新疆分心木体外抗肿瘤作用及其化学成分的研究

发布时间：2018-04-10 13:24

  本文选题：分心木 + 体外抗肿瘤　； 参考：《新疆医科大学》2016年硕士论文

【摘要】：目的:1.研究分心木不同提取物的体外抗肿瘤活性,筛选出体外抗肿瘤作用的活性部位。2.对活性部位的化学成分进行系统研究。3.优化分心木总皂苷的提取纯化工艺。4.探究分心木总皂苷、总黄酮和总多糖的体外抗氧化活性。方法:1.95%乙醇回流提取分心木,收集提取液旋转蒸发成浸膏后,依次用石油醚、氯仿、乙酸乙酯、水饱和正丁醇对乙醇提取物进行萃取;采用MTT法测定分心木各提取物对人肝癌细胞7404、人非小细胞肺癌细胞A549及人结肠癌细胞Hct116、Caco-2增殖的影响,初步筛选出体外抗肿瘤的活性部位;流式细胞仪测定分心木活性部位对4种癌细胞凋亡的影响,进一步确认其活性部位。2.采用溶剂萃取、大孔吸附树脂、硅胶柱层析、反相硅胶柱及重结晶等方法对活性部位的化学成分进行分离和纯化,根据理化性质,波谱学数据以及文献对照确定所得化合物的结构。3.以齐墩果酸为对照品,利用紫外可见分光光度法测定分心木中总皂苷的含量;利用单因素实验结果,设计正交实验,研究分心木总皂苷最佳提取工艺;利用大孔吸附树脂对分心木总皂苷进行纯化工艺的研究。4.以VC作为阳性对照,检测分心木中总皂苷、总黄酮和总多糖对超氧阴离子(-O2)、羟基自由基(-OH)、DPPH自由基的清除能力及其铁还原能力测定。结果:1.分心木不同提取物对4种癌细胞的增殖均具有一定的抑制作用,但分心木乙酸乙酯和正丁醇提取物对4种癌细胞增殖的抑制作用最强,初步判断乙酸乙酯和正丁醇提取物为分心木体外抗肿瘤活性的主要活性部位。通过流式细胞仪测定乙酸乙酯和正丁醇提取物对4种癌细胞凋亡的影响,在不同浓度下两种提取物诱导4种癌细胞凋亡,其给药浓度在400μg/mL时,4种癌细胞的凋亡率都大于50%。2.对体外抗肿瘤活性部位(正丁醇部位)进行分离纯化,从中分离出3个化合物,分别为:对苯二甲酸二甲酯(I)、槲皮苷(II)、Jugnaphthalenoside A(III)。3.总皂苷最佳提取工艺为25倍量的80%乙醇,80℃回流提取2次,每次2 h;最佳纯化工艺为采用D101大孔吸附树脂,最大上样量为180 mL,水洗脱量为8BV,洗脱剂为50%乙醇,洗脱体积为7BV,总皂苷纯度由27.4%上升到50.8%,总皂苷的洗脱率可达到82.4%。4.体外抗氧化实验结果表明总皂苷、总黄酮和总多糖均具有较好的对DPPH自由基,羟基自由基,超氧阴离子自由基清除能力和铁还原能力,并在一定的质量浓度范围内成量效关系。结论:1.乙酸乙酯和正丁醇提取物是分心木发挥体外抗肿瘤作用的主要活性部位。2.本研究首次从分心木正丁醇提取物中分离得到化合物I和III。3.实验表明优选出的提取纯化工艺稳定可行,适用分心木总皂苷的提取纯化。4.分心木总皂苷、总黄酮及总多糖具有体外抗氧化活性。
[Abstract]:Purpose 1.To study the antitumor activity of different extracts of distraction tree in vitro, and to screen out the active site of anti-tumor effect. 2. 2.The chemical constituents of active parts were systematically studied.Optimization of extraction and purification process of total saponins. 4.To explore the anti-oxidation activity of total saponins, flavonoids and polysaccharides of distraction wood in vitro.Methods 1. 95% ethanol was refluxed to extract distraction wood. The extract was then extracted by petroleum ether, chloroform, ethyl acetate and water saturated n-butanol.The proliferation of human hepatoma cell line (7404), human non-small cell lung cancer cell line A549 and human colon cancer cell line Hct116Caco-2 was determined by MTT assay.Flow cytometry was used to determine the effect of the active site of distraction tree on apoptosis of four kinds of cancer cells, and to confirm the active site. 2. 2.The active parts were separated and purified by solvent extraction, macroporous adsorption resin, silica gel column chromatography, reverse phase silica gel column and recrystallization.The structure of the compound was determined by spectroscopic data and literature comparison.The content of total saponins in distraction wood was determined by ultraviolet visible spectrophotometry with oleanolic acid as reference substance, and the optimum extraction process of total saponins was studied by orthogonal experiment.Study on purification of total saponins by macroporous resin. 4.The scavenging ability of total saponins, flavonoids and polysaccharides in distraction trees against superoxide anion (O _ 2), hydroxyl radical (OH) -OHH _ (2) and their iron reduction ability were determined with VC as a positive control.The result is 1: 1.Different extracts of distraction tree had certain inhibitory effects on the proliferation of four kinds of cancer cells, but ethyl acetate and n-butanol extracts had the strongest inhibitory effect on the proliferation of four kinds of cancer cells.It was preliminarily determined that ethyl acetate and n-butanol extract were the main active sites of antitumor activity in vitro.The effects of ethyl acetate and n-butanol extract on the apoptosis of four kinds of cancer cells were determined by flow cytometry. The apoptotic rates of four kinds of cancer cells were higher than 50. 2 at the concentration of 400 渭 g/mL.The antitumor active site (n-butanol) was isolated and purified in vitro. Three compounds were isolated, I. E. dimethyl terephthalate, Quercetin II, Jugnaphthalenoside Ajii. 3.The optimum extraction process of total saponins was 25 times of 80% ethanol at 80 鈩，

本文编号：1731377