基于UHPLC-Q-TOF-MS/MS的升麻素苷在正常大鼠和牙周炎模型大鼠体内外代谢研究

In vivo and in vitro metabolism study of prim-O-glucosylcimifugin in normal rats and periodontitis model rats based on UHPLC-Q-TOF-MS/MS

目的采用超高效液相色谱-四极杆-飞行时间质谱联用技术(UHPLC-Q-TOF-MS/MS)并结合多种数据后处理技术分析鉴定升麻素苷在正常大鼠及牙周炎模型大鼠体内外代谢物,并比较代谢差异。方法收集正常大鼠和模型大鼠新鲜粪便,探究升麻素苷在肠道菌孵育体系中的体外代谢轮廓。正常及模型大鼠ig升麻素苷(100 mg/kg)后,分别于不同时间收集血浆、尿液、粪便、胆汁样本,合并相同样本,经适当方法进行处理后,采用COSMOCORE C_(18)柱(150 mm×2.1 mm,2.6μm),以0.1%甲酸水溶液(A)-甲醇(B)为流动相,梯度洗脱。在电喷雾离子源(electro-spray ionization,ESI)正离子模式下采集各生物样品的质谱数据,同时以右美沙芬为内标,采用峰面积相对定量法评估不同生物样品中代谢物的相对含量。结果初步鉴定出正常大鼠中代谢产物25个(其中I相代谢产物21个、II相代谢产物4个),牙周炎模型大鼠中代谢产物27个(其中I相代谢产物22个、II相代谢产物5个)。通过峰面积相对定量比较发现在不同病理状态下及不同生物样本中各代谢物含量存在显著差异,其中升麻素在各生物样品中的含量普遍较高。结论在不同病理状态下,同种化合物产生的代谢物种类及含量存在显著差异,模型动物的代谢研究更符合临床实际用药。升麻素可能是其在体内发挥抗炎、抗菌作用的药效物质。

Objective To analyze and identify the metabolites of prim-O-glucosylcimifugin between normal and periodontitis model rats both in vivo and in vitro by ultra-high performance liquid chromatography coupled to quadrupole time-of-flight mass spectrometry(UHPLC-Q-TOF-MS/MS)combined with various data post-processing techniques and compare the metabolic differences.Methods Fresh feces of normal and model rats were collected to explore the in vitro metabolic profile of prim-O-glucosylcimifugin in intestinal bacteria incubation system.After the normal and model rats were given prim-O-glucosylcimifugin(100 mg/kg),plasma,urine,feces and bile samples were collected at different times,and the same samples were combined.After sample pre-treatment,COSMOCORE C_(18) column(150 mm×2.1 mm,2.6μm)was used and 0.1%formic acid(A)-acetonitrile(B)solution were as the mobile phase for gradient elution.The mass spectrometry data of biological samples were acquired in positive ion mode with electro-spray ionization(ESI).At the same time,dextromethorphan was used as the internal standard to evaluate the relative content of metabolites in different biological samples by peak area relative quantitative method.Results A total of 25 metabolites were preliminarily identified in normal rats(including 21 phaseⅠmetabolites and four phaseⅡmetabolites),and a total of 27 metabolites in periodontitis model rats were identified(including 22 phaseⅠmetabolites and five phaseⅡmetabolites).Through the relative quantitative comparison of peak area,it was found that there were significant differences in the contents of metabolites in different pathological conditions and different biological samples,and the content of cimifugin was generally higher in all biological samples.Conclusion Under different pathological conditions,there are significant differences in the type and content of metabolites produced by the same compound,and the metabolic study of model animals is more in line with the clinical use of drugs.Cimifugin may be a pharmacological substance that exerts anti-inflammatory and antibacterial effects in vivo.