Exploring the Potential of Water-Soluble Squid Ink Melanin: Stability, Free Radical Scavenging, and Cd2+ Adsorption Abilities.

Squid ink melanin can be efficiently extracted from the byproduct ink sac generated during squid processing. As a natural food colorant, it possesses inherent antioxidant properties and the capability to adsorb heavy metals. This study aims to investigate the solubility of water-soluble squid ink melanin (WSSM) obtained from the ink sac, as well as its stability under various conditions including temperature, pH, salt, sugar, potassium sorbate, metal ions, sodium benzoate, sodium sulfite (reducing agent), and hydrogen peroxide (oxidizing agent). Moreover, it explores the scavenging effects of WSSM on free radicals and cadmium ions. The findings suggest that WSSM's stability is insignificantly affected by high temperature, sucrose, and salt. However, acidity, sodium benzoate, potassium sorbate, sodium sulfite (Na2SO3), and hydrogen peroxide (H2O2) significantly influence its stability. Most metal ions do not impact the stability of WSSM, except for Fe2+, Fe3+, Al3+, and Cu2+, which result in the precipitation of WSSM. Additionally, WSSM exhibits remarkable antioxidant activity with IC50 values of 0.91, 0.56, and 0.52 mg/mL for scavenging superoxide anion radicals (O2-·), hydroxyl radicals (·OH), and DPPH radicals, respectively. It also demonstrates the ability to adsorb the heavy metal Cd2+, with the adsorption rate gradually increasing with a higher temperature and larger amounts of WSSM added. Infrared spectroscopy analysis reveals the weakening of characteristic peaks (-COOH and -OH) during the process of Cd2+ adsorption by WSSM, while SEM confirms surface roughening and structural damage after Cd2+ adsorption. This study provides valuable insights for the utilization of squid melanin products as natural antioxidants and heavy metal adsorbents in the food industry.

Affinity Purification and Molecular Characterization of Angiotensin-Converting Enzyme (ACE)-Inhibitory Peptides from Takifugu flavidus.

An affinity chromatography filler of CNBr-activated Sepharose 4B-immobilized ACE was used to purify ACE-inhibitory peptides from Takifugu flavidus protein hydrolysate (<1 kDa). Twenty-four peptides with an average local confidence score (ALC) ≥ 80% from bounded components (eluted by 1 M NaCl) were identified by LC-MS/MS. Among them, a novel peptide, TLRFALHGME, with ACE-inhibitory activity (IC50 = 93.5 µmol·L-1) was selected. Molecular docking revealed that TLRFALHGME may interact with the active site of ACE through H-bond, hydrophobic, and electrostatic interactions. The total binding energy (ΔGbinding) of TLRFALHGME was estimated to be -82.7382 kJ·mol-1 by MD simulations, indicating the favorable binding of peptides with ACE. Furthermore, the binding affinity of TLRFALHGME to ACE was determined by surface plasmon resonance (SPR) with a Kd of 80.9 µmol, indicating that there was a direct molecular interaction between them. TLRFALHGME has great potential for the treatment of hypertension.

Combined Effects of Cold and Hot Air Drying on Physicochemical Properties of Semi-Dried Takifugu obscurus Fillets.

The physicochemical properties of semi-dried Takifugu obscurus fillets in cold air drying (CAD), hot air drying (HAD), and cold and hot air combined drying (CHACD) were analyzed based on pH, water state, lipid oxidation, protein degradation, and microstructure, using a texture analyzer, low-field nuclear magnetic resonance, thiobarbituric acid, frozen sections, sodium dodecyl sulfate polyacrylamide gel electrophoresis, and differential scanning calorimetry. Water binding to the samples was enhanced by all three drying methods, and the immobilized water content of CHACD was between that of HAD and CAD. The pH of the semi-dried fillets was improved by CHACD. When compared to HAD and CAD, CHACD improved the springiness and chewiness of the fillets, especially cold air drying for 90 min (CAD-90), with values of 0.97 and 59.79 g, respectively. The muscle fibers were arranged compactly and clearly in CAD-90, having higher muscle toughness. CHACD reduced the drying time and degree of lipid oxidation compared to HAD and CAD. CAD better preserved protein composition, whereas HAD and CHACD promoted actin production; CHACD had a higher protein denaturation temperature (74.08-74.57 °C). CHACD results in better physicochemical properties than HAD or CAD, including shortened drying time, reduced lipid oxidation, enhanced protein stability, and denser tissue structure. These results provide a theoretical basis for selecting the appropriate drying method for T. obscurus in industrial applications.

Effects of astaxanthin on microRNA expression in a rat cardiomyocyte anoxia-reoxygenation model.

Introduction: The protective effects of astaxanthin against myocardial ischemia-reperfusion injuries are well documented, although the mechanisms are not defined. Methods: The anoxia-reoxygenation injury model was established after astaxanthin treated H9c2 cells for 24 h. Cell viability, lactate dehydrogenase, oxidative stress level and western blot were tested. Secondly, measured the effects of astaxanthin pretreatment on microRNA expression in a rat myocardial cell anoxia-reoxygenation injury model. Results: After anoxia-reoxygenation injury, in a dose dependent manner, astaxanthin increased cell viability, superoxide dismutase and glutathione peroxidase activity, decreased lactate dehydrogenase and malondialdehyde levels, downregulated protein expression of caspase-3, caspase-8, nuclear factor erythroid-2-related factor 2 and heme oxygenase-1, and upregulated the Bcl-2/Bax ratio. High-throughput sequencing and qPCR showed that microRNAs rno-miR-125b-5p and rno-let-7c-1-3p were differentially expressed (|log2| ≥ 0.585, q < 0.1) between the normal, anoxia-reoxygenation, and astaxanthin (1.25 µM) groups. Kyoto Encyclopedia of Genes and Genomes and GO Gene ontology pathway enrichment analyses showed that TNF signaling, axon guidance, NF-κB signaling pathway, and other pathways displayed differentially expressed microRNA target genes associated with myocardial injuries. Discussion: These results suggested that thetarget genes of rno-miR-125b-5p were enriched in inflammation and apoptosis-related signaling pathways. Also, the results imply that simultaneous targeting of these related signaling pathways could significantly prevent myocardial anoxia-reoxygenation injury in the presence of astaxanthin.

Screening and Molecular Mechanisms of Novel ACE-Inhibitory Peptides from Gracilariopsis lemaneiformis.

Candidate peptides with novel angiotensin-I-converting enzyme (ACE) inhibitor activity were obtained from hydrolysates of Gracilariopsis lemaneiformis by virtual screening method. Our results showed that G. lemaneiformis peptides (GLP) could significantly lower blood pressure in spontaneously hypertensive rats (SHR). At least 101 peptide sequences of GLP were identified by LC-MS/MS analysis and subjected to virtual screening. A total of 20 peptides with the highest docking score were selected and chemically synthesized in order to verify their ACE-inhibitory activities. Among them, SFYYGK, RLVPVPY, and YIGNNPAKG showed good effects with IC50 values of 6.45 ± 0.22, 9.18 ± 0.42, and 11.23 ± 0.23 µmoL/L, respectively. Molecular docking studies revealed that three peptides interacted with the active center of ACE by hydrogen bonding, hydrophobic interactions, and electrostatic forces. These peptides could form stable complexes with ACE. Furthermore, SFYYGK, RLVPVPY, and YIGNNPAKG significantly reduced systolic blood pressure (SBP) in SHR. YIGNNPAKG exhibited the highest antihypertensive effect, with the largest decrease in SBP (approximately 23 mmHg). In conclusion, SFYYGK, RLVPVPY, and YIGNNPAKG can function as potent therapeutic candidates for hypertension treatment.

Effects of Collagen Hydrolysate From Large Hybrid Sturgeon on Mitigating Ultraviolet B-Induced Photodamage.

Ultraviolet B (UVB) radiation leads to the excessive accumulation of reactive oxygen species (ROS), which subsequently promote inflammation, degradation of the extracellular matrix, and photoaging in skin. Thus antioxidant activity is particularly important when screening for active substances to prevent or repair photodamage. Marine fish-derived bioactive peptides have become a trend in cosmetics and functional food industries owing to their potential dermatological benefits. In this study, 1-diphenyl- 2-pycryl-hydrazyl (DPPH) scavenging activity was selected to optimize the hydrolysis conditions of sturgeon skin collagen peptides with antioxidant activity. The optimal hydrolysis conditions for sturgeon skin collagen hydrolysate (SSCH) were determined by response surface methodology, which comprised an enzyme dosage of flavorzyme at 6,068.4 U/g, temperature of 35.5°C, pH of 7, and hydrolysis time of 6 h. SSCH showed good radical-scavenging capacities with a DPPH scavenging efficiency of 95%. Then, the effect of low-molecular-weight SSCH fraction (SSCH-L) on UVB irradiation-induced photodamage was evaluated in mouse fibroblast L929 cells and zebrafish. SSCH-L reduced intracellular ROS levels and the malondialdehyde content, thereby alleviating the oxidative damage caused by UVB radiation. Moreover SSCH-L inhibited the mRNA expression of genes encoding the pro-inflammatory cytokines IL-1ß, IL-6, TNF-α, and Cox-2. SSCH-L treatment further increased the collagen Ⅰα1 content and had a significant inhibitory effect on matrix metalloproteinase expression. The phosphorylation level of JNK and the expression of c-Jun protein were significantly reduced by SSCH-L. Additionally, SSCH-L increased the tail fin area at 0.125 and 0.25 mg/ml in a zebrafish UVB radiation model, which highlighted the potential of SSCH-L to repair UVB-irradiated zebrafish skin damage. Peptide sequences of SSCH-L were identified by liquid chromatography-tandem mass spectrometry. Based on the 3D-QSAR modeling prediction, six total peptides were selected to test the UVB-protective activity. Among these peptides, DPFRHY showed good UVB-repair activity, ROS-scavenging activity, DNA damage-protective activity and apoptosis inhibition activity. These results suggested that DPFRHY has potential applications as a natural anti-photodamage material in cosmetic and functional food industries.

A Novel Angiotensin-I-Converting Enzyme (ACE) Inhibitory Peptide from Takifugu flavidus.

Alcalase, neutral protease, and pepsin were used to hydrolyze the skin of Takifugu flavidus. The T. flavidus hydrolysates (TFHs) with the maximum degree of hydrolysis (DH) and angiotensin-I-converting enzyme (ACE)-inhibitory activity were selected and then ultra-filtered to obtain fractions with components of different molecular weights (MWs) (<1, 1-3, 3-10, 10-50, and >50 kDa). The components with MWs < 1 kDa showed the strongest ACE-inhibitory activity with a half-maximal inhibitory concentration (IC50) of 0.58 mg/mL. Purification and identification using semi-preparative liquid chromatography, Sephadex G-15 gel chromatography, RP-HPLC, and LC-MS/MS yielded one new potential ACE-inhibitory peptide, PPLLFAAL (non-competitive suppression mode; IC50 of 28 µmmol·L-1). Molecular docking and molecular dynamics simulations indicated that the peptides should bind well to ACE and interact with amino acid residues and the zinc ion at the ACE active site. Furthermore, a short-term assay of antihypertensive activity in spontaneously hypertensive rats (SHRs) revealed that PPLLFAAL could significantly decrease the systolic blood pressure (SBP) and diastolic blood pressure (DBP) of SHRs after intravenous administration. These results suggested that PPLLFAAL may have potential applications in functional foods or pharmaceuticals as an antihypertensive agent.

ACE Inhibitory Peptide from Skin Collagen Hydrolysate of Takifugu bimaculatus as Potential for Protecting HUVECs Injury.

Angiotensin-I-converting enzyme (ACE) is a crucial enzyme or receptor that catalyzes the generation of potent vasopressor angiotensin II (Ang II). ACE inhibitory peptides from fish showed effective ACE inhibitory activity. In this study, we reported an ACE inhibitory peptide from Takifugu bimaculatus (T. bimaculatus), which was obtained by molecular docking with acid-soluble collagen (ASC) hydrolysate of T. bimaculatus. The antihypertensive effects and potential mechanism were conducted using Ang-II-induced human umbilical vein endothelial cells (HUVECs) as a model. The results showed that FNLRMQ alleviated the viability and facilitated apoptosis of Ang-II-induced HUVECs. Further research suggested that FNLRMQ may protect Ang-II-induced endothelial injury by regulating Nrf2/HO-1 and PI3K/Akt/eNOS signaling pathways. This study, herein, reveals that collagen peptide FNLRMQ could be used as a potential candidate compound for antihypertensive treatment, and could provide scientific evidence for the high-value utilization of marine resources including T. bimaculatus.

Molecular Characterization of the Von Willebrand Factor Type D Domain of Vitellogenin from Takifugu flavidus.

The von Willebrand factor type D (VWD) domain in vitellogenin has recently been found to bind tetrodotoxin. The way in which this protein domain associates with tetrodotoxin and participates in transporting tetrodotoxin in vivo remains unclear. A cDNA fragment of the vitellogenin gene containing the VWD domain from pufferfish (Takifugu flavidus) (TfVWD) was cloned. Using in silico structural and docking analyses of the predicted protein, we determined that key amino acids (namely, Val115, ASP116, Val117, and Lys122) in TfVWD mediate its binding to tetrodotoxin, which was supported by in vitro surface plasmon resonance analysis. Moreover, incubating recombinant rTfVWD together with tetrodotoxin attenuated its toxicity in vivo, further supporting protein-toxin binding and indicating associated toxicity-neutralizing effects. Finally, the expression profiling of TfVWD across different tissues and developmental stages indicated that its distribution patterns mirrored those of tetrodotoxin, suggesting that TfVWD may be involved in tetrodotoxin transport in pufferfish. For the first time, this study reveals the amino acids that mediate the binding of TfVWD to tetrodotoxin and provides a basis for further exploration of the molecular mechanisms underlying the enrichment and transfer of tetrodotoxin in pufferfish.