莲原花青素对皮肤的保护作用及其分子机制研究
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摘要
本文是在本课题组发现莲中富含原花青素，并已证实莲房原花青素(LSPC)能有效清除自由基、抗脂质过氧化、调节血脂、保护心脏等研究基础上，旨在利用现代医学、分子生物学技术和药理实验方法及参考保健食品功能测定方法，从细胞膜中V_E含量和膜脂流动性、酶活性、免疫调节等角度，探讨莲房活性因子—原花青素对正常皮肤的保护作用，在此基础上，从整体动物水平、细胞水平和基因水平，进一步深入系统地研究LSPC对肿瘤和辐射致损伤皮肤的保护和修复作用。并着重从抗氧化、病理形态、细胞凋亡、基因调控等方面探明LSPC对皮肤黑色素瘤B16的抑制作用及其分子机制。为LSPC作为预防和治疗黑色素瘤的药物或辅助药物及保护皮肤的保健食品和化妆品的开发利用提供理论基础和科学依据。同时也为系统深入了解原花青素对皮肤保护作用机制提供可借鉴的思路和方法。
主要研究结果如下：
1．莲房原花青素对大鼠红细胞膜维生素E含量及膜脂流动性的影响
LSPC是有效的羟基自由基清除剂和很强的天然抗氧化剂。对Fenton体系产生的羟基自由基有很强的清除作用；体系酸碱度、金属离子(Cu~(2+)、Ca~(2+)、Al~(3+)、Mg~(2+))对LSPC清除羟基自由基有很大影响。当有20mmol／L Al~(3+)存在时，使得LSPC的清除率从63.1％升高到81.6％。说明LSPC鳌合Cu~(2+)、Ca~(2+)、Al~(3+)、Mg~(2+)金属离子后，能使其清除羟基自由基的作用增效。
LSPC可有效防止红细胞膜中不饱和脂肪酸的过氧化，抑制膜中脂质过氧化产物MDA生成，保持细胞膜的完整性和功能；在保护红细胞膜中天然抗氧化剂V_E的同时，并使其再生。当LSPC浓度为5.0μg／mL时，红细胞受氧化损伤的膜得以恢复，其膜的荧光偏振度(p值)接近正常红细胞膜p值水平，从而降低了红细胞膜因氧化造成的各向异性增加。
2．莲房原花青素对皮肤的抗氧化作用研究
LSPC能加快皮肤和血清中抗氧化酶的生物合成和提高酶的活力，从而提高机体的抗氧化能力。给大鼠连续口服灌胃100mg／kg．bw LSPC 35d，血清和皮肤中MDA含量明显低于对照组(P＜0.05)；而SOD和GSH-Px酶活力显著高于对照组；皮肤和血清中Hyp含量也显著高于对照组(P＜0.05)，使皮肤中胶原蛋白和水分含量分别提高2.7％和3.8个百分点。提示，LSPC是通过提高皮肤抗氧化酶系统活力和降低MDA生成，增强胶原蛋白的生物合成及提高皮肤组织的保水性能来延缓皮肤衰老。
段玉清华中农业大学2004届博士学位论文
3.莲房原花青素对小鼠免疫功能的影响
连续灌胃30~gomg/k g.bw LSPC对小鼠体内腹腔巨噬细胞吞噬功能有较强
的增强作用，能使单核一巨噬细胞的吞噬能力明显提高:提高DNFB诱导的小鼠
耳肿胀率和小鼠半数溶血值(HC50)，且存在明显的剂量依赖关系;体外MTT
法测得LSPC能刺激T、B淋巴细胞增殖，并存在剂量一效应关系。半体内法测
得LSPC能显著激活NK细胞活性。表明，LSPC对免疫功能有较强的调节作用。
4莲房原花青素对酪氨酸酶及黑色素生物合成抑制作用初步研究
LsPC对黑色素的生物合成有显著的抑制作用。7.8 X10一49/L一5.09几LsPc
对酪氨酸酶活力有抑制作用，其中，7.8 X10“49/L一0.19/L时，随LsPC浓度增
大抑制率下降;0.lg/L~4.og/L时，随着LSPC浓度增大抑制率逐渐增高，最大
抑制率高达99.9%，且该抑制作用属于可逆的竞争性抑制。对多巴色素自动转化
黑色素有显著的抑制作用，其半抑制浓度ICS。1.7509/L，最大抑制率为69.8%。
5.莲房原花青素对皮肤黑色素瘤B16的抑制作用及其分子机制
LSPC具有抗黑色素瘤的作用。连续灌胃1 20mg/kg.bw LSPC14d抑瘤率达到
55.3%。用含25~100pg/mL的LSpC培养小鼠黑色素瘤Bx6细胞72h，可显著
抑制其生长增殖，对B16有细胞毒，半数抑制浓度(IC50)为32.4协g/mL，最
大抑制率高达84.5%。
LSPC抑制黑色素瘤细胞分裂增殖，机制有两点:
第一，LSPC可促进B16细胞凋亡。显微形态观察表明，LSPC能引起B16细
胞形态发生改变，使细胞缩小、核固缩，胞质凝缩，线粒体膨胀聚集，染色质
逐渐凝集边移，附在核膜周边:荧光强度明显增加。细胞凋亡分析，GO/GI期前
出现亚二倍峰;细胞周期分析，肿瘤细胞生长阻滞于S期。通过激光扫描共聚焦
显微镜的静态和动态观察，经100林g/mL LsPc处理B16细胞后，可使细胞内ca，+
堆积和重新分布，细胞核内ca2+荧光强度从133.1士24.a增加到187.8士29.2，达到
显著性差异(P<0.05)。这可能是LSPC促进B16细胞凋亡的机制之一;LSPC可下
调Bcl一2基因表达，抑制突变型p53基因和c一myc基因表达，这可能是LsPc诱导B16
细胞凋亡的另一重要机制。
第二，LsPc对B16细胞有分化诱导作用。体内连续灌胃1 20mg/k g.bw LSPC
和体外用100“岁mL LSPC培养B16细胞都能使B16瘤细胞出现功能和分化改
变，表现为酪氨酸酶活力分别提高28.5%和“.6%，黑色素生成能力分别提高
23.8%和44.4%;体外培养的B16细胞形态上出现分化改变，如细胞变大，变长，
多数细胞具有树突样结构，并向正常上皮样细胞分化。
一一一一一一一一一止墅巡竺墅过些鲍f究.
以上研究表明，LSPC对皮肤黑色素瘤B16有较强的抑制作用，其主要机制
是通过增加细胞
Proanthocyanidins from Lotus Seedpod(LSPC) show excellent performance in scavenging free radicals, anti-lipid peroxidation, adjusting serum lipid, and protecting the heart, which is confirmed by our lab's previous studies. And on this basis, the modern medical and molecular biological technology and pharmacological methods, as well as technical standards for testing and assessment for health food were used in the dissertation. From the aspects of Vitamin E content in cell membrane, cell membrane lipid fluidity, correlative enzymes activities and immunity adjustment, the protecting effect of LSPC on normal skin was discussed. Further more, its protection and repairing effects on damnification caused by tumor and radiation were investigated at animals level, cell level and molecular level in this paper. Specifically, the growth inhibition effect, inducing apoptosis effect of LSPC on melanoma B16 cell and its molecular mechanism were researched from the aspects of antioxidation, pathological morphology, immune
function, apoptosis and gene regulation. The studies provide theoretical foundation and scientific bases for using LSPC to develop medicines for preventing or curing skin melanoma and health food or cosmetics for protecting skin. Thoughts and methods to investigate protection mechanism of Proanthocyanidins on skin can also be as reference in further systematic study.
The following are the main results:
1. Effect of LSPC on the content of Vitamin E in red cell membrane of rats and on membrane lipid fluidity
LSPC is known as effective natural antioxidant and free radical scavenger ,which can clear up OH from Fenton system, while pH, concentration of metal ions(Cu2+ Ca2+ A13+ Mg2+) of the reaction system can affect the scavenging rate remarkably. LSPC chelated with metal ions can reinforce its free radical scavenging ability, as can be seen that the free radical scavenging rate of LSPC was 63.1% in the reaction system without
metal ions, and then increased to 81.6% in system with 20mmol/L A13+.
LSPC can effectively prevent unsaturated fatty acid (UFA) in red cell membrane from peroxidation as well as inhibit the formation of MDA and keep the integrity of cell membrane and its function. Meanwhile, it can protect the Vitamin E in cell membrane and show cooperating effect with Vitamin E in antioxidation. Red cell membrane damnified by oxidation can be recovered with LSPC at 5.0 g/mL, the fluorescence polarization degree (p) of the membrane was similar to normal level, indicate that increased anisotropic by membrane oxidation decreased with LSPC.
2 Antioxidation Effect of LSPC on skin
LSPC can improve body antioxidation ability by increasing the antioxidase synthesis and its activity in skin and serum. In the skin and serum of rats fed LSPC at the dosage of 100mg/kg.bw for 35 days(ig.), MDA level was significantly lower than the comparison control group (p<0.05), while SOD and GSH-Px activity, Hyp level remarkably higher than the comparison control group (p<0.05). In the meantime, collagen and water content in skin increased by 2.7% and 3.8% respectively. All above indicate that LSPC postpones skin senescence by increasing antioxidase activity, collagen synthesis, water content and decreasing MDA formation.
3 Effect of LSPC on mice immune function
LSPC can adjust immune function effectively. Fed mice LSPC at the dosage of 30~ 90mg/kg.bw (ig.) continuously can remarkably increase its M $ cell activity, ear tumefaction rate induced by DNFB, half hemolysis value(HC50), which was obviously dosage-dependent; LSPC can activate the proliferation of T,B cells by MTT method analysis in vivo, which was dosage-dependent too. And also LSPC can activate NK cell noticeably.
4 Inhibition effect of LSPC on synthesize of tyrosinase and melanin
LSPC can significantly inhibit the synthesis of melanin. LSPC at the concentration of 7.8 10g/L~5.0g/L can inhibit tyrosinase activity. The inhibition rate decreased with the increasing of LSPC at 7.8 10g/L-.01g/L but increased with the increasing of LSPC at 0.1g/L~4.0g/L, t
主要研究结果如下：
1．莲房原花青素对大鼠红细胞膜维生素E含量及膜脂流动性的影响
LSPC是有效的羟基自由基清除剂和很强的天然抗氧化剂。对Fenton体系产生的羟基自由基有很强的清除作用；体系酸碱度、金属离子(Cu~(2+)、Ca~(2+)、Al~(3+)、Mg~(2+))对LSPC清除羟基自由基有很大影响。当有20mmol／L Al~(3+)存在时，使得LSPC的清除率从63.1％升高到81.6％。说明LSPC鳌合Cu~(2+)、Ca~(2+)、Al~(3+)、Mg~(2+)金属离子后，能使其清除羟基自由基的作用增效。
LSPC可有效防止红细胞膜中不饱和脂肪酸的过氧化，抑制膜中脂质过氧化产物MDA生成，保持细胞膜的完整性和功能；在保护红细胞膜中天然抗氧化剂V_E的同时，并使其再生。当LSPC浓度为5.0μg／mL时，红细胞受氧化损伤的膜得以恢复，其膜的荧光偏振度(p值)接近正常红细胞膜p值水平，从而降低了红细胞膜因氧化造成的各向异性增加。
2．莲房原花青素对皮肤的抗氧化作用研究
LSPC能加快皮肤和血清中抗氧化酶的生物合成和提高酶的活力，从而提高机体的抗氧化能力。给大鼠连续口服灌胃100mg／kg．bw LSPC 35d，血清和皮肤中MDA含量明显低于对照组(P＜0.05)；而SOD和GSH-Px酶活力显著高于对照组；皮肤和血清中Hyp含量也显著高于对照组(P＜0.05)，使皮肤中胶原蛋白和水分含量分别提高2.7％和3.8个百分点。提示，LSPC是通过提高皮肤抗氧化酶系统活力和降低MDA生成，增强胶原蛋白的生物合成及提高皮肤组织的保水性能来延缓皮肤衰老。
段玉清华中农业大学2004届博士学位论文
3.莲房原花青素对小鼠免疫功能的影响
连续灌胃30~gomg/k g.bw LSPC对小鼠体内腹腔巨噬细胞吞噬功能有较强
的增强作用，能使单核一巨噬细胞的吞噬能力明显提高:提高DNFB诱导的小鼠
耳肿胀率和小鼠半数溶血值(HC50)，且存在明显的剂量依赖关系;体外MTT
法测得LSPC能刺激T、B淋巴细胞增殖，并存在剂量一效应关系。半体内法测
得LSPC能显著激活NK细胞活性。表明，LSPC对免疫功能有较强的调节作用。
4莲房原花青素对酪氨酸酶及黑色素生物合成抑制作用初步研究
LsPC对黑色素的生物合成有显著的抑制作用。7.8 X10一49/L一5.09几LsPc
对酪氨酸酶活力有抑制作用，其中，7.8 X10“49/L一0.19/L时，随LsPC浓度增
大抑制率下降;0.lg/L~4.og/L时，随着LSPC浓度增大抑制率逐渐增高，最大
抑制率高达99.9%，且该抑制作用属于可逆的竞争性抑制。对多巴色素自动转化
黑色素有显著的抑制作用，其半抑制浓度ICS。1.7509/L，最大抑制率为69.8%。
5.莲房原花青素对皮肤黑色素瘤B16的抑制作用及其分子机制
LSPC具有抗黑色素瘤的作用。连续灌胃1 20mg/kg.bw LSPC14d抑瘤率达到
55.3%。用含25~100pg/mL的LSpC培养小鼠黑色素瘤Bx6细胞72h，可显著
抑制其生长增殖，对B16有细胞毒，半数抑制浓度(IC50)为32.4协g/mL，最
大抑制率高达84.5%。
LSPC抑制黑色素瘤细胞分裂增殖，机制有两点:
第一，LSPC可促进B16细胞凋亡。显微形态观察表明，LSPC能引起B16细
胞形态发生改变，使细胞缩小、核固缩，胞质凝缩，线粒体膨胀聚集，染色质
逐渐凝集边移，附在核膜周边:荧光强度明显增加。细胞凋亡分析，GO/GI期前
出现亚二倍峰;细胞周期分析，肿瘤细胞生长阻滞于S期。通过激光扫描共聚焦
显微镜的静态和动态观察，经100林g/mL LsPc处理B16细胞后，可使细胞内ca，+
堆积和重新分布，细胞核内ca2+荧光强度从133.1士24.a增加到187.8士29.2，达到
显著性差异(P<0.05)。这可能是LSPC促进B16细胞凋亡的机制之一;LSPC可下
调Bcl一2基因表达，抑制突变型p53基因和c一myc基因表达，这可能是LsPc诱导B16
细胞凋亡的另一重要机制。
第二，LsPc对B16细胞有分化诱导作用。体内连续灌胃1 20mg/k g.bw LSPC
和体外用100“岁mL LSPC培养B16细胞都能使B16瘤细胞出现功能和分化改
变，表现为酪氨酸酶活力分别提高28.5%和“.6%，黑色素生成能力分别提高
23.8%和44.4%;体外培养的B16细胞形态上出现分化改变，如细胞变大，变长，
多数细胞具有树突样结构，并向正常上皮样细胞分化。
一一一一一一一一一止墅巡竺墅过些鲍f究.
以上研究表明，LSPC对皮肤黑色素瘤B16有较强的抑制作用，其主要机制
是通过增加细胞
Proanthocyanidins from Lotus Seedpod(LSPC) show excellent performance in scavenging free radicals, anti-lipid peroxidation, adjusting serum lipid, and protecting the heart, which is confirmed by our lab's previous studies. And on this basis, the modern medical and molecular biological technology and pharmacological methods, as well as technical standards for testing and assessment for health food were used in the dissertation. From the aspects of Vitamin E content in cell membrane, cell membrane lipid fluidity, correlative enzymes activities and immunity adjustment, the protecting effect of LSPC on normal skin was discussed. Further more, its protection and repairing effects on damnification caused by tumor and radiation were investigated at animals level, cell level and molecular level in this paper. Specifically, the growth inhibition effect, inducing apoptosis effect of LSPC on melanoma B16 cell and its molecular mechanism were researched from the aspects of antioxidation, pathological morphology, immune
function, apoptosis and gene regulation. The studies provide theoretical foundation and scientific bases for using LSPC to develop medicines for preventing or curing skin melanoma and health food or cosmetics for protecting skin. Thoughts and methods to investigate protection mechanism of Proanthocyanidins on skin can also be as reference in further systematic study.
The following are the main results:
1. Effect of LSPC on the content of Vitamin E in red cell membrane of rats and on membrane lipid fluidity
LSPC is known as effective natural antioxidant and free radical scavenger ,which can clear up OH from Fenton system, while pH, concentration of metal ions(Cu2+ Ca2+ A13+ Mg2+) of the reaction system can affect the scavenging rate remarkably. LSPC chelated with metal ions can reinforce its free radical scavenging ability, as can be seen that the free radical scavenging rate of LSPC was 63.1% in the reaction system without
metal ions, and then increased to 81.6% in system with 20mmol/L A13+.
LSPC can effectively prevent unsaturated fatty acid (UFA) in red cell membrane from peroxidation as well as inhibit the formation of MDA and keep the integrity of cell membrane and its function. Meanwhile, it can protect the Vitamin E in cell membrane and show cooperating effect with Vitamin E in antioxidation. Red cell membrane damnified by oxidation can be recovered with LSPC at 5.0 g/mL, the fluorescence polarization degree (p) of the membrane was similar to normal level, indicate that increased anisotropic by membrane oxidation decreased with LSPC.
2 Antioxidation Effect of LSPC on skin
LSPC can improve body antioxidation ability by increasing the antioxidase synthesis and its activity in skin and serum. In the skin and serum of rats fed LSPC at the dosage of 100mg/kg.bw for 35 days(ig.), MDA level was significantly lower than the comparison control group (p<0.05), while SOD and GSH-Px activity, Hyp level remarkably higher than the comparison control group (p<0.05). In the meantime, collagen and water content in skin increased by 2.7% and 3.8% respectively. All above indicate that LSPC postpones skin senescence by increasing antioxidase activity, collagen synthesis, water content and decreasing MDA formation.
3 Effect of LSPC on mice immune function
LSPC can adjust immune function effectively. Fed mice LSPC at the dosage of 30~ 90mg/kg.bw (ig.) continuously can remarkably increase its M $ cell activity, ear tumefaction rate induced by DNFB, half hemolysis value(HC50), which was obviously dosage-dependent; LSPC can activate the proliferation of T,B cells by MTT method analysis in vivo, which was dosage-dependent too. And also LSPC can activate NK cell noticeably.
4 Inhibition effect of LSPC on synthesize of tyrosinase and melanin
LSPC can significantly inhibit the synthesis of melanin. LSPC at the concentration of 7.8 10g/L~5.0g/L can inhibit tyrosinase activity. The inhibition rate decreased with the increasing of LSPC at 7.8 10g/L-.01g/L but increased with the increasing of LSPC at 0.1g/L~4.0g/L, t
引文
1．国植，徐莉．原花青素：具有广阔发展前景的植物药[J]．国外医药·植物药分册，1996：11(5)．196-203
2．韩炯，李莹，刘新平等．葡萄籽提取物原花青素诱导乳腺癌MCF-7细胞脱落凋亡[J]．中草药，2003；34(8)：722-725
3．焦淑萍，陈彪，李佳．野生山葡多酚对小鼠组织的抗氧化作用研究[J]．中国公共卫生，2003a，19(5)：569-570
4．焦淑萍，姜虹，陈彪等．野生山葡萄皮渣多酚类物质对老龄大鼠脑脂褐素的影响[J]．北华大学学报(自然科学版)，2003b，4(2)：127-129
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7．凌智群，谢笔钧．莲房原花青素对氧自由基和脂质过氧化的作用[J]．营养学报2002a，24(2)：121-125
8．凌智群，谢笔钧．莲房原花青素抗氧化损伤作用的研究[J]．食品科学，2002b，23(7)：98-100
9．刘玉娟，钟进义．葡多酚对辐射后淋巴细胞凋亡的影响[J]．齐鲁医学杂志，2003，18(4)：373-374
10．陆茵，孙志广，赵万湘等．原花青素抗促癌物诱发H_2O_2释放及脂质过氧化[J]．中国药理学通报，2001，17(5)：562-565
11．吕丽爽，曹栋．葡萄籽中低聚原花青素的抗氧化性的研究[J]．食品科技，2000，4：41-42
12．舒啸尘，李悠慧，严卫星等．葡萄籽多酚在拟衰老模型小鼠中抗氧化功能的
研究[J]．卫生研究，2002，31(3)：191-192，196
13．张志军．葡萄酒的有效成分原花色素．红葡萄酒为什么对人体有益[J]．国外医学中医中药分册，2000，22(1)：18-20
14．张丁，张杰，孟光等．低聚原花青素及银杏黄酮对小鼠体内脂质过氧化和延缓果蝇衰老实验研究[J]．河南预防医学杂志，2002，13(5)：257-260
15．赵超英，姚小曼．葡萄籽提取物原花青素的营养保健功能[J]．中国食品卫生杂志，2000，12(6)：38-41
16．赵万洲，陆茵，阎新琦等．葡萄籽原花青素抗促癌作用的实验研究[J]．中草药，2000，3 1(12)：917-920
17．钟进义，栗世如．葡多酚与辐照对小鼠S180肉瘤的协同抑制作用研究[J]．癌变·畸变·突变，1999a，11(5)：264-266
18．钟进义和栗世如．葡多酚对小鼠DNA与染色体放射性损伤的防护作用[J]．中华劳动卫生职业病杂志，1999b，17(4)：211-213
19．朱振勤，翟万银，陈季武等．葡萄籽原花青素提取物抗氧化作用研究[J]．华东师范大学学报(自然科学版)，2003，1：98-102
20. Agarwal C,Sharma Y, Agarwal R. Studies of procyanidins on DU145 cells of human prostatic tauter[J]. Mol Carcinog,2000,28 (3) :129
21. Akyoshi R, Kimie K, Toshali Aet al., 日本公开特许公报94 336418
22. Bagchi D, et al., Oxygen Free Radical Scavenging Abilities of Vitamins C and E, and a Grape Seed Proanthocyanidin Extract In Vitro. Research Communications in Molecular Pathology and Pharmacology, 95:170-189, 1997
23. Bagchi D, et al., Recent Advances in Grape Seed Proanthocyanidin Research: Its Biological Role in Diverse Multicellular Organisms. 1999 SFRR Europe Winter Meeting on Bio-Flavonoids & Polyphenols in Health & Disease, Dinard, France, Abstract OP25, 1999
24. Bagchi D,et al., Oxygen free radical scavenging abilities of vitamins C and Vitamins E,and agrape seed proanthocyanidins extract in vitro research[J]. Commications in Molecular Pathology and Pharmacology, 1986,25:223
25. Bombardelli E,et al., Fitoterapia, 1995,56(4):297
26. Castillo J,Benavente G O,Alcaraz M et al., Antioxidant activity and radioprotective effects against chromosomal damage induced in vivo by X-rays of flavan-3-ols (proeyanidins) from grape seeds (Vitis vinifera): comparative study versus other phenolic and organic compounds. Journal of Agricultural & Food Chemistry[Washington, D.C. : American Chemical Society] , 2000, 48 (5): 1738-1745
27. Counet, Christine; Collin, Sonia. Effect of the number of flavanol units on the
antioxidant activity of procyanidin fractions isolated from chocolate. Journal of Agricultural and Food Chemistry,2003, 51 (23) : 6816-6822
28. Deters A,Dauer A, Schnetz E et al., High molecular compounds (polysaccharides and proanthocyanidins) from Hamamelis virginiana bark: influence on human skin keratinocyte proliferation and differentiation and influence on irritated skin. Phytochemistry. [Oxford:Elsevier Science Ltd],2001,58 (6):. 949-958
29. Dragsted, Lars O. Antioxidant actions of polyphenols in humans. International Journal for Vitamin and Nutrition Research ,2003,73 (2) : 112-119
30. Duenas M,Hernandez T, Estrella I et al., Phenolic composition and antioxidant activity of mocan seeds (Visnea mocanera L.f) [J]. Food Chemistry, 2003,82 (3) : 373-379
31. Gali H,et al.Planta Med, 1994, 60(3):236
32. Gerardo G M,Fernando C,José M D et al., Epicatechin, catechin, and dimeric procyanidins inhibit PMA-induced NF-κB activation at multiple steps in Jurkat T cells[M]. The FASEB Journal Express Article doi, 2003, 10
33. Hashimoto F, Ono M, Masuoka C et al., Evaluation of the anti-oxidative effect (in vitro) of tea polyphenols. Bioscience Biotechnology and Biochemistry, 2003, 67 (2) : 396-401
34. Huynh T H,Teel R W. Selective induction of apoptosis in human mammary cancer cell [MCF-7] by pycnogenol[J]. Anticancer Res,2000,20(4):2417
35. Katarina H,Juergen R, Silvia B et al., Hyperforin a constituent of St John,s wort(Hypericum perforatum L.)extract induces apoptosis by triggerin activation of caspases and with hypericin synergistically exerts cytotoxicity towards human malignant cell lines[J]. Earopean Jouranl of Pharmaceutics and Biopharmaceutics, 2003,56:121-132
36. Keiji Takagi, Tohru Mitsunaga .Tyrosinase inhibitory activity of proanthocyanidins from woody plants. Journal of Wood Science.Springer-Verlag Toky, 2003, Pages: 461-465
37. Koga T, Moro K,Nakumori et al. Increase of antioxidative potential of rat plasma by oral administration of proanthocyanidins-rich extract from grape seeds. J Agric Food Chem, 1999,47(5): 1892
38. Kobayashi MS,et al., Radical scavenger activity of three flavonoid metabolites studied by inhibition of chemiluminecence in human PMNS. Free Radic Res. 2000,32(2): 115
38. Maffei F R, Carini M,Akdinj G et al., Sparing effect of procyanidins from Vitis vinifera on vitamin E. Planta Mel ,1998,61(1) :343
39. Mao TK, Van De Water J,Keen C L et al., Cocoa Flavonols and Procyanidins Promote Transforming Growth Factor-β_1 Homeostasis in Peripheral Blood Mononuclear Cells [J]. Experimental Biology and Medicine,2003,228: 93-99
40. Narayanan N,Supriya M,Rarn C et al., Grape Seed Extract Activates Thl Cells In Vitro[J]. Clinical and Diagnostic Laboratory Immunology, 2002, 9(2):470-476
41. Laparra J,Michaud J,Masquelier J. Etude pharmacocinetique des. Oligomeres flavanoliques[J]. Plantes , edicinales, et Phytoterapie, 1977,11:133-142
42. Maritim A,Dene B A, Sanders R A et al., Effects of pycnogenol treatment on oxidative stress in streptozotocin-induced diabetic rats. Journal of Biochemical and Molecular Toxicology,2003,17 (3) : 193-199
43. Masqudier, Jack pycnogenols: Recent Advances in the Therapeutical Activity of procyanidins,Naturalproducts as Medicinal Agents,Beal,J.1,and Reinhard,E. Eds., Supplement of plant Medica, Journal of Medicinal plant.Research and Journal of Natural Roducts, Lloydia, July. 1980, pp.243-255
44. Masquelier J,Laparra J., Kadical scavenger Effbet(WE) of proanthocyanidins, Proan-thocyanidine. Et Radicaux Libres, 1985
45. Matito C,Mastorakou F, Centelles J et al., Antiproliferative effect of antioxidant polyphenols from grape in murine Hepa-1e1c7. J European Journal of Nutrition, 2003,42 (1) : 43-49
46. Mittal A., Elmets C A., Katiyar S K. Dietary feeding ofprocyanidins from grape seeds prevents photocarcinogenesis in mouse skin: Prevention of photocarcinogenesis associated with the inhibition of ultraviolet radiation-induced oxidative stress and cellular signaling. Journal of Investigative Dermatology ,2003,121 ( 1 ) : 1086
47. Noda Y, Anzai K,Mori A et al., Hydroxyl and superoxide anion radical scavenging activities of natural source antioxidants using the computerized JES-FR30 RSR spectrometer system. Biochem Mol Biol Int, 1997,42(1):35
48. Masaru O, Tooru K. 日本公开特许公报 94 256160
49. Oligomoric Proanthocyanidins Complexes: History, Structure,and phytopharmaceutical Application. Anme Marie Fine. CPA, No Candidatate 2000
50. Patent: US No 4698360
51. Patent: US 4797421 (1989)
52. Patent: WO973963
53. Patent: US05972999(1999)
54. Plumb O W et al., Antioxidant properties of catechins and proanthocyanidins: effect of polymerization,galloylation and glycosylation. Free Radic Res, 1998,
29(4):35
55. Porto P, Andreia L S,Laranjinha J. Antioxidant protection of low density lipoprotein by procyanidins: Structure/activity relationships. Biochemical Pharmacology, 2003,66 (6) : 947-954
56. Ray SD et al., IH636 Grape Seed Proanthocyanidin Extract (GSPE) ExPosure Significantly Attenuates Dimethylnitrosamine (DMN)-Induced Liver Cancer and Mortality in ICR Mice. 91st Annual Meeting of the American Association for Cancer Research, 41: Abstract 2928, 460, 2000
57. Roin R,Cascon E,Arola L et al., Procyanidins protect Fao cells against hydrogen peroxide-induced oxidative stress. Biochimica et Biophysica Acta. [Amsterdam : Elsevier Science B.V]. 2002.1572 (1) :25-30.
58. Shafiee M, Carbonneau M A, Urban, N et al., Grape and grape seed extract capacities at protecting LDL against oxidation generated by Cu~(2+), AAPH or SIN-1 and at decreasing superoxide THP-1 cell production. A comparison to other extracts or compounds. Free Radical Research, 2003,37 (5) : 573-584
59. Simonetti P, Ciapellano S,Gardana C et al., Procyanidins from Vitis vinifera seeds: in vivo effects on oxidative stress. Journal of Agricultural & Food Chemistry[Washington, D.C:American Chemical Society], 2002,50 (21): 6217-6221.
60. Takahata, Y, Ohnishi K M,Furuta S et al., Highly polymerized procyanidins in brown soybean seed coat with a high radical-scavenging activity. Report of National Food Research Institute,2003, 67 : 124
61. Tin M, Judy Van de Water, Carl L K et al., Cocoa Procyanidins and Human Cytokine Transcription and Secretion[J]. Journal of Nutrition, 2000,130: 2093-2099
62. Tixier J M et al., Biochemical.Pharmacology ,1984,33(24):3933-3939
63. Venna B et al., Oral freeze-drid forms of procyanidins. Pharm Acta Helv 1994,64(11):316
64. Ye X et al., Cytotoxic Effects of a Novel IH636 Grape Seed Proanthocyanidin Extract (GSPE) on Cultured Human Cancer Cells[J], Molecular and Cellular Biochemistry, 1999,196:99-108
62. Zhu Q Y, Schramm D D,Kwik U, Catherine L et al., Influence of cocoa flavanols on free radical-induced erythrocyte hemolysis. FASEB Meeting on Experimental Biology: Translating the Genome, April 11-15, 2003, San Diego, CA, USA
2．韩炯，李莹，刘新平等．葡萄籽提取物原花青素诱导乳腺癌MCF-7细胞脱落凋亡[J]．中草药，2003；34(8)：722-725
3．焦淑萍，陈彪，李佳．野生山葡多酚对小鼠组织的抗氧化作用研究[J]．中国公共卫生，2003a，19(5)：569-570
4．焦淑萍，姜虹，陈彪等．野生山葡萄皮渣多酚类物质对老龄大鼠脑脂褐素的影响[J]．北华大学学报(自然科学版)，2003b，4(2)：127-129
5．凌智群，谢笔钧等．莲房原花青素对大鼠实验性心肌缺血的保护作用[J]．中国药理学通报，2001a，17(6)：687-690
6．凌智群，谢笔钧等．莲房原花青素对家兔血脂及肝组织形态的影响[J]．天然产物研究与开发，2001b，13(4)：62-64
7．凌智群，谢笔钧．莲房原花青素对氧自由基和脂质过氧化的作用[J]．营养学报2002a，24(2)：121-125
8．凌智群，谢笔钧．莲房原花青素抗氧化损伤作用的研究[J]．食品科学，2002b，23(7)：98-100
9．刘玉娟，钟进义．葡多酚对辐射后淋巴细胞凋亡的影响[J]．齐鲁医学杂志，2003，18(4)：373-374
10．陆茵，孙志广，赵万湘等．原花青素抗促癌物诱发H_2O_2释放及脂质过氧化[J]．中国药理学通报，2001，17(5)：562-565
11．吕丽爽，曹栋．葡萄籽中低聚原花青素的抗氧化性的研究[J]．食品科技，2000，4：41-42
12．舒啸尘，李悠慧，严卫星等．葡萄籽多酚在拟衰老模型小鼠中抗氧化功能的
研究[J]．卫生研究，2002，31(3)：191-192，196
13．张志军．葡萄酒的有效成分原花色素．红葡萄酒为什么对人体有益[J]．国外医学中医中药分册，2000，22(1)：18-20
14．张丁，张杰，孟光等．低聚原花青素及银杏黄酮对小鼠体内脂质过氧化和延缓果蝇衰老实验研究[J]．河南预防医学杂志，2002，13(5)：257-260
15．赵超英，姚小曼．葡萄籽提取物原花青素的营养保健功能[J]．中国食品卫生杂志，2000，12(6)：38-41
16．赵万洲，陆茵，阎新琦等．葡萄籽原花青素抗促癌作用的实验研究[J]．中草药，2000，3 1(12)：917-920
17．钟进义，栗世如．葡多酚与辐照对小鼠S180肉瘤的协同抑制作用研究[J]．癌变·畸变·突变，1999a，11(5)：264-266
18．钟进义和栗世如．葡多酚对小鼠DNA与染色体放射性损伤的防护作用[J]．中华劳动卫生职业病杂志，1999b，17(4)：211-213
19．朱振勤，翟万银，陈季武等．葡萄籽原花青素提取物抗氧化作用研究[J]．华东师范大学学报(自然科学版)，2003，1：98-102
20. Agarwal C,Sharma Y, Agarwal R. Studies of procyanidins on DU145 cells of human prostatic tauter[J]. Mol Carcinog,2000,28 (3) :129
21. Akyoshi R, Kimie K, Toshali Aet al., 日本公开特许公报94 336418
22. Bagchi D, et al., Oxygen Free Radical Scavenging Abilities of Vitamins C and E, and a Grape Seed Proanthocyanidin Extract In Vitro. Research Communications in Molecular Pathology and Pharmacology, 95:170-189, 1997
23. Bagchi D, et al., Recent Advances in Grape Seed Proanthocyanidin Research: Its Biological Role in Diverse Multicellular Organisms. 1999 SFRR Europe Winter Meeting on Bio-Flavonoids & Polyphenols in Health & Disease, Dinard, France, Abstract OP25, 1999
24. Bagchi D,et al., Oxygen free radical scavenging abilities of vitamins C and Vitamins E,and agrape seed proanthocyanidins extract in vitro research[J]. Commications in Molecular Pathology and Pharmacology, 1986,25:223
25. Bombardelli E,et al., Fitoterapia, 1995,56(4):297
26. Castillo J,Benavente G O,Alcaraz M et al., Antioxidant activity and radioprotective effects against chromosomal damage induced in vivo by X-rays of flavan-3-ols (proeyanidins) from grape seeds (Vitis vinifera): comparative study versus other phenolic and organic compounds. Journal of Agricultural & Food Chemistry[Washington, D.C. : American Chemical Society] , 2000, 48 (5): 1738-1745
27. Counet, Christine; Collin, Sonia. Effect of the number of flavanol units on the
antioxidant activity of procyanidin fractions isolated from chocolate. Journal of Agricultural and Food Chemistry,2003, 51 (23) : 6816-6822
28. Deters A,Dauer A, Schnetz E et al., High molecular compounds (polysaccharides and proanthocyanidins) from Hamamelis virginiana bark: influence on human skin keratinocyte proliferation and differentiation and influence on irritated skin. Phytochemistry. [Oxford:Elsevier Science Ltd],2001,58 (6):. 949-958
29. Dragsted, Lars O. Antioxidant actions of polyphenols in humans. International Journal for Vitamin and Nutrition Research ,2003,73 (2) : 112-119
30. Duenas M,Hernandez T, Estrella I et al., Phenolic composition and antioxidant activity of mocan seeds (Visnea mocanera L.f) [J]. Food Chemistry, 2003,82 (3) : 373-379
31. Gali H,et al.Planta Med, 1994, 60(3):236
32. Gerardo G M,Fernando C,José M D et al., Epicatechin, catechin, and dimeric procyanidins inhibit PMA-induced NF-κB activation at multiple steps in Jurkat T cells[M]. The FASEB Journal Express Article doi, 2003, 10
33. Hashimoto F, Ono M, Masuoka C et al., Evaluation of the anti-oxidative effect (in vitro) of tea polyphenols. Bioscience Biotechnology and Biochemistry, 2003, 67 (2) : 396-401
34. Huynh T H,Teel R W. Selective induction of apoptosis in human mammary cancer cell [MCF-7] by pycnogenol[J]. Anticancer Res,2000,20(4):2417
35. Katarina H,Juergen R, Silvia B et al., Hyperforin a constituent of St John,s wort(Hypericum perforatum L.)extract induces apoptosis by triggerin activation of caspases and with hypericin synergistically exerts cytotoxicity towards human malignant cell lines[J]. Earopean Jouranl of Pharmaceutics and Biopharmaceutics, 2003,56:121-132
36. Keiji Takagi, Tohru Mitsunaga .Tyrosinase inhibitory activity of proanthocyanidins from woody plants. Journal of Wood Science.Springer-Verlag Toky, 2003, Pages: 461-465
37. Koga T, Moro K,Nakumori et al. Increase of antioxidative potential of rat plasma by oral administration of proanthocyanidins-rich extract from grape seeds. J Agric Food Chem, 1999,47(5): 1892
38. Kobayashi MS,et al., Radical scavenger activity of three flavonoid metabolites studied by inhibition of chemiluminecence in human PMNS. Free Radic Res. 2000,32(2): 115
38. Maffei F R, Carini M,Akdinj G et al., Sparing effect of procyanidins from Vitis vinifera on vitamin E. Planta Mel ,1998,61(1) :343
39. Mao TK, Van De Water J,Keen C L et al., Cocoa Flavonols and Procyanidins Promote Transforming Growth Factor-β_1 Homeostasis in Peripheral Blood Mononuclear Cells [J]. Experimental Biology and Medicine,2003,228: 93-99
40. Narayanan N,Supriya M,Rarn C et al., Grape Seed Extract Activates Thl Cells In Vitro[J]. Clinical and Diagnostic Laboratory Immunology, 2002, 9(2):470-476
41. Laparra J,Michaud J,Masquelier J. Etude pharmacocinetique des. Oligomeres flavanoliques[J]. Plantes , edicinales, et Phytoterapie, 1977,11:133-142
42. Maritim A,Dene B A, Sanders R A et al., Effects of pycnogenol treatment on oxidative stress in streptozotocin-induced diabetic rats. Journal of Biochemical and Molecular Toxicology,2003,17 (3) : 193-199
43. Masqudier, Jack pycnogenols: Recent Advances in the Therapeutical Activity of procyanidins,Naturalproducts as Medicinal Agents,Beal,J.1,and Reinhard,E. Eds., Supplement of plant Medica, Journal of Medicinal plant.Research and Journal of Natural Roducts, Lloydia, July. 1980, pp.243-255
44. Masquelier J,Laparra J., Kadical scavenger Effbet(WE) of proanthocyanidins, Proan-thocyanidine. Et Radicaux Libres, 1985
45. Matito C,Mastorakou F, Centelles J et al., Antiproliferative effect of antioxidant polyphenols from grape in murine Hepa-1e1c7. J European Journal of Nutrition, 2003,42 (1) : 43-49
46. Mittal A., Elmets C A., Katiyar S K. Dietary feeding ofprocyanidins from grape seeds prevents photocarcinogenesis in mouse skin: Prevention of photocarcinogenesis associated with the inhibition of ultraviolet radiation-induced oxidative stress and cellular signaling. Journal of Investigative Dermatology ,2003,121 ( 1 ) : 1086
47. Noda Y, Anzai K,Mori A et al., Hydroxyl and superoxide anion radical scavenging activities of natural source antioxidants using the computerized JES-FR30 RSR spectrometer system. Biochem Mol Biol Int, 1997,42(1):35
48. Masaru O, Tooru K. 日本公开特许公报 94 256160
49. Oligomoric Proanthocyanidins Complexes: History, Structure,and phytopharmaceutical Application. Anme Marie Fine. CPA, No Candidatate 2000
50. Patent: US No 4698360
51. Patent: US 4797421 (1989)
52. Patent: WO973963
53. Patent: US05972999(1999)
54. Plumb O W et al., Antioxidant properties of catechins and proanthocyanidins: effect of polymerization,galloylation and glycosylation. Free Radic Res, 1998,
29(4):35
55. Porto P, Andreia L S,Laranjinha J. Antioxidant protection of low density lipoprotein by procyanidins: Structure/activity relationships. Biochemical Pharmacology, 2003,66 (6) : 947-954
56. Ray SD et al., IH636 Grape Seed Proanthocyanidin Extract (GSPE) ExPosure Significantly Attenuates Dimethylnitrosamine (DMN)-Induced Liver Cancer and Mortality in ICR Mice. 91st Annual Meeting of the American Association for Cancer Research, 41: Abstract 2928, 460, 2000
57. Roin R,Cascon E,Arola L et al., Procyanidins protect Fao cells against hydrogen peroxide-induced oxidative stress. Biochimica et Biophysica Acta. [Amsterdam : Elsevier Science B.V]. 2002.1572 (1) :25-30.
58. Shafiee M, Carbonneau M A, Urban, N et al., Grape and grape seed extract capacities at protecting LDL against oxidation generated by Cu~(2+), AAPH or SIN-1 and at decreasing superoxide THP-1 cell production. A comparison to other extracts or compounds. Free Radical Research, 2003,37 (5) : 573-584
59. Simonetti P, Ciapellano S,Gardana C et al., Procyanidins from Vitis vinifera seeds: in vivo effects on oxidative stress. Journal of Agricultural & Food Chemistry[Washington, D.C:American Chemical Society], 2002,50 (21): 6217-6221.
60. Takahata, Y, Ohnishi K M,Furuta S et al., Highly polymerized procyanidins in brown soybean seed coat with a high radical-scavenging activity. Report of National Food Research Institute,2003, 67 : 124
61. Tin M, Judy Van de Water, Carl L K et al., Cocoa Procyanidins and Human Cytokine Transcription and Secretion[J]. Journal of Nutrition, 2000,130: 2093-2099
62. Tixier J M et al., Biochemical.Pharmacology ,1984,33(24):3933-3939
63. Venna B et al., Oral freeze-drid forms of procyanidins. Pharm Acta Helv 1994,64(11):316
64. Ye X et al., Cytotoxic Effects of a Novel IH636 Grape Seed Proanthocyanidin Extract (GSPE) on Cultured Human Cancer Cells[J], Molecular and Cellular Biochemistry, 1999,196:99-108
62. Zhu Q Y, Schramm D D,Kwik U, Catherine L et al., Influence of cocoa flavanols on free radical-induced erythrocyte hemolysis. FASEB Meeting on Experimental Biology: Translating the Genome, April 11-15, 2003, San Diego, CA, USA