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Objective:To establish a quality evaluation method for the simultaneous determination of Calycosin-7-O-β-D-Glucopyranoside and Lobetyolin in Danqi Xinmaikang boiled powders and pieces.Methods:Quantitative analysis of multi-components was performed to determine contents of Calycosin-7-O-β-D-Glucopyranoside and Lobetyolin with Calycosin-7-O-β-D-Glucopyranoside as the reference substance by single-maker (QAMS). The chromatogram conditions were established, with C18 column as solid phase, acetonitrile-water as flowing phase, 268 nm as detecting wavelength, 1.0 ml/min as flowing rate, 30 ℃ as column temperature, and 10 μl as injection volume.Results:The relative correction factor between Calycosin-7-O-β-D-Glucopyranoside and Lobetyolin was 1.14. There was no significant difference of measured values between the external standard method and QAMS ( P>0.05). With Calycosin-7-O-β-D-Glucopyranoside retention time of 1.00, the relative retention time of Lobetyolin was 1.51 and RSD was less than 5%. Conclusion:It is feasible and accurate to evaluate the quality of Danqi Xinmaikang boiled powders and pieces by QAMS.
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Objective: To establish a UPLC method to simultaneously determine 10 active ingredients in Yiganning Granules (YG) and provide scientific basis for the quality control, evaluation and standard revision of YG preparations. Methods: A UPLC method was used with an Acquity UPLC BEH C18 column (100 mm × 2.1 mm, 1.7 μm). The mobile phase was acetonitrile-mehanol-0.15% phosphoric acid solution with gradient elution. The flow rate was 0.3 mL/min. The column temperature was 45 ℃. The injection volume was 2 μL. Results: Ten active ingredients (chlorogenic acid, atractylenolide I, paeoniflorin, calycosin 7-O-β-D- glucopyranoside, stilbene glycoside, caffeic acid, toosendanin, kaempferol, paeonol and tanshinone ⅡA) in YG were simultaneously determined. The linearity was good (r ≥ 0.999 0), the limit of detection and quantification were 0.006-0.017 μg/mL and 0.017-0.510 μg/mL. The average recoveries were 98.8%-102.5% with RSDs of 1.13%-5.37%. Through the determination of 16 batches of samples, the average content of the above 10 ingredients was in turn (5.724 ± 0.017), (0.273 ± 0.003), (0.854 ± 0.005), (1.228 ± 0.004), (0.496 ± 0.003), (1.287 ± 0.004), (0.137 ± 0.004), (3.624 ± 0.014), (7.366 ± 0.032) and (1.754 ± 0.005) mg/g, respectively. Conclusion The established UPLC method is simple, specific, sensitive, stable, precise, accurate, and reproducible, which can be used for quality control and evaluation of YG.
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Objective: To establish HPLC fingerprints of Qiwei Tangmaishu tablet and simultaneously determine the contents of the six constituents, acteoside, martynoside, calycosin 7-O-β- D-glucopyranoside, schisandrin, formononetin and deoxyschizan- drin. Methods: The analysis of 50% methanol extract of this drug was performed on a 30℃ thermostatic Waters Symmetry C18 column, with the mobile phase comprising of acetonitrile(A)-0.2% formic acid solution(B)flowing at 1.0 ml/min in a gradient elution manner. The detection wavelength was set at 330 nm for acteoside and martynoside, 254 nm for calycosin 7-O-β-D-glucopyranoside, schisandrin, formononetin and deoxyschizandrin. Results: There were thirteen common peaks in the fingerprints of ten batches of samples with the similarities more than 0.9. Acteoside, martynoside, calycosin 7-O-β-D-glucopyranoside, schisandrin, formononetin and deoxyschizandrin showed good linear relationship within the ranges of 1.47-36.75, 0.66-16.50, 0.89-22.25, 2.58-64.50, 1.91-47.75 and 0.77-19.25 μg/ml(r≥0.9993), and their average recoveries were 97.57%, 99.22%, 96.69%, 100.01%, 98.79% and 96.77%, with the RSD of 0.79%, 1.54%, 0.61%, 0.64%, 0.83% and 0.36%, respectively. Conclusion: The established method is easily operable and repeatable, which could be used for quality control of Qiwei Tangmaishu tablet.
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Objective To determine the optimum extraction and purification technology of Huangqi Baihe Granules (HBG) by using orthogonal design and combination empowerment based on G1-entropy method, so as to provide a reference for the industrial production. Methods With the ethanol extraction amount and paste-forming rate of calycosin7-O-β-D-glucopyranoside, hesperidin, crude polysaccharide as evaluation index, and with the extraction times, extraction time and the water adding amount as investigate factors, then the combination empowerment method based on G1-entropy method and orthogonal design were used to optimize the extraction technology of HBG. The same combined methods were used to optimize the purification technology of HBG with retention rate and removal rate of impurity of ethanol extract of calycosin7-O-β-D-glucopyranoside, hesperidin and crude polysaccharide, as evaluation index, and with the membrane pore diameter, operating pressure and filtration temperature of multi-channel tubular ceramic ultrafiltration membrane as investigate factors. Results The test results showed that the optimum extraction technology was as follows: Extracted 2 times with 20 times the amount of water and each for 75 min. The optimum ultrafiltration technology was as follows: Multi-channel tubular inorganic ceramic membrane at 50 nm, operating pressure at 0.10 MPa, filtration temperature at 45 ℃. Under such condition, there was no significant difference between verification groups of three batches. Conclusion The optimized extraction and purification process is stable and feasible by verification, which can provide experimental basis for industrial production of HBG.
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Objective To establish fingerprint of Jinsangqi Kangdu Dropping Pills by HPLC; To control the quality of the preparation. Methods Waters XSELECT CSH-C18 chromatographic column (4.6 mm × 150 mm, 5 μm) was used and eluted with acetonitrile - 0.1% phosphoric acid solution gradient at the flow rate of 1.0 mL/min. The detection wavelength was 260 nm with column temperature of 30 ℃. Using calycosin-7-O-β-D-glucopyranoside, rutin, liquiritin, hyperoside, quercetin and ammonium glycyrrhizinate as the object references, ten batches of Jinsangqi Kangdu Dropping Pills were tested and analyzed by similarity comparison.Results Fringerprint spectrum of Jinsangqi Kangdu Dropping Pills had 24 common peaks in total, and characteristic spectrums of Hypericum Perforatum, Mori Cortex, Astragali Radix and Glycyrrhizae Radix et Rhizoma had been found, while similarity of HPLC fingerprint was more than 0.9 among those batches of samples. Conclusion Using HPLC fingerprint can evaluate the Jinsangqi Kangdu Dropping Pills quality totality,which can provide references for improving the quality control of the preparation.
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Objective: To establish HPLC-DAD method for the simultaneous determination of 23-acetate alisol B, ferulic acid, verbascoside, ginsenoside Rg1, ginsenoside Re, ginsenoside Rb1, gastrodin, chrysophanol, aurantio-obtusin, calycosin 7-O-β-D-glucopyranoside, and hyperoside in Shenqi Shiyiwei Granule (SSG). Methods: The chromatographic separation was achieved on an Waters XBridge-C18 (250 mm × 4.6 mm, 5.0 μm) column with methanol-acetonitrile-water (15:80:5) and methanol-0.1% phosphoric acid (10:90) as mobile phases for gradient elution, at the flow rate of 1.0 mL/min; The column temperature was 35℃. Results: The linear ranges of 23-acetate alisol B, ferulic acid, verbascoside, ginsenoside Rg1, ginsenoside Re, ginsenoside Rb1, gastrodin, chrysophanol, aurantio-obtusin, calycosin 7-O-β-D-glucopyranoside, and hyperoside were 0.4-8.0 μg/mL (r = 0.999 2), 0.2-4.0 μg/mL (r = 0.999 5), 0.2-4.0 μg/mL (r = 0.999 5), 0.1-2.0 μg/mL (r = 0.999 6), 0.1-2.0 μg/mL (r = 0.999 7), 0.1-2.0 μg/mL (r = 0.999 4), 0.5-10 μg/mL (r = 0.999 2), 0.6-12 μg/mL (r = 0.999 2), 0.4-8.0 μg/mL (r = 0.999 4), 1.0-20 μg/mL (r = 0.999 6), and 0.8-16 μg/mL (r = 0.9993). The average recoveries (n = 6) were 98.1% (RSD = 0.9%), 98.1% (RSD = 1.6%), 99.1% (RSD = 1.6%), 98.3% (RSD = 1.8%), 99.5% (RSD = 1.5%), 99.9% (RSD = 0.6%), 98.5% (RSD = 0.7%), 100.4 (RSD = 0.8%), 101.6% (RSD = 0.4%), 99.7% (RSD = 0.9%), and 101.2% (RSD = 1.1%), respectively. The contents of nine batches of 23-acetate alisol B, ferulic acid, verbascoside, ginsenoside Rg1, ginsenoside Re, ginsenoside Rb1, gastrodin, chrysophanol, aurantio-obtusin, calycosin 7-O-β-D-glucopyranoside, and hyperoside were 0.081-0.089, 0.261-0.269, 0.060-0.069, 0.038-0.047, 0.030-0.037, 0.042-0.049, 0.420-0.428, 0.141-0.151, 0.178-0.189, 0.107-0.117, and 0.069-0.078 mg/g. The results showed that there was little difference among the batches. Conclusion: The method is accurate, sensitive, credible, and repeatable. It can be applied to the quality control of SSG.
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Objective: To establish an HPLC-DAD method for the simultaneous determination of 10 components in Shendan Sanjie Capsule including tanshinone IIA, magnolol, honokiol, naringin, neohesperidin, ginsenoside Rg1, ginsenoside Re, ginsenoside Rb1, calycosin 7-O-β-D-glucopyranoside, and atractylenolide I. Methods: The chromatographic separation was performed on a Hypersil BDS column (150 mm × 4.6 mm, 3.5 μm) with acetonitrile-merhanol-0.1% phosphate acid solution as mobile phase at the flow rate of 1.0 mL/min for gradient elution and the column temperature was 40 ℃. The detection wavelength was set at 270 nm for tanshinone IIA, 294 nm for magnolol and honokiol, 283 nm for naringin and neohesperidin, 203 nm for ginsenoside Rg1, ginsenoside Re, and ginsenoside Rb1, 260 nm for calycosin 7-O-β-D-glucopyranoside, and 220 nm for atractylenolide I. The volume of sample injection was 10 μL. Results: Ten compounds were well separated under the determined chromatographic conditions. The RSD values of precision and repeatability experiment were all less than 2% and the sample solution was stable during 10 h. All the compounds had a wide linear range and good linearity: the linear range of tanshinone IIA, magnolol, honokiol, naringin, neohesperidin, ginsenoside Rg1, ginsenoside Re, ginsenoside Rb1, calycosin 7-O-β-D-glucopyranoside, and atractylenolide I were 112-560 μg/mL (r = 0.999 6), 64-320 μg/mL (r = 0.999 1), 48-240 μg/mL (r = 0.999 3), 80-400 μg/mL(r = 0.999 4), 80-400 μg/mL (r = 0.999 5), 16-80 μg/mL (r = 0.999 2), 16-80 μg/mL (r = 0.999 1), 16-80 μg/mL (r = 0.999 1), 40-200 μg/mL (r = 0.999 2), and 56-280 μg/mL (r = 0.999 3), respectively. The average recoveries were in the range of 98.43%-101.52% and the RSD values were all less than 2.0%. The content ranges of tanshinone IIA, magnolol, honokiol, naringin, neohesperidin, ginsenoside Rg1, ginsenoside Re, ginsenoside Rb1, calycosin 7-O-β-D-glucopyranoside, and atractylenolide I in six batches of Shendan Sanjie Capsule were 0.829-0.840 mg/g, 0.538-0.548 mg/g, 0.360-0.369 mg/g, 0.210-0.219 mg/g, 0.111-0.118 mg/g, 0.081-0.089 mg/g, 0.070-0.078 mg/g, 0.111-0.117 mg/g, 0.072-0.080 mg/g, and 0.130-0.137 mg/g, respectively. Conclusion: The method is simple and convenient, the methodology validation shows that determination result of the method is accurate and reliable and it can be an effective approach for the quality control of Shendan Sanjie Capsule.
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Objective: To study the metabolites from rats urine after ig administration of calycosin-7-O-β-D-glucopyranoside. Methods: The constituents were isolated and purified by preparative liquid chromatographic technique, and the structures were identified by spectroscopic analyses including ESI-MS, NMR, and 2D-NMR. Results: Five metabolites were isolated from rats urine after ig administration of calycosin-7-O-β-D-glucopyranoside. They were identified as calycosin (M1), 3', 4', 7-trihydroxyisoflavone (M2), daidzein (M3), calycosin-3'-O-β-D-glucuronide (M4), and calycosin-3'-O-β-D-glucuronide methyl ester (M5). Conclusion: M5 is a new compound.
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The isoflavone calycosin-7-O-β-d-glucopyranoside (CG) is a principal constituent of Astragalus membranaceus (AR) and has been reported to inhibit osteoclast development in vitro and bone loss in vivo. The aim of this study was to investigate the osteogenic effects of CG and its underlying mechanism in ST2 cells. The results show that exposure of cells to CG in osteogenic differentiation medium increases ALP activity, osteocalcin (Ocal) mRNA expression and the osteoblastic mineralization process. Mechanistically, CG treatment increased the expression of bone morphogenetic protein 2 (BMP-2), p-Smad 1/5/8, β-catenin and Runx2, all of which are regulators of the BMP- or wingless-type MMTV integration site family (WNT)/β-catenin-signaling pathways. Moreover, the osteogenic effects of CG were inhibited by Noggin and DKK-1 which are classical inhibitors of the BMP and WNT/β-catenin-signaling pathways, respectively. Taken together, the results indicate that CG promotes the osteoblastic differentiation of ST2 cells through regulating the BMP/WNT signaling pathways. On this basis, CG may be a useful lead compound for improving the treatment of bone-decreasing diseases and enhancing bone regeneration.
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Objective: To optimize the percolation extraction technology for Qixue Shuangbu Tinctura by response surface methodology (RSM). Methods: The single factor experiment combined with Box-Behnken design was used to optimize the extracting technology, with six major characteristic components (ginsenoside Rb1, calycosin 7-O-β-D-glucopyranoside, ferulic acid, stilbene glucoside, paeoniflorin, hesperidin) and extract of Qixue Shuangbu Tinctura as indexes, in order to detect three factors, including the alcohol volume fraction, the dosage of ethanol, and the percolate speed, and optimize the extraction process of Qixue Shuangbu Tinctura. Results: Optimum percolation process was added 10 times of the amount of 48% ethanol with percolation speed of 1.4 mL/min. Conclusion: This optimized extraction technology of Qixue Shuangbu Tinctura is reasonable, stable, and feasible, and with high accuracy, which could be extended to the applications of large-scale production.