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Objective:To establish high performance liquid chromatography （HPLC） fingerprint and multi-component determination for the substance benchmark of Yiweitang， and to evaluate its quality in combination with chemical pattern recognition method. Method:Fifteen batches of substance benchmark of Yiweitang were prepared， the "Chinese medicine chromatographic fingerprint similarity evaluation system" （2012 edition） was used to calculate similarity. Cluster analysis， principal component analysis and orthogonal partial least squares-discriminant analysis were employed to handle the common peaks for evaluating the quality difference among 15 batches of the substance benchmark. The contents of catalpol， verbascoside and methylophiopogonanone A were determined with mobile phase system of acetonitrile-phosphoric acid solution at detection wavelengths of 210 nm and 334 nm. Result:There were 22 common peaks in HPLC fingerprint of the substance benchmark， among them， peaks 1， 9， 12， 14-17， 19 and 20 belonged to Rehmanniae Radix， peaks 3， 4， 6， 7 and 21 belonged to Glehniae Radix， peaks 5 and 22 belonged to Ophiopogonis Radix， peaks 2 and 18 belonged to Polygonati Odorati Rhiaoma， peak 8 was the common peak of Ophiopogonis Radix and Rehmanniae Radix， peak 10 was shared by Ophiopogonis Radix， Polygonati Odorati Rhiaoma<italic> </italic>and<italic> </italic>Rehmanniae Radix， peak 11 was the common peak of these four herbs， and peak 13 was shared by Polygonati Odorati Rhiaoma and Rehmanniae Radix. The similarities between HPLC fingerprints of 15 batches of the substance benchmark and the control fingerprint were all >0.90， the samples could be divided into four categories by three chemical pattern recognition methods. Quantitative analysis showed that the contents of catalpol， verbascoside and methylophiopogonanone A among 15 batches of samples ranged from 0.37% to 1.14%， 0.002% to 0.054% and 0.016% to 0.079%， respectively. Conclusion:The established fingerprint and determination for the substance benchmark of Yiweitang have good separation and high accuracy， which reflect the overall chemical composition characteristics of Yiweitang， and can provide experimental basis for the further development and quality control of the compound preparations of this famous classical formula.
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Objective: To explore the change features of Ophiopogonis Radix in the development process, and to find the accumulation rules of effective components in Ophiopogonis Radix. Methods: The method of chlorfluazuron formulation was analyzed by HPLC. DAD detector, column temperature was set at 30 ℃ and detection wavelength was 203 nm; Flow rate was set at 0.8 mL/min and sample size was 15 μL and the mobile phase was acetonitrile (A)-water (B) with gradient elution mode (0~20 min, 25% A →40% A; 20~50 min, 40%~50% A; 50~60 min, 50%~70% A; 60~70 min, 70% A; 70~80 min, 70%~100% A, 80~90 min, 100% A), at the volume flow rate of 1.0 mL/min. ELSD detector, column temperature was set at 35 ℃, drift tube temperature at 100 ℃ and the gas flow rate was set at 3.0 L/min. The sample size was 15 μL and the mobile phase was acetonitrile (A)-0.1% phosphoric acid water (B) with gradient elution mode: 0~60 min, 35%~65% A, at the volume flow rate of 1.0 mL/min. Results: The main components in the chromatogram were both separated well under the two conditions. At 203 nm, there were 14 effective peaks, the main components were flavones whose representative was methylophiopogonanone A. There were 11 effective peaks in the condition of ELSD, and the main peak represent saponins whose representative was Ophiopogonin D. It could be judged that the accumulation law of the each component in Ophiopogonis Radix was inconsistent in the different growth phase according to the peaks area. The overall change tendency of the components is increase to peak value and keep balance and to a slight decrease after the balance. Conclusion: The accumulation rules of effective components in Ophiopogonis Radix could be reflected comprehensively by HPLC specific chromatogram.It could provide abundant information for the produce of Ophiopogonis Radix and quality control.
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Objective: Using chemical component content determination and fingerprint analysis to evaluate the effects of different drying methods on quality of Ophiopogonis Radix. Methods: The contents of total saponin, total flavonoids, and total polysaccharide were determined by ultraviolet spectrophotometry, and ophiopogonin D and methylophiopogonanone A were determined using HPLC; the chromatograms fingerprint was established by HPLC. Results: The influence of different drying methods on the chemical components in Ophiopogonis Radixa was significantly different, and the coefficient of variation fell in the range of 6.9%-20.8%. Among them, the damage degree of dried in the shade, dried in the sun, freeze drying and dried after semi-died in the sun is small on chemical component of Ophiopogon japonicus. However, the far infrared drying and microwave drying have the most serious effect; Ophiopogonis Radix samples have 18 characteristic peaks through HPLC. The 14 kinds of drying methods were divided into three types by cluster analysis, and a class of drying methods has similar drying conditions. Conclusion: The difference of drying method can be effectively reflected by the change of the content of chemical compositions and the feature of HPLC spectrum, which can be used as indicators of O. japonicus origin screening method. In the producing area, it is best to combine drying in the sun with in air source heat pump dryer in processing of Ophiopogonis Radix.
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Objective: To compare the contents of three homoisoflavones and their anti-oxidative activity of Ophiopogon japonicus in Sichuan (OJS) and Zhejiang (OJZ). Methods: The determination was performed on ACQUITYTM UPLC BEH C18 column (100 mm×2.1 mm, 1.7 μm), the Ultra Performance Liquid Chromatography-Photo-Diode Array (UPLC-PDA) technology was applied, and the mobile phase consisted of acetonitrile and 0.2% formic acid aqueous solution (55:45) with gradient elution program, flow rate was 0.20 mL/min with 2 μL of sample quantity at 296 nm, and the anti-DPPH radical efficiency of water extract from Ophiopogonis Radix were evaluated by UV-photometer. Results: The average values of methylophiopogonone A, methylophiopogonanone A, and methylophiopogonanone B were (3.06±0.54), (40.10±5.63), and (29.51±5.06) μg/g in OJS, respectively; while those in OJZ were (9.22±3.52), (106.63±27.56), and (256.97±61.79) μg/g, separately. The IC50 value was 16.59 mg/mL in OJS, while that in OJZ was 14.48 mg/mL. The IC50 value of positive control VC was 7.06 μg/mL. Conclution: Compared with OJS, the contents of homoisoflavones in OJZ are higher, and the anti-radical efficiency of water extract from OJZ is stronger. It provides the basis for the quality evaluation and geo-authentic research of Ophiopogonis Radix.
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Objective: To develop a UPLC-MS/MS method for simultaneously determining harpagide, liquiritin, harpagoside, platycodin D, ammonium glycyrrhetate, ophiopogonin D, methylophiopogonanone A, and methylophiopogonanone B in Xuanmai Ganjie Granules (composed with Scrophulariae Radix, Ophiopogonis Radix, Glycyrrhizae Radix et Rhizoma, and Platycodonis Radix) from different pharmaceutical companies. Methods: The chromatographic separation was achieved on Phenomenex Kenetix C18 column (50 mm × 2.1 mm, 5 μm) by using a mobile phase consisted of acetonitrile and 0.1% formic acid water at the flow rate of 0.3 mL/min for gradient elution. Simultaneous monitoring of positive and negative ions and multiple reaction monitoring (MRM) scan mode were applied to the quantification of the components in Xuanmai Ganjie Granules; Sample volume was 5 μL. Results: There was good linearity between the absorption peak area and the concentration for harpagide, liquiritin, harpagoside, platycodin D, ammonium glycyrrhetate, ophiopogonin D, methylophiopogonanone A, and methylophiopogonanone B in the ranges of 9-2250, 8-2000, 3.4-850, 96-24000, 12.4-3100, 3.6-1900, 1.7-425, and 1.5-375 ng/mL, respectively. The average recoveries were ranged from 97.2% to 102.8% (RSD ≤ 2.7%). The contents of harpagide, liquiritin, harpagoside, platycodin D, ammonium glycyrrhetate, ophiopogonin D, methylophiopogonanone A, and methylophiopogonanone B in eight batches of samples were in the ranges of 32.8-107.6, 54.8-178.0, 14.6-70.7, 31.2-280.0, 106.4-287.9, 0.1-0.6, 0.01-0.07, and 0.03-0.17 μg/g, respectively. Conclusion: The developed method is simple, effective, and credible for determining the eight components in Xuanmai Ganjie Granules. It provides more helpful information for the comprehensive quality evaluation of Xuanmai Ganjie Granules.
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Objective: To develop a liquid chromatography-tandem mass spectrometry (HPLC-MS/MS) method for simultaneously analyzing seven components (ginsenosides Rg1, ginsenosides Re, ginsenosides Rb1, ophiopogonin D, ophiopogonin D', methylophiopogonanone A, and methylophiopogonanone B) in Shenmai Injection. Methods: Multiple reaction monitoring (MRM) scan mode was used for the quantification of five saponins and two flavones. The seven constituents were separated within 15 min on a Phenomenex Luna C18 column (150 mm × 2.1 mm, 5 μm) using a mobile phase consisted of acetonitrile and 0.03% acetic acid water solution with gradient elution. Results: The linear relationships between the concentration and peak areas of the seven target components were ginsenoside Rg1 Y=15.6 X + 1.63 × 104, ginsenoside Re Y=14 X + 5.36 × 103, ginsenoside Rb1 Y=2.46 X + 4.74 × 103, ophiopogonin D Y=11 X + 9.73 × 103, ophiopogonin D' Y=5.56 X + 1.64 × 103, methylophiopogonanone A Y=3.58 × 103 X + 2.33 × 104, and methylophiopogonanone B Y=4.87 × 103 X + 2.72 × 104, respectively. The precisions, repeatabilities, and stabilities of the method were good for the seven components. The average recovery ranged from 95.3%-104.3%, and the precision in terms of RSD was less than 2.4%. Conclusion: The method is rapid and reliable for the determination of the seven constituents in Shenmai Injection. Among these constituents, ophiopogonin D, ophiopogonin D', methylophiopogonanone A, and methylophiopogonanone B are quantified in the Shenmai Injection for the first time.
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Objective To determine the contents of methylophiopogonanone A (MOA) and methylophiopogonanone B (MOB) in Radix Ophiopogonis and its extracts. Methods An HPLC-UV method was used for determining the contents of MOA and MOB in all samples. Analytical column was Kromasil C18 (250 mm?4.6 mm, 5 ?m). Mobile phase was acetonitrle-water (55∶45) and detection wavelength was 298 nm. The flow rate of mobile phase was 1 mL/min, and temperature was 30 ℃. Results The contents of MOA in Radix Ophiopogonis cropped in Sichuan and Zhejiang Provinces were 0.004 0%-0.009 6%, 0.006 7%-0.013 4%, and the contents of MOB were 0.002 1%-0.006 2%, 0.015 9%-0.028 2%, respectively. The contents of MOA in the extract of Radix Ophiopogonis cropped in Sichuan and Zhejiang Provinces were 0.007 5%-0.008 8%, 0.011 2%-0.012 6%, and the contents of MOB were 0.003 8%-0.005 1%, 0.020 7%-0.023 8%, respectively. Conclusion The contents of MOA and MOB in Radix Ophiopogonis cropped in Zhejiang Province and its extracts are more than those in Sichuan Province and its extrouts. The method can be used for the purpose of the quality control of Radix Ophiopogonis and its extracts.