中药橘叶抗乳腺炎的药效机制研究及基于多策略数据挖掘系统的"谱－效"关系构建

Mastitis，one of highest incidence diseases for dairy cows，made a great economic loss for dairy breeding industry . Traditional Chinese veterinary often use Chinese herbal medicines extensively used for mankind to cure bovine mastitis, which restrict these herbs as veterinary medicines because of limited resources. China has largest planting area of citrus in the world, so citrus leavse are abundant, which had treated mastitis for more than 500 years with unacquainted mechanism and rarely used for human. Models based on the infection of live animals suffer from variations introduced by multifold interference factors. Therefore, we would establish inflammatory model in mammary gland epithelial cell for the study of mechanism of action of citrus leaves at cellular level. The varieties of citrus are various, causing chemical ingredients and efficacy of citrus leaves to change with the variety. Due to herbs' efficacy coming from chemical integrity, High Performance Liquid Chromatography (HPLC), High Performance Capillary Electrophoresis (HPCE), Gas Chromatography (GC) and structural determination coupling techniques would used in this research project to establish multiple and holistic chemical fingerprints of citrus leaves from different varieties. In addition, we would conform the chemical information to the efficacy information based on multi-strategy data mining system to construct spectrum-effect relationship at integral level, which would provide an assistance for determining medicinal quality of citrus leaves quickly and normatively.

乳腺炎是奶牛发病率最高的疾病之一，给奶牛养殖业造成了严重的经济损失。中兽医目前治疗该疾病所用的中药材多为人用大宗药材，因资源有限，限制了其作为兽药的使用。中国柑橘种植面积居世界首位，橘叶资源丰富，为人用小宗药材，治疗乳腺炎已有五百余年历史，但机制不明。鉴于以整体动物为模型的试验结果易受多种因素的干扰，本课题将通过小鼠乳腺上皮细胞炎症模型的建立，在细胞水平探讨橘叶的药效机制，可望为防治奶牛乳腺炎提供新的思路与药用资源。柑橘品种繁多，品种变化会引起橘叶的化学组分与功效随之而变。本课题将基于中药起效的整体性观念，采用HPLC、HPCE、GC等分析方法与结构测定联用技术，建立不同品种橘叶的多元整体性化学成分指纹表达谱。借助多策略数据挖掘系统，对橘叶的多元化学信息与多参数药效信息进行有效沟通与整合，从整体层次上构建能充分表达"谱-效"关系的数学模型，为快速、规范化确定不同品种橘叶的药用品质提供支持。

本项目从抗氧化、体外抑菌、对炎性奶牛乳腺上皮细胞的干预几个方面入手，探讨柑橘属植物叶抗乳腺炎的作用机制。体外抗氧化实验抑或HPLC-DPPH在线检测结果都提示柑橘属植物叶的抗氧化作用均不强。不同品种样品的体外抑菌效果有较大差异。采用MTT比色法考察了不同品种柑橘属植物叶提取物对奶牛乳腺上皮细胞的毒性作用及细胞发生炎症后的细胞活力。利用金黄色葡萄球菌的培养基构建了炎性奶牛乳腺上皮细胞模型，加入柑橘属植物叶提取物共培养后，提取细胞的总RNA，观察TNF-α、IL-1β、IL-6、SOD、ACACA、FASN、CSN2、CSN3等8种基因表达情况。还测定了在柑橘属植物叶提取物的干预下，TNF-α、NO、 IL-6、IL-1β等4种炎症细胞因子的表达水平。本项目采用HPLC、HPCE等技术获取了不同采集时间、不同采集地点、不同品种的柑橘属植物叶的化学成分表达谱，对某些品种进行了挥发油成分分析、HPLC指纹图谱研究或活性成分的含量测定。在此基础上，对“谱-效”关系进行了初步研究。