Marine Drugs

17 pages, 2907 KiB

Open AccessArticle

The Microbial Community of Tetrodotoxin-Bearing and Non-Tetrodotoxin-Bearing Ribbon Worms (Nemertea) from the Sea of Japan

by Daria I. Melnikova and Timur Yu. Magarlamov

Mar. Drugs 2020, 18(3), 177; https://doi.org/10.3390/md18030177 - 23 Mar 2020

Cited by 5 | Viewed by 3219

Abstract

A potent marine toxin, tetrodotoxin (TTX), found in a great variety of marine and some terrestrial species, leaves intriguing questions about its origin and distribution in marine ecosystems. TTX-producing bacteria were found in the cultivable microflora of many TTX-bearing hosts, thereby providing strong [...] Read more.

A potent marine toxin, tetrodotoxin (TTX), found in a great variety of marine and some terrestrial species, leaves intriguing questions about its origin and distribution in marine ecosystems. TTX-producing bacteria were found in the cultivable microflora of many TTX-bearing hosts, thereby providing strong support for the hypothesis that the toxin is of bacterial origin in these species. However, metagenomic studies of TTX-bearing animals addressing the whole microbial composition and estimating the contribution of TTX-producing bacteria to the overall toxicity of the host were not conducted. The present study is the first to characterize and compare the 16S rRNA gene data obtained from four TTX-bearing and four non-TTX-bearing species of marine ribbon worms. The statistical analysis showed that different nemertean species harbor distinct bacterial communities, while members of the same species mostly share more similar microbiomes. The bacterial species historically associated with TTX production were found in all studied samples but predominated in TTX-bearing nemertean species. This suggests that deeper knowledge of the microbiome of TTX-bearing animals is a key to understanding the origin of TTX in marine ecosystems. Full article

(This article belongs to the Special Issue Tetrodotoxin: Chemistry, Toxicity, Source, Distribution and Detection)

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Taxonomic assignment at the phylum (a) and class (b) levels showing the relative abundance (%) of the 16S rRNA gene sequences from individual nemertean samples. Full article ">Figure 2
Venn diagram representing the number of stable and exclusive OTUs from a total of 32 OTUs for tetrodotoxin (TTX)-bearing (a) and non-TTX-bearing (b) nemerteans. Full article ">Figure 3
Measures of alpha diversity metrics for nemerteans microbiomes. Box-and-whisker plots comparing the numbers of observed OTUs (a), Faith’s phylogenetic diversity index (PD) (b), and Shannon diversity index (c). The boxes represent the interquartile range between the 75th and 25th percentiles and the internal lines the median value (50th percentile). The whiskers show the range. The ordinates: conventional units of the respective metrics. Full article ">Figure 4
Principal coordinate analysis (PCoA) of the nemertean microbial communities based on the Bray Curtis dissimilarity distance matrix (a) and Jaccard distance (b). The plots show the distances between the communities in the samples examined (n = 24). Full article ">Figure 5
Principal coordinate analysis (PCoA) of the nemertean microbial communities based on the weighted (a) and unweighted (b) UniFrac distance matrices. The plots show the distances between the communities in the samples studied (n = 24). Full article ">

10 pages, 2080 KiB

Open AccessCommunication

Design, Synthesis and Biological Evaluation of Jahanyne Analogs as Cell Cycle Arrest Inducers

by Baijun Ye, Jianmiao Gong, Qiuying Li, Shiqi Bao, Xuemei Zhang, Jing Chen, Qing Meng, Bolin Chen, Peng Jiang, Liang Wang and Yue Chen

Mar. Drugs 2020, 18(3), 176; https://doi.org/10.3390/md18030176 - 23 Mar 2020

Cited by 4 | Viewed by 2593

Abstract

Jahanyne, a lipopeptide with a unique terminal alkynyl and OEP (2-(1-oxo-ethyl)-pyrrolidine) moiety, exhibits anticancer activity. We synthesized jahanyne and analogs modified at the OEP moiety, employing an α-fluoromethyl ketone (FMK) strategy. Preliminary bioassays indicated that compound 1b (FMK–jahanyne) exhibited decreased activities to varying [...] Read more.

Jahanyne, a lipopeptide with a unique terminal alkynyl and OEP (2-(1-oxo-ethyl)-pyrrolidine) moiety, exhibits anticancer activity. We synthesized jahanyne and analogs modified at the OEP moiety, employing an α-fluoromethyl ketone (FMK) strategy. Preliminary bioassays indicated that compound 1b (FMK–jahanyne) exhibited decreased activities to varying degrees against most of the cancer cells tested, whereas the introduction of a fluorine atom to the α-position of a hydroxyl group (2b) enhanced activities against all lung cancer cells. Moreover, jahanyne and 2b could induce G0/G1 cell cycle arrest in a concentration-dependent manner. Full article

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15 pages, 3017 KiB

Open AccessArticle

Marine Microalgae, Spirulina maxima-Derived Modified Pectin and Modified Pectin Nanoparticles Modulate the Gut Microbiota and Trigger Immune Responses in Mice

by H.P.S.U. Chandrarathna, T.D. Liyanage, S.L. Edirisinghe, S.H.S. Dananjaya, E.H.T. Thulshan, Chamilani Nikapitiya, Chulhong Oh, Do-Hyung Kang and Mahanama De Zoysa

Mar. Drugs 2020, 18(3), 175; https://doi.org/10.3390/md18030175 - 21 Mar 2020

Cited by 33 | Viewed by 5572

Abstract

This study evaluated the modulation of gut microbiota, immune responses, and gut morphometry in C57BL/6 mice, upon oral administration of S. maxima-derived modified pectin (SmP, 7.5 mg/mL) and pectin nanoparticles (SmPNPs; 7.5 mg/mL). Metagenomics analysis was conducted using fecal samples, and mice [...] Read more.

This study evaluated the modulation of gut microbiota, immune responses, and gut morphometry in C57BL/6 mice, upon oral administration of S. maxima-derived modified pectin (SmP, 7.5 mg/mL) and pectin nanoparticles (SmPNPs; 7.5 mg/mL). Metagenomics analysis was conducted using fecal samples, and mice duodenum and jejunum were used for analyzing the immune response and gut morphometry, respectively. The results of metagenomics analysis revealed that the abundance of Bacteroidetes in the gut increased in response to both modified SmP and SmPNPs (75%) as compared with that in the control group (66%), while that of Firmicutes decreased in (20%) as compared with that in the control group (30%). The mRNA levels of mucin, antimicrobial peptide, and antiviral and gut permeability-related genes in the duodenum were significantly (p < 0.05) upregulated (> 2-fold) upon modified SmP and SmPNPs feeding. Protein level of intestinal alkaline phosphatase was increased (1.9-fold) in the duodenum of modified SmPNPs feeding, evidenced by significantly increased goblet cell density (0.5 ± 0.03 cells/1000 µm²) and villi height (352 ± 10 µm). Our results suggest that both modified SmP and SmPNPs have the potential to modulate gut microbial community, enhance the expression of immune related genes, and improve gut morphology. Full article

(This article belongs to the Special Issue Nano-Marine Drugs: Relevance of Nanoformulations in Cancer Therapies)

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Growth performance and blood glucose level of mice during 4 weeks of modified SmP and SmPNPs treatment. (A) Body weight (g); (B) Body weight gain percentage (%); (C) Blood glucose levels (mg/dL). 4* denoted the fasting glucose level at the end of the experiment (4th week); (means ± standard deviation, n = 4 per replicate, 3 replicates per group). Full article ">Figure 2
Metagenomics analysis of mouse fecal matter. (A) Sequencing reads, Operational Taxonomic Units (OTUs) count and diversity indices; (B) Venn diagram of observed OTUs; (C) Rarefaction curves of control, modified SmP and SmPNPs treated mice (n = 4 per replicate, 3 replicates per each group). Full article ">Figure 3
Diet-specific changes of taxonomic composition of fecal microbial community of control, modified SmP, and modified SmPNPs treated mice. (A) Comparison of relative abundance of metagenomics based gut microbial phyla; (B) Comparison of relative abundance of metagenomics based gut microbial families; (C) Principal component analysis (PCA) of relative abundance of gut microbiota families. The percentage variance of PC1 and PC2 are represented in x and y axis, respectively, (Software version: R 3.6.1, packages. Vegan). (n = 4 per replicate, 3 replicates per each group). Full article ">Figure 4
Transcriptional responses of immune related genes in modified SmP and SmPNPs supplemented mice. (A) Relative mRNA expression in control, modified SmP, and modified SmPNPs treatment groups. Relative expression-fold was presented as mean ± standard error. Asterisk mark is used to indicate the significant difference between the pectin treatments and control (3 replicates/group); (B) Comparison of relative expression fold between the treatment groups by color schematic representation. Basal expression level was considered as 1.0-fold; upregulated and down regulated expression were considered as >1.0- and <1.0-folds, respectively. Full article ">Figure 5
Immunoblot analysis of duodenum intestine alkaline phosphatase expression (IAP) of mice fed with modified SmP and SmPNPs supplemented diet and the control. (A) IAP expression. β-actin was used to confirm equal loading of proteins; (B) Quantitative analyses of IAP expression in modified SmP and SmPNPs, which normalized to β-actin and compared with the control. Data are expressed as the mean ± standard error (n = 3). Full article ">Figure 6
Histological analysis of jejunum morphometry of mice fed with modified SmP and SmPNPs supplemented diet as compared with control. (A) Light micrographs of Alcian blue and periodic acid=Schiff (AB-PAS) stained histological sections; (B) Comparison of goblet cell density; (C) Comparison of villi height in control, modified SmP, and modified SmPNPs treated mouse gut. a, villus goblet cells; b, crypt goblet cells; c, paneth cells. * Significantly (p < 0.05) higher goblet cell density and villi height as compared with the control; # significantly (p < 0.05) higher villi height in modified SmPNPs supplemented group as compared with modified SmP group. Full article ">

13 pages, 1892 KiB

Open AccessArticle

Identification of a Key Enzyme for the Hydrolysis of β-(1→3)-Xylosyl Linkage in Red Alga Dulse Xylooligosaccharide from Bifidobacterium Adolescentis

by Manami Kobayashi, Yuya Kumagai, Yohei Yamamoto, Hajime Yasui and Hideki Kishimura

Mar. Drugs 2020, 18(3), 174; https://doi.org/10.3390/md18030174 - 20 Mar 2020

Cited by 21 | Viewed by 4697

Abstract

Red alga dulse possesses a unique xylan, which is composed of a linear β-(1→3)/β-(1→4)-xylosyl linkage. We previously prepared characteristic xylooligosaccharide (DX3, (β-(1→3)-xylosyl-xylobiose)) from dulse. In this study, we evaluated the prebiotic effect of DX3 on enteric bacterium. Although DX3 was utilized by Bacteroides [...] Read more.

Red alga dulse possesses a unique xylan, which is composed of a linear β-(1→3)/β-(1→4)-xylosyl linkage. We previously prepared characteristic xylooligosaccharide (DX3, (β-(1→3)-xylosyl-xylobiose)) from dulse. In this study, we evaluated the prebiotic effect of DX3 on enteric bacterium. Although DX3 was utilized by Bacteroides sp. and Bifidobacterium adolescentis, Bacteroides Ksp. grew slowly as compared with β-(1→4)-xylotriose (X3) but B. adolescentis grew similar to X3. Therefore, we aimed to find the key DX3 hydrolysis enzymes in B. adolescentis. From bioinformatics analysis, two enzymes from the glycoside hydrolase family 43 (BAD0423: subfamily 12 and BAD0428: subfamily 11) were selected and expressed in Escherichia coli. BAD0423 hydrolyzed β-(1→3)-xylosyl linkage in DX3 with the specific activity of 2988 mU/mg producing xylose (X1) and xylobiose (X2), and showed low activity on X2 and X3. BAD0428 showed high activity on X2 and X3 producing X1, and the activity of BAD0428 on DX3 was 1298 mU/mg producing X1. Cooperative hydrolysis of DX3 was found in the combination of BAD0423 and BAD0428 producing X1 as the main product. From enzymatic character, hydrolysis of X3 was completed by one enzyme BAD0428, whereas hydrolysis of DX3 needed more than two enzymes. Full article

(This article belongs to the Collection Marine Polysaccharides)

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Effect of carbohydrate on bacterial growth and pH. The bacterial strains were cultured at 37 °C for 96 h in PYF (Peptone−Yeast extract−Fildes) medium containing 0.5% carbohydrate samples under anaerobic conditions. The data were obtained as ΔOD600 and ΔpH by the subtraction of each bacterial growth without carbohydrate samples. Error bars indicate SD (n = 3). Growth data of C. paraputrificum and E. limosum for β-(1→4)-xylotriose (X3) were not determined. Symbols: black bar, glucose; gray bar, xylose; red bar, X3; and blue bar, β-(1→3)/β-(1→4)-xylotriose (DX3). Full article ">Figure 2
Time course growth rate of B. vulgatus (a) and B. adolescentis (b). Bacteria grow in PYF medium containing 0.5% carbohydrates at 37 °C in anaerobic condition. Symbols: ●, glucose (G1); ◯, xylose (X1); ▲, X3; △, DX3. Mean ± standard deviation of three replicate determinations and the error bars were within the symbols. Full article ">Figure 3
Relationship between bacteria and the number of enzymes containing β-xylosidase (EC 3.2.1.37) in glycoside hydrolase families (GHs). The colors are related to the number of enzymes from pink (low) to red (high). The bold and underlined number means that GHs contain β-xylosidase. Full article ">Figure 4
Relationship between bacteria and the number of enzymes in the GH43 subfamily. The colors are related to the number of enzymes from pink (low) to purple (high). The bold and underlined number shows subfamilies contain β-xylosidase. Full article ">Figure 5
Hydrolysis products of oligosaccharides (XOS). Ten mM XOS was hydrolyzed by 50 μg/mL BAD0423, BAD0428, and BAD1527 at pH 6.5 and 37 °C for 1 h. The products were analyzed by HPLC. Black and gray indicate the amount of X1 and X2, respectively. Full article ">Figure 6
The putative hydrolysis mechanism of DX3 and X3. (a) DX3; (b) X3. Full article ">

28 pages, 2406 KiB

Open AccessReview

Marine Toxins Targeting Kv1 Channels: Pharmacological Tools and Therapeutic Scaffolds

by Rocio K. Finol-Urdaneta, Aleksandra Belovanovic, Milica Micic-Vicovac, Gemma K. Kinsella, Jeffrey R. McArthur and Ahmed Al-Sabi

Mar. Drugs 2020, 18(3), 173; https://doi.org/10.3390/md18030173 - 20 Mar 2020

Cited by 36 | Viewed by 6012

Abstract

Toxins from marine animals provide molecular tools for the study of many ion channels, including mammalian voltage-gated potassium channels of the Kv1 family. Selectivity profiling and molecular investigation of these toxins have contributed to the development of novel drug leads with therapeutic potential [...] Read more.

Toxins from marine animals provide molecular tools for the study of many ion channels, including mammalian voltage-gated potassium channels of the Kv1 family. Selectivity profiling and molecular investigation of these toxins have contributed to the development of novel drug leads with therapeutic potential for the treatment of ion channel-related diseases or channelopathies. Here, we review specific peptide and small-molecule marine toxins modulating Kv1 channels and thus cover recent findings of bioactives found in the venoms of marine Gastropod (cone snails), Cnidarian (sea anemones), and small compounds from cyanobacteria. Furthermore, we discuss pivotal advancements at exploiting the interaction of κM-conotoxin RIIIJ and heteromeric Kv1.1/1.2 channels as prevalent neuronal Kv complex. RIIIJ’s exquisite Kv1 subtype selectivity underpins a novel and facile functional classification of large-diameter dorsal root ganglion neurons. The vast potential of marine toxins warrants further collaborative efforts and high-throughput approaches aimed at the discovery and profiling of Kv1-targeted bioactives, which will greatly accelerate the development of a thorough molecular toolbox and much-needed therapeutics. Full article

(This article belongs to the Special Issue Ion Channels as Marine Drug Targets)

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15 pages, 2745 KiB

Open AccessArticle

Exploring Ultrasound, Microwave and Ultrasound–Microwave Assisted Extraction Technologies to Increase the Extraction of Bioactive Compounds and Antioxidants from Brown Macroalgae

by Marco Garcia-Vaquero, Viruja Ummat, Brijesh Tiwari and Gaurav Rajauria

Mar. Drugs 2020, 18(3), 172; https://doi.org/10.3390/md18030172 - 20 Mar 2020

Cited by 149 | Viewed by 12160

Abstract

This study aims to determine the influence of (1) ultrasound-assisted extraction (UAE), (2) microwave-assisted extraction (MAE) and (3) a combination of ultrasound–microwave-assisted extraction (UMAE) on the yields of fucose-sulphated polysaccharides (FSPs), total soluble carbohydrates and antioxidants extracted from A. nodosum. Scanning electron [...] Read more.

This study aims to determine the influence of (1) ultrasound-assisted extraction (UAE), (2) microwave-assisted extraction (MAE) and (3) a combination of ultrasound–microwave-assisted extraction (UMAE) on the yields of fucose-sulphated polysaccharides (FSPs), total soluble carbohydrates and antioxidants extracted from A. nodosum. Scanning electron microscopy (SEM) was used to evaluate the influence of the extraction technologies on the surface of macroalgae while principal component analysis was used to assess the influence of the extraction forces on the yields of compounds. UMAE generated higher yields of compounds compared to UAE and MAE methods separately. The maximum yields of compounds achieved using UMAE were: FSPs (3533.75 ± 55.81 mg fucose/100 g dried macroalgae (dm)), total soluble carbohydrates (10408.72 ± 229.11 mg glucose equivalents/100 g dm) and phenolic compounds (2605.89 ± 192.97 mg gallic acid equivalents/100 g dm). The antioxidant properties of the extracts showed no clear trend or extreme improvements by using UAE, MAE or UMAE. The macroalgal cells were strongly altered by the application of MAE and UMAE, as revealed by the SEM images. Further research will be needed to understand the combined effect of sono-generated and microwave-induced modifications on macroalgae that will allow us to tailor the forces of extraction to target specific molecules. Full article

(This article belongs to the Special Issue Marine Nutraceuticals and Functional Foods)

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Full article ">Figure 1
Scheme summarizing the preparation, pre-treatments and extraction conditions applied to A. nodosum to generate macroalgal extracts together with the chemical analyses performed. Full article ">Figure 2
Yields of fucose-sulphated polysaccharides (FSPs), total soluble carbohydrates, phenolic compounds and antioxidant activities (FRAP and DPPH) of extracts from A. nodosum obtained by UAE at ultrasonic amplitudes (20%, 50% and 100%). Light and dark bars represent ultrasound-assisted extraction (UAE) treatments of 2 or 5 min, respectively. Results are expressed as average ± standard deviation of the mean (n = 6). The statistical differences between different treatment times for each UAE combination are expressed as follows: * P < 0.05, ** P < 0.01, *** P < 0.001. Full article ">Figure 3
Yields of FSPs, total soluble carbohydrates, phenolic compounds and antioxidant activities (FRAP and DPPH) of extracts from A. nodosum obtained by microwave-assisted extraction (MAE) at microwave powers (250, 600 and 1000 W). Light and dark bars represent MAE treatments of 2 or 5 min, respectively. Results are expressed as average ± standard deviation of the mean (n = 6). The statistical differences between different treatment times for each MAE combination are expressed as follows: * P < 0.05, ** P < 0.01, *** P < 0.001. Full article ">Figure 4
Yields of FSPs, total soluble carbohydrates and phenolic compounds of extracts from A. nodosum obtained by ultrasound–microwave-assisted extraction (UMAE). The recoveries were explored using multiple combinations of ultrasonic amplitude (20%, 50% and 100%) and microwave power (250, 600 and 1000 W). Light and dark bars represent the yields of each compound obtained when extracting macroalgae for 2 or 5 min, respectively. Results are expressed as average ± standard deviation of the mean (n = 6). The statistical differences between different treatment times for each UMAE combination are expressed as follows: * P < 0.05, ** P < 0.01, *** P < 0.001. Full article ">Figure 5
Antioxidant activities (FRAP and DPPH) of extracts from A. nodosum obtained by UMAE at multiple combinations of ultrasonic amplitude (20%, 50% and 100%) and microwave power (250, 600 and 1000 W). Light and dark bars represent the yields of each compound obtained when extracting macroalgae for 2 or 5 min, respectively. Results are expressed as average ± standard deviation of the mean (n = 6). The statistical differences between different treatment times for each UMAE combination are expressed as follows: * P < 0.05, ** P < 0.01, *** P < 0.001. Full article ">Figure 6
Principal component analysis scatter plot representing the scores for the extraction yields of FSPs, total soluble carbohydrates, phenols and antioxidant activities (FRAP and DPPH) of extracts from A. nodosum obtained using sonication and microwave technological treatments. Full article ">Figure 7
Scanning electron microscopy images of (I) dried and milled A. nodosum biomass before extraction, (II) macroalgal residue after MAE (250 W, 2 min) and (III) macroalgal biomass after the process of UMAE (1000 W, 100%, 5 min). Scale bars (A) 200 µm (magnification: 250×) and (B) 50 µm (magnification: 1000×). Full article ">Figure 8
Schemes showing the technological designs (I) ultrasound-assisted extraction (UAE), (II) microwave-assisted extraction (MAE) and (III) ultrasound–microwave-assisted extraction (UMAE) used to generate macroalgal extracts. Full article ">

10 pages, 2313 KiB

Open AccessArticle

Clavukoellians G–K, New Nardosinane and Aristolane Sesquiterpenoids with Angiogenesis Promoting Activity from the Marine Soft Coral Lemnalia sp.

by Qi Wang, Xuli Tang, Hui Liu, Xiangchao Luo, Ping Jyun Sung, Pinglin Li and Guoqiang Li

Mar. Drugs 2020, 18(3), 171; https://doi.org/10.3390/md18030171 - 20 Mar 2020

Cited by 16 | Viewed by 3461

Abstract

The chemical examination of the marine soft coral Lemnalia sp., collected at the Xisha islands in the South China Sea, resulted in the isolation of four new nardosinane-type sesquiterpenoids, namely clavukoellians G–J (1–4), and one new aristolane sesquiterpene, namely [...] Read more.

The chemical examination of the marine soft coral Lemnalia sp., collected at the Xisha islands in the South China Sea, resulted in the isolation of four new nardosinane-type sesquiterpenoids, namely clavukoellians G–J (1–4), and one new aristolane sesquiterpene, namely clavukoellian K (5), together with five known compounds, 6–10. The structure elucidation of the isolated natural products was based on various spectroscopic techniques including HRESIMS and NMR, while their absolute configurations were resolved on the basis of comparisons of the ECD spectra with the calculated ECD data. The isolated new compounds 1–5 were evaluated for their anti- and pro- angiogenesis activities in a transgenic fluorescent zebrafish (Tg(vegfr2:GFP)) model. Quantitative analysis revealed that compound 5 displayed pro-angiogenesis activity in a PTK787-induced vascular injury zebrafish model at 2.5 μM. Data showed that compound 5 significantly promoted the angiogenesis in a dose-dependent manner. Full article

(This article belongs to the Special Issue Advancements in Purification, Isolation and Chemical Profiling to Expedite the Discovery of Marine Natural Products)

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22 pages, 1180 KiB

Open AccessReview

Fucoidans: Downstream Processes and Recent Applications

by Ahmed Zayed and Roland Ulber

Mar. Drugs 2020, 18(3), 170; https://doi.org/10.3390/md18030170 - 18 Mar 2020

Cited by 66 | Viewed by 8234

Abstract

Fucoidans are multifunctional marine macromolecules that are subjected to numerous and various downstream processes during their production. These processes were considered the most important abiotic factors affecting fucoidan chemical skeletons, quality, physicochemical properties, biological properties and industrial applications. Since a universal protocol for [...] Read more.

Fucoidans are multifunctional marine macromolecules that are subjected to numerous and various downstream processes during their production. These processes were considered the most important abiotic factors affecting fucoidan chemical skeletons, quality, physicochemical properties, biological properties and industrial applications. Since a universal protocol for fucoidans production has not been established yet, all the currently used processes were presented and justified. The current article complements our previous articles in the fucoidans field, provides an updated overview regarding the different downstream processes, including pre-treatment, extraction, purification and enzymatic modification processes, and shows the recent non-traditional applications of fucoidans in relation to their characters. Full article

(This article belongs to the Special Issue Fucoidans)

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18 pages, 3312 KiB

Open AccessArticle

Autotrophic and Heterotrophic Growth Conditions Modify Biomolecole Production in the Microalga Galdieria sulphuraria (Cyanidiophyceae, Rhodophyta)

by Roberto Barone, Lorenzo De Napoli, Luciano Mayol, Marina Paolucci, Maria Grazia Volpe, Luigi D’Elia, Antonino Pollio, Marco Guida, Edvige Gambino, Federica Carraturo, Roberta Marra, Francesco Vinale, Sheridan Lois Woo and Matteo Lorito

Mar. Drugs 2020, 18(3), 169; https://doi.org/10.3390/md18030169 - 18 Mar 2020

Cited by 21 | Viewed by 5350

Abstract

Algae have multiple similarities with fungi, with both belonging to the Thallophyte, a polyphyletic group of non-mobile organisms grouped together on the basis of similar characteristics, but not sharing a common ancestor. The main difference between algae and fungi is noted [...] Read more.

Algae have multiple similarities with fungi, with both belonging to the Thallophyte, a polyphyletic group of non-mobile organisms grouped together on the basis of similar characteristics, but not sharing a common ancestor. The main difference between algae and fungi is noted in their metabolism. In fact, although algae have chlorophyll-bearing thalloids and are autotrophic organisms, fungi lack chlorophyll and are heterotrophic, not able to synthesize their own nutrients. However, our studies have shown that the extremophilic microalga Galderia sulphuraria (GS) can also grow very well in heterotrophic conditions like fungi. This study was carried out using several approaches such as scanning electron microscope (SEM), gas chromatography/mass spectrometry (GC/MS), and infrared spectrophotometry (ATR-FTIR). Results showed that the GS, strain ACUF 064, cultured in autotrophic (AGS) and heterotrophic (HGS) conditions, produced different biomolecules. In particular, when grown in HGS, the algae (i) was 30% larger, with an increase in carbon mass that was 20% greater than AGS; (ii) produced higher quantities of stearic acid, oleic acid, monounsaturated fatty acids (MUFAs), and ergosterol; (iii) produced lower quantities of fatty acid methyl esters (FAMEs) such as methyl palmytate, and methyl linoleate, saturated fatty acids (SFAs), and poyliunsaturated fatty acids (PUFAs). ATR-FTIR and principal component analysis (PCA) statistical analysis confirmed that the macromolecular content of HGS was significantly different from AGS. The ability to produce different macromolecules by changing the trophic conditions may represent an interesting strategy to induce microalgae to produce different biomolecules that can find applications in several fields such as food, feed, nutraceutical, or energy production. Full article

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Galdieria sulphuraria strain ACUF 064 cultured in (a) heterotrophic (FOV: 62.5 µm, mode: 15kV-point, detector: BSD full) and (b) autotrophic conditions (FOV 39.5 µm, mode: 15kV-point, detector: BSD full). Full article ">Figure 2
Different content of elements in Galdieria sulphuraria strain ACUF 064 cultured in heterotrophic (a) and autotrophic (b) conditions. Percentages are reported in <a href="#marinedrugs-18-00169-t001" class="html-table">Table 1</a>. Full article ">Figure 3
Infrared spectrophotometry (ATR-FTIR) spectra of Galdieria sulphuraria strain ACUF 064 cultured in autotrophic (____) and heterotrophic (___) conditions. (___) Spirulina platensis. Full article ">Figure 4
(A) Representative ATR-FTIR spectra of Galdieria sulphuraria strain ACUF 064 cultured in autotrophic (___) and heterotrophic (___) conditions and the substraction spectrum (___). (B) Second derivatives of Galdieria sulphuraria strain ACUF 064 cultured in autotrophic (___) and heterotrophic (___) conditions. (___) Spirulina platensis. Full article ">Figure 5
Three-dimensional principal component analysis score plot of Galdieria sulphuraria strain ACUF 064 cultivated in autotrophic (▄) and heterotrophic conditions (▄), plus Spirulina platensis in autotrophic conditions (▄). Data analysis was performed in the spectrum ranges reported in the rectangles above each plot. Full article ">

29 pages, 2151 KiB

Open AccessReview

Advanced Technologies for the Extraction of Marine Brown Algal Polysaccharides

by Ana Dobrinčić, Sandra Balbino, Zoran Zorić, Sandra Pedisić, Danijela Bursać Kovačević, Ivona Elez Garofulić and Verica Dragović-Uzelac

Mar. Drugs 2020, 18(3), 168; https://doi.org/10.3390/md18030168 - 18 Mar 2020

Cited by 181 | Viewed by 13472

Abstract

Over the years, brown algae bioactive polysaccharides laminarin, alginate and fucoidan have been isolated and used in functional foods, cosmeceutical and pharmaceutical industries. The extraction process of these polysaccharides includes several complex and time-consuming steps and the correct adjustment of extraction parameters (e.g., [...] Read more.

Over the years, brown algae bioactive polysaccharides laminarin, alginate and fucoidan have been isolated and used in functional foods, cosmeceutical and pharmaceutical industries. The extraction process of these polysaccharides includes several complex and time-consuming steps and the correct adjustment of extraction parameters (e.g., time, temperature, power, pressure, solvent and sample to solvent ratio) greatly influences the yield, physical, chemical and biochemical properties as well as their biological activities. This review includes the most recent conventional procedures for brown algae polysaccharides extraction along with advanced extraction techniques (microwave-assisted extraction, ultrasound assisted extraction, pressurized liquid extraction and enzymes assisted extraction) which can effectively improve extraction process. The influence of these extraction techniques and their individual parameters on yield, chemical structure and biological activities from the most current literature is discussed, along with their potential for commercial applications as bioactive compounds and drug delivery systems. Full article

(This article belongs to the Collection Marine Polysaccharides)

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14 pages, 1672 KiB

Open AccessArticle

Antimalarial Peptide and Polyketide Natural Products from the Fijian Marine Cyanobacterium Moorea producens

by Anne Marie Sweeney-Jones, Kerstin Gagaring, Jenya Antonova-Koch, Hongyi Zhou, Nazia Mojib, Katy Soapi, Jeffrey Skolnick, Case W. McNamara and Julia Kubanek

Mar. Drugs 2020, 18(3), 167; https://doi.org/10.3390/md18030167 - 18 Mar 2020

Cited by 34 | Viewed by 5195

Abstract

A new cyclic peptide, kakeromamide B (1), and previously described cytotoxic cyanobacterial natural products ulongamide A (2), lyngbyabellin A (3), 18E-lyngbyaloside C (4), and lyngbyaloside (5) were identified from an antimalarial [...] Read more.

A new cyclic peptide, kakeromamide B (1), and previously described cytotoxic cyanobacterial natural products ulongamide A (2), lyngbyabellin A (3), 18E-lyngbyaloside C (4), and lyngbyaloside (5) were identified from an antimalarial extract of the Fijian marine cyanobacterium Moorea producens. Compounds 1 and 2 exhibited moderate activity against Plasmodium falciparum blood-stages with EC₅₀ values of 0.89 and 0.99 µM, respectively, whereas 3 was more potent with an EC₅₀ value of 0.15 nM. Compounds 1, 4, and 5 displayed moderate liver-stage antimalarial activity against P. berghei liver schizonts with EC₅₀ values of 1.1, 0.71, and 0.45 µM, respectively. The threading-based computational method FINDSITE^comb2.0 predicted the binding of 1 and 2 to potentially druggable proteins of Plasmodium falciparum, prompting formulation of hypotheses about possible mechanisms of action. Kakeromamide B (1) was predicted to bind to several Plasmodium actin-like proteins and a sortilin protein suggesting possible interference with parasite invasion of host cells. When 1 was tested in a mammalian actin polymerization assay, it stimulated actin polymerization in a dose-dependent manner, suggesting that 1 does, in fact, interact with actin. Full article

(This article belongs to the Special Issue Compounds from Cyanobacteria III)

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11 pages, 1983 KiB

Open AccessCommunication

Lysophosphatidylcholines and Chlorophyll-Derived Molecules from the Diatom Cylindrotheca closterium with Anti-Inflammatory Activity

by Chiara Lauritano, Kirsti Helland, Gennaro Riccio, Jeanette H. Andersen, Adrianna Ianora and Espen H. Hansen

Mar. Drugs 2020, 18(3), 166; https://doi.org/10.3390/md18030166 - 17 Mar 2020

Cited by 58 | Viewed by 5069

Abstract

Microalgae have been shown to be excellent producers of lipids, pigments, carbohydrates, and a plethora of secondary metabolites with possible applications in the pharmacological, nutraceutical, and cosmeceutical sectors. Recently, various microalgal raw extracts have been found to have anti-inflammatory properties. In this study, [...] Read more.

Microalgae have been shown to be excellent producers of lipids, pigments, carbohydrates, and a plethora of secondary metabolites with possible applications in the pharmacological, nutraceutical, and cosmeceutical sectors. Recently, various microalgal raw extracts have been found to have anti-inflammatory properties. In this study, we performed the fractionation of raw extracts of the diatom Cylindrotheca closterium, previously shown to have anti-inflammatory properties, obtaining five fractions. Fractions C and D were found to significantly inhibit tumor necrosis factor alpha (TNF-⍺) release in LPS-stimulated human monocyte THP-1 cells. A dereplication analysis of these two fractions allowed the identification of their main components. Our data suggest that lysophosphatidylcholines and a breakdown product of chlorophyll, pheophorbide a, were probably responsible for the observed anti-inflammatory activity. Pheophorbide a is known to have anti-inflammatory properties. We tested and confirmed the anti-inflammatory activity of 1-palmitoyl-sn-glycero-3-phosphocholine, the most abundant lysophosphatidylcholine found in fraction C. This study demonstrated the importance of proper dereplication of bioactive extracts and fractions before isolation of compounds is commenced. Full article

(This article belongs to the Special Issue Bioactive Molecules from Marine Microorganisms)

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10 pages, 989 KiB

Open AccessCommunication

Sesquiterpenes and Cyclodepsipeptides from Marine-Derived Fungus Trichoderma longibrachiatum and Their Antagonistic Activities against Soil-Borne Pathogens

by Feng-Yu Du, Guang-Lin Ju, Lin Xiao, Yuan-Ming Zhou and Xia Wu

Mar. Drugs 2020, 18(3), 165; https://doi.org/10.3390/md18030165 - 16 Mar 2020

Cited by 36 | Viewed by 3531

Abstract

Soil-borne pathogens, including phytopathogenic fungi and root-knot nematodes, could synergistically invade vegetable roots and result in serious economic losses. The genus of Trichoderma has been proven to be a promising reservoir of biocontrol agents in agriculture. In this study, the search for antagonistic [...] Read more.

Soil-borne pathogens, including phytopathogenic fungi and root-knot nematodes, could synergistically invade vegetable roots and result in serious economic losses. The genus of Trichoderma has been proven to be a promising reservoir of biocontrol agents in agriculture. In this study, the search for antagonistic metabolites from a marine-derived fungus, Trichoderma longibrachiatum, obtained two structural series of sesquiterpenes 1–6 and cyclodepsipeptides 7–9. Notably, the novel 1 was a rare norsesquiterpene characterized by an unprecedented tricyclic-6/5/5-[4.3.1.0^1,6]-decane skeleton. Their structures were elucidated by extensive spectroscopic analyses, while the absolute configuration of novel 1 was determined by the comparison of experimental and calculated ECD spectra. The novel 1 and known 2 and 3 showed significant antifungal activities against Colletotrichum lagrnarium with MIC values of 8, 16, and 16 μg/mL respectively, even better than those of the commonly used synthetic fungicide carbendazim with 32 μg/mL. They also exhibited antifungal potential against carbendazim-resistant Botrytis cinerea. Cyclodepsipeptides 7–9 showed moderate nematicidal activities against the southern root-knot nematode (Meloidogyne incognita). This study constitutes the first report on the antagonistic effects of metabolites from T. Longibrachiatum against soil-borne pathogens, also highlighting the integrated antagonistic potential of marine-derived T. Longibrachiatum as a biocontrol agent. Full article

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9 pages, 2257 KiB

Open AccessArticle

Terpenoids from the Deep-Sea-Derived Fungus Penicillium thomii YPGA3 and Their Bioactivities

by Zhongbin Cheng, Wan Liu, Runzhu Fan, Shouye Han, Yuanli Li, Xiaoyun Cui, Jia Zhang, Yinan Wu, Xin Lv, Yun Zhang, Zhuhua Luo, Siti Aisyah Alias, Wei Xu and Qin Li

Mar. Drugs 2020, 18(3), 164; https://doi.org/10.3390/md18030164 - 16 Mar 2020

Cited by 16 | Viewed by 3483

Abstract

A chemical study of the ethyl acetate (EtOAc) extract from the deep-sea-derived fungus Penicillium thomii YPGA3 led to the isolation of a new austalide meroterpenoid (1) and seven known analogues (2−8), two new labdane-type diterpenoids (9 [...] Read more.

A chemical study of the ethyl acetate (EtOAc) extract from the deep-sea-derived fungus Penicillium thomii YPGA3 led to the isolation of a new austalide meroterpenoid (1) and seven known analogues (2−8), two new labdane-type diterpenoids (9 and 10) and a known derivative (11). The structures of new compounds 1, 9, and 10 were determined by comprehensive analyses via nuclear magnetic resonance (NMR) and mass spectroscopy (MS) data. The absolute configurations of 1, 9, and 10 were determined by comparisons of experimental electronic circular dichroism (ECD) with the calculated ECD spectra. Compound 1 represented the third example of austalides bearing a hydroxyl group at C-5 instead of the conserved methoxy in other known analogues. To our knowledge, diterpenoids belonging to the labdane-type were discovered from species of Penicillium for the first time. Compound 1 showed cytotoxicity toward MDA-MB-468 cells with an IC₅₀ value of 38.9 μM. Compounds 2 and 11 exhibited inhibition against α-glucosidase with IC₅₀ values of 910 and 525 μM, respectively, being more active than the positive control acarbose (1.33 mM). Full article

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Full article ">Figure 1
Structures of compounds 1–11 from Penicillium thomii YPGA3. Full article ">Figure 2
Key correlation spectroscopy (COSY, <img alt="Marinedrugs 18 00164 i001" src="/marinedrugs/marinedrugs-18-00164/article_deploy/html/images/marinedrugs-18-00164-i001.png"/>) and heteronuclear multiple-bond correlations (HMBC, <img alt="Marinedrugs 18 00164 i002" src="/marinedrugs/marinedrugs-18-00164/article_deploy/html/images/marinedrugs-18-00164-i002.png"/>) of compounds 1, 9, and 10. Full article ">Figure 3
Key nuclear Overhauser effect spectroscopy (NOESY) correlations ( <img alt="Marinedrugs 18 00164 i003" src="/marinedrugs/marinedrugs-18-00164/article_deploy/html/images/marinedrugs-18-00164-i003.png"/>) of compounds 1 and 9. Full article ">Figure 4
Experimental electronic circular dichroism (ECD) spectra (200–400 nm) of compounds 1 and 2 in methanol and the calculated ECD spectrum of 11S, 14R, 20S, 21R-1 at the B3LYP/6-31+G(d,p) level in methanol. Full article ">Figure 5
Experimental ECD spectra (200–300 nm) of 9 and 10 in methanol and the calculated ECD spectrum of 3S, 4R, 5R, 9S, 10R-9 at the B3LYP/6-31+G(d,p) level in methanol. Full article ">

17 pages, 4826 KiB

Open AccessArticle

Synthesis and Characterization of N,N,N-trimethyl-O-(ureidopyridinium)acetyl Chitosan Derivatives with Antioxidant and Antifungal Activities

by Jingjing Zhang, Wenqiang Tan, Qing Li, Fang Dong and Zhanyong Guo

Mar. Drugs 2020, 18(3), 163; https://doi.org/10.3390/md18030163 - 16 Mar 2020

Cited by 16 | Viewed by 2984

Abstract

Chitosan is an active biopolymer, and the combination of it with other active groups can be a valuable method to improve the potential application of the resultant derivatives in food, cosmetics, packaging materials, and other industries. In this paper, a series of N [...] Read more.

Chitosan is an active biopolymer, and the combination of it with other active groups can be a valuable method to improve the potential application of the resultant derivatives in food, cosmetics, packaging materials, and other industries. In this paper, a series of N,N,N-trimethyl-O-(ureidopyridinium)acetyl chitosan derivatives were synthesized. The combination of chitosan with ureidopyridinium group and quaternary ammonium group made it achieve developed water solubility and biological properties. The structures of chitosan and chitosan derivatives were confirmed by FTIR, ¹H NMR spectra, and elemental analysis. The prepared chitosan derivatives were evaluated for antioxidant property by 1,1-diphenyl-2-picrylhydrazyl (DPPH) radical scavenging ability, hydroxyl radical scavenging ability, and superoxide radical scavenging ability. The results revealed that the synthesized chitosan derivatives exhibited improved antioxidant activity compared with chitosan. The chitosan derivatives were also investigated for antifungal activity against Phomopsis asparagus as well as Botrytis cinerea, and they showed a significant inhibitory effect on the selected phytopathogen. Meanwhile, CCK-8 assay was used to test the cytotoxicity of chitosan derivatives, and the results showed that most derivatives had low toxicity. These data suggested to develop analogs of chitosan derivatives containing ureidopyridinium group and quaternary ammonium group, which will provide a new kind of promising biomaterials having decreased cytotoxicity as well as excellent antioxidant and antimicrobial activity. Full article

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38 pages, 4661 KiB

Open AccessReview

Terpenoids in Marine Heterobranch Molluscs

by Conxita Avila

Mar. Drugs 2020, 18(3), 162; https://doi.org/10.3390/md18030162 - 14 Mar 2020

Cited by 29 | Viewed by 6503

Abstract

Heterobranch molluscs are rich in natural products. As other marine organisms, these gastropods are still quite unexplored, but they provide a stunning arsenal of compounds with interesting activities. Among their natural products, terpenoids are particularly abundant and diverse, including monoterpenoids, sesquiterpenoids, diterpenoids, sesterterpenoids, [...] Read more.

Heterobranch molluscs are rich in natural products. As other marine organisms, these gastropods are still quite unexplored, but they provide a stunning arsenal of compounds with interesting activities. Among their natural products, terpenoids are particularly abundant and diverse, including monoterpenoids, sesquiterpenoids, diterpenoids, sesterterpenoids, triterpenoids, tetraterpenoids, and steroids. This review evaluates the different kinds of terpenoids found in heterobranchs and reports on their bioactivity. It includes more than 330 metabolites isolated from ca. 70 species of heterobranchs. The monoterpenoids reported may be linear or monocyclic, while sesquiterpenoids may include linear, monocyclic, bicyclic, or tricyclic molecules. Diterpenoids in heterobranchs may include linear, monocyclic, bicyclic, tricyclic, or tetracyclic compounds. Sesterterpenoids, instead, are linear, bicyclic, or tetracyclic. Triterpenoids, tetraterpenoids, and steroids are not as abundant as the previously mentioned types. Within heterobranch molluscs, no terpenoids have been described in this period in tylodinoideans, cephalaspideans, or pteropods, and most terpenoids have been found in nudibranchs, anaspideans, and sacoglossans, with very few compounds in pleurobranchoideans and pulmonates. Monoterpenoids are present mostly in anaspidea, and less abundant in sacoglossa. Nudibranchs are especially rich in sesquiterpenes, which are also present in anaspidea, and in less numbers in sacoglossa and pulmonata. Diterpenoids are also very abundant in nudibranchs, present also in anaspidea, and scarce in pleurobranchoidea, sacoglossa, and pulmonata. Sesterterpenoids are only found in nudibranchia, while triterpenoids, carotenoids, and steroids are only reported for nudibranchia, pleurobranchoidea, and anaspidea. Many of these compounds are obtained from their diet, while others are biotransformed, or de novo biosynthesized by the molluscs. Overall, a huge variety of structures is found, indicating that chemodiversity correlates to the amazing biodiversity of this fascinating group of molluscs. Full article

(This article belongs to the Special Issue Secondary Metabolites from Marine Molluscs: Chemistry, Biosynthesis, Synthesis and Bioactivity)

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11 pages, 1238 KiB

Open AccessArticle

Establishment of Novel High-Standard Chemiluminescent Assay for NTPase in Two Protozoans and Its High-Throughput Screening

by Masamitsu Harada, Jun Nagai, Riho Kurata, Kenji Shimizu, Xiaofeng Cui, Takayuki Isagawa, Hiroaki Semba, Jun Ishihara, Yasuhiro Yoshida, Norihiko Takeda, Koji Maemura and Tomo Yonezawa

Mar. Drugs 2020, 18(3), 161; https://doi.org/10.3390/md18030161 - 13 Mar 2020

Cited by 4 | Viewed by 3037

Abstract

Toxoplasma gondii is a major protozoan parasite and infects human and many other warm-blooded animals. The infection leads to Toxoplasmosis, a serious issue in AIDS patients, organ transplant recipients and pregnant women. Neospora caninum, another type of protozoa, is closely related to [...] Read more.

Toxoplasma gondii is a major protozoan parasite and infects human and many other warm-blooded animals. The infection leads to Toxoplasmosis, a serious issue in AIDS patients, organ transplant recipients and pregnant women. Neospora caninum, another type of protozoa, is closely related to Toxoplasma gondii. Infections of the protozoa in animals also causes serious diseases such as Encephalomyelitis and Myositis-Polyradiculitis in dogs or abortion in cows. Both Toxoplasma gondii and Neospora caninum have similar nucleoside triphosphate hydrolases (NTPase), NcNTPase and TgNTPase-I in Neospora caninum and Toxoplasma gondii, respectively. These possibly play important roles in propagation and survival. Thus, we targeted the enzymes for drug discovery and tried to establish a novel high-standard assay by a combination of original biochemical enzyme assay and fluorescent assay to determine ADP content. We then validated whether or not it can be applied to high-throughput screening (HTS). Then, it fulfilled criterion to carry out HTS in both of the enzymes. In order to identify small molecules having inhibitory effects on the protozoan enzyme, we also performed HTS using two synthetic compound libraries and an extract library derived from marine bacteria and then, identified 19 compounds and 6 extracts. Nagasaki University collected many extracts from over 18,000 marine bacteria found in local Omura bay, and continues to compile an extensive collection of synthetic compounds from numerous drug libraries established by Japanese chemists. Full article

(This article belongs to the Special Issue High-Throughput Screening of Marine Resources)

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Amino acid sequences of Toxoplasma gondii and Neospora caninum NTPase and structural information of their active mutants. (A) Amino acid sequences of TgNTPase-I (lower raw) and NcNTPase (upper raw). Red indicates two important cysteine residues for locking enzymatic activity at 258 and 268 of TgNTPase-I, or at 234 and 244 of NcNTPase. (B) Structural information of disulfide-bond on TgNTPase inactive (left), TgNTPase active mutant (middle) and NcNTPase active mutant (right). Full article ">Figure 2
Establishment of novel high-standard assay to determine NTPase activity by combination of classical enzymatic assay and fluorescent assay to measure ADP content. Enzymatic activity of NcNTPase (0.002 μg/mL) was measured by classical absorbance assay (A) or novel assay combination of classical enzymatic and fluorescence assay by measuring ADP content (B). (C) The activities of various concentration of NcNTPase at 0.00002, 0.0002, 0.002, 0.02 or 2 μg/mL. (D) The activities of various concentration of TgNTPase at 0.0002, 0.002, 0.02 or 2 μg/mL. The data are expressed as means ± SEM (n = 3 or 4). Asterisk: Was considered to be statistically significant if their P values were P < 0.05. Full article ">Figure 3
High-throughput screening (HTS) using a synthetic compound library, which is provided by the University of Tokyo, in order to identify compounds inhibiting NcNTPase. (A) The results of HTSs. Green dashed line indicates threshold as average of positive control ± 30% of its activity. Red rhombus indicates less than the threshold. The data of positive control (Yellow rhombus, 0.002 μg/mL NcNTPase) or negative control (Black rhombus, 0.5% DMSO) are expressed as means ± SEM (n = 16). (B) Structural information and inhibited activities of hit compounds. Full article ">Figure 4
HTS using a synthetic compound library, which is provided by Nagasaki University, in order to identify compounds inhibiting TgNTPase. (A) The results of HTSs. Dashed line indicates threshold as average of positive control—3× SD. Red rhombus indicates less than the threshold. The data of positive control (Yellow rhombus, 0.002 μg/mL TgNTPase) or negative control (Black rhombus, 0.5% DMSO) are expressed as means ± SEM (n = 16). (B) Structural information and inhibited activities of hit compounds. Full article ">Figure 5
HTS using an extract library derived from marine bacteria in order to identify extracts inhibiting TgNTPase. (A) The results of HTSs. Dashed line indicates threshold as average of positive control—3× SD. Red rhombus indicates less than the threshold. The data from positive control (Yellow rhombus, 0.002 μg/mL TgNTPase) or negative control (Black rhombus, 0.5% DMSO) are expressed as means ± SEM (n = 16). (B) Extracts identified by 1st HTS inhibited TgNTPase activity in a concentration-dependent manner. The data are expressed as means ± SEM (n = 8 or 16). Asterisk: Was considered to be statistically significant if their P values were P < 0.05. Full article ">

12 pages, 2041 KiB

Open AccessArticle

Cytotoxic Thiodiketopiperazine Derivatives from the Deep Sea-Derived Fungus Epicoccum nigrum SD-388

by Lu-Ping Chi, Xiao-Ming Li, Li Li, Xin Li and Bin-Gui Wang

Mar. Drugs 2020, 18(3), 160; https://doi.org/10.3390/md18030160 - 13 Mar 2020

Cited by 28 | Viewed by 3652

Abstract

Four new thiodiketopiperazine alkaloids, namely, 5’-hydroxy-6’-ene-epicoccin G (1), 7-methoxy-7’-hydroxyepicoccin G (2), 8’-acetoxyepicoccin D (3), and 7’-demethoxyrostratin C (4), as well as a pair of new enantiomeric diketopiperazines, (±)-5-hydroxydiphenylalazine A (5), along with five [...] Read more.

Four new thiodiketopiperazine alkaloids, namely, 5’-hydroxy-6’-ene-epicoccin G (1), 7-methoxy-7’-hydroxyepicoccin G (2), 8’-acetoxyepicoccin D (3), and 7’-demethoxyrostratin C (4), as well as a pair of new enantiomeric diketopiperazines, (±)-5-hydroxydiphenylalazine A (5), along with five known analogues (6–10), were isolated and identified from the culture extract of Epicoccum nigrum SD-388, a fungus obtained from deep-sea sediments (−4500 m). Their structures were established on the basis of detailed interpretation of the NMR spectroscopic and mass spectrometric data. X-ray crystallographic analysis confirmed the structures and established the absolute configurations of compounds 1–3, while the absolute configurations for compounds 4 and 5 were determined by ECD calculations. Compounds 4 and 10 showed potent activity against Huh7.5 liver tumor cells, which were comparable to that of the positive control, sorafenib, and the disulfide bridge at C-2/C-2’ is likely essential for the activity. Full article

(This article belongs to the Special Issue Bioactive Molecules from Marine Microorganisms)

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30 pages, 3035 KiB

Open AccessFeature PaperArticle

Types and Distribution of Bioactive Polyunsaturated Aldehydes in a Gradient from Mesotrophic to Oligotrophic Waters in the Alborán Sea (Western Mediterranean)

by Ana Bartual, María Hernanz-Torrijos, Iria Sala, María J. Ortega, Cristina González-García, Marina Bolado-Penagos, Angel López-Urrutia, Leonardo Romero-Martínez, Luís M. Lubián, Miguel Bruno, Fidel Echevarría and Carlos M. García

Mar. Drugs 2020, 18(3), 159; https://doi.org/10.3390/md18030159 - 12 Mar 2020

Cited by 11 | Viewed by 3756

Abstract

Polyunsaturated aldehydes (PUAs) are bioactive molecules suggested as chemical defenses and infochemicals. In marine coastal habitats, diatoms reach high PUA production levels during bloom episodes. Two fractions of PUA can usually be analyzed: pPUA obtained via artificial breakage of collected phytoplankton cells and [...] Read more.

Polyunsaturated aldehydes (PUAs) are bioactive molecules suggested as chemical defenses and infochemicals. In marine coastal habitats, diatoms reach high PUA production levels during bloom episodes. Two fractions of PUA can usually be analyzed: pPUA obtained via artificial breakage of collected phytoplankton cells and dissolved PUA already released to the environment (dPUA). In nature, resource supply arises as a main environmental controlling factor of PUA production. In this work, we monitored the vertical distribution and daily variation of pPUA associated with large-size phytoplankton and dPUA, at three sites located in the Alborán Sea from mesotrophic to oligotrophic waters. The results corroborate the presence of large-size PUA producers in oligotrophic and mesotrophic waters with a significant (58%–85%) diatom biomass. In addition to diatoms, significant correlations between pPUA production and dinoflagellate and silicoflagellate abundance were observed. 2E,4E/Z-Heptadienal was the most abundant aldehyde at the three sites with higher values (17.1 fg·cell⁻¹) at the most oligotrophic site. 2E,4E/Z-Decadienal was the least abundant aldehyde, decreasing toward the oligotrophic site. For the first time, we describe the daily fluctuation of pPUA attributable to cellular physiological state and not exclusively to taxonomical composition. Our results demonstrate the persistence of threshold levels of dPUA deep in the water column, as well as the different chromatographic profiles of dPUA compared with pPUA. We propose different isomerization processes that alter the chemical structure of the released PUAs with unknown effects on their stability, biological function, and potential bioactivity. Full article

(This article belongs to the Special Issue Bioactive Compounds Derived from Marine Microalgae 2.0)

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Map showing the location of the different sampling sites named Coast, Jet, and Gyre sites, with overlaid mean chlorophyll a concentration (mg·m−3) for the sampling period (from 7–9 October 2015). Chlorophyll a data were downloaded from the Copernicus Marine Environmental Monitoring Service (CMEMS; <a href="http://marine.copernicus.eu/" target="_blank">http://marine.copernicus.eu/</a>). Full article ">Figure 2
Vertical distribution of nutrients concentrations along the sampling day (μM) (NO3−, (A–C); PO43−, (D–F); SiO4, (G–I)) from surface to 200 m at the Coast, Jet, and Gyre sites. Full article ">Figure 3
Different fractions of polyunsaturated aldehydes (PUAs) at the three sites: (A) averaged particulate PUA (pPUA of large-size phytoplankton) expressed as pmol from cells in 1 L at 5 m and deep chlorophyll maximum (DCM). (B) pPUA normalized by large-size phytoplankton cell abundance (fg PUA·cell−1) at 5 m and DCM; (C) averaged vertical dPUA (pM) at the three sites. C7: 2E,4E/Z-heptadienal; C8: 2E,4E/Z-octadienal; C10: 2E,4E/Z-decadienal; TPUA: total PUA. Full article ">Figure 4
Daily vertical distribution of total dissolved PUA (dTPUA) at the three sites. Please note that depth scales are different for each site for a better observation of dPUA patterns. The black dots indicate sampling depths. Full article ">Figure 5
Vertical distribution scaled from 0 to 200 m of in situ fluorescence at the Coast, Jet, and Gyre sites. Full article ">Figure 6
Box plot representing ranges of dissolved total PUA (dTPUA) and rates of dissipation of turbulent kinetic energy (ε) at the different sites. Full article ">Figure 7
Combined illustration showing the temporal variation along the day of TChla (mg·m−3), pPUA (pg·cell−1), and large phytoplankton abundance at the DCM of the different sites. Upper pie charts show the percentage of TpPUA of pC7 (2E,4E/Z-heptadienal), pC8 (2E,4E/Z-octadienal), and pC10 (2E,4E/Z-decadienal). Lower pie charts show the percentage of abundance of large-size phytoplankton fraction categories. Gray areas indicate the night period. Full article ">

17 pages, 1385 KiB

Open AccessArticle

Antioxidant, Hypolipidemic and Hepatic Protective Activities of Polysaccharides from Phascolosoma esculenta

by Yaqing Wu, Hongying Jiang, Jyuan-Siou Lin, Jia Liu, Chang-Jer Wu and Ruian Xu

Mar. Drugs 2020, 18(3), 158; https://doi.org/10.3390/md18030158 - 12 Mar 2020

Cited by 16 | Viewed by 3455

Abstract

The aims of this study were to investigate the antioxidant, hypolipidemic and hepatic protective effects of Phascolosoma esculenta polysaccharides (PEP). PEP was prepared from Phascolosoma esculenta by enzyme hydrolysis and its characterization was analyzed. The antioxidant activities of PEP were evaluated by the [...] Read more.

The aims of this study were to investigate the antioxidant, hypolipidemic and hepatic protective effects of Phascolosoma esculenta polysaccharides (PEP). PEP was prepared from Phascolosoma esculenta by enzyme hydrolysis and its characterization was analyzed. The antioxidant activities of PEP were evaluated by the assays of scavenging 1,1-Diphenyl-2-picrylhydrazyl (DPPH), superoxide anion, hydroxyl radicals and chelating ferrous ion in vitro. It showed that PEP could scavenge radicals effectively and had favorable antioxidant activities. In the meantime, the hypolipidemic effect of PEP was investigated in vivo by using mice model fed with high-fat diet with or without PEP treatment. Compared with the hyperlipidemic mice without treatment, the serum levels of total cholesterol (TC) (30.1–35.7%, p < 0.01), triglyceride (TG) (24.5–50.8%, p < 0.01 or p < 0.05), low-density lipoprotein cholesterol (LDL-C) (49.6–56.8%, p < 0.01) and liver levels of TC (21.0–28.4%, p < 0.01), TG (23.8–37.0%, p < 0.01) decreased significantly, whereas serum high-density lipoprotein cholesterol (HDL-C) (47.7–59.9%, p < 0.01 or p < 0.05) increased significantly after treatment with different dosage of PEP (0.2, 0.4 and 0.8 g per kg body weight, respectively). In addition, superoxide dismutase (SOD) (10.2–22.2% and 18.8–26.9%, p < 0.05), glutathione peroxidase (GSH-Px) (11.9–15.4% and 26.6–30.4%, p < 0.05) activities in serum and liver enhanced markedly while aspartate aminotransferase (AST) (18.7–29.6% and 42.4–58.0%, p < 0.05), alanine transaminase (ALT) (42.7–46.0% and 31.2–42.2%, p < 0.05) activities, as well as the levels of malondialdehyde (MDA) (15.9–24.4% and 15.0–16.8%, p < 0.01 or p < 0.05) in serum and liver reduced markedly. Moreover, the histopathological observation of livers indicated that PEP could attenuate liver cell injury. The animal experimental results demonstrated that PEP exerted hypolipidemic and hepatoprotective roles in hyperlipidemic mice. In summary, our results above suggest that PEP might be a potential natural antioxidant and utilized as a therapeutic candidate for hyperlipidemia. Full article

(This article belongs to the Special Issue Marine-Derived Anti-hyperlipidemic Drugs)

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The monosaccharide composition of P. esculenta polysaccharide (PEP) detected by pre-column derivatization high performance liquid chromatography (HPLC). The number indicated the corresponding monosaccharide. 1, mannose; 2, ribose; 3, rhamnose; 4, glucuronic acid; 5, galacturonic acid; 6, glucose; 7, galactose; 8, xylose; 9, arabinose; 10, fucose. Full article ">Figure 2
Antioxidant activities in vitro of PEP. (a) DPPH free radical scavenging activity; (b) Ferrous ion chelating activity; (c) Superoxide anion radical scavenging activity; (d) Hydroxyl radical scavenging activity. L-ascorbic acid and EDTA used as positive control. Data are expressed as a mean ± standard deviation (n = 3). ** p < 0.01: compared with the positive control at the same concentration. Full article ">Figure 3
Changes in body weight of the mice after fed with different foods for 75 days. The normal (N) group was fed the basic diet; the negative control (NC) group was fed with a high-fat diet with 78.8% basal feed, 1% cholesterol, 10% egg yolk power, 10% lard, 0.2% bile salts; the positive control (PC) group was fed with a high-fat diet feed plus Soybean lecithin soft capsule 0.6 g·kg−1 body weight; the PE group was fed with a high-fat diet feed plus PE powder 1.35 g·kg−1 body weight; the low-dose PEP (PEP-L) group was fed a high-fat diet feed plus PEP 0.2 g·kg−1 body weight; the middle-dose PEP (PEP-M) group was fed with a high-fat diet feed plus PEP 0.4 g·kg−1 body weight; the high-dose PEP (PEP-H) group was fed with a high-fat diet feed plus PEP 0.8 g·kg−1 body weight. Full article ">Figure 4
Changes in morphology and histopathology of liver. (a) Effect of PEP on liver morphologies in different groups, (b) Effect of PEP on histological structure of livers in different groups. Haematoxylin and eosin staining, Original mignification 400×. Full article ">

13 pages, 606 KiB

Open AccessArticle

Effect of a Laminarin Rich Macroalgal Extract on the Caecal and Colonic Microbiota in the Post-Weaned Pig

by Stafford Vigors, John V O’Doherty, Ruth Rattigan, Mary J McDonnell, Gaurav Rajauria and Torres Sweeney

Mar. Drugs 2020, 18(3), 157; https://doi.org/10.3390/md18030157 - 11 Mar 2020

Cited by 36 | Viewed by 3945

Abstract

Dietary supplementation with 300 ppm of a laminarin rich macroalgal extract reduces post-weaning intestinal dysfunction in pigs. A comprehensive analysis of the impact of laminarin on the intestinal microbiome during this period is essential to inform on the mode of action of this [...] Read more.

Dietary supplementation with 300 ppm of a laminarin rich macroalgal extract reduces post-weaning intestinal dysfunction in pigs. A comprehensive analysis of the impact of laminarin on the intestinal microbiome during this period is essential to inform on the mode of action of this bioactivity. The objective of this study was to evaluate the effects of supplementing the diet of newly weaned pigs with 300 ppm of a laminarin rich extract, on animal performance, volatile fatty acids, and the intestinal microbiota using 16S rRNA gene sequencing. Pigs fed the laminarin-supplemented diet had higher average daily feed intake, growth rate, and body weight compared to pigs fed the control diet (p < 0.05). Pigs fed the laminarin-supplemented diet had reduced abundance of OTUs assigned to Enterobacteriaceae and increased abundance of OTUs assigned to the genus Prevotella (p < 0.05) compared to pigs fed the control diet. Enterobacteriaceae had negative relationships (p < 0.05) with average daily feed intake (ADFI), average daily gain (ADG), and butyric acid concentrations. In contrast, Prevotellaceae were positively correlated (p < 0.05) with ADFI, ADG, total VFA, acetic, propionic, butyric acids, and negatively correlated with isovaleric acid. Hence supplementation with a laminarin enriched extract potentially improves performance during the post-weaning period by promoting the proliferation of bacterial taxa such as Prevotella that favourably enhance nutrient digestion while reducing the load of potentially pathogenic bacterial taxa including Enterobacteriaceae. Full article

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19 pages, 3665 KiB

Open AccessArticle

Antioxidant Peptides from Collagen Hydrolysate of Redlip Croaker (Pseudosciaena polyactis) Scales: Preparation, Characterization, and Cytoprotective Effects on H₂O₂-Damaged HepG2 Cells

by Wan-Yi Wang, Yu-Qin Zhao, Guo-Xu Zhao, Chang-Feng Chi and Bin Wang

Mar. Drugs 2020, 18(3), 156; https://doi.org/10.3390/md18030156 - 11 Mar 2020

Cited by 81 | Viewed by 5588

Abstract

Bioactive peptides from fish collagens with antioxidant properties have become a topic of great interest for health, food, and processing/preservation industries. To explore the high-value utilized way of scales produced during the fish processing, collagen hydrolysates of redlip croaker (Pseudosciaena polyactis) [...] Read more.

Bioactive peptides from fish collagens with antioxidant properties have become a topic of great interest for health, food, and processing/preservation industries. To explore the high-value utilized way of scales produced during the fish processing, collagen hydrolysates of redlip croaker (Pseudosciaena polyactis) scales were prepared using six different proteases, and the hydrolysate (RSCH) prepared using neutrase showed the highest degree of hydrolysis (21.36 ± 1.18%) and 2,2-diphenyl-1-picrylhydrazyl (DPPH·) radical scavenging activity (30.97 ± 1.56%) among the six hydrolysates. Subsequently, six antioxidant peptides were purified from RSCH using membrane ultrafiltration and serial chromatography, and their amino acid sequences were identified as DGPEGR, GPEGPMGLE, EGPFGPEG, YGPDGPTG, GFIGPTE, and IGPLGA with molecular masses of 629.61, 885.95, 788.96, 762.75, 733.80, and 526.61 Da, respectively. Among six collagen peptides, GPEGPMGLE, EGPFGPEG, and GFIGPTE exhibited the strongest scavenging activities on DPPH· radical (EC₅₀ 0.59, 0.37, and 0.45 mg/mL), hydroxyl radical (EC₅₀ 0.45, 0.33, and 0.32 mg/mL), and superoxide anion radical (EC₅₀ 0.62, 0.47, and 0.74 mg/mL). GPEGPMGLE, EGPFGPEG, and GFIGPTE showed high inhibiting ability on lipid peroxidation in a linoleic acid model system and protective activities on oxidation-damaged DNA. More importantly, GPEGPMGLE, EGPFGPEG, and GFIGPTE could protect HepG2 cells from H₂O₂-induced oxidative damage through decreasing the levels of reactive oxygen species (ROS) and MDA and activating intracellular antioxidant enzymes of superoxide dismutase (SOD), catalase (CAT), and glutathione peroxidase (GSH-Px). These results suggested that six collagen peptides (RCP1–RCP6), especially GPEGPMGLE, EGPFGPEG, and GFIGPTE, might serve as potential antioxidants applied in nutraceutical and pharmaceutical products. Full article

(This article belongs to the Special Issue Nutraceuticals and Pharmaceuticals from Marine Fish and Invertebrates)

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14 pages, 1230 KiB

Open AccessArticle

Immunoadjuvant Activity of Fucoidans from the Brown Alga Fucus evanescens

by Tatyana A. Kuznetsova, Tatyana P. Smolina, Ilona D. Makarenkova, Lydmila A. Ivanushko, Elena V. Persiyanova, Svetlana P. Ermakova, Artem S. Silchenko, Tatyana S. Zaporozhets, Natalya N. Besednova, Lydmila N. Fedyanina and Sergey P. Kryzhanovsky

Mar. Drugs 2020, 18(3), 155; https://doi.org/10.3390/md18030155 - 11 Mar 2020

Cited by 21 | Viewed by 3892

Abstract

The study presents the results of a comparative evaluation of the effect of structural modifications of fucoidans from the brown alga Fucus evanescens (native, highly purified product of fucoidan enzymatic hydrolysis, a new regular 1→3;1→4-α-L-fucan, sulphated mainly at C2 and acetylated at C4 [...] Read more.

The study presents the results of a comparative evaluation of the effect of structural modifications of fucoidans from the brown alga Fucus evanescens (native, highly purified product of fucoidan enzymatic hydrolysis, a new regular 1→3;1→4-α-L-fucan, sulphated mainly at C2 and acetylated at C4 of the fucose residue) on the effector functions of innate and adaptive immunity cells in vitro and in vivo. Using flow cytometry, we found that all examined fucoidans induce the maturation of dendritic cells, enhance the ability of neutrophils to migrate and adhere, activate monocytes and enhance their antigen-presenting functions, and increase the cytotoxic potential of natural killers. Fucoidans increase the production of hepatitis B virus (HBs) specific IgG and cytokine Th1 (IFN-γ, TNF-α) and Th2 (IL-4) profiles in vivo. The data obtained suggest that in vitro and in vivo adjuvant effects of the products of fucoidan enzymatic hydrolysis with regular structural characteristics are comparable to those of the native fucoidan. Based on these data, the products of fucoidan enzymatic hydrolysis can be considered as an effective and safe candidate adjuvant to improve the efficacy of prophylactic and therapeutic vaccines. Full article

(This article belongs to the Special Issue Marine-Derived Vaccine Adjuvants)

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13 pages, 2358 KiB

Open AccessArticle

Improvement of Psoriasis by Alteration of the Gut Environment by Oral Administration of Fucoidan from Cladosiphon Okamuranus

by Masanobu Takahashi, Kento Takahashi, Sunao Abe, Kosuke Yamada, Manami Suzuki, Mai Masahisa, Mari Endo, Keiko Abe, Ryo Inoue and Hiroko Hoshi

Mar. Drugs 2020, 18(3), 154; https://doi.org/10.3390/md18030154 - 10 Mar 2020

Cited by 22 | Viewed by 5188

Abstract

Psoriasis is a chronic autoimmune inflammatory disease for which there is no cure; it results in skin lesions and has a strong negative impact on patients’ quality of life. Fucoidan from Cladosiphon okamuranus is a dietary seaweed fiber with immunostimulatory effects. The present [...] Read more.

Psoriasis is a chronic autoimmune inflammatory disease for which there is no cure; it results in skin lesions and has a strong negative impact on patients’ quality of life. Fucoidan from Cladosiphon okamuranus is a dietary seaweed fiber with immunostimulatory effects. The present study reports that the administration of fucoidan provided symptomatic relief of facial itching and altered the gut environment in the TNF receptor-associated factor 3-interacting protein 2 (Traf3ip2) mutant mice (m-Traf3ip2 mice); the Traf3ip2 mutation was responsible for psoriasis in the mouse model used in this study. A fucoidan diet ameliorated symptoms of psoriasis and decreased facial scratching. In fecal microbiota analysis, the fucoidan diet drastically altered the presence of major intestinal opportunistic microbiota. At the same time, the fucoidan diet increased mucin volume in ileum and feces, and IgA contents in cecum. These results suggest that dietary fucoidan may play a significant role in the prevention of dysfunctional immune diseases by improving the intestinal environment and increasing the production of substances that protect the immune system. Full article

(This article belongs to the Special Issue Fucoidans)

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Graphical abstract

Graphical abstract
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Effects of fucoidan diet on m-Traf3ip2 mouse faces by PASI score analysis and scratching actions. (A) Aspects of m-Traf3ip2 typical mouse face over time. (a), (b), (c) Sections are normal diet mice and (d), (e), (f) sections are fucoidan diet mice. (B) Scratching behavior of individual mice. Closed triangle shows normal diet group and closed circle shows fucoidan diet group. PASI test scored 5 persons and each value. The calculated values for the in vitro experiments are mean ± SD (fucoidan diet group n = 14 and normal diet group n = 9). (C) The severity of the psoriasis-like skin condition. (D) Histological analysis by HE staining of faces. Normal diet mice and fucoidan diet mice epidermal sections are shown on left and right, respectively. Bars show 0.2 mm. Asterisk (*) shows significant difference (p < 0.05). Full article ">Figure 2
Fecal microbiota analysis. 16S rRNA genome-wide screening analysis. Time dependence of fecal microbiota analyses were performed on the phylum (A), family (B), (C), and genus (D), (E), levels of normal diet group (left) and fucoidan diet group (right). White bar shows normal diet group and black bar shows fucoidan group. The calculated values for the in vitro experiments are means ± SD (n =5). Full article ">Figure 2 Cont.
Fecal microbiota analysis. 16S rRNA genome-wide screening analysis. Time dependence of fecal microbiota analyses were performed on the phylum (A), family (B), (C), and genus (D), (E), levels of normal diet group (left) and fucoidan diet group (right). White bar shows normal diet group and black bar shows fucoidan group. The calculated values for the in vitro experiments are means ± SD (n =5). Full article ">Figure 3
Quantification of mouse fecal mucin. Volume concentrations of (A) m-Traf3ip2 and (B) wild type mouse in fecal mucin. White bar shows normal diet group and black bar shows fucoidan group from fecal. The calculated values for the in vitro experiments are mean ± SD (n = 5). Asterisk (*) shows significant difference (p < 0.05). Full article ">Figure 4
Quantification of total IgA in cecal contents of fucoidan diet group and normal diet group. Volume concentrations of (A) m-Traf3ip2 and (B) wild type mouse total IgA were quantified from 63 days and 31 days, respectively, in cecum contents by ELISA. White bar shows normal diet group and black bar shows fucoidan diet group from cecum. The absorbance of reaction products was determined at 450 nm, and the production was quantified. The calculated values for the in vitro experiments are expressed as mean ± SD (n = 5). Asterisk (*) shows significant difference (p < 0.05). Full article ">

21 pages, 30807 KiB

Open AccessArticle

Antioxidant Peptides from the Protein Hydrolysate of Monkfish (Lophius litulon) Muscle: Purification, Identification, and Cytoprotective Function on HepG2 Cells Damage by H₂O₂

by Xiao-Meng Hu, Yu-Mei Wang, Yu-Qin Zhao, Chang-Feng Chi and Bin Wang

Mar. Drugs 2020, 18(3), 153; https://doi.org/10.3390/md18030153 - 10 Mar 2020

Cited by 94 | Viewed by 5364

Abstract

In the work, defatted muscle proteins of monkfish (Lophius litulon) were separately hydrolyzed by pepsin, trypsin, and in vitro gastrointestinal (GI) digestion methods, and antioxidant peptides were isolated from proteins hydrolysate of monkfish muscle using ultrafiltration and chromatography processes. The antioxidant [...] Read more.

In the work, defatted muscle proteins of monkfish (Lophius litulon) were separately hydrolyzed by pepsin, trypsin, and in vitro gastrointestinal (GI) digestion methods, and antioxidant peptides were isolated from proteins hydrolysate of monkfish muscle using ultrafiltration and chromatography processes. The antioxidant activities of isolated peptides were evaluated using radical scavenging and lipid peroxidation assays and H₂O₂-induced model of HepG2 cells. In which, the cell viability, reactive oxygen species (ROS) content, and antioxidant enzymes and malondialdehyde (MDA) levels were measured for evaluating the protective extent on HepG2 cells damaged by H₂O₂. The results indicated that the hydrolysate (MPTH) prepared using in vitro GI digestion method showed the highest degree of hydrolysis (27.24 ± 1.57%) and scavenging activity on a 2,2-diphenyl-1-picrylhydrazyl (DPPH) radical (44.54 ± 3.12%) and hydroxyl radical (41.32 ± 2.73%) at the concentration of 5 mg protein/mL among the three hydrolysates. Subsequently, thirteen antioxidant peptides (MMP-1 to MMP-13) were isolated from MPTH. According to their DPPH radical and hydroxyl radical scavenging activity, three peptides with the highest antioxidant activity were selected and identified as EDIVCW (MMP-4), MEPVW (MMP-7), and YWDAW (MMP-12) with molecular weights of 763.82, 660.75, and 739.75 Da, respectively. EDIVCW, MEPVW, and YWDAW showed high scavenging activities on DPPH radical (EC₅₀ 0.39, 0.62, and 0.51 mg/mL, respectively), hydroxyl radical (EC₅₀ 0.61, 0.38, and 0.32 mg/mL, respectively), and superoxide anion radical (EC₅₀ 0.76, 0.94, 0.48 mg/mL, respectively). EDIVCW and YWDAW showed equivalent inhibiting ability on lipid peroxidation with glutathione in the linoleic acid model system. Moreover, EDIVCW, MEPVW, and YWDAW had no cytotoxicity to HepG2 cells at the concentration of 100.0 µM and could concentration-dependently protect HepG2 cells from H₂O₂-induced oxidative damage through decreasing the levels of reactive oxygen species (ROS) and MDA and activating intracellular antioxidant enzymes of superoxide dismutase (SOD), catalase (CAT), and glutathione peroxidase (GSH-Px). These present results indicated that the protein hydrolysate and isolated antioxidant peptides from monkfish muscle, especially YWDAW could serve as powerful antioxidants applied in the treatment of some liver diseases and healthcare products associated with oxidative stress. Full article

(This article belongs to the Special Issue Marine Natural Product and Oxidative Stress)

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18 pages, 8299 KiB

Open AccessArticle

Laminarin-Derived from Brown Algae Suppresses the Growth of Ovarian Cancer Cells via Mitochondrial Dysfunction and ER Stress

by Hyocheol Bae, Gwonhwa Song, Jin-Young Lee, Taeyeon Hong, Moon-Jeong Chang and Whasun Lim

Mar. Drugs 2020, 18(3), 152; https://doi.org/10.3390/md18030152 - 9 Mar 2020

Cited by 33 | Viewed by 4463

Abstract

Ovarian cancer (OC) is difficult to diagnose at an early stage and leads to the high mortality rate reported in the United States. Standard treatment for OC includes maximal cytoreductive surgery followed by platinum-based chemotherapy. However, relapse due to chemoresistance is common in [...] Read more.

Ovarian cancer (OC) is difficult to diagnose at an early stage and leads to the high mortality rate reported in the United States. Standard treatment for OC includes maximal cytoreductive surgery followed by platinum-based chemotherapy. However, relapse due to chemoresistance is common in advanced OC patients. Therefore, it is necessary to develop new anticancer drugs to suppress OC progression. Recently, the anticancer effects of laminarin, a beta-1,3-glucan derived from brown algae, have been reported in hepatocellular carcinoma, colon cancer, leukemia, and melanoma. However, its effects in OC are not reported. We confirmed that laminarin decreases cell growth and cell cycle progression of OC cells through the regulation of intracellular signaling. Moreover, laminarin induced cell death through DNA fragmentation, reactive oxygen species generation, induction of apoptotic signals and endoplasmic reticulum (ER) stress, regulation of calcium levels, and alteration of the ER-mitochondria axis. Laminarin was not cytotoxic in a zebrafish model, while in a zebrafish xenograft model, it inhibited OC cell growth. These results suggest that laminarin may be successfully used as a novel OC suppressor. Full article

(This article belongs to the Special Issue Seaweeds Secondary Metabolites: Successes In and/or Probable Therapeutic Applications 2.0)

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Cell viability and cell cycle progression in laminarin-treated ES2 and OV90 cells. (A) Structure of laminarin derived from Laminaria digitate. (B,C) Cell proliferation analysis performed using BrdU reveals that laminarin (0.1, 0.25, 0.5, 1 and 2 mg/mL) decreased cell proliferation in a dose-dependent manner in ES2 (B) and OV90 (C) cells. Results are calculated compared with vehicle-treated control (viability considered as 100%). (D,E) Alterations in cell cycle progression were analyzed by flow cytometry using propidium iodide (PI) staining in laminarin-treated ES2 (D) and OV90 (E) cells. The percentages in each cell cycle stage are shown. *** p < 0.001, ** p < 0.01, * p < 0.05 compared with vehicle-treated control cells. Full article ">Figure 2
Laminarin inhibited intracellular signal transduction in ovarian cancer (OC) cells. (A–G) Immunoblotting showing the phosphorylation of cyclin D1 (A), AKT (B), P70S6K (C), S6 (D), extracellular signal-regulated kinase 1/2 (ERK1/2) (E), c-Jun N-terminal kinase (JNK) (F), and P38 (G) proteins in laminarin (0.5, 1, and 2 mg/mL)-treated OC cells. Phosphoprotein intensities were normalized to the total protein levels compared with vehicle-treated controls. *** p < 0.001, ** p < 0.01, and * p < 0.05 indicate statistical significance compared with non-treated cells. Full article ">Figure 3
Inhibition of PI3K and MAPK signals in laminarin-treated OC cells. OC cells were treated with inhibitors, including LY294002 (20 μΜ, AKT), U0126 (10 µM, ERK1/2), SB203580 (20 µM, p38), SP600125 (20 µM, JNK), and laminarin (2 mg/mL) for western blot analysis to determine levels of AKT (A), P70S6K (B), S6 (C), ERK1/2 (D), JNK (E), and P38 (F). Phosphoprotein intensities were normalized to the total protein levels compared with vehicle-treated controls. *** p < 0.001, ** p < 0.01, and * p < 0.05 indicate statistical significance compared with non-treated cells. Full article ">Figure 4
Laminarin induced apoptosis of human OC cells. (A,B) DNA fragmentation was observed using terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) staining (red). The nuclei of cells were counterstained using 4′,6-diamidino-2-phenylindole (DAPI) (blue). The scale bar represents 20 μm (in the first horizontal panel set) and 5 μm (in the second horizontal panel set). The apoptotic ES2 (C) and OV90 (D) cells treated with laminarin were measured using annexin V and propidium iodide (PI) fluorescent dyes. Reactive oxygen species (ROS) production in laminarin-treated ES2 (E) and OV90 (F) cells was observed using dichlorofluorescein (DCF) fluorescence by flow cytometry compared with vehicle-treated cells. (G,H) Apoptotic signals induced by laminarin were observed using western blot analysis in OC cells. Apoptotic protein intensities were normalized to alpha-tubulin (TUBA) compared with vehicle-treated cells. *** p < 0.001, ** p < 0.01, and * p < 0.05 indicate statistical significance compared with non-treated cells. Full article ">Figure 5
Effects of laminarin on calcium homeostasis and mitochondria membrane potential (MMP) in ES2 and OV90 cells. (A,B) Flow cytometry analysis using fluo-4 showed cytosolic calcium in response to laminarin (0.1, 0.25, 0.5, 1, and 2 mg/mL). Flow cytometry analysis using rhod-2 revealed mitochondrial calcium levels in ES2 (C) and OV90 (D) cells following laminarin treatment for 48 h. Flow cytometry analysis indicated MMP in ES2 (E) and OV90 (F) cells that were treated with various laminarin concentrations. Loss of MMP was analyzed using JC-1 red and green fluorescence ratios. *** p < 0.001, ** p < 0.01, and * p < 0.05 indicate statistical significance compared with non-treated cells. Full article ">Figure 6
Inhibition of calcium influx in laminarin-treated OC cells. The number of late apoptotic ES2 (A) and OV90 (B) cells co-treated with laminarin and calcium chelators (2-aminoethoxydiphenyl borate (2-APB), 1,2-bis(2-aminophenoxy)ethane-N,N,N′,N′-tetraacetic acid tetrakis(acetoxymethyl ester) (BAPTA), and ruthenium red) was measured using annexin V and PI staining. (C,D) Flow cytometry analysis using fluo-4 showed cytosolic calcium in OC cells co-treated with laminarin and calcium chelators. (E,F) Flow cytometry analysis using rhod-2 revealed mitochondrial calcium levels in ES2 and OV90 cells following treatment with laminarin and calcium chelators. (G,H) Loss of MMP was analyzed using JC-1 red and green fluorescence ratios by treatment of ES2 and OV90 cells with laminarin and a calcium chelator. *** p < 0.001, ** p < 0.01, and * p < 0.05 indicate statistical significance compared with non-treated cells. ‘a’ indicates statistical significance (p < 0.05) compared with laminarin-treated cells. Full article ">Figure 7
Laminarin induced ER stress in OC cells. (A–F) Activation of ER stress-associated proteins, including inositol-requiring enzyme-1α (IRE1α), activating transcription factor 6α (ATF6α), growth arrest and DNA damage 153 (GADD153), protein kinase R (PKR)-like endoplasmic reticulum kinase (PERK), eukaryotic translation initiation factor 2α (eIF2α), and 78-kDa glucose-regulated protein (GRP78), was analyzed by western blotting of ES2 and OV90 cells treated with various concentrations of laminarin (0.5, 1 and 2 mg/mL). Target protein intensity was detected and analyzed relative to total protein or TUBA protein. *** p < 0.001, ** p < 0.01, and * p < 0.05 indicate statistical significance compared with non-treated cells. Full article ">Figure 8
Laminarin altered intracellular signaling related to ER-mitochondria contact proteins and autophagy. (A–D) Expression of ER-mitochondrial tethering proteins, including IP3R1, IP3R2, GRP75, and MFN2, was analyzed by western blot analysis of ES2 and OV90 cells treated with various concentrations of laminarin (0.5, 1, and 2 mg/mL). (E,F) Expression of autophagic proteins, including ULK1 and p62, was analyzed by western blot analysis of ES2 and OV90 cells. Target protein intensity was detected and analyzed relative to total protein or TUBA protein. *** p < 0.001, ** p < 0.01, and * p < 0.05 indicate statistical significance compared with non-treated cells. Full article ">Figure 9
Effects of laminarin on cytotoxicity and tumor formation in vivo. (A) Zebrafish embryos, with their egg shells removed, were treated with various concentrations of laminarin (0.5, 1, and 2 mg/mL) for 24 h. Under light microscopy, normal zebrafish viability and development were observed following laminarin treatment. (B,C) Laminarin-treated OC cells were injected into zebrafish yolks to form a xenograft model. Zebrafish were incubated in 24-well plates containing Danieau’s solution with 0.003% phenylthiourea at 28.5 °C for 72 h. CM-Dil dye stained the tumor cells, and they were quantified by ImageJ software. *** p < 0.001, ** p < 0.01, and * p < 0.05 indicate statistical significance compared with non-treated embryos. Full article ">

17 pages, 2988 KiB

Open AccessArticle

The Nutritional and Pharmacological Potential of New Australian Thraustochytrids Isolated from Mangrove Sediments

by Thi Linh Nham Tran, Ana F. Miranda, Adarsha Gupta, Munish Puri, Andrew S. Ball, Benu Adhikari and Aidyn Mouradov

Mar. Drugs 2020, 18(3), 151; https://doi.org/10.3390/md18030151 - 6 Mar 2020

Cited by 27 | Viewed by 4772

Abstract

Mangrove sediments represent unique microbial ecosystems that act as a buffer zone, biogeochemically recycling marine waste into nutrient-rich depositions for marine and terrestrial species. Marine unicellular protists, thraustochytrids, colonizing mangrove sediments have received attention due to their ability to produce large amounts of [...] Read more.

Mangrove sediments represent unique microbial ecosystems that act as a buffer zone, biogeochemically recycling marine waste into nutrient-rich depositions for marine and terrestrial species. Marine unicellular protists, thraustochytrids, colonizing mangrove sediments have received attention due to their ability to produce large amounts of long-chain ω3-polyunsaturated fatty acids. This paper represents a comprehensive study of two new thraustochytrids for their production of valuable biomolecules in biomass, de-oiled cakes, supernatants, extracellular polysaccharide matrixes, and recovered oil bodies. Extracted lipids (up to 40% of DW) rich in polyunsaturated fatty acids (up to 80% of total fatty acids) were mainly represented by docosahexaenoic acid (75% of polyunsaturated fatty acids). Cells also showed accumulation of squalene (up to 13 mg/g DW) and carotenoids (up to 72 µg/g DW represented by astaxanthin, canthaxanthin, echinenone, and β-carotene). Both strains showed a high concentration of protein in biomass (29% DW) and supernatants (2.7 g/L) as part of extracellular polysaccharide matrixes. Alkalinization of collected biomass represents a new and easy way to recover lipid-rich oil bodies in the form of an aqueous emulsion. The ability to produce added-value molecules makes thraustochytrids an important alternative to microalgae and plants dominating in the food, pharmacological, nutraceutical, and cosmetics industries. Full article

(This article belongs to the Special Issue Marine Nutraceuticals and Functional Foods)

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Phylogenetic analysis of thraustochytrids strains. Full article ">Figure 2
Growth of thraustochytrids cells in media supplemented with different carbon sources. (A,B): Growth rates. (C,D): Consumption rates of glucose molecules. Glu: glucose; Gly: glycerol; Fru: fructose; SUC: sucrose; * Significance levels: p < 0.05. Full article ">Figure 3
Lipid yields in thraustochytrids cells. (A,B): MAN65; C,D: MAN70. Glu: glucose; Gly: glycerol; Fru: fructose. * Significance levels: p < 0.05. Full article ">Figure 4
Concentrations of protein, carotenoids, carbohydrates, and squalene in thraustochytrids cells. (A,B): protein; (C,D): carotenoids; (E,F): carbohydrates: (G,H): squalene. (A,C,E,G): MAN65; (B,D,F,H): MAN70. Glu: glucose; Gly: glycerol; Fru: fructose. * Significance levels: p < 0.05. Full article ">Figure 5
Accumulation of lipids, proteins, and carbohydrates in supernatants. (A,B): lipids; (C,D): carbohydrates; (E,F): proteins. (A,C,E): MAN65; B,D,F: MAN70. Glu: glucose; Gly: glycerol; Fru: fructose. * Significance levels: p < 0.05. Full article ">Figure 6
Recovery of oil bodies. (A–D): Oil bodies’ accumulation in cells; (E,F): cells before pH 12 treatment; (G–H): oil bodies 15 min after pH 12 treatment; (I–K: oil droplets and solidified oil droplets in the supernatant after 3–12 h of pH 12 treatment. (L): Accumulation of OBs (shown by arrow) after centrifugation; (A,C,E,G): bright-field microscopy; (B,D,F,H–K): fluorescent microscopy after staining lipids with Nile Red. OD: oil droplets; SOD: solidified oil droplets. Scale bars: (A–H, K) = 20 µm; (I,J) = 100 µm; (L) = 1 cm. Full article ">

14 pages, 2604 KiB

Open AccessArticle

Synthesis, Pharmacological and Structural Characterization of Novel Conopressins from Conus miliaris

by Julien Giribaldi, Lotten Ragnarsson, Tom Pujante, Christine Enjalbal, David Wilson, Norelle L. Daly, Richard J. Lewis and Sebastien Dutertre

Mar. Drugs 2020, 18(3), 150; https://doi.org/10.3390/md18030150 - 6 Mar 2020

Cited by 10 | Viewed by 3678

Abstract

Cone snails produce a fast-acting and often paralyzing venom, largely dominated by disulfide-rich conotoxins targeting ion channels. Although disulfide-poor conopeptides are usually minor components of cone snail venoms, their ability to target key membrane receptors such as GPCRs make them highly valuable as [...] Read more.

Cone snails produce a fast-acting and often paralyzing venom, largely dominated by disulfide-rich conotoxins targeting ion channels. Although disulfide-poor conopeptides are usually minor components of cone snail venoms, their ability to target key membrane receptors such as GPCRs make them highly valuable as drug lead compounds. From the venom gland transcriptome of Conus miliaris, we report here on the discovery and characterization of two conopressins, which are nonapeptide ligands of the vasopressin/oxytocin receptor family. These novel sequence variants show unusual features, including a charge inversion at the critical position 8, with an aspartate instead of a highly conserved lysine or arginine residue. Both the amidated and acid C-terminal analogues were synthesized, followed by pharmacological characterization on human and zebrafish receptors and structural investigation by NMR. Whereas conopressin-M1 showed weak and only partial agonist activity at hV1bR (amidated form only) and ZFV1a1R (both amidated and acid form), both conopressin-M2 analogues acted as full agonists at the ZFV2 receptor with low micromolar affinity. Together with the NMR structures of amidated conopressins-M1, -M2 and -G, this study provides novel structure-activity relationship information that may help in the design of more selective ligands. Full article

(This article belongs to the Special Issue Marine Toxins from Venom: Potential Pharmaceutical and Medicinal Application Exploration)

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RP-HPLC/ESI-MS analyses of the synthesized conopressins and alignment of conopressin-related sequences. (A) Alignment of conopressin-related sequences. The asterisks * indicate an amidated C-terminal. Conopressin-M1 and M2 with γ-conopressin-vil are the only sequences that display a negatively charged amino acid at position 8. Interestingly, conopressin-M1 also displays an unusual proline residue at position 3. The highly conserved glycine residue at position 9 is replaced by a serine residue in conopressin-M1 and M2. (B) RP-HPLC/ESI-MS analyses of the synthesized conopressins. Acetonitrile (ACN) gradient from 0% to 30% over 30 min. For Con-M1 the two peaks display the same mass, possibly caused by the two proline residues inducing cis-trans isomerization causing dynamic conformational exchange leading to the splitting of the UV chromatogram peak [<a href="#B16-marinedrugs-18-00150" class="html-bibr">16</a>,<a href="#B17-marinedrugs-18-00150" class="html-bibr">17</a>,<a href="#B18-marinedrugs-18-00150" class="html-bibr">18</a>]. The asterisk (*) on ESI-MS insets indicate an ion resulting from in source fragmentation of the proline residue [<a href="#B19-marinedrugs-18-00150" class="html-bibr">19</a>]. Full article ">Figure 2
Three-dimensional structures of Con-G, Con-M1, Con-M2 and Con-T. The 20 lowest NMR structures are superimposed over the backbone atoms. The backbone is shown in ribbon format and the side-chains as sticks. Proline residues bringing constraints to the structures are highlighted in yellow. Full article ">Figure 3
Representative concentration-response curves measuring increasing concentrations of intracellular calcium using a FLIPR assay for the hOTR, hV1aR and hV1bR, and representative concentration-response curves measuring accumulation of cAMP using a cAMP signaling assay for the hV2R of all tested compounds. Each point represents the mean of measurements from one experiment performed in triplicate. Error bars represent S.E.M. Full article ">Figure 4
Representative concentration-response curves measuring increasing concentrations of intracellular calcium using a FLIPR assay of all tested compounds against Danio rerio (zebrafish) oxytocin-vasopressin related receptors. Each point represents the mean of measurements from one experiment performed in triplicate. Error bars represent S.E.M. Full article ">Figure 5
Alignment between hV2R (uniprot entry P30518) and cloned ZF V2R. The substitution of D297 in hV2R with S275 in ZF V2R is bordered in black. Asterisks (*) indicate amino-acid residues that have been suggested to participate and to be important in receptor–ligand interaction. Arrows indicate the seven putative transmembrane domains (TM 1–7). Full article ">

14 pages, 2888 KiB

Open AccessArticle

A Phenotarget Approach for Identifying an Alkaloid Interacting with the Tuberculosis Protein Rv1466

by Yan Xie, Yunjiang Feng, Angela Di Capua, Tin Mak, Garry W. Buchko, Peter J. Myler, Miaomiao Liu and Ronald J. Quinn

Mar. Drugs 2020, 18(3), 149; https://doi.org/10.3390/md18030149 - 5 Mar 2020

Cited by 14 | Viewed by 4482

Abstract

In recent years, there has been a revival of interest in phenotypic-based drug discovery (PDD) due to target-based drug discovery (TDD) falling below expectations. Both PDD and TDD have their unique advantages and should be used as complementary methods in drug discovery. The [...] Read more.

In recent years, there has been a revival of interest in phenotypic-based drug discovery (PDD) due to target-based drug discovery (TDD) falling below expectations. Both PDD and TDD have their unique advantages and should be used as complementary methods in drug discovery. The PhenoTarget approach combines the strengths of the PDD and TDD approaches. Phenotypic screening is conducted initially to detect cellular active components and the hits are then screened against a panel of putative targets. This PhenoTarget protocol can be equally applied to pure compound libraries as well as natural product fractions. Here we described the use of the PhenoTarget approach to identify an anti-tuberculosis lead compound. Fractions from Polycarpa aurata were identified with activity against Mycobacterium tuberculosis H37Rv. Native magnetic resonance mass spectrometry (MRMS) against a panel of 37 proteins from Mycobacterium proteomes showed that a fraction from a 95% ethanol re-extraction specifically formed a protein-ligand complex with Rv1466, a putative uncharacterized Mycobacterium tuberculosis protein. The natural product responsible was isolated and characterized to be polycarpine. The molecular weight of the ligand bound to Rv1466, 233 Da, was half the molecular weight of polycarpine less one proton, indicating that polycarpine formed a covalent bond with Rv1466. Full article

(This article belongs to the Special Issue Selected Papers from XVI MaNaPro and XI ECMNP)

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15 pages, 2354 KiB

Open AccessArticle

Spirulina Lipids Alleviate Oxidative Stress and Inflammation in Mice Fed a High-Fat and High-Sucrose Diet

by Yuhong Yang, Lei Du, Masashi Hosokawa and Kazuo Miyashita

Mar. Drugs 2020, 18(3), 148; https://doi.org/10.3390/md18030148 - 4 Mar 2020

Cited by 24 | Viewed by 4803

Abstract

High-fat and high-sucrose diet (HFHSD)-induced obesity leads to oxidative stress and chronic inflammatory status. However, little is known about the beneficial effects of total lipids extracted from Spirulina. Hence, in the present study, Spirulina lipids were extracted with chloroform/methanol (SLC) or ethanol [...] Read more.

High-fat and high-sucrose diet (HFHSD)-induced obesity leads to oxidative stress and chronic inflammatory status. However, little is known about the beneficial effects of total lipids extracted from Spirulina. Hence, in the present study, Spirulina lipids were extracted with chloroform/methanol (SLC) or ethanol (SLE) and then their effects on oxidative stress and inflammation in the mice fed a HFHSD were investigated. The results show that the major lipid classes and fatty acid profiles of SLC and SLE were almost similar, but the gamma-linolenic acid (GLA) and carotenoid contents in SLE was a little higher than that in SLC. Dietary 4% SLC or SLE for 12 weeks effectively decreased the hepatic lipid hydroperoxide levels as well as increased the activities and mRNA levels of antioxidant enzymes in the mice fed a HFHSD. In addition, supplementation with SLC and SLE also markedly decreased the levels of serum pro-inflammatory cytokines and the mRNA expression of pro-inflammatory cytokines in the liver and epididymal white adipose tissue of mice fed a HFHSD, and the effects of SLC and SLE were comparable. These findings confirm for the first time that dietary Spirulina lipids could alleviate HFHSD-induced oxidative stress and inflammation. Full article

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Full article ">Figure 1
A flow chart of the present study design. Full article ">Figure 2
Lipid hydroperoxide level in the liver of mice fed experimental diets. The values are expressed as mean ± SE (n = 7) and different letters indicate a significant difference at P < 0.05. Full article ">Figure 3
Enzyme activities of the superoxide dismutase (SOD) (A), catalase (CAT) (B), and GSSG/GSH (C) ratios in the liver of mice fed experimental diets. The values are expressed as mean ± SE (n = 7) and different letters indicate a significant difference at P < 0.05. Full article ">Figure 4
Enzyme activities of SOD (A), CAT (B), and GSSG/GSH (C) ratios in the eWAT of mice fed experimental diets. The values are expressed as mean ± SE (n = 7) and different letters indicate a significant difference at P < 0.05. Full article ">Figure 5
Relative mRNA expression of antioxidant enzymes in the liver (A) and the eWAT (B) of mice fed experimental diets. Data normalization was accomplished using the endogenous reference GAPDH. The values are expressed as mean ± SE (n = 7) and different letters indicate a significant difference at P < 0.05. Full article ">Figure 6
Serum pro-inflammatory cytokines’ levels in the mice fed experimental diets. A: TNF-α; B: IL-6; C: MCP-1. The values are expressed as mean ± SE (n = 7) and different letters indicate a significant difference at P < 0.05. Full article ">Figure 7
Relative mRNA expression of pro-inflammatory cytokines in the liver (A) and the eWAT (B) of mice fed experimental diets. Data normalization was accomplished using the endogenous reference GAPDH. The values are expressed as mean ± SE (n = 7) and different letters indicate a significant difference at P < 0.05. Full article ">

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Mar. Drugs, Volume 18, Issue 3 (March 2020) – 47 articles

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