Peptide identification, molecular docking, and a zebrafish model of constipation were used to screen for peptides with laxative activity from an enzymatic hydrolysate of walnut meal. The most promising peptide, FGGDSTHPFN (FN-10), was further evaluated in a mouse model of constipation, and its physicochemical stability was characterized. The results revealed that 10 bioactive peptides with potential laxative effects were obtained, with FN-10 being the most effective among them. After administering FN-10 to constipated mice, we found that compared with the model group, the time to first black stool excretion of mice was significantly shortened by (46.93 ± 3.40)% and (34.65 ± 6.05)% (P < 0.05) in the low-dose (5 mg/kg m_b) and high-dose (30 mg/kg m_b) FN-10 groups, respectively. The number of fecal pellets at 5 h increased significantly by (102.78 ± 14.09)% and (43.98 ± 11.16)% (P < 0.05), respectively. The wet mass of feces increased significantly by (105.89 ± 14.23)% and (42.39 ± 15.12)% (P < 0.05), respectively, and the dry mass by (149.03 ± 23.00)% and (55.41 ± 17.22)% (P < 0.05), respectively. Additionally, FN-10 exhibited excellent physicochemical stability. Walnut peptide FN-10 is a natural bioactive peptide with laxative effects. This study provides a theoretical foundation for developing functional peptide products to alleviate constipation.

The blood-brain barrier (BBB) is a major challenge in drug delivery for the treatment of central nervous system diseases. Walnut derived peptide TWLPLPR (TW-7) has been proved to promote neuronal mitochondrial autophagy and enhance hippocampal neuronal synaptic plasticity, thereby improving learning and memory abilities in mice. We investigated the internalization mechanism and intracellular transport pathway for the walnut-derived peptide, TW-7, using bEnd.3 cells in an in vitro BBB model system. TW-7 was taken up by the bEnd.3 cells in a concentration-, temperature-, and energy-dependent manner; this involved increases in caveolin-1 and caveolin-2 protein expression and phosphorylation and inhibition of P-glycoprotein-mediated efflux. Subcellular localization of TW-7 in bEnd.3 cells was observed, indicating that the plasma membrane, endoplasmic reticulum, Golgi apparatus, lysosomes, and mitochondria participated in intracellular trafficking and that the peptide escaped from lysosomes over time. Caveolae may be critical for TW-7 uptake by brain microvascular endothelial cells, assisting TW-7 to cross the BBB. The results of this study provide a theoretical basis for the mechanism of active peptide penetrating the BBB, and provide a reference for developing neuroprotective active peptide products.

In study, we investigated the effect of treatment with combination of walnut peptides with molecular weight < 3 kDa and ginsenoside Rg1 (< 3 kDa + Rg1) on scopolamine-induced cognitive impairment in mice and the mechanism of brain-derived neurotrophic factor (BDNF)/tyrosine kinase B (TrKB)/cAMP response element-binding protein (CREB) signaling pathway in PC12 cells. In behavioral experiments, < 3 kDa + Rg1 treatment improved the memorizing ability of mice. Treatment with < 3 kDa + Rg1 significantly regulated the function of neurotransmitters and effectively improved the morphology of the neurons determined by hematoxylin and eosin (H&E), Nissl, and Golgi staining. Additionally, immunohistochemistry showed that the < 3 kDa + Rg1 treatment significantly decreased acetylcholinesterase (AChE) activity and increased choline acetyl transferase (ChAT) content in the hippocampus. The treatment upregulated vesicular acetylcholine transporter (VAChT), activated the BDNF/TrKB/CREB signaling pathway, improved the remodeling of dendritic spines, and enhanced cholinergic functions. In the scopolamine-induced PC12 cells, combination treatment increased thioredoxin-1 (Trx-1) expression after administering TrKB and activated signaling pathway. The results showed combination of < 3 kDa + Rg1 activated the BDNF/TrKB/CREB signaling pathway by regulating function of neurotransmitters and enhanced cholinergic function to decrease cognitive impairment.