赤芍与白芍的药动学研究与化学成分的液质联用分析
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摘要
赤芍与白芍为我国常用中药材。赤芍为毛茛科植物芍药Paeonia lactiflora Pall.或川赤芍Paeonia veitchii Lynch直接使用的干燥根;白芍为毛茛科植物芍药Paeonia lactiflora Pall.水煮去皮后晒干使用的根。二者来源相近,但临床功效却不尽相同。赤芍具有清热凉血,散瘀止痛的功效,用于温毒发斑,吐血衄血,目赤肿痛,肝郁胁痛,经闭痛经,癥瘕腹痛,跌扑损伤,痈肿疮疡。而白芍具有平肝止痛,养血调经,敛阴止汗的功能,主要用于头痛眩晕,胁痛,腹痛,四肢挛痛,血虚萎黄,月经不调,自汗和盗汗。
本研究即从化学成分与药动学两方面着手,探讨两药材基源相近却功效迥异的原因。在提取分离制备白芍和赤芍有效成分芍药苷、芍药内酯苷和苯甲酰芍药苷的基础上,采用液相色谱-质谱联用技术研究大鼠灌胃赤芍或白芍提取物后芍药苷、芍药内酯苷和氧化芍药苷的药物动力学特征,并初步探讨两药材的药动学特征的差异。对赤芍、白芍、川赤芍与常用混淆品新疆芍药的化学成分进行HPLC-MS定性分析,并比较化学成分的差异。本研究对阐明赤芍、白芍的作用机制和药效物质基础做出了有价值的探索,对指导临床用药具有重要意义。
第一部分川赤芍化学成分研究(芍药苷、芍药内酯苷和苯甲酰芍药苷的制备)
目的:从川赤芍中分离制备芍药苷、芍药内酯苷和苯甲酰芍药苷3种成分。
方法:采用硅胶柱色谱与ODS柱色谱的方法对川赤芍醇提物进行系统分离纯化,通过薄层、UV和HPLC等方法对其分离产物进行鉴定。
结果:提取分离得到3个化合物,分别鉴定为芍药苷、芍药内酯苷和苯甲酰芍药苷。所得芍药苷、芍药内酯苷和苯甲酰芍药苷用归一化法定量,纯度均大于98%。
结论:本法简便,制备成本低,芍药苷、芍药内酯苷和苯甲酰芍药苷可作为定性定量分析使用的对照品,也可用于药物代谢动力学的研究。
第二部分赤芍与白芍提取物的药动学研究
目的:建立高效液相色谱-质谱联用(HPLC-MS)法同时测定大鼠血浆中芍药苷、芍药内酯苷和氧化芍药苷的浓度,并将其用于SD大鼠灌胃赤芍或白芍提取物后血浆中3种成分的药动学研究,初步比较赤芍和白芍两种药材药动学特征的差异。
方法:以0.2g·g-1相同生药量灌胃给与大鼠赤芍或白芍提取物,分别于给药后5,10,15,20,30,60,90,120,180,240,360和540min眼内眦静脉丛取血,制备血浆样品后,采用甲醇沉淀蛋白的方法进行预处理,采用HPLC-MS法,以栀子苷为内标进行分析:(1)色谱条件:色谱柱为Agilent Zorbax-C18柱(150mm×4.6mm,5μm),流动相为甲醇-0.1%甲酸水(33:67),流速为0.7mL·min-1。(2)质谱条件:ESI源,源电压- 4500 V,负离子检测,源温度650℃,雾化气(gas 1) 60 psi,加热气(gas 2)65 psi,帘气25 psi。检测方式为多反应监测(MRM)模式,用于定量分析检测的离子及各自的解簇电压(DP)和裂解电压(CE)分别为m/z 525.2→121.0(芍药苷),DP -22V,CE -37eV;m/z 525.2→121.0(芍药内酯苷),DP -30V,CE -37eV;m/z 495.2→137.0(氧化芍药苷),DP -65V,CE -40eV和m/z 433.2→225.1(内标栀子苷),DP -20V,CE -19eV。
结果:芍药苷、芍药内酯苷和氧化芍药苷的线性范围分别为0.9262~185.2,1.858~178.6和0.7689~38.22ng·mL-1,最低定量限分别为0.9262,1.858和0.7689 ng。日内和日间精密度(RSD)在0.5%和6.3%之间。其提取回收率在85.6%和105.2%之间。大鼠灌胃赤芍提取物后,3个主要成分芍药苷、芍药内酯苷和氧化芍药苷的药物动力学参数如下: T1/2分别为1.86±0.27,1.17±0.33和1.59±0.53 h,Tmax分别为0.67±0.07,0.33±0.15和0.33±0.06 h,ke分别为0.35±0.10,0.59±0.09和0.44±0.06 h,Cmax分别为185.24±26.24,122.08±30.41和16.64±4.53 ng·ml-1,AUC0-t分别为48572.38±480.18,7796.00±125.33和69.73±5.52 ng·h·ml-1,AUC0-∞分别为52274.73±489.32,8804.31±100.92和83.92±2.99 ng·h·ml-1。大鼠灌胃白芍提取物后,芍药内酯苷出现了双峰现象,Tmax1和Tmax2分别为0.33±0.05和0.67±0.07 h,Cmax1和Cmax2分别为178.60±35.39和180.86±29.77 ng·ml-1,芍药苷和氧化芍药苷的Tmax分别为0.33±0.02和0.67±0.23 h,Cmax分别为34.44±13.42和19.22±3.81 ng·ml-1,3个主要成分芍药苷、芍药内酯苷和氧化芍药苷的其他药物动力学参数如下:T1/2分别为0.85±0.11,1.06±0.13和2.21±0.78 h,ke分别为0.79±0.29,0.59±0.09和0.44±0.06 h,AUC0-t分别为288.66±80.38,14865.04±239.59和79.03±9.31 ng·h·ml-1,AUC0-∞分别为315.08±85.44,15646.40±228.44和99.27±8.76 ng·h·ml-1。白芍和赤芍中3个成分的AUC0-t、AUC0-∞之和分别为56438.11、61162.96 ng·h·ml-1和15232.73、16060.76 ng·h·ml-1。
结论:本文建立了同时测定血浆中芍药苷、芍药内酯苷和氧化芍药苷的HPLC-MS法,并将其应用于SD大鼠灌胃赤芍或白芍提取物后3种成分的药动学研究,并将得到的药动学参数进行综合,初步研究了赤芍和白芍两种药材药动学性质的差异。
第三部分赤芍与白芍化学成分的HPLC-MS分析
目的:建立HPLC-MS法定性分析赤芍、白芍、川赤芍和新疆芍药的化学成分,考察4种药材的化学成分差异。
方法:对芍药苷、芍药内酯苷、氧化芍药苷和苯甲酰芍药苷对照品进行一级质谱(Q1)、子离子(Product ion)、母离子(Precusor ion)等多种扫描模式,分析其电喷雾电离质谱裂解途径,并总结单萜苷类成分的质谱裂解特征。采用超声提取法制备4种药材的50%乙醇提取液,高速离心取上清液进行HPLC-MS分析:(1)色谱条件:Agilent Zorbax C18柱(250mm×4.6mm,5μm),流动相为甲醇-0.1%甲酸水,梯度洗脱(0~8min,0~8%甲醇;8~10min,8~20%甲醇;10~30min,20~30%甲醇;30~40min,30~40%甲醇;40~65min,40~60%甲醇;65~75min,60~90%甲醇;75~80min,90%甲醇),流速为1mL·min-1。(2)质谱条件:ESI源;源电压- 4500 V;负离子检测;源温度(TEM):650℃;雾化气(gas 1) :65 psi,加热气(gas 2): 65 psi,帘气(CUR):25 psi。检测方式为多离子监测-自动信息关联-增强型子离子(MRM-IDA-EPI)模式,扫描质量范围m/z 50~1000。
结果:在负离子模式下,芍药单萜苷类结构中蒎烷基本骨架产生的m/z 165碎片离子较常见,这一离子可作为芍药苷类化合物的特征性碎片离子。同时葡萄糖或蒎烷骨架结构连有不同的取代基时,相应的碎片离子也会出现。根据化合物的特征碎片离子,推测出11个芍药苷类化合物, 4种糖类化合物以及2种酸或酯类化合物共17种。其中大部分化合物在这4种药材中广泛存在。白芍中未能检测到去苯甲酰基芍药苷及其异构体;芍药苷亚硫酸酯仅出现在白芍和赤芍药材中;川赤芍中未能检测到没食子酸甲酯;四没食子酰基葡萄糖仅赤芍药材中未能检出。
结论:本文建立的HPLC-MS方法可用于赤芍、白芍、川赤芍和新疆芍药的化学成分定性研究,为白芍和赤芍药效物质基础的阐明奠定了基础。
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 Alba
Objective: 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 method
Objective: 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.
本研究即从化学成分与药动学两方面着手,探讨两药材基源相近却功效迥异的原因。在提取分离制备白芍和赤芍有效成分芍药苷、芍药内酯苷和苯甲酰芍药苷的基础上,采用液相色谱-质谱联用技术研究大鼠灌胃赤芍或白芍提取物后芍药苷、芍药内酯苷和氧化芍药苷的药物动力学特征,并初步探讨两药材的药动学特征的差异。对赤芍、白芍、川赤芍与常用混淆品新疆芍药的化学成分进行HPLC-MS定性分析,并比较化学成分的差异。本研究对阐明赤芍、白芍的作用机制和药效物质基础做出了有价值的探索,对指导临床用药具有重要意义。
第一部分川赤芍化学成分研究(芍药苷、芍药内酯苷和苯甲酰芍药苷的制备)
目的:从川赤芍中分离制备芍药苷、芍药内酯苷和苯甲酰芍药苷3种成分。
方法:采用硅胶柱色谱与ODS柱色谱的方法对川赤芍醇提物进行系统分离纯化,通过薄层、UV和HPLC等方法对其分离产物进行鉴定。
结果:提取分离得到3个化合物,分别鉴定为芍药苷、芍药内酯苷和苯甲酰芍药苷。所得芍药苷、芍药内酯苷和苯甲酰芍药苷用归一化法定量,纯度均大于98%。
结论:本法简便,制备成本低,芍药苷、芍药内酯苷和苯甲酰芍药苷可作为定性定量分析使用的对照品,也可用于药物代谢动力学的研究。
第二部分赤芍与白芍提取物的药动学研究
目的:建立高效液相色谱-质谱联用(HPLC-MS)法同时测定大鼠血浆中芍药苷、芍药内酯苷和氧化芍药苷的浓度,并将其用于SD大鼠灌胃赤芍或白芍提取物后血浆中3种成分的药动学研究,初步比较赤芍和白芍两种药材药动学特征的差异。
方法:以0.2g·g-1相同生药量灌胃给与大鼠赤芍或白芍提取物,分别于给药后5,10,15,20,30,60,90,120,180,240,360和540min眼内眦静脉丛取血,制备血浆样品后,采用甲醇沉淀蛋白的方法进行预处理,采用HPLC-MS法,以栀子苷为内标进行分析:(1)色谱条件:色谱柱为Agilent Zorbax-C18柱(150mm×4.6mm,5μm),流动相为甲醇-0.1%甲酸水(33:67),流速为0.7mL·min-1。(2)质谱条件:ESI源,源电压- 4500 V,负离子检测,源温度650℃,雾化气(gas 1) 60 psi,加热气(gas 2)65 psi,帘气25 psi。检测方式为多反应监测(MRM)模式,用于定量分析检测的离子及各自的解簇电压(DP)和裂解电压(CE)分别为m/z 525.2→121.0(芍药苷),DP -22V,CE -37eV;m/z 525.2→121.0(芍药内酯苷),DP -30V,CE -37eV;m/z 495.2→137.0(氧化芍药苷),DP -65V,CE -40eV和m/z 433.2→225.1(内标栀子苷),DP -20V,CE -19eV。
结果:芍药苷、芍药内酯苷和氧化芍药苷的线性范围分别为0.9262~185.2,1.858~178.6和0.7689~38.22ng·mL-1,最低定量限分别为0.9262,1.858和0.7689 ng。日内和日间精密度(RSD)在0.5%和6.3%之间。其提取回收率在85.6%和105.2%之间。大鼠灌胃赤芍提取物后,3个主要成分芍药苷、芍药内酯苷和氧化芍药苷的药物动力学参数如下: T1/2分别为1.86±0.27,1.17±0.33和1.59±0.53 h,Tmax分别为0.67±0.07,0.33±0.15和0.33±0.06 h,ke分别为0.35±0.10,0.59±0.09和0.44±0.06 h,Cmax分别为185.24±26.24,122.08±30.41和16.64±4.53 ng·ml-1,AUC0-t分别为48572.38±480.18,7796.00±125.33和69.73±5.52 ng·h·ml-1,AUC0-∞分别为52274.73±489.32,8804.31±100.92和83.92±2.99 ng·h·ml-1。大鼠灌胃白芍提取物后,芍药内酯苷出现了双峰现象,Tmax1和Tmax2分别为0.33±0.05和0.67±0.07 h,Cmax1和Cmax2分别为178.60±35.39和180.86±29.77 ng·ml-1,芍药苷和氧化芍药苷的Tmax分别为0.33±0.02和0.67±0.23 h,Cmax分别为34.44±13.42和19.22±3.81 ng·ml-1,3个主要成分芍药苷、芍药内酯苷和氧化芍药苷的其他药物动力学参数如下:T1/2分别为0.85±0.11,1.06±0.13和2.21±0.78 h,ke分别为0.79±0.29,0.59±0.09和0.44±0.06 h,AUC0-t分别为288.66±80.38,14865.04±239.59和79.03±9.31 ng·h·ml-1,AUC0-∞分别为315.08±85.44,15646.40±228.44和99.27±8.76 ng·h·ml-1。白芍和赤芍中3个成分的AUC0-t、AUC0-∞之和分别为56438.11、61162.96 ng·h·ml-1和15232.73、16060.76 ng·h·ml-1。
结论:本文建立了同时测定血浆中芍药苷、芍药内酯苷和氧化芍药苷的HPLC-MS法,并将其应用于SD大鼠灌胃赤芍或白芍提取物后3种成分的药动学研究,并将得到的药动学参数进行综合,初步研究了赤芍和白芍两种药材药动学性质的差异。
第三部分赤芍与白芍化学成分的HPLC-MS分析
目的:建立HPLC-MS法定性分析赤芍、白芍、川赤芍和新疆芍药的化学成分,考察4种药材的化学成分差异。
方法:对芍药苷、芍药内酯苷、氧化芍药苷和苯甲酰芍药苷对照品进行一级质谱(Q1)、子离子(Product ion)、母离子(Precusor ion)等多种扫描模式,分析其电喷雾电离质谱裂解途径,并总结单萜苷类成分的质谱裂解特征。采用超声提取法制备4种药材的50%乙醇提取液,高速离心取上清液进行HPLC-MS分析:(1)色谱条件:Agilent Zorbax C18柱(250mm×4.6mm,5μm),流动相为甲醇-0.1%甲酸水,梯度洗脱(0~8min,0~8%甲醇;8~10min,8~20%甲醇;10~30min,20~30%甲醇;30~40min,30~40%甲醇;40~65min,40~60%甲醇;65~75min,60~90%甲醇;75~80min,90%甲醇),流速为1mL·min-1。(2)质谱条件:ESI源;源电压- 4500 V;负离子检测;源温度(TEM):650℃;雾化气(gas 1) :65 psi,加热气(gas 2): 65 psi,帘气(CUR):25 psi。检测方式为多离子监测-自动信息关联-增强型子离子(MRM-IDA-EPI)模式,扫描质量范围m/z 50~1000。
结果:在负离子模式下,芍药单萜苷类结构中蒎烷基本骨架产生的m/z 165碎片离子较常见,这一离子可作为芍药苷类化合物的特征性碎片离子。同时葡萄糖或蒎烷骨架结构连有不同的取代基时,相应的碎片离子也会出现。根据化合物的特征碎片离子,推测出11个芍药苷类化合物, 4种糖类化合物以及2种酸或酯类化合物共17种。其中大部分化合物在这4种药材中广泛存在。白芍中未能检测到去苯甲酰基芍药苷及其异构体;芍药苷亚硫酸酯仅出现在白芍和赤芍药材中;川赤芍中未能检测到没食子酸甲酯;四没食子酰基葡萄糖仅赤芍药材中未能检出。
结论:本文建立的HPLC-MS方法可用于赤芍、白芍、川赤芍和新疆芍药的化学成分定性研究,为白芍和赤芍药效物质基础的阐明奠定了基础。
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 Alba
Objective: 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 method
Objective: 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.
引文
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1 Yang HO, Ko WK, Kim JY, et al. Paeoniflorin: an antihyperlipidemic agent from Paeonia lactiflora. Firorerapia, 2004, 75:45-49
2 Ye G, Li YZ, Li YY, et al. SPE-HPLC method for the determination and pharmacokinetic studies on paeoniflorin in rat serum after oral administration of traditional Chinese medicinal preparation Guan-Xin-Er-Hao decoction. Journal of Pharmaceutical and Biomedical Analysis 2003, 33:521-527
3张晓燕,李铣.白芍的化学研究进展.沈阳药科大学学报,2002,19(1):70-73
4张晓燕,王金辉,李铣.芍药单萜苷的NMR谱学研究.沈阳药科大学学报,2001,18(1):27-29
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2陈海生,徐一新,廖时萱,等.川赤芍化学成分的研究.第二军医大学学报,1994,15(1):72-73
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1 Yang HO, Ko WK, Kim JY, et al. Paeoniflorin: an antihyperlipidemic agent from Paeonia lactiflora. Firorerapia, 2004, 75:45-49
2 Ye G, Li YZ, Li YY, et al. SPE-HPLC method for the determination and pharmacokinetic studies on paeoniflorin in rat serum after oral administration of traditional Chinese medicinal preparation Guan-Xin-Er-Hao decoction. Journal of Pharmaceutical and Biomedical Analysis 2003, 33:521-527
3张晓燕,李铣.白芍的化学研究进展.沈阳药科大学学报,2002,19(1):70-73
4张晓燕,王金辉,李铣.芍药单萜苷的NMR谱学研究.沈阳药科大学学报,2001,18(1):27-29
5武洁,姚楠,王大为. HPLC-MS/MS测定大鼠血浆中的芍药苷及其药动学研究.中国中药杂志,2008,33(20):2369-2373
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7 Wang CH, Wang R, Cheng XM, et al. Comparative pharmacokinetic study of paeoniflorin after oral administration of decoction of Radix Paeoniae Rubra and Radix Paeoniae Alba in rats. Journal of Ethnopharmacology, 2008, 117:467-472
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