摘要
白花蛇舌草为茜草科植物白花蛇舌草Hedyotis diffusa Willd.[Oldenlandia diffusa(Willd.)Roxb.]的干燥全草。本品作为具有抗癌活性的中草药被广泛用于治疗各种肿瘤,尤其用于消化系统肿瘤,如食道癌、胃癌、肝癌、直肠癌等的治疗。本文以白花蛇舌草为研究对象,对其化学成分、体外药理活性、质量控制方法和体内药物动力学进行了初步研究。具体研究内容如下:
采用硅胶柱色谱、反相制备液相色谱、聚酰胺柱色谱和葡聚糖凝胶色谱等多种分离技术从白花蛇舌草药材的90%乙醇提取物中分离得到17个化合物,根据理化反应和光谱、波谱数据共鉴定了15个。其中1个新化合物:2,7-二羟基-3-甲基蒽醌;1个属首分化合物:3,4-二羟基苯甲酸甲酯。
采用液相色谱/飞行时间质谱联用技术(HPLC-ESI/TOF-MS)对白花蛇舌草提取液中的化学成分进行定性分析。在正、负离子扫描方式下,以各色谱峰的一级和二级质谱裂解信息为依据,经文献检索或与对照品对照共鉴定了8个成分,分别为:去乙酰基车叶草苷酸、去乙酰基车叶草苷、鸡屎藤次苷、10-乙酰基鸡屎藤次苷、车叶草苷、对香豆酸、反式-6-O-对香豆酰鸡屎藤苷甲酯、顺式-6-O-对香豆酰鸡屎藤苷甲酯。
采用MTT比色法,以肝癌BEL-7402、胃癌SGC-7901和乳腺癌MCF-7为实验细胞株,分别考察了白花蛇舌草60%乙醇提取物以及从白花蛇舌草中分离得到的8个单体化合物对三种肿瘤细胞株的增殖抑制作用。实验结果表明:白花蛇舌草60%乙醇提取物对三种肿瘤细胞株均表现出不同程度的增殖抑制作用,其中对胃癌细胞SGC-7901的抑制活性最强,IC_(50)值为4.81μg/mL。八个单体化合物中,仅2,7-二羟基-3-甲基蒽醌对MCF-7、BEL-7402、SGC-7901三种肿瘤细胞株均表现出中等强度的增殖抑制活性,且抑制率呈明显浓度依赖关系。该化合物对三种肿瘤细胞株的IC_(50)值分别为53.72μM,54.22μM,84.40μM。
蒽醌类化合物和环烯醚萜类化合物为白花蛇舌草中含量较高的两类化合物。本文采用HPLC-UV法,建立了白花蛇舌草药材中四种蒽醌类成分的含量测定方法;采用高效液相色谱梯度洗脱方式,二极管阵列检测法,建立了白花蛇舌草药材中对香豆酸和三种环烯醚萜类成分的含量测定方法,并用于不同产地白花蛇舌草中葸醌类和环烯醚萜类化合物的含量测定。本文所建立的方法专属、准确、灵敏,为白花蛇舌草药材的质量控制提供了新的方法。
分别选取2-羟基-3-甲基蒽醌和去乙酰基车叶草苷酸作为白花蛇舌草中蒽醌类和环烯醚萜类的代表化合物,对其在大鼠体内的药物动力学过程进行了初步研究。①建立了大鼠血浆中2-羟基-3-甲基蒽醌的HPLC-UV分析方法,以炔雌醇为内标,采用乙腈沉淀蛋白法处理血浆样品,流动相为甲醇-水(78:22,v/v),检测波长为265 nm。血浆样品中2-羟基-3-甲基蒽醌的浓度在0.10～4.0μg/mL范围内线性关系良好,方法的定量下限为0.10μg/mL,日内精密度RSD≤7.1%,日间精密度RSD≤9.3%,准确度RE%在-5.1～5.4%之间。利用所建立的HPLC-UV法对大鼠腹腔注射给予2-羟基-3-甲基蒽醌的药动学进行了研究。大鼠腹腔注射给予6 mg/kg2-羟基-3-甲基葸醌后的C_(max)为2.89μg/mL,药时曲线下面积AUC_(O-∞)为159μg·min·mL~(-1),表观消除速率常数K_e为0.0164 min~(-1),消除半衰期T_(1/2)为44.3 min。②建立了大鼠血浆中去乙酰基车叶草苷酸的HPLC-UV分析方法,以鸡屎藤次苷为内标,采用甲醇沉淀蛋白法处理血浆样品,流动相为甲醇-5 mM乙酸铵溶液(3:97,v/v,冰醋酸调节水相pH至3.7),检测波长为230 nm。血浆样品中去乙酰基车叶草苷酸的浓度在0.20～40μg/mL范围内线性关系良好,方法的定量下限为0.20μg/mL,日内精密度RSD≤6.6%,日间精密度RSD≤10.1%,准确度RE%在-2.5～0.75%之间。利用所建立的HPLC-UV法对大鼠尾静脉注射给予去乙酰基车叶草苷酸的药动学进行了研究。大鼠尾静脉注射给予4.2 mg/kg去乙酰基车叶草苷酸后的C_(max)为17.66μg/mL,药时曲线下面积AUC_(O-∞)为619μg·min·mL~(-1),表观消除速率常数K_e为0.0244min~(-1),消除半衰期T_(1/2)为28.6 min。
本论文综合运用多学科专业知识和多种分析手段,研究了白花蛇舌草药材的化学成分,考察了白花蛇舌草药材提取物和单体化合物在体外对肿瘤细胞的增殖抑制活性,建立了白花蛇舌草药材中葸醌类和环烯醚萜类成分的含量测定方法,初步探讨了2-羟基-3-甲基蒽醌和去乙酰基车叶草苷酸在大鼠体内的药物动力学过程,为白花蛇舌草抗肿瘤药效物质基础的阐明、质量控制方法的研究和药材的进一步开发、利用奠定了基础。
Hedyotis diffusa Willd.[Oldenlandia diffusa(Willd.) Roxb.],which belongs to the family of Rubiaceae,is well-known in Chinese folk medicine for the treatment of cancer, especially for the therapies against esophagus,stomach,liver and rectum malignant tumors. In this study,the chemical constituents,pharmacological activity in vitro,quality control methods and preliminary pharmacokinetics of Hedyotis diffusa were investigated by means described as follows.
The chemical constituents of 90%ethanol extract of Hedyotis diffusa were isolated by using multiple column chromatographic techniques,including silica gel column chromatography,reversed-phased preparative high performance liquid chromatography (RP-PHPLC),Polyamide column chromatography and Sephadex LH-20,and 17 compounds were obtained.The structures of 15 compounds were fully elucidated by chemical and spectroscopic methods(MS,UV,NMR).Among them,one is a new compound, 2,7-dihydroxy-3-methyl anthraquinone;one compound,3,4-dihydroxy methyl benzoate,was isolated from Hedyotis diffusa for the first time.
An HPLC-ESI/TOF-MS method in both positive and negative modes was developed and applied to study the chemical constituents of extract of Hedyotis diffusa.According to the retention time and fragmentation patterns of the four standard compounds,as well as the structure information obtained from the literatures,eight constituents were unambiguous/tentatively identified.The chemical names of the identified compounds are deacetylasperulosidic acid,deacetyl asperuloside,scandoside,10-acetyl scandoside, asperuloside,p-coumaric acid,E-6-O-p-coumaroyl scandoside methyl ester, Z-6-O-p-coumaroyl scandoside methyl ester.
Both the crude extract and eight compounds isolated from Hedyotis diffusa were tested for their anticancer activity in vitro by an MTT assay.The growth inhibition study showed that the crude extract of Hedyotis diffusa exerted a significant toxic effect against MCF-7, BEL-7402 and SGC-7901 cell lines.Compared with the other two cancer cell lines, SGC-7901 cells were more sensitive to the cytotoxic action of Hedyotis diffusa extract,which exhibited an IC_(50 value of 4.81μg/mL.Among the compounds tested,only 2,7-dihydroxy-3-methyl anthraquinone exhibited dose dependent growth inhibition effect and showed a IC50 values of 53.72μM,54.22μM and 84.40μM against MCF-7,BEL-7402 and SGC-7901 cancer cell lines,respectively.
Anthraquinones and iridoids are two major kinds of constituents in Hedyotis diffusa.An HPLC-UV method was developed for simultaneous determination of four anthraquinones in Hedyotis diffusa.An HPLC-DAD method using gradient elution was established for the simultaneous determination of p-coumaric acid and three iridoids in Hedyotis diffusa.These methods were applied to determine the amounts of these bioactive compounds in Hedyotis diffusa.The results indicated that the contents of those constituents in samples from different sources varied significantly.The methods established in this paper were specific,accurate, sensitive and were suitable for the quality control of Hedyotis diffusa.
2-Hydroxy-3-methyl anthraquinone and deacetylasperulosidic acid were selected respectively as the representative compounds of anthraquinone and iridoid constituents in Hedyotis diffusa for the in vivo pharmacokinetic study.An HPLC method coupled with UV detection was developed and validated for the determination of 2-hydroxy-3-methyl anthraquinone in rat plasma.Ethinylestradiol was chosen as the internal standard. Chromatographic separation was conducted on a Diamonsil C_(18) column(200 mm×4.6 mm,5μm),using a mixture of methanol-water(78:22,v/v) as mobile phase with detection wavelength at 265 nm.The calibration curve of 2-hydroxy-3-methyl anthraquinone was linear over the range of 0.10-4.0μg/mL in rat plasma.The lower limit of quantification (LLOQ) was 0.10μg/mL.The intra- and inter-day precisions(RSDs) were less than 7.1%and 9.3%,respectively.The intra-and inter-day accuracies(REs) were within-5.1-5.4%.The developed method was applied to the pharmacokinetic study of 2-hydroxy-3-methyl anthraquinone in rats after intraperitoneal administration.The pharmacokinetic parameters obtained after intraperitoneal administration of 6mg/kg 2-hydroxy-3-methyl anthraquinone were as follows:C_(max) 2.89μg/mL,area under concentration-time curve(A∪C_(0-∞)) 159μg·min/mL,apparent elimination rate constant(K_e) 0.0164 min~(-1),biological half life(T_(1/2)) 44.3min.An HPLC method coupled with UV detection was developed and validated for the determination of deacetylasperulosidic acid in rat plasma.Scandoside was chosen as the internal standard.Chromatographic separation was performed on a pinnacleⅡC_(18) column (250mm×4.6mm,5μm),using a mixture of methanol-5 mM ammonium acetate(3:97,v/v, pH adjusted to 3.7 with glacial acetic acid) as mobile phase with detection wavelength at 230 nm.The calibration curve of deacetylasperulosidic acid was linear over the range of 0.20-40μg/mL in rat plasma.The lower limit of quantification(LLOQ) was 0.20μg/mL.The intra and inter-day precisions(RSDs) were less than 6.6%and 10.1%,respectively.The intra- and inter-day accuracies(REs) were within-2.5-0.75%.The developed method was applied to the pharmacokinetic study of deacetylasperulosidic acid in rats after intravenous administration.The pharmacokinetic parameters obtained after intravenous administration of 4.2mg/kg deacetylasperulosidic acid were as follows:C_(max) 17.66μg/mL,A∪C_(0-∞) 619μg·min/mL,K_e 0.0244min~(-1),T_(1/2) 28.6min。
In conclusion,by utilizing various professional knowledge and analytical techniques,the chemical constituents of Hedyotis diffusa were investigated,the crude extract and some compounds isolated from Hedyotis diffusa were tested for their growth inhibition effect against cancer cell lines in vitro,the quantitative determination of anthraquinones and iridoids in Hedyotis diffusa were developed,and pharmacokinetic processes of some representative constituents had also been studied.This study provided useful information for the interpretation of antitumor activity-related substance,quality control,exploration and utilization of Hedyotis diffusa.
引文
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