目的 研究达立通颗粒主要活性成分在正常和功能性消化不良(FD)模型大鼠胃肠道组织中的分布差异。方法 采用夹尾刺激结合不规律饮食双因素干预方法建立FD模型。12只大鼠(对照、模型各6只)用于FD模型验证,进行胃、小肠排空实验,HE染色后进行胃肠组织病理学观察;36只大鼠(对照、模型各18只)用于胃肠道组织差异分布研究,禁食不禁水18 h,按22.4 g·kg^-1剂量(8倍临床等效剂量) ig给予达立通颗粒1次,于给药后1、3、6 h收集各组大鼠的胃、十二指肠、空肠、回肠和结肠。应用超高效液相色谱-串联四级杆/线性离子阱质谱(UPLC-QTRAP-MS/MS)法检测各组织牡荆素-4''-O-葡糖苷、原阿片碱、黄连碱、小檗碱、甜橙黄酮、川陈皮素、桔皮素、去甲基川陈皮素、木香烃内酯、去氢木香内酯、原儿茶酸、伞形花内酯、木犀草苷、橙皮苷、木犀草素、柚皮素、橙皮素、白杨素含量,采用ACQUITY HPLC^TMHSS T₃(50 mm×2.1 mm,1.8 μm)色谱柱,柱温为40℃,0.1 %甲酸水-乙腈为流动相,梯度洗脱,体积流量为0.3 mL·min^-1,进样量为2 μL。结果 与对照组比较,模型组大鼠体质量显著降低(P<0.001),胃、小肠排空率显著降低(P<0.001),十二指肠肠黏膜的紧密连接被破坏,但未出现胃肠组织的器质性病变,符合FD的病理特征。建立的UPLC-QTRAP-MS/MS方法中,各化合物线性关系良好,R² ≥ 0.990 0。对建立的检测方法进行专属性、精密度与准确度、提取回收率、稳定性和基质效应等方面的考察,均符合要求。各成分在对照和模型组大鼠胃肠道组织中浓度差异明显,且在各组织中达到最高浓度的时间点差异较大,模型组出现达峰时间延后的现象;与对照组比较,模型组大鼠胃组织中化合物的暴露水平降低,可能与FD引起胃肠道蠕动减慢有关。橙皮苷、橙皮素、柚皮素、原儿茶酸、去氢木香内酯、川陈皮素、去甲基川陈皮素、小檗碱和桔皮素在胃、十二指肠、空肠、回肠和结肠中暴露水平较高,且在对照组和模型组中各时间点下的浓度差异较显著。结论 经方法学验证,所建立的UPLC-QTRAP-MS/MS法灵敏、专属性强、准确性高,适用于大鼠胃肠道组织中达立通颗粒主要活性成分的胃肠组织分布研究。达立通颗粒主要活性成分在正常和FD模型大鼠胃肠组织分布具有显著性差异。

Objective The aim of this study was to explore the gastrointestinal tissue distribution differences of the main active compounds from Dalitong granules in normal and functional dyspepsia model rats. Method The rat model of FD was established by tail clipping stimulation combined with irregular diet (alternate day fasting). 12 rats (half of them as controls and half as models) were used to validate the FD model, and gastric and small intestinal emptying experiments were conducted. After HE staining, pathological observations were made on the gastrointestinal tissues. 36 rats (half as controls and half as models) were used for the study of differential distribution of gastrointestinal tissues, and they were fasted but not water-restricted for 18 hours. They were given Daliting Granules at a dose of 22.4 g·kg^-1 (8 times the clinical equivalent dose) once, and the gastric, duodenum, jejunum, ileum, and colon were collected from each group of rats at 1, 3, and 6 hours after the administration of the drug. The contents of vitexin-4'' -O-glucoside, protopine, coptisine, berberine, sinensetin, nobiletin, tangeretin, 5-O-demethylnobiletin, costunolide, dehydrocostuslactone, protocatechuic acid, umbelliferone, cynaroside, hesperidin, luteolin, naringin, hesperetin, and chrysin were detected by UPLC-QTRAP-MS/MS method in the gastric, duodenum, jejunum, ileum, and colon of each group of rats. UPLCQTRAP-MS/MS was used. Column was an ACQUITY HPLC^TM HSS T₃ (50 mm×2.1 mm, 1.8 μm) and set at 40℃. Gradient elution was performed using 0.1 % formic acid water and acetonitrile as the mobile phase, the flow rate was 0.3 mL·min-1 and the injection volume was 2 μL. Results Compared with the control group, the body mass of rats in the model group was significantly decreased (P < 0.001), the emptying rate of the stomach and small intestine was significantly decreased (P < 0.001), the close connection of the duodenal mucosa was destroyed, but no organic lesions of the gastrointestinal tissue were found, which was consistent with the pathological characteristics of FD. For the established UPLC-QTRAP-MS/MS method, the correlation between the response and concentration of the compounds in gastrointestinal tissues met R² ≥ 0.990 0. The results including the specificity, precision and accuracy, recovery rate, stability and matrix effects of the established method all met the requirements. The results showed that the concentration of the components varied greatly in the control group and the functional dyspepsia model group, and there were significant differences in the time points at which the highest concentration was reached in different tissues. In the control gastric tissue of rats, the tested compounds quickly reached the highest concentration within 1 h. However, the concentration of compounds decreased significantly, which is partially due to slow gastrointestinal motility in the model group. Hesperidin, hesperetin, naringin, protocatechuic acid, dehydrocostuslactone, nobiletin, 5-O-demethylnobiletin, berberine, and tangeretin were found to be exposed at higher levels in the stomach, duodenum, jejunum, ileum and colon, and the concentration differences between the control group and the model group at each time point were significant. Conclusion The established method has high sensitivity, specificity and high accuracy, which was suitable for the tissue distribution study of the main active compounds of Dalitong granules in the rat gastrointestinal tissues. Significant differences were showed in the tissue of the functional dyspepsia model rats compared to control.

R969.1

中国医学科学院医学与健康科技创新工程项目(CIFMS:2021-I2M-5-011);江苏省前沿引领技术基础研究专项拟立项目(BK20192005)