皂角刺抗肿瘤活性成分与含量测定方法研究

【作者】 徐哲；

【导师】 毕开顺； 陈晓辉；

【作者基本信息】 沈阳药科大学 ， 中药学， 2007， 硕士

【摘要】 本研究以皂角刺为研究对象，用MTT法测定抗肿瘤活性，采用体外抗肿瘤活性跟踪与化学分离相结合的方法，确定了皂角刺的抗肿瘤活性成分，并以其为指标对皂角刺药材的质量进行控制。首先在确定皂角刺乙醇提取物具有抗肿瘤活性的前提下，依次用石油醚、氯仿、乙酸乙酯、正丁醇萃取，分别得到不同萃取层，抗肿瘤活性测定显示：70％乙醇提取物的乙酸乙酯萃取层对Bel-7402、Hela和KB细胞株的抑制作用较强，IC₅₀值分别为122.7μg·mL⁽-1_、118.3μg·mL^-1和145.9μg·mL^-1。因此确定皂角刺70％乙醇提取物的乙酸乙酯萃取层为抗肿瘤活性部位。取皂角刺9kg，用70％乙醇回流提取，乙酸乙酯萃取得到活性部位后，对其进行硅胶柱层析，以氯仿-甲醇（100∶0～0∶100）为洗脱溶剂，以TLC为指导合并相同流分，对得到的七个流分进行抗肿瘤活性试验。结果表明：流分Ⅱ、Ⅲ和Ⅳ对肿瘤细胞增殖有较好的抑制作用，其对Bel-7402和Hela细胞株的IC₅₀分别为80.2μg·mL^-1、76.5μg·mL^-1、76.3μg·mL^-1和69.2μg·mL^-1、79.9μg·mL^-1、88.8μg·mL^-1。进一步采用硅胶柱层析、Sephadex LH-20柱层析等多种手段对流分Ⅱ和Ⅳ的化学成分进行了分离、纯化和鉴定。从该活性部位中分离得到了8个化合物，利用理化性质和光谱分析方法将其分别鉴定为：黄颜木素（1）、槲皮素（2）、3β-acetoxyolean-12-en-28-oic acid （3）、木栓酮（4）、棕榈酸（5）、白桦醇（6）、β-谷甾醇（7）和胡萝卜苷（8）。其中化合物2、3、4、5、6和8均为首次从皂角刺中分离。采用MTT法对以上8个化合物进行抗肿瘤活性分析，结果表明：化合物1和3对多种体外培养的肿瘤细胞具有明显的抗肿瘤活性。其中化合物3对Bel-7402、Hela、HT1080、KB、A549、SGC-7901和Heps等7种肿瘤细胞株生长增殖具有良好的抑制作用，IC₅₀为11.6-18.7μg·mL^-1。化合物1对除A549之外的6种肿瘤细胞株抑制作用良好，IC₅₀为11.3-19.3μg·mL^-1。由此可以推测化合物1和3为皂角刺抗肿瘤作用的活性成分。本实验对皂角刺中黄颜木素和槲皮素提取工艺进行研究。采用正交试验设计，以黄颜木素和槲皮素的含量为考察指标，对提取溶剂用量、提取时间和提取次数3个因素进行优化，确定皂角刺中黄颜木素和槲皮素的最佳提取工艺为加15倍量70％乙醇，回流提取2次，每次1h。首次建立了同时测定皂角刺中黄颜木素和槲皮素的LC-MS分析方法。以Kromasil-C₁₈（250×4.6 mm i．d．，5μm）为色谱柱，乙腈-1％冰乙酸水溶液（30：70，v／v）为流动相，流速0.8 mL·min^-1，柱温35℃，采用ESI源，扫描方式为选择离子监测，检测方式为正离子检测，以黄豆苷元为内标，定量分析m／z 287（黄颜木素），m／z 303（槲皮素），m／z 255（黄豆苷元）三个离子，对所收集的17批不同产地的皂角刺药材中黄颜木素和槲皮素进行含量测定。结果表明黄颜木素和槲皮素线性范围分别是：黄颜木素，0.084-2.688μg·mL^-1，r=0.9990；槲皮素，0.84-26.88μg·mL^-1，r=0.9993。平均回收率（n=9）黄颜木素为97.7％，RSD为1.9％；槲皮素为98.5％，RSD为2.4％。该方法简便快速，重复性好，为今后皂角刺的进一步研究和开发利用奠定了基础。更多还原

【Abstract】 Gleditsia sinensis spine is the thorns of Gleditsia sinensis Lam., which is belonging to the pulse family. In modern research, the studies on Gleditsia sinensis Lam. are mostly about its chemical compositions other than its important anti-tumor efficacy. Now we know that, flavones, phenols and amino acids are the main components of it. However, the bioactivity components that attribute to its anti-tumor activity haven’t been well investigated up to now. Thus, we have undertaken an investigation on the anti-tumor constituents, as well as a development of the quality assessment of the plant.In order to find out active constituents from Gleditsia sinensis Lam. alcoholic extract, which has been found an anti-tumor capacity with, we dissolved it in water and fractionated it sequentially with petroleum ether, chloroform, ethyl acetate and n-butanol. Different fractions had variable cytotoxin effects on different tumor cell lines. Ethyl acetate part had remarkable anti-tumor activity in vitro, and its IC₅₀ against Bel-7402, Hela or KB cell lines were 122.67μg·mL^-1, 118.3μg·mL^-1 and 145.91μg·mL^-1, respectively. Chloroform part provided some cytotoxin effect on Bel-7402, Hela or KB cell lines, with the IC₅₀ of 189.66βg·mL^-1, 196.43μg·mL^-1 and 191.77μg·mL^-1. N-butanol part provided a little inhibitory effect on these cell lines. The water-soluble part had no effect on any cell lines in test. That is to say, ethyl acetate part is confirmed to be the anti-tumor active part of Gleditsia sinensis Lam. with considerable cytotoxin capacity.The thorns of Gleditsia sinensis Lam. （ 9 Kg ） were extracted with 70 % alcohol water solution into an alcoholic extract, which was extracted with ethyl acetate to afford an active ethyl acetate extract. The ethyl acetate fraction was chromatographed on silica gel and obtained seven subfractions. The anti-tumor tests showed that the subfractionⅡ,ⅢandⅣwere active subfractions of the ethyl acetate extract.Monitoring isolation guided by bioassay, the active subfractionⅡand subfractionⅣwere chromatographed on silica gel and Sephadex LH-20, respectively, giving out 8 compounds. They were elucidated on the basis of physi-chemical and spectral methods and identified as Fisetin （1）, Quercetin （2）, 3β-acetoxyolean-12-en-28-oic acid （3）, Friedelin （4）, palmitic acid （5）, Betulin （6）,β-sitosterol （7） and Daucosterol （8）. Among them, compounds 2, 3, 4, 5, 6 and 8 are isolated from the plant for the first time.The cytotoxic activities of 8 isolated compounds against seven tumor cell lines were evaluated by MTT assay. The results showed that compound 1 and 3 exhibited conspicuous cytotoxic activity. Compound 3 had significant cytotoxic activities against Bel cells, Hela cells, HT1080 cells, KB cells, A549 cells, SGC-7901 cells and Heps cells in a concentration-dependent manner, with IC₅₀ 11.6-18.7μg·mL^-1; Compound 1 had significant cytotoxic activities against all cells except A549 cells in a concentration-dependent manner, with IC₅₀ 11.3-19.3μg·mL^-1.The optimal extraction process of Gleditsia sinensis Lam. extract was studied by orthogonal design with the content of fisetin and quercetin as quality assessment index. Three factors were studied in this experiment, including the solvent consumption, duration of extraction and times of extraction. The result showed that the optimal extracting condition was 70% alcohol consumed fifteen times the amount of material, and twice for 1 hour each time.We established a method for the determination of fisetin and quercetin in the thorns of Gleditsia sinensis Lam. by LC-MS means for the first time. The contents of fisetin and quercetin in 17 batches of Gleditsia sinensis Lam. were analyzed. The chromatographic condition included a Hypersil-C₁₈ （250 x 4.6 mm i.d., 5μm ） and the mobile phase consisting of acetonitrile- water within 1% glacial acetic acid （ 30:70, v/v ）. The calibration curves were linear over the range of 0.084-2.688μg·mL^-1, r = 0.9990 and 0.84-26.88μg·mL^-1, r = 0.9993. The average recoveries of fisetin and quercetin were 97.7% （RSD = 1.9%） and 98.5%（RSD = 2.4 % ） respectively. The LC-MS assay was found to be simple and accurate to measure the concentrations of fisetin and quercetin in the thorns of Gleditsia sinensis Lam.更多还原