【作者】 段玉清；

【导师】 谢笔钧；

【作者基本信息】 华中农业大学 ， 农产品加工及贮藏工程， 2004， 博士

【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(Cu²⁺、Ca²⁺、Al³⁺、Mg²⁺)对LSPC清除羟基自由基有很大影响。当有20mmol／L Al³⁺存在时，使得LSPC的清除率从63.1％升高到81.6％。说明LSPC鳌合Cu²⁺、Ca²⁺、Al³⁺、Mg²⁺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/L⁴.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25¹00pg/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更多还原

【Abstract】 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 fluidityLSPC 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 withoutmetal 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 skinLSPC 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 functionLSPC 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 melaninLSPC 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更多还原