evaluate the quality of Shirebi Tablet from different batches based on the results of fingerprint common peak area. Results: The fingerprint of Shirebi Tablet was established. Sixteen common peaks were identified. The similarity of fingerprints of 10 batches of Shirebi Tablet was more than 0.95. Nine components had good linear relationship in the range of mass concentration (r2 ≥ 0.999 1), and the average recoveries were 98.87%, 97.44%, 97.94%, 98.39%, 100.13%, 99.06%, 96.80%, 98.44% and 99.15% with the RSDs of 1.42%, 1.17%, 1.30%, 0.91%, 0.86%, 1.23%, 1.08%, 1.37% and 0.79%, respectively. The concentrations of mulberroside A, mulberroside F, moracin M, forsythoside B, forsythoside A, forsythin, atractylodinol, atractylenolide II, and atractylodin in 10 batches were 0.192—0.289, 0.057—0.095, 0.113—0.158, 0.309—0.375, 1.537—1.916, 0.478—0.596, 0.049—0.072, 0.279—0.354, and 0.629—0.759 mg/g, respectively. The results of cluster analysis showed that 10 batches of Shirebi Tablet were clustered into two groups, and the results of principal component analysis showed that the principal components 1—6 were the main factor affecting the quality evaluation of Shirebi Tablets. Conclusion: The method is simple, accurate and reproducible, which can be used for the quality control and evaluation of Shirebi Tablet.
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Objective: To establish HPLC-ELSD fingerprint of Zhenwu Decoction(ZWD), screen out the signature components of ZWD through chemical pattern recognition, so as to establish the content determination method of ZWD based on this index. Methods: The fingerprint of 16 batches of ZWD was established by HPLC-ELSD method. The similarity evaluation system of traditional Chinese medicine chromatographic fingerprint (2012 Version) was used for similarity evaluation to determine the common peaks and its attribution. Cluster analysis (CA), principal component analysis (PCA) and orthogonal partial least squares discriminant analysis (OPLS-DA) were used to select the index components of ZWD. Results: The fingerprint of ZWD was established, 38 common peaks were confirmed, and the similarity was > 0.95. The results of CA, PCA and OPLS-DA were consistent and the samples were divided into three categories. Benzoylmesaconine, benzoylaconitine, benzoylhypacoitine, polyporenic acid C, pachymic acid, atractylenolide II, atractylenolide III, oxypaeoniflorin, albiflorin, paeoniflorin and benzoylpaeoniflorin were identified as the 11 index components with significant difference contribution in different batches of ZWD samples. 6-Gingerol and 6-shogaol were the main active components of ginger, so the above 13 components were taken as the index components of ZWD. The chromatographic peak separation degree and linear relationship were good. The average recovery rate was 96.46%-99.80%, RSD ≤ 3.15%. The mass fraction range of benzoylmesaconine, benzoylaconitine, benzoylhypacoitine, polyporenic acid C, pachymic acid, atractylenolide II, atractylenolide III, oxypaeoniflorin, albiflorin, paeoniflorin, benzoylpaeoniflorin, 6-gingerol, 6-shogaol in 16 batches were 283.93-576.86, 25.05-147.39, 62.96-303.37, 31.24-131.27, 9.76-44.04, 32.15-83.55, 76.55-333.13, 17.48-146.61, 456.58-1554.14, 3 322.48-5 590.01, 158.21-556.50, 525.85-582.92 and 68.52-74.73 mg/g, respectively. Conclusion: The fingerprint combined with PCA, CA and OPLS-DA can comprehensively evaluate the quality of ZWD. This method is stable and reliable, providing reference for the quality evaluation.
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To explore the role of miR-31-5p/STAT3 in the pathogenesis of colitis-assosiated cancer (CAC) and the intervention mechanism of Huangqi Baizhu decoction. Methods The CAC model of C57BL/6 mice was established by AOM/DSS method. The differential expression of MicroRNA in control and CAC mice was detected. qPCR was used to screen differential MicroRNAs; Western blot was used to detect the expression levels of STAT3 , p-STAT3 and IL-6Ra. Results AOM was injected into abdominal cavity once, combined with 3% DSS free drinking for three cycles to establish model. After eight weeks, moderate and/or severe dysplasia of colonic mucosa appeared in model mice. After detection, the expression of miR-31-5p and STAT3 and p-STAT3 proteins significantly increased. After intervention with Huangqi Baizhu decoction, the expressions of miR-31-5p and STAT3 and p-STAT3 proteins were down-regulated. In vitro, miR-31-5p over-expression in RAW264. 7 by transfect method, and the expression of STAT3 protein increased significantly compared with that of control. On the other hand, stimulated by IL-6 or TNF-a for 24 h, miR-31-5p levels were up-regulated significantly, suggesting that inflammatory factors could cause a positive correlation between miR-31-5p/STAT3. At the same time, ATRII + AST (atractylodesin II + astragaloside) mixture significantly down-regulated miR-31-5p, STAT3, IL-6Ra increase induced by IL-6 stimulation. Conclusions miR-31-5p/STAT3 forms a positive feedback loop, which plays an important role in the pathogenesis of CAC. miR-31-5p/STAT3 may continue to amplify the inflammation effect and eventually lead to the dysplasia of colonic epithelium and even tumorigenesis.
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Objective To study the effects of atractylodes I, II, and III against rotavirus in vitro and in vivo. Methods An in vitro study model was established using Caco-2 cells. The cytopathic effect (CPE) and MTT staining were used to determine the toxicity of atractylenolide I, II, and III to cells for the inhibition of rotavirus biosynthesis, direct inactivation of rotavirus, and antiviral adsorption, with ribavirin as a positive drug. With half of the therapeutic concentration (EC50) and half of the cytotoxic concentration (TC50), the treatment index TI value was obtained and used as the evaluation index. An RV-infected model of suckling diarrhea was established in vivo to observe the signs and symptoms of the suckling mice, and the in vivo anti-rotavirus effect was preliminarily determined according to the diarrhea score and the weight gain. Results In vitro studies found that atractylenolide III had the direct inactivation effect on rotavirus with TI value of 8; atractylodes III medium-dose group has the best anti-rotavirus effect in vivo. Conclusion Atractylodes III, the main active component of Atractylodes macrocephala, has significant anti-rotavirus effect in vitro and in vivo; Atractylenolide III mainly works by directly inactivating rotavirus in vitro.
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Objective To comparative analyze the contents of 20 active ingredients in two dosage forms of Zhizhu Pills (ZP) and Zhizhu Granules (ZG). Methods Three batches of raw materials were used for the preparation of ZP and ZG according to the preparation process of China Pharmacopoeia (2015 Editon). Twenty active ingredients in the two dosage forms were detected simultaneously using high-performance liquid chromatography-triple quadrupole mass spectrometry (HPLC-QqQ-MS). The separation was performed on a Poroshell 120 SB-C18 (100 mm × 4.6 mm, 2.7 μm) column with a flow rate of 0.5 mL/min. Mobile phase consisted of 0.1% formic acid in water (A)-0.1% formic acid in acetonitrile (B). The column temperature was set at 30 ℃. The gradient elution conditions: 0-7 min, 23% B; 7-17 min, 23%-80% B; 17-20 min, 80%-100% B, 20-30 min, 100% B; flow rate was 0.5 mL/min. Results The contents of 20 active ingredients in the pills were 1.48-13.37 times that of the granules, the total difference of them perday were 493.02-615.08 mg. Conclusion There were significant differences in the content of active components in the two dosage forms of ZP and ZG, especially those components with poor solubility in water and thermal instability.
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Objective: To optimize the processing technology of Atractylodis Macrocephalae Rhizoma prepared by rice-washed water rinsing, and provide a scientific basis for producing specification in processing. Methods: Design the processing with central composite design-response surface methodology and take the factors of volume of rice-washed water, rinsing time, and rinsing temperature as independent variables. The contents of atractylenolide I, II, and III were determined by HPLC and the comprehensive scores of the three components were regarded as the response index or OD. By analyzing with Design Expert, the best processing parasite for the experiment could be induced. Results: The best processing conditions were 9-time volume of rice-washed water, 55 h for rinsing, and at the temperature of 26℃. On the selected condition, the value of OD was at 0.960. Conclusion: The rice-washed water rinsing processing technology for Atractylodis Macrocephalae Rhizoma is stable and feasible under the condition selected, which can be used as reference for its production and quality control.
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Objective To establish a quality assessment method for Ermiao Pills (EP) based on HPLC fingerprint and qualitatively analyze the chemical constituents. Methods The chromatographic column Shiseido C18 (250 mm × 4.6 mm, 5 μm) was used, acetonitrile-0.1% formic acid as mobile phase with gradient elution at the flow rate of 1.0 mL/min, and the detection wavelength was 330 nm. The standard chromatographic fingerprint was synthesized from chromatogram of the mixed standard herbs of Phellodendron Rupr. and Atractylodes DC, and the similarity evaluation of Ermiao Pills samples was carried out by the Similarity Evaluation System for Chromatographic Fingerprints of TCM (Chinese Pharmacopoeia Commission, version 2012A). Ultra high-performance liquid chromatography coupled with linear ion trap-Orbitrap Elite mass spectrometer (UPLC-LTQ-Orbitrap) was used to characterize the chemical constituents of Ermiao Pills. A Thermo Scientific Syncronis C18 (100 mm × 2.1 mm, 1.9 μm) column and a gradient elution of acetonitrile-0.1% formic acid were used for UPLC separation. The combination of ESI-LTQ-Orbitrap mass analyzer with a linear ion trap was applied for high resolution mass spectrometry and collision-induced dissociation (CID). Results The chromatographic fingerprints were generated with 10 common peaks. The similarity scores of 20 samples between each material batch and the reference fingerprint ranged from 0.869-0.992. Twenty-one components were identified via referring to reference components and literatures and analyzing MS data, they were neo-chlorogenic acid, magnocurarine, xanthoplanine, magnoflorine, 3-O-feruloylquinic acid, menisperine, demethyleneberberine, oxyberberine, columbamine, jatrorrhizine, berberubine, palmatine, berberine, syringic acid, caffeic acid, (E)-4-(3-hydroxyprop-1-en1yl)-2-methoxyphenol, atractylenolide II, acetosyringone, atractylenolide I, selina-4(14), 7(11)-dien-8-one, and atractylodin. Conclusion The established method of fingerprint is specific, combined with LC-MS qualitative analysis, can be used for the quality evaluation of Ermiao Pills, giving support to quality control comprehensively.
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Objective: To establish and identify the HPLC-PDA fingerprint of Atractylodis Macrocephalae Rhizoma (AMR) and provide a reference for the comprehensive control of the quality of AMR. Methods: AMR was extracted with 70% methanol by sonicating for 60 min. The analysis of AMR extract was performed on Inertsil® ODS-SP column (150 mm × 4.6 mm, 5 μm), column temperature was maintained at 40 ℃, flow rate was 1.0 mL/min, and detector was Waters 2998 UV detector with detection wavelength 235 nm. Mobile phase was acetonitrile (B)-water (A) with the elution gradient 0 -10 min, 30%-45% B, 10-25 min, 45% B, 25-50 min, 45%-70% B, 50-55 min, 70% B, 55-62 min, 70%-30% B, 62-75 min, 30% B. Time-of-flight mass spectrometer (TOF/MS) and electro-spray ion (ESI) source were used for the qualitative analysis in a positive ion mode, and mass scan range was m/z 50-1 500. Results: Comparing and fitting the peaks of AMR from different habitats (Zhejiang, Anhui, and Hunan Provinces), the HPLC-PDA fingerprint was set up with six common peaks, and they were identified by UFLC-Q-TOF/MS as 5-(hydroxymethyl)-2-furaldehyde, atractylenolide III, atractylenolide I, atractylenolide II, atractylenolide VI, and biatractylenolide. System suitability, extraction, and chromatographic conditions of AMR were optimized. RSD of accuracy, stability and repeatability was all less than 2%. Measuring ten batches and fitting fingerprint similarity, the values were all greater than 0.95. Conclusion: The HPLC fingerprint can be used as standard uniformity and stability of quality control methods for AMR slice.
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To optimize the processing technology of Atractylodis Macrocephalae Rhizoma fried with honey rice chaff in Jian Chang Bang. With the contents of atractylenolide I, atractylenolide II, atractylenolide III, atractylone, and alcohol extract as indexes, which were determined by HPLC and alcohol extract method in Pharmacopoeia 2010 edition, L9 (34) orthogonal test was used to determine the best processing technology. The optimum processing technology of Atractylodis Macrocephalae Rhizoma fried with honey rice chaff was as following: the amount of honey rice chaff 50%, frying temperature 200℃, and frying time 5 min. The optimum processing technology is stable and feasible by verification.