背景:从复杂的天然产物中筛选新型胰脂肪酶抑制剂是一项有意义的任务。
目的:通过准确筛选和分离金曲柳铁线莲中的胰脂肪酶抑制剂(C.tangutica),发现新的减肥先导化合物,加快藏药资源的开发利用。
方法:结合了亲和超滤和高效液相色谱-四极杆飞行时间质谱(AU-HPLC-QTOFMS)的综合策略,有针对性的分离,体外验证,并开发了分子对接技术来筛选丹氏梭菌的胰脂肪酶抑制剂。进行了AU-HPLC-QTOFMS技术,以寻找潜在的活性物质。大孔树脂,制备型液相色谱,并采用高速逆流色谱法对活性化合物进行准确、有针对性的分离。通过体外抑制实验检测目标化合物对胰脂肪酶的抑制活性。使用分子对接分析结合亲和力和结合位点。
结果:从唐氏梭菌中筛选出11种胰脂肪酶抑制物质。首次筛选出7种三萜皂苷作为脂肪酶抑制剂,并成功制备出纯度高于97%的三萜皂苷。TanguticosideB,铁皮糖苷J,hederosideH1和芦丁显示出更强的抑制作用,IC50值分别为1.539±0.048、1.661±0.092、1.793±0.069和1.792±0.094mmol/l。此外,它们对胰脂肪酶的最低ΔG值为-10.84,-9.97,-10.87和-9.39kcal/mol。
结论:使用AU-HPLC-QTOFMS的综合策略,有针对性的分离,体外验证,分子对接技术对于从唐氏梭菌中快速筛选和定向分离胰脂肪酶抑制剂是可行的。
BACKGROUND: Screening of novel pancreatic lipase inhibitors from complex natural products is a meaningful task.
OBJECTIVE: Through accurately screening and separating pancreatic lipase inhibitors from Clematis tangutica (C. tangutica), to discover new leading compounds for slimming and accelerate the development and utilization of Tibetan medicine resources.
METHODS: An integrated strategy that combines affinity ultrafiltration and high-performance liquid chromatography-quadrupole time-of-flight mass spectrometry (AU-HPLC-QTOFMS), targeted separation, in vitro validation, and molecular docking was developed to screen pancreatic lipase inhibitors from C. tangutica. The AU-HPLC-QTOFMS technique was performed to fish for the potential active substances. Macroporous resin, preparative liquid chromatography, and high-speed countercurrent chromatography were implemented for the accurate and targeted separation of active compounds. The inhibitory activities of target compounds to pancreatic lipase were detected by the inhibition experiments in vitro. The binding affinities and binding sites were analyzed using molecular docking.
RESULTS: A total of eleven kinds of pancreatic lipase inhibitory substances were screened from C. tangutica. Seven triterpenoid saponins were screened for the first time as lipase inhibitors and successfully prepared with purities higher than 97%. Tanguticoside B, clematangoticoside J, hederoside H1, and rutin showed stronger inhibitory effects with IC50 values of 1.539 ± 0.048, 1.661 ± 0.092, 1.793 ± 0.069, and 1.792 ± 0.094 mmol/l. Moreover, they have the lowest ΔG values of -10.84, -9.97, -10.87, and -9.39 kcal/mol to pancreatic lipase.
CONCLUSIONS: The integrated strategy using AU-HPLC-QTOFMS, targeted separation, in vitro validation, and molecular docking was feasible for rapidly screening and directionally isolating pancreatic lipase inhibitors from C. tangutica.