磁珠固定化酶和LC-MS/MS筛选鉴定虎杖中的胰脂肪酶抑制剂

Screening and Identification of Pancreatic Lipase Inhibitors in Polygonum cuspidatum with Enzyme-Immobilized Magnetic Nanoparticles and LC-MS/MS

肥胖严重危害着人类的健康,各种减肥药物和替代疗法正在不断研发之中。本文首先结合单因素实验,利用响应面分析法,优化了羧基磁珠固定化胰脂肪酶合成条件,对其结构、稳定性、专一性及重复性进行了验证,然后通过该固定化酶筛选出了天然植物虎杖中的胰脂肪酶抑制剂,最后通过分子模拟对不同抑制类型抑制剂结合位置进行了预测。结果表明:缓冲液p H值7.48、PL浓度20.32 mg/m L、温度35.40℃为最佳合成条件,胰脂肪酶成功固定于羧基磁珠之上,且储存2周后酶活仍保持80%以上,可特异性筛选出阳性抑制剂,重复利用五次后酶活仍保持50%以上。从虎杖醇提水沉物中共筛选出6个胰脂肪酶抑制剂,经LC-MS/MS鉴定和混合标准品确认,共鉴定出4个胰脂肪酶抑制剂,其IC50值和抑制类型分别为大黄素(72.3±2.4μg/m L,非竞争性抑制)、白藜芦醇(195.5±3.6μg/m L,混合型抑制)、大黄素甲醚(168.8±3.4μg/m L,反竞争性抑制)和大黄素-8-O-β-D-葡萄糖(234.3±5.2μg/m L,非竞争性抑制),其中白藜芦醇、大黄素-8-O-β-D-葡萄糖是新发现的胰脂肪酶抑制剂。分子模拟预测出不同抑制类型抑制剂分别结合于胰脂肪酶的两个位置。

Obesity is seriously harmful to human health,therefore,a variety of anti-obesity drugs and alternative therapies are in constant development. In this study,pancreatic lipase immobilized on carboxylic acid-terminated magnetic nanoparticles synthesis conditions were optimized by response surface analysis firstly. Next,pancreatic lipase inhibitors were screened and identified from Polygonum cuspidatum. At last,binding locations of different type inhibitors and complex of pancreatic lipase and colipase were predicted. The results showed that the optimal synthetic conditions were pH 7. 48,PL20. 32 mg/mL and 35. 40 ℃,pancreatic lipase had been successfully immobilized on magnetic nanoparticles. And after 2weeks,the enzyme activity still remained over 80%,which can specifically screen out positive inhibitors. In addtion,the enzyme activity kept over 50% after it had been reused for five times. Altogther 6 inhibitors on pancreatic lipase had been screened out from P. cuspidatum,and 4 inhibitors were identified by LC-MS/MS and mixed stantand sample. The IC50 and inhibition types were respectively emodin（ 72. 3 ± 2. 4 μg/mL,non-competitive inhibition）,resveratrol（ 195. 5± 3. 6 μg/mL,mixed inhibition）,physcion（ 168. 8 ± 3. 4 μg/mL,anti-competitive inhibition） and emodin-8-O-β-Dglucose（ 234. 3 ± 5. 2 μg/mL,non-competitive inhibition）. In 4 inhibitors,resveratrol and emodin-8-O-β-D-glucose inhibitors were firstly discovered. Two binding location of different type inhibitors on pancreatic lipase and colipase complex were predicted.