维药意大利牛舌草抗补体活性成分的分离与定性定量研究
发布时间：2018-12-29 14:50
【摘要】：目的:研究意大利牛舌草抗补体的活性成分,并对其进行了初步的质量研究,为寻找天然来源的高效低毒的补体抑制剂奠定物质基础以及该药材进一步的开发利用提供科学依据。方法:采用体外细胞溶血试验,以补体经典途径的溶血活性(CH50)为指标考察了意大利牛舌草醇提物的不同萃取部位对补体系统的抑制作用,确定了活性部位;利用硅胶柱色谱、Sephadex LH-20色谱、ODS等柱色谱和半制备HPLC技术,从意大利牛舌草乙酸乙酯萃取部位分离得到10个单体化合物,根据理化性质和1H-NMR、13C-NMR等波谱学数据进行结构鉴定;以补体经典途径(CH50)和旁路途径(AP50)的溶血活性为指标,测定单体成分的活性;采用UPLC-PDA法,色谱柱为ACQUITY UPLC BEH C18(100mm × 2.1 mm,1.7 μm);流动相为甲醇(A)-0.4%甲酸(B);洗脱程序(0-3 min,5-31%A;3-10 min,31%-37%A;10-12 min,37%-40%A;12-30 min,40%-40%A),柱温30℃;检测波长366 nm,测定意大利牛舌草中芦丁和山奈酚的含量。结果:(1)体外细胞溶血试验结果表明意大利牛舌草醇提物对补体系统的经典途径的激活有较强的抑制作用,进一步对醇提物进行萃取,所得到的乙酸乙酯萃取部位和正丁醇萃取部位的活性均较强,而石油醚萃取部位较弱,水提取物无抗补体活性。因此,确定乙酸乙酯萃取部位和正丁醇萃取部位为意大利牛舌草的抗补体活性部位。(2)以抗补体活性为导向,采用多种色谱分离方法,结合波谱分析技术从乙酸乙酯部位活性较强的4个组分中分离得到了 10个单体化合物,鉴定出8个化合物的结构。(3)单体化合物的抗补体活性测试结果表明,山奈酚、芦丁、2,6,10,14-tetramethyl-18-butanecarboxymethylene-henecos-12-en-17β-ol和苯甲酸壬酯对补体系统显示了不同程度的抑制活性(CH500.085～1.23 mg/mL;AP50 0.45～0.96 mg/mL);而邻苯二甲酸二丁酯、邻苯二甲酸二(2-甲基)丙酯和邻苯二甲酸二(2-乙基)癸酯对补体系统的抑制作用较弱;2-糠酸则无补体抑制作用。(4)意大利牛舌草中芦丁和山奈酚的含量分别在1.25～3.34 mg/g和2.86～6.05 mg/g范围内,平均回收率分别为100.09%和97.78%,精密度、重复性、稳定性的RSD均小于2.0%。结论:得到的8个化合物中,邻苯二甲酸二(2-乙基)癸酯为首次从该植物中分离得到,2-糠酸、2,6,10,14-tetramethyl-18-butanecarboxymethylene-henecos-12-en-17β-ol和苯甲酸壬酯均为首次从本属植物中分离得到。萜类化合物 2,6,10,14-tetramethyl-18-butanecarboxy-methylene-henecos-12-en-17β-ol的体外抗补体活性最好,接近于阳性药肝素钠,其次是2个黄酮类化合物芦丁和山奈酚,这为寻找天然来源的新型补体抑制剂奠定了基础。2个主要活性成分芦丁和山奈酚的含量同地区之间差异较小,不同地区之间存在差异,为进一步完善该药材的质量标准提供参考。
[Abstract]:Objective: to study the anti-complement active components of Tauricus Italia, and to study its quality. It provides a scientific basis for searching for natural sources of high efficiency and low toxicity complement inhibitors and for the further development and utilization of this medicine. Methods: in vitro cell hemolysis test was used to investigate the inhibitory effect of different extraction sites of ethanol extract of Tauricus Italia on complement system using the hemolytic activity (CH50) of the classical pathway of complement as the index, and the active sites were determined. Ten monomers were isolated by silica gel column chromatography, Sephadex LH-20 chromatography, ODS column chromatography and semi-preparative HPLC. 13C-NMR isospectral data were used to identify the structure. The hemolytic activity of monomers was determined by using the hemolytic activity of the classical complement pathway (CH50) and the bypass pathway (AP50) as the index, and the ACQUITY UPLC BEH C18 (100mm 脳 2.1 mm,1.7 渭 m);) column was used for the determination of the activity of the monomers by UPLC-PDA. Mobile phase methanol (A) 0.4% formic acid (B); elution process (0-3 min,5-31%A;3-10 min,31%-37%A;10-12 min,37%-40%A;) The column temperature was 30 鈩，
本文编号：2394975
[Abstract]:Objective: to study the anti-complement active components of Tauricus Italia, and to study its quality. It provides a scientific basis for searching for natural sources of high efficiency and low toxicity complement inhibitors and for the further development and utilization of this medicine. Methods: in vitro cell hemolysis test was used to investigate the inhibitory effect of different extraction sites of ethanol extract of Tauricus Italia on complement system using the hemolytic activity (CH50) of the classical pathway of complement as the index, and the active sites were determined. Ten monomers were isolated by silica gel column chromatography, Sephadex LH-20 chromatography, ODS column chromatography and semi-preparative HPLC. 13C-NMR isospectral data were used to identify the structure. The hemolytic activity of monomers was determined by using the hemolytic activity of the classical complement pathway (CH50) and the bypass pathway (AP50) as the index, and the ACQUITY UPLC BEH C18 (100mm 脳 2.1 mm,1.7 渭 m);) column was used for the determination of the activity of the monomers by UPLC-PDA. Mobile phase methanol (A) 0.4% formic acid (B); elution process (0-3 min,5-31%A;3-10 min,31%-37%A;10-12 min,37%-40%A;) The column temperature was 30 鈩，
本文编号：2394975
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