基于响应面法的加味塔布森⁃2水提工艺优化及体外药效考察

Optimization of water extraction process of modified Tabusen⁃2 based on response surface methodology and investigation of its in vitro efficacy

目的:采用Box⁃Behnken设计⁃响应面法优化加味塔布森⁃2水提工艺,并对其体外药效进行考察。方法:采用响应面法,以咖啡酸、京尼平苷酸2种成分的质量分数为综合得分,优选出浸泡时间、料液比、提取时间和提取次数的最佳值。将最优工艺下提取物与核因子⁃κB受体活化因子配体(receptor activator for nuclear factor⁃κB ligand,RANKL)诱导的RAW264.7细胞共培养5 d,随后以抗酒石酸酸性磷酸酶(TRAP)染色,显微镜下观察TRAP(mL·g^(-1))阳性细胞状态,评价提取物的体外药效。结果:选取提取时间、浸泡时间以及料液比为考察因素,以咖啡酸和京尼平苷酸含量的综合评分为评价指标,经响应面法优化,得到的最佳提取条件:浸泡时间为540 min,料液比为1∶12(g·mL^(-1)),提取时间为44 min,提取次数为2次。此条件下所得的提取物对RANKL诱导的RAW264.7细胞有明显抑制作用,可减少TRAP阳性细胞,并抑制多核生成。结论:运用响应面分析法优选的加味塔布森⁃2水提工艺重复性好、稳定可行,且具有良好的体外药效活性,为后期体内药效实验研究奠定可靠的基础。

Objective:To optimize the water extraction process of modified Tabusen⁃2 by response surface methodology(Box⁃Behnken)and investigate its in vitro efficacy.Methods:Taking the mass fractions of caffeic acid and geniposide as the comprehensive score,response surface methodology was used to optimize the soaking time,material⁃to⁃liquid ratio,extraction time and extraction times.The RAW 264.7 cells induced by nuclear factor⁃κB receptor activator ligand(RANKL)were co⁃cultured with the optimal extracts for 5 d,and then treated with tartrate⁃resistant acid phosphatase(TRAP).The TRAP⁃positive cells were stained and observed under a microscope to evaluate the in vitro efficacy of the extract.Results:The extraction time,soaking time,and material⁃to⁃liquid ratio were selected as the inspection factors,and the comprehensive score of caffeic acid and geniposide content was used as the evaluation index.After optimization by response surface method,the optimal extraction condition was 540 min;the liquid ratio was 1∶12(g·mL^(-1));the extraction time was 44 min,and the extraction times were two times.The extract obtained under this condition had a significant inhibitory effect on RAW264.7 cells induced by RANKL,could reduce TRAP⁃positive cells,and inhibited multinucleation.Conclusion:The water extraction process of modified Tabusen⁃2 optimized by response surface analysis method is reproducible,stable and feasible,and has good in vitro pharmacodynamic activity,laying a reliable foundation for future in vivo pharmacodynamic experimental research.