Characterization, mechanisms, structure-activity relationships, and antihypertensive effects of ACE inhibitory peptides: rapid screening from sufu hydrolysate.

This study investigates the characterization, mechanisms of action, structure-activity relationships, and in vivo antihypertensive effects of ACE inhibitory peptides derived from sufu hydrolysate following simulated gastrointestinal digestion. Sufu was enzymatically digested using pepsin, trypsin, and chymotrypsin to mimic gastrointestinal conditions, followed by ultrafiltration to fractionate the peptides based on molecular weight. The fraction under 1 kDa exhibited the highest ACE inhibitory activity. LC-MS/MS analysis identified 119 peptide fragments, with bioinformatics screening highlighting 41 peptides with potential ACE inhibitory properties. Among these, two peptides, AWR and LLR, were selected and synthesized for in vitro validation, displaying IC50 values of 98.04 ± 2.56 µM and 94.01 ± 5.07 µM, respectively. Stability tests showed that both peptides maintained their ACE inhibitory activity across various temperatures and pH levels. Molecular docking and Highest Occupied Molecular Orbital analysis indicated strong binding interactions between these peptides and ACE, with the second-position tryptophan in AWR and the N-terminal leucine in LLR identified as key bioactive sites. These findings were further supported by molecular dynamics simulations, which confirmed the stability of the peptide-ACE complexes. In vivo studies using spontaneously hypertensive rats demonstrated significant reductions in both systolic and diastolic blood pressure, indicating that AWR and LLR have strong antihypertensive potential. This study illustrates that ultrafiltration, combined with LC-MS/MS and bioinformatics analysis, is an effective approach for the rapid screening of ACE inhibitory peptides. These results not only enhance our understanding of sufu-derived peptides but also offer promising implications for hypertension management.

A novel ACE inhibitory peptide from Douchi hydrolysate: Stability, inhibition mechanism, and antihypertensive potential in spontaneously hypertensive rats.

Angiotensin I-converting enzyme (ACE) regulates blood pressure through the renin-angiotensin system. Douchi, a traditional fermented soybean condiment, may have antihypertensive effects, but research on ACE inhibitory peptides from Douchi hydrolysates is limited. We hypothesized that enzymatic treatment could enhance ACE inhibitory peptide diversity and efficacy. We tested ten single enzymes and four combinations, finding pepsin-trypsin-chymotrypsin most effective. Hydrolysates were purified using Sephadex G-15 and reversed-phase HPLC, and peptides were identified via LC-MS/MS. Five peptides (LF, VVF, VGAW, GLFG, NGK) were identified, with VGAW as the most potent ACE inhibitor (IC50 46.6 ± 5.2 µM) showing excellent thermal and pH stability. Lineweaver-Burk plots confirmed competitive inhibition, and molecular docking revealed eight hydrogen bonds between VGAW and ACE. In hypertensive rats, VGAW significantly reduced blood pressure at 12.5, 25, and 50 mg/kg. These findings highlight Douchi as a source of ACE inhibitory peptides and suggest VGAW as a promising functional food ingredient.

Physicochemical, functional, and antioxidant properties of black soldier fly larvae protein.

This study explores the multifaceted attributes of black soldier fly larvae protein (BSFLP), focusing on its physicochemical, functional, and antioxidant properties. BSFLP is characterized by 16 amino acids, with a predominant random coil secondary structure revealed by circular dichroism spectra. Differential scanning calorimetry indicates a substantial thermal denaturation temperature of 97.63°C. The protein exhibits commendable solubility, emulsification, and foaming properties in alkaline and low-salt environments, albeit with reduced water-holding capacity and foam stability under elevated alkaline and high-temperature conditions. In vitro assessments demonstrate that BSFLP displays robust scavenging proficiency against 2,2-diphenyl-1-picrylhydrazyl, 2,2'-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid), and hydroxyl radicals, with calculated EC50 values of 1.90 ± 0.57, 0.55 ± 0.01, and 1.14 ± 0.02 mg/mL, respectively, along with notable reducing capabilities. Results from in vivo antioxidant experiments reveal that BSFLP, administered at doses of 300 and 500 mg/kg, significantly enhances the activities of antioxidant enzymes (superoxide dismutase, catalase, and glutathione peroxidase) (p < 0.05) while simultaneously reducing malondialdehyde levels in both serum and tissues of d-galactose-induced oxidative stress in mice. Moreover, the protein effectively attenuates oxidative damage in liver and hippocampal tissues. These findings underscore the potential utility of BSFLP as a natural antioxidant source, with applications spanning the food, pharmaceutical, and cosmetic industries. PRACTICAL APPLICATION: Black soldier fly larvae protein emerges as an environmentally sustainable reservoir of natural antioxidants, holding significant promise for the food, pharmaceutical, and cosmetic sectors. Its advantageous amino acid composition, robust thermal resilience, and impressive functional attributes position it as a compelling subject for continued investigation and advancement in various applications.

Different effects of 4ß-hydroxywithanolide E and withaferin A, two withanolides from Solanaceae plants, on the Akt signaling pathway in human breast cancer cells.

BACKGROUND: Triple-negative breast cancer (TNBC) represents a clinical challenge because it lacks sensitivity to hormone therapy or other available molecule-targeted agents. In addition, TNBC frequently exhibits over-activation of the PI3K/Akt survival pathway that can contribute to chemotherapy resistance. 4ß-Hydroxywithanolide E (4-HW) and withaferin A (WA) are two withanolides from Solanaceae plants that exhibit promising anticancer activity in vitro and in vivo. PURPOSE: The aim of this study is to investigate and compare the effects of 4-HW and WA on TNBC cells and underling mechanisms. STUDY DESIGN/METHODS: The anticancer effects of 4-HW and WA were evaluated by cell viability, cell cycle arrest, and apoptosis assays. PI3K/Akt signaling and the expression of survivin, Bcl-2 family proteins and cyclin-dependent kinase inhibitors were evaluated by Western blot. The role of PI3K/Akt signaling in the withanolides-induced anticancer effects was examined by using a PI3K inhibitor and overexpression of a constitutively active form of Akt. RESULTS: In TNBC MDA-MB-231 cells, 4-HW and WA displayed different kinetic effect on cell availability. Cell cycle analysis revealed that 4-HW induced the G1-phase arrest while WA caused the G2/M-phase block. Both withanolides induced apoptosis, but WA also caused necrosis. 4-HW inhibited the PI3K/Akt pathway and survivin expression as well as up-regulated the cyclin-dependent kinase inhibitors p21 and p27. In contrast, WA is a more potent inhibitor of Hsp90 and elicited Akt activation at low doses but inhibited Akt signaling at higher doses by depleting the Akt protein. The PI3K inhibitor LY294002 mimicked the effects of 4-HW and potentiated the cytotoxic activity of WA. In contrast, overexpressing a constitutively active form of myristoylated Akt rescue cancer cells from 4-HW-induced cell death. CONCLUSION: The withanolides 4-HW and WA potently inhibit the viability of TNBC cells through induction of cell cycle arrest and apoptosis/necrosis. The PI3K/Akt pathway plays distinct roles in cancer cells respond to 4-HW and WA. These results suggest the potential applications of the withanolides for the treatment of TNBC.