Qin Luping, Han Ting, Zhang Qiaoyan, Cao Dapeng, Nian Hua, Rahman Khalid, Zheng Hanchen
School of Pharmacy, Second Military Medical University, 325 Guohe Road, Shanghai 200433, PR China.
J Ethnopharmacol. 2008 Jul 23;118(2):271-9. doi: 10.1016/j.jep.2008.04.009. Epub 2008 Apr 15.
Er Xian Decoction (EXD), a traditional Chinese medicine formula, has long been used for the treatment of osteoporosis and menopausal syndrome in China. The present study was designed to investigate the antiosteoporotic constituents of EXD, and evaluate their antiosteoporotic effects in ovariectomized rats.
Osteoblasts in neonatal calvaria cultures and osteoclasts derived from rat marrow cells were used to bioactivity-guided screen the active constituents. The proliferation of osteoblast was assayed by MTT methods. The activity of ALP and TRAP was measured by p- nitrophenyl sodium phosphate assay. The antiosteoporotic effects of icariin (1), anemarsaponin B II (8) and berberine (6) were verified by using OVX rats model. The bone mineral density (BMD) was measured by dual-energy X-ray absorptiometry using the small animal scan mode. The undecalcified longitudinal proximal tibial metaphysical (PTM) sections were cut and stained for the bone histomorphometric analysis.
Bioactivity-guided fractionation has led to the successful isolation of antiosteoporotic constituents, i.e., icariin (1), icariside I (2), baohuoside I (3), mangiferin (4), neomangiferin (5), berberine (6), anemarsaponin B (7), anemarsaponin BII (8), anemarsaponin C (9), anemarrhenasaponin I (10), rubiadin-1-methyl ether (11) and obaculactone (12) from EXD. Further study showed that icariin (1), anemarsaponin BII (8) and berberine (6) increased the BMD in ovariectomized rats, and icariin (1) not only increased the bone formation, but also inhibited bone resorption; anemarsaponin BII (8) mainly increased bone formation and berberine (6) only inhibited the bone resorption in ovariectomized rats.
Our findings demonstrate that multiple ingredients are responsible for antiosteoporotic activity in traditional Chinese medicine formula Er-Xian decoction.
二仙汤（EXD）是一种中药方剂，在中国长期用于治疗骨质疏松症和更年期综合征。本研究旨在研究二仙汤的抗骨质疏松成分，并评估其对去卵巢大鼠的抗骨质疏松作用。
采用新生颅骨培养的成骨细胞和大鼠骨髓细胞来源的破骨细胞进行生物活性导向筛选活性成分。采用MTT法检测成骨细胞的增殖。采用对硝基苯磷酸钠法检测碱性磷酸酶（ALP）和抗酒石酸酸性磷酸酶（TRAP）的活性。采用去卵巢大鼠模型验证淫羊藿苷（1）、朝藿定B II（8）和小檗碱（6）的抗骨质疏松作用。采用小动物扫描模式通过双能X线吸收法测量骨密度（BMD）。切取未脱钙的胫骨近端干骺端（PTM）纵切片并进行染色，用于骨组织形态计量学分析。
通过生物活性导向分离，成功从二仙汤中分离出抗骨质疏松成分，即淫羊藿苷（1）、淫羊藿次苷I（2）、宝藿苷I（3）、芒果苷（4）、新芒果苷（5）、小檗碱（6）、朝藿定B（7）、朝藿定B II（8）、朝藿定C（9）、知母皂苷I（10）、茜草素-1-甲醚（11）和澳咔内酯（12）。进一步研究表明，淫羊藿苷（1）、朝藿定B II（8）和小檗碱（6）可增加去卵巢大鼠的骨密度，淫羊藿苷（1）不仅增加骨形成，还抑制骨吸收；朝藿定B II（8）主要增加骨形成，而小檗碱（6）仅抑制去卵巢大鼠的骨吸收。
我们的研究结果表明，中药方剂二仙汤中的多种成分具有抗骨质疏松活性。
