西兰苔叶黄酮的提取、分离、鉴定及体外抗癌活性研究
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摘要
本实验以西兰苔叶黄酮的提取和分离纯化为目的,运用乙醇超声法为提取手段,实现黄酮类化合物最大化利用。借助HPLC、LC-ESI-MS、GC-MS等现代仪器对粗提物进行分析和结构鉴定,将黄酮粗提物以及纯化物进行抗癌活性研究。
1.采用乙醇超声波提取方法,考察提取温度、乙醇浓度、料液比因素对西兰苔叶黄酮的提取率的影响。通过响应面设计确定最佳工艺条件:提取温度60℃、浓度70%、料液比1:27。黄酮得率为11.96mg/g。
2.运用HPLC、LC-ESI-MS、GC-MS等分析手段,对西兰苔叶黄酮粗提物成分进行分析,GC-MS结果显示西兰苔叶含有丰富的挥发油成分,其中富含脂肪酸(棕榈酸和硬脂酸)。以LC-MS为检测方法,初步鉴定出粗提物中含有四种黄酮类物质。分别是槲皮素、芦丁、芹菜素、山奈酚。
3.粗提物通过乙酸乙酯、正丁醇萃取后,经过聚酰胺树脂分离出三种物质,乙酸乙酯层经50%乙醇洗脱出的溶液含有槲皮素,含量为85.4%。正丁醇层分别用50%、70%乙醇洗脱,50%乙醇洗脱出的溶液含有山奈酚,含量为78.5%,经70%乙醇洗脱出的溶液是山奈酚和芹菜素,含量为82.6%。
4.通过MTT实验检测EOF(黄酮粗提物)和纯化物对细胞生长抑制率,Annexi-V/PI双染流式细胞仪检测细胞凋亡,来探讨西兰苔叶黄酮对癌细胞增殖抑制作用以及凋亡机制。结果显示,EOF对结肠癌SW480细胞、肝癌HepG2细胞、宫颈癌Hela细胞、肺癌A549细胞这几种细胞株均表现出抑制作用,抑制率随药物浓度的增加而增大,呈明显的量效关系,其IC50值分别为88.14、99.65、104.43、79.77μg/mL。对SW480细胞的作用最为明显。以SW480为受试细胞株,比较EOF、SFB1(粗黄酮分离产物1)、SFB2(粗黄酮分离产物2)、SFB3(粗黄酮分离产物3)、Que(槲皮素)对SW480抑制作用强弱,结果表明这几种物质表现出不同程度的抑制作用,IC50分别为88.14μg/mL、65.06μg/mL、72.62μg/mL、79.42μg/mL、54.64μg/mL。通过流式细胞仪得出SW480细胞的凋亡率分别为12.74%、16.41 %、19.61%、28.28%、50.66%。实验结果表明EOF是通过抑制增殖和诱导细胞凋亡来发挥对肿瘤的治疗作用。
With the aim of extraction,separation and purification flavonoids from Broccolini,the technological condition of extracting flavonoid of the total flavonoids from Broccolini was studied in this thesis. HPLC、LC-ESI-MS、GC-MS and other modern instruments were used to reasearch the structure and anti-tumor activity of the flavonoid extracts.
(1)Effects of ethanol concentration,extraction temperature,solid-liquid ration on extraction yields of flavonoids were determined individually;By response surface design,the results showed that optimum conditions: 70% ethanol concentration, extraction temperature 60℃,solid-liquid of 1:27,the yields of flavonoids was 11.9589g/mg.
(2)The contents of flavonoid from Broccolini were detected by HPLC、LC-ESI-MS、GC-MS.GC-MS results showed Broccolini leaves are rich in volatile oil,expecially Hexadecanoic acid and Octadecanoic acid.Take LC-MS as the identification method,four kinds of flavonoid were determined.They are quercetin,rutin,apigenin,kaempferol.
(3) Crude extract was purified by ethyl acetate,butanol.To sparate flavonoid by the column chromatography of polyamid resin.ethyl acetate layer with 50% ethanol as eluent,the elution solution contains quercetin,the purity was 85.4%, butanol layer with 50%,70% ethanol as eluent resper respectively.50% elution solution contains kaempferol,the purity was 78.5% ,70% elution solution contains kaempferol and apigenin,the purity was 82.6%.
(4)To investigate the mechanism of EOF on cell proliferation and apoptosis of human cancer cell line in vitro. The cell growth inhibiting ratio was assessed by the MTT assay ,the cell apoptotic was detected with Annexi-V/PI doubled staining flow cytometry(FCM).Human cancer cells line SW480、HepG2、Hela、A549 were treated with different concentrations of EOF respectively. MTT assay showed EOF inhibited the proliferation of human cancer cell line in a dose-dependent manner(P<0.01). The MTT assay results showed that the IC50 of EOF for SW480、HepG2、Hela、A549 were 88.14、99.65、104.43、79.77μg/mL respectively. EOF、SFB1、SFB2、SFB3、Que inhibited the proliferation of SW480, and the IC50 were 88.14μg/mL、65.06μg/mL、72.62μg/mL、79.42μg/mL、54.64μg/mL.Que inhibitory effects of EOF on the growth of cell line are the most remarkable. Compared with the control group, the apoptosis rate in SW480 cells was found to be 12.74%、16.41 %、19.61%、28.28%、50.66 %.Experimental results show that the EOF plays a role in tumor therapy by inhibiting proliferation and inducing apoptosis.
1.采用乙醇超声波提取方法,考察提取温度、乙醇浓度、料液比因素对西兰苔叶黄酮的提取率的影响。通过响应面设计确定最佳工艺条件:提取温度60℃、浓度70%、料液比1:27。黄酮得率为11.96mg/g。
2.运用HPLC、LC-ESI-MS、GC-MS等分析手段,对西兰苔叶黄酮粗提物成分进行分析,GC-MS结果显示西兰苔叶含有丰富的挥发油成分,其中富含脂肪酸(棕榈酸和硬脂酸)。以LC-MS为检测方法,初步鉴定出粗提物中含有四种黄酮类物质。分别是槲皮素、芦丁、芹菜素、山奈酚。
3.粗提物通过乙酸乙酯、正丁醇萃取后,经过聚酰胺树脂分离出三种物质,乙酸乙酯层经50%乙醇洗脱出的溶液含有槲皮素,含量为85.4%。正丁醇层分别用50%、70%乙醇洗脱,50%乙醇洗脱出的溶液含有山奈酚,含量为78.5%,经70%乙醇洗脱出的溶液是山奈酚和芹菜素,含量为82.6%。
4.通过MTT实验检测EOF(黄酮粗提物)和纯化物对细胞生长抑制率,Annexi-V/PI双染流式细胞仪检测细胞凋亡,来探讨西兰苔叶黄酮对癌细胞增殖抑制作用以及凋亡机制。结果显示,EOF对结肠癌SW480细胞、肝癌HepG2细胞、宫颈癌Hela细胞、肺癌A549细胞这几种细胞株均表现出抑制作用,抑制率随药物浓度的增加而增大,呈明显的量效关系,其IC50值分别为88.14、99.65、104.43、79.77μg/mL。对SW480细胞的作用最为明显。以SW480为受试细胞株,比较EOF、SFB1(粗黄酮分离产物1)、SFB2(粗黄酮分离产物2)、SFB3(粗黄酮分离产物3)、Que(槲皮素)对SW480抑制作用强弱,结果表明这几种物质表现出不同程度的抑制作用,IC50分别为88.14μg/mL、65.06μg/mL、72.62μg/mL、79.42μg/mL、54.64μg/mL。通过流式细胞仪得出SW480细胞的凋亡率分别为12.74%、16.41 %、19.61%、28.28%、50.66%。实验结果表明EOF是通过抑制增殖和诱导细胞凋亡来发挥对肿瘤的治疗作用。
With the aim of extraction,separation and purification flavonoids from Broccolini,the technological condition of extracting flavonoid of the total flavonoids from Broccolini was studied in this thesis. HPLC、LC-ESI-MS、GC-MS and other modern instruments were used to reasearch the structure and anti-tumor activity of the flavonoid extracts.
(1)Effects of ethanol concentration,extraction temperature,solid-liquid ration on extraction yields of flavonoids were determined individually;By response surface design,the results showed that optimum conditions: 70% ethanol concentration, extraction temperature 60℃,solid-liquid of 1:27,the yields of flavonoids was 11.9589g/mg.
(2)The contents of flavonoid from Broccolini were detected by HPLC、LC-ESI-MS、GC-MS.GC-MS results showed Broccolini leaves are rich in volatile oil,expecially Hexadecanoic acid and Octadecanoic acid.Take LC-MS as the identification method,four kinds of flavonoid were determined.They are quercetin,rutin,apigenin,kaempferol.
(3) Crude extract was purified by ethyl acetate,butanol.To sparate flavonoid by the column chromatography of polyamid resin.ethyl acetate layer with 50% ethanol as eluent,the elution solution contains quercetin,the purity was 85.4%, butanol layer with 50%,70% ethanol as eluent resper respectively.50% elution solution contains kaempferol,the purity was 78.5% ,70% elution solution contains kaempferol and apigenin,the purity was 82.6%.
(4)To investigate the mechanism of EOF on cell proliferation and apoptosis of human cancer cell line in vitro. The cell growth inhibiting ratio was assessed by the MTT assay ,the cell apoptotic was detected with Annexi-V/PI doubled staining flow cytometry(FCM).Human cancer cells line SW480、HepG2、Hela、A549 were treated with different concentrations of EOF respectively. MTT assay showed EOF inhibited the proliferation of human cancer cell line in a dose-dependent manner(P<0.01). The MTT assay results showed that the IC50 of EOF for SW480、HepG2、Hela、A549 were 88.14、99.65、104.43、79.77μg/mL respectively. EOF、SFB1、SFB2、SFB3、Que inhibited the proliferation of SW480, and the IC50 were 88.14μg/mL、65.06μg/mL、72.62μg/mL、79.42μg/mL、54.64μg/mL.Que inhibitory effects of EOF on the growth of cell line are the most remarkable. Compared with the control group, the apoptosis rate in SW480 cells was found to be 12.74%、16.41 %、19.61%、28.28%、50.66 %.Experimental results show that the EOF plays a role in tumor therapy by inhibiting proliferation and inducing apoptosis.
引文
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