基于“咸入肾"、"肾主骨”理论研究青娥丸中杜仲、补骨脂盐炙增效机理

The prescription of Qing’e Pills was originated from “Formularies of the Bureau of People's Welfare Pharmacies”, a classical works of TCM, which was compiled in the Song Dynasty. Its clinic effect on osteoporosis is positive and clear. Our previous research also revealed that after salt-processing, the anti-osteoporosis effect of Eucommia Bark and Scurfpea Fruit was significantly enhanced, nevertheless the mechanism still remained unclear. . In view of the fact that presently, the research of herb processing cannot meet the demand of its clinic usage, we proposed a hypothesis that after salt-processing, the dissolution and bio-availability of the main components of Eucommia Bark and Scurfpea Fruit, such as lignanoids, iridoids, coumarins and flavonoids etc. will be enhanced; while the main active principles achieve their anti-osteoporesis effect via Wnt/β-catenin and OPG/RANKL/RANK path way, a way that adjusts bone metabolism. . Guiding by the TCM theory “salty flavor entering the kidney” and “kidney be in charge of bones”, and with the help of advanced analytic equipment and molecular biological technology, this project will try to reveal the relationship between salt-processing of Eucammia Bark and Scurfpea Fruit with the anti-ostroporosis effect of Qing’e Pills by using comprehensive research methodology, which involves Crude herb processing, Chinese herbal property theory, Pharmacological activities, Active components and their ADME in animals. Hopefully, the results of this project can reveal the effect enhancing mechanism of salt-processing on Eucammia Bark and Scurfpea Fruit, so that can guide rational clinic usage of this 2 herbs and redevelop Qing’e Pills. In the meantime, we also hope to pave a new way for the research of Chinese crude drug processing in future.

青娥丸源于宋·《太平惠民和剂局方》，治疗骨质疏松症的疗效确切。课题组前期研究结果表明：杜仲、补骨脂盐炙增强了青娥丸抗骨质疏松药效，但增效机理不明确。针对中药炮制研究与中医临床用药脱节的现状，提出假说：“杜仲、补骨脂盐炙能提高其木脂素类、环烯醚萜类、香豆素类及黄酮类等成分的体外溶出和体内生物利用度；主要活性成分是通过Wnt/β-catenin和OPG /RANKL/RANK信号通路调节骨代谢，增强青娥丸抗骨质疏松功效”。课题在“咸入肾”、“肾主骨”中医药理论指导下，通过仪器分析技术及分子生物学手段，采用“中药炮制-药性理论-药理作用-效应成分-ADME”综合研究方法，揭示杜仲、补骨脂盐炙与青娥丸抗骨质疏松临床功效的相关性，阐明杜仲、补骨脂盐炙增效机理，指导临床合理用药及青娥丸的二次开发，开辟中药炮制研究新思路。

课题在“咸入肾”、“肾主骨”的中医药理论指导下开展：（1）通过UHPLC-Q-TOF/MS技术发现青娥丸盐炙品与生品的差异性成分为异补骨脂素、补骨脂定、补骨脂苷、补骨脂异黄酮醇、Corylifol A 及松脂醇二葡萄糖苷；（2）以去卵巢大鼠模型，验证青娥丸抗骨质疏松的药效作用，青娥丸盐炙品的抗骨质疏松作用较生品更为显著。通过青娥丸含药血清对成骨细胞、破骨细胞活性的影响研究，发现青娥丸生、盐品均有显著促进成骨细胞分化、抑制破骨细胞增殖的作用，且盐炙品作用强于生品；（3）利用各时间点大鼠含药血清中各成分的丰度值及其对成骨细胞增殖率、破骨细胞抑制率进行谱效相关性分析，发现京尼平苷酸、松脂醇二葡萄糖苷、补骨脂二氢黄酮和Corylifol A既有促进成骨细胞增殖又有抑制破骨细胞作用；（4）研究盐炙对青娥丸中活性成分在大鼠体内组织分布和代谢的影响，盐炙能促进青娥丸中补骨脂苷等12种成分靶向“肾经”（肾、子宫、卵巢、睾丸），且这12种成分在代谢过程中存在相互转化。（5）在整体动物、器官、细胞三个层次考察了盐炙对活性成分吸收的影响，发现盐炙促进了青娥丸中补骨脂苷、补骨脂素、异补骨脂素和京尼平苷酸等4种主要活性成分的吸收，从而增强疗效。（6）青娥丸中主要活性成分抗骨质疏松的作用机制为：补骨脂酚和补骨脂二氢黄酮通过上调Wnt/β-catenin信号通路促进成骨细胞的分化；而补骨脂二氢黄酮甲醚主要通过PI3k/AKT信号通路AKT磷酸化以及MAPK信号通路p38、JNK、ERK的磷酸化来抑制破骨细胞生成、分化及功能的发挥。.课题揭示了杜仲、补骨脂盐炙与青娥丸抗骨质疏松临床功效的相关性，阐明了杜仲、补骨脂盐炙增效机理，开辟了中药炮制研究新思路。