目的 以大麻素2型受体（cannabinoid receptor type 2，CB2R）为靶点，从补肾中药覆盆子中分离、鉴定、筛选得到具有激动CB2R的激动剂，并探讨其抗骨质疏松作用的机制。方法 以激动CB2R活性为导向进行分离并筛选，采用正相硅胶柱色谱、凝胶柱色谱、大孔树脂、重结晶和半制备型高效液相色谱对二氯甲烷层和醋酸乙酯层浸膏进行分离，结合化合物理化性质和核磁共振波谱对分离得到的化合物进行结构鉴定。通过双荧光素酶方法筛选出具有可特异性激动CB2R的化合物，并通过测定转染CB2R的人胚胎肾细胞HEK293-CB2内环磷酸腺苷（cyclic adenosine monophosphate，cAMP）积聚水平和CB2R蛋白表达情况来验证筛选的化合物对CB2R的特异性激动活性，最后评价筛选得到的化合物对破骨细胞骨代谢方面的调节作用。结果 以激动CB2R为活性导向进行分离和筛选，共得到24个化合物，分别鉴定为山柰酚（1）、木犀草苷（2）、银椴苷（3）、金丝桃苷（4）、橙皮素（5）、刺芒柄花素（6）、槲皮素（7）、对羟基苯甲酸（8）、迷迭香酸（9）、对羟基苯甲酸乙酯（10）、水杨酸（11）、没食子酸（12）、咖啡酸（13）、原儿茶酸（14）、鞣花酸（15）、香草酸（16）、loliolide（17）、对羟基苯甲酸甲酯（18）、ethyl dioxindole-3-acetate（19）、苦莓苷F1（20）、科罗索酸（21）、委陵菜酸（22）、山楂酸（23）、野鸦椿酸（24）。双荧光素酶筛选体系从24个化合物中筛选得到2个新的CB2R激动剂（化合物5和8）。cAMP和蛋白免疫印迹表明筛选得到化合物5和8对CB2R具有特异性激动活性和较好的亲和力，可显著抑制破骨细胞的活性并影响其骨吸收功能。结论 化合物2、5、6、9、13、14、17、19为首次从中药覆盆子中分离得到。2个新的CB2R激动剂（化合物5和8）能特异性地激动CB2R并增加CB2R蛋白的表达，抑制给药刺激后HEK293-CB2细胞内cAMP的积累；以CB2R依赖性对破骨细胞功能的正常发挥产生影响。

Objective Cannabinoid type 2 receptor (CB2R) was used as a target to isolate, identify and screen agonists that stimulate CB2R from Fupenzi (Rubi Fructus), a traditional Chinese medicine for tonifying the kidney, and to explore the mechanism of its anti-osteoporosis effect. Methods Under the guidance of activating CB2R activity, dichloromethane layer and ethyl acetate layer extract were separated by normal phase silica gel column chromatography, microporous resin, recrystallization and semi-preparative high performance liquid chromatography. The structures of the isolated compounds were identified by physicochemical properties and nuclear magnetic resonance spectroscopy. The compounds which can specifically activate CB2R were screened by double luciferase method, and the activity of the selected compounds on the specific activation of CB2R was verified by measuring the accumulation level of cAMP and the expression of CB2R protein in HEK293-CB2. Finally, the regulatory effect of the selected compounds on bone metabolism of osteoclasts was evaluated. Results The isolation and screening was carried out using agonist CB2R, and a total of 24 compounds were obtained, which were identified as kaempferol (1), luteolin-7-O-glucoside (2), tiliroside (3), hyperoside (4), hesperetin (5), formononetin (6), quercetin (7), 4-hydroxybenzoic acid (8), rosmarinic acid (9), ethyl 4-hydroxybenzoate (10), salicylic acid (11), gallic acid (12), caffeic acid (13), protocatechuic acid (14), ellagic acid (15), vanillic acid (16), loliolide (17), methyl 4-hydroxybenzoate (18), ethyl dioxindole-3-acetate (19), nigaichigoside F1 (20), corosolic acid (21), tormentic acid (22), maslinic acid (23), and euscaphic acid (24). Two CB2R agonists (hesperetin and methyl 4-hydroxybenzoate) were screened from 24 compounds by double luciferase screening system. cAMP and Western blotting showed that the two compounds had specific activation activity and good affinity for CB2R, which could significantly inhibit the activity of osteoclasts and affect their bone resorption function. Conclusion Compounds 2, 5, 6, 9, 13, 14, 17, 19 were isolated from Rubi Fructus for the first time. Two new CB2R agonists (hesperetin and 4-hydroxybenzoic acid) can specifically activate CB2R and increase the expression of CB2R protein, inhibit the accumulation of cAMP in HEK293-CB2 cells after administration, and affect the normal function of osteoclasts in a CB2R-dependent manner.

R284.1

国家自然科学基金资助项目（81803794）；浙江中医药大学“5151远志人才工程”经费（No.112123A12201/001/004/019）