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.     

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.     

Affinity Purification of Angiotensin Converting Enzyme Inhibitory Peptides from Wakame (Undaria Pinnatifida) Using Immobilized ACE on Magnetic Metal Organic Frameworks

Angiotensin-I-converting enzyme (ACE) inhibitory peptides derived from marine organism have shown a blood pressure lowering effect with no side effects. A new affinity medium of Fe₃O₄@ZIF-90 immobilized ACE (Fe₃O₄@ZIF-90-ACE) was prepared and used in the purification of ACE inhibitory peptides from Wakame (Undaria pinnatifida) protein hydrolysate (<5 kDa). The Fe₃O₄@ZIF-90 nanoparticles were prepared by a one-pot synthesis and crude ACE extract from pig lung was immobilized onto it, which exhibited excellent stability and reusability. A novel ACE inhibitory peptide, KNFL (inhibitory concentration 50, IC₅₀ = 225.87 μM) was identified by affinity purification using Fe₃O₄@ZIF-90-ACE combined with reverse phase-high performance liquid chromatography (RP-HPLC) and MALDI-TOF mass spectrometry. Lineweaver–Burk analysis confirmed the non-competitive inhibition pattern of KNFL, and molecular docking showed that it bound at a non-active site of ACE via hydrogen bonds. This demonstrates that affinity purification using Fe₃O₄@ZIF-90-ACE is a highly efficient method for separating ACE inhibitory peptides from complex protein mixtures and the purified peptide KNFL could be developed as a functional food ingredients against hypertension.     

Bioactive Chaetoglobosins from the Mangrove Endophytic Fungus Penicillium chrysogenum

A novel chaetoglobosin named penochalasin I (1) with a unprecedented six-cyclic 6/5/6/5/6/13 fused ring system, and another new chaetoglobosin named penochalasin J (2), along with chaetoglobosins G, F, C, A, E, armochaetoglobosin I, and cytoglobosin C (3–9) were isolated from the culture of Penicillium chrysogenum V11. Their structures were elucidated by 1D, 2D NMR spectroscopic analysis and high resolution mass spectroscopic data. The absolute configuration of compounds 1 and 2 were determined by comparing the theoretical electronic circular dichroism (ECD) calculation with the experimental CD. Compound 1 was the first example, with a six-cyclic fused ring system formed by the connection of C-5 and C-2′ of the chaetoglobosin class. Compounds 5–8 remarkably inhibited the plant pathogenic fungus R. solani (minimum inhibitory concentrations (MICs) = 11.79–23.66 μM), and compounds 2, 6, and 7 greatly inhibited C. gloeosporioides (MICs = 23.58–47.35 μM), showing an antifungal activity higher than that of carbendazim. Compound 1 exhibited marked cytotoxicity against MDA-MB-435 and SGC-7901 cells (IC₅₀ < 10 μM), and compounds 6 and 9 showed potent cytotoxicity against SGC-7901 and A549 cells (IC₅₀ < 10 μM).     

Furobenzotropolones A,B and 3-Hydroxyepicoccone B with Antioxidative Activity from Mangrove Endophytic Fungus Epicoccum nigrum MLY-3

Three new metabolites, furobenzotropolones A, B (1–2) with unusual benzene and dihydrofuran moieties and 3-hydroxyepicoccone B (3), together with seven known compounds (4–10) were obtained from the endophytic fungus Epicoccum nigrum MLY-3 isolated from the fresh leaf of mangrove plant Bruguiear gymnorrhiza collected from Zhuhai. Their structures were assigned by the analysis of UV, IR, NMR, and mass spectroscopic data. Compound 1 was further confirmed by single-crystal X-ray diffraction experiment using Cu Kα radiation. In antioxidant activities in vitro, compounds 2, 3, 5, and 8 showed promising DPPH· scavenging activity with IC₅₀ values ranging from 14.7 to 29.3 µM. Compounds 2, 3, 5, 7, and 8 exhibited promising potent activity in scavenging ABTS· with IC₅₀ values in the range of 18–29.2 µM, which was stronger than that of the positive control ascorbic acid (IC₅₀ = 33.6 ± 0.8 µM).     

Polyhydroxy p-Terphenyls from a Mangrove Endophytic Fungus Aspergillus candidus LDJ-5

Six undescribed polyhydroxy p-terphenyls, namely asperterphenyllins A–F, were isolated from an endophytic fungus Aspergillus candidus LDJ-5. Their structures were determined by NMR and MS data. Differing from the previously reported p-terphenyls, asperterphenyllin A represents the first p-terphenyl dimer connected by a C-C bond. Asperterphenyllin A displayed anti-influenza virus A (H1N1) activity and protein tyrosine phosphatase 1B (PTP1B) inhibitory activity with IC₅₀ values of 53 μM and 21 μM, respectively. The anti-influenza virus A (H1N1) activity and protein tyrosine phosphatase 1B (PTP1B) inhibitory activity of p-terphenyls are reported for the first time. Asperterphenyllin G exhibited cytotoxicity against nine cell lines with IC₅₀ values ranging from 0.4 to 1.7 μM. Asperterphenyllin C showed antimicrobial activity against Proteus species with a MIC value of 19 μg/mL.     

Three New Resveratrol Derivatives from the Mangrove Endophytic Fungus Alternaria sp.

Three new resveratrol derivatives, namely, resveratrodehydes A–C (1–3), were isolated from the mangrove endophytic fungus Alternaria sp. R6. The structures of these compounds were elucidated by analysis of their MS, 1D and 2D NMR spectroscopic data. All compounds showed broad-spectrum inhibitory activities against three human cancer cell lines including human breast MDA-MB-435, human liver HepG2, and human colon HCT-116 by MTT assay (IC₅₀ < 50 μM). Among them, compounds 1 and 2 both exhibited marked cytotoxic activities against MDA-MB-435 and HCT-116 cell lines (IC₅₀ < 10 μM). Additionally, compounds 1 and 3 showed moderate antioxidant activity by DPPH radical scavenging assay.     

Design,Semisynthesis, Insecticidal and Antibacterial Activities of a Series of Marine-Derived Geodin Derivatives and Their Preliminary Structure–Activity Relationships

To enhance the biological activity of the natural product geodin (1), isolated from the marine-derived fungus Aspergillus sp., a series of new ether derivatives (2–37) was designed and semisynthesized using a high-yielding one-step reaction. In addition, the insecticidal and antibacterial activities of all geodin congeners were evaluated systematically. Most of these derivatives showed better insecticidal activities against Helicoverpa armigera Hübner than 1. In particular, 15 showed potent insecticidal activity with an IC₅₀ value of 89 μM, comparable to the positive control azadirachtin (IC₅₀ = 70 μM). Additionally, 5, 12, 13, 16, 30 and 33 showed strong antibacterial activity against Staphylococcus aureus and Aeromonas salmonicida with MIC values in the range of 1.15–4.93 μM. The preliminary structure–activity relationships indicated that the introduction of halogenated benzyl especially fluorobenzyl, into 1 and substitution of 4-OH could be key factors in increasing the insecticidal and antibacterial activities of geodin.     

Six New Diterpene Glycosides from the Soft Coral Lemnalia bournei

A chemical study on the extracts of soft coral Lemnalia bournei resulted in the isolation and identification of six new bicyclic diterpene glycosides including three new lemnaboursides E–G (1–3), and three new lemnadiolboursides A–C (4–6), along with three known lemnaboursides (7–9). Their structures were elucidated by detailed spectroscopic analysis, ECD analysis, chemical methods, and comparison with the literature data. Lemnadiolboursides A–C (4–6) contained a lemnal-1(10)-ene-7,12-diol moiety compared with the lemnaboursides. All these compounds were evaluated for antibacterial activity; cell growth inhibition of A549, Hela, HepG2, and CCRF-CEM cancer cell lines; and inhibition of LPS-induced NO production in RAW264.7 macrophages. The results indicated that compounds 1, 2, and 4–6 exhibited antibacterial activity against Staphylococcus aureus and Bacillus subtilis (MIC 4–16 μg/mL); compounds 1–9 displayed low cytotoxicity on the CCRF-CEM cell lines (IC₅₀ 10.44–27.40 µM); and compounds 1, 2, and 5 showed weak inhibition against LPS-induced NO production (IC₅₀ 21.56–28.06 μM).     

Cyclic Peptides from the Soft Coral-Derived Fungus Aspergillus sclerotiorum SCSIO 41031

Three novel cyclic hexapeptides, sclerotides C–E (1–3), and a new lipodepsipeptide, scopularide I (4), together with a known cyclic hexapeptide sclerotide A (5), were isolated from fermented rice cultures of a soft coral-derived fungus: Aspergillus sclerotiorum SCSIO 41031. The structures of the new peptides were determined by 1D and 2D NMR spectroscopic analysis, Marfey’s method, ESIMS/MS analysis, and single crystal X-ray diffraction analysis. Scopularide I (4) exhibited acetylcholinesterase inhibitory activity with an IC₅₀ value of 15.6 μM, and weak cytotoxicity against the human nasopharyngeal carcinoma cell line HONE-EBV with IC₅₀ value of 10.1 μM.     

Anti-Cancer and Anti-Inflammatory Activities of Three New Chromone Derivatives from the Marine-Derived Penicillium citrinum

Three new and uncommon chromone analogs, epiremisporine F (1), epiremisporine G (2), and epiremisporine H (3), were isolated from marine-origin Penicillium citrinum. Among the isolated compounds, compounds 2–3 remarkably suppressed fMLP-induced superoxide anion generation by human neutrophils, with IC₅₀ values of 31.68 ± 2.53, and 33.52 ± 0.42 μM, respectively. Compound 3 exhibited cytotoxic activities against human colon carcinoma (HT-29) and non-small lung cancer cell (A549) with IC₅₀ values of 21.17 ± 4.89 and 31.43 ± 3.01 μM, respectively, and Western blot assay confirmed that compound 3 obviously induced apoptosis of HT-29 cells, via Bcl-2, Bax, and caspase 3 signaling cascades.     

Thioester-Containing Benzoate Derivatives with α-Glucosidase Inhibitory Activity from the Deep-Sea-Derived Fungus Talaromyces indigoticus FS688

Eurothiocins C–H (1–6), six unusual thioester-containing benzoate derivatives, were isolated from the deep-sea-derived fungus Talaromyces indigoticus FS688 together with a known analogue eurothiocin A (7). Their structures were elucidated through spectroscopic analysis and the absolute configurations were determined by X-ray diffraction and ECD calculations. In addition, compound 1 exhibited significant inhibitory activity against α-glucosidase with an IC₅₀ value of 5.4 μM, while compounds 4 and 5 showed moderate effects with IC₅₀ values of 33.6 and 72.1 μM, respectively. A preliminary structure–activity relationship is discussed and a docking analysis was performed.     

Scalarane-Type Sesterterpenoids from the Marine Sponge Lendenfeldia sp. Alleviate Inflammation in Human Neutrophils

Sponge-derived scalaranes are remarkable sesterterpenoids previously found to exhibit profound inhibitory effects against neutrophilic inflammation. In our current work, we constructed the metabolomic profile of marine sponge Lendenfeldia sp. for the first time using a tandem mass spectrometry (MS/MS) molecular networking approach. The results highlighted the rich chemical diversity of these scalaranes, motivating us to conduct further research to discover novel scalaranes targeting neutrophilic inflammation. MS- and NMR-assisted isolation and elucidation led to the discovery of seven new homoscalaranes, lendenfeldaranes K–Q (1–7), characterized by methylation at C-24, together with five known derivatives, lendenfeldarane B (8), 25-nor-24-methyl-12,24-dioxoscalar-16-en-22-oic acid (9), 24-methyl-12,24,25-trioxoscalar-16-en-22-oic acid (10), felixin B (11), and 23-hydroxy-20-methyldeoxoscalarin (12). Scalaranes 1–4 and 6–12 were assayed against superoxide anion generation and elastase release, which represented the neutrophilic inflammatory responses of respiratory burst and degranulation, respectively. The results indicated that 1–3 and 6–12 exhibited potential anti-inflammatory activities (IC₅₀ for superoxide anion scavenging: 0.87~6.57 μM; IC₅₀ for elastase release: 1.12~6.97 μM).     

Lyngbyastatins 8–10, Elastase Inhibitors with Cyclic Depsipeptide Scaffolds Isolated from the Marine Cyanobacterium Lyngbya semiplena

Investigation of an extract from the marine cyanobacterium Lyngbya semiplena, collected in Tumon Bay, Guam, led to the identification of three new cyclodepsipeptides, lyngbyastatins 8–10 (1–3). The structures of 1–3 were determined by NMR, MS, ESIMS fragmentation and chemical degradation. Compounds 1–3 are closely related to lyngbyastatins 4–7. Like the latter compounds, we found 1–3 to inhibit porcine pancreatic elastase, with IC₅₀ values of 123 nM, 210 nM and 120 nM, respectively.     

Discovery and Evaluation of Thiazinoquinones as Anti-Protozoal Agents

Pure compound screening has identified the dioxothiazino-quinoline-quinone ascidian metabolite ascidiathiazone A (2) to be a moderate growth inhibitor of Trypanosoma brucei rhodesiense (IC₅₀ 3.1 μM) and Plasmodium falciparum (K1 dual drug resistant strain) (IC₅₀ 3.3 μM) while exhibiting low levels of cytotoxicity (L6, IC₅₀ 167 μM). A series of C-7 amide and Δ²⁽³⁾ analogues were prepared that explored the influence of lipophilicity and oxidation state on observed anti-protozoal activity and selectivity. Little variation in anti-malarial potency was observed (IC₅₀ 0.62–6.5 μM), and no correlation was apparent between anti-malarial and anti-T. brucei activity. Phenethylamide 7e and Δ²⁽³⁾-glycine analogue 8k exhibited similar anti-Pf activity to 2 but with slightly enhanced selectivity (SI 72 and 93, respectively), while Δ²⁽³⁾-phenethylamide 8e (IC₅₀ 0.67 μM, SI 78) exhibited improved potency and selectivity towards T. brucei rhodesiense compared to the natural product hit. A second series of analogues were prepared that replaced the quinoline ring of 2 with benzofuran or benzothiophene moieties. While esters 10a/10b and 15 were once again found to exhibit cytotoxicity, carboxylic acid analogues exhibited potent anti-Pf activity (IC₅₀ 0.34–0.035 μM) combined with excellent selectivity (SI 560–4000). In vivo evaluation of a furan carboxylic acid analogue against P. berghei was undertaken, demonstrating 85.7% and 47% reductions in parasitaemia with ip or oral dosing respectively.     

Fusarins G–L with Inhibition of NO in RAW264.7 from Marine-Derived Fungus Fusarium solani 7227

Six new fusarin derivatives, fusarins G–L (1–6), together with five known compounds (5–11) were isolated from the marine-derived fungus Fusarium solani 7227. The structures of the new compounds were elucidated by means of comprehensive spectroscopic methods (1D and 2D NMR, HRESIMS, ECD, and ORC) and X-ray crystallography. Compounds 5–11 exhibited potent anti-inflammatory activity by inhibiting the production of NO in RAW264.7 cells activated by lipopolysaccharide, with IC₅₀ values ranging from 3.6 to 32.2 μM. The structure–activity relationships of the fusarins are discussed herein.     

Inhibitory Activity of Marine Sponge-Derived Natural Products against Parasitic Protozoa

In this study, thirteen sponge-derived terpenoids, including five linear furanoterpenes: furospinulosin-1 (1), furospinulosin-2 (2), furospongin-1 (3), furospongin-4 (4), and demethylfurospongin-4 (5); four linear meroterpenes: 2-(hexaprenylmethyl)-2-methylchromenol (6), 4-hydroxy-3-octaprenylbenzoic acid (7), 4-hydroxy-3-tetraprenyl-phenylacetic acid (8), and heptaprenyl-p-quinol (9); a linear triterpene, squalene (10); two spongian-type diterpenes dorisenone D (11) and 11β-acetoxyspongi-12-en-16-one (12); a scalarane-type sesterterpene; 12-epi-deoxoscalarin (13), as well as an indole alkaloid, tryptophol (14) were screened for their in vitro activity against four parasitic protozoa; Trypanosoma brucei rhodesiense, Trypanosoma cruzi, Leishmania donovani and Plasmodium falciparum. Cytotoxic potential of the compounds on mammalian cells was also assessed. All compounds were active against T. brucei rhodesiense, with compound 8 being the most potent (IC₅₀ 0.60 μg/mL), whereas 9 and 12 were the most active compounds against T. cruzi, with IC₅₀ values around 4 μg/mL. Compound 12 showed the strongest leishmanicidal activity (IC₅₀ 0.75 μg/mL), which was comparable to that of miltefosine (IC₅₀ 0.20 μg/mL). The best antiplasmodial effect was exerted by compound 11 (IC₅₀ 0.43 μg/mL), followed by compounds 7, 10, and 12 with IC₅₀ values around 1 μg/mL. Compounds 9, 11 and 12 exhibited, besides their antiprotozoal activity, also some cytotoxicity, whereas all other compounds had low or no cytotoxicity towards the mammalian cell line. This is the first report of antiprotozoal activity of marine metabolites 1–14, and points out the potential of marine sponges in discovery of new antiprotozoal lead compounds.     

Chitosan Nanoparticles Attenuate Hydrogen Peroxide-Induced Stress Injury in Mouse Macrophage RAW264.7 Cells

This study was carried out to investigate the protective effects of chitosan nanoparticles (CNP) against hydrogen peroxide (H₂O₂)-induced oxidative damage in murine macrophages RAW264.7 cells. After 24 h pre-incubation with CNP (25–200 μg/mL) and chitosan (CS) (50–200 μg/mL, as controls), the viability loss in RAW264.7 cells induced by H₂O₂ (500 μM) for 12 h was markedly restored in a concentration-dependent manner as measured by MTT assay (P < 0.05) and decreased in cellular LDH release (P < 0.05). Moreover, CNP also exerted preventive effects on suppressing the production of lipid peroxidation such as malondialdehyde (MDA) (P < 0.05), restoring activities of endogenous antioxidant including superoxide dismutase (SOD), and glutathione peroxidase (GSH-Px) (P < 0.05), along with increasing total antioxidant capacity (T-AOC) (P < 0.05). In addition, pre-incubation of CNP with RAW264.7 cells for 24 h resulted in the increase of the gene expression level of endogenous antioxidant enzymes, such as MnSOD and GSH-Px (P < 0.05). At the same concentration, CNP significantly decreased LDH release and MDA (P < 0.05) as well as increased MnSOD, GSH-Px, and T-AOC activities (P < 0.05) as compared to CS. Taken together, our findings suggest that CNP can more effectively protect RAW264.7 cells against oxidative stress by H₂O₂ as compared to CS, which might be used as a potential natural compound-based antioxidant in the functional food and pharmaceutical industries.     

Rare Chromone Derivatives from the Marine-Derived Penicillium citrinum with Anti-Cancer and Anti-Inflammatory Activities

Three new and rare chromone derivatives, epiremisporine C (1), epiremisporine D (2), and epiremisporine E (3), were isolated from marine-derived Penicillium citrinum, together with four known compounds, epiremisporine B (4), penicitrinone A (5), 8-hydroxy-1-methoxycarbonyl-6-methylxanthone (6), and isoconiochaetone C (7). Among the isolated compounds, compounds 2–5 significantly decreased fMLP-induced superoxide anion generation by human neutrophils, with IC₅₀ values of 6.39 ± 0.40, 8.28 ± 0.29, 3.62 ± 0.61, and 2.67 ± 0.10 μM, respectively. Compounds 3 and 4 exhibited cytotoxic activities with IC₅₀ values of 43.82 ± 6.33 and 32.29 ± 4.83 μM, respectively, against non-small lung cancer cell (A549), and Western blot assay confirmed that compounds 3 and 4 markedly induced apoptosis of A549 cells, through Bcl-2, Bax, and caspase 3 signaling cascades.     

Chevalones H–M: Six New α-Pyrone Meroterpenoids from the Gorgonian Coral-Derived Fungus Aspergillus hiratsukae SCSIO 7S2001

Six new α-pyrone meroterpenoid chevalones H–M (1–6), together with six known compounds (7–12), were isolated from the gorgonian coral-derived fungus Aspergillus hiratsukae SCSIO 7S2001 collected from Mischief Reef in the South China Sea. Their structures, including absolute configurations, were elucidated on the basis of spectroscopic analysis and X-ray diffraction data. Compounds 1–5 and 7 showed different degrees of antibacterial activity with MIC values of 6.25–100 μg/mL. Compound 8 exhibited potent cytotoxicity against SF-268, MCF-7, and A549 cell lines with IC₅₀ values of 12.75, 9.29, and 20.11 μM, respectively.