【作者】 任丹丹；

【导师】 张声华；

【作者基本信息】 华中农业大学 ， 食品科学， 2007， 博士

【摘要】 菹草（Potamogeton crispus L．）是一种多年生沉水维管束植物，分布十分广泛。广大湖区的蛋鸭采食菹草后能产下优质的红心鸭蛋。已有研究表明，蛋黄色泽与饲料中类胡萝卜素的种类和含量密切相关。本研究室研究结果揭示，菹草中含有大量的类胡萝卜素，它不仅能加深蛋黄色泽，同时关于它的抗氧化、免疫调节、抗肿瘤等生物活性更是成为当今研究的热点之一。菹草作为一种新的类胡萝卜素资源，无论是其类胡萝卜素的结构分类研究、还是生物活性的系统探讨以及资源的深度开发利用均鲜见报道，因此深入研究菹草中类胡萝卜素将对菹草资源的开发利用具有重要的战略意义。本课题综合运用了色谱及波谱等现代仪器手段和化学分析方法，对菹草中类胡萝卜素的结构进行了分类研究，同时采用生物学前沿技术和手段，探讨了菹草类胡萝卜素的抗氧化活性及体内外抗肿瘤的作用与机制。研究结果如下：1菹草类胡萝卜素的结构鉴定与分类采用有机溶剂石油醚：丙酮（1∶1，v／v）提取菹草中类胡萝卜素，通过完全皂化处理得到菹草总类胡萝卜素TCPC。采用柱层析和薄层层析对TCPC进行分离纯化，得到组分y₁，y₂，y₃和y₄。为鉴定菹草类胡萝卜素y₁、y₂、y₃和y₄的化学结构，本文系统运用了色谱、波谱技术，结合化学分类反应，鉴定y₁，y₂，y₃和y₄分别为新黄质、紫黄质、叶黄素和β-胡萝卜素（菹草中红色素已由本实验室上届博士生鉴定为紫杉紫素的顺反异构体）。菹草中含有胡萝卜素和叶黄素两大类类胡萝卜素，主要为新黄质、紫黄质、叶黄素、紫杉紫素和β-胡萝卜素等五种类胡萝卜素。本文采用分光光度法测定了菹草中类胡萝卜素总含量（以干基计算）为230.8μg／g。用HPLC法测定了菹草中各种类胡萝卜素的相对含量（以干基计算）：菹草中β-胡萝卜素的含量为80.2μg／g；新黄质的含量为2.4μg／g；紫黄质的含量为24.8μg／g；叶黄素的含量为84.4μg／g；紫杉紫素的含量为36.0μg／g。2菹草类胡萝卜素的生物活性本文研究了菹草总类胡萝卜素TCPC和菹草紫杉紫素RPC的体外抗氧化能力，并从免疫学和细胞凋亡角度对菹草类胡萝卜素的抗癌活性进行了研究，探讨了类胡萝卜素抗癌的作用机理。（1）菹草类胡萝卜素体外抗氧化活性研究本实验通过对羟自由基、红细胞溶血、肝脏与肝线粒体中的脂质过氧化以及肝线粒体肿胀度的测定，结果发现菹草总类胡萝卜素TCPC能够有效地清除羟自由基，抑制由羟自由基引起的脂质过氧化，保护生物膜，减少红细胞溶血，减轻肝线粒体肿胀程度，提示TCPC具有很强的抗氧化能力。通过对TCPC和RPC在油脂中的抗氧化作用研究，结果发现在油脂自动氧化实验中单独使用RPC无抗氧化作用，而使用TCPC具有抗氧化作用。TCPC和RPC对VC的抗油脂氧化性能均有一定的协同增效作用，且TCPC的增效作用比RPC强。油脂光敏氧化实验结果表明，TCPC和RPC均可抑制由亚甲基蓝所引发的油脂光敏氧化，且抑制效果随着类胡萝卜素浓度的升高而增强。（2）TCPC对H₂₂荷瘤小鼠的抗肿瘤作用研究以肝癌H₂₂荷瘤小鼠为模型，分别灌胃5mg／kg．d、10mg／kg．d和20mg／kg．d TCPC 10d后均可显著抑制肿瘤的生长，其抑瘤率分别为37.4％，50.7％，52.3％；TCPC各剂量组均能显著提高荷瘤小鼠迟发型超敏反应的强度，促进T淋巴细胞增殖和NK细胞活性；增强肝脏中SOD的活性，降低MDA的含量；TCPC还可诱导小鼠肝癌细胞发生凋亡，将肿瘤细胞的细胞周期阻滞于G0／G1期。结果提示TCPC抗肿瘤的作用机理可能涉及到对机体免疫功能的调节、抗氧化作用以及诱导癌细胞发生凋亡，改变细胞周期等方面。（3）TCPC和RPC诱导人宫颈癌Hela细胞凋亡的研究首次提出TCPC和RPC诱导人宫颈癌Hela细胞凋亡是类胡萝卜素抗肿瘤活性的作用机制之一。其凋亡机制可能与改变细胞周期、降低线粒体跨膜电位、增加胞内钙离子浓度、增强iNOS活性，升高NO浓度等方面有关。MTT实验发现2.5～10μg／mL TCPC和5～20μg／mL RPC对Hela细胞的生长有显著的抑制作用，且具有时间-效应和剂量-效应关系。采用倒置显微镜、荧光显微镜、透射电镜和激光扫描共聚焦显微镜观察到TCPC和RPC作用4d后的Hela细胞呈典型的凋亡形态。与对照组比较，流式细胞仪检测到TCPC和RPC作用Hela细胞后使其细胞周期发生改变，线粒体跨膜电位降低。激光扫描共聚焦显微镜检测经TCPC和RPC处理4d后的细胞胞内Ca²⁺浓度显著升高。TCPC和RPC还可使HeLa细胞培养液中NO含量显著增加，NOS及iNOS活性显著增强，细胞膜中唾液酸含量明显降低。经国内外文献检索表明：以上有关菹草中类胡萝卜素的结构鉴定与分类、菹草总类胡萝卜素TCPC和菹草紫杉紫素RPC的抗氧化活性及抗肿瘤活性的研究均未见报道。更多还原

【Abstract】 Potamogeton crispus L. （P. crispus） is a submersed herbaceous perennial plant, which is distributed very widely. It was found that ducks fed with P. crispus could lay natural red-yolk eggs with good quality in vast lake areas. It has been proved that the color of egg yolk is closely related with the type and concentration of carotenoids in feed. The studies in our lab showed that there are abundant carotenoids in the P. crispus. Carotenoids not only can enhance the color of egg yolk, but also play an important role in health care such as antioxidation, immunity-regulation and antieaneer etc.. These bioaetivities have become one of Hotpoint in today’s researches. P. crispus is a new resource of carotenoids, while there are few reprots about the systematic researches of structure indentifieation, classification, bioaetivities and deep exploitation and use of its carotenoids at home and abroad. So it has very important strategic significance to make comprehensive studies on carotenoids of P. crispus for its exploitation and use.In this study, modern apparatus means of chromatogram and spectrum and the chemical analysis are used to research the structures and classification of carotenoids in P. crispus. And some advanced technologies of cellular and molecular biology were used to study the bioactivities （including antioxidation activity in vitro, anticancer activity both in vitro and in vivo） and anticancer mechartism of carotenoids. The results are as follows:1 Structure identification and Classification of carotenoids in P. crispusCarotenoids in P. crispus, were extracted with a mixture of acetone and petroleum ether （1:1, v/v）. Total. carotenoids from P. crispus （TCPC） were obtained after complete saponification by 40 % （w/v） KOH in methanol. Then fractions of y₁, y₂, y₃ and y₄ were separated and purified by column chromatography and thin layer chromatography （TLC）. In order to identify the structures of y₁, y₂, y₃ and y₄, technologies of chromatogram and spectrum combining with the chemical analysis were systemically used. The results showed that y₁, y₂, y₃ and y₄ were neoxanthin, violaxanthin, lutein andβ-carotene, respectively. （The red pigments which were identified by a graduated Ph.D. from our lab were rhodoxanthin and its cis-trans isomers.） So P. crispus contains two kinds of carotenoids, i.e. carotenes and xanthophylls, which include neoxanthin, violaxanthin, lutein, rhodoxanthin andβ-carotene.The content of total carotenoids in P crispus which was 230.8μg/g （dry weight） by spectrophotometer. The relative contents of various carotenoids in P. crispus （dry weight） were examined by means of HPLC. The results showed that there were 80.2μg ofβ-carotene, 2.4μg of neoxanthin, 24.8μg of violaxanthin, 84.4μg of lutein and 36.0μg of rhodoxanthin in per gram of P crispus respectively.2 Bioactivities of carotenoids in P. crispusTo clarify the bioactivities of total carotenoids （TCPC） and/or rhodoxanthin （RPC） from P. crispus, the test samples, animals and cells were exposed to TCPC and/or RPC respectively and the effects in antioxidation, antitumor activities and apoptosis in Hela cells were examined.（1） Antioxidation activities of carotenoids in P. crispus in vitroThe effects of TCPC on·OH, red blood cells （RBC） hemolysis, liver mitochondria swelling and lipid peroxidation in liver homogenate and liver mitochondria were studied in this part. The results showed that TCPC could eliminate .OH effectively and inhibit lipid peroxidation induced by·OH. It could protect the biomembrane, reduce RBC hemolysis and lighten the degree of liver mitochondria swelling. So TCPC had very strong antioxidation activities.The effects of TCPC and RPC on lipid peroxidation in oil were also studied. Only RPC had little antioxidation in both bean oil and rape oil, while TCPC could protect lipid against peroxidation in both bean oil and rape oil. Both TCPC and RPC had antioxidation effects in both oils when cooperating with VC. And the effect of TCPC was better than RPC. In the test of photosensitive oxidation of both oils, TCPC and RPC could inhibit the photosensitive oxidation induced by methylene blue. And the antioxidation effects increased with the increase of the concentration of TCPC and RPC.（2） Antitumor activities of TCPC on hepatocarcinoma H₂₂ tumor-bearing miceIn this research, the hepatocarcinoma H₂₂ tumor-beating mice were used as the model. After the mice were fed with TCPC at doses of 10mg/kg.d, 20mg/kg.d and 30mg/kg.d for 10d respectively, it was found that TCPC could obviously inhibit the growth of transplanted hepatoearcinoma H₂₂ tumor and inhibition rates of tumor are 37.4%, 50.7% and 52.3% respectively. TCPC of each group all increased the delayed-type hypersensitivity （DTH） response, promoted the T lymphocyte proliferation and NK cell activity significantly. The antitumor activities of TCPC were accompanied by the significant increase in SOD activity and decrease in MDA content. H₂₂ tumor cell apoptosis induced by TCPC and the change of tumor cell cycle were detected by FCM. The antitumor mechanism of TCPC might involve the enhanced cellular immune function, the improved antioxidant capability, apoptosis induced by TCPC and the change of tumor cell cycle in H₂₂ tumor-bearing mice.（3） Apoptosis induced by TCPC and RPC in Hela cellsIt was found for the first time that the apoptosis induced by TCPC and RPC in Hela cells （human cervical carcinoma） was one of anticancer mechanisms of carotenoids. In addition, the change of cell cycle, the decrease of mitochondrial transmembrane potential （MTP）, the increase of intracellular Ca²⁺ level and activity or content of NOS and NO might participate in the process of apoptosis induced by TCPC and RPC. 2.5-10μg/mL TCPC and 5-20μg/mL RPC could inhibit cell proliferation of Hela cells in a dose and time-dependent manner by MTT assay. After Hela cells were treated with TCPC and RPC for 4d, typical apoptotic morphological: changes could be observed by inverted microscope, fluorescence microscope, transmission electron microscope （TEM）and LSCM. After treatment of TCPC and RPC for 4d to Hela cells, compared with the control group, the change of cell cycle distribution and loss of mitoehondrial transmembrane potential were detected by FCM; intracellular Ca²⁺ level examined under LSCM increased obviously; NO content and NOS and iNOS activity in the culture supernatant of Hela cells increased and SA content in the cell membrane also increased.There are few reports about structure identification and classification of carotenoids in P. crispus, bioactivities including antioxidation and anticancer activities of TCPC and RPC until now.更多还原