冬枣黄酮的提取分离及抗氧化、抑瘤活性研究
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摘要
冬枣(Zyzyhpus jujuha dates)为鼠李科枣属植物枣树(Zyzyhpus jujuba Mill)的果实,原产于我国渤海地区、黄河三角洲腹地,现在山东、河北等地大面积种植,是我国特有的一种鲜食果品。冬枣营养价值很高,富含各种维生素、黄酮类、环腺苷酸等。冬枣季节性很强,皮薄、水分含量高,不易保存。多项流行病学研究结果表明,蔬菜和水果的摄入量与心血管疾病、癌症等慢性疾病的发生率呈负相关。水果和蔬菜中的植物化学物质尤其是黄酮类作为主要的生物活性物质,在保护机体健康方面起着重要的作用。黄酮类物质广泛存在于水果、蔬菜、茶叶等各类植物之中,具有较强的抗氧化、抗过敏、消炎、抑菌、抑制肿瘤生长等作用。越来越多的证据表明,低浓度的黄酮或2-羟基黄酮可以改善阿霉素的疗效。黄烷酮和2-羟基黄酮可能具有抑制体内肺肿瘤发展的作用,而没有任何明显的毒性迹象。冬枣富含黄酮类物质,实验室研究显示冬枣黄酮提取物具有很强的抗氧化活性,可以减少脂质过氧化,但迄今为止鲜见有关的冬枣活性的研究。
本研究在提取分离、纯化冬枣黄酮的基础上,采用模拟体系观察了冬枣黄酮的抗氧化活性及作用机理;并通过对荷瘤小鼠补充不同剂量冬枣黄酮提取物,观察其抑瘤作用,并探讨其可能的作用机制,为肿瘤的营养干预和开发利用冬枣资源提供理论指导。
1.以总抗氧化活性作为冬枣黄酮提取效果的指标,以乙醇为提取剂,通过加热振荡提取法、超声波辅助提取法以及微波辅助提取法的单因素实验,分别筛选出影响三种提取法提取效率的主要因素,并采用正交试验法优化出三种提取法的最佳提取条件,通过比较三种方法的提取效率优化出冬枣黄酮的提取工艺。优化的总黄酮提取工艺为:萃取时间20s×9(每次提取20s,共提取9次),微波功率468W,乙醇浓度75%,料液比1:20。在此条件下,总黄酮的提取率为1.46%。
2.用5种国产大孔树脂对冬枣黄酮进行了静态吸附与解析试验。结果表明,X-5型树脂最适于纯化冬枣黄酮。通过动态吸附试验确定了X-5型树脂纯化冬枣黄酮的最佳工艺为:先用2倍柱体积的50%乙醇液洗脱、再用1.5倍柱体积的70%乙醇液进行梯度洗脱,洗脱率达到82.42%。
3.冬枣黄酮乙醇萃取物(ECF),乙酸乙酯萃取物(ETF)、正丁醇萃取物(BAF)和萃余水溶物(WF)对DPPH·、O_2~-和·OH均具有一定的清除作用,且在一定浓度范围内呈良好的量效关系。ECF、ETF和WF均能有效的抑制Fe~(2+)诱导的大豆磷脂模拟膜脂质体过氧化,减少丙二醛的生成;且均能有效地推迟Cu~(2+)诱导的血清脂蛋白氧化修饰的启动时间,抑制脂蛋白的氧化进程,在一定浓度范围内呈良好的量效关系。
4.DJF可有效抑制H_2O_2诱导的大鼠血淋巴细胞DNA、VECs氧化损伤,有助于维持细胞的正常结构和功能;DJF可有效抑制Fe~(2+)+H_2O_2诱导的小鼠肝组织MDA的生成,提高GSH-Px、Na~+—K~+—ATPase、Ca~(2+)—Mg~+—ATPase的活力;降低肝线粒体的肿胀度;降低H_2O_2诱导的小鼠RBC溶血。剂量一效应关系较明显。
5.DJF对小鼠S_(180)移植瘤的生长有一定的抑制作用,在100-400 mg/Kg·d剂量范围内,抑瘤率为20.15%一30.01%。DJF可促进荷瘤小鼠肿瘤组织凋亡促进基因Bax的表达,同时减少凋亡抑制基因Bcl一2的表达,升高Bax与Bcl一2的比值,促进肿瘤细胞凋亡。与荷瘤对照组比较,DJF能显著提高荷瘤小鼠脾脏指数、胸腺指数,增加小鼠脾淋巴细胞的增殖率,升高小鼠血浆IL-2含量。
6.体内安全性(LD_(50))实验结果表明,DJF组动物经14天观察未见任何中毒症状、无死亡、不伴有降低体重或其他毒副作用。LD_(50)＞5000mg/kg,属实际无毒级。
研究结果表明,冬枣黄酮使用安全,具有一定的抑瘤作用,其作用机制可能与冬枣黄酮具有诱导肿瘤细胞凋亡,提高机体的免疫功能,并具有一定的抗氧化作用有关。对冬枣果实黄酮的制备及其抗氧化、抑瘤活性的研究填补了国内外学术界的空白。
Phytochemicals,especially flavonoids,in fruits and vegetables are thought to be the major bioactive compounds beneficial for human health.Flavonoids,originated in fruits,vegetables and tea,were shown to have antioxidant activity,antianaphylaxis, bacteriostasis and inhibiting cancer cell proliferation,which may therefore play a key role in reducing chronic diseases.In recent years,accumulating evidence show that treatment with a low concentration of flavanone or 2-OH flavanone can improve the efficacy of doxorubicin.Flavanone and 2-OH flavanone may have an in vivo inhibitory effect on the lung tumor development without any apparent sign of toxicity primarily based on the data of body weight.There are abundant flavonoids in Dongzao Jujube,but few anti-tumor researches have been done using Dongzao jujube before.This study was designed to optimize the extraction and purification conditions of flavonoids in Dongzao jujube(DJF),to study it's antioxidant effect and possible mechanisms,as well as to investigate the inhibition of DJF on S180 sarcoma,to explore therapeutic and commercial potentials of Dongzao jujube.The main research content and results are as follows:
1.Using ethanol as the extractant,through the single test of oscillation by heating extraction,ultrasonic-assisted extraction and microwave-assisted extraction methods, main impact factors of the three methods were determined,respectively.And then the extraction condition of each method was optimized by using orthogonal test.The results showed that 75%of the ethanol solutions under the microwave power at 468W each extract 20s and extracted 9 times was the optimized condition for obtaining DJF with the extraction ratio of DJF about 1.46%.
2.By comparing the five kinds of macroporous resin adsorption and desorption ability for DJF,the optimal separation and purification condition were determined. Results show that X-5 macroporous resin was the most appropriate resin in all for the preliminary purification of DJF.The best technology of preliminary purification using X-5 macroporous resin is as follows:first 2-times the column volume of 50%ethanol solution to elute and then 1.5-times the column volume of 70%ethanol solution for gradient elution.Under the optimized process conditions,the elution rate is 82.42%.
3.The capacity of DJF scavenging DPPH·,superoxide radical(O·_2~-) and·OH of DJF were evaluated with DPPH·system,O·_2~- system and·OH system.The inhibition ability of DJF to lipid oxidation and serum protein oxidative modificated ability were evaluated with the ferric chloride-ascorbic acid system and the Cu2+ catalyst system. Results indicate that different solvent extracts of DJF have effect of scavenging DPPH·,O·_2~- and·OH,showing good dose-effect relationship at a certain concentration range.The ethanol extract(ECF),ethyl acetate extract(ETF) and extracted more than water-soluble material(WF) can effectively inhibit the Fe~(2+)-induced membrane analog soybean phospholipid liposome peroxidation, reducing the generation of malondialdehyde,showing good dose-response relationship.ECF,ETF,WF can effectively postpone the launch of oxidized lipoprotein time,inhibit oxidation process.Results showed that DJF could scavenge reactive oxygen species and inhibit lipid peroxidation in some modified chemical systems.The capabilities of quenching free radicals and reduction or chelate iron ion and copper ion linked with oxidation were revealed to be the mechanism of inhibiting oxidation of DJF.
4.Dongzao jujube flavonoids' antioxidant activity in vitro was evaluated with HaOa-induced oxidation of model.MDA content and GSH-Px,ATP-ase of vitality were determined with malondialdehyde(MDA) Kit,GSH-Px Kit and ATP-ase Kit. RBC hemolysis and liver mitochondrial swelling of the mice was determined with Spectrophotometry.Effects of DJF on blood lymphocyte DNA,and VECs of oxidative damage were measured with Comet assay,cell culture method separately. The results showed that DJF could inhibit H_2O_2-induced blood lymphocyte DNA, VECs oxidative damage,inhibit the swelling of mice liver mitochondria and the hemolysis of mice RBC induced by H_2O_2 as well.DJF could also heighten the GSH-Px,Na~+—K~+—ATPase、Ca~(2+)—Mg~+—ATPase of vitality,and inhibit the formation of MDA in mice liver homogenate induced by Fe~(2+)+ H_2O_2.These results showed that DJF has the antioxidant effect on mice/rat,cell and tissue levels in vitro.
5.S_(180) tumor-bearing mice models were established to explore the inhibitory effects of the DJF and its possible mechanism.Sarcoma weight showed that DJF could inhibit S_(180) sarcoma growth in vivo and the inhibiting rate of 100-400 mg/Kg·d DJF were between 20.23%and 30.01%.DJF may promote tumor-bearing mice to promote apoptosis of tumor tissue gene expression of Bax.At the same time,DIF can reduce the inhibitor of apoptosis gene Bcl-2 expression,increased Bax and Bcl-2 ratio, thereby promoting tumor cell apoptosis.DJF can enhance the spleen index,thymus index,serum IL-2 content,and increase splenocyte proliferation of tumor-bearing mice.
6.Horn method was used to measure the DJF half lethal dose(LD50).Results show that DJF does not have evident toxicity with its LD50 more than 5000mg·Kg~(-1).
DJF is safe.It can inhibit the growth of S_(180) sarcom.The anti-tumor effect of DJF to S_(180) tumor-bearing mice related to promoting tumor cell apoptosis,significantly strengthening the immune system and antioxidant activities.
本研究在提取分离、纯化冬枣黄酮的基础上,采用模拟体系观察了冬枣黄酮的抗氧化活性及作用机理;并通过对荷瘤小鼠补充不同剂量冬枣黄酮提取物,观察其抑瘤作用,并探讨其可能的作用机制,为肿瘤的营养干预和开发利用冬枣资源提供理论指导。
1.以总抗氧化活性作为冬枣黄酮提取效果的指标,以乙醇为提取剂,通过加热振荡提取法、超声波辅助提取法以及微波辅助提取法的单因素实验,分别筛选出影响三种提取法提取效率的主要因素,并采用正交试验法优化出三种提取法的最佳提取条件,通过比较三种方法的提取效率优化出冬枣黄酮的提取工艺。优化的总黄酮提取工艺为:萃取时间20s×9(每次提取20s,共提取9次),微波功率468W,乙醇浓度75%,料液比1:20。在此条件下,总黄酮的提取率为1.46%。
2.用5种国产大孔树脂对冬枣黄酮进行了静态吸附与解析试验。结果表明,X-5型树脂最适于纯化冬枣黄酮。通过动态吸附试验确定了X-5型树脂纯化冬枣黄酮的最佳工艺为:先用2倍柱体积的50%乙醇液洗脱、再用1.5倍柱体积的70%乙醇液进行梯度洗脱,洗脱率达到82.42%。
3.冬枣黄酮乙醇萃取物(ECF),乙酸乙酯萃取物(ETF)、正丁醇萃取物(BAF)和萃余水溶物(WF)对DPPH·、O_2~-和·OH均具有一定的清除作用,且在一定浓度范围内呈良好的量效关系。ECF、ETF和WF均能有效的抑制Fe~(2+)诱导的大豆磷脂模拟膜脂质体过氧化,减少丙二醛的生成;且均能有效地推迟Cu~(2+)诱导的血清脂蛋白氧化修饰的启动时间,抑制脂蛋白的氧化进程,在一定浓度范围内呈良好的量效关系。
4.DJF可有效抑制H_2O_2诱导的大鼠血淋巴细胞DNA、VECs氧化损伤,有助于维持细胞的正常结构和功能;DJF可有效抑制Fe~(2+)+H_2O_2诱导的小鼠肝组织MDA的生成,提高GSH-Px、Na~+—K~+—ATPase、Ca~(2+)—Mg~+—ATPase的活力;降低肝线粒体的肿胀度;降低H_2O_2诱导的小鼠RBC溶血。剂量一效应关系较明显。
5.DJF对小鼠S_(180)移植瘤的生长有一定的抑制作用,在100-400 mg/Kg·d剂量范围内,抑瘤率为20.15%一30.01%。DJF可促进荷瘤小鼠肿瘤组织凋亡促进基因Bax的表达,同时减少凋亡抑制基因Bcl一2的表达,升高Bax与Bcl一2的比值,促进肿瘤细胞凋亡。与荷瘤对照组比较,DJF能显著提高荷瘤小鼠脾脏指数、胸腺指数,增加小鼠脾淋巴细胞的增殖率,升高小鼠血浆IL-2含量。
6.体内安全性(LD_(50))实验结果表明,DJF组动物经14天观察未见任何中毒症状、无死亡、不伴有降低体重或其他毒副作用。LD_(50)＞5000mg/kg,属实际无毒级。
研究结果表明,冬枣黄酮使用安全,具有一定的抑瘤作用,其作用机制可能与冬枣黄酮具有诱导肿瘤细胞凋亡,提高机体的免疫功能,并具有一定的抗氧化作用有关。对冬枣果实黄酮的制备及其抗氧化、抑瘤活性的研究填补了国内外学术界的空白。
Phytochemicals,especially flavonoids,in fruits and vegetables are thought to be the major bioactive compounds beneficial for human health.Flavonoids,originated in fruits,vegetables and tea,were shown to have antioxidant activity,antianaphylaxis, bacteriostasis and inhibiting cancer cell proliferation,which may therefore play a key role in reducing chronic diseases.In recent years,accumulating evidence show that treatment with a low concentration of flavanone or 2-OH flavanone can improve the efficacy of doxorubicin.Flavanone and 2-OH flavanone may have an in vivo inhibitory effect on the lung tumor development without any apparent sign of toxicity primarily based on the data of body weight.There are abundant flavonoids in Dongzao Jujube,but few anti-tumor researches have been done using Dongzao jujube before.This study was designed to optimize the extraction and purification conditions of flavonoids in Dongzao jujube(DJF),to study it's antioxidant effect and possible mechanisms,as well as to investigate the inhibition of DJF on S180 sarcoma,to explore therapeutic and commercial potentials of Dongzao jujube.The main research content and results are as follows:
1.Using ethanol as the extractant,through the single test of oscillation by heating extraction,ultrasonic-assisted extraction and microwave-assisted extraction methods, main impact factors of the three methods were determined,respectively.And then the extraction condition of each method was optimized by using orthogonal test.The results showed that 75%of the ethanol solutions under the microwave power at 468W each extract 20s and extracted 9 times was the optimized condition for obtaining DJF with the extraction ratio of DJF about 1.46%.
2.By comparing the five kinds of macroporous resin adsorption and desorption ability for DJF,the optimal separation and purification condition were determined. Results show that X-5 macroporous resin was the most appropriate resin in all for the preliminary purification of DJF.The best technology of preliminary purification using X-5 macroporous resin is as follows:first 2-times the column volume of 50%ethanol solution to elute and then 1.5-times the column volume of 70%ethanol solution for gradient elution.Under the optimized process conditions,the elution rate is 82.42%.
3.The capacity of DJF scavenging DPPH·,superoxide radical(O·_2~-) and·OH of DJF were evaluated with DPPH·system,O·_2~- system and·OH system.The inhibition ability of DJF to lipid oxidation and serum protein oxidative modificated ability were evaluated with the ferric chloride-ascorbic acid system and the Cu2+ catalyst system. Results indicate that different solvent extracts of DJF have effect of scavenging DPPH·,O·_2~- and·OH,showing good dose-effect relationship at a certain concentration range.The ethanol extract(ECF),ethyl acetate extract(ETF) and extracted more than water-soluble material(WF) can effectively inhibit the Fe~(2+)-induced membrane analog soybean phospholipid liposome peroxidation, reducing the generation of malondialdehyde,showing good dose-response relationship.ECF,ETF,WF can effectively postpone the launch of oxidized lipoprotein time,inhibit oxidation process.Results showed that DJF could scavenge reactive oxygen species and inhibit lipid peroxidation in some modified chemical systems.The capabilities of quenching free radicals and reduction or chelate iron ion and copper ion linked with oxidation were revealed to be the mechanism of inhibiting oxidation of DJF.
4.Dongzao jujube flavonoids' antioxidant activity in vitro was evaluated with HaOa-induced oxidation of model.MDA content and GSH-Px,ATP-ase of vitality were determined with malondialdehyde(MDA) Kit,GSH-Px Kit and ATP-ase Kit. RBC hemolysis and liver mitochondrial swelling of the mice was determined with Spectrophotometry.Effects of DJF on blood lymphocyte DNA,and VECs of oxidative damage were measured with Comet assay,cell culture method separately. The results showed that DJF could inhibit H_2O_2-induced blood lymphocyte DNA, VECs oxidative damage,inhibit the swelling of mice liver mitochondria and the hemolysis of mice RBC induced by H_2O_2 as well.DJF could also heighten the GSH-Px,Na~+—K~+—ATPase、Ca~(2+)—Mg~+—ATPase of vitality,and inhibit the formation of MDA in mice liver homogenate induced by Fe~(2+)+ H_2O_2.These results showed that DJF has the antioxidant effect on mice/rat,cell and tissue levels in vitro.
5.S_(180) tumor-bearing mice models were established to explore the inhibitory effects of the DJF and its possible mechanism.Sarcoma weight showed that DJF could inhibit S_(180) sarcoma growth in vivo and the inhibiting rate of 100-400 mg/Kg·d DJF were between 20.23%and 30.01%.DJF may promote tumor-bearing mice to promote apoptosis of tumor tissue gene expression of Bax.At the same time,DIF can reduce the inhibitor of apoptosis gene Bcl-2 expression,increased Bax and Bcl-2 ratio, thereby promoting tumor cell apoptosis.DJF can enhance the spleen index,thymus index,serum IL-2 content,and increase splenocyte proliferation of tumor-bearing mice.
6.Horn method was used to measure the DJF half lethal dose(LD50).Results show that DJF does not have evident toxicity with its LD50 more than 5000mg·Kg~(-1).
DJF is safe.It can inhibit the growth of S_(180) sarcom.The anti-tumor effect of DJF to S_(180) tumor-bearing mice related to promoting tumor cell apoptosis,significantly strengthening the immune system and antioxidant activities.
引文
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