二仙汤抗骨质疏松有效组分的指纹图谱、多成分含量测定及其镇痛和调节骨代谢作用研究

Establishment of fingerprint and content determination of multi-components for antiosteoporotic active fraction from Er-xian decoction and research on its analgesic and bone metabolism regulating effects

目的建立二仙汤抗骨质疏松有效组分的指纹图谱及多成分含量测定方法,为基于二仙汤的抗骨质疏松组分中药的研制奠定基础。方法HPLC指纹图谱检测采用Agilent ZorBax Extend C18色谱柱(250 mm×4.6 mm,5μm);流动相为乙腈0.1%磷酸水溶液(含0.025%十二烷基硫酸钠),梯度洗脱:0~30 min,4%~30%乙腈;30~50 min,30%~50%乙腈;50~60 min,50%~90%乙腈;体积流量1 mL/min;柱温25℃;检测波长270 nm。运用“中药色谱指纹图谱相似度评价系统”进行相似度评价;通过对照品比对,对共有峰进行指认。样品含量测定的梯度洗脱调整为0~25 min,10%~18%乙腈;25~35 min,18%~19%乙腈;35~40 min,19%~25%乙腈;40~50 min,25%~32%乙腈;50~60 min,32%~55%乙腈;并测定8种有效成分的含量。以小鼠醋酸扭体和成骨细胞和破骨细胞为模型评价二仙汤有效组分的镇痛和对骨代谢的调控作用。结果建立了二仙汤有效组分的HPLC指纹图谱,确定了16个共有峰,16批样品与对照指纹谱比较的相似度均在0.9以上。指认出其中8个共有峰(1、4、10~12、13、15、16号峰),分别为新芒果苷、芒果苷、阿魏酸、仙茅苷、朝藿定B、淫羊藿苷、盐酸巴马汀和盐酸小檗碱。含量测定的线性关系、重复性、稳定性、精密度及加样回收率考察符合要求。二仙汤抗骨质疏松有效组分在小鼠醋酸扭体模型上具有显著的镇痛作用,并可促进成骨细胞的形成和骨基质矿化,抑制破骨细胞的骨吸收。结论建立的二仙汤抗骨质疏松有效组分HPLC指纹图谱及多成分含量测定方法可准确、高效、全面的对其质量进行评价,为其质量控制提供依据。

Objective To establish the fingerprint and multi-component content determination method of antiosteoporotic active fraction from Er-xian decoction,and lay a foundation for the development of antiosteoporotic component Chinese medicine based on Er-xian decoction.Methods A Agilent ZorBax Extend C18 column(250 mm×4.6 mm,5μm)was selected for HPLC fingerprint detection;the mobile phase is consist of acetonitrile-0.1%aqueous phosphoric acid solution(containing 0.025%sodium dodecyl sulfate);gradient elution:0—30 min,4%—30%acetonitrile;30—50 min,30%—50%acetonitrile;50—60 min,50%—90%acetonitrile;flow rate is 1 mL/min;column temperature is 25℃;detection wavelength is 270 nm.The“traditional Chinese medicine chromatography fingerprint similarity evaluation system”was used to evaluate the similarity;the common peaks were identified by comparison with reference compounds.The gradient elution for content determination was adjusted to:0—25 min,10%—18%acetonitrile;25—35 min,18%—19%acetonitrile;35—40 min,19%—25%acetonitrile;40—50 min,25%—32%acetonitrile;50—60 min,32%—55%acetonitrile and the contents of eight active components were determined.The analgesic effect of the effective components of Er-xian decoction and the regulation of bone metabolism were evaluated in mice with acetic acid twist body,osteoblasts and osteoclasts.Results The HPLC fingerprints of active fraction from Er-xian decoction were established,and 16 common peaks were identified.The similarity of 16 batches of samples comparison with reference fingerprint was more than 0.9.Eight common peaks(peaks 1,4,10—12,13,15,16)were identified as neomangiferin,mangiferin,ferulic acid,curculigoside,epimedin B,icariin,palmatine hydrochloride and berberine hydrochloride.The linear relationship,repeatability,stability,precision and recovery in content determination meet the requirements.The antiosteoporotic active fraction from Er-xian decoction had a significant analgesic effect in the mouse acetate twist body model,and increased the formation of osteoblasts and mineralization of bone matrix,and inhibited the bone resorption of osteoclasts.Conclusion The established HPLC fingerprint and multi-component content determination method of antiosteoporotic active fraction from Er-xian decoction could accurately,efficiently and comprehensively evaluate its quality,and provide a basis for its quality control.