Evaluating Peptides of Picrorhiza kurroa and Their Inhibitory Potential against ACE, DPP-IV, and Oxidative Stress

Picrorhiza kurroa Royle ex Benth. is a high-altitude plant having great medicinal value. However, its medicinal value at the peptide level is still unknown, which limits its utility in the development of peptide-based therapeutics. Here, we identify 65 peptides fromP. kurroa hydrolysate. Sequence analysis suggests that one novel bioactive peptide, ASGLCPEEAVPRR (BP1), has antioxidant potential and shows angiotensin-converting enzyme (ACE) and dipeptidyl peptidase-IV (DPP-IV) inhibitory activities. The molecular docking study showed that BP1 has a lower binding energy and strong affinity toward active pockets of ACE and DPP-IV, which explains its higher ACE [IC50 = 59.90 ± 9.52 μg/mL (43.40 μM)] and DPP-IV [IC50 = 3.04 ± 0.26 μg/mL (2.2 μM)] inhibitory activities. BP1 protects HEK293 cells from H2O2-induced oxidative damage by inhibiting intracellular reactive oxygen species (ROS) and malondialdehyde accumulation and activating the intrinsic antioxidant defense system. Additionally, phase-contrast microscopy studies revealed that pre-treatment of BP1 to HEK293 cells before exposure to H2O2 retains the normal morphology and blocks apoptosis. Furthermore, it also suppresses ROS-induced mitochondrial apoptosis via restoring the mitochondrial membrane potential (ΔΨm) and inhibiting caspase 3/7 activity. Therefore, BP1 has antioxidant potential and ACE and DPP-IV inhibitory activities that could be used for peptide-based formulation(s) in pharmaceuticals to treat diabetes, cardiovascular diseases, and other diseases associated with ROS.胡黄连（Picrorhiza kurroa Royle ex Benth.）是一种高海拔药用植物，具有极高的药用价值。然而，其在肽层面的药用价值仍未明确，这限制了其在肽类治疗药物开发中的应用潜力。本研究从胡黄连水解物中鉴定出65种肽段。序列分析显示，其中一种新型生物活性肽ASGLCPEEAVPRR（BP1）具备抗氧化潜能，同时可抑制血管紧张素转换酶（ACE）与二肽基肽酶-IV（DPP-IV）的活性。分子对接研究表明，BP1与ACE和DPP-IV的活性口袋结合能更低、亲和力更强，这也解释了其较高的ACE抑制活性[IC₅₀ = 59.90 ± 9.52 μg/mL (43.40 μM)]与DPP-IV抑制活性[IC₅₀ = 3.04 ± 0.26 μg/mL (2.2 μM)]。BP1可通过抑制细胞内活性氧（ROS）与丙二醛积累、激活内源性抗氧化防御系统，保护HEK293细胞免受H₂O₂诱导的氧化损伤。此外，相差显微镜研究显示，在H₂O₂处理前预先用BP1孵育HEK293细胞，可维持细胞正常形态并阻断细胞凋亡。进一步研究发现，BP1还可通过恢复线粒体膜电位（ΔΨm）、抑制半胱天冬酶3/7活性，抑制ROS诱导的线粒体凋亡。综上，BP1兼具抗氧化活性与ACE、DPP-IV抑制活性，可用于开发肽类药物制剂，用于治疗糖尿病、心血管疾病及其他与ROS相关的疾病。