Fang Ke-Xin, Sun Xi, Chen Liang-Ke, Wang Kun, Yang Chao-Jie, Mei Shan-Shan, Huang Chu-Ying, Yang Yao-Jun
School of Chinese Materia Medica, Beijing University of Chinese Medicine, Beijing 102488, China.
Molecules. 2025 Jul 30;30(15):3184. doi: 10.3390/molecules30153184.
Medicinal plants and animal-derived proteins represent valuable natural sources of bioactive components with pharmaceutical potential. Whilst some medicinal plants and animal-derived proteins also offer rich sources of anticoagulant bioactive peptides, their development faces multiple challenges: anticoagulant evaluation relies on single-parameter assays with limited reliability, native proteins demonstrate suboptimal activity without enzymatic treatment, and few researchers investigate bioavailable peptides. Our study establishes an innovative framework using the leech as a case study to overcome these barriers. A novel anticoagulant evaluation model was first established with the Critic-G1 weighting method. And we optimized the enzymatically hydrolyzed extracts with high activity using Box-Behnken response surface methodology. Subsequently, the everted gut sac model was implemented to simulate intestinal absorption and screen for absorbable peptide fractions. Furthermore, peptidomics was employed to identify the bioactive peptides. Lastly, we identified the bioactivity using anticoagulation assays. Results indicated that the optimal hydrolysis conditions were achieved with trypsin at 50.48 °C, an enzyme-to-substrate ratio of 6.78%, 7.51 h, and pH of 8.06. The peptide DLRWM was identified through integrated peptidomics and molecular docking approaches, with subsequent activity validation demonstrating its potent anticoagulant effects. This study has successfully identified a novel anticoagulant peptide (DLRWM) with confirmed intestinal absorption properties and provides a template for unlocking the pharmaceutical potential of medicinal animal proteins.
药用植物和动物源蛋白是具有药用潜力的生物活性成分的宝贵天然来源。虽然一些药用植物和动物源蛋白也提供了丰富的抗凝血生物活性肽来源，但其开发面临多重挑战：抗凝血评估依赖于可靠性有限的单参数测定，天然蛋白在未经酶处理时活性欠佳，且很少有研究人员对生物可利用肽进行研究。我们的研究以水蛭为案例建立了一个创新框架来克服这些障碍。首先用Critic-G1加权法建立了一种新型抗凝血评估模型。然后我们使用Box-Behnken响应面法优化了具有高活性的酶解提取物。随后，采用外翻肠囊模型模拟肠道吸收并筛选可吸收肽段。此外，利用肽组学鉴定生物活性肽。最后，我们通过抗凝血测定来鉴定生物活性。结果表明，在胰蛋白酶作用下，50.48℃、酶与底物比例为6.78%、7.51小时、pH值为8.06时可达到最佳水解条件。通过整合肽组学和分子对接方法鉴定出肽DLRWM，随后的活性验证证明了其强大的抗凝血作用。本研究成功鉴定出一种具有确认肠道吸收特性的新型抗凝血肽（DLRWM），并为挖掘药用动物蛋白的药用潜力提供了一个模板。
