Influence of Amino Acid Compositions and Peptide Profiles on Antioxidant Capacities of Two Protein Hydrolysates from Skipjack Tuna (Katsuwonus pelamis) Dark Muscle

Influence of amino acid compositions and peptide profiles on antioxidant capacities of two protein hydrolysates from skipjack tuna (Katsuwonus pelamis) dark muscle was investigated. Dark muscles from skipjack tuna were hydrolyzed using five separate proteases, including pepsin, trypsin, Neutrase, papain and Alcalase. Two hydrolysates, ATH and NTH, prepared using Alcalase and Neutrase, respectively, showed the strongest antioxidant capacities and were further fractionated using ultrafiltration and gel filtration chromatography. Two fractions, Fr.A3 and Fr.B2, isolated from ATH and NTH, respectively, showed strong radical scavenging activities toward 2,2-diphenyl-1-picrylhydrazyl radicals (EC₅₀ 1.08% ± 0.08% and 0.98% ± 0.07%), hydroxyl radicals (EC₅₀ 0.22% ± 0.03% and 0.48% ± 0.05%), and superoxide anion radicals (EC₅₀ 1.31% ± 0.11% and 1.56% ± 1.03%) and effectively inhibited lipid peroxidation. Eighteen peptides from Fr.A3 and 13 peptides from Fr.B2 were isolated by reversed-phase high performance liquid chromatography, and their amino acid sequences were determined. The elevated antioxidant activity of Fr.A3 might be due to its high content of hydrophobic and aromatic amino acid residues (181.1 and 469.9 residues/1000 residues, respectively), small molecular sizes (3–6 peptides), low molecular weights (524.78 kDa), and amino acid sequences (antioxidant score 6.11). This study confirmed that a smaller molecular size, the presence of hydrophobic and aromatic amino acid residues, and the amino acid sequences were the key factors that determined the antioxidant activities of the proteins, hydrolysates and peptides. The results also demonstrated that the derived hydrolysates and fractions from skipjack tuna (K. pelamis) dark muscles could prevent oxidative reactions and might be useful for food preservation and medicinal purposes.     

Identification of the Major ACE-Inhibitory Peptides Produced by Enzymatic Hydrolysis of a Protein Concentrate from Cuttlefish Wastewater

The aim of this work was the purification and identification of the major angiotensin converting enzyme (ACE) inhibitory peptides produced by enzymatic hydrolysis of a protein concentrate recovered from a cuttlefish industrial manufacturing effluent. This process consisted on the ultrafiltration of cuttlefish softening wastewater, with a 10 kDa cut-off membrane, followed by the hydrolysis with alcalase of the retained fraction. Alcalase produced ACE inhibitors reaching the highest activity (IC₅₀ = 76.8 ± 15.2 μg mL⁻¹) after 8 h of proteolysis. Sequential ultrafiltration of the 8 h hydrolysate with molecular weight cut-off (MWCO) membranes of 10 and 1 kDa resulted in the increased activity of each permeate, with a final IC₅₀ value of 58.4 ± 4.6 μg mL⁻¹. Permeate containing peptides lower than 1 kDa was separated by reversed-phase high performance liquid chromatography (RP-HPLC). Four fractions (A–D) with potent ACE inhibitory activity were isolated and their main peptides identified using high performance liquid chromatography coupled to an electrospray ion trap Fourier transform ion cyclotron resonance-mass spectrometer (HPLC-ESI-IT-FTICR) followed by comparison with databases and de novo sequencing. The amino acid sequences of the identified peptides contained at least one hydrophobic and/or a proline together with positively charged residues in at least one of the three C-terminal positions. The IC₅₀ values of the fractions ranged from 1.92 to 8.83 μg mL⁻¹, however this study fails to identify which of these peptides are ultimately responsible for the potent antihypertensive activity of these fractions.     

Encapsulation of Salmon Peptides in Marine Liposomes: Physico-Chemical Properties,Antiradical Activities and Biocompatibility Assays

Salmon byproducts (Salmo salar) generated by the food chain represent a source of long-chain polyunsaturated fatty acids (eicosapentaenoic acid (EPA): 20:5n-3; docosahexaenoic acid (DHA): 22:6n-3) and peptides that can be used as supplements in food for nutraceutical or health applications, such as in the prevention of certain pathologies (e.g., Alzheimer’s and cardiovascular diseases). The extraction of polar lipids naturally rich in PUFAs by enzymatic processes without organic solvent (controlled by pH-Stat method), coupled with the production of 1 kDa salmon peptides by membrane filtration, allowed the formulation of nanocarriers. The physicochemical properties of the nanoliposomes (size ranging from 120 to 140 nm, PDI of 0.27, zeta potential between −32 and −46 mV and encapsulation efficiency) were measured, and the bioactivity of salmon hydrolysate peptides was assessed (antioxidant and antiradical activity: ABTS, ORAC, DPPH; iron metal chelation). Salmon peptides exhibited good angiotensin-conversion-enzyme (ACE) inhibition activity, with an IC₅₀ value of 413.43 ± 13.12 µg/mL. Cytotoxicity, metabolic activity and proliferation experiments demonstrated the harmlessness of the nanostructures in these experimental conditions.     

Angiotensin I Converting Enzyme Inhibitory Peptides Derived from Phycobiliproteins of Dulse Palmaria palmata

We examined the inhibitory activity of angiotensin I converting enzyme (ACE) in protein hydrolysates from dulse, Palmaria palmata. The proteins extracted from dulse were mainly composed of phycoerythrin (PE) followed by phycocyanin (PC) and allophycocyanin (APC). The dulse proteins showed slight ACE inhibitory activity, whereas the inhibitory activity was extremely enhanced by thermolysin hydrolysis. The ACE inhibitory activity of hydrolysates was hardly affected by additional pepsin, trypsin and chymotrypsin treatments. Nine ACE inhibitory peptides (YRD, AGGEY, VYRT, VDHY, IKGHY, LKNPG, LDY, LRY, FEQDWAS) were isolated from the hydrolysates by reversed-phase high-performance liquid chromatography (HPLC), and it was demonstrated that the synthetic peptide LRY (IC₅₀: 0.044 μmol) has remarkably high ACE inhibitory activity. Then, we investigated the structural properties of dulse phycobiliproteins to discuss the origin of dulse ACE inhibitory peptides. Each dulse phycobiliprotein possesses α-subunit (Mw: 17,477–17,638) and β-subunit (Mw: 17,455–18,407). The sequences of YRD, AGGEY, VYRT, VDHY, LKNPG and LDY were detected in the primary structure of PE α-subunit, and the LDY also exists in the APC α- and β-subunits. In addition, the LRY sequence was found in the β-subunits of PE, PC and APC. From these results, it was suggested that the dulse ACE inhibitory peptides were derived from phycobiliproteins, especially PE. To make sure the deduction, we carried out additional experiment by using recombinant PE. We expressed the recombinant α- and β-subunits of PE (rPEα and rPEβ, respectively), and then prepared their peptides by thermolysin hydrolysis. As a result, these peptides showed high ACE inhibitory activities (rPEα: 94.4%; rPEβ: 87.0%). Therefore, we concluded that the original proteins of dulse ACE inhibitory peptides were phycobiliproteins.     

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.     

Cytotoxic,Cytostatic and HIV-1 PR Inhibitory Activities of the Soft Coral Litophyton arboreum

Bioassay-guided fractionation using different chromatographic and spectroscopic techniques in the analysis of the Red Sea soft coral Litophyton arboreum led to the isolation of nine compounds; sarcophytol M (1), alismol (2), 24-methylcholesta-5,24(28)-diene-3β-ol (3), 10-O-methyl alismoxide (4), alismoxide (5), (S)-chimyl alcohol (6), 7β-acetoxy-24-methylcholesta-5-24(28)-diene-3,19-diol (7), erythro-N-dodecanoyl-docosasphinga-(4E,8E)-dienine (8), and 24-methylcholesta-5,24(28)-diene-3β,7β,19-triol (9). Some of the isolated compounds demonstrated potent cytotoxic- and/or cytostatic activity against HeLa and U937 cancer cell lines and inhibitory activity against HIV-1 protease (PR). Compound 7 was strongly cytotoxic against HeLa cells (CC₅₀ 4.3 ± 0.75 µM), with selectivity index of SI 8.1, which was confirmed by real time cell electronic sensing (RT-CES). Compounds 2, 7, and 8 showed strong inhibitory activity against HIV-1 PR at IC₅₀s of 7.20 ± 0.7, 4.85 ± 0.18, and 4.80 ± 0.92 µM respectively. In silico docking of most compounds presented comparable scores to that of acetyl pepstatin, a known HIV-1 PR inhibitor. Interestingly, compound 8 showed potent HIV-1 PR inhibitory activity in the absence of cytotoxicity against the cell lines used. In addition, compounds 2 and 5 demonstrated cytostatic action in HeLa cells, revealing potential use in virostatic cocktails. Taken together, data presented here suggest Litophyton arboreum to contain promising compounds for further investigation against the diseases mentioned.     

Purification and Identification of Novel Xanthine Oxidase Inhibitory Peptides Derived from Round Scad (Decapterus maruadsi) Protein Hydrolysates

The objective of the present study was to investigate the xanthine oxidase (XO) inhibitory effects of peptides purified and identified from round scad (Decapterus maruadsi) hydrolysates (RSHs). In this study, RSHs were obtained by using three proteases (neutrase, protamex and alcalase). Among them, the RSHs of 6-h hydrolysis by neutrase displayed the strongest XO inhibitory activity and had an abundance of small peptides (<500 Da). Four novel peptides were purified by immobilized metal affinity chromatography and identified by nano-high-performance liquid chromatography mass/mass spectrometry. Their amino acid sequences were KGFP (447.53 Da), FPSV (448.51 Da), FPFP (506.59 Da) and WPDGR (629.66 Da), respectively. Then the peptides were synthesized to evaluate their XO inhibitory activity. The results indicated that the peptides of both FPSV (5 mM) and FPFP (5 mM) exhibited higher XO inhibitory activity (22.61 ± 1.81% and 20.09 ± 2.41% respectively). Fluorescence spectra assay demonstrated that the fluorescence quenching mechanism of XO by these inhibitors (FPSV and FPFP) was a static quenching procedure. The study of inhibition kinetics suggested that the inhibition of both FPSV and FPFP was reversible, and the type of their inhibition was a mixed one. Molecular docking revealed the importance of π-π stacking between Phe residue (contained in peptides) and Phe⁹¹⁴ (contained in the XO) in the XO inhibitory activity of the peptides.     

Novel Antioxidant Collagen Peptides of Siberian Sturgeon (Acipenser baerii) Cartilages: The Preparation,Characterization, and Cytoprotection of H2O2-Damaged Human Umbilical Vein Endothelial Cells (HUVECs)

For making full use of aquatic by-products to produce high value-added products, Siberian sturgeon (Acipenser baerii) cartilages were degreased, mineralized, and separately hydrolyzed by five kinds of proteases. The collagen hydrolysate (SCH) generated by Alcalase showed the strongest 2,2-diphenyl-1-picrylhydrazyl radical (DPPH·) and hydroxide radical (HO·) scavenging activity. Subsequently, thirteen antioxidant peptides (SCP1-SCP3) were isolated from SCH, and they were identified as GPTGED, GEPGEQ, GPEGPAG, VPPQD, GLEDHA, GDRGAEG, PRGFRGPV, GEYGFE, GFIGFNG, PSVSLT, IELFPGLP, LRGEAGL, and RGEPGL with molecular weights of 574.55, 615.60, 583.60, 554.60, 640.64, 660.64, 885.04, 700.70, 710.79, 602.67, 942.12, 714.82, and 627.70 Da, respectively. GEYGFE, PSVSLT, and IELFPGLP showed the highest scavenging activity on DPPH· (EC₅₀: 1.27, 1.05, and 1.38 mg/mL, respectively) and HO· (EC₅₀: 1.16, 0.97, and 1.63 mg/mL, respectively), inhibiting capability of lipid peroxidation, and protective functions on H₂O₂-damaged plasmid DNA. More importantly, GEYGFE, PSVSLT, and IELFPGLP displayed significant cytoprotection on HUVECs against H₂O₂ injury by regulating the endogenous antioxidant enzymes of superoxide dismutase (SOD) and glutathione peroxidase (GSH-Px) to decrease the contents of reactive oxygen species (ROS) and malondialdehyde (MDA). Therefore, the research provided better technical assistance for a higher-value utilization of Siberian sturgeon cartilages and the thirteen isolated peptides—especially GEYGFE, PSVSLT, and IELFPGLP—which may serve as antioxidant additives for generating health-prone products to treat chronic diseases caused by oxidative stress.     

Optimization of hydrolysis conditions for the production of angiotensin-I converting enzyme-inhibitory peptides and isolation of a novel peptide from lizard fish (Saurida elongata) muscle protein hydrolysate

Lizard fish (Saurida elongata) muscle protein was hydrolyzed using neutral protease to produce protein hydrolysate (LFPH), and the hydrolysis conditions were investigated using response-surface methodology. The optimum conditions for producing peptides with the highest angiotensin-I converting enzyme (ACE)-inhibitory activity were the following: enzyme-to-substrate ratio of 10,000 U/g, temperature of 48 °C, pH 7.0, and hydrolysis time of 2 h. Under these conditions, the ACE-inhibitory activity of LFPH and the degree of hydrolysis were 84% and 24%, respectively. A novel ACE-inhibitory peptide was isolated from LFPH using ultrafiltration, Sephadex G-15, and high-performance liquid chromatography. The amino acid sequence of the ACE-inhibitory peptide was identified as Ser-Pro-Arg-Cys-Arg (SPRCR), and its IC₅₀ was 41 ± 1 µM.     

Magnificines A and B,Antimicrobial Marine Alkaloids Featuring a Tetrahydrooxazolo[3,2-a]azepine-2,5(3H,6H)-dione Backbone from the Red Sea Sponge Negombata magnifica

Investigation of the Red Sea sponge Negombata magnifica gave two novel alkaloids, magnificines A and B (1 and 2) and a new β-ionone derivative, (±)-negombaionone (3), together with the known latrunculin B (4) and 16-epi-latrunculin B (5). The analysis of the NMR and HRESIMS spectra supported the planar structures and the relative configurations of the compounds. The absolute configurations of magnificines A and B were determined by the analysis of the predicted and experimental ECD spectra. Magnificines A and B possess a previously unreported tetrahydrooxazolo[3,2-a]azepine-2,5(3H,6H)-dione backbone and represent the first natural compounds in this class. (±)-Negombaionone is the first β-ionone of a sponge origin. Compounds 1-3 displayed selective activity against Escherichia coli in a disk diffusion assay with inhibition zones up to 22 mm at a concentration of 50 µg/disc and with MIC values down to 8.0 µM. Latrunculin B and 16-epi-latrunculin B inhibited the growth of HeLa cells with IC₅₀ values down to 1.4 µM.     

Characterization of Protein Hydrolysates from Fish Discards and By-Products from the North-West Spain Fishing Fleet as Potential Sources of Bioactive Peptides

Fish discards and by-products can be transformed into high value-added products such as fish protein hydrolysates (FPH) containing bioactive peptides. Protein hydrolysates were prepared from different parts (whole fish, skin and head) of several discarded species of the North-West Spain fishing fleet using Alcalase. All hydrolysates had moisture and ash contents lower than 10% and 15%, respectively. The fat content of FPH varied between 1.5% and 9.4% and had high protein content (69.8–76.6%). The amino acids profiles of FPH are quite similar and the most abundant amino acids were glutamic and aspartic acids. All FPH exhibited antioxidant activity and those obtained from Atlantic horse mackerel heads presented the highest 2,2-diphenyl-1-picrylhydrazyl (DPPH) radical scavenging activity, reducing power and Cu²⁺ chelating activity. On the other hand, hydrolysates from gurnard heads showed the highest ABTS radical scavenging activity and Fe²⁺ chelating activity. In what concerns the α-amylase inhibitory activity, the IC₅₀ values recorded for FPH ranged between 5.70 and 84.37 mg/mL for blue whiting heads and whole Atlantic horse mackerel, respectively. α-Glucosidase inhibitory activity of FPH was relatively low but all FPH had high Angiotensin Converting Enzyme (ACE) inhibitory activity. Considering the biological activities, these FPH are potential natural additives for functional foods or nutraceuticals.     

First Total Syntheses and Antimicrobial Evaluation of Penicimonoterpene,a Marine-Derived Monoterpenoid,and Its Various Derivatives

The first total synthesis of marine-derived penicimonoterpene (±)-1 has been achieved in four steps from 6-methylhept-5-en-2-one using a Reformatsky reaction as the key step to construct the basic carbon skeleton. A total of 24 new derivatives of 1 have also been designed and synthesized. Their structures were characterized by analysis of their ¹H NMR, ¹³C NMR and HRESIMS data. Some of them showed significant antibacterial activity against Aeromonas hydrophila, Escherichia coli, Micrococcus luteus, Staphylococcus aureus, Vibrio anguillarum, V. harveyi and/or V. parahaemolyticus, and some showed activity against plant-pathogenic fungi (Alternaria brassicae, Colletotrichum gloeosporioides and/or Fusarium graminearum). Some of the derivatives exhibited antimicrobial MIC values ranging from 0.25 to 4 μg/mL, which were stronger than those of the positive control. Notably, Compounds 3b and 10 showed extremely high selectively against plant-pathogenic fungus F. graminearum (MIC 0.25 μg/mL) and pathogenic bacteria E. coli (MIC 1 μg/mL), implying their potential as antimicrobial agents. SAR analysis of 1 and its derivatives indicated that modification of the carbon-carbon double bond at C-6/7, of groups on the allylic methylene unit and of the carbonyl group at C-1, effectively enhanced the antimicrobial activity.     

Characterization of Neoagarooligosaccharide Hydrolase BpGH117 from a Human Gut Bacterium Bacteroides plebeius

α-Neoagarobiose (NAB)/neoagarooligosaccharide (NAO) hydrolase plays an important role as an exo-acting 3,6-anhydro-α-(1,3)-L-galactosidase in agarose utilization. Agarose is an abundant polysaccharide found in red seaweeds, comprising 3,6-anhydro-L-galactose (AHG) and D-galactose residues. Unlike agarose degradation, which has been reported in marine microbes, recent metagenomic analysis of Bacteroides plebeius, a human gut bacterium, revealed the presence of genes encoding enzymes involved in agarose degradation, including α-NAB/NAO hydrolase. Among the agarolytic enzymes, BpGH117 has been partially characterized. Here, we characterized the exo-acting α-NAB/NAO hydrolase BpGH117, originating from B. plebeius. The optimal temperature and pH for His-tagged BpGH117 activity were 35 °C and 9.0, respectively, indicative of its unique origin. His-tagged BpGH117 was thermostable up to 35 °C, and the enzyme activity was maintained at 80% of the initial activity at a pre-incubation temperature of 40 °C for 120 min. K_m and V_max values for NAB were 30.22 mM and 54.84 U/mg, respectively, and k_cat/K_m was 2.65 s⁻¹ mM⁻¹. These results suggest that His-tagged BpGH117 can be used for producing bioactive products such as AHG and agarotriose from agarose efficiently.     

Territrem and Butyrolactone Derivatives from a Marine-Derived Fungus Aspergillus Terreus

Seventeen lactones including eight territrem derivatives (1–8) and nine butyrolactone derivatives (9–17) were isolated from a marine-derived fungus Aspergillus terreus SCSGAF0162 under solid-state fermentation of rice. Compounds 1–3 and 9–10 were new, and their structures were elucidated by spectroscopic analysis. The acetylcholinesterase inhibitory activity and antiviral activity of compounds 1–17 were evaluated. Among them, compounds 1 and 2 showed strong inhibitory activity against acetylcholinesterase with IC₅₀ values of 4.2 ± 0.6, 4.5 ± 0.6 nM, respectively. This is the first time it has been reported that 3, 6, 10, 12 had evident antiviral activity towards HSV-1 with IC₅₀ values of 16.4 ± 0.6, 6.34 ± 0.4, 21.8 ± 0.8 and 28.9 ± 0.8 μg·mL⁻¹, respectively. Antifouling bioassay tests showed that compounds 1, 11, 12, 15 had potent antifouling activity with EC₅₀ values of 12.9 ± 0.5, 22.1 ± 0.8, 7.4 ± 0.6, 16.1 ± 0.6 μg·mL⁻¹ toward barnacle Balanus amphitrite larvae, respectively.     

Microwave-Assisted Extraction of Phlorotannins from Fucus vesiculosus

Microwave-assisted extraction (MAE) was carried out to maximize the extraction of phlorotannins from Fucus vesiculosus using a hydroethanolic mixture as a solvent, as an alternative to the conventional method with a hydroacetonic mixture. Optimal MAE conditions were set as ethanol concentration of 57% (v/v), temperature of 75 °C, and time of 5 min, which allowed a similar recovery of phlorotannins from the macroalgae compared to the conventional extraction. While the phlorotannins richness of the conventional extract was slightly superior to that of MAE (11.1 ± 1.3 vs. 9.8 ± 1.8 mg PGE/g DW_extract), both extracts presented identical phlorotannins constituents, which included, among others, tetrafucol, pentafucol, hexafucol, and heptafucol structures. In addition, MAE showed a moderate capacity to scavenge ABTS^•+ (IC₅₀ of 96.0 ± 3.4 µg/mL) and to inhibit the activity of xanthine oxidase (IC₅₀ of 23.1 ± 3.4 µg/mL) and a superior ability to control the activity of the key metabolic enzyme α-glucosidase compared to the pharmaceutical drug acarbose.     

In Vitro Bioactivity of Astaxanthin and Peptides from Hydrolisates of Shrimp (Parapenaeus longirostris) By-Products: From the Extraction Process to Biological Effect Evaluation,as Pilot Actions for the Strategy “From Waste to Profit”

Non-edible parts of crustaceans could be a rich source of valuable bioactive compounds such as the carotenoid astaxanthin and peptides, which have well-recognized beneficial effects. These compounds are widely used in nutraceuticals and pharmaceuticals, and their market is rapidly growing, suggesting the need to find alternative sources. The aim of this work was to set up a pilot-scale protocol for the reutilization of by-products of processed shrimp, in order to address the utilization of this valuable biomass for nutraceutical and pharmaceuticals application, through the extraction of astaxanthin-enriched oil and antioxidant-rich protein hydrolysates. Astaxanthin (AST) was obtained using “green extraction methods,” such as using fish oil and different fatty acid ethyl esters as solvents and through supercritical fluid extraction (SFE), whereas bioactive peptides were obtained by protease hydrolysis. Both astaxanthin and bioactive peptides exhibited bioactive properties in vitro in cellular model systems, such as antioxidant and angiotensin I converting enzyme (ACE) inhibitory activities (IA). The results show higher astaxanthin yields in ethyl esters fatty acids (TFA) extraction and significant enrichment by short-path distillation (SPD) up to 114.80 ± 1.23 µg/mL. Peptide fractions of <3 kDa and 3–5 kDa exhibited greater antioxidant activity while the fraction 5–10 kDa exhibited a better ACE-IA. Lower-molecular-weight bioactive peptides and astaxanthin extracted using supercritical fluids showed protective effects against oxidative damage in 142BR and in 3T3 cell lines. These results suggest that “green” extraction methods allow us to obtain high-quality bioactive compounds from large volumes of shrimp waste for nutraceutical and pharmaceutical applications.     

Interaction of Thalassia testudinum Metabolites with Cytochrome P450 Enzymes and Its Effects on Benzo(a)pyrene-Induced Mutagenicity

The aim of the present work was to evaluate the effects of Thalassia testudinum hydroethanolic extract, its polyphenolic fraction and thalassiolin B on the activity of phase I metabolizing enzymes as well as their antimutagenic effects. Spectrofluorometric techniques were used to evaluate the effect of tested products on rat and human CYP1A and CYP2B activity. The antimutagenic effect of tested products was evaluated in benzo[a]pyrene (BP)-induced mutagenicity assay by an Ames test. Finally, the antimutagenic effect of Thalassia testudinum (100 mg/kg) was assessed in BP-induced mutagenesis in mice. The tested products significantly (p < 0.05) inhibit rat CYP1A1 activity, acting as mixed-type inhibitors of rat CYP1A1 (Ki = 54.16 ± 9.09 μg/mL, 5.96 ± 1.55 μg/mL and 3.05 ± 0.89 μg/mL, respectively). Inhibition of human CYP1A1 was also observed (Ki = 197.1 ± 63.40 μg/mL and 203.10 ± 17.29 μg/mL for the polyphenolic fraction and for thalassiolin B, respectively). In addition, the evaluated products significantly inhibit (p < 0.05) BP-induced mutagenicity in vitro. Furthermore, oral doses of Thalassia testudinum (100 mg/kg) significantly reduced (p < 0.05) the BP-induced micronuclei and oxidative damage, together with an increase of reduced glutathione, in mice. In summary, Thalassia testudinum metabolites exhibit antigenotoxic activity mediated, at least, by the inhibition of CYP1A1-mediated BP biotransformation, arresting the oxidative and mutagenic damage. Thus, the metabolites of T. testudinum may represent a potential source of chemopreventive compounds for the adjuvant therapy of cancer.     

Metabolic Profiling and In Vitro Assessment of the Biological Activities of the Ethyl Acetate Extract of Penicillium chrysogenum “Endozoic of Cliona sp. Marine Sponge” from the Red Sea (Egypt)

Marine sponge-derived endozoic fungi have been gaining increasing importance as promising sources of numerous and unique bioactive compounds. This study investigates the phytochemical profile and biological activities of the ethyl acetate extract of Penicillium chrysogenum derived from Cliona sp. sponge. Thirty-six compounds were tentatively identified from P. chrysogenum ethyl acetate extract along with the kojic acid (KA) isolation. The UPLC-ESI-MS/MS positive ionization mode was used to analyze and identify the extract constituents while 1D and 2D NMR spectroscopy were used for kojic acid (KA) structure confirmation. The antimicrobial, antioxidant, and cytotoxic activities were assessed in vitro. Both the extract and kojic acid showed potent antibacterial activity against Staphylococcus aureus and Pseudomonas aeruginosa with MIC 250 ± 0.82 µg/mL. Interestingly, the extract showed strong antifungal activity against Candida albicans and Cryptococcus neoformans with MIC 93.75 ± 0.55 and 19.53 ± 0.48 µg/mL, respectively. Furthermore, KA showed the same potency against Fusarium oxysporum and Cryptococcus neoformans with MIC 39.06 ± 0.85 and 39.06 ± 0.98 µg/mL, respectively. Ultimately, KA showed strong antioxidant activity with IC₅₀ 33.7 ± 0.8 µg/mL. Moreover, the extract and KA showed strong cytotoxic activity against colon carcinoma (with IC₅₀ 22.6 ± 0.8 and 23.4 ± 1.4 µg/mL, respectively) and human larynx carcinoma (with equal IC₅₀ 30.8 ± 1.3 and ± 2.1 µg/mL, respectively), respectively. The current study represents the first insights into the phytochemical profile and biological properties of P. chrysoenum ethyl acetate extract, which could be a promising source of valuable secondary metabolites with potent biological potentials.     

Characterization of ACE Inhibitory Peptides from Mactra veneriformis Hydrolysate by Nano-Liquid Chromatography Electrospray Ionization Mass Spectrometry (Nano-LC-ESI-MS) and Molecular Docking

Food-derived bioactive compounds are gaining increasing significance in life sciences. In the present study, we identified angiotensin I-converting enzyme (ACE)-inhibitory peptides from Mactra veneriformis hydrolysate using a nano-LC-MS/MS method. Mactra veneriformis hydrolysate was first separated into four fractions (F1–F4) based on molecular weight by ultrafiltration. The fraction with molecular weight lower than 1 kDa (F1) showed the highest ACE inhibitory activity. F1 was then analyzed by a high throughput nano-LC-MS/MS method and sequences of peptides in F1 were calculated accordingly. The 27 peptides identified as above were chemically synthesized and tested for ACE-inhibitory activity. The hexapeptide VVCVPW showed the highest potency with an IC₅₀ value of 4.07 μM. We then investigated the interaction mechanism between the six most potent peptides and ACE by molecular docking. Our docking results suggested that the ACE inhibitory peptides bind to ACE via interactions with His383, His387, and Glu411 residues. Particularly, similar to the thiol group of captopril, the cysteine thiol group of the most potent peptide VVCVPW may play a key role in the binding of this peptide to the ACE active site.     

Two Novel Antioxidant Nonapeptides from Protein Hydrolysate of Skate (Raja porosa) Muscle

In the current study, the preparation conditions of neutrase hydrolysate (SMH) from skate (Raja porosa) muscle protein were optimized using orthogonal L₉(3)⁴ tests, and R values indicated that pH was the most important factor affecting HO· scavenging activity of SMH. Under the optimum conditions of pH 7.0, enzymolysis temperature 60 °C, enzyme/substrate ratio (E/S) 2%, and enzymolysis time 5 h, EC₅₀ of SMH on HO· was 2.14 ± 0.17 mg/mL. Using ultrafiltration, gel filtration chromatography, and RP-HPLC, two novel antioxidant nonapeptides (SP-A and SP-B) were isolated from SMH and their amino acid sequences were found to be APPTAYAQS (SP-A) and NWDMEKIWD (SP-B) with calculated molecular masses of 904.98 Da and 1236.38 Da, respectively. Both showed strong antioxidant activities. SP-A and SP-B exhibited good scavenging activities on HO· (EC₅₀ 0.390 and 0.176 mg/mL), DPPH· (EC₅₀ 0.614 and 0.289 mg/mL), and O₂⁻· (EC₅₀ 0.215 and 0.132 mg/mL) in a dose-dependent manner. SP-B was also effective against lipid peroxidation in the model system. The aromatic (2Trp), acidic (2Asp and Glu), and basic (Lys) amino acid residues within the sequences of SP-B might account for its pronounced antioxidant activity. The results of this study suggested that protein hydrolysate and peptides from skate muscle might be effective as food additives for retarding lipid peroxidation occurring in foodstuffs.