白头翁质量评价与五环三萜皂苷类成分的药物代谢动力学研究
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摘要
白头翁(Pulsatilla chinensis (Bunge) Regel)是毛莨科植物白头翁的干燥根,主要分布于亚洲、欧洲和北美洲等地区。白头翁具有清热解毒、凉血止痢的功效,临床上被广泛用于阿米巴性痢疾、疟疾、阴道滴虫、细菌感染和恶性肿瘤等疾病的治疗。近年来,由于白头翁药材具有明显的抗肿瘤作用而越来越被广泛关注。故本课题对白头翁药材进行了系统地分离、质量评价和药物代谢动力学研究。
本实验采用大孔吸附树脂法,硅胶柱色谱法、葡聚糖凝胶和反相柱色谱法对白头翁药材进行系统分离,并建立了一个简单、快速的HPLC-ESI-MS法同时定量测定白头翁药材中9种化学成分。在对其进行定性研究中,利用HPLC-ESI-MS/MS联用技术分析研究了8个五环三萜皂苷类化合物的特征碎片离子,推测其电喷雾质谱裂解规律,最终在白头翁提取物中推导出了37个五环三萜皂苷化合物的结构。在药代动力学研究中,首次建立了一种灵敏的、可靠的多次切换监测模式HPLC-MS方法同时测定大鼠灌胃给予白头翁提取物后大鼠血浆中4种三萜皂苷的含量,并应用于药代动力学研究。首次对大鼠口服白头翁提取物后胆汁中6种五环三萜皂苷类化合物进行分析测定,并对这些三萜皂苷的胆汁排泄轮廓进行研究。综上对白头翁药材的系统研究将为其进一步开发应用提供全面的理论依据。
第一部分白头翁化学成分研究
目的：研究药材白头翁(Pulsatilla chinensis (Bunge) Regel)中的化学成分。
方法：取粉碎后的白头翁约10kg,用70%乙醇冷浸提取3次,采用AB-8大孔吸附树脂进行分离,收集70%乙醇洗脱液部分,得浸膏(总苷)约500g。采用大孔吸附树脂法,硅胶柱色谱法、葡聚糖凝胶和反相柱色谱法对白头翁药材进行系统分离,并用波谱和化学方法鉴定化合物的结构。
结果：从白头翁的70%乙醇提取物中分离得到9个单体化合物,对其进行了结构鉴定,其中包括5个五环三萜皂苷,2个三萜酸,2个甾酮类化合物。分别鉴定为齐墩果酸、白头翁皂苷A3、白头翁皂苷B4、23-羟基白桦脂酸、刺人参皂苷S、白头翁皂苷B、白头翁皂苷C、筋骨草甾酮C和p-蜕皮甾酮。
结论：从白头翁中分离鉴定了9个化合物,其中3个化合物为首次从该植物中发现,分别为刺人参皂苷S,筋骨草甾酮C和p-蜕皮甾酮。第二部分液质联用技术同时测定白头翁中9种化学成分及其质量表征
目的：建立HPLC-MS法测定白头翁中白头翁皂苷B4(1),白头翁皂苷A3(2),23-羟基白桦脂酸(3),刺人参苷S(4),白头翁皂苷C(5),白头翁皂苷B(6),齐墩果酸(7),p-蜕皮甾酮(8)和筋骨草甾酮C(9)含量的方法。
方法：采用Sapphire C18柱(250mm×4.6mm,5μm);流动相：A(甲醇含0.1%甲酸)-B(0.1%甲酸水溶液),梯度洗脱程序为0-6min(75%-95%A),6-14min(95%A),随后回到最初75%A并持续到21min；流速为0.8mL.min-1。采用电喷雾离子源进行正离子和负离子模式检测,多反应监测模式(MRM)用于定量测定。正离子监测模式下源喷射电压设置为5500V；负离子监测模式下源喷射电压设置为-4500V；离子源温度为650℃。雾化气(Gas1):40psi,加热气(Gas2):50psi,接口持续加热,帘气25psi,全程氮气通入状态。扫描方式采用多重反应监测(MRM)；碰撞气(CAD,N2)压力为medium;Q1和Q3分辨率均为UNIT。
结果：峰面积与浓度呈良好的线性关系(r2>0.9948)；日内和日间精密度分别小于2.78%和2.68%,并应用于20批次不同来源白头翁药材的含量测定。
结论：本研究中建立的分析方法灵敏度高、专属性好,可为白头翁药材质量控制提供一个强有力的工具。有助于规范白头翁药材的质量,并能够在一定程度上指导中医临床用药的合理性。
第三部分液质联用技术定性鉴定白头翁中五环三萜皂苷类成分
目的：对白头翁进行超声提取后即进行HPLC-MS分析,研究8个五环三萜皂苷类化合物的特征碎片离子,推测其电喷雾质谱裂解规律,再将此规律应用于白头翁中未知羽扇豆烷型和齐墩果烷型三萜皂苷类化合物的结构推测,根据已知裂解规律尽可能多的推测出白头翁中五环三萜皂苷的化学结构。
方法：采用了MRM-IDA-EPI和PREC-IDA-EPI两种模式分析未知五环三萜皂苷类化合物。取白头翁药材粉末(40目)5g,加70%甲醇25mL,称重,冰浴超声提取1h,称重,用70%甲醇补足减失的重量,0.45μm微孔滤膜滤过,滤液减压蒸干,残渣用2mL的70%甲醇溶解,0.45μm微孔滤膜滤过,进行HPLC-MS分析。色谱柱为Sapphire C18(250mm×4.6mm,5μm),采用0.1%甲酸甲醇(A)-0.1%甲酸水(B)为流动相,梯度洗脱75-75%A (0-15min);75-80%A (15-30min);80-95%A (30-40min);95-95%A (40-50min),流速为700μL·min-1。质谱：ESI源；负离子检测源电压-4500V；正离子检测源电压5500V；源温度(TEM)：650。C；雾化气(gas1):40psi,加热气(gas2):50psi,帘气(CUR)：25psi。
结果：通过与对照品的保留时间、分子量和质谱数据比较,分别对8个已知五环三萜化合物色谱峰进行归属。化合物6,10,14,18,20,21,35和36分别鉴定为白头翁皂苷B4,白头翁皂苷C,23-羟基羽扇R-20(29)-烯-28-酸-3-O-[吡喃鼠李糖基-吡喃阿拉伯糖基]28-O-[吡喃葡萄糖酯苷],白头翁皂苷B,23-羟基羽扇豆-20(29)-烯-28-酸-3-O-[吡喃阿拉伯糖基]-28-O-[呲喃葡萄糖酯苷],常春藤皂苷元28-0-[吡喃葡萄糖基-吡喃葡萄糖酯苷],白头翁皂苷A3和常春藤皂苷。同时,通过由对照品裂解行为总结得出的规律推断出了29个五环三萜皂苷类化合物的结构。
结论：本实验首次建立了利用HPLC-ESI-MS联用技术分析白头翁中五环三萜皂苷类成分的方法。本法灵敏度高、专属性强,成功用于白头翁中五环三萜皂苷类化合物的结构鉴定。通过联合MRM-IDA-EPI和PREC-IDA-EPI扫描模式对化合物结构分析方面具有明显优越性,对控制中药材质量具有重要意义。
第四部分HPLC-MS法同时测定大鼠血浆中4种五环三萜皂苷类成分及其药物代谢动力学研究
目的：建立HPLC-MS/MS同时测定大鼠血浆中白头翁提取物4个五环三萜皂苷类化合物(白头翁皂苷B4,白头翁皂苷B,白头翁皂苷A3和23-羟基白桦脂酸)的分析方法,并计算其在大鼠体内的药动学参数。
方法：血浆样品采用了Strata-X固相萃取柱预处理,采用Sapphire C18(250mm×4.6mm,5μm)色谱柱；以A(甲醇含0.1%甲酸)-B(0.1%甲酸水溶液)为流动相,梯度洗脱程序为：0-6min,75-95%A;6-15min,保持95%A不变,预平衡时间为6min,流速为800μL·min-1。色谱柱后溶液全部进入离子源,无分流,分析时间为15min。质谱以电喷雾离子源进行正离子模式和负离子模式检测,采用多反应监测模式,并首次应用了多次切换检测模式。白头翁皂苷B4,白头翁皂苷B,白头翁皂苷A3,23-羟基白桦脂酸和内标的监测离子对分别为m/z1219.7/749.4, m/z819.4/347.2, m/z749.6/471.2, m/z471.4/471.4和m/z461.1/285.0。本研究将4种五环三萜皂苷类成分和内标化合物的检测过程划分为5个阶段：0.0-6.998min,内标和白头翁皂苷B4在负离子模式下监测；6.998-7.028min,负离子扫描模式被切换至正离子扫描模式；7.028-10.027min,正离子模式下监测白头翁皂苷B；10.027-10.057min,正离子扫描模式被切换至负离子扫描模式；10.057-15.059min,负离子模式下监测白头翁皂苷A3和23-羟基白桦脂酸。
结果：4种五环三萜皂苷类成分在大鼠血浆中专属性、线性、准确度、精密度、提取回收率和基质回收率均被验证。4种待测化合物的线性关系良好(r2>0.99)；日内、日间精密度的相对标准偏差(RSD)均小于9%；在室温储存24h,5次冻融和-20。C储存30天的条件下各待测化合物的稳定性良好,准确度在-6.82%-6.18%之间；高、中、低三个浓度水平的平均提取回收率分别为82.16%-104.5%；基质效应均值在77.69%-108.2%之间。
结论：该方法特异、快速、灵敏,结合固相萃取的预处理方法和HPLC-MS的新检测手段对白头翁中4种五环三萜皂苷类化合物进行了药物代谢动力学研究。本研究中首次应用了多次切换检测模式,并首次在五环三萜皂苷类化合物体内测定时采用了Strata-X固相小柱进行生物样品预处理。此方法为五环三萜皂苷类化合物的药物代谢动力学研究提供了一个新的思路。
第五部分液质联用技术测定大鼠胆汁中6种五环三萜皂苷类成分及其排泄动力学研究
目的：建立一种可同时测定大鼠胆汁中6种主要五环三萜皂苷类化合物(白头翁皂苷B4,刺人参苷S,白头翁皂苷C,白头翁皂苷B,白头翁皂苷A3和23-羟基白桦脂酸)含量的HPLC-MS方法,并用于大鼠灌胃给予白头翁提取物后其6种待测化合物的胆汁排泄动力学研究。
方法：色谱柱为Sapphire-C18柱(250mm×4.6mm,5μm);柱温为25℃;流动相为(0.1%甲酸)甲醇A-0.1%甲酸水溶液B,梯度洗脱,洗脱程序如下：0.0-6.0min,75-95%A;6.0-15.0min,保持95%A；进样前预平衡6min。流速为800μL·min-1。色谱柱后溶液全部进入离子源,无分流,进样量为10μL。电喷雾离子源(ESI源),Turbo V离子源,源温度为650℃。源喷射电压为5500V和-4500V,源内气体1(Gas1, N2)压力：40psi；气体2(Gas2, N2)压力：50psi；气帘气体(N2)压力：25psi；正负离子分别检测模式；扫描方式为多重反应监测(MRM)；碰撞气(CAD,N2)压力为medium; Q1分辨率为UNIT, Q3分辨率为UNIT. SD大鼠8只,随机分成2组。其中2只为空白对照组,实验组6只大鼠灌胃给予白头翁提取物(10mL·kg-1)后,收集给药后0-1h,1-3h,3-5h,5-7h,7-9h,9-12h和12-24h时间段的胆汁并测定体积。测定大鼠灌胃给予白头翁提取物后胆汁中6种五环三萜皂苷类化合物的累积排泄率。
结果：大鼠胆汁中内源性物质不干扰6种待测化合物和内标的测定；6种五环三萜皂苷类成分标准曲线的相关系数(r2)均大于0.9973；日内RSD和日间RSD分别小于9.1%和9.0%,RE在-9.5%到9.2%之间；提取回收率的范围为80.03%～115.6%；基质效应的范围为78.43%-109.7%。
结论：该方法简便、快速、灵敏度高、专属性强,可满足大鼠胆汁中微量五环三萜类皂苷成分的测定要求。待测6种五环三萜皂苷类化合物在1-3h内排泄率最高,在3-5h时间段时略有减少,随着时间的推移,排泄率逐渐降低,并趋于平缓。结果表明,只有不到14%的待测物以原型从胆汁排泄,结果表明其五环三萜皂苷类化合物在体内可能存在某种生物转化和体内代谢。
第六部分白桦脂酸血浆蛋白结合率测定
目的：建立白桦脂酸在大鼠血浆、人血浆和牛血清白蛋白中蛋白结合率的测定方法。
方法：采用平衡透析法测定白桦脂酸血浆蛋白结合率,利用HPLC法测定血浆中白桦脂酸浓度,测定并比较不同血浆中白桦脂酸的血浆蛋白结合率。
结果：在50～100μg/mL,药物浓度对血浆蛋白结合率无显著影响,白桦脂酸与大鼠和人的血浆蛋白结合率存在显著性差异,白桦脂酸与人血浆蛋白结合率高于与大鼠血浆蛋白结合率。
结论：白桦脂酸与血浆蛋白具有较强的结合,且在实验设计的浓度范围内蛋白结合率与透析液中药物浓度无关。只有少量游离药物作为活性药物分子参与体内药物处置,从而产生药理效应。第七部分白桦脂酸大鼠在体肠吸收动力学研究
目的：建立同时测定肠循环液中白桦脂酸和酚红浓度的HPLC-DAD法,并探讨白桦脂酸在大鼠各肠段的吸收动力学特征及不同药物浓度对吸收的影响。
方法：采用大鼠在体肠吸收实验模型,并考察吸收部位、药物浓度和pH值对药物吸收的影响。色谱柱为Diamonsil ODS C18色谱柱(250mm×4.6mm,5μm);流动相为乙腈-0.2%乙酸(75:25)；流速1.0mL.min-1;柱温30℃；检测波长203nm(白桦脂酸)和430nm(酚红)；进样量20μL。
结果：在75～125μg·mL-1内白桦脂酸的吸收速率与质量浓度呈线性关系,Ka值基本保持不变；各肠段的吸收速率无显著性差异,十二指肠、空肠.回肠和结肠的Ka值分别为(0.151±0.0049),(0.156±0.0056),(0.149±0.0083),(0.159±0.0041)h-1。
结论：白桦脂酸在小肠中吸收良好,没有特定吸收部位；不同浓度对白桦脂酸在大鼠全肠道的吸收无显著影响,其在肠道的吸收呈一级吸收动力学特征,吸收机制为被动扩散。白桦脂酸是难溶性药物,可以通过增加药物的溶出度,进而提高药物的生物利用度。
Pulsatilla chinensis, the dried roots of Pulsatilla chinensis (Thunb.) Bercht. et Opiz., has been widely used as traditional Chinese medicine (TCM) for thousands of years in China. Modern pharmacological studies demonstrated that Pulsatilla chinensis extracts had a number of effects, such as hypoglycaemic, antitumour, cognition-enhancing, neuroprotective, cvtotoxic and antiendotoxin activities. Pulsatilla chinensis is known for containing a large number of compounds, including triterpenoidal saponins, phytosterone and anthocyanins. Among these, triterpenoidal saponins are generally considered to be the major components, and many of them have been found to have biological activities. In recent years, more and more triterpenoidal saponins have been isolated from Pulsatilla chinensis and they exhibit a variety of bioactivities.
Therefore, quality control analysis of the active components is an important issue for the safe and effective use of herbal medicines and their preparations. The constituents in Pulsatilla chinensis are complex, and some of them usually are of low content. Therefore, a more sensitive, selective and rapid method is demanded. The emergence of high performance liquid chromatography connected with tandem mass spectrometry (HPLC-MS/MS) makes the determination possible.
In this research, the nine main components were isolated and identified from Pulsatilla chinensis. A rapid, efficient and accurate HPLC-ESI-MS/MS method provided a new basis for overall assessment on quality of Pulsatilla chinensis and should be considered as a suitable quality control method. Then37triterpenoid saponins in Pulsatilla chinensis were identified by HPLC-ESI-MS/MS. Finally, a novel method was developed to determined triterpenoidal saponins in rat plasma and rat bile by HPLC-MS. At the same time, drug-protein binding and absorption kinetics of betulic acid were studies.
Part one Studies on chemical components of Pulsatilla chinensis
Objective:To study the chemical constituents of Pulsatilla chinensis.
Methods:Air-dried herbal of Pulsatilla chinensis (10kg) were extracted by diffusion for three times with70%alcohol.The residue was pretreated by AB-8macroporous resin, and then collected the part of70%alcohol eluant. Finally we got the extractum for500g. The compounds were isolated and purified by macroporous resin, silicagel, dextrane gel and reversed-phase column chromatographies. Their structures were identified by various spectral data.
Results:Nine compounds were isolated from the70%ethanol extract of Pulsatilla chinensis, including5triterpenoidal saponins,2triterpenic acid and2phytosterone. Their structures were elucidated as oleanolic acid, anemoside A3, anemoside B4,23-hydroxybetulinic acid, cirenshenoside S, pulsatilloside B, pulsatilloside C, ajugasterone C and (3-ecdysterone, respectively.
Conclusion:Nine compounds were isolated and identified from Pulsatilla chinensis. Compounds5,8and9were isolated from Pulsatilla chinensis for the first time.
Part two Quantitative determination of9components in Pulsatilla chinensis by HPLC-ESI-MS/MS
Objective:The present study describes the development, validation and a practical application of a fully automated analytical method based on high performance liquid chromatography coupled to electrospray ionization tandem mass spectrometry (HPLC-ESI-MS/MS) for the simultaneous determination of7triterpenoidal saponins, anemoside B4(1), anemoside A3(2),23-Hydroxybetulinic acid (3), cirenshenoside S (4), pulsatilloside C (5), pulsatilloside B (6) and oleanolic acid (7), together with two other phytosterone named β-ecdysterone (8) and ajugasterone C (9) in Pulsatilla chinensis.
Methods:A Sapphire C18(250mm×4.6mm,5μm) and gradient elution were used during the analysis. A binary gradient elution was performed:0min (75%A),0-6min (95%A),6-14min (95%A). Then quickly returned to initial75%A and maintained until21min for column balance. The identification and quantification of those analytes were achieved on a hybrid quadrupole linear ion trap mass spectrometer. Multiple-reaction monitoring scanning was employed for quantification with switching electrospray ion source polarity between positive and negative modes in a single run. The ion spray voltage was set to5500V and-4500V, respectively. The turbo spray temperature was maintained at500℃. Nebulizer gas (gas1) and heater gas (gas2) was set at40and50psi, respectively.
Results:All calibration curves showed good linearity (r2>0.9948) within the test ranges. The intra-day and inter-day variations were less than2.78%and2.68%, respectively. The developed method was successfully applied to determine the investigated compounds in20samples of Pulsatilla chinensis.
Conclusion:Compared with the reported analytical methods of Pulsatilla chinensis, this rapid, efficient and accurate HPLC-ESI-MS/MS method provided a new basis for overall assessment on quality of Pulsatilla chinensis and should be considered as a suitable quality control method.
Part three Characterization of triterpenoid saponins in Pulsatilla chinensis using liquid chromatography coupled with hybrid triple quadruple linear ion trap mass spectrometry
Objective:Eight triterpenoid saponins in crude extracts from Pulsatilla chinensis have been investigated by electrospray ionization multi-stage tandem mass spectrometry. To summarize fragmentation rules and develop a high sensitive and efficient liquid chromatography-mass spectrometry (LC-MS) method for detection and characterization of the trace triterpenoid saponins in Pulsatilla chinensis.
Methods:The triterpenoidal saponins in a crude extract of Pulsatilla chinensis were characterized by the combined use of the MRM-IDA-EPI and PREC-IDA-EPI modes on a hybrid triple quadrupole-linear ion trap mass spectrometer. The chromatographic separation was performed on a Sapphire C18column (250mm×4.6mm,5μm) with linear gradient elution with methanol containing0.1%formic acid-0.1%aqueous formic acid at a flow rate of0.8mL·min-1. The ion spray voltage was set to5500V and-4500V, respectively. The turbo spray temperature was maintained at650℃. Nebulizer gas (gas1) and heater gas (gas2) was set at40and50psi, respectively. The curtain gas was kept at25psi and interface heater was on. Nitrogen was used in all cases.
Results:According to the retention times, molecular weight and characteristic fragment ions, compounds6,10,14,18,20,21,35and36were identified anemoside B4, pulchinenoside C,23-hydroxy-lup-20(29)-en-28-oic acid3-0-[rhamnopyranosyl-arabinopyranosyl]28-0-[glucopyranoside], pulchinenoside B,23-hydroxy-lup-20(29)-en-28-oic acid3-O-[arabinopyranosyl]28-O-[glucopyranoside], hederagenin28-O-[glucopyranosyl-glucopyranoside], anemoside A3and hederagenin, respectively. Six fragmentation rules were summarized. Finally,29triterpenoid saponins in a crude extract of Pulsatilla chinensis were characterized.
Conclusion:In this study, a high sensitive, accurate and effective HPLC-MS method for on-line qualitative analysis of the trace triterpenoid saponins in Pulsatilla chinensis has been developed. This study has demonstrated the unprecedented advantage of the combination use of the MRM-IDA-EPI and PREC-IDA-EPI mode. It will play an important role in controlling the quality of medicinal herb in the future.
Part four Development of a novel method for triterpenoidal saponins in rat plasma by HPLC-MS
Objective:A novel method using high-performance liquid chromatography tandem mass spectrometry (HPLC-MS/MS) has been developed for the quantification of four triterpenoidal saponins (anemoside B4, pulsatilloside B, anemoside A3and23-hydroxybetulinic acid) in rat plasma following solid phase extraction (SPE).
Methods:The optimized procedure utilized off-line extraction of the analytes from plasma using polymeric (Strata-X) SPE cartridges. Detection and quantitation was performed by MS/MS using electrospray ionization (ESI) and multiple reaction monitoring (MRM) in a novel multi-switching monitoring mode. The analytes and internal standard (scutellarin) were analyzed using a Sapphire C18column (250mm×4.6mm,5μm) with a linear gradient elution. The mass transition ion pairs of the triterpenoidal saponins were executed as the following:m/z1219.7/749.4for anemoside B4, m/z819.4/347.2for pulsatilloside B, m/z749.6/471.2for anemoside A3, m/z471.4/471.4for23-hydroxybetulinic acid and m/z461.1/285.0for the internal standard. the negative ionization mode was switched to the negative mode from0.0to6.998min to detect the IS and anemoside B4. Then, the negative ionization mode was switched to the positive mode from6.998to7.028min. Within the time range of7.028to10.027min, pulsatilloside B was detected in the positive ionization mode. Subsequently, the ESI converted back to the negative ionization mode once again from10.027to10.057min. In the last section, anemoside A3and23-hydroxybetulinic acid were detected in the negative mode from10.057to15.059min.
Results:The specificity, linearity, accuracy, precision, recovery, matrix effect, and stabilities were validated for all analytes in the plasma samples.the four analytes showed good linearity over the low and high concentration range with r2>0.99. The LLOQ of the assay was defined as the lowest concentration of the standard curve that could be quantified accurately within a20%bias of the nominal concentration, with the precision not exceeding20%. Both the precision and accuracy of the four analytes at LLOQ were less than9%. The analytes in rat plasma were all stable for24h at room temperature, three cycles of freeze-thaw,30days at-20℃with accuracy in the range from-6.82to6.18%. At three concentration levels of these analytes, the extraction recoveries were all between82.16and104.5%. The matrix effects of this study were evaluated by analyzing samples at three concentration levels (low, medium and high), which were in the ranges of77.69-108.2%.
Conclusion:A novel analytical approach, multiswitching monitoring mode, was presented that applies different ESI modes in a single HPLC ESI-MS/MS run. In addition, the use of polymeric (Strata-X) SPE cartridges has been applied to the off-line extraction of triterpenoidal saponins from rat plasma samples for the first time. Combining the rapid and efficient off-line drug SPE with the specific and sensitive multiswitching monitoring mode, a new, robust analytical method for the analysis of triterpenoidal saponins in rat plasma has been developed and validated. This validated method is a novel technique for sample preparation and quantitation and was successfully applied to estimate the pharmacokinetics of triterpenoidal saponins.
Part five Quantitative analysis of six triterpenoidal saponins in rat bile after oral administration of Pulsatilla chinensis extract by high performance liquid chromatography-electrospray ionization tandem mass spectrometry
Objective:The aim of this study was to develop a liquid chromatography-tandem mass spectrometry (HPLC-MS/MS) method for the simultaneous quantitative determination of the following triterpenoidal saponins:anemoside A3, anemoside B4,23-hydroxybetulinic acid, pulsatilloside B, pulsatilloside C and cirenshenoside S in rat bile.
Methods:The chromatographic separation was performed using a Sapphire C18column (250mm×4.6mm,5μm) and gradient elution was used during the analysis. Anemoside A3, anemoside B4and23-hydroxybetulinic acid was detected with the mass spectrometer in negative ion mode monitoring at m/z749.6/471.2,1219.7/749.4,471.4/471.4and pulsatilloside B, pulsatilloside C and cirenshenoside S were in positive ion mode monitoring at m/z819.4/347.2,965.5/493.2and1097.9/493.1, respectively. As an internal standard, scutellarin was used.
Results:The calibration curves were indicative of good linearity (r2≥0.9973) in the range of interest for each analyte. Intra-day and inter-day precision (C.V.,%) was less than9.1%and9.0%, respectively. Accuracy was between-9.5%and9.2%. Recovery was80.03%～115.6%. The method is very rapid, simple, reliable and suitable for pharmacokinetic analysis. It can be routinely used for simultaneous determination of six triterpenoidal saponins in rat bile.
Conclusion:These results indicated that the newly developed method can also be applied to studies after administration of extraction of saponins from Pulsatilla chinensis to rats. Part six Determination of plasma protein binding rate of betulic acid
Objective:To develop high performance liquid chromatography (HPLC) method to determine the protein binding rates of betulic acid in human plasma, rat plasma and bovine serum albumin (BSA).
Methods:Plasma protein binding rate studies were conducted by equilibrium dialysis method. The influence of drug concentration and plasma in different species on plasma protein binding rate were studied.
Results:There was no significant difference in the plasma protein binding rates at low, middle and high betulic acid concentrations in dilution medium. The protein binding rate of betulic acid in human plasma was higher than in the rat plasma.
Conclusion:Betulic acid has higher protein binding extent with both rat plasma and human plasma. Part seven Studies on Absorption Kinetics of Betulic acid in Rat's Intestines
Objective:To investigate the in vivo absorption kinetics of betulic acid at different intestine segments in rats and develop a high performance liquid chromatography coupled with diode array detection (HPLC-DAD) method to determine the contents of betulic acid and phenolsulfonphthalein.
Methods:The rat single-pass intestinal perfusion technique was applied. The effects of absorption site, drug concentration and pH value on betulic acid absorption were studied. The samples were separated on a Diamonsil ODS C18column (250mm×4.6mm,5μm), by a gradient elution using acetonitrile and0.1%v/v aqueous acetic acid as mobile phase at a flow rate of1.0mL·min-1. The column temperature was30℃
Results:When the concentration was increased from75to125μg·mL the uptake of betulic acid in was increased linearly. The concentration had no effect on the permeability coefficient. The permeability coefficients of betulic acid at duodenum, jejunum, ileum and colon were (0.151±0.0049),(0.156±0.0056),(0.149±0.0083),(0.159±0.0041) h-1, respectively.
Conclusion:Betulic acid has no specific absorption site in the intestine. The absorption mechanism of the drug conforms to passive transport mechanism and first-order kinetics. The bioavailability of betulic acid preparation can be increased by increasing the dissolution rate and the solubility.
本实验采用大孔吸附树脂法,硅胶柱色谱法、葡聚糖凝胶和反相柱色谱法对白头翁药材进行系统分离,并建立了一个简单、快速的HPLC-ESI-MS法同时定量测定白头翁药材中9种化学成分。在对其进行定性研究中,利用HPLC-ESI-MS/MS联用技术分析研究了8个五环三萜皂苷类化合物的特征碎片离子,推测其电喷雾质谱裂解规律,最终在白头翁提取物中推导出了37个五环三萜皂苷化合物的结构。在药代动力学研究中,首次建立了一种灵敏的、可靠的多次切换监测模式HPLC-MS方法同时测定大鼠灌胃给予白头翁提取物后大鼠血浆中4种三萜皂苷的含量,并应用于药代动力学研究。首次对大鼠口服白头翁提取物后胆汁中6种五环三萜皂苷类化合物进行分析测定,并对这些三萜皂苷的胆汁排泄轮廓进行研究。综上对白头翁药材的系统研究将为其进一步开发应用提供全面的理论依据。
第一部分白头翁化学成分研究
目的：研究药材白头翁(Pulsatilla chinensis (Bunge) Regel)中的化学成分。
方法：取粉碎后的白头翁约10kg,用70%乙醇冷浸提取3次,采用AB-8大孔吸附树脂进行分离,收集70%乙醇洗脱液部分,得浸膏(总苷)约500g。采用大孔吸附树脂法,硅胶柱色谱法、葡聚糖凝胶和反相柱色谱法对白头翁药材进行系统分离,并用波谱和化学方法鉴定化合物的结构。
结果：从白头翁的70%乙醇提取物中分离得到9个单体化合物,对其进行了结构鉴定,其中包括5个五环三萜皂苷,2个三萜酸,2个甾酮类化合物。分别鉴定为齐墩果酸、白头翁皂苷A3、白头翁皂苷B4、23-羟基白桦脂酸、刺人参皂苷S、白头翁皂苷B、白头翁皂苷C、筋骨草甾酮C和p-蜕皮甾酮。
结论：从白头翁中分离鉴定了9个化合物,其中3个化合物为首次从该植物中发现,分别为刺人参皂苷S,筋骨草甾酮C和p-蜕皮甾酮。第二部分液质联用技术同时测定白头翁中9种化学成分及其质量表征
目的：建立HPLC-MS法测定白头翁中白头翁皂苷B4(1),白头翁皂苷A3(2),23-羟基白桦脂酸(3),刺人参苷S(4),白头翁皂苷C(5),白头翁皂苷B(6),齐墩果酸(7),p-蜕皮甾酮(8)和筋骨草甾酮C(9)含量的方法。
方法：采用Sapphire C18柱(250mm×4.6mm,5μm);流动相：A(甲醇含0.1%甲酸)-B(0.1%甲酸水溶液),梯度洗脱程序为0-6min(75%-95%A),6-14min(95%A),随后回到最初75%A并持续到21min；流速为0.8mL.min-1。采用电喷雾离子源进行正离子和负离子模式检测,多反应监测模式(MRM)用于定量测定。正离子监测模式下源喷射电压设置为5500V；负离子监测模式下源喷射电压设置为-4500V；离子源温度为650℃。雾化气(Gas1):40psi,加热气(Gas2):50psi,接口持续加热,帘气25psi,全程氮气通入状态。扫描方式采用多重反应监测(MRM)；碰撞气(CAD,N2)压力为medium;Q1和Q3分辨率均为UNIT。
结果：峰面积与浓度呈良好的线性关系(r2>0.9948)；日内和日间精密度分别小于2.78%和2.68%,并应用于20批次不同来源白头翁药材的含量测定。
结论：本研究中建立的分析方法灵敏度高、专属性好,可为白头翁药材质量控制提供一个强有力的工具。有助于规范白头翁药材的质量,并能够在一定程度上指导中医临床用药的合理性。
第三部分液质联用技术定性鉴定白头翁中五环三萜皂苷类成分
目的：对白头翁进行超声提取后即进行HPLC-MS分析,研究8个五环三萜皂苷类化合物的特征碎片离子,推测其电喷雾质谱裂解规律,再将此规律应用于白头翁中未知羽扇豆烷型和齐墩果烷型三萜皂苷类化合物的结构推测,根据已知裂解规律尽可能多的推测出白头翁中五环三萜皂苷的化学结构。
方法：采用了MRM-IDA-EPI和PREC-IDA-EPI两种模式分析未知五环三萜皂苷类化合物。取白头翁药材粉末(40目)5g,加70%甲醇25mL,称重,冰浴超声提取1h,称重,用70%甲醇补足减失的重量,0.45μm微孔滤膜滤过,滤液减压蒸干,残渣用2mL的70%甲醇溶解,0.45μm微孔滤膜滤过,进行HPLC-MS分析。色谱柱为Sapphire C18(250mm×4.6mm,5μm),采用0.1%甲酸甲醇(A)-0.1%甲酸水(B)为流动相,梯度洗脱75-75%A (0-15min);75-80%A (15-30min);80-95%A (30-40min);95-95%A (40-50min),流速为700μL·min-1。质谱：ESI源；负离子检测源电压-4500V；正离子检测源电压5500V；源温度(TEM)：650。C；雾化气(gas1):40psi,加热气(gas2):50psi,帘气(CUR)：25psi。
结果：通过与对照品的保留时间、分子量和质谱数据比较,分别对8个已知五环三萜化合物色谱峰进行归属。化合物6,10,14,18,20,21,35和36分别鉴定为白头翁皂苷B4,白头翁皂苷C,23-羟基羽扇R-20(29)-烯-28-酸-3-O-[吡喃鼠李糖基-吡喃阿拉伯糖基]28-O-[吡喃葡萄糖酯苷],白头翁皂苷B,23-羟基羽扇豆-20(29)-烯-28-酸-3-O-[吡喃阿拉伯糖基]-28-O-[呲喃葡萄糖酯苷],常春藤皂苷元28-0-[吡喃葡萄糖基-吡喃葡萄糖酯苷],白头翁皂苷A3和常春藤皂苷。同时,通过由对照品裂解行为总结得出的规律推断出了29个五环三萜皂苷类化合物的结构。
结论：本实验首次建立了利用HPLC-ESI-MS联用技术分析白头翁中五环三萜皂苷类成分的方法。本法灵敏度高、专属性强,成功用于白头翁中五环三萜皂苷类化合物的结构鉴定。通过联合MRM-IDA-EPI和PREC-IDA-EPI扫描模式对化合物结构分析方面具有明显优越性,对控制中药材质量具有重要意义。
第四部分HPLC-MS法同时测定大鼠血浆中4种五环三萜皂苷类成分及其药物代谢动力学研究
目的：建立HPLC-MS/MS同时测定大鼠血浆中白头翁提取物4个五环三萜皂苷类化合物(白头翁皂苷B4,白头翁皂苷B,白头翁皂苷A3和23-羟基白桦脂酸)的分析方法,并计算其在大鼠体内的药动学参数。
方法：血浆样品采用了Strata-X固相萃取柱预处理,采用Sapphire C18(250mm×4.6mm,5μm)色谱柱；以A(甲醇含0.1%甲酸)-B(0.1%甲酸水溶液)为流动相,梯度洗脱程序为：0-6min,75-95%A;6-15min,保持95%A不变,预平衡时间为6min,流速为800μL·min-1。色谱柱后溶液全部进入离子源,无分流,分析时间为15min。质谱以电喷雾离子源进行正离子模式和负离子模式检测,采用多反应监测模式,并首次应用了多次切换检测模式。白头翁皂苷B4,白头翁皂苷B,白头翁皂苷A3,23-羟基白桦脂酸和内标的监测离子对分别为m/z1219.7/749.4, m/z819.4/347.2, m/z749.6/471.2, m/z471.4/471.4和m/z461.1/285.0。本研究将4种五环三萜皂苷类成分和内标化合物的检测过程划分为5个阶段：0.0-6.998min,内标和白头翁皂苷B4在负离子模式下监测；6.998-7.028min,负离子扫描模式被切换至正离子扫描模式；7.028-10.027min,正离子模式下监测白头翁皂苷B；10.027-10.057min,正离子扫描模式被切换至负离子扫描模式；10.057-15.059min,负离子模式下监测白头翁皂苷A3和23-羟基白桦脂酸。
结果：4种五环三萜皂苷类成分在大鼠血浆中专属性、线性、准确度、精密度、提取回收率和基质回收率均被验证。4种待测化合物的线性关系良好(r2>0.99)；日内、日间精密度的相对标准偏差(RSD)均小于9%；在室温储存24h,5次冻融和-20。C储存30天的条件下各待测化合物的稳定性良好,准确度在-6.82%-6.18%之间；高、中、低三个浓度水平的平均提取回收率分别为82.16%-104.5%；基质效应均值在77.69%-108.2%之间。
结论：该方法特异、快速、灵敏,结合固相萃取的预处理方法和HPLC-MS的新检测手段对白头翁中4种五环三萜皂苷类化合物进行了药物代谢动力学研究。本研究中首次应用了多次切换检测模式,并首次在五环三萜皂苷类化合物体内测定时采用了Strata-X固相小柱进行生物样品预处理。此方法为五环三萜皂苷类化合物的药物代谢动力学研究提供了一个新的思路。
第五部分液质联用技术测定大鼠胆汁中6种五环三萜皂苷类成分及其排泄动力学研究
目的：建立一种可同时测定大鼠胆汁中6种主要五环三萜皂苷类化合物(白头翁皂苷B4,刺人参苷S,白头翁皂苷C,白头翁皂苷B,白头翁皂苷A3和23-羟基白桦脂酸)含量的HPLC-MS方法,并用于大鼠灌胃给予白头翁提取物后其6种待测化合物的胆汁排泄动力学研究。
方法：色谱柱为Sapphire-C18柱(250mm×4.6mm,5μm);柱温为25℃;流动相为(0.1%甲酸)甲醇A-0.1%甲酸水溶液B,梯度洗脱,洗脱程序如下：0.0-6.0min,75-95%A;6.0-15.0min,保持95%A；进样前预平衡6min。流速为800μL·min-1。色谱柱后溶液全部进入离子源,无分流,进样量为10μL。电喷雾离子源(ESI源),Turbo V离子源,源温度为650℃。源喷射电压为5500V和-4500V,源内气体1(Gas1, N2)压力：40psi；气体2(Gas2, N2)压力：50psi；气帘气体(N2)压力：25psi；正负离子分别检测模式；扫描方式为多重反应监测(MRM)；碰撞气(CAD,N2)压力为medium; Q1分辨率为UNIT, Q3分辨率为UNIT. SD大鼠8只,随机分成2组。其中2只为空白对照组,实验组6只大鼠灌胃给予白头翁提取物(10mL·kg-1)后,收集给药后0-1h,1-3h,3-5h,5-7h,7-9h,9-12h和12-24h时间段的胆汁并测定体积。测定大鼠灌胃给予白头翁提取物后胆汁中6种五环三萜皂苷类化合物的累积排泄率。
结果：大鼠胆汁中内源性物质不干扰6种待测化合物和内标的测定；6种五环三萜皂苷类成分标准曲线的相关系数(r2)均大于0.9973；日内RSD和日间RSD分别小于9.1%和9.0%,RE在-9.5%到9.2%之间；提取回收率的范围为80.03%～115.6%；基质效应的范围为78.43%-109.7%。
结论：该方法简便、快速、灵敏度高、专属性强,可满足大鼠胆汁中微量五环三萜类皂苷成分的测定要求。待测6种五环三萜皂苷类化合物在1-3h内排泄率最高,在3-5h时间段时略有减少,随着时间的推移,排泄率逐渐降低,并趋于平缓。结果表明,只有不到14%的待测物以原型从胆汁排泄,结果表明其五环三萜皂苷类化合物在体内可能存在某种生物转化和体内代谢。
第六部分白桦脂酸血浆蛋白结合率测定
目的：建立白桦脂酸在大鼠血浆、人血浆和牛血清白蛋白中蛋白结合率的测定方法。
方法：采用平衡透析法测定白桦脂酸血浆蛋白结合率,利用HPLC法测定血浆中白桦脂酸浓度,测定并比较不同血浆中白桦脂酸的血浆蛋白结合率。
结果：在50～100μg/mL,药物浓度对血浆蛋白结合率无显著影响,白桦脂酸与大鼠和人的血浆蛋白结合率存在显著性差异,白桦脂酸与人血浆蛋白结合率高于与大鼠血浆蛋白结合率。
结论：白桦脂酸与血浆蛋白具有较强的结合,且在实验设计的浓度范围内蛋白结合率与透析液中药物浓度无关。只有少量游离药物作为活性药物分子参与体内药物处置,从而产生药理效应。第七部分白桦脂酸大鼠在体肠吸收动力学研究
目的：建立同时测定肠循环液中白桦脂酸和酚红浓度的HPLC-DAD法,并探讨白桦脂酸在大鼠各肠段的吸收动力学特征及不同药物浓度对吸收的影响。
方法：采用大鼠在体肠吸收实验模型,并考察吸收部位、药物浓度和pH值对药物吸收的影响。色谱柱为Diamonsil ODS C18色谱柱(250mm×4.6mm,5μm);流动相为乙腈-0.2%乙酸(75:25)；流速1.0mL.min-1;柱温30℃；检测波长203nm(白桦脂酸)和430nm(酚红)；进样量20μL。
结果：在75～125μg·mL-1内白桦脂酸的吸收速率与质量浓度呈线性关系,Ka值基本保持不变；各肠段的吸收速率无显著性差异,十二指肠、空肠.回肠和结肠的Ka值分别为(0.151±0.0049),(0.156±0.0056),(0.149±0.0083),(0.159±0.0041)h-1。
结论：白桦脂酸在小肠中吸收良好,没有特定吸收部位；不同浓度对白桦脂酸在大鼠全肠道的吸收无显著影响,其在肠道的吸收呈一级吸收动力学特征,吸收机制为被动扩散。白桦脂酸是难溶性药物,可以通过增加药物的溶出度,进而提高药物的生物利用度。
Pulsatilla chinensis, the dried roots of Pulsatilla chinensis (Thunb.) Bercht. et Opiz., has been widely used as traditional Chinese medicine (TCM) for thousands of years in China. Modern pharmacological studies demonstrated that Pulsatilla chinensis extracts had a number of effects, such as hypoglycaemic, antitumour, cognition-enhancing, neuroprotective, cvtotoxic and antiendotoxin activities. Pulsatilla chinensis is known for containing a large number of compounds, including triterpenoidal saponins, phytosterone and anthocyanins. Among these, triterpenoidal saponins are generally considered to be the major components, and many of them have been found to have biological activities. In recent years, more and more triterpenoidal saponins have been isolated from Pulsatilla chinensis and they exhibit a variety of bioactivities.
Therefore, quality control analysis of the active components is an important issue for the safe and effective use of herbal medicines and their preparations. The constituents in Pulsatilla chinensis are complex, and some of them usually are of low content. Therefore, a more sensitive, selective and rapid method is demanded. The emergence of high performance liquid chromatography connected with tandem mass spectrometry (HPLC-MS/MS) makes the determination possible.
In this research, the nine main components were isolated and identified from Pulsatilla chinensis. A rapid, efficient and accurate HPLC-ESI-MS/MS method provided a new basis for overall assessment on quality of Pulsatilla chinensis and should be considered as a suitable quality control method. Then37triterpenoid saponins in Pulsatilla chinensis were identified by HPLC-ESI-MS/MS. Finally, a novel method was developed to determined triterpenoidal saponins in rat plasma and rat bile by HPLC-MS. At the same time, drug-protein binding and absorption kinetics of betulic acid were studies.
Part one Studies on chemical components of Pulsatilla chinensis
Objective:To study the chemical constituents of Pulsatilla chinensis.
Methods:Air-dried herbal of Pulsatilla chinensis (10kg) were extracted by diffusion for three times with70%alcohol.The residue was pretreated by AB-8macroporous resin, and then collected the part of70%alcohol eluant. Finally we got the extractum for500g. The compounds were isolated and purified by macroporous resin, silicagel, dextrane gel and reversed-phase column chromatographies. Their structures were identified by various spectral data.
Results:Nine compounds were isolated from the70%ethanol extract of Pulsatilla chinensis, including5triterpenoidal saponins,2triterpenic acid and2phytosterone. Their structures were elucidated as oleanolic acid, anemoside A3, anemoside B4,23-hydroxybetulinic acid, cirenshenoside S, pulsatilloside B, pulsatilloside C, ajugasterone C and (3-ecdysterone, respectively.
Conclusion:Nine compounds were isolated and identified from Pulsatilla chinensis. Compounds5,8and9were isolated from Pulsatilla chinensis for the first time.
Part two Quantitative determination of9components in Pulsatilla chinensis by HPLC-ESI-MS/MS
Objective:The present study describes the development, validation and a practical application of a fully automated analytical method based on high performance liquid chromatography coupled to electrospray ionization tandem mass spectrometry (HPLC-ESI-MS/MS) for the simultaneous determination of7triterpenoidal saponins, anemoside B4(1), anemoside A3(2),23-Hydroxybetulinic acid (3), cirenshenoside S (4), pulsatilloside C (5), pulsatilloside B (6) and oleanolic acid (7), together with two other phytosterone named β-ecdysterone (8) and ajugasterone C (9) in Pulsatilla chinensis.
Methods:A Sapphire C18(250mm×4.6mm,5μm) and gradient elution were used during the analysis. A binary gradient elution was performed:0min (75%A),0-6min (95%A),6-14min (95%A). Then quickly returned to initial75%A and maintained until21min for column balance. The identification and quantification of those analytes were achieved on a hybrid quadrupole linear ion trap mass spectrometer. Multiple-reaction monitoring scanning was employed for quantification with switching electrospray ion source polarity between positive and negative modes in a single run. The ion spray voltage was set to5500V and-4500V, respectively. The turbo spray temperature was maintained at500℃. Nebulizer gas (gas1) and heater gas (gas2) was set at40and50psi, respectively.
Results:All calibration curves showed good linearity (r2>0.9948) within the test ranges. The intra-day and inter-day variations were less than2.78%and2.68%, respectively. The developed method was successfully applied to determine the investigated compounds in20samples of Pulsatilla chinensis.
Conclusion:Compared with the reported analytical methods of Pulsatilla chinensis, this rapid, efficient and accurate HPLC-ESI-MS/MS method provided a new basis for overall assessment on quality of Pulsatilla chinensis and should be considered as a suitable quality control method.
Part three Characterization of triterpenoid saponins in Pulsatilla chinensis using liquid chromatography coupled with hybrid triple quadruple linear ion trap mass spectrometry
Objective:Eight triterpenoid saponins in crude extracts from Pulsatilla chinensis have been investigated by electrospray ionization multi-stage tandem mass spectrometry. To summarize fragmentation rules and develop a high sensitive and efficient liquid chromatography-mass spectrometry (LC-MS) method for detection and characterization of the trace triterpenoid saponins in Pulsatilla chinensis.
Methods:The triterpenoidal saponins in a crude extract of Pulsatilla chinensis were characterized by the combined use of the MRM-IDA-EPI and PREC-IDA-EPI modes on a hybrid triple quadrupole-linear ion trap mass spectrometer. The chromatographic separation was performed on a Sapphire C18column (250mm×4.6mm,5μm) with linear gradient elution with methanol containing0.1%formic acid-0.1%aqueous formic acid at a flow rate of0.8mL·min-1. The ion spray voltage was set to5500V and-4500V, respectively. The turbo spray temperature was maintained at650℃. Nebulizer gas (gas1) and heater gas (gas2) was set at40and50psi, respectively. The curtain gas was kept at25psi and interface heater was on. Nitrogen was used in all cases.
Results:According to the retention times, molecular weight and characteristic fragment ions, compounds6,10,14,18,20,21,35and36were identified anemoside B4, pulchinenoside C,23-hydroxy-lup-20(29)-en-28-oic acid3-0-[rhamnopyranosyl-arabinopyranosyl]28-0-[glucopyranoside], pulchinenoside B,23-hydroxy-lup-20(29)-en-28-oic acid3-O-[arabinopyranosyl]28-O-[glucopyranoside], hederagenin28-O-[glucopyranosyl-glucopyranoside], anemoside A3and hederagenin, respectively. Six fragmentation rules were summarized. Finally,29triterpenoid saponins in a crude extract of Pulsatilla chinensis were characterized.
Conclusion:In this study, a high sensitive, accurate and effective HPLC-MS method for on-line qualitative analysis of the trace triterpenoid saponins in Pulsatilla chinensis has been developed. This study has demonstrated the unprecedented advantage of the combination use of the MRM-IDA-EPI and PREC-IDA-EPI mode. It will play an important role in controlling the quality of medicinal herb in the future.
Part four Development of a novel method for triterpenoidal saponins in rat plasma by HPLC-MS
Objective:A novel method using high-performance liquid chromatography tandem mass spectrometry (HPLC-MS/MS) has been developed for the quantification of four triterpenoidal saponins (anemoside B4, pulsatilloside B, anemoside A3and23-hydroxybetulinic acid) in rat plasma following solid phase extraction (SPE).
Methods:The optimized procedure utilized off-line extraction of the analytes from plasma using polymeric (Strata-X) SPE cartridges. Detection and quantitation was performed by MS/MS using electrospray ionization (ESI) and multiple reaction monitoring (MRM) in a novel multi-switching monitoring mode. The analytes and internal standard (scutellarin) were analyzed using a Sapphire C18column (250mm×4.6mm,5μm) with a linear gradient elution. The mass transition ion pairs of the triterpenoidal saponins were executed as the following:m/z1219.7/749.4for anemoside B4, m/z819.4/347.2for pulsatilloside B, m/z749.6/471.2for anemoside A3, m/z471.4/471.4for23-hydroxybetulinic acid and m/z461.1/285.0for the internal standard. the negative ionization mode was switched to the negative mode from0.0to6.998min to detect the IS and anemoside B4. Then, the negative ionization mode was switched to the positive mode from6.998to7.028min. Within the time range of7.028to10.027min, pulsatilloside B was detected in the positive ionization mode. Subsequently, the ESI converted back to the negative ionization mode once again from10.027to10.057min. In the last section, anemoside A3and23-hydroxybetulinic acid were detected in the negative mode from10.057to15.059min.
Results:The specificity, linearity, accuracy, precision, recovery, matrix effect, and stabilities were validated for all analytes in the plasma samples.the four analytes showed good linearity over the low and high concentration range with r2>0.99. The LLOQ of the assay was defined as the lowest concentration of the standard curve that could be quantified accurately within a20%bias of the nominal concentration, with the precision not exceeding20%. Both the precision and accuracy of the four analytes at LLOQ were less than9%. The analytes in rat plasma were all stable for24h at room temperature, three cycles of freeze-thaw,30days at-20℃with accuracy in the range from-6.82to6.18%. At three concentration levels of these analytes, the extraction recoveries were all between82.16and104.5%. The matrix effects of this study were evaluated by analyzing samples at three concentration levels (low, medium and high), which were in the ranges of77.69-108.2%.
Conclusion:A novel analytical approach, multiswitching monitoring mode, was presented that applies different ESI modes in a single HPLC ESI-MS/MS run. In addition, the use of polymeric (Strata-X) SPE cartridges has been applied to the off-line extraction of triterpenoidal saponins from rat plasma samples for the first time. Combining the rapid and efficient off-line drug SPE with the specific and sensitive multiswitching monitoring mode, a new, robust analytical method for the analysis of triterpenoidal saponins in rat plasma has been developed and validated. This validated method is a novel technique for sample preparation and quantitation and was successfully applied to estimate the pharmacokinetics of triterpenoidal saponins.
Part five Quantitative analysis of six triterpenoidal saponins in rat bile after oral administration of Pulsatilla chinensis extract by high performance liquid chromatography-electrospray ionization tandem mass spectrometry
Objective:The aim of this study was to develop a liquid chromatography-tandem mass spectrometry (HPLC-MS/MS) method for the simultaneous quantitative determination of the following triterpenoidal saponins:anemoside A3, anemoside B4,23-hydroxybetulinic acid, pulsatilloside B, pulsatilloside C and cirenshenoside S in rat bile.
Methods:The chromatographic separation was performed using a Sapphire C18column (250mm×4.6mm,5μm) and gradient elution was used during the analysis. Anemoside A3, anemoside B4and23-hydroxybetulinic acid was detected with the mass spectrometer in negative ion mode monitoring at m/z749.6/471.2,1219.7/749.4,471.4/471.4and pulsatilloside B, pulsatilloside C and cirenshenoside S were in positive ion mode monitoring at m/z819.4/347.2,965.5/493.2and1097.9/493.1, respectively. As an internal standard, scutellarin was used.
Results:The calibration curves were indicative of good linearity (r2≥0.9973) in the range of interest for each analyte. Intra-day and inter-day precision (C.V.,%) was less than9.1%and9.0%, respectively. Accuracy was between-9.5%and9.2%. Recovery was80.03%～115.6%. The method is very rapid, simple, reliable and suitable for pharmacokinetic analysis. It can be routinely used for simultaneous determination of six triterpenoidal saponins in rat bile.
Conclusion:These results indicated that the newly developed method can also be applied to studies after administration of extraction of saponins from Pulsatilla chinensis to rats. Part six Determination of plasma protein binding rate of betulic acid
Objective:To develop high performance liquid chromatography (HPLC) method to determine the protein binding rates of betulic acid in human plasma, rat plasma and bovine serum albumin (BSA).
Methods:Plasma protein binding rate studies were conducted by equilibrium dialysis method. The influence of drug concentration and plasma in different species on plasma protein binding rate were studied.
Results:There was no significant difference in the plasma protein binding rates at low, middle and high betulic acid concentrations in dilution medium. The protein binding rate of betulic acid in human plasma was higher than in the rat plasma.
Conclusion:Betulic acid has higher protein binding extent with both rat plasma and human plasma. Part seven Studies on Absorption Kinetics of Betulic acid in Rat's Intestines
Objective:To investigate the in vivo absorption kinetics of betulic acid at different intestine segments in rats and develop a high performance liquid chromatography coupled with diode array detection (HPLC-DAD) method to determine the contents of betulic acid and phenolsulfonphthalein.
Methods:The rat single-pass intestinal perfusion technique was applied. The effects of absorption site, drug concentration and pH value on betulic acid absorption were studied. The samples were separated on a Diamonsil ODS C18column (250mm×4.6mm,5μm), by a gradient elution using acetonitrile and0.1%v/v aqueous acetic acid as mobile phase at a flow rate of1.0mL·min-1. The column temperature was30℃
Results:When the concentration was increased from75to125μg·mL the uptake of betulic acid in was increased linearly. The concentration had no effect on the permeability coefficient. The permeability coefficients of betulic acid at duodenum, jejunum, ileum and colon were (0.151±0.0049),(0.156±0.0056),(0.149±0.0083),(0.159±0.0041) h-1, respectively.
Conclusion:Betulic acid has no specific absorption site in the intestine. The absorption mechanism of the drug conforms to passive transport mechanism and first-order kinetics. The bioavailability of betulic acid preparation can be increased by increasing the dissolution rate and the solubility.
引文
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12 Dai L, Wang H, Chen Y, The Immune-enhancing Effect of PcG2A-a Glycoprotein Isolated from Dried Root of Pulsatilla Chinensis(Bunge) Regel. Chin. J. Biochem. Pharm.,2000,21:230-231
13 Long SJ, Fu PJ, Tan RC, Study on Antioidant Activity of 17 traditional Chinese medicine. Chin. Tradit. Herb Drugs,199930:40-42
14 Weng FH, Hu P, Jiao JJ, Effect of anti-inflammatory action of chinese herbs on the synthsis of LTB4 and 5-hete of peritoneal macrophages in rats. J. Tianjin Med. Univ.,1996,2:1-3
15 Zhang G.D, Zhang YZ, Fast D.M, et al Ultra sensitive quantitation of endogenous oxytocin in rat and human plasma using a two-dimensional liquid chromatography-tandem mass spectrometry assay. Anal. Biochem. 2011,416:45-52
16 Hewavitharana A, Lee S, Dawson PA, et al Development of an HPLC-MS/MS method for the selective determination of paracetamol metabolites in mouse urine. Anal. Biochem.,2008,374:106-111
17 Joo KM, Lee JH, Jeon HY, et al Pharmacokinetic study of ginsenoside Re with pure ginsenoside Re and ginseng berry extracts in mouse using ultra performance liquid chromatography/mass spectrometric method. J. Pharm. Biomed. Anal.,2010,51:278-283
18 Chen CY, Qi LW, Yi L, et al Liquid chromatography-mass spectrometry analysis of macranthoidin B, macranthoidin A, dipsacoside B, and macranthoside B in rat plasma for the pharmacokinetic investigation. J. Chromatogr. B,2009,877:159-165
19 Wang W, Wang GJ, Xie HT, Sun JG, et al Determination of ginsenoside Rd in dog plasma by liquid chromatography-mass spectrometry after solid-phase extraction and its application in dog pharmacokinetics studies. J. Chromatogr. B,2007,852:8-14
1 Weber L, Multi-component reactions and evolutionary chemistry. Drug Discov. Today,2002,7:143-147
2 Ueberall F, Fuchs D, Vennos C, Anti-inflammatory potential of Padma 28-review of experimental data on the antiatherogenic activity and discussion of the multi-component principle, Forsch. Komplementmed, 2006,13 (Suppl.1):7-12
3 China Pharmacopoeia Committee, Pharmacopoeia of the People's Republic of China, China Chemical Industry Press, Beijing,2010
4 Cheng L, Zhang M, Zhang P, et al Silver complexation and tandem mass spectrometry for differentiation of triterpenoid saponins from the roots of Pulsatilla chinensis (Bunge) Regel. Rapid Commun. Mass Spectrom,2008, 22:3783-3790
5 Jiangsu New Medical College, Dictionary of Chinese Medicinal Materials, Scientific and Technological Press, Shanghai,1977
6 Wang XF, Geng BQ, Zhang RH, The antitumor effect of radix pulsatillae alcohol extract. J. Zhejiang Med. Univ,1998,27:204-206
7 Cai Y, Tang YM, Liang BW, Antitumor effect of Pulsatilla chinensis in vitro. Chin. Tradit. Herb Drugs,1999,30:441-443
8 Cai Y, Tang YM, Liang BW, Antitumor effect of Pulsatilla chinensis in vivo. Chin. Tradit. Herb Drugs,1999,30:929-931
9 Zhuang XH, Geng BQ, Yong BG, Studies on antitumor effect of Pulsatilla chinensis. J. Pract. Oncol,1999,13:1494-1496
10 Zhang RH, Zhang ZM, Geng BQ, The preventive and therapeutic effect of radix pulsatillae on mice colorectal cancer induced by dimethylhydrazine. Pharmacol. Clin. Chin. Mater. Med,1999,15:33-35
1 中华人民共和国药典委员会.中华人民共和国药典.一部[M].北京：中国医药科技出版社,2010：96
2 Chang SY, Nohara T. Two new lupane-triterpene glycosides from leaves of Acanthoparar Koreanum[J]. Chem Pharm Bull,1998,46(1):163-165
3 Fujioka T, Kashiwada Y, Kilkuskic RE. Anti-AIDS agents 11, betulinic acid and platonic acid as anti-HIV principles from syzigium claviflorum and the anti-HIV activity of structurally related triterpenoids [J]. J Nat Prod,1994,57(2):243-247
4 Sun IC, Chen CH, Kashiwada Y. Anti-AIDS agents 49 synthesis, anti-HIV, and anti-fusion activeties of IC9564 analogues based on betulinic acid [J]. J Med Chem,2002,45(19):4271-4275
5 Srivastava AK, Shakeel M, Khan AA. QSAR studies on anti HIV-1 N-subtituted Betulinic acid amides [J]. Ind J Chem,2002,41B(2):436-439
6 Pisha E, Chai H, Lee IS. Discovery of betulinic acid as a selective inhibitor of human melanoma that function by induction of apoptosis [J]. J Nat Med,1995,1(10):1046-1051
7卢来春,蒋学华,杨俊毅,等.阿莫西林对格列美脲血浆蛋白结合率的影响[J].华西药学杂志,2003,18(4)：250-254
8郭宾,李川.药物与血浆蛋白结合的药理学基础及其研究进展[J].中国临床药理学与治疗学,2005,10(3)：2411
1 Dasgupta TK, Pezzuto JM, Use of betulinic acid and its derivatives for inhibiting malignant melanoma growth and method of monitoring this [P]. W.96/29068.1996-09-26
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3 Kuo HL, Yoshiki K, Fumio H, et al Inventors Betulinic acid derivatives and uses therefor [P]. W.96/39033.1996-12-12
4 Srivastava AK, Shakeel M, Khan AA, et al QSAR studies on anti HIV-1 N-subtituted Betulinic acid amides [J]. Ind J Chem,2002,41B(2):436
5 Sun IC, Chen CH, Kashiwada Y, Anti-AIDS agents 49 synthesis, anti-HIV, and anti-fusion activeties of IC9564 analogues based on betulinic acid [J]. J Med Chem,2002,45(19):4271
6 Fujioka T, Kashiwada Y, Kilkuskic RE, et al Anti-AIDS agents 11, betulinic acid and platonic acid as anti-HIV principles from syzigium claviflorum and the anti-HIV activity of structurally related triterpenoids [J]. J Nat Prod,1994,57(2):243
7 Pisha E, Chai H, Lee IS, et al Discovery of betulinic acid as a selective inhibitor of human melanoma that function by induction of apoptosis [J]. J Nat Med,1995,1(10):1046
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1 Lu XM, Wang XY, Wang JG, The effects of Pulsatilla chinensis on the immune functions of mice, J Gansu College of TCM,1998,15:32-35
2 Dai L, Wang H, Chen Y, The Immune enhancing Effect of PcG2A-a Glycoprotein Isolated from Dried Root of Pulsatilla Chinensis(Bunge) Regel, Chinese Journal of Biochemical Pharmaceutics,2000,21:230-231
3 Chen SH, Chang L, Sun X, Zhao HY, Study of the antibacterial activity of Pulsatillae decoction in vivo. Institute of Traditional Chinese Veterinary Medicine, Jilin Agricultural University, Zhongguo Shouyi Zazhi,2008,44: 14-15
4 Liu ZP, Pang HM, Zhao YX, et al Preliminary study on antimutagenesis and antioxidation of aqueous extract of Pulsatilla chinensis Regel. Tubian, 2008,20:481-482
5 Michiko O, Masaki T, Kumiko N, et al Relevance of anti-reactive oxygen species activity to anti-inflammatory activity of components of Eviprostat, a phytotherapeutic agent for benign prostatic hyperplasia, Phytomedicine, 2007,14:465-472
6 Wang XF, Geng BQ, Zhang RH, The Antitumor Effect of Radix Pulsatillae Alcohol Extract, Journal of Zhejiang Medical University 1998,27(5): 204-206
7 Cai Y, Tang YM, Liang BW, Antitumor effect of Pulsatilla chinensis in vitro, Chinese Traditional And Herbal Drugs,1999,30:441-443
8 Cai Y, Tang YM, Liang BW, Antitumor effect of Pulsatilla chinensis in vivo, Chinese Traditional And Herbal Drugs,1999,30:929-931
9 Zhuang XH, Geng BQ, Yong BG, Experimental study on the effect of Pulsatilla chinensis on antitumor, Journal of Practical Oncology,1999,14: 94-96
10 Zhang RH, Zhang ZM, Geng BQ, The Preventive and Therapeutic Effect of Radix Pulsatillae on Mice Colorectal Cancer Induced by Dimethylhydrazine, Pharmacology and Clinics of Chinese Materia Medica, 1999,15:33-35
11 Zhang M, Song ZH, Wang D, et al The anti-tumor effect of the light petroleum extract from Pulsatilla chinensis (Bunge) Regel. Natural Product Communications,2008,3:819-822
12 Wang Y, Wang GC, Zhao HN, et al RP-HPLC determination of 23-Hydroxybetulinic acid in the roots of Pulsatilla chinensis Regel., Chin. J. Pharm.Anal.2007,27(1):13-15
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1 Weber L, Multi-component reactions and evolutionary chemistry. Drug Discov. Today,2002,7:143-147
2 Ueberall F, Fuchs D, Vennos C, Anti-inflammatory potential of Padma 28--review of experimental data on the antiatherogenic activity and discussion of the multi-component principle. Forsch Komplementmed. (Suppl. 1),2006,13:7-12
3 China Pharmacopoeia Committee, Pharmacopoeia of the People's Republic of China. China Chemical Industry Press, Beijing,2010
4 Cheng L, Zhang M, Zhang P, et al Silver complexation and tandem mass spectrometry for differentiation of triterpenoid saponins from the roots of Pulsatilla chinensis (Bunge) Regel. Rapid Commun. Mass Spectrom., 2008,22:3783-3790
5 Jiangsu New Medical College, Dictionary of Chinese Medicinal Materials. Scientific and Technological Press, Shanghai,1977
6 Wang XF, Geng BQ, Zhang RH, The Antitumor Effect of Radix Pulsatillae Alcohol Extract. J. Zhejiang Med. Univ.27 (1998) 204-206
7 Cai Y, Tang Y., Liang BW, Antitumor effect of Pulsatilla chinensis in vitro. Chin. Tradit. Herb Drugs,1999,30:441-443
8 Cai Y, Tang YM, Liang BW, Antitumor effect of Pulsatilla chinensis in vivo. Chin. Tradit. Herb Drugs,1999,30:929-931
9 Zhuang XH, Geng BQ, Yong BG, Studies on antitumor effect of Pulsatilla chinensis J. Pract. Oncol.,1999,13:1494-96
10 Zhang RH, Zhang ZM, Geng BQ, The Preventive and Therapeutic Effect of Radix Pulsatillae on Mice Colorectal Cancer Induced by Dimethylhydrazine.Pharmacol. Clin. Chin. Mater. Medica,1999,15: 33-35
11 Lu XM, Wang XY, Wang JG, studies on immune function effect of extracts from Pulsatilla Chinensis in mice. J. Gansu College of TCM.,1998,15: 32-34
12 Dai L, Wang H, Chen Y, The Immune-enhancing Effect of PcG2A-a Glycoprotein Isolated from Dried Root of Pulsatilla Chinensis(Bunge) Regel. Chin. J. Biochem. Pharm.,2000,21:230-231
13 Long SJ, Fu PJ, Tan RC, Study on Antioidant Activity of 17 traditional Chinese medicine. Chin. Tradit. Herb Drugs,199930:40-42
14 Weng FH, Hu P, Jiao JJ, Effect of anti-inflammatory action of chinese herbs on the synthsis of LTB4 and 5-hete of peritoneal macrophages in rats. J. Tianjin Med. Univ.,1996,2:1-3
15 Zhang G.D, Zhang YZ, Fast D.M, et al Ultra sensitive quantitation of endogenous oxytocin in rat and human plasma using a two-dimensional liquid chromatography-tandem mass spectrometry assay. Anal. Biochem. 2011,416:45-52
16 Hewavitharana A, Lee S, Dawson PA, et al Development of an HPLC-MS/MS method for the selective determination of paracetamol metabolites in mouse urine. Anal. Biochem.,2008,374:106-111
17 Joo KM, Lee JH, Jeon HY, et al Pharmacokinetic study of ginsenoside Re with pure ginsenoside Re and ginseng berry extracts in mouse using ultra performance liquid chromatography/mass spectrometric method. J. Pharm. Biomed. Anal.,2010,51:278-283
18 Chen CY, Qi LW, Yi L, et al Liquid chromatography-mass spectrometry analysis of macranthoidin B, macranthoidin A, dipsacoside B, and macranthoside B in rat plasma for the pharmacokinetic investigation. J. Chromatogr. B,2009,877:159-165
19 Wang W, Wang GJ, Xie HT, Sun JG, et al Determination of ginsenoside Rd in dog plasma by liquid chromatography-mass spectrometry after solid-phase extraction and its application in dog pharmacokinetics studies. J. Chromatogr. B,2007,852:8-14
1 Weber L, Multi-component reactions and evolutionary chemistry. Drug Discov. Today,2002,7:143-147
2 Ueberall F, Fuchs D, Vennos C, Anti-inflammatory potential of Padma 28-review of experimental data on the antiatherogenic activity and discussion of the multi-component principle, Forsch. Komplementmed, 2006,13 (Suppl.1):7-12
3 China Pharmacopoeia Committee, Pharmacopoeia of the People's Republic of China, China Chemical Industry Press, Beijing,2010
4 Cheng L, Zhang M, Zhang P, et al Silver complexation and tandem mass spectrometry for differentiation of triterpenoid saponins from the roots of Pulsatilla chinensis (Bunge) Regel. Rapid Commun. Mass Spectrom,2008, 22:3783-3790
5 Jiangsu New Medical College, Dictionary of Chinese Medicinal Materials, Scientific and Technological Press, Shanghai,1977
6 Wang XF, Geng BQ, Zhang RH, The antitumor effect of radix pulsatillae alcohol extract. J. Zhejiang Med. Univ,1998,27:204-206
7 Cai Y, Tang YM, Liang BW, Antitumor effect of Pulsatilla chinensis in vitro. Chin. Tradit. Herb Drugs,1999,30:441-443
8 Cai Y, Tang YM, Liang BW, Antitumor effect of Pulsatilla chinensis in vivo. Chin. Tradit. Herb Drugs,1999,30:929-931
9 Zhuang XH, Geng BQ, Yong BG, Studies on antitumor effect of Pulsatilla chinensis. J. Pract. Oncol,1999,13:1494-1496
10 Zhang RH, Zhang ZM, Geng BQ, The preventive and therapeutic effect of radix pulsatillae on mice colorectal cancer induced by dimethylhydrazine. Pharmacol. Clin. Chin. Mater. Med,1999,15:33-35
1 中华人民共和国药典委员会.中华人民共和国药典.一部[M].北京：中国医药科技出版社,2010：96
2 Chang SY, Nohara T. Two new lupane-triterpene glycosides from leaves of Acanthoparar Koreanum[J]. Chem Pharm Bull,1998,46(1):163-165
3 Fujioka T, Kashiwada Y, Kilkuskic RE. Anti-AIDS agents 11, betulinic acid and platonic acid as anti-HIV principles from syzigium claviflorum and the anti-HIV activity of structurally related triterpenoids [J]. J Nat Prod,1994,57(2):243-247
4 Sun IC, Chen CH, Kashiwada Y. Anti-AIDS agents 49 synthesis, anti-HIV, and anti-fusion activeties of IC9564 analogues based on betulinic acid [J]. J Med Chem,2002,45(19):4271-4275
5 Srivastava AK, Shakeel M, Khan AA. QSAR studies on anti HIV-1 N-subtituted Betulinic acid amides [J]. Ind J Chem,2002,41B(2):436-439
6 Pisha E, Chai H, Lee IS. Discovery of betulinic acid as a selective inhibitor of human melanoma that function by induction of apoptosis [J]. J Nat Med,1995,1(10):1046-1051
7卢来春,蒋学华,杨俊毅,等.阿莫西林对格列美脲血浆蛋白结合率的影响[J].华西药学杂志,2003,18(4)：250-254
8郭宾,李川.药物与血浆蛋白结合的药理学基础及其研究进展[J].中国临床药理学与治疗学,2005,10(3)：2411
1 Dasgupta TK, Pezzuto JM, Use of betulinic acid and its derivatives for inhibiting malignant melanoma growth and method of monitoring this [P]. W.96/29068.1996-09-26
2 Chang SY, Nohara T, Two new lupane-triterpene glycosides from leaves of Acanthoparar Koreanum [J]. Chem Pharm Bull,1998,46(1):163
3 Kuo HL, Yoshiki K, Fumio H, et al Inventors Betulinic acid derivatives and uses therefor [P]. W.96/39033.1996-12-12
4 Srivastava AK, Shakeel M, Khan AA, et al QSAR studies on anti HIV-1 N-subtituted Betulinic acid amides [J]. Ind J Chem,2002,41B(2):436
5 Sun IC, Chen CH, Kashiwada Y, Anti-AIDS agents 49 synthesis, anti-HIV, and anti-fusion activeties of IC9564 analogues based on betulinic acid [J]. J Med Chem,2002,45(19):4271
6 Fujioka T, Kashiwada Y, Kilkuskic RE, et al Anti-AIDS agents 11, betulinic acid and platonic acid as anti-HIV principles from syzigium claviflorum and the anti-HIV activity of structurally related triterpenoids [J]. J Nat Prod,1994,57(2):243
7 Pisha E, Chai H, Lee IS, et al Discovery of betulinic acid as a selective inhibitor of human melanoma that function by induction of apoptosis [J]. J Nat Med,1995,1(10):1046
1刘文冯,果刘梦,莹林昶,等.白头翁汤有效成分提取工艺的研究[J].中国民族民间医药,2008,17(1)：60-63
2 Ye WC, Ji NN, Zhao SX, et al Triterpenoids from Pulsatilla chinensis[J]. Phytochemistry,1996,42 (3):799-802
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