中药复方清肝散结颗粒的药效物质基础及质量控制研究
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摘要
原发性肝癌是我国常见的恶性肿瘤之一，发病率高，近年来发病率有逐年上升的趋势。中医药治疗已成为肝癌综合治疗的一个重要组成部分，在改善患者症状、提高生存率方面具有一定的优势，已经日益受到重视和关注。中药复方“清肝散结方”是东方肝胆外科医院中西医结合治疗科经长期临床实践经验总结得出的抗肝癌基本方，由猫人参、石见穿、白花蛇舌草及半枝莲等十四味中药组成，具有良好的改善患者肝功能、减轻患者症状的作用，还有良好的抑制肝癌生长，抗肿瘤转移与复发，提高患者预期生存率的作用。
中药复方是中医用药的主要形式，它基于辨证的思想观点，按照君臣佐使的组方原则，选择合适的中药饮片、按一定的组方比例配伍而成，是中医辨证论治理论的具体体现。中药及复方药效物质基础研究是阐明中药药效作用的关键，是中药质量控制的基础与核心，是中药材及其产品安全、有效和质量稳定、可控的保障。为阐明清肝散结方中的药效物质基础并对其制剂质量进行有效的控制，本研究采用HepG2和SMMC-7721肝癌细胞模型，以氟尿嘧啶为阳性对照，对清肝散结方提取物、君药中的主要药效成分齐墩果酸、熊果酸及二者混合物在抑制肝癌细胞生长的药效作用进行了研究。清肝散结方提取物、齐墩果酸、熊果酸及二者混合物对HepG2细胞的IC50分别为9.255mg/mL、7.53μg/mL（16.51μM）、34.16μg/mL（74.91μM）及36.63μg/mL；对SMMC-7721细胞的IC50分别为8.970mg/mL、15.11μg/mL（33.13μM）、55.06μg/mL（120.7μM）及32.23μg/mL，结果表明清肝散结方具有明显的抗癌作用，复方的药效作用要优于齐墩果酸及熊果酸单独给药，而齐墩果酸及熊果酸的药效作用也优于氟尿嘧啶，为临床应用清肝散结方治疗肝癌提供了有力的体外实验数据。
根据清肝散结方组方中各单方药材的研究报道及国内专业数据库，建立了清肝散结方中各组方药材已报道的千余种已知化学成分数据库，采用6倍量70％乙醇对清肝散结方中的化学成分进行超声提取，结合LC-TOF/MS技术实现了对复方提取物中复杂化学成分的快速定性分析，借助数据库自动及手动匹配，在正、负离子模式下共鉴别出清肝散结方中163种化学成分，并对其来源的药材进行了归属，其中三萜酸、黄酮及皂苷类成分种类较多，相对含量较高，主要来源于君药猫人参、石见穿、白花蛇舌草与半枝莲、佐药黄芩与柴胡及使药甘草等，定性结果表征了清肝散结方的复杂化学物质基础，为阐明该方药效物质基础及质量控制提供依据。
由于清肝散结方中所含化学成分复杂，极性由强到弱分布广泛，含量高低分布不均，很多组分在色谱柱上具有相似的保留行为。采用加速溶剂萃取对其中多种化学成分进行快速提取，利用正交实验设计优化了影响提取结果的七个影响因素，包括提取溶剂、萃取温度，静态萃取时间，提取体积、循环次数、冲洗体积和吹洗时间，并以待测成分中含量较高的11个成分的提取总量为观察指标，优化最佳提取参数。采用LC-TOF/MS建立了复方提取物中复杂化学成分的快速分离分析方法，进行了完整的方法学考察。对复方中30个化学成分同时进行含量测定，明确其中主要药效成分的含量。各化学成分的含量如下：原儿茶酸55.65±1.80μg/g、岩白菜素830.5±6.82μg/g、儿茶素11.74±0.43μg/g、绿原酸174.6±8.41μg/g、没食子酸儿茶素酯27.40±0.52μg/g、表儿茶素5.413±0.1μg/g、甘草苷55.91±1.02μg/g、野黄芩苷174.9±3.68μg/g、柚皮苷54.34±2.29μg/g、迷迭香酸38.430±1.73μg/g、异甘草苷7.727±0.32μg/g、甘草素3.633±0.16μg/g、黄芩苷4440±193.17μg/g、斛皮素24.83±0.64μg/g、柚皮素9.995±0.25μg/g、木犀草素24.79±0.77μg/g、山奈酚4.425±0.19μg/g、柴胡皂苷C7.298±0.26μg/g、汉黄芩苷1161±47.64μg/g、芹菜素29.66±0.76μg/g、黄芩素669.0±31.35μg/g、汉黄芩素208.6±4.89μg/g、甘草酸188.9±4.58μg/g、柴胡皂苷A131.1±4.63μg/g、积雪草酸148.2±6.68μg/g、柴胡皂苷B21.871±0.07μg/g、柴胡皂苷D79.60±1.63μg/g、齐墩果酸227.4±7.82μg/g及熊果酸428.2±13.26μg/g，甘草次酸未检测到。该方法前处理方便、重复性好、分离及检测灵敏度高、便于自动化，对分析复杂体系具有明显的优势，能够满足对多组分的同时分析，为清肝散结方中药效成分的体内代谢动力学研究提供了数据支持。
根据复方中化学成分含量测定结果，对其中含量相对较高的化学成分进行大鼠体内的药代动力学研究。采用有机溶剂萃取结合LC-MS测定方法，对清肝散结方不同组合（君药、君药与臣药、君药，臣药与佐药及复方）提取物给药后大鼠血浆中齐墩果酸及熊果酸的浓度进行测定，研究其在大鼠体内的药代动力学。发现君药中所含药效成分齐墩果酸及熊果酸在不同组方配伍给药后，大鼠体内的半衰期T1/2约增加了一倍，分别为君药8.94±2.19h及4.78±1.07h、君药与臣药17.14±6.6h及7.31±3.70h、君药，臣药与佐药18.56±13.67h及9.52±4.41h、复方15.03±7.36h及8.64±1.36h。统计检验结果显示存在显著差异，说明组方中臣、佐及使药的加入能够延长了二者在大鼠体内的代谢处置过程，导致半衰期延长。采用有机溶剂直接沉淀法结合LC-MS/MS技术对复方中所含的岩白菜素、绿原酸、野黄芩苷、黄芩苷、汉黄芩苷及汉黄芩素等多种药效成分同时进行大鼠体内的药代动力学研究，结果显示岩白菜素及绿原酸受其他成分竞争吸收的影响较小，呈现典型的血管外给药处置过程，而黄酮类化合物则出现双峰现象，Tmax2分别为野黄芩苷8.00±3.79h、黄芩苷9.67±2.66h、汉黄芩苷16.33±8.62h、汉黄芩素9.00±2.54h，比文献报道出现双峰的时间晚，推测复方中的复杂化学成分竞争性延长了黄酮类成分在肠道内的代谢转化过程，导致二次吸收时间推迟，使得这些成分在给药12h后仍维持较高的血浆浓度，提高了生物利用度。
在对清肝散结方中药效物质进行研究的基础上，采用高效液相色谱法分别对清肝散结颗粒中君药所含的三萜酸类齐墩果酸与熊果酸，以及君药与佐药中所含的黄酮类药效成分黄芩苷、汉黄芩苷、野黄芩苷、黄芩素与汉黄芩素进行含量测定，建立了清肝散结颗粒中主要药效成分的质量控制方法，并采用正交设计实验对前处理方法进行了优化。清肝散结颗粒中齐墩果酸、熊果酸、黄芩苷、汉黄芩苷、野黄芩苷、黄芩素及汉黄芩素的含量分别为7.29±0.18μg/g、16.37±0.47μg/g、3798±1.93μg/g、829.9±0.33μg/g、180.5±0.05μg/g、158.9±0.04μg/g及72.6±0.06μg/g，为其制剂质量控制提供了简便快速、准确可靠、便于普及的可行方法。
Hepatic Cell Carcinoma (HCC) is one of the common malignant tumors with a highmortality rate in our country, and the incidence of HCC was in an increasing trend inrecently years. Traditional Chinese Medicine (TCMs) has become an important part in thetreatment of HCC; TCMs have certain advantages in improving patient symptoms andimproving the survival rate, and have been growing attentions and concerns. Qinggansanjiedecoction was the primary anti-hepatoma prescription basic the long-term clinical practiceexperience against liver cancer in the department of integrative traditional Chinese andwestern medicine treatment, Eastern Hepatobiliary Surgery Hospital. It was formed byfourteen Chinese medical herbs including Actinidia valvata Dunn, Chinese Sage Herb,Herba Hedyotis and Herba Scutellariae Barbatae, etc, with the role of improving liverfunction, relieveing the patient's symptoms, inhibition growth of hepatocellular carcinoma,resistance to tumor metastasis and recurrence, and improve survival of patientexpectations.
TCMs were the main form in the treatment of desease, which was based on dialecticalideas, in accordance with the formula principle of monarch, minister, assistant and guide,choose the appropriate Chinese Herbal Pieces, and according to to a certain proportion ofprescription compatibility, was concrete manifestation of the theory of traditional Chinesemedicine. The effect of material basic research of TCMs was the role of Chinese medicinesand compound prescription, and the base and core of quality control to ensure the safe,effective and stable quality of TCMs and their preparations. In order to clarify the effectivein antitumor and control the quality of Qinggansanjie prescription and the preparation,hepatoma cell model of HepG2，SMMC-7721with fluorouracil as a positive control wereused to evaluate the effect in inhibiting the growth of liver cancer cells after oraladministration of Qinggansanjie extract, oleanolic acid, ursolic acid and the two mixtures.The IC50in HepG2cells were9.255mg/mL,7.53μg/mL (16.51μM),34.16μg/mL (74.91μM)and36.63μg/mL, respectively. The IC50in SMMC-7721cells were8.970mg/mL,15.11μg/mL (33.13μM),55.06μg/mL (120.7μM) and32.23μg/mL, respectively. The resultsshowed that the Qinggansanjie decoction has significant anti-cancer effect rather than thecompound of the single administration of oleanolic acid, ursolic acid and the two mixtures,oleanolic acid and ursolic acid were better than fluorouracil for treatment of HCC, thus itproved the effect of Qinggansanjie prescription in clinical applications with the strong experimental data in vitro.
We established a constituent database of known chemical compositions been reportedand data in Chinese medicine ingredient database, including more than thousand ofcomponds in the herbs to compose Qinggansanjie prescription. With six times the amountof70%ethanol by ultrasonic extraction of the chemical composition in Qinggansanjieprescription and combined with LC-TOF/MS, the complex chemical composition in theextracts were qualitative analysis rapid with automatically and manually match to thedatabase in positive and negative mode. We found163chemical ingredients in the extractand its source of medicinal materials of belonging were interpreted. There were types oftriterpene acids, flavonoids and saponins with higher content relatively, mainly derivedfrom monarch herbs including Actinidia valvata Dunn, Chinese Sage Herb, HerbaHedyotis and Herba Scutellariae Barbatae, Radix Scutellariae and Chinese Thorowax Rootof assistant and Radix Glycyrrhizae of guide, the qualitative results of constituents in theextract clarified the chemical material base and provide basis for quality control ofQinggansanjie prescription.
Because of the complex chemical compositions in Qinggansanjie prescription, thepolarity of compounds distribution were in a wide range, and the content of them weredefferent, many components in the chromatography have similar retention behavior. Wedesign an orthogonal experimental to optimize the impact of seven influential factorsincluding the extraction solvent, extraction temperature, static extraction time, extractionvolume, number of cycles, flush volume and purge time in the extraction procession ofQinggansanjie prescription by accelerated solvent extraction, and the total content of the11components in the higher levels were observed to conclude the parameters of extraction.AnLC-TOF/MS method was established to rapid separate and quantity of the complexchemical composition in the extracts, the content of30chemical compositions weredetermined as follows: protocatechuic55.65±1.80μg/g, Bergenin830.5±6.82μg/gcatechins11.74±0.43μg/g, chlorogenic acid174.6±8.41μg/g, gallic acid catechin ester27.40±0.52μg/g, epicatechin5.413±0.1μg/g, glycyrrhizin55.91±1.02μg/g, scutellarin174.±3.68μg/g, naringin54.34±2.29μg/g, rosmarinic acid38.430±1.73μg/g, isoliquiritin7.727±0.32μg/g, glycyrrhizin3.633±0.16μg/g, baicalin4440±193.17μg/g, quercetin24.83±0.64μg/g, naringenin9.995±0.25μg/g, luteolin24.79±0.77μg/g, kaempferol4.425±0.19μg/g, saikosaponin C7.298±0.26μg/g, wogonoside1161±47.64μg/g, apigenin29.66±0.76μg/g, baicalein669.0±31.35μg/g, wogonin208.6±4.89μg/g, glycyrrhizic acid188.9±4.58μg/g, tsaikosaponin A131.1±4.63μg/g, asiatic acid148.2±6.68μg/g,saikosaponin B21.871±0.07μg/g, saikosaponin D79.60±1.63μg/g, oleanolic acid227.4±7.82μg/g and ursolic acid428.2±13.26μg/g, glycyrrhetinic acid was not detected.The method was easy, good in repeatability, high separation and detection sensitivity, easyto automation, which has obviously advantages for quantitive analysis themulticomponents in TCMs simultaneously.
According to the determination by LC-TOF/MS, the chemical compositions with highcontent relatively were chosen to pharmacokinetic study in rats. Using organic solventextraction combined with LC-MS determination method, the concentration of oleanolicacid and ursolic acid in rat plasma were measured after oral administration of differentcombinations extraction of Qinggansanjie prescription (monarch; monarch and minister;monarch, minister and assistant; compound recipe), and its pharmacokinetics parameters inrats were investigated. We found that the T1/2of oleanolic acid and ursolic acid weredifferent after administration of different combination. The T1/2of oleanolic acid andursolic acid in four groups was8.94±2.19h and4.78±1.07h,17.14±6.6h and7.31±3.70h,18.56±13.67h and9.52±4.41h,15.03±7.36h and8.64±1.36h, respectively. There weresignificant differences by statistical test, indicating that T1/2were prolonged to double afteradding the monarch, minister and guide group. The method of precipitation by organicsolvent combined with LC-MS/MS was used to simultaneously determine theconcentration of bergenin, chlorogenic acid, scutellarin, baicalin, wogonoside and wogoninafter administration of extratct of Qinggansanjie prescription. The results ofpharmacokinetic study shown that bergenin and chlorogenic acid have a typical disposalprocess of extravascular administration, but the flavonoids had double peak in the curve ofconcentration vs time, Tmax2of scutellarin was8.00±3.79h, baicalin was9.67±2.66h,wogonoside was16.33±8.62h, wogonin was9.00±2.54h, respectively. It was later than thereports in the literatures, which indicated that the complex chemical composition in theextract of Qinggansanjie prescription could prolong metabolic transformation process ofthe flavonoids in the intestine, result in secondary absorption time been delayed, theseflavonoids ingredients still maintain high concentration in12h rat plasma afteradministration, and the bioavailability were improved.
Based in the effect material foundation study of Qinggansanjie prescription, weestablished a high performance liquid chromatography to determinate the content oftriterpene acids (oleanolic acid and ursolic acid) and flavonoids (scutellarin, baicalin, wogonoside, baicalein and wogonin) in Qinggansanjie granules, orthogonal design wasused to optimize the parameters in the process of extraction by ultrasonic. The content ofoleanolic acid, ursolic acid, baicalin, wogonoside, scutellarin, baicalein and wogoninin inQinggansanjie granules were7.29±0.18μg/g,16.37±0.47μg/g,3798±1.93μg/g,829.9±0.33μg/g,180.5±0.05μg/g,158.9±0.04μg/g and72.6±0.06μg/g, respectively. Itprovides a simple, rapidly, accurate, reliable, easy to popularize method for the qualitycontrol of Qinggansanjie granules.
中药复方是中医用药的主要形式，它基于辨证的思想观点，按照君臣佐使的组方原则，选择合适的中药饮片、按一定的组方比例配伍而成，是中医辨证论治理论的具体体现。中药及复方药效物质基础研究是阐明中药药效作用的关键，是中药质量控制的基础与核心，是中药材及其产品安全、有效和质量稳定、可控的保障。为阐明清肝散结方中的药效物质基础并对其制剂质量进行有效的控制，本研究采用HepG2和SMMC-7721肝癌细胞模型，以氟尿嘧啶为阳性对照，对清肝散结方提取物、君药中的主要药效成分齐墩果酸、熊果酸及二者混合物在抑制肝癌细胞生长的药效作用进行了研究。清肝散结方提取物、齐墩果酸、熊果酸及二者混合物对HepG2细胞的IC50分别为9.255mg/mL、7.53μg/mL（16.51μM）、34.16μg/mL（74.91μM）及36.63μg/mL；对SMMC-7721细胞的IC50分别为8.970mg/mL、15.11μg/mL（33.13μM）、55.06μg/mL（120.7μM）及32.23μg/mL，结果表明清肝散结方具有明显的抗癌作用，复方的药效作用要优于齐墩果酸及熊果酸单独给药，而齐墩果酸及熊果酸的药效作用也优于氟尿嘧啶，为临床应用清肝散结方治疗肝癌提供了有力的体外实验数据。
根据清肝散结方组方中各单方药材的研究报道及国内专业数据库，建立了清肝散结方中各组方药材已报道的千余种已知化学成分数据库，采用6倍量70％乙醇对清肝散结方中的化学成分进行超声提取，结合LC-TOF/MS技术实现了对复方提取物中复杂化学成分的快速定性分析，借助数据库自动及手动匹配，在正、负离子模式下共鉴别出清肝散结方中163种化学成分，并对其来源的药材进行了归属，其中三萜酸、黄酮及皂苷类成分种类较多，相对含量较高，主要来源于君药猫人参、石见穿、白花蛇舌草与半枝莲、佐药黄芩与柴胡及使药甘草等，定性结果表征了清肝散结方的复杂化学物质基础，为阐明该方药效物质基础及质量控制提供依据。
由于清肝散结方中所含化学成分复杂，极性由强到弱分布广泛，含量高低分布不均，很多组分在色谱柱上具有相似的保留行为。采用加速溶剂萃取对其中多种化学成分进行快速提取，利用正交实验设计优化了影响提取结果的七个影响因素，包括提取溶剂、萃取温度，静态萃取时间，提取体积、循环次数、冲洗体积和吹洗时间，并以待测成分中含量较高的11个成分的提取总量为观察指标，优化最佳提取参数。采用LC-TOF/MS建立了复方提取物中复杂化学成分的快速分离分析方法，进行了完整的方法学考察。对复方中30个化学成分同时进行含量测定，明确其中主要药效成分的含量。各化学成分的含量如下：原儿茶酸55.65±1.80μg/g、岩白菜素830.5±6.82μg/g、儿茶素11.74±0.43μg/g、绿原酸174.6±8.41μg/g、没食子酸儿茶素酯27.40±0.52μg/g、表儿茶素5.413±0.1μg/g、甘草苷55.91±1.02μg/g、野黄芩苷174.9±3.68μg/g、柚皮苷54.34±2.29μg/g、迷迭香酸38.430±1.73μg/g、异甘草苷7.727±0.32μg/g、甘草素3.633±0.16μg/g、黄芩苷4440±193.17μg/g、斛皮素24.83±0.64μg/g、柚皮素9.995±0.25μg/g、木犀草素24.79±0.77μg/g、山奈酚4.425±0.19μg/g、柴胡皂苷C7.298±0.26μg/g、汉黄芩苷1161±47.64μg/g、芹菜素29.66±0.76μg/g、黄芩素669.0±31.35μg/g、汉黄芩素208.6±4.89μg/g、甘草酸188.9±4.58μg/g、柴胡皂苷A131.1±4.63μg/g、积雪草酸148.2±6.68μg/g、柴胡皂苷B21.871±0.07μg/g、柴胡皂苷D79.60±1.63μg/g、齐墩果酸227.4±7.82μg/g及熊果酸428.2±13.26μg/g，甘草次酸未检测到。该方法前处理方便、重复性好、分离及检测灵敏度高、便于自动化，对分析复杂体系具有明显的优势，能够满足对多组分的同时分析，为清肝散结方中药效成分的体内代谢动力学研究提供了数据支持。
根据复方中化学成分含量测定结果，对其中含量相对较高的化学成分进行大鼠体内的药代动力学研究。采用有机溶剂萃取结合LC-MS测定方法，对清肝散结方不同组合（君药、君药与臣药、君药，臣药与佐药及复方）提取物给药后大鼠血浆中齐墩果酸及熊果酸的浓度进行测定，研究其在大鼠体内的药代动力学。发现君药中所含药效成分齐墩果酸及熊果酸在不同组方配伍给药后，大鼠体内的半衰期T1/2约增加了一倍，分别为君药8.94±2.19h及4.78±1.07h、君药与臣药17.14±6.6h及7.31±3.70h、君药，臣药与佐药18.56±13.67h及9.52±4.41h、复方15.03±7.36h及8.64±1.36h。统计检验结果显示存在显著差异，说明组方中臣、佐及使药的加入能够延长了二者在大鼠体内的代谢处置过程，导致半衰期延长。采用有机溶剂直接沉淀法结合LC-MS/MS技术对复方中所含的岩白菜素、绿原酸、野黄芩苷、黄芩苷、汉黄芩苷及汉黄芩素等多种药效成分同时进行大鼠体内的药代动力学研究，结果显示岩白菜素及绿原酸受其他成分竞争吸收的影响较小，呈现典型的血管外给药处置过程，而黄酮类化合物则出现双峰现象，Tmax2分别为野黄芩苷8.00±3.79h、黄芩苷9.67±2.66h、汉黄芩苷16.33±8.62h、汉黄芩素9.00±2.54h，比文献报道出现双峰的时间晚，推测复方中的复杂化学成分竞争性延长了黄酮类成分在肠道内的代谢转化过程，导致二次吸收时间推迟，使得这些成分在给药12h后仍维持较高的血浆浓度，提高了生物利用度。
在对清肝散结方中药效物质进行研究的基础上，采用高效液相色谱法分别对清肝散结颗粒中君药所含的三萜酸类齐墩果酸与熊果酸，以及君药与佐药中所含的黄酮类药效成分黄芩苷、汉黄芩苷、野黄芩苷、黄芩素与汉黄芩素进行含量测定，建立了清肝散结颗粒中主要药效成分的质量控制方法，并采用正交设计实验对前处理方法进行了优化。清肝散结颗粒中齐墩果酸、熊果酸、黄芩苷、汉黄芩苷、野黄芩苷、黄芩素及汉黄芩素的含量分别为7.29±0.18μg/g、16.37±0.47μg/g、3798±1.93μg/g、829.9±0.33μg/g、180.5±0.05μg/g、158.9±0.04μg/g及72.6±0.06μg/g，为其制剂质量控制提供了简便快速、准确可靠、便于普及的可行方法。
Hepatic Cell Carcinoma (HCC) is one of the common malignant tumors with a highmortality rate in our country, and the incidence of HCC was in an increasing trend inrecently years. Traditional Chinese Medicine (TCMs) has become an important part in thetreatment of HCC; TCMs have certain advantages in improving patient symptoms andimproving the survival rate, and have been growing attentions and concerns. Qinggansanjiedecoction was the primary anti-hepatoma prescription basic the long-term clinical practiceexperience against liver cancer in the department of integrative traditional Chinese andwestern medicine treatment, Eastern Hepatobiliary Surgery Hospital. It was formed byfourteen Chinese medical herbs including Actinidia valvata Dunn, Chinese Sage Herb,Herba Hedyotis and Herba Scutellariae Barbatae, etc, with the role of improving liverfunction, relieveing the patient's symptoms, inhibition growth of hepatocellular carcinoma,resistance to tumor metastasis and recurrence, and improve survival of patientexpectations.
TCMs were the main form in the treatment of desease, which was based on dialecticalideas, in accordance with the formula principle of monarch, minister, assistant and guide,choose the appropriate Chinese Herbal Pieces, and according to to a certain proportion ofprescription compatibility, was concrete manifestation of the theory of traditional Chinesemedicine. The effect of material basic research of TCMs was the role of Chinese medicinesand compound prescription, and the base and core of quality control to ensure the safe,effective and stable quality of TCMs and their preparations. In order to clarify the effectivein antitumor and control the quality of Qinggansanjie prescription and the preparation,hepatoma cell model of HepG2，SMMC-7721with fluorouracil as a positive control wereused to evaluate the effect in inhibiting the growth of liver cancer cells after oraladministration of Qinggansanjie extract, oleanolic acid, ursolic acid and the two mixtures.The IC50in HepG2cells were9.255mg/mL,7.53μg/mL (16.51μM),34.16μg/mL (74.91μM)and36.63μg/mL, respectively. The IC50in SMMC-7721cells were8.970mg/mL,15.11μg/mL (33.13μM),55.06μg/mL (120.7μM) and32.23μg/mL, respectively. The resultsshowed that the Qinggansanjie decoction has significant anti-cancer effect rather than thecompound of the single administration of oleanolic acid, ursolic acid and the two mixtures,oleanolic acid and ursolic acid were better than fluorouracil for treatment of HCC, thus itproved the effect of Qinggansanjie prescription in clinical applications with the strong experimental data in vitro.
We established a constituent database of known chemical compositions been reportedand data in Chinese medicine ingredient database, including more than thousand ofcomponds in the herbs to compose Qinggansanjie prescription. With six times the amountof70%ethanol by ultrasonic extraction of the chemical composition in Qinggansanjieprescription and combined with LC-TOF/MS, the complex chemical composition in theextracts were qualitative analysis rapid with automatically and manually match to thedatabase in positive and negative mode. We found163chemical ingredients in the extractand its source of medicinal materials of belonging were interpreted. There were types oftriterpene acids, flavonoids and saponins with higher content relatively, mainly derivedfrom monarch herbs including Actinidia valvata Dunn, Chinese Sage Herb, HerbaHedyotis and Herba Scutellariae Barbatae, Radix Scutellariae and Chinese Thorowax Rootof assistant and Radix Glycyrrhizae of guide, the qualitative results of constituents in theextract clarified the chemical material base and provide basis for quality control ofQinggansanjie prescription.
Because of the complex chemical compositions in Qinggansanjie prescription, thepolarity of compounds distribution were in a wide range, and the content of them weredefferent, many components in the chromatography have similar retention behavior. Wedesign an orthogonal experimental to optimize the impact of seven influential factorsincluding the extraction solvent, extraction temperature, static extraction time, extractionvolume, number of cycles, flush volume and purge time in the extraction procession ofQinggansanjie prescription by accelerated solvent extraction, and the total content of the11components in the higher levels were observed to conclude the parameters of extraction.AnLC-TOF/MS method was established to rapid separate and quantity of the complexchemical composition in the extracts, the content of30chemical compositions weredetermined as follows: protocatechuic55.65±1.80μg/g, Bergenin830.5±6.82μg/gcatechins11.74±0.43μg/g, chlorogenic acid174.6±8.41μg/g, gallic acid catechin ester27.40±0.52μg/g, epicatechin5.413±0.1μg/g, glycyrrhizin55.91±1.02μg/g, scutellarin174.±3.68μg/g, naringin54.34±2.29μg/g, rosmarinic acid38.430±1.73μg/g, isoliquiritin7.727±0.32μg/g, glycyrrhizin3.633±0.16μg/g, baicalin4440±193.17μg/g, quercetin24.83±0.64μg/g, naringenin9.995±0.25μg/g, luteolin24.79±0.77μg/g, kaempferol4.425±0.19μg/g, saikosaponin C7.298±0.26μg/g, wogonoside1161±47.64μg/g, apigenin29.66±0.76μg/g, baicalein669.0±31.35μg/g, wogonin208.6±4.89μg/g, glycyrrhizic acid188.9±4.58μg/g, tsaikosaponin A131.1±4.63μg/g, asiatic acid148.2±6.68μg/g,saikosaponin B21.871±0.07μg/g, saikosaponin D79.60±1.63μg/g, oleanolic acid227.4±7.82μg/g and ursolic acid428.2±13.26μg/g, glycyrrhetinic acid was not detected.The method was easy, good in repeatability, high separation and detection sensitivity, easyto automation, which has obviously advantages for quantitive analysis themulticomponents in TCMs simultaneously.
According to the determination by LC-TOF/MS, the chemical compositions with highcontent relatively were chosen to pharmacokinetic study in rats. Using organic solventextraction combined with LC-MS determination method, the concentration of oleanolicacid and ursolic acid in rat plasma were measured after oral administration of differentcombinations extraction of Qinggansanjie prescription (monarch; monarch and minister;monarch, minister and assistant; compound recipe), and its pharmacokinetics parameters inrats were investigated. We found that the T1/2of oleanolic acid and ursolic acid weredifferent after administration of different combination. The T1/2of oleanolic acid andursolic acid in four groups was8.94±2.19h and4.78±1.07h,17.14±6.6h and7.31±3.70h,18.56±13.67h and9.52±4.41h,15.03±7.36h and8.64±1.36h, respectively. There weresignificant differences by statistical test, indicating that T1/2were prolonged to double afteradding the monarch, minister and guide group. The method of precipitation by organicsolvent combined with LC-MS/MS was used to simultaneously determine theconcentration of bergenin, chlorogenic acid, scutellarin, baicalin, wogonoside and wogoninafter administration of extratct of Qinggansanjie prescription. The results ofpharmacokinetic study shown that bergenin and chlorogenic acid have a typical disposalprocess of extravascular administration, but the flavonoids had double peak in the curve ofconcentration vs time, Tmax2of scutellarin was8.00±3.79h, baicalin was9.67±2.66h,wogonoside was16.33±8.62h, wogonin was9.00±2.54h, respectively. It was later than thereports in the literatures, which indicated that the complex chemical composition in theextract of Qinggansanjie prescription could prolong metabolic transformation process ofthe flavonoids in the intestine, result in secondary absorption time been delayed, theseflavonoids ingredients still maintain high concentration in12h rat plasma afteradministration, and the bioavailability were improved.
Based in the effect material foundation study of Qinggansanjie prescription, weestablished a high performance liquid chromatography to determinate the content oftriterpene acids (oleanolic acid and ursolic acid) and flavonoids (scutellarin, baicalin, wogonoside, baicalein and wogonin) in Qinggansanjie granules, orthogonal design wasused to optimize the parameters in the process of extraction by ultrasonic. The content ofoleanolic acid, ursolic acid, baicalin, wogonoside, scutellarin, baicalein and wogoninin inQinggansanjie granules were7.29±0.18μg/g,16.37±0.47μg/g,3798±1.93μg/g,829.9±0.33μg/g,180.5±0.05μg/g,158.9±0.04μg/g and72.6±0.06μg/g, respectively. Itprovides a simple, rapidly, accurate, reliable, easy to popularize method for the qualitycontrol of Qinggansanjie granules.
引文
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