Gypenoside L和gypenoside LI的制备及其对不同癌细胞毒性筛选与作用机制

发布时间：2019-07-09 14:31

【摘要】：目的Gypenoside L和gypenoside LI是壮药国虾薄(绞股蓝)中的皂苷类化合物。本论文旨在制备gypenoside L和gypenoside LI这两个化合物,筛选二者对不同癌细胞的抗癌活性,并研究其对癌细胞的作用机制。方法1.用液-液萃取、硅胶柱色谱、快速制备色谱和半制备HPLC等方法对热处理国虾薄总提物进行分离、纯化,用UV、ESI-MS、NMR等数据对所分离得到的化合物进行结构鉴定。2.CCK 8法检测绞股蓝皂苷gypenoside L和gypenoside LI对人非小细胞肺癌A549细胞、人肝癌HepG2细胞、人食管癌EC-109细胞以及人前列腺癌PC-3细胞增殖的抑制作用,计算其半数抑制浓度IC50值,比较不同构型的同分异构体gypenoside L和gypenoside LI对癌细胞的影响。3.CCK 8 法检测 gypenoside L 和 gypenoside LI 对 A549 细胞增殖抑制活性与作用时间及作用剂量的关系;比较gypenoside L和gypenoside LI与阳性对照药人参皂苷Rg3对A549细胞增殖的抑制作用。4.用 PI/RNase 染色、Annexin V-FITC/PI双染、JC-1 及 DCFH-DA染色法及流式细胞术检测gypenoside L和gypenoside LI对A549细胞周期时相分布、细胞凋亡的影响。5.通过划痕实验和 Transwell 检测 gypenoside L 和 gypenoside LI对A549细胞迁移和侵袭能力的影响。6.Western Blot方法从蛋白水平上验证gypenoside L和gypenoside LI对A549细胞凋亡及迁移、侵袭的影响。结果1.从热处理国虾薄80%乙醇总提物中分离得到绞股蓝皂苷gypenoside L(2.2 g)和 gypenoside LI(0.9 g),纯度达到 98%以上。2.Gypenoside L 和 gypenoside LI 对 A549 细胞、HepG2 细胞、EC-109细胞以及PC-3细胞增殖均呈现浓度依赖性抑制关系。作用24 h 时,gypenoside L 的 IC50值依次为 21.09 ± 3.60、28.31 ± 1.48、78.70士 3.06 和 96.16 ± 2.43 μg/mL,gypenoside LI 的 IC50 值依次为 17.09 ±0.63、17.70 ±1.15、19.05 ±2.82 和 46.03 ±6.54 μg/mL。3.Gypenoside L和gypenoside LI对A549细胞增殖抑制作用呈作用剂量及作用时间依赖关系;且在作用时间为24 h,浓度为30 μg/mL时,gypenoside L和gypenoside LI比人参皂苷Rg3对A549增殖的抑制率要高。4.经 24 μg/mL gypenoside L 处理后,G0/G1 期 A549 细胞的比例由(66.42 ± 0.15)%升高至(76.23 ± 1.41)%;A549 细胞凋亡率由(0.57 ± 0.18)%增加到(24.62 土 0.70)%;线粒体膜电势去极化明显,红绿荧光相对比例由(15.44 ± 2.29)%下降到(4.09 ± 0.23)%;细胞内活性氧浓度明显增加,平均荧光强度由14.3 ± 0.14增加到142.5± 4.03。用 17 μg/mL gypenoside LI 处理后,G2/M 期细胞比例由(6.9± 0.32)%升高至(25.83 ± 1.63)%。;A549 细胞凋亡率由(0.57 ±0.18)%增加到(22.44 ± 0.71)%;线粒体膜电势去极化明显,红绿荧光相对比例由(15.44 ± 2.29)%下降到(4.43 土 0.02)%;细胞内活性氧浓度明显增加,平均荧光强度由14.3 ± 0.14增加到152.0 ±5.59。5·用 20 μg/mL gypenoside L 和 15 μg/mL gypenoside LI 处理后A549细胞的划痕愈合率分别比对照组降低了 30.45%和19.87%,细胞侵袭率分别比对照组降低了 39.03%和51.22%。6.经低、中、高剂量组的gypenosideL和gypenoside LI处理后,A549细胞内IL-24蛋白表达呈上升趋势,MMP-2和MMP-9蛋白表达明显下降。结论1.从国虾薄热处理产物中得到大量高纯度的C20位为R构型的gypenoside L 和 C20 位为 S 构型的 gypenoside LI。2.通过CCK 8法对人的非小细胞肺癌A549、肝癌HepG2、食管癌EC-109、前列腺癌PC-3细胞进行活性筛选,发现gypenosideL和gypenoside LI对A549细胞和HepG2细胞的抑制作用较强,而对PC-3细胞则表现出较弱的抑制作用;Gypenoside LI对EC-109细胞的抑制活性比gypenoside L大。3.Gypenoside L和gypenoside LI对A549细胞的可能作用机制如下:① Gypenoside L 将 A549 细胞阻滞于 G0/G1 期,而 gypenoside LI将A549细胞阻滞于G2/M期。②二者均能降低A549细胞线粒体膜电位,增加细胞内活性氧的浓度,说明gypenoside L和gypenoside LI可能通过线粒体信号介导的途径诱导A549细胞凋亡。③通过降低划痕愈合率、侵袭率及下调MMP-2和MMP-9蛋白表达说明gypenoside L和gypenoside LI能够抑制A549细胞的迁移和侵袭。④二者均能上调A549细胞IL-24蛋白的表达,说明二者可能通过IL-24相关途径抑制A549细胞增殖、诱导细胞凋亡、抑制细胞迁移及侵袭。
[Abstract]:The purpose of this study is that Gypenoside L and Gypenoside LI are soap-like compounds in the shrimp thin (Gynostemma pentaphyllum) of the Zhuang nationality. The purpose of this study was to prepare the two compounds of Gypenoside L and Gypenoside LI, to screen the anti-cancer activity of the two compounds against different cancer cells and to study its mechanism of action on cancer cells. Method 1. The method comprises the following steps of: carrying out separation and purification on the thin total extract of the heat-treated Chinese shrimp by liquid-liquid extraction, silica gel column chromatography, rapid preparation chromatography and semi-preparative HPLC and the like, and using UV and ESI-MS, 2. The inhibition of the proliferation of human non-small cell lung cancer A549 cells, human liver cancer HepG2 cells, human esophageal cancer EC-109 cells and human prostate cancer PC-3 cells was detected by means of the CCK 8 method. The effect of gynoside L and Gypenoside LI on the proliferation of A549 cells was measured by means of the CCK 8 method, and the relationship between the inhibitory activity and the time of action and the action dose of the cell line A549 cells was detected by the CCK 8 method. The effect of Gypenoside L and gypenoside LI and positive control drug, ginseng soap, Rg3, on the proliferation of A549 cells was compared. The cell cycle phase distribution and apoptosis were measured by PI/ RNase staining, Annexin V-FITC/ PI double staining, JC-1 and DCFH-DA staining and flow cytometry. The effects of Gypenoside L and Gypenoside LI on the cell migration and invasion of A549 cells were measured by scratch test and Transwell.6. Western Blot method was used to verify the effect of gynoside L and gynoside LI on the apoptosis and migration and invasion of A549 cells. Results 1. The concentration-dependent inhibition relationship of gynostemma pentaphylla soap, gypenoside L (2.2 g) and gynoside LI (0.9 g) was obtained from the 80% ethanol total extract of the heat-treated Chinese shrimp. The purity of the gynostemma pentaphyllum L (2.2 g) and the gynoside LI (0.9 g) was over 98%. The IC50 values of Gypenoside L were 21.09, 3.60, 28.31, 1.48, 78.70, 3.06 and 96.16, 2.43 & mu; g/ mL, respectively. The IC50 values of gynoside LI were 17.09-0.63, 17.70-1.15, 19.05-2.82 and 46.03-6.54. mu.g/ mL. At the concentration of 30. m u.g/ mL, the inhibition rate of gynoside L and gynoside LI on the proliferation of A549 was higher than that of ginseng soap and Rg3. The percentage of A549 cells in G0/ G1 phase increased from (66.42-0.15)% to (76.23-1.41)%, and the apoptotic rate of A549 cells increased from (0.57-0.18)% to (24.62-0.70)% after treatment with 24. m u.g/ mL of Gypenoside L. The membrane potential of the mitochondria was significant. The relative proportion of red and green fluorescence decreased from 15.44 (2.29)% to (4.09-0.23)%; the concentration of active oxygen in the cells increased significantly, and the average fluorescence intensity increased from 14.3 to 0.14 to 142.5-4.03. The ratio of G2/ M cells increased from (6.9% 0.32)% to (25.83% 1.63)% after treatment with 17. m u.g/ mL of the Gypenoside LI. The apoptosis rate of A549 cells was increased from (0.57-0.18)% to (22.44-0.71)%; the membrane potential of the mitochondria was significant, the relative proportion of red-green fluorescence decreased from (15.44-2.29)% to (4.43-0.02)%, and the concentration of active oxygen in the cells increased significantly. The average fluorescence intensity increased from 14.3 to 0.14 to 152.0 to 5.59.5. The healing rate of the scratch was 30.45% and 19.87%, respectively, compared with the control group, and the cell invasion rate was 39.03% and 51.22%, respectively, compared with the control group. The expression of IL-24 in A549 cells was on the rise, and the expression of MMP-2 and MMP-9 in A549 cells decreased significantly. Conclusion 1. A large number of high-purity C20-bits of the prehenoside L and C20-bits of the S-configuration were obtained from the heat-treated product of the shrimp. By using CCK 8 method, the activity of human non-small cell lung cancer A549, HepG2, EC-109 and PC-3 cells was selected. The inhibitory activity of Gypenoside LI on the EC-109 cells is greater than that of the Gypenoside L.3. The possible mechanism of the Gypenoside L and the Gypenoside LI on the A549 cells is as follows: The human A549 cells are blocked in the G0/ G1 phase, while the gynoside LI blocks the A549 cells in the G2/ M phase. Both of them can reduce the mitochondrial membrane potential of A549 cells and increase the concentration of active oxygen in the cells. The expression of MMP-2 and MMP-9 could inhibit the migration and invasion of A549 cells by reducing the healing rate of the scratch, the invasion rate, and the expression of MMP-2 and MMP-9. Both of them can increase the expression of IL-24 in A549 cells, which suggests that they can inhibit the proliferation of A549 cells through IL-24-related pathway, induce cell apoptosis, and inhibit cell migration and invasion.
【学位授予单位】：中央民族大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：R29

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