红豆越橘多酚对氧化诱导损伤及癌细胞增殖抑制作用研究
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摘要
自由基医学研究表明自由基引发的氧化损伤与人类近百余种疾病密切相关，因此评价和筛选具有强抗氧化活性的天然产物成分已成为医学、生物学和食品科学研究的新趋势。本论文选取红豆越橘为研究对象，采用活性跟踪的方法，获得了活性最强的红豆越橘多酚（LBP），并对其主要成分进行分析鉴定。系统的分析了LBP的体外抗氧化活性，对辐射诱导机体损伤的防护作用及对肿瘤细胞增殖的抑制活性，并对抗氧化活性和抗癌细胞增殖活性进行了相关性分析。结果显示，LBP具有较强的抗氧化和抗癌细胞增殖活性且具有良好的相关性，说明浆果中天然存在的植物化合物的联合作用在抗氧化以及肿瘤预防方面至关重要。
采用活性跟踪的方法分离纯化野生红豆越橘，首先用80%丙酮冷冻提取游离型酚类和类黄酮，浓缩后用大孔吸附树脂X-5富集分离红豆越橘多酚，多酚含量和ABTS抗氧化做指标确定乙醇梯度洗脱有效组分，并命名为LB-1，LB-2，LB-3，LB-4，其总洗脱物命名为LB-MIX（LBP）。用MTS法检测以上5个组分抗结肠癌HT29、卵巢癌Hela、肺癌A549和肝癌HepG-2细胞增殖活性，结果显示，LBP具有较好的抑制几株癌细胞的增殖活性。选取抗癌活性较强的顺铂为阳性对照。为了鉴定此纯化物的化学组成，本实验采用UPLC-ESI-MS联用的方法定性分析红豆越橘多酚的组成成分。结果显示，其主要成分有9种花色苷，4种原花青素和5种酚酸。
系统的研究了LBP的体外抗氧化活性作用。如清除生理自由基、非生理自由基、还原能力、螯合能力、抗脂质过氧化能力和辐射诱导氧化损伤的防护能力等，结果显示，LBP具有较高的抗氧化活性，并呈良好的剂量效应关系，其氧化能力略低于或与抗氧化剂抗坏血酸相当。并具有较高的金属离子螯合能力和还原能力，对于脂质过氧化和辐射诱导的氧化损伤具有一定的保护。比较了10种浆果与红豆越橘的多酚、黄酮和花色苷含量，不同浆果中多酚、黄酮、花色苷含量差异性显著（p<0.01）。红豆越橘的多酚、黄酮和花色苷含量在11果中排前4位，同时对ABTS+和DPPH的清除活性也居于前列。
构建了辐射诱导的氧化损伤模型，实验表明，LBP各剂量组可有效的促进造血和免疫细胞增殖和生长，能够明显的提高小鼠各脏器中超氧化物歧化酶（SOD）活性，过氧化氢酶（CAT）活性，乳酸脱氢酶（LDH）活性和增加还原型谷胱甘肽（GSH）的含量，降低髓过氧化物酶（POM）活性，减少丙二醛（MDA）含量，能够有效的激活抗氧化酶系，抑制氧化酶系，减少脂质过氧化，保护细胞膜的损伤，减少微核和染色体畸变率，细胞周期分布显示与模型组比较，LBP加药组使阻滞于G0/G1期的细胞减少而S期和G2/M期细胞增多，呈极显著性差异（p<0.01），细胞增殖能力得到恢复。单细胞凝胶电泳显示辐射前灌胃LBP，可以有效的较少DNA的损伤，使彗星尾长度明显减少。透射电镜再一次证明LBP对脾组织和肠绒毛的保护作用。
研究了LBP对癌细胞的增殖抑制活性，结果显示，11种浆果中红豆越橘对HT29和HepG2两种癌细胞的抑制活性最强，蓝靛果对癌细胞的抑制活性较弱，11种浆果对癌细胞的抑制活性与其多酚含量没有显著的相关性。进一步采用琼脂糖凝胶电泳验证了LBP可以使细胞膜损伤产生DNA-Ladder碎片，同时用流式细胞仪对LBP及顺铂作用后的几种癌细胞进行了细胞周期分析，用单细胞凝胶电泳验证了提取物对癌细胞的DNA损伤程度。结果显示，红豆越橘多酚可以从不同角度诱导细胞凋亡。建立了体外抗氧化和抗癌细胞增殖的相关性，揭示了抗氧化剂的抗氧化、清除ROS作用与其防癌抑癌作用之间存在关联，进一步证实了红豆越橘多酚具有较好的功能活性，对于揭示浆果多酚预防由自由基引发的氧化损伤的作用机制以及开发研制新型抗癌药物具有重要的理论和实际意义。
The medical research on free radicals has proved that the oxidation damageinduced by free radical is closely related to nearly100kinds of human diseases. Inrecent20years, the studies on reactive oxygen species and free radicals have becomethe hotspots in modern life science. Evaluation and selection of natural resources withstrong antioxidant activity has become a new tendency in biology, medicine and foodscience. In this study, we prepared high-activity polyphenol in Lingonberry (LBP) bybioactivity-guided isolation, and analyzed its main components. We systematicallyanalyzed antioxidant activities of LBP in vitro, its protective effects on radiation-induced oxidation damage, inhibition on tumor cell proliferation. The results showedthat LBP had stronger antioxidant and antiproliferative activities, with a goodcorrelation, indicating that the combined effects of natural plant compounds play crucialroles in antioxidant and cancer prevention.
Wild lingonberry was isolated and purified by active tracking method. Free phenolsand flavonoid in fresh fruits were extracted with chilled80%acetone (1:2, w/v) using achilled JJ-2Tissue disintegrator. The homogenates were filtered and evaporated.Residue was diluted in distilled water. After separating the precipitate by filtration, theremaining solution was further separated on an adsorptive resin X-5column. Theeffective constituents were determined by ethanol gradient elution using contents ofpolyphenol and ABTS as an index, named LB-1, LB-2, LB-3, LB-4; the total elutioncomponents were named as LB-MIX (LBP). The antiproliferative activities of the abovefive effective constituents on HT29, Hela, A549and HepG-2cells were measured byMTT assay. The results showed that LBP had good antiproliferative activities on cancercell. Under the condition, the cisplatin was chosen as positive control for its highanticancer activity. The constituent of LBP was determined by UPLC-ESI-MS. Theresults showed that main constituent of LBP consisted of nine kinds of anthocyanins,four kinds of proanthocyanidins and five kinds of phenolic acids.
Antioxidant activities of LBP were studied in vitro, such as removal ofphysiological free radicals and non-physiological free radicals, reduction, chelation,antilipidperoxidation and protective effect on radiation-induced damages. The resultsshowed that LBP had high antioxidant activities, which were slightly lower thanascorbic acid or equivalent, with good doseresponse relationships. LBP also had higheffect of reduction, chelation and protective effect on radiation-induced damages. The11kinds of fruits including lingonberry in China were evaluated for total contents ofphenolics, flavonoids and anthocyanins.
We constructed a model of radiation-induced oxidative damage. All the LBP dosessignificantly increased the proliferation of hematopoietic cell and immunological cell;enhanced the activities of superoxide dismutase (SOD), catlase (CAT), lactatedehydrogenase (LDH) and increased the content of glutathione (GSH); lowered theactivities of myeloperoxidase (POM) and the content of malonyldiadehyde (MDA).Moreover, LBP plays functions in the activation of antioxidant enzyme system,inhibition of oxidase system, the decrease of lipid peroxidation, protection of cellmembrane, and generation inhibition on production of radiation-induced micronuclei.Analysis on cell cycle distribution and model groups showed significant differencesbetween different LBP dosage groups (p<0.01). The LBP medicine group had less cellsin the G0/G1stages and more cells in G2/M stages. This result implies that thetreatment play a role in the recovery of cell proliferation.
LBP has significantly inhibitory effect on cancer cell proliferation. Among the11fruits, the inhibitory effect of lingonberry extracts on both HT-29and HepG2cancercells was the highest, blue honeysuckle the lowest. There was no correlation betweenthe antiproliferative activity and the total phenolic contents in fruit extracts. Agarose gelelectrophoresis analysis demonstrated that LBP resulted in the damage of cancermembrane as indicated by the generation of DNA-Ladder fragment. The effects of LBPand cisplatin treatment on cancer cell cycles were measured by using flow cytometer.Single cell gel electrophoresis was used to detect the DNA molecular damage. Theresults indicated that LBP can induce the cell apoptosis in different pathways. Thepresent study showed a close correlation between LBP activity and anti-proliferation ofcancer cell. This result demonstrated that lingonberry has effective functional activity.The study has importantly theoretical and practical significance to reveal mechanism ofpreventing various damages induced by free radical and develop new anticancer drugs.
采用活性跟踪的方法分离纯化野生红豆越橘，首先用80%丙酮冷冻提取游离型酚类和类黄酮，浓缩后用大孔吸附树脂X-5富集分离红豆越橘多酚，多酚含量和ABTS抗氧化做指标确定乙醇梯度洗脱有效组分，并命名为LB-1，LB-2，LB-3，LB-4，其总洗脱物命名为LB-MIX（LBP）。用MTS法检测以上5个组分抗结肠癌HT29、卵巢癌Hela、肺癌A549和肝癌HepG-2细胞增殖活性，结果显示，LBP具有较好的抑制几株癌细胞的增殖活性。选取抗癌活性较强的顺铂为阳性对照。为了鉴定此纯化物的化学组成，本实验采用UPLC-ESI-MS联用的方法定性分析红豆越橘多酚的组成成分。结果显示，其主要成分有9种花色苷，4种原花青素和5种酚酸。
系统的研究了LBP的体外抗氧化活性作用。如清除生理自由基、非生理自由基、还原能力、螯合能力、抗脂质过氧化能力和辐射诱导氧化损伤的防护能力等，结果显示，LBP具有较高的抗氧化活性，并呈良好的剂量效应关系，其氧化能力略低于或与抗氧化剂抗坏血酸相当。并具有较高的金属离子螯合能力和还原能力，对于脂质过氧化和辐射诱导的氧化损伤具有一定的保护。比较了10种浆果与红豆越橘的多酚、黄酮和花色苷含量，不同浆果中多酚、黄酮、花色苷含量差异性显著（p<0.01）。红豆越橘的多酚、黄酮和花色苷含量在11果中排前4位，同时对ABTS+和DPPH的清除活性也居于前列。
构建了辐射诱导的氧化损伤模型，实验表明，LBP各剂量组可有效的促进造血和免疫细胞增殖和生长，能够明显的提高小鼠各脏器中超氧化物歧化酶（SOD）活性，过氧化氢酶（CAT）活性，乳酸脱氢酶（LDH）活性和增加还原型谷胱甘肽（GSH）的含量，降低髓过氧化物酶（POM）活性，减少丙二醛（MDA）含量，能够有效的激活抗氧化酶系，抑制氧化酶系，减少脂质过氧化，保护细胞膜的损伤，减少微核和染色体畸变率，细胞周期分布显示与模型组比较，LBP加药组使阻滞于G0/G1期的细胞减少而S期和G2/M期细胞增多，呈极显著性差异（p<0.01），细胞增殖能力得到恢复。单细胞凝胶电泳显示辐射前灌胃LBP，可以有效的较少DNA的损伤，使彗星尾长度明显减少。透射电镜再一次证明LBP对脾组织和肠绒毛的保护作用。
研究了LBP对癌细胞的增殖抑制活性，结果显示，11种浆果中红豆越橘对HT29和HepG2两种癌细胞的抑制活性最强，蓝靛果对癌细胞的抑制活性较弱，11种浆果对癌细胞的抑制活性与其多酚含量没有显著的相关性。进一步采用琼脂糖凝胶电泳验证了LBP可以使细胞膜损伤产生DNA-Ladder碎片，同时用流式细胞仪对LBP及顺铂作用后的几种癌细胞进行了细胞周期分析，用单细胞凝胶电泳验证了提取物对癌细胞的DNA损伤程度。结果显示，红豆越橘多酚可以从不同角度诱导细胞凋亡。建立了体外抗氧化和抗癌细胞增殖的相关性，揭示了抗氧化剂的抗氧化、清除ROS作用与其防癌抑癌作用之间存在关联，进一步证实了红豆越橘多酚具有较好的功能活性，对于揭示浆果多酚预防由自由基引发的氧化损伤的作用机制以及开发研制新型抗癌药物具有重要的理论和实际意义。
The medical research on free radicals has proved that the oxidation damageinduced by free radical is closely related to nearly100kinds of human diseases. Inrecent20years, the studies on reactive oxygen species and free radicals have becomethe hotspots in modern life science. Evaluation and selection of natural resources withstrong antioxidant activity has become a new tendency in biology, medicine and foodscience. In this study, we prepared high-activity polyphenol in Lingonberry (LBP) bybioactivity-guided isolation, and analyzed its main components. We systematicallyanalyzed antioxidant activities of LBP in vitro, its protective effects on radiation-induced oxidation damage, inhibition on tumor cell proliferation. The results showedthat LBP had stronger antioxidant and antiproliferative activities, with a goodcorrelation, indicating that the combined effects of natural plant compounds play crucialroles in antioxidant and cancer prevention.
Wild lingonberry was isolated and purified by active tracking method. Free phenolsand flavonoid in fresh fruits were extracted with chilled80%acetone (1:2, w/v) using achilled JJ-2Tissue disintegrator. The homogenates were filtered and evaporated.Residue was diluted in distilled water. After separating the precipitate by filtration, theremaining solution was further separated on an adsorptive resin X-5column. Theeffective constituents were determined by ethanol gradient elution using contents ofpolyphenol and ABTS as an index, named LB-1, LB-2, LB-3, LB-4; the total elutioncomponents were named as LB-MIX (LBP). The antiproliferative activities of the abovefive effective constituents on HT29, Hela, A549and HepG-2cells were measured byMTT assay. The results showed that LBP had good antiproliferative activities on cancercell. Under the condition, the cisplatin was chosen as positive control for its highanticancer activity. The constituent of LBP was determined by UPLC-ESI-MS. Theresults showed that main constituent of LBP consisted of nine kinds of anthocyanins,four kinds of proanthocyanidins and five kinds of phenolic acids.
Antioxidant activities of LBP were studied in vitro, such as removal ofphysiological free radicals and non-physiological free radicals, reduction, chelation,antilipidperoxidation and protective effect on radiation-induced damages. The resultsshowed that LBP had high antioxidant activities, which were slightly lower thanascorbic acid or equivalent, with good doseresponse relationships. LBP also had higheffect of reduction, chelation and protective effect on radiation-induced damages. The11kinds of fruits including lingonberry in China were evaluated for total contents ofphenolics, flavonoids and anthocyanins.
We constructed a model of radiation-induced oxidative damage. All the LBP dosessignificantly increased the proliferation of hematopoietic cell and immunological cell;enhanced the activities of superoxide dismutase (SOD), catlase (CAT), lactatedehydrogenase (LDH) and increased the content of glutathione (GSH); lowered theactivities of myeloperoxidase (POM) and the content of malonyldiadehyde (MDA).Moreover, LBP plays functions in the activation of antioxidant enzyme system,inhibition of oxidase system, the decrease of lipid peroxidation, protection of cellmembrane, and generation inhibition on production of radiation-induced micronuclei.Analysis on cell cycle distribution and model groups showed significant differencesbetween different LBP dosage groups (p<0.01). The LBP medicine group had less cellsin the G0/G1stages and more cells in G2/M stages. This result implies that thetreatment play a role in the recovery of cell proliferation.
LBP has significantly inhibitory effect on cancer cell proliferation. Among the11fruits, the inhibitory effect of lingonberry extracts on both HT-29and HepG2cancercells was the highest, blue honeysuckle the lowest. There was no correlation betweenthe antiproliferative activity and the total phenolic contents in fruit extracts. Agarose gelelectrophoresis analysis demonstrated that LBP resulted in the damage of cancermembrane as indicated by the generation of DNA-Ladder fragment. The effects of LBPand cisplatin treatment on cancer cell cycles were measured by using flow cytometer.Single cell gel electrophoresis was used to detect the DNA molecular damage. Theresults indicated that LBP can induce the cell apoptosis in different pathways. Thepresent study showed a close correlation between LBP activity and anti-proliferation ofcancer cell. This result demonstrated that lingonberry has effective functional activity.The study has importantly theoretical and practical significance to reveal mechanism ofpreventing various damages induced by free radical and develop new anticancer drugs.
引文
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