Gypenoside L和gypenoside LI的制備及其對不同癌細(xì)胞毒性篩選與作用機制
發(fā)布時間：2019-07-09 14:31
【摘要】：目的Gypenoside L和gypenoside LI是壯藥國蝦薄(絞股藍(lán))中的皂苷類化合物。本論文旨在制備gypenoside L和gypenoside LI這兩個化合物,篩選二者對不同癌細(xì)胞的抗癌活性,并研究其對癌細(xì)胞的作用機制。方法1.用液-液萃取、硅膠柱色譜、快速制備色譜和半制備HPLC等方法對熱處理國蝦薄總提物進(jìn)行分離、純化,用UV、ESI-MS、NMR等數(shù)據(jù)對所分離得到的化合物進(jìn)行結(jié)構(gòu)鑒定。2.CCK 8法檢測絞股藍(lán)皂苷gypenoside L和gypenoside LI對人非小細(xì)胞肺癌A549細(xì)胞、人肝癌HepG2細(xì)胞、人食管癌EC-109細(xì)胞以及人前列腺癌PC-3細(xì)胞增殖的抑制作用,計算其半數(shù)抑制濃度IC50值,比較不同構(gòu)型的同分異構(gòu)體gypenoside L和gypenoside LI對癌細(xì)胞的影響。3.CCK 8 法檢測 gypenoside L 和 gypenoside LI 對 A549 細(xì)胞增殖抑制活性與作用時間及作用劑量的關(guān)系;比較gypenoside L和gypenoside LI與陽性對照藥人參皂苷Rg3對A549細(xì)胞增殖的抑制作用。4.用 PI/RNase 染色、Annexin V-FITC/PI雙染、JC-1 及 DCFH-DA染色法及流式細(xì)胞術(shù)檢測gypenoside L和gypenoside LI對A549細(xì)胞周期時相分布、細(xì)胞凋亡的影響。5.通過劃痕實驗和 Transwell 檢測 gypenoside L 和 gypenoside LI對A549細(xì)胞遷移和侵襲能力的影響。6.Western Blot方法從蛋白水平上驗證gypenoside L和gypenoside LI對A549細(xì)胞凋亡及遷移、侵襲的影響。結(jié)果1.從熱處理國蝦薄80%乙醇總提物中分離得到絞股藍(lán)皂苷gypenoside L(2.2 g)和 gypenoside LI(0.9 g),純度達(dá)到 98%以上。2.Gypenoside L 和 gypenoside LI 對 A549 細(xì)胞、HepG2 細(xì)胞、EC-109細(xì)胞以及PC-3細(xì)胞增殖均呈現(xiàn)濃度依賴性抑制關(guān)系。作用24 h 時,gypenoside L 的 IC50值依次為 21.09 ± 3.60、28.31 ± 1.48、78.70士 3.06 和 96.16 ± 2.43 μg/mL,gypenoside LI 的 IC50 值依次為 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對A549細(xì)胞增殖抑制作用呈作用劑量及作用時間依賴關(guān)系;且在作用時間為24 h,濃度為30 μg/mL時,gypenoside L和gypenoside LI比人參皂苷Rg3對A549增殖的抑制率要高。4.經(jīng) 24 μg/mL gypenoside L 處理后,G0/G1 期 A549 細(xì)胞的比例由(66.42 ± 0.15)%升高至(76.23 ± 1.41)%;A549 細(xì)胞凋亡率由(0.57 ± 0.18)%增加到(24.62 土 0.70)%;線粒體膜電勢去極化明顯,紅綠熒光相對比例由(15.44 ± 2.29)%下降到(4.09 ± 0.23)%;細(xì)胞內(nèi)活性氧濃度明顯增加,平均熒光強度由14.3 ± 0.14增加到142.5± 4.03。用 17 μg/mL gypenoside LI 處理后,G2/M 期細(xì)胞比例由(6.9± 0.32)%升高至(25.83 ± 1.63)%。;A549 細(xì)胞凋亡率由(0.57 ±0.18)%增加到(22.44 ± 0.71)%;線粒體膜電勢去極化明顯,紅綠熒光相對比例由(15.44 ± 2.29)%下降到(4.43 土 0.02)%;細(xì)胞內(nèi)活性氧濃度明顯增加,平均熒光強度由14.3 ± 0.14增加到152.0 ±5.59。5·用 20 μg/mL gypenoside L 和 15 μg/mL gypenoside LI 處理后A549細(xì)胞的劃痕愈合率分別比對照組降低了 30.45%和19.87%,細(xì)胞侵襲率分別比對照組降低了 39.03%和51.22%。6.經(jīng)低、中、高劑量組的gypenosideL和gypenoside LI處理后,A549細(xì)胞內(nèi)IL-24蛋白表達(dá)呈上升趨勢,MMP-2和MMP-9蛋白表達(dá)明顯下降。結(jié)論1.從國蝦薄熱處理產(chǎn)物中得到大量高純度的C20位為R構(gòu)型的gypenoside L 和 C20 位為 S 構(gòu)型的 gypenoside LI。2.通過CCK 8法對人的非小細(xì)胞肺癌A549、肝癌HepG2、食管癌EC-109、前列腺癌PC-3細(xì)胞進(jìn)行活性篩選,發(fā)現(xiàn)gypenosideL和gypenoside LI對A549細(xì)胞和HepG2細(xì)胞的抑制作用較強,而對PC-3細(xì)胞則表現(xiàn)出較弱的抑制作用;Gypenoside LI對EC-109細(xì)胞的抑制活性比gypenoside L大。3.Gypenoside L和gypenoside LI對A549細(xì)胞的可能作用機制如下:① Gypenoside L 將 A549 細(xì)胞阻滯于 G0/G1 期,而 gypenoside LI將A549細(xì)胞阻滯于G2/M期。②二者均能降低A549細(xì)胞線粒體膜電位,增加細(xì)胞內(nèi)活性氧的濃度,說明gypenoside L和gypenoside LI可能通過線粒體信號介導(dǎo)的途徑誘導(dǎo)A549細(xì)胞凋亡。③通過降低劃痕愈合率、侵襲率及下調(diào)MMP-2和MMP-9蛋白表達(dá)說明gypenoside L和gypenoside LI能夠抑制A549細(xì)胞的遷移和侵襲。④二者均能上調(diào)A549細(xì)胞IL-24蛋白的表達(dá),說明二者可能通過IL-24相關(guān)途徑抑制A549細(xì)胞增殖、誘導(dǎo)細(xì)胞凋亡、抑制細(xì)胞遷移及侵襲。
[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.
【學(xué)位授予單位】：中央民族大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2017
【分類號】：R29
本文編號：2512211
[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.
【學(xué)位授予單位】：中央民族大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2017
【分類號】：R29
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