苦丁茶中苦味物质的鉴定及其化学性肝损伤保护作用研究

Ilex latifolia Thunb (I. latifolia), a medicine edible species, is a kind of traditional Chinese beverage with health care function. The unique bitter taste gives its unique medicinal value. However, so far the bitterness ingredients in I. latifolia are still not clear. Studies showed that much bitter taste traditional Chinese medicine and food have good effects of injured hepatocytes prevention and treatment. Besides, I. latifolia has been proved to have good hepatic protection after a long practice. Three bitter-tasting monomeric compounds that have obviously protective effect on injured hepatocytes, which had been isolated and identified from I. latifolia in our previous work. This project will further to isolate and identify the bitter active ingredients in I. latifolia comprehensively, and then intervene the injured hepatocytes, to investigate their influence on the protection of the injured hepatocytes, and at the same time, to analysis the hepatocyte’s gene expression level of the key enzymes and proteins and other regulators in different metabolic pathways, to determine the effect of the bitter active ingredients against the injured hepatocytes in energy metabolism and cell apoptosis, etc., so as to reveal the main pathways and regulators that bitter active ingredients would act on. This project will have the obvious originality in the research on the identification of bitterness ingredients from Ilex latifolia Thunb and its liver protection effect, providing scientific basis for the rational utilization of I. latifolia resources, and will show inspiration and demonstration significance to study and develop the nutrient composition of functional health food.

药食同源品种苦丁茶是我国传统的纯天然保健饮品，其成品茶特有的苦味口感赋予它独特的药用价值，然而苦丁茶中的苦味物质成分至今不明确。相关研究表明，许多苦味中药和苦味食品对肝功能损伤相关疾病颇有预防和治疗效果。苦丁茶经过长期实践证明具有良好的护肝保健功效。课题组前期从苦丁茶中获得三个具有苦味的单体化合物对肝损伤细胞有明显的保护作用。本研究进一步全面对苦丁茶中苦味物质进行分离和鉴定，用苦味物质对肝损伤细胞进行干预，考察苦味物质对肝损伤细胞保护的影响，同时分析肝细胞代谢途径中关键酶和蛋白等调控因子的基因表达水平，确定苦味物质对损伤肝细胞能量代谢和细胞凋亡等途径的作用效果，揭示苦味物质作用的主要途径和调控因子。本项目在苦丁茶中苦味物质的鉴定及其护肝研究方面有原创性，研究成果将为苦丁茶资源的合理利用提供科学依据，对我国功能保健食品中营养成分的研究与开发有示范借鉴意义。

苦丁茶是我国一种传统的纯天然保健饮品，同时也是卫生部公布的药食同源品种之一，具有抗衰老、解酒护肝、防治心血管疾病等多种功效，素有“保健茶”、“益寿茶”等美称。传统中医理论认为，苦味食物中苦味物质即是主要药效成分，有关苦丁茶中苦味物质所具有的保健功效一直未见报道，对其苦味成分及活性进行研究，为其在更多领域应用提供一定的理论依据，有利于苦丁茶资源的进一步开发。本项目建立了苦丁茶原材料的HPLC指纹图谱，能较好的反映出苦丁茶化学成分质与量的分布情况。本项目采用乙醇加热回流和有机溶剂分级萃取的方法提取苦丁茶中的有效成分，利用柱色谱和重结晶等手段对苦丁茶有效成分进行分离纯化，得到了3种苦味物质分别为：19-羟基熊果酸（PA）、3,4-O-二咖啡酰基奎宁酸甲酯、3,5-O-二咖啡酰基奎宁酸甲酯。本项目研究了苦丁茶中苦味化合物PA对人正常肝细胞HL-7702化学性损伤的保护作用，结果表明：PA对CCl4损伤后的HL-7702细胞的保护作用具有一定的剂量效应关系，在一定程度上能够抑制损伤细胞中的谷丙转氨酶（ALT）和谷草转氨酶（AST）活性、降低丙二醛（MDA）含量、提高超氧化物歧化酶（SOD）活力；PA对CCl4导致的HL-7702细胞凋亡有一定的保护作用，PA能有效的降低CCl4导致的HL-7702细胞发生凋亡，对CCl4导致的HL-7702细胞发生凋亡的保护作用可能是通过下调Caspase-3蛋白的表达来实现的。本项目对苦丁茶中苦味活性成分茶多酚抑制人肝癌Hep-G2细胞增殖及诱导其凋亡的作用进行了研究，结果表明：苦丁茶多酚对肝癌细胞的增殖有明显的抑制作用；苦丁茶多酚可以显著提高Hep-G2细胞内ROS含量，进而打破细胞内氧的代谢平衡来抑制肝癌细胞的生长和增殖；苦丁茶多酚通过下调抑凋亡蛋白Bcl-2的表达、上调促凋亡蛋白Bax的表达来诱导肝癌细胞发生凋亡。