| Traditional Chinese Medicine (TCM) is the oldest continuously practiced system of herbal medicine in the world and has been applied by TCM practitioners for thousands of years. Radix Paeoniae Rubra and Radix Paeoniae Alba are both TCMs commonly used in clinic for a long history in China. Radix Paeoniae Rubra is the dried root of either Paeonia lactiflora Pall. or Paeonia veitchii Lynch and Radix Paeoniae Alba is the dried root of Paeonia lactiflora Pall.. Though these two herbs have almost the same source, the processing procedures are different. The former is the dried and directly used root of either Paeonia lactiflora Pall. or Paeonia veitchii Lynch. The latter is the decorticated and boiled dried root of Paeonia lactiflora Pall. Although being from almost the same origins, the clinical efficacies of the two medicines are different. Radix Paeoniae Rubra is considered as a medicine which could reduce fever, cool blood, eliminate stasis, activate blood circulation and relieve pain, while Radix Paeoniae Alba is often used to calm liver wind, stop pain, nourish the blood, regulate the menstrual function, astringe yin and suppress sweating. In other words, the two TCMs are regarded as two independent medicines. Thus investigations of the pharmacokinetic properties of these two herbs may be important in the study on their totally different clinical use.In the present study, an investigation on the pharmacokinetics and components analysis of Radix Paeoniae Rubra and Radix Paeoniae Alba by using HPLC-MS method was carried out. A separation method of paeoniflorin, albiflorin and benzoylpaeoniflorin from Paeonia veitchii L. was established. Pharmacokinetics of paeoniflorin, albiflorin and oxypaeoniflorin in rat plasma after oral gavage of extractions of Radix Paeoniae Rubra and Radix Paeoniae Alba were investigated, and a new way to describe the pharmacokinetic properties of these two herbs was developed. At last, a qualitative study by HPLC-MS was carried out to find out the differences of chemical constituents of these medicines. The research provided a significant exploration for pharmacokinetic and therapeutic basis of TCM.Part one Studies on chemical consituents of Paeonia vtitchii L (Seperation of paeoniflorin, albiflorin and benzoylpaeoniflorin)Objective: To establish a separation method of paeoniflorin, albiflorin and benzoylpaeoniflorin from Paeonia veitchii L.Methods: After extracted with alcohol and precipitated with water from Paeonia veitchii L, the extraction was isolated and puried by silica gel column chromatography and ODS column chromatography. Paeoniflorin, albiflorin and benzoylpaeoniflorin were identified by thin-layer chromatography and high performance liquid chromatography.Results: Three chemical constituents were separaed from Paeonia veitchii L and identified as: paeoniflorin, albiflorin and benzoylpaeoniflorin. The purities of these three compounds were all higher than 98% by normalization method of HPLC.Conclusion: The method we established was proved to be convenient and cost less. The products acquired could be used to carry out the pharmacokinetic study and qualitative study.Part two Studies on pharmacokinetic properties of Radix Paeoniae Rubra and Radix Paeoniae AlbaObjective: To establish a HPLC-MS method for simultaneously determination of paeoniflorin, albiflorin and oxypaeoniflorin in rat plasma for studying the pharmacokinetic properties of Radix Paeoniae Rubra and Radix Paeoniae Alba.Methods: Aqueous solutions of Radix Paeoniae Rubra and Radix Paeoniae Alba were orally gavaged to rats at a dose containing 0.2 g/g crude drug. After dosing for 5, 10, 15, 20, 30, 60, 90, 120, 180, 240, 360 and 540min, venous blood samples were collected in heparinized 1.5ml tubes by eye puncture. Sample was pretreated by a single-step protein precipitation with methanol and geniposide was internal standard. (1) Chromatographic conditions: Agilent Zorbax SB-C18 column (150 mm×4.6 mm, 5μm), and the column temperature was kept at room temperature. The mobile phase was composed of 33% methanol and 67% of 0.1% formic acid in water. The flow rate was 700μl/min. (2) Mass conditions: ion spray voltage -4.5 kV; turbo spray temperature 650°C; nebulizer gas (gas 1), 60 psi; heater gas (gas 2), 65 psi; curtain gas 25 psi. Analytes were quantificated by multiple-reaction-monitoring (MRM) mode employing the following precursor-to-product ion pair: paeoniflorin, m/z 525.2→121.0 with declustering potential (DP) -22 V and collision energy (CE) -37 eV; albiflorin, m/z 525.2→121.0 with DP -30 V and CE -37 eV; oxypaeoniflorin, m/z 495.2→137.0 with DP -65 V and CE -40 eV; geniposide, m/z 433.2→225.1 with DP -20 V and CE -19 eV.Results: Calibration curves of paeoniflorin, albiflorin and oxypaeoniflorin were ranged over 0.9262~185.2, 1.858~178.6 and 0.7689~38.22 ng·mL-1, respectively. The lower limits of detection (LLOD) of these three compounds were 0.9262,1.858 and 0.7689 ng/mL-1, respectively. RSD values of intra- and inter-day precisions were between 0.5% and 6.3% and the extraction recoveries were all between 85.6% and 105.2%. Main pharmacokinetic parameters for paeoniflorin, albiflorin and oxypaeoniflorin after oral gavage of Radix Paeoniae Rubra were T1/2 1.86±0.27, 1.17±0.33 and 1.59±0.53 h, Tmax 0.67±0.07, 0.33±0.15 and 0.33±0.06 h, ke 0.35±0.10, 0.59±0.09 and 0.44±0.06 h, Cmax 185.24±26.24, 122.08±30.41 and 16.64±4.53 ng·ml-1, AUC0-t 48572.38±480.18, 7796.00±125.33 and 69.73±5.52 ng·h·ml-1, AUC0-∞52274.73±489.32, 8804.31±100.92 and 83.92±2.99 ng·h·ml-1. Bimodal phenomenon was appeared for albiflorin in rats after oral gavage of Radix Paeoniae Alba, its parameters were Tmax1 0.33±0.05 h and Tmax2 0.67±0.07 h, Cmax1 178.60±35.39 ng·ml-1 and Cmax2 180.86±29.77 ng·ml-1. For paeoniflorin and oxypaeoniflorin, the parameters were Tmax 0.33±0.02 h and 0.67±0.23 h, Cmax 34.44±13.42 ng·ml-1 and 19.22±3.81 ng·ml-1. Other parameters were T1/2 0.85±0.11, 1.06±0.13 and 2.21±0.78 h,ke 0.79±0.29, 0.59±0.09 and 0.44±0.06 h, AUC0-t 288.66±80.38, 14865.04±239.59 and 79.03±9.31 ng·h·ml-1, AUC0-∞315.08±85.44, 15646.40±228.44 and 99.27±8.76 ng·h·ml-1. Total AUC0-t and AUC0-∞for Radix Paeoniae Rubra and Radix Paeoniae Alba was 56438.11, 61162.96 ng·h·ml-1 and 15232.73, 16060.76 ng·h·ml-1, respectively.Conclusion: A sensitive, specific and accurate HPLC-ESI-MS method was developed for the analysis of paeoniflorin, albiflorin and oxypaeoniflorin in SD rat plasma. The method had advantages of satisfactory selectivity, recovery, precision, stability, and simple preparation. The analytical procedure was then successfully applied to the pharmacokinetic study of paeoniflorin, albiflorin and oxypaeoniflorin in rats after oral gavage of extraction of Radix Paeoniae Rubra or Radix Paeoniae Alba. It played an important role in investigating the action mechanism of Radix Paeoniae Rubra or Radix Paeoniae Alba and provided a useful tool for clinical application of traditional Chinese medicine.Part three Studies on chemical constituents of Radix Paeoniae Rubra and Radix Paeoniae Alba by HPLC-MS methodObjective: Establish a qualitative method to analyse the chemical constituents of Radix Paeoniae Rubra and Radix Paeoniae Alba in order to investigate the different compositions of these herbs and explore the possible reasons of their different efficacies.Methods: Reference substances of paeoniflorin, albiflorin, oxypaeoniflorin and benzoylpaeoniflorin were used to carry out MS detections applying Q1 scan, product ion scan and precursor scan. 50% alcohol extractions of four different kinds of Radix Paeoniae were acquired by ultrasonic extracting and the supernatant was injected into the HPLC-MS instrument after high speed centrifuging. (1) Chromatographic conditions: Agilent Zorbax SB-C18 column (250 mm×4.6 mm, 5μm), and the column temperature was kept at room temperature. The mobile phase was composed of methanol (A)– 0.1% formic acid, using gradient elution (0~8 min, 0~8%A; 8~10 min, 8~20%A; 10~30 min, 20~30%A; 30~40 min, 30~40%A; 40~65 min, 40~60%A; 65~75 min, 60~90%A; 75~80 min, 90%A). The flow rate was 1 ml/min. (2) Mass conditions: ion spray voltage -4.5 kV; turbo spray temperature 650°C; nebulizer gas (gas 1), 65 psi; heater gas (gas 2), 65 psi; curtain gas 25 psi. Analytes were monitored by multiple ion monitoring (MRM)-information dependent acquisition (IDA)-enhanced product ion (EPI) mode. Mass range being tested was m/z 50~1000.Results: The chemical constituents of these four herbs were well isolated by HPLC, including monoterpene glycosides, saccharides and phenolic compounds. Under the negative mode, on the basis of feature fragment ions of each compound, 17 kinds of possible structures were inferred. Chemical constituents had some differences in these four herbs.Conclusion: Establish a HPLC-MS method to carry out the qualitative research about four kinds of Radix Paeonia. We supply a new way for the investigation of TCM.
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