目的 研究達立通顆粒主要活性成分在正常和功能性消化不良(FD)模型大鼠胃腸道組織中的分布差異。方法 采用夾尾刺激結(jié)合不規(guī)律飲食雙因素干預(yù)方法建立FD模型。12只大鼠(對照、模型各6只)用于FD模型驗證,進行胃、小腸排空實驗,HE染色后進行胃腸組織病理學觀察;36只大鼠(對照、模型各18只)用于胃腸道組織差異分布研究,禁食不禁水18 h,按22.4 g·kg^-1劑量(8倍臨床等效劑量) ig給予達立通顆粒1次,于給藥后1、3、6 h收集各組大鼠的胃、十二指腸、空腸、回腸和結(jié)腸。應(yīng)用超高效液相色譜-串聯(lián)四級桿/線性離子阱質(zhì)譜(UPLC-QTRAP-MS/MS)法檢測各組織牡荊素-4''-O-葡糖苷、原阿片堿、黃連堿、小檗堿、甜橙黃酮、川陳皮素、桔皮素、去甲基川陳皮素、木香烴內(nèi)酯、去氫木香內(nèi)酯、原兒茶酸、傘形花內(nèi)酯、木犀草苷、橙皮苷、木犀草素、柚皮素、橙皮素、白楊素含量,采用ACQUITY HPLC^TMHSS T₃(50 mm×2.1 mm,1.8 μm)色譜柱,柱溫為40℃,0.1 %甲酸水-乙腈為流動相,梯度洗脫,體積流量為0.3 mL·min^-1,進樣量為2 μL。結(jié)果 與對照組比較,模型組大鼠體質(zhì)量顯著降低(P<0.001),胃、小腸排空率顯著降低(P<0.001),十二指腸腸黏膜的緊密連接被破壞,但未出現(xiàn)胃腸組織的器質(zhì)性病變,符合FD的病理特征。建立的UPLC-QTRAP-MS/MS方法中,各化合物線性關(guān)系良好,R² ≥ 0.990 0。對建立的檢測方法進行專屬性、精密度與準確度、提取回收率、穩(wěn)定性和基質(zhì)效應(yīng)等方面的考察,均符合要求。各成分在對照和模型組大鼠胃腸道組織中濃度差異明顯,且在各組織中達到最高濃度的時間點差異較大,模型組出現(xiàn)達峰時間延后的現(xiàn)象;與對照組比較,模型組大鼠胃組織中化合物的暴露水平降低,可能與FD引起胃腸道蠕動減慢有關(guān)。橙皮苷、橙皮素、柚皮素、原兒茶酸、去氫木香內(nèi)酯、川陳皮素、去甲基川陳皮素、小檗堿和桔皮素在胃、十二指腸、空腸、回腸和結(jié)腸中暴露水平較高,且在對照組和模型組中各時間點下的濃度差異較顯著。結(jié)論 經(jīng)方法學驗證,所建立的UPLC-QTRAP-MS/MS法靈敏、專屬性強、準確性高,適用于大鼠胃腸道組織中達立通顆粒主要活性成分的胃腸組織分布研究。達立通顆粒主要活性成分在正常和FD模型大鼠胃腸組織分布具有顯著性差異。

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

中國醫(yī)學科學院醫(yī)學與健康科技創(chuàng)新工程項目(CIFMS:2021-I2M-5-011);江蘇省前沿引領(lǐng)技術(shù)基礎(chǔ)研究專項擬立項目(BK20192005)