尾巨桉树皮中防治福寿螺的先导化合物的发现及其作用机理研究

Guangxi, the largest province of eucalyptus plantations in China. Eucalyptus bark, a waste product from the wood board industry and pulp industry, were studied as source of bioactive compounds. These by-products could be re-used to reduce their environmental impact and also to obtain an economic benefit. Howtever, eucalyptus barks were thrown away or simply burned for energy production in China. Our previously studies suggested that the ethanol extract of the barks of Eucalyptus urophylla×Eucalyptus grandis exhibited potent molluscicidal activity against Pomacea canaliculata. However, the chemical constituents in the bark of E. urophylla×E. grandis were still not investigated, which is worthy of further investigation. A procedure based on bioactivity screening and chemical screening was designed to investigate the novel bioactive constituents from the barks of E. urophylla×E. grandis. The bark was extracted with ethanol and compounds in the active fractions were isolated and purified by silica gel, Sephadex LH-20 column chromatographies, and preparative HPLC. Chemical structures of compounds were elucidated by physicochemical properties and spectroscopic analysis. To study the action mechanism of the bark extract and active compounds, such as ultrastructure change, carbohydrate change, amino acid and protein change, enzymatic activity change, apoptosis of liver cell, proteome analysis on differential expression of liver proteins, identification of different expression genes of liver. This study provided theory basis to researching green molluscicides of plant origin for control Pomacea canaliculata, and provide a compound synthesis template for new molluscicides creating. It was also a significant attempt to re-use the eucalyptus barks from wood board industry.

广西是我国桉树种植面积最大的省，树皮是其加工业产生的主要废料，国外已有研究证明树皮是可重复利用的生物资源，含有多种活性成分，但在中国尚未得到很好的利用，不是废弃就是被焚烧，造成极大的资源浪费和环境污染。在前期研究中我们发现尾巨桉树皮的乙醇提取物对福寿螺有较强的毒杀活性，但其化学成分至今未有研究。本项目在前期研究基础上，拟采用活性筛选和化学筛选相结合的方法，对尾巨桉树皮中的活性成分进行靶向分离，利用现代波谱技术及化学方法鉴定活性化合物的结构并研究其构效关系，从中筛选出结构新颖的先导化合物；从福寿螺超微结构、总蛋白质和糖原含量、酶活性、细胞凋亡、蛋白质差异表达、基因差异表达等方面深入研究活性成分的作用机理，为研发新的植物源杀螺剂提供科学依据。本研究通过筛选桉树树皮中的活性先导化合物，为新农药的创制提供了化合物合成模版，同时，为桉树树皮废物开发再利用做出了有意义的探索。

广西是我国桉树种植面积最大的省，树皮是其加工业产生的主要废料，是可重复利用的生物资源，含有多种活性成分，但在中国尚未得到很好的利用。尾巨桉是广西目前主要种植的树种，在前期研究中我们发现尾巨桉树皮提取物（EBE）对福寿螺有较强的毒杀活性，但其化学成分及杀螺作用机理至今未有研究。本项目采用活性筛选和化学筛选相结合的方法，对尾巨桉树皮中的化学成分进行分析和结构鉴定。从分析结果来看，自然脱落树皮和新鲜树皮中总三萜含量为11.70%和4.01%，总黄酮含量分别为6.41%和2.95%，总单宁含量分别为5.05%和6.49%。UPLC-Q-TOF-MS分析结果表明，尾巨桉树皮中主要含有齐墩果酸、熊果酸、betulinic acid、betulonic acid及其衍生物。并从福寿螺总蛋白质含量、酶活性、蛋白质差异表达、基因差异表达等方面深入研究EGE及化学成分的作用机理。研究发现，EBE能明显抑制福寿螺头足中LDH、AKP 和 AST/GOT 的活性以及肝脏中 ALT/GPT 和 AST/GOT 的活性，显著提高福寿螺肝脏中SOD、CAT、GSH-Px活性和MDA水平以及头足中的GSH-Px活性，且未发现该提取物具有任何毒副作用。此外，尾巨桉树皮中齐墩果酸能提高福寿螺肝脏和头足中CAT、T-SOD和GSH酶活性，提高肝脏AST/GOT酶活性，升高头足中AChE酶活性，降低了头足中NOS酶活性。齐墩果酸胁迫下福寿螺蛋白表达差异，共鉴定出274个DEPS，其中上调168个，下调106个。参与了糖代谢、能量合成和蛋白质合成的破环，并触发了一系列的包含许多参与免疫过程的关键蛋白的分子途径，以保护福寿螺抵御齐墩果酸的胁迫。综上，本研究明确了EBE具有显著的杀螺活性，明确了其主要化学成分，初步探索了EBE及其化学成分杀螺作用机理，为其在植物源杀螺剂方面的应用提供实验及理论依据。