α-葡萄糖苷酶是治疗糖尿病与肥胖症的关键靶标酶,抑制其活性可有效实现治疗作用。为高效发掘天然产物中靶向α-葡萄糖苷酶的抑制剂,该研究采用磁性纳米颗粒与α-葡萄糖苷酶结合的策略,选择性捕获潜在生物活性化合物,并利用超高效液相色谱-四极杆-飞行时间串联质谱(ultra-high performance liquid chromatography-quadrupole-time-of-flight tandem mass spectrometry, UHPLC-Q-TOF/MS)对捕获成分进行快速鉴定。通过单因素试验确定,当戊二醛体积分数为15%、α-葡萄糖苷酶浓度为7 U/mL、固定反应时间为4 h时,固定化酶的相对活性达最大值。基于优化后的固定化酶体系,经UHPLC-Q-TOF/MS鉴定从青钱柳(Cyclocarya paliurus)提取物中筛选出14种化合物,其中10种活性成分对α-葡萄糖苷酶具有较强的抑制作用,各活性化合物和阿卡波糖IC50值如下:异绿原酸A(870.3 μg/mL)、杨梅素-3-O-β-D-葡萄糖苷(123.9 μg/mL)、槲皮素-3-O-葡萄糖醛酸苷(587.2 μg/mL)、槲皮苷(320.9 μg/mL)、杨梅素(25.7 μg/mL)、金丝桃苷(394.1 μg/mL)、阿福豆苷(272.7 μg/mL)、山柰酚-3-O-β-D-吡喃葡萄糖醛酸苷(524.9 μg/mL)、山柰酚(21.6 μg/mL)、槲皮素(31.0 μg/mL)和阿卡波糖(0.62 μg/mL)。结果表明,固定化酶技术在药用植物潜在治疗化合物的高效发掘中具有重要应用前景。
α-Glucosidase is a key enzyme target for diabetes and obesity treatment.Inhibiting its activity can produce therapeutic effects.To efficiently discover natural product-derived α-glucosidase inhibitors, this study employed a strategy combining magnetic nanoparticles with α-glucosidase to selectively capture potential bioactive compounds, followed by rapid identification of captured components using ultra-high performance liquid chromatography-quadrupole-time-of-flight tandem mass spectrometry (UHPLC-Q-TOF/MS).Single-factor experiments determined optimal immobilization conditions:15% glutaraldehyde concentration, 7 U/mL α-glucosidase concentration, and 4 h immobilization time, achieving maximum relative enzyme activity.This optimized immobilized enzyme system was used to screen and identify 14 compounds from Cyclocarya paliurus extracts, the IC50 values of 10 active compounds and acarbose are as follows:isochlorogenic acid A(870.3 μg/mL), myricetin-3-O-β-D-glucoside(123.9 μg/mL), quercetin-3-O-glucuronide(587.2 μg/mL), quercitrin(320.9 μg/mL), myricetin(25.7 μg/mL), hyperoside(394.1 μg/mL), afzelin(272.7 μg/mL), kaempferol-3-O-β-D-glucuronopyranoside(524.9 μg/mL), kaempferol(21.6 μg/mL), quercetin(31.0 μg/mL), acarbose(0.62 μg/mL).The results demonstrate that immobilized enzyme technology holds strong potential for efficiently discovering therapeutic compounds in medicinal plants.
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