摘要
鹿衔草为鹿蹄草科植物鹿蹄草Pyrola calliantha H．Andres和普通鹿蹄草Pyrola decorata H．Andres的干燥全草，收载于《中国药典》2005年版一部。具有补虚益肾、驱风除湿、活血调经的功效，用于治疗虚弱咳嗽、劳伤吐血、风湿痹痛、腰膝无力、月经过多、外伤出血等。
中药鹿衔草的应用历史久远，可与多种中药配伍使用，已有研究多集中于药理活性和临床应用等方面。本论文主要从化学成分、药效物质基础、活性成分的质量控制和药物动力学等方面对该药的一个品种—普通鹿蹄草进行了深入的研究。
对普通鹿蹄草的95％乙醇提取物进行了系统的化学成分研究，采用硅胶柱色谱、聚酰胺柱色谱、Sephadex LH-20分子排阻色谱、ODS柱色谱以及制备HPLC等多种色谱技术分离得到22个化合物，通过理化性质和波谱学分析鉴定了它们的结构。6，6′-dihydroxy-4，5′-dimethyl-[1，1′-biphenyl]-3，3′-diyl bis-β-D-glucopyranoside为新化合物；梅笠草素(chimaphilin)、齐墩果酸(oleanolic acid)、熊果酸(ursolic acid)、鹿蹄草素(toluhydroquinone)、香草酸(vanillic acid)、pomolic acid、maslinic acid、colosic acid、槲皮素(quercetin)、异槲皮苷(isoquercitrin)、槲皮苷(quercitrin)、pyrolaside A、异高熊果苷(isohomoarbutin)、没食子酸(gallic acid)、3-β-O-α—L-arabinopyranosylsiaresinolic acid-28-O-β-D-glucopyranosyl ester和ziyuglycosideⅠ等16个化合物为首次从普通鹿蹄草种植物中分离得到；金丝桃苷(hyperin)、槲皮素3-O-呋喃阿拉伯糖苷(quercetin-3-O-α-L-arabinofuranoside)、高熊果苷(homoarbutin)、2″-O-galloylhyperin和鹿蹄草苷(pirolatin)等为分离得到的已知化合物。
根据鹿衔草的传统用药记载以及现代临床应用，对其抗肿瘤和抗菌两方面生物活性进行了研究。采用MTT法，首先利用人肝癌Bel-7402细胞株，以细胞增殖抑制率为评价指标，对鹿衔草抗肿瘤活性部位进行追踪，确定了活性部位为该药乙酸乙酯萃取层经硅胶柱层析得到的甲醇-氯仿(1：9)洗脱流份，活性部位中化学成分协同发挥抗癌活性，对Bel-7402细胞的增殖抑制量效关系较好，IC_(50)为3.2 g生药量·mL~(-1)；进一步利用人肝癌Bel-7402、人肝癌HepG-2、人宫颈癌HeLa、人胃癌SGC-7901和人纤维肉瘤HT-1080等五种细胞株，对从活性部位分离得到的化学成分进行抗肿瘤活性初步评价，结果表明：醌类成分梅笠草素、酚酸类成分鹿蹄草素和三萜类成分熊果酸、pomolic acid、colosic acid对考察的5种人癌细胞都表现出不同强度的增殖抑制活性，IC_(50)分别在7.64～19.14μg·mL~(-1)，14.87～28.52μg·mL~(-1)，1.36～5.84μg·mL~(-1)，17.34～23.99μg·mL~(-1)和12.69～28.75μg·mL~(-1)之间，三萜类成分maslinic acid对3种人癌细胞具有选择性抑制作用，IC_(50)在24.24～47.29μg·mL~(-1)之间。采用纸片法和微量肉汤稀释法，初步考察了普通鹿蹄草中化学成分的体外抗菌活性。结果表明，醌类成分梅笠草素和酚酸类成分鹿蹄草素具有一定的广谱抗菌活性，它们对革兰氏阳性细菌金黄色葡萄球菌、肺炎链球菌和革兰氏阴性细菌大肠埃希氏菌的MIC分别为4～64μg·mL~(-1)和8～32μg·mL~(-1)；黄酮类成分2″-O-galloylhyperin具有抗真菌活性，对白色念珠菌的MIC为32μg·mL~(-1)。以上研究结果初步阐明了鹿衔草抗肿瘤和抗菌的药效物质基础，为鹿衔草的临床应用和进一步开发提供了实验依据。
建立了鹿衔草中多组分的同时定量分析方法，定量指标涉及该药中三萜、黄酮和酚酸等三大类主要成分。采用LC-MS法同时定量分析了3-β-O-α-L-arabinopyranosylsiaresinolic acid-28-O-β-D-glucopyranosyl ester、ziyuglycosideⅠ、pomolic acid、maslinic acid、colosic acid、齐墩果酸和熊果酸等7种三萜类成分，选用Hypersil C_(18)色谱柱，以甲醇-水为流动相进行梯度洗脱，大气压电离源(APCI)负离子方式电离，SIM法检测，各组分线性关系良好，平均回收率为94.5～103.3％，RSD≤4.6％；采用HPLC-UV法同时定量分析了金丝桃苷、异槲皮苷、2″-O-galloylhyperin、槲皮素-3-O-呋喃阿拉伯糖苷和槲皮苷等5种黄酮苷类成分，选用Zobax Extend C_(18)色谱柱，以乙腈-水(14：86，v/v)为流动相，在350 nm处检测，各组分线性关系良好，平均回收率为96.3～104.2％，RSD≤4.2％；采用HPLC-UV法同时定量分析了异高熊果苷、高熊果苷和鹿蹄草素等3种酚酸类成分，选用Zobax Extend C_(18)色谱柱，以甲醇-水为流动相进行梯度洗脱，在280 nm处检测，各组分线性关系良好，平均回收率为97.2～101.3％，RSD≤2.7％。建立的分析方法简便、灵敏、准确，为鹿衔草药材的质量控制提供了技术保证。
建立了鹿衔草中醌类成分梅笠草素，酚酸类成分鹿蹄草素、异高熊果苷、高熊果苷和三萜类成分齐墩果酸、熊果酸在大鼠血浆中的分析方法。采用LC-MS法测定梅笠草素，以联苯甲酰为内标，乙醚为提取溶剂，梅笠草素在10～1000 ng·mL~(-1)范围内线性良好，方法的定量下限为10 ng·mL~(-1)，日内精密度RSD≤11.5％，日间精密度RSD≤7.6％，准确度在88.4％～113.0％之间，平均回收率为85.5％；采用GC法测定鹿蹄草素，以对硝基苯乙酮为内标，血浆经乙醚-异丙醇(9：1)提取，以氢火焰离子化检测器(FID)检测，色谱柱为HP-5毛细管柱，鹿蹄草素在1.0～50μg·mL~(-1)范围内线性良好，方法的定量下限为1.0μg·mL~(-1)，日内精密度RSD≤11.7％，日间精密度RSD≤15.6％，准确度在88.1～105.4％之间，平均回收率为94.1％；采用HPLC-UV法测定异高熊果苷和高熊果苷，以阿昔洛韦为内标，血浆经甲醇沉淀蛋白处理，异高熊果苷和高熊果苷的线性范围均为0.6～60μg·mL~(-1)，定量下限均为0.6μg·mL~(-1)，日内精密度分别为RSD≤5.5％和RSD≤4.5％，日间精密度分别为RSD≤10.0％和RSD≤6.3％，准确度分别在90.1％～111.4％和92.7％～104.2％之间，平均回收率分别为92.7％和94.9％；采用LC-MS法测定齐墩果酸和熊果酸，以白桦酯酸为内标，乙酸乙酯为提取溶剂，齐墩果酸和熊果酸的线性范围分别为2～160 ng·mL~(-1)和10～800 ng·mL~(-1)定量下限分别为2 ng·mL~(-1)和10 ng·mL~(-1)，日内精密度分别为RSD≤11.0％和RSD≤9.8％，日间精密度分别为RSD≤12.7％和RSD≤6.2％，准确度分别在89.7％～115.8％和94.2％～114.7％之间，平均回收率分别为75.0％和73.8％。
研究了大鼠口服鹿衔草提取液后各指标成分的药动学行为，结果表明，大鼠血浆中梅笠草素(30 mg·kg~(-1))吸收快、消除较平缓，T_(max)和MRT分别为1.2h和5.0 h，组织分布广，V为44.5 L·kg~(-1)，AUC_(0-∞)为3467 ng·h·mL~(-1)，药时曲线存在双峰现象；异高熊果苷(20.2 mg·kg~(-1))和高熊果苷(20.0 mg·kg~(-1))具有明显的吸收，T_(max)分别为0.6 h和0.5 h，t_(1/2)分别为1.5 h和1.2 h，C_(max)分别为18.5μg·mL~(-1)和35.1μg·mL~(-1)，AUC_(0-∞)分别为37.7μg·h·mL~(-1)和52.7μg·h·mL~(-1)，高熊果苷的吸收和消除速度均略快于异高熊果苷，吸收量大于异高熊果苷，AUC/Dose值约为异高熊果苷的1.4倍；齐墩果酸(20.8 mg·Kg~(-1))和熊果酸(88.4mg·Kg~(-1))在大鼠体内具有一定吸收，C_(max)分别为93.5 ng·mL~(-1)和178.9 ng·mL~(-1)，AUC_(0-∞)分别为215.2 ng·h·mL~(-1)和1686 ng·h·mL~(-1)；口服鹿衔草提取液后，大鼠血浆中未检测到鹿蹄草素。进一步研究了大鼠股静脉注射鹿蹄草素单体的药动学行为，结果表明，鹿蹄草素(20 mg·kg~(-1))的药动学过程均符合二室模型，吸收和消除速度均较快，t_(1/2α)和t_(1/2β)分别为1.6 min和31.6 min，C_(max)为23.9μg·mL~(-1)，在体内维持最小抑菌浓度MIC(8～32μg·mL~(-1))的时间仅为20 min。以上药动学结果可为鹿衔草作用机制研究、药效学评价以及临床合理用药提供实验依据。
本研究在中医药学理论和实践的指导下，综合运用中药化学、分析化学、药理学、微生物学和计算机技术，对鹿衔草的化学成分和质量控制方法进行了较系统的研究，初步探讨了鹿衔草抗肿瘤和抗菌的药效物质基础和活性成分的体内药动学过程。本研究为鹿衔草的开发利用提供了科学依据，为中药现代化做了有意义的探索。
Lu xian cao(Herba Pyrolae) is commonly used herbal drug as tonics, sedatives, hemostatics, anti-inflammation, and analgesics against rheumatoid arthritis in China since the antiquity. According to the Pharmacopoeia of the PRC(edition 2005), lu xian cao is the dried whole plant of Pyrola decorata H. Andres. In this dissertation, the chemical components, therapeutic basis, quality control method, as well as pharmacokinetics of lu xian cao were studied systematically.
From the whole plant of Pyrola decorata H. Andres, 22 compounds were isolated by various chromatographic techniques and structurally elucidated by chemical and spectroscopic methods. One of them was a new compound, 6,6'-dihydroxy-4,5'-dimethyl-[1,1'-biphenyl]-3,3'-diyl bis-β-D-glucopyranoside, and sixteen of them were isolated from this plant for the first time: chimaphilin, oleanolic acid(OA), ursolic acid(UA), toluhydroquinone, vanillic acid, pomolic acid(PA), maslinic acid(MA), colosic acid(CA), quercetin(QG1), isoquercitrin(QRh), quercitrin, pyrolaside A, isohomoarbutin, gallic acid, 3-β-O-α-L-arabinopyranosylsiaresinolic acid-28-O-β-D-glucopyranosyl ester(ASGE) and ziyuglycoside I(APGE). Five known ones, hyperin(QGa), quercetin-3-O-α-L-arabinofuranoside(QAr), homoarbutin, 2"-O-galloylhyperin(GQGa) and pirolatin were also isolated.
The therapeutic bases of lu xian cao were explored by antitumor and antibacterial activity evaluation.①The antitumor activity of lu xian cao was firstly studied on screening of active ingredients with their cytotoxicity against Bel-7402 hepatoma cell line by using MTT method. As a result, the ethyl acetate soluble extract of lu xian cao showed intense activity. Further fractionation of the ethyl acetate soluble extract on silica gel column lead to two main parts, namely fraction eluted with chloroform and fraction eluted with menthol-chloroform(1:9). These two fractions acted only when they were combined and the activity of the combination was not significantly different from the acetate soluble extract by ANOVA test. The cytotoxicitiy of compounds isolated from the two main effect parts was further investigated using five human tumor cell lines, namely, Bel-7402 hepatoma ceil line, HepG-2 hepatoma cell line, Hela epithelial carcinoma cell line, SGC-7901 gastric carcinoma cell line, and HT-1080 fibrosarcoma cell line. Chimaphilin, toluhydroquinone, ursolic acid, pomolic acid, and colosic acid showed significant cytotoxicities against the aforementioned cell lines and gave IC_(50) values in the range of 7.64～19.14μg·mL~(-1), 14.87～28.52μg·mL~(-1), 1.36～5.84μg·mL~(-1), 17.34～23.99μg·mL~(-1) and 12.69～28.75μg·mL~(-1), respectively. Maslinic acid showed slelective activity on three tumor cell lines with IC_(50) values in the range of 24.24～47.29μg·mL~(-1).②The in vitro antimicrobial activity of the isolated compounds from lu xian cao was tested against gram-positive(Staphylococcus aureus and Streptococcus pneumoniae), gram-negative(Escherichia coli and Pseudomonas aeruginosa) bacteria and the yeast(Candida albicans). The MIC was estimated by filter paper mensuration and the broth micro-dilution method in 96-well microtiter plates. As a result, chimaphiiin and toluhydroquinone exhibited significant inhibitory effect against S. aureus, S. pneumoniae and E. coli with MIC values in the range of 4～64μg·mL~(-1) and 8～32μg·mL~(-1), respectively. 2"-O-galloylhyperin exhibited significant inhibitory effect against C. albicans and the MIC was 32μg·mL~(-1)
Simultaneous quantification methods of multi-constituents in lu xian cao were developed and validated in this paper. The selected indices involved three main kinds of constituents in lu xian cao. A LC-MS method was established for the determination of seven major triterpenoids ASGE, APGE, PA, MA, CA, OA and UA. LC separation was performed on Hypersil C_(18) column using gradient elution of methanol-water as mobile phase. The analytes were ionized by APCI source and determined on SIM mode. All analytes showed good linearity within the test ranges and the recovery rates were 94.5～103.3%(RSD<4.6%). A HPLC-UV method was developed for the determination of five major flavonoids QGa, QGl, GQGa, QAr and QRh, the separation was carried on Zobax Extend C_(18) column using acetonitrile-water(14:86, v/v) as mobile phase. The absorbance was monitored at 350 nm. All analytes show good linearity within the test ranges and the recovery rates were 96.3～104.2%(RSD<4.2%). A HPLC-UV method was developed for the determination of major phonolic compounds toluhydroquinone, isohomoarbutin and homoarbutin. Separation was achieved on Zobax Extend C_(18) column using gradient elution of methanol-water as mobile phase. The absorbance was monitored at 280 nm. All analytes show good linearity within the test ranges and the recovery rates were 97.2～101.3%(RSD<2.7%). The established assay methods were simple, accurate and performed well in application to the determination of twenty commercial samples of lu xian cao collected from different regions of China. It can be further used for the quality control of both plant materials and preparations of lu xian cao.
Pharmacokinetic study of lu xian cao was accomplished on the constituent toluhydroquinone, isohomoarbutin and homoarbutin, oleanolic acid and ursolic acid. Chimaphilin: The LC separation was carried on Cromasil C_(18) column using methanol-water(75:25, v/v) as mobile phase after the plasma sample was extracted with diethyl ether. APCI-MS in SIM mode was used to determine[M]~-at 186 and 210 for chimaphilin and benzyl(internal standard). Toluhydroquinone: The GC separation was achieved on HP-5 capillary column. After extracted with diethyl ether-isopropanol(9:1, v/v), the plasma sample was analyzed by FID with p-nitroacetophenone as internal standard. Isohomoarbutin and homoarbutin: The HPLC separation was achieved on Zobax Extend C_(18) column using methanol-water(6:94, v/v) as mobile phase after the plasma sample was deproteinized by methanol, acyclovir was used as internal standard and the UV detector was set at 280 nm. Oleanolic acid and ursolic acid: The LC separation was carried on Hypersil C_(18) column using methanol-water(82:12, v/v) as mobile phase after the plasma sample was extracted with ethyl acetate. APCI-MS in SIM mode was used to determine[M-H]~-at 455 for oleanolic acid, ursolic acid and betulinic acid(internal standard). Validation assays indicated that the established methods were simple and performs well in terms of selectivity, linearity, precision, and accuracy.
After oral administration of Lu Xian Cao Decoction, the main pharmacokinetic parameters for aforementioned constituents were as follows: chimaphilin(30 mg·kg~(-1)): T_(max), 1.2 h; MRT, 5.0 h; V, 44.5 L·kg(-1), AUC_(0-∞), 3467 ng·h·mL(-1); isohomoarbutin(20.2 mg·kg(-1)): T_(max), 0.6 h; C_(max), 18.5μg·mL~(-1); AUC_(0-∞), 37.7μg·h·mL~(-1); homoarbutin(20.0 mg·kg(-1)): T_(max), 0.5 h; C_(max), 35.1μg·mL~(-1); AUC_(0-∞), 52.7μg·h·mL~(-1); oleanolic acid(88.4 mg·Kg(-1)): C_(max), 93.5 ng·mL(-1);AUC_(0-∞), 215.2 ng·h·mL(-1); t(1/2), 1.7 h; ursolic acid(20.8 mg·Kg(-1)): C_(max), 178.9 ng·mL(-1); AUC_(0-∞), 1685.9 ng·h·mL(-1); t_(1/2),6.9 h; toluhydroquinone was not founded in plasma due to its minor content in lu xian cao. After intravenous administration of toluhydroquinone(20 mg·Kg(-1)) injection, the pharmacokinetic behavior of toluhydroquinone was found to be in line with the two-compartment intravenous model. The t_(1/2α), t_(1/2β),C_(max) and AUC_(0-∞) were 1.6 min, 31.6 min, 23.9μg·mL~(-1),37.7μg·h·mL~(-1), respectively.
Conclusively, under the theory and methodologies of traditional Chinese medicine, utilizing the knowledge of phytochemistry, analytical chemistry, pharmacology, microbiology, pharmacokinetics and chemometrics, the quality control methods for lu xian cao was developed, the pharmacokinetic study of this medicinal herb was also investigated to some extend. The present research provided valuable academic evidences for the future research and development of lu xian cao.
引文
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