蓝靛果花色苷提取及其抗肿瘤功能机理的初步研究
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摘要
花色苷具有消除自由基、抗衰老、抗异变、抗炎症、改善视力、预防和治疗心血管疾病、抗癌、提高免疫力等多种生理活性功能。从蓝靛果(Lonicera edulis Turcz.)中提取花色苷并对其生理活性功能进行研究,对蓝靛果的进一步开发和利用具有十分重要的实际意义。本论文利用蓝靛果为原料,对蓝靛果花色苷的提取条件进行研究,并对蓝靛果花色苷进行分离纯化,对蓝靛果花色苷的抗氧化活性和抗肿瘤功能机理进行了研究。
采用溶剂浸提法提取蓝靛果中具有抗氧化活性的花色苷,通过正交试验确定其最佳提取工艺条件。分别采用紫外分光光度法、H202/Fe体系和DPPH法测定花色苷的提取量及其抗氧化活性。结果表明：各因素对蓝靛果花色苷体外抗氧化作用影响程度的大小关系为：提取剂浓度>料液比>提取温度>提取时间。提取溶剂乙醇浓度为70%,料液比1:15(W:V),提取温度60℃，提取时间90min, pH值2,提取3次为最佳提取工艺参数。
将不同浓度的蓝靛果花色苷作用于人结肠癌HT29、人肝癌HepG2和人肉瘤U-20S三种肿瘤细胞24 h、48h和72h,采用四甲基偶氮唑盐(MTT)比色法检测细胞增殖。结果表明蓝靛果花色苷对三种肿瘤细胞都具有抑制作用,且呈现量效和时效依赖关系,即随着花色苷浓度的增大,作用时间的延长,抑制率显著增加。
不同浓度蓝靛果花色苷作用结肠癌HT29细胞48h,用Annexin V-FITC/PI流式细胞术检测细胞凋亡。结果表明随着花色苷浓度的增大,各组细胞凋亡数随之增大。加药组(0.25、0.5、1.0、2.0mg/mL)的凋亡率分别是(10.11±1.84)%、(28.87±2.32)%、(54.74_+±4.69)%、(73.56+±8.34)%,与对照组相比,加药组的凋亡率差异有统计学意义(P<0.05)。结肠癌HT29细胞分别出现了细胞数量明显减少,体积缩小,皱缩变形,细胞表面有许多的球状突起,进一步有凋亡小体产生,细胞间连接逐渐断裂,细胞浆出现较多空泡,胞浆浓缩,核物质致密,呈斑块状,或呈新月形边集于核膜,核形不规整等典型的凋亡细胞形态学特征。DNA凝胶电泳结果可以看到细胞凋亡的梯形条带,单细胞凝胶电泳显示细胞出现明显的拖尾现象。线粒体膜电位水平逐渐下降,且显著低于正常细胞水平；Caspase-3酶活性随着花色苷浓度的增加而升高,且与对照组细胞酶活性相比,差异性显著(P<0.05)。
综上所述,蓝靛果花色苷可能通过诱导肿瘤细胞凋亡来显著抑制肿瘤细胞的增殖；蓝靛果花色苷诱导肿瘤细胞凋亡的机制可能与线粒体介导的细胞凋亡通路有关。
Anthocyanins has many kinds of physiological activity function, such as elimination free radical, resist caducity and variation, resisting inflammation, improvement eyesight, prevention and treatment of cardiovascular diseases, cancer prevention, enhancement immunity. Study the physiological activity function of anthocyanins from Lonicera edulis (Lonicera edulis Turcz.) has very important practical significance.This paper study the extraction conditions, the oxidation resistance and antitumor function mechanism of anthocyanins after isolation and purification from Lonicera edulis.
The optimum condition of extract anthocyanins from Lonicera edulis was discussed by the orthogonal experiment through solvent digesting. The UV spectrophotometer was used to determine the content of anthocyanin of extraction, while H2O2/Fe system and DPPH assay method was adopted to determine the antioxidative capacity of extraction. The results indicated that solvent showed the remarkable effect on antioxidant of anthocyanin from Lonicera edulis, while the ratio of material to liquid, extraction temperature and time showed minor effects. Optimum extraction processing parameter was determined as follow:ethanol concentration was 70%, ratio of material to liquid was 1:15(W:V), temperature was 60℃, time was 90min, pH value was 2, extraction times was third.
Anthocyanins from Lonicera edulis of different concentrations was administered to human colon HT29 Cells, lier cancer HepG2 cells and U-2OS cells of human osteosarcoma for 24h,48h and 72h, respectively. The proliferation of cells was measured by MTT assay. The results showed that anthocyanins from Lonicera edulis can inhibit three types of tumor cells showed dose-dependent and time-dependent. The inhibition rate increased significantly with the increased of the concentration of anthocyanins and treatment time.
HT29 cells were treated with anthocyanins in different concentration for 48 h, flow cytometry with annexin V FITC/PI double staining was used to detect the apoptosis of the cells. Results:The number of apoptotic cells increases with the increase of anthocyanin concentration. The apoptosis rate of the groups (0.25,0.5,1,2 mg/mL) was (10.11±1.84)%, (28.87±2.32)%, (54.74±4.69)%(73.56±8.34)%. Compared with the control group, the apoptosis rate of the drug groups were significantly (P<0.05).Many typical apoptotic morphology were observed, such as:the number of cells decreased, the volume shrunk, many spherules heaved out cells surface, apoptotic bodies appear, connections between cells gradually fracture, the vacuole inside plasm obviously increased, cytoplasm condense, dense nuclear matter was patchy or crescent-shaped on boundary of the nuclear membrane, nuclear shape was irregular. A typical DNA degradation associated with apoptosis was determined by DNA agarose electrophoresis. Single-celled gel electrophoresis showed cells visible delay phenomenon. Mitochondrial membrane potential was decreased and significantly lower than normal cellular level; Caspase-3 activity was increased with the increase of anthocyanins concentration and the difference was significantly compared to control cells(P<0.05).
The results suggest that anthocyanins from Lonicera edulis inhibit cancer cells proliferation maybe was related with apoptosis through the mitochondrial apoptotic signaling pathway.
采用溶剂浸提法提取蓝靛果中具有抗氧化活性的花色苷,通过正交试验确定其最佳提取工艺条件。分别采用紫外分光光度法、H202/Fe体系和DPPH法测定花色苷的提取量及其抗氧化活性。结果表明：各因素对蓝靛果花色苷体外抗氧化作用影响程度的大小关系为：提取剂浓度>料液比>提取温度>提取时间。提取溶剂乙醇浓度为70%,料液比1:15(W:V),提取温度60℃，提取时间90min, pH值2,提取3次为最佳提取工艺参数。
将不同浓度的蓝靛果花色苷作用于人结肠癌HT29、人肝癌HepG2和人肉瘤U-20S三种肿瘤细胞24 h、48h和72h,采用四甲基偶氮唑盐(MTT)比色法检测细胞增殖。结果表明蓝靛果花色苷对三种肿瘤细胞都具有抑制作用,且呈现量效和时效依赖关系,即随着花色苷浓度的增大,作用时间的延长,抑制率显著增加。
不同浓度蓝靛果花色苷作用结肠癌HT29细胞48h,用Annexin V-FITC/PI流式细胞术检测细胞凋亡。结果表明随着花色苷浓度的增大,各组细胞凋亡数随之增大。加药组(0.25、0.5、1.0、2.0mg/mL)的凋亡率分别是(10.11±1.84)%、(28.87±2.32)%、(54.74_+±4.69)%、(73.56+±8.34)%,与对照组相比,加药组的凋亡率差异有统计学意义(P<0.05)。结肠癌HT29细胞分别出现了细胞数量明显减少,体积缩小,皱缩变形,细胞表面有许多的球状突起,进一步有凋亡小体产生,细胞间连接逐渐断裂,细胞浆出现较多空泡,胞浆浓缩,核物质致密,呈斑块状,或呈新月形边集于核膜,核形不规整等典型的凋亡细胞形态学特征。DNA凝胶电泳结果可以看到细胞凋亡的梯形条带,单细胞凝胶电泳显示细胞出现明显的拖尾现象。线粒体膜电位水平逐渐下降,且显著低于正常细胞水平；Caspase-3酶活性随着花色苷浓度的增加而升高,且与对照组细胞酶活性相比,差异性显著(P<0.05)。
综上所述,蓝靛果花色苷可能通过诱导肿瘤细胞凋亡来显著抑制肿瘤细胞的增殖；蓝靛果花色苷诱导肿瘤细胞凋亡的机制可能与线粒体介导的细胞凋亡通路有关。
Anthocyanins has many kinds of physiological activity function, such as elimination free radical, resist caducity and variation, resisting inflammation, improvement eyesight, prevention and treatment of cardiovascular diseases, cancer prevention, enhancement immunity. Study the physiological activity function of anthocyanins from Lonicera edulis (Lonicera edulis Turcz.) has very important practical significance.This paper study the extraction conditions, the oxidation resistance and antitumor function mechanism of anthocyanins after isolation and purification from Lonicera edulis.
The optimum condition of extract anthocyanins from Lonicera edulis was discussed by the orthogonal experiment through solvent digesting. The UV spectrophotometer was used to determine the content of anthocyanin of extraction, while H2O2/Fe system and DPPH assay method was adopted to determine the antioxidative capacity of extraction. The results indicated that solvent showed the remarkable effect on antioxidant of anthocyanin from Lonicera edulis, while the ratio of material to liquid, extraction temperature and time showed minor effects. Optimum extraction processing parameter was determined as follow:ethanol concentration was 70%, ratio of material to liquid was 1:15(W:V), temperature was 60℃, time was 90min, pH value was 2, extraction times was third.
Anthocyanins from Lonicera edulis of different concentrations was administered to human colon HT29 Cells, lier cancer HepG2 cells and U-2OS cells of human osteosarcoma for 24h,48h and 72h, respectively. The proliferation of cells was measured by MTT assay. The results showed that anthocyanins from Lonicera edulis can inhibit three types of tumor cells showed dose-dependent and time-dependent. The inhibition rate increased significantly with the increased of the concentration of anthocyanins and treatment time.
HT29 cells were treated with anthocyanins in different concentration for 48 h, flow cytometry with annexin V FITC/PI double staining was used to detect the apoptosis of the cells. Results:The number of apoptotic cells increases with the increase of anthocyanin concentration. The apoptosis rate of the groups (0.25,0.5,1,2 mg/mL) was (10.11±1.84)%, (28.87±2.32)%, (54.74±4.69)%(73.56±8.34)%. Compared with the control group, the apoptosis rate of the drug groups were significantly (P<0.05).Many typical apoptotic morphology were observed, such as:the number of cells decreased, the volume shrunk, many spherules heaved out cells surface, apoptotic bodies appear, connections between cells gradually fracture, the vacuole inside plasm obviously increased, cytoplasm condense, dense nuclear matter was patchy or crescent-shaped on boundary of the nuclear membrane, nuclear shape was irregular. A typical DNA degradation associated with apoptosis was determined by DNA agarose electrophoresis. Single-celled gel electrophoresis showed cells visible delay phenomenon. Mitochondrial membrane potential was decreased and significantly lower than normal cellular level; Caspase-3 activity was increased with the increase of anthocyanins concentration and the difference was significantly compared to control cells(P<0.05).
The results suggest that anthocyanins from Lonicera edulis inhibit cancer cells proliferation maybe was related with apoptosis through the mitochondrial apoptotic signaling pathway.
引文
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