基于肺纤维化模型的五味参芩汤配比筛选及提取工艺研究

Formulation Optimization and Extraction Technology of Wuwei Shenqin Decoction(五味参芩汤)Based on Pulmonary Fibrosis Model

目的:研究五味参芩汤的最佳提取工艺,基于肺纤维化模型研究最佳配伍。方法:采用高效液相色谱法(HPLC)和正交实验法探究五味参芩汤的最佳提取工艺,使用博来霉素诱导建立肺纤维化大鼠模型,研究五味参芩汤最佳配比。通过HE染色观察各组肺组织病理变化;采用DSI/BUXCO气道阻力和肺顺应性监测系统仪器,计算机实时监测并记录大鼠潮气量、气道阻力及动态肺顺应性;试剂盒检测肺纤维化模型大鼠血清中III型前胶原氨基端前肽(PIIINP)、基质金属蛋白酶7(MMP-7)、表面活性蛋白A(SP-A)、表面活性蛋白-D(SP-D)、涎液化糖链抗原6(KL-6)、血红素氧化酶-1(HO-1)及肺组织中相关指标羟脯氨酸(HYP)含量,计算各组综合评分以此从16组配比组合中筛选出最优配比。以五味参芩汤中人参皂苷Rg1、黄芩苷、五味子醇甲、阿魏酸、桔梗皂苷D的含量以及出膏率为综合考察指标,以乙醇浓度、液料比、煎煮时间和煎煮次数为考察因素设计出L9(3^(4))正交实验表,计算各组综合评分以此初步优选五味参芩汤的提取工艺。结果:与正常对照组比较,模型对照组潮气量、动态肺顺应性肺功能指标显著降低(P<0.01),气道阻力及PIIINP、MMP-7、SP-A、SP-D、KL-6、HO-1及HYP含量显著升高(P<0.01);与模型对照组比较,五味参芩汤给药16组潮气量、动态肺顺应性增加(P<0.05,P<0.01),气道阻力及PIIINP、MMP-7、SP-A、SP-D、KL-6、HO-1以及HYP含量明显降低(P<0.05或P<0.01)。五味参芩汤最佳配比为:人参9 g,黄芩15 g,五味子6 g,川芎12 g,桔梗9 g。最佳提取工艺为:10倍量90%乙醇,煎煮1.5 h,提取1次。结论:以博来霉素成功复制了肺纤维化大鼠模型,且五味参芩汤可以用于肺纤维化的治疗;筛选出了五味参芩汤最佳配比;建立了此方中主要成分含量测定的液相方法,且优选的提取工艺方法重现性、稳定性、精密度、加样回收均良好,方法稳定可靠。

Objective:To explore the optimal extraction process of Wuwei Shenqin Decoction(五味参芩汤,WSD),to screen the optimal proportion of WSD based on a rat model of pulmonary fibrosis.Methods:High-performance liquid chromatography(HPLC)and orthogonal assay were used to explore the optimal extraction process of WSD,and bleomycin was used to establish a rat model of pulmonary fibrosis to screen the optimal proportion of WSD.The pathological changes of lung tissues were observed by HE staining.The DSI/BUXCO airway resistance and lung compliance monitoring system was used to monitor and record the tidal volume,airway resistance,and dynamic lung compliance of rats in real time.Kits were used to detect the levels of procollagen type Ⅲ aminoterminal propeptide(PIIINP),matrix metalloproteinase 7(MMP-7),surfactant protein A(SP-A),surfactant protein D(SP-D),Krebs von den Lungen-6(KL-6),heme oxygenase-1(HO-1)in serum,and hydroxyproline(HYP)in lung tissue of model rats with pulmonary fibrosis.The comprehensive score of each group was calculated to select the optimal proportion from the 16 combinations.With the content of ginsenoside Rg1,baicalin,schisandrin A,ferulic acid,and platycodon D in WSD and the extraction rate as comprehensive indexes,the L9(3^(4))orthogonal experiment table was designed with ethanol concentration,liquid-to-material ratio,decocting time,and decocting times as factors,and the comprehensive score of each group was calculated to optimize the extraction process of WSD.Results:Compared with the results in the blank control group,the tidal volume and dynamic lung compliance of the model control group were significantly decreased(P<0.01),while airway resistance and the content of PIIINP,MMP-7,SP-A,SP-D,KL-6,HO-1,and HYP were significantly increased(P<0.01).Compared with the results in the model control group,the tidal volume and dynamic lung compliance were significantly increased(P<0.05,P<0.01),while the airway resistance and content of PIIINP,MMP-7,SP-A,SP-D,KL-6,HO-1,and HYP were significantly decreased(P<0.05,P<0.01).The optimal proportion of WSD was Ginseng Radix et Rhizoma 9 g,Scutellariae Radix 15 g,Schisandrae Chinensis Fructus 6 g,Chuanxiong Rhizoma 12 g,and Platycodonis Radix 9 g.The optimal extraction process was as follows:10 times the amount of 90%ethanol was used for decocting for 1.5 hours with once extraction.Conclusion:The rat model of pulmonary fibrosis was successfully established using bleomycin,and WSD can be used in the treatment of pulmonary fibrosis.In addition,the optimal proportion of WSD was selected,and the liquid-phase method for determining the main components in this formula was established.The optimal extraction process shows good reproducibility,stability,precision,sample addition,and recovery,and the method is stable and reliable.