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@#To establish a quantitative LC-MS/MS method for the simultaneous detection of components of Erlong Zuoci Pill in rat plasma: verbascoside, oxypaeoniflorin, echinacoside and benzoylpaeoniflorin, and to evaluate the pharmacokinetic characteristics of Erlong Zuoci Pill in rats, plasma samples were purified by protein precipitation using methanol as a protein precipitant.Methanol was used as the organic phase and aqueous solution containing 0.1% formic acid was used as the water phase.The quantitative analysis method of verbascoside, oxypaeoniflorin, echinacoside and benzoylpaeoniflorin was established in negative ion mode, and the validation of bioanalytical method was carried out.Healthy SD rats were selected, and 20 mL/kg (equivalent to the original drug 10 g/kg dose) of Erlong Zuoci Pill extract was administered by intragastric administration.The plasma concentration of the target compounds at different time intervals after administration was determined using the established method, and the pharmacokinetic parameters was calculated by the Phoenix WinNonlin8.3 software using the non-compartmental model.The method validation results showed that verbascoside (r = 0.993 7) and oxypaeoniflorin (r = 0.994 6) had good linear relationship in the concentration range of 0.5-50 ng/mL, echinacoside (r = 0.993 6) and benzoylpaeoniflorin (r = 0.992 6) had good linear relationship in the concentration range of 1-100 ng/mL.The relative standard deviations of the inter- and intra- batch precision of the four compounds were all less than 15%, and the inter- batch and intra- accuracies were between 85% and 115%.Extraction recovery, matrix effect and stability met the relevant requirements.After a single gavage of Erlong Zuoci Pill extract in rats, all the four compounds were rapidly absorbed and eliminated.Oxypaeoniflorin, echinacoside, and benzoylpaeoniflorin showed two peaks in their drug concentration-time curves.Compared with the other three compounds, oxypaeoniflorin has the highest concentration in rat plasma with cmax1 of (24.40 ± 4.78) ng/mL and cmax2 of (22.50 ± 2.70) ng/mL. The results show that the validation results of this method are in line with the guiding principles of biological sample analysis methods, and it can be used to evaluate the pharmacokinetic characteristics of Erlong Zuoci Pill extract in rats.
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Objective: To establish an HPLC method for simultaneous determination of 15 monoterpene glycosides pyrindyl- paeoniflorin, mudanpioside F, oxyalbiflorin, oxypaeoniflorin, 10-hydroxypaeoniflorin, albiflorin, paeoniflorin, oxypaeonidanin, 4-O- methyl-oxypaeoniflorin, galloylpaeoniflorin, 4-O-methyl-paeoniflorin, albiflorin R1, paeonidanin, benzoyloxypaeoniflorin, and benzoylpaeoniflorin in Paeoniae Rubra Radix Formula Granule, in order to compare the quality of Paeoniae Rubia Radix Formula Granule from different manufacturers and provide the basis for the establishment of a unified quality control method. Methods The separation was performed on an Agilent Zorbax SB-Aq C18 column (250 mm × 4.6 mm, 5 µm), using acetonitrile and potassium dihydrogen phosphate solution (pH 2.8) as the mobile phase at the flow rate of 1.0 mL/min for a gradient elution. The detection wavelength was set at 260 nm. Results: Paeoniflorin, albiflorin, and oxypaeoniflorin were the three main monoterpene glycosides with the highest content in the Paeoniae Rubra Radix Formula Granule. There were extremely significant difference among the contents of 15 monoterpene glycosides in Paeoniae Rubra Radix Formula Granule produced by different manufacturers. The sample of CSPFKL-KRT was remarkable for the highest content of paeoniflorin and oxypaeoniflorin (73.214 mg and 16.935 mg per gram sample, respectively) and the lowest content of albiflorin among all samples (2.343 mg per gram sample), while sample CSPFKL-XLS was remarkable for the lowest content of paeoniflorin and oxypaeoniflorin (26.327 mg and 4.165 mg per gram sample, respectively) and the highest content of albiflorin among all samples (18.893 mg per gram sample). Conclusion: There were extremely significant difference among the contents of main monoterpene glycosides in Paeoniae Rubra Radix Formula Granule produced by different manufacturers, which may affect the clinical use. The establishment of a unified quality standard plays an important role in the quality control of Paeoniae Rubia Radix Formula Granule.
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OBJECTIVE: To establish a quantitative analysis of multi-components by single-marker (QAMS) method for simultaneous determination of senkyunolide H, senkyunolide I, senkyunolide A, ligustilide, oxypaeoniflorin, albiflorin, paeoniflorin, benzoylpaeoniflorin and α-cyperone in Renshen Nüjin pills, in order to provide basis for studying its quality standards.METHODS: The analysis of the methanol extract of Renshen Nüjin pills was performed on Agilent Zorbax SB C18 column (4.6 mm×250 mm,5 μm), with mobile phase composed of methanol-acetonitrile (2∶1)-0.1% phosphoric acid solution at a flow rate of 0.9 mL•min-1 in gradient elution mode. The column temperature was maintained at 30 ℃, and the detection wavelengths were set at 280, 230 and 242 nm. Paeoniflorin was selected as the internal standard, and the relative correlation factors (RCF) of senkyunolide H, senkyunolide I, senkyunolide A, ligustilide, oxypaeoniflorin, albiflorin, paeoniflorin, benzoylpaeoniflorin and α-cyperone were determined by HPLC. The accuracy and feasibility of the method were validated by comparing the results of QAMS method and external standard method.RESULTS: The standard curves of senkyunolide H, senkyunolide I, senkyunolide A, ligustilide, oxypaeoniflorin, albiflorin, paeoniflorin, benzoylpaeoniflorin and α-cyperone had good linear relationship in the ranges of the tested concentrations. The precision, stability and repeatability complied with the requirements of methodology. The recoveries were 97.38%, 98.16%, 98.84%, 99.63%, 97.04%, 99.14%, 100.04%, 96.93% and 98.48%, RSDs were 1.38%, 1.18%, 0.97%, 0.86%, 1.68%, 1.30%, 0.57%, 1.32% and 1.19%, respectively. No significant differences were observed in the determination results by QAMS method and external standard method.CONCLUSION: The QAMS method can be used for the content determination and quality control of the nine components in Renshen Nüjin pills.
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Objective: To establish a UPLC fingerprint of the compounds in Huoxuetongluo Injection, and to make a quantitative analysis. Methods: The Thermo C18 (100 mm × 3 mm, 1.7 μm) column was used with a mobile phase of methyl alcohol-0.05% formic acid gradient elution, the flow rate was 0.4 mL/min, the column temperature was 20℃, and the detection wavelength was 260 nm. Results: The fingerprint chromatography included 21 mutual peaks, of which 11 mutual peaks from Paeoniae Radix Rubra, 7 mutual peaks from Chuanxiong Rhizoma, and 3 mutual peaks from Persicae semen. The similarity among the batches was more than 0.98. Based on the retention time, and UV absorption spectra of reference compounds, five components, amygdalin, oxypaeoniflorin, albiflorin, paeoniflorin, and ferulic acid, were identified and quantified. Conclusion: The method is rapid, simple, and accurate, and can be used for the quality control of Huoxuetongluo Injection.
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Objective: To optimize the best processing technology for bran-fried Paeoniae Alba Radix (PAR). Methods: Taking the contents and extract yields of paeoniflorin, oxypaeoniflorin, albiflorin, and gallic acid in PAR as the indexes, the affecting factors on the processing technology of bran-fried PAR was investigated by orthogonal design test and multi-index comprehensive weighted mark method. Results: The best processing technology was to add 10% bran and fry for 8 min at 200°C. Conclusion: The optimal processing technology is stable, and could provide the references for the further study of PAR.
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Objective: To study the absorption and transport characteristic of paeoniflorin (PF), oxypaeoniflorin (OP), benzoylpaeoniflorin (BP), tetraacetylpaeoniflorin (TP), pentaacetylpaeoniflorin (PP), and pentacacetylalbiflorin (PA) in human colon adenocarcinoma cell line Caco-2 cell monolayer model. Methods: The Caco-2 cell monolayers were used as an intestinal epithelial cell model. The permeability of the tested compounds from apical (AP) side to basolateral (BL) side or from BL side to AP side was evaluated. The concentration of the tested compounds was measured by HPLC coupled with UV detector. The transport parameters and apparent permeability coefficients (Papp) were calculated, and the Papp values were compared with the reported values for model compounds, Propranolol and Atenolol. Results: The Papp values of PF in the bi-directional transport and atenolol were at the quantitative degree of 10-7 cm/s. Whereas those of OP, BP, TP, PP, and PA were between atenolol and propranolol used as a control substance for low and high permeability, respectively. The absorption and transport of the tested compounds were concentration-dependent at the concentration range of 10-200 μmol/L for PF, OP, and BP, 10-150 μmol/L for TP and PA, and 10-100 μmol/L for PP. Conclusion: The six tested compounds could be absorbed across the intestinal epithelial cells by passive diffusion mechanism. PF is poorly absorbed compound and OP, BP, TP, PP, and PA are moderately absorbed compounds. BP has a role to promote atenolol uptake transporters in Caco-2 cell monolayer model.
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Objective To determine and compare the contents of oxypaeoniflorin and paeoniflorin of Paeoniae Radix,and to improve the quality standard of Radix Paeonia.Methods The contents of oxypaeoniflorin and paeoniflorin in different kind of White and Red Peony Root were successfully analyzed by HPLC.Results The content of oxypaeoniflorin in Red Peony Root is more than White Peony Root at 0.8361％ ;The content of paeoniflorin in White Peony Root is more than Red Peony Root at 0.2157％.The proportion of paeoniflorin and oxypaeoniflorin in White Peony Root is more than Red Peony Root.Conclusion With HPLC technology,quantitative analysis of active constituents in Paeoniae Radix was performed accurately.