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16 pages, 2291 KiB  
Article
Prevention of Sunlight-Induced Cell Damage by Selective Blue-Violet-Light-Filtering Lenses in A2E-Loaded Retinal Pigment Epithelial Cells
by Coralie Barrau, Mélanie Marie, Camille Ehrismann, Pauline Gondouin, José-Alain Sahel, Thierry Villette and Serge Picaud
Antioxidants 2024, 13(10), 1195; https://doi.org/10.3390/antiox13101195 - 1 Oct 2024
Viewed by 327
Abstract
Blue light accelerates retinal aging. Previous studies have indicated that wavelengths between 400 and 455 nm are most harmful to aging retinal pigment epithelia (RPE). This study explored whether filtering these wavelengths can protect cells exposed to broad sunlight. Primary porcine RPE cells [...] Read more.
Blue light accelerates retinal aging. Previous studies have indicated that wavelengths between 400 and 455 nm are most harmful to aging retinal pigment epithelia (RPE). This study explored whether filtering these wavelengths can protect cells exposed to broad sunlight. Primary porcine RPE cells loaded with 20 µM A2E were exposed to emulated sunlight filtered through eye media at 1.8 mW/cm2 for 18 h. Filters selectively filtering out light over 400–455 nm and a dark-yellow filter were interposed. Cell damage was measured by apoptosis, hydrogen peroxide (H2O2) production, and mitochondrial membrane potential (MMP). Sunlight exposure increased apoptosis by 2.7-fold and H2O2 by 4.8-fold, and halved MMP compared to darkness. Eye Protect SystemTM (EPS) technology, filtering out 25% of wavelengths over 400–455 nm, reduced apoptosis by 44% and H2O2 by 29%. The Multilayer Optical Film (MOF), at 80% of light filtered, reduced apoptosis by 91% and H2O2 by 69%, and increased MMP by 73%, overpassing the dark-yellow filter. Photoprotection increased almost linearly with blue-violet light filtering (400–455 nm) but not with total blue filtering (400–500 nm). Selective filters filtering out 25% (EPS) to 80% (MOF) of blue-violet light offer substantial protection without affecting perception or non-visual functions, making them promising for preventing light-induced retinal damage with aesthetic acceptance for permanent wear. Full article
(This article belongs to the Special Issue Mitochondrial Oxidative Stress in Aging and Disease—2nd Edition)
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Figure 1

Figure 1
<p>Tailored light set-up for simulating sunlight exposure at the retinal level. The light device consists of four units: (<b>A</b>) the lighting unit housing the light source; (<b>B</b>) the digital micromirror device which regulates both power and spectrum; (<b>C</b>) the optical unit separating the beam into four identical beams with the same optical power; and (<b>D</b>) the homogenizing unit. All units but the homogenizing unit are placed outside of the cell incubator to avoid heating and vibrations. The device enables exposure of a 96-well plate, divided into four sections of 16 wells each. Irradiance levels and homogeneity among the subdivision of the cell plate were monitored before and after each experiment, using a calibrated spectroradiometer. Additionally, light delivered by the custom-made device was continuously monitored using a calibrated photodiode. The current of the Xenon source was adjusted to stabilize the light level. The custom-made visible-light source can be spectrally adjusted to replicate the solar spectrum across 400–600 nm, weighted by eye media filtering (<b>E</b>).</p>
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<p>Evaluation of markers of cell damage in A2E-loaded cells exposed to simulated daylight. A2E-loaded RPE cells were exposed to simulated daylight at retinal level for 18 h before evaluation of apoptosis (<span class="html-italic">n</span> = 21 experiments; per experiment, 1 condition averaged on at least 12 wells). For each condition, applied light irradiances are expressed as mean +/− SEM in mW/cm<sup>2</sup>; with the grey symbol ο. (<b>A</b>,<b>B</b>), hydrogen peroxide (<span class="html-italic">n</span> = 8 experiments; per experiment, 1 condition averaged on at least 12 wells) (H<sub>2</sub>O<sub>2</sub>, <b>C</b>,<b>D</b>) and mitochondrial membrane potential (<span class="html-italic">n</span> = 4 experiments; 1 condition averaged on at least 12 wells) (MMP, <b>E</b>,<b>F</b>). Each marker was evaluated in darkness with and without A2E incubation and after light exposure in RPE cells incubated with 0 or 20 µM A2E. Data were either normalized to dark control with 0 µM A2E (<b>A</b>,<b>C</b>,<b>D</b>) or to dark control with 20 µM A2E (<b>B</b>,<b>D</b>,<b>F</b>). Data are expressed as mean +/− SEM. Two-way ANOVA with repeated measures and Tukey post hoc tests were used to compare variances between groups at each A2E concentration. Differences between sample and dark control were considered significant when <span class="html-italic">p</span> &lt; 0.05 (*), or <span class="html-italic">p</span> &lt; 0.001 (***).</p>
Full article ">Figure 3
<p>Characterization of five blue-light-filtering lenses. Filters were characterized by their appearance (<b>A</b>), their technology and optical properties (<b>B</b>), and their spectral transmittance across 400–600 nm (<b>C</b>). EPS and BA40 utilize narrow absorptive dyes, PUV functions as a longpass blue-light absorber, MOF is an interferential bandstop filter, and Y-IOL is a broadband absorptive filter. EPS, PUV, BA40, and MOF are selective blue-violet light filters (400–455 nm), whereas Y-IOL absorbs light smoothly across the entire blue-light range (400–500 nm).</p>
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<p>Photoprotection of filters in A2E-loaded cells exposed to simulated daylight. A2E-loaded RPE cells were exposed to simulated daylight at retinal level for 18 h without filter or with filter (EPS, PUV, BA40, MOF, or Y-IOL) before evaluation of apoptosis (<b>A</b>), hydrogen peroxide (H<sub>2</sub>O<sub>2</sub>, <b>B</b>), and mitochondrial membrane potential (MMP, <b>C</b>). Data are expressed as mean +/− SEM. Data are normalized to dark control with 20 µM A2E. One-way ANOVA with repeated measures and post hoc Dunnett unilateral test were used to compare variance of all light-exposed groups with filters to the light condition without filter at 20 µM of A2E. Differences between sample and light without filter were considered significant when <span class="html-italic">p</span> &lt; 0.05 (*), <span class="html-italic">p</span> &lt; 0.01 (**), or <span class="html-italic">p</span> &lt; 0.001 (***). One-way ANOVA with repeated measures and Tukey post hoc tests were used to compare variances between light conditions with filters. Differences between filters were considered significant when <span class="html-italic">p</span> &lt; 0.05 (#), or <span class="html-italic">p</span> &lt; 0.01 (##).</p>
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<p>Photoprotection as a function of filtering properties. Photoprotection rates obtained with each filter against light-induced apoptosis (dotted lines in <b>A</b>,<b>B</b>) and H<sub>2</sub>O<sub>2</sub> (gray lines in <b>A</b>,<b>B</b>) were plotted as a function of (<b>A</b>) their blue-violet reduction across 400–455 nm, expressed as BVC(B’), and (<b>B</b>) their average blue-light reduction across 400–500 nm. Each selective blue-violet filter is represented by a circle (EPS, PUV, BA40, and MOF), while Y-IOL is represented by a triangle to distinguish it as the only broadband filter. Each filter is depicted with a distinct color: EPS in light violet, PUV in gray, BA40 in vivid violet, MOF in blue, and Y-IOL in orange. (<b>A</b>) Increasing blue-violet reduction (400–455 nm) resulted in higher photoprotection, positioning MOF last after Y-IOL on the curves. (<b>B</b>) Conversely, an increasing average blue reduction (400–500 nm) did not consistently lead to increased photoprotection, as evidenced by MOF being positioned ahead of Y-IOL on the curve.</p>
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16 pages, 2905 KiB  
Article
Lactobacillus delbrueckii subsp. bulgaricus 1.0207 Exopolysaccharides Attenuate Hydrogen Peroxide-Induced Oxidative Stress Damage in IPEC-J2 Cells through the Keap1/Nrf2 Pathway
by Deyu Liu, Yingxue Yue, Lijun Ping, Cuicui Sun, Tingting Zheng, Yang Cheng, Guicheng Huo and Bailiang Li
Antioxidants 2024, 13(9), 1150; https://doi.org/10.3390/antiox13091150 - 23 Sep 2024
Viewed by 401
Abstract
Lactobacillus delbrueckii subsp. bulgaricus (L. bulgaricus) is one of the most commonly employed Lactobacillus in the food industry. Exopolysaccharides (EPS) of Lactobacillus, which are known to exhibit probiotic properties, are secondary metabolites produced during the growth of Lactobacillus. This [...] Read more.
Lactobacillus delbrueckii subsp. bulgaricus (L. bulgaricus) is one of the most commonly employed Lactobacillus in the food industry. Exopolysaccharides (EPS) of Lactobacillus, which are known to exhibit probiotic properties, are secondary metabolites produced during the growth of Lactobacillus. This study identified the structure of the EPS produced by L. bulgaricus 1.0207 and investigated the mitigation of L. bulgaricus 1.0207 EPS on H2O2-induced oxidative stress in IPEC-J2 cells. L. bulgaricus 1.0207 EPS consisted of glucose and galactose and possessed a molecular weight of 4.06 × 104 Da. L. bulgaricus 1.0207 EPS exhibited notable scavenging capacity against DPPH, hydroxyl radicals, superoxide anions, and ABTS radicals. Additionally, L. bulgaricus 1.0207 EPS enhanced cell proliferation, reduced intracellular reactive oxygen species (ROS) accumulation, increased activity of superoxide dismutase (SOD), glutathione peroxidase (GSH-Px), catalase (CAT), and total antioxidant capacity (T-AOC) elevated the relative expression of CAT, SOD, HO-1, NQO1, ZO-1, and Occludin genes. Moreover, L. bulgaricus 1.0207 EPS improved the expression of Nrf2, pNrf2, pNrf2/Nrf2, and Bcl-2 proteins, while decreasing the expression of Keap1, Caspase3, and Bax proteins, with the best effect at a concentration of 100 μg/mL. L. bulgaricus 1.0207 EPS mitigated H2O2-induced oxidative stress injury in IPEC-J2 cells by activating the Keap1/Nrf2 pathway. Meanwhile, L. bulgaricus 1.0207 EPS exhibited the potential to decrease apoptosis and restore the integrity of the gut barrier. The findings establish a theoretical foundation for the development and application of L.bulgaricus 1.0207 and its EPS. Full article
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Figure 1

Figure 1
<p>The structural composition of <span class="html-italic">L. bulgaricus</span> 1.0207 EPS. (<b>A</b>) Results of the determination of the monosaccharide composition of EPS by HPLC; (<b>B</b>) Results of molecular weight determination of EPS by GPC; (<b>C</b>) Evaluation of characteristic groups and glycosidic bonds in EPS molecules by FT-IR.</p>
Full article ">Figure 2
<p>Antioxidant properties of <span class="html-italic">L. bulgaricus</span> 1.0207 EPS. (<b>A</b>) The EPS sample was mixed with DPPH ethanol solution, reacted for 30 min, centrifuged, and then the absorbance at 517 nm was measured to calculate DPPH scavenging activity; (<b>B</b>) The EPS sample was incubated with H<sub>2</sub>O<sub>2</sub>, PBS, 1,10-phenanthroline, and FeSO<sub>4</sub> for 90 min, and hydroxyl radical scavenging was assessed by measuring absorbance at 536 nm; (<b>C</b>) The EPS sample was mixed with Tris-HCl buffer and o-toluidine, shaken for 4 min, then HCl was added to immediately terminate the reaction. Superoxide anion scavenging activity was assessed by measuring absorbance at 325 nm; (<b>D</b>) EPS was added to ABTS radical cation solution, and the absorbance at 734 nm was measured after 5 min to determine ABTS scavenging activity.</p>
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<p>Effect of different EPS and H<sub>2</sub>O<sub>2</sub> concentrations on the activity of IPEC-J2 cells as determined by CCK-8 dye. (<b>A</b>) effect of different concentrations of EPS on cell activity; (<b>B</b>) effect of different concentrations of H<sub>2</sub>O<sub>2</sub> on cell activity; (<b>C</b>) effect of different EPS concentrations on cell activity recovery after oxidative stress caused by H<sub>2</sub>O<sub>2</sub>. Different lowercase letters indicate significant differences between groups (<span class="html-italic">p</span> &lt; 0.05).</p>
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<p>Effect of EPS on antioxidant indexes in oxidatively stressed IPEC-J2 cells. (<b>A</b>) DCF-DA staining to observe the changes of ROS levels in each group of cells at different multiplicities; ROS in cells showed green fluorescence under DCFH-DA staining (<b>B</b>) Collection of adherent cells for ultrasonic disruption followed by measurement of their MDA levels using kits; (<b>C</b>) Collection of adherent cells for ultrasonic disruption followed by measurement of their SOD levels using kits; (<b>D</b>) Detection of GSH-Px levels in cell supernatants using kits; (<b>E</b>) Collection of adherent cells for ultrasonic disruption followed by measurement of their CAT levels using kits; (<b>F</b>) Collection of adherent cells for ultrasonic disruption followed by measurement of their T-AOC levels using kits; (<b>G</b>) Detection of LDH levels in cell supernatants using kits. Different lowercase letters indicate significant differences between groups (<span class="html-italic">p</span> &lt; 0.05).</p>
Full article ">Figure 5
<p>Effect of EPS on the expression of antioxidant genes in oxidatively stressed IPEC-J2 cells detected by qPCR. (<b>A</b>) Relative expression of SOD mRNA; (<b>B</b>) Relative expression of CAT mRNA; (<b>C</b>) Relative expression of HO-1 mRNA; (<b>D</b>) Relative expression of GPX mRNA; (<b>E</b>) Relative expression of NQO1 mRNA. Different lowercase letters indicate significant differences between groups (<span class="html-italic">p</span> &lt; 0.05).</p>
Full article ">Figure 6
<p>Effect of EPS on Keap1/Nrf2 pathway in oxidatively stressed IPEC-J2 cells. (<b>A</b>) Detection of protein expression related to the Keap1/Nrf2 signaling pathway using Western blotting; (<b>B</b>) Relative expression of Nrf2 mRNA; (<b>C</b>) Relative expression of Keap1 mRNA; (<b>D</b>) Relative protein expression of Nrf2; (<b>E</b>) Relative protein expression of pNrf2; (<b>F</b>) Relative protein expression of Keap1; (<b>G</b>) Relative protein expression of pNrf2/Nrf2. Different lowercase letters indicate significant differences between groups (<span class="html-italic">p</span> &lt; 0.05).</p>
Full article ">Figure 7
<p>Effect of EPS on the expression of apoptotic proteins and genes of tight junction proteins in oxidatively stressed IPEC-J2 cells. (<b>A</b>) Detection of protein expression of apoptotic genes using Western blotting; (<b>B</b>) Relative protein expression of Bcl-2; (<b>C</b>) Relative protein expression of Caspase3; (<b>D</b>) Relative protein expression of Bax. (<b>E</b>) Relative protein expression of ZO-1; (<b>F</b>) Relative protein expression of Occludin; (<b>G</b>) Relative protein expression of Claudin1. Different lowercase letters indicate significant differences between groups (<span class="html-italic">p</span> &lt; 0.05).</p>
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26 pages, 8096 KiB  
Article
Analysis of Composition, Antioxidation, and Immunoregulation for Exopolysaccharide Produced by Dellaglioa algida
by Yao Sun, Tianhui Wei, Tongqing Ma, Jiaqi Guan, Zhiwei Wang, Zhiying Fan and Jinzhu Song
Fermentation 2024, 10(9), 491; https://doi.org/10.3390/fermentation10090491 - 22 Sep 2024
Viewed by 446
Abstract
Lactobacillus is a recognized probiotic and has been widely used in food and medicine. As a new type of low-temperature resistant Lactobacillus, the fermentation products of Dellaglioa algida have multiple physiological activities. This study focuses on the exopolysaccharide (EPS) produced by Dellaglioa [...] Read more.
Lactobacillus is a recognized probiotic and has been widely used in food and medicine. As a new type of low-temperature resistant Lactobacillus, the fermentation products of Dellaglioa algida have multiple physiological activities. This study focuses on the exopolysaccharide (EPS) produced by Dellaglioa algida. The composition of the EPS is analyzed by FTIR, UV, GPC, HPLC, NMR, and SEM, and its antioxidant and immune activities are explored. The experimental results show that the EPS is a polymer composed of nine monosaccharides such as rhamnose, glucose, and mannose, connected by α- and β-glycosidic bonds, with an average molecular weight of 2.163×104 Da. When the EPS concentration reaches 100 μg/mL, the scavenging activities of DPPH and ABTS+ are 60.0% and 51.2%, respectively. The EPS promotes the secretion of NO by regulating the iNOS/NO pathway, reduces oxidative damage, and reduces the secretion of inflammatory factors such as IL-6, IL-1β, and TNF-α, and downregulates the mRNA expression of inflammatory factors, thereby alleviating the cell inflammation stimulated by the cold-resistant bacteria Pseudomonas fluorescens and Pseudomonas fragi. By virtue of these properties, the EPS produced by Dellaglioa algida fermentation has the potential to act as an antioxidant and immunomodulator. Full article
(This article belongs to the Section Microbial Metabolism, Physiology & Genetics)
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Figure 1

Figure 1
<p>Molecular weight analysis of the EPS produced by <span class="html-italic">Del. algida</span>.</p>
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<p>Analysis of monosaccharide composition of the EPS produced by <span class="html-italic">Del. algida</span>.</p>
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<p>Pie chart of monosaccharide percentages of the EPS produced by <span class="html-italic">Del. algida</span>.</p>
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<p>FTIR analysis of the EPS produced by <span class="html-italic">Del. algida</span>.</p>
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<p>Ultraviolet full-wavelength scanning of EPS produced by <span class="html-italic">Del. algida</span>.</p>
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<p>Appearance of lyophilized EPS produced by <span class="html-italic">Del. algida</span>.</p>
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<p>SEM observations of EPS produced by <span class="html-italic">Del. algida</span> at different multiples, where subfigures (<b>A</b>–<b>F</b>) stand for the SEM images at 1200-, 2500-, 5000-, 20,000-, 40,000-, and 80,000-time magnifications, respectively.</p>
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<p><sup>1</sup>H NMR spectra of EPS produced by <span class="html-italic">Del. algida</span>.</p>
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<p><sup>13</sup>C NMR spectra of EPS produced by <span class="html-italic">Del. algida</span>.</p>
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<p>Antioxidant capacity of the EPS produced by <span class="html-italic">Del. algida</span> in vitro, where subfigures (<b>A</b>–<b>D</b>) are the scavenging capacity of ABTS<sup>+</sup>, DPPH, superoxide anion, and hydroxyl.</p>
Full article ">Figure 11
<p>Effects of <span class="html-italic">Pseudomonas</span> on the survival rate and inhibition rate of RAW264.7 cells, where subfigures (<b>A</b>,<b>B</b>) are the effects of <span class="html-italic">P. fluorescens</span> on the survival rate and inhibition rate, respectively, while subfigures (<b>C</b>,<b>D</b>) are the effects of <span class="html-italic">P. fragi</span> on the survival rate and inhibition rate, respectively. * <span class="html-italic">p</span> &lt; 0.05, *** <math display="inline"><semantics> <mrow> <mi>p</mi> <mo>&lt;</mo> <mn>0.001</mn> </mrow> </semantics></math>, <sup>#</sup> <math display="inline"><semantics> <mrow> <mi>p</mi> <mo>&lt;</mo> <mn>0.05</mn> </mrow> </semantics></math>, <sup>##</sup> <math display="inline"><semantics> <mrow> <mi>p</mi> <mo>&lt;</mo> <mn>0.01</mn> </mrow> </semantics></math>, <sup>###</sup> <math display="inline"><semantics> <mrow> <mi>p</mi> <mo>&lt;</mo> <mn>0.001</mn> </mrow> </semantics></math>, ns: <math display="inline"><semantics> <mrow> <mi>p</mi> <mo>&gt;</mo> <mn>0.05</mn> </mrow> </semantics></math>, where * represents the significant difference compared with the blank CK group and <sup>#</sup> represents the significant difference compared with the 0.01% group.</p>
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<p>Effects of different concentrations of EPS on the survival rate and inhibition rate of RAW264.7 cells, where subfigures (<b>A</b>,<b>B</b>) show the effects of different concentrations of EPS on the survival rate and inhibition rate of RAW264.7 cells, respectively. * <math display="inline"><semantics> <mrow> <mi>p</mi> <mo>&lt;</mo> <mn>0.05</mn> </mrow> </semantics></math>, ns: <math display="inline"><semantics> <mrow> <mi>p</mi> <mo>&gt;</mo> <mn>0.05</mn> </mrow> </semantics></math>.</p>
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<p>Effects of EPS on the survival rate and inhibition rate of RAW264.7 cells stimulated by <span class="html-italic">Pseudomonas</span>, where subfigures (<b>A</b>,<b>B</b>) are the survival rate and inhibition rate, respectively. The CK, PB, EPS-L, and EPS-H groups stand for blank group, <span class="html-italic">Pseudomonas</span> CFS treatment group, low-dose group, and high-dose group, respectively. *** <math display="inline"><semantics> <mrow> <mi>p</mi> <mo>&lt;</mo> <mn>0.001</mn> </mrow> </semantics></math>, <sup>#</sup> <math display="inline"><semantics> <mrow> <mi>p</mi> <mo>&lt;</mo> <mn>0.05</mn> </mrow> </semantics></math>, <sup>###</sup> <math display="inline"><semantics> <mrow> <mi>p</mi> <mo>&lt;</mo> <mn>0.001</mn> </mrow> </semantics></math>, ns: <math display="inline"><semantics> <mrow> <mi>p</mi> <mo>&gt;</mo> <mn>0.05</mn> </mrow> </semantics></math>, where * represents the significant difference compared with the blank CK group, and <sup>#</sup> represents the significant difference compared with the 0.01% group.</p>
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<p>Effects of EPS on the secretion of NO by inflammatory RAW264.7 cells stimulated by <span class="html-italic">Pseudomonas</span> (400×), where subfigures (<b>A</b>–<b>D</b>) are the CK, PB, EPS-L and EPS-H groups, respectively, stimulated by <span class="html-italic">P. fluorescens</span>, and subfigures (<b>E</b>–<b>H</b>) are the CK, PB, EPS-L and EPS-H groups, respectively, stimulated by <span class="html-italic">P. fragi</span>.</p>
Full article ">Figure 15
<p>Effects of EPS on NO content of inflammatory RAW264.7 cells stimulated by <span class="html-italic">Pseudomonas</span>, where subfigures (<b>A</b>,<b>B</b>) refer to <span class="html-italic">P. fluorescens</span> and <span class="html-italic">P. fragi.</span> The CK, PB, EPS-L, and EPS-H groups stand for the blank group, <span class="html-italic">Pseudomonas</span> CFS treatment group, low-dose group, and high-dose group, respectively. * <math display="inline"><semantics> <mrow> <mi>p</mi> <mo>&lt;</mo> <mn>0.05</mn> </mrow> </semantics></math>, <sup>##</sup> <math display="inline"><semantics> <mrow> <mi>p</mi> <mo>&lt;</mo> <mn>0.01</mn> </mrow> </semantics></math>, <sup>###</sup> <math display="inline"><semantics> <mrow> <mi>p</mi> <mo>&lt;</mo> <mn>0.001</mn> </mrow> </semantics></math>, ns: <math display="inline"><semantics> <mrow> <mi>p</mi> <mo>&gt;</mo> <mn>0.05</mn> </mrow> </semantics></math>, where * represents the significant difference compared with the blank CK group, and <sup>#</sup> represents the significant difference compared with the PB group.</p>
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<p>Effects of EPS on ROS secretion of inflammatory cells stimulated by <span class="html-italic">Pseudomonas</span> (400×), where subfigures (<b>A</b>–<b>D</b>) are the CK, PB, EPS-L and EPS-H groups, respectively, stimulated by <span class="html-italic">P. fluorescens</span>, and subfigures (<b>E</b>–<b>H</b>) are the CK, PB, EPS-L and EPS-H groups, respectively, stimulated by <span class="html-italic">P. fragi</span>.</p>
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<p>Percentage of ROS secreted by EPS in inflammatory RAW264.7 cells stimulated by <span class="html-italic">Pseudomonas</span>, where subfigures (<b>A</b>,<b>B</b>) stand for using <span class="html-italic">P. fluorescens</span> CFS and <span class="html-italic">P. fragi</span> CFS, respectively. The CK, PB, EPS-L, and EPS-H groups stand for the blank group, <span class="html-italic">Pseudomonas</span> CFS treatment group, low-dose group, and high-dose group, respectively. *** <math display="inline"><semantics> <mrow> <mi>p</mi> <mo>&lt;</mo> <mn>0.001</mn> </mrow> </semantics></math>, <sup>###</sup> <math display="inline"><semantics> <mrow> <mi>p</mi> <mo>&lt;</mo> <mn>0.001</mn> </mrow> </semantics></math>, ns: <math display="inline"><semantics> <mrow> <mi>p</mi> <mo>&gt;</mo> <mn>0.05</mn> </mrow> </semantics></math>, where * represents the significant difference compared with the blank CK group, and <sup>#</sup> represents the significant difference compared with the PB group.</p>
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<p>Effects of different concentrations of EPS on the secretion of TNF-<math display="inline"><semantics> <mi>α</mi> </semantics></math> by RAW264.7 cells stimulated by <span class="html-italic">Pseudomonas</span>, where subfigures (<b>A</b>,<b>B</b>) stand for using <span class="html-italic">P. fluorescens</span> CFS and <span class="html-italic">P. fragi</span> CFS, respectively. The CK, PB, EPS-L, and EPS-H groups stand for the blank group, <span class="html-italic">Pseudomonas</span> CFS treatment group, low-dose group, and high-dose group, respectively. *** <math display="inline"><semantics> <mrow> <mi>p</mi> <mo>&lt;</mo> <mn>0.001</mn> </mrow> </semantics></math>, ** <math display="inline"><semantics> <mrow> <mi>p</mi> <mo>&lt;</mo> <mn>0.01</mn> </mrow> </semantics></math>, ns: <math display="inline"><semantics> <mrow> <mi>p</mi> <mo>&gt;</mo> <mn>0.05</mn> </mrow> </semantics></math>, where * represents the significant difference compared with the blank CK group.</p>
Full article ">Figure 19
<p>The effect of the EPS at different concentrations on the secretion of iNOS by RAW264.7 cells stimulated by <span class="html-italic">Pseudomonas</span>, where subfigures (<b>A</b>,<b>B</b>) stand for using <span class="html-italic">P. fluorescens</span> CFS and <span class="html-italic">P. fragi</span> CFS, respectively. The CK, PB, EPS-L, and EPS-H groups stand for the blank group, <span class="html-italic">Pseudomonas</span> CFS treatment group, low-dose group, and high-dose group, respectively. *** <math display="inline"><semantics> <mrow> <mi>p</mi> <mo>&lt;</mo> <mn>0.001</mn> </mrow> </semantics></math>, <sup>#</sup> <math display="inline"><semantics> <mrow> <mi>p</mi> <mo>&lt;</mo> <mn>0.05</mn> </mrow> </semantics></math>, <sup>##</sup> <math display="inline"><semantics> <mrow> <mi>p</mi> <mo>&lt;</mo> <mn>0.01</mn> </mrow> </semantics></math>, <sup>###</sup> <math display="inline"><semantics> <mrow> <mi>p</mi> <mo>&lt;</mo> <mn>0.001</mn> </mrow> </semantics></math>, where * represents the significant difference compared with the blank CK group, and <sup>#</sup> represents the significant difference compared with the PB group.</p>
Full article ">Figure 20
<p>Effects of different concentrations of EPS on IL-6 secretion of RAW264.7 cells stimulated by <span class="html-italic">Pseudomonas</span>, where subfigures (<b>A</b>,<b>B</b>) stand for using <span class="html-italic">P. fluorescens</span> CFS and <span class="html-italic">P. fragi</span> CFS, respectively. The CK, PB, EPS-L, and EPS-H groups stand for the blank group, <span class="html-italic">Pseudomonas</span> CFS treatment group, low-dose group, and high-dose group, respectively. *** <math display="inline"><semantics> <mrow> <mi>p</mi> <mo>&lt;</mo> <mn>0.001</mn> </mrow> </semantics></math>, <sup>#</sup> <math display="inline"><semantics> <mrow> <mi>p</mi> <mo>&lt;</mo> <mn>0.05</mn> </mrow> </semantics></math>, <sup>##</sup> <math display="inline"><semantics> <mrow> <mi>p</mi> <mo>&lt;</mo> <mn>0.01</mn> </mrow> </semantics></math>, <sup>###</sup> <math display="inline"><semantics> <mrow> <mi>p</mi> <mo>&lt;</mo> <mn>0.001</mn> </mrow> </semantics></math>, ns: <math display="inline"><semantics> <mrow> <mi>p</mi> <mo>&gt;</mo> <mn>0.05</mn> </mrow> </semantics></math>, where * represents the significant difference compared with the blank CK group, and <sup>#</sup> represents the significant difference compared with the PB group.</p>
Full article ">Figure 21
<p>Effects of different concentrations of EPS on IL-1<math display="inline"><semantics> <mi>β</mi> </semantics></math> secretion of RAW264.7 cells stimulated by <span class="html-italic">Pseudomonas</span>, where subfigures (<b>A</b>,<b>B</b>) stand for using <span class="html-italic">P. fluorescens</span> CFS and <span class="html-italic">P. fragi</span> CFS, respectively. The CK, PB, EPS-L, and EPS-H groups stand for the blank group, <span class="html-italic">Pseudomonas</span> CFS treatment group, low-dose group, and high-dose group, respectively. * <math display="inline"><semantics> <mrow> <mi>p</mi> <mo>&lt;</mo> <mn>0.05</mn> </mrow> </semantics></math>, ** <math display="inline"><semantics> <mrow> <mi>p</mi> <mo>&lt;</mo> <mn>0.01</mn> </mrow> </semantics></math>, <sup>#</sup> <math display="inline"><semantics> <mrow> <mi>p</mi> <mo>&lt;</mo> <mn>0.05</mn> </mrow> </semantics></math>, <sup>##</sup> <math display="inline"><semantics> <mrow> <mi>p</mi> <mo>&lt;</mo> <mn>0.01</mn> </mrow> </semantics></math>, where * represents the significant difference compared with the blank CK group and <sup>#</sup> represents the significant difference compared with the PB group.</p>
Full article ">Figure 22
<p>Effects of different concentrations of EPS on mRNA expression of inflammatory factors in RAW264.7 cells stimulated by <span class="html-italic">P. fluorescens</span> and <span class="html-italic">P. fragi</span>, where subfigures (<b>A</b>–<b>E</b>) stand for TNF-<math display="inline"><semantics> <mi>α</mi> </semantics></math>, COX-2, IL-1<math display="inline"><semantics> <mi>β</mi> </semantics></math>, IL-6, and iNOS, respectively. The CK, PB, EPS-L, and EPS-H groups stand for the blank group, <span class="html-italic">Pseudomonas</span> CFS treatment group, low-dose group, and high-dose group, respectively. ** <math display="inline"><semantics> <mrow> <mi>p</mi> <mo>&lt;</mo> <mn>0.01</mn> </mrow> </semantics></math>, *** <math display="inline"><semantics> <mrow> <mi>p</mi> <mo>&lt;</mo> <mn>0.001</mn> </mrow> </semantics></math>, <sup>#</sup> <math display="inline"><semantics> <mrow> <mi>p</mi> <mo>&lt;</mo> <mn>0.01</mn> </mrow> </semantics></math>, <sup>##</sup> <math display="inline"><semantics> <mrow> <mi>p</mi> <mo>&lt;</mo> <mn>0.01</mn> </mrow> </semantics></math>, <sup>###</sup> <math display="inline"><semantics> <mrow> <mi>p</mi> <mo>&lt;</mo> <mn>0.001</mn> </mrow> </semantics></math>, ns: <math display="inline"><semantics> <mrow> <mi>p</mi> <mo>&gt;</mo> <mn>0.05</mn> </mrow> </semantics></math>, where * represents the significant difference compared with CK in the blank group and <sup>#</sup> represents the significant difference compared with the PB group.</p>
Full article ">
38 pages, 7020 KiB  
Article
Antidiabetic and Antihyperlipidemic Activities and Molecular Mechanisms of Phyllanthus emblica L. Extract in Mice on a High-Fat Diet
by Hsing-Yi Lin, Cheng-Hsiu Lin, Yueh-Hsiung Kuo and Chun-Ching Shih
Curr. Issues Mol. Biol. 2024, 46(9), 10492-10529; https://doi.org/10.3390/cimb46090623 - 20 Sep 2024
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Abstract
We planned to explore the protective activities of extract of Phyllanthus emblica L. (EPE) on insulin resistance and metabolic disorders including hyperlipidemia, visceral obesity, and renal dysfunction in high-fat diet (HFD)-progressed T2DM mice. Mice treatments included 7 weeks of HFD induction followed by [...] Read more.
We planned to explore the protective activities of extract of Phyllanthus emblica L. (EPE) on insulin resistance and metabolic disorders including hyperlipidemia, visceral obesity, and renal dysfunction in high-fat diet (HFD)-progressed T2DM mice. Mice treatments included 7 weeks of HFD induction followed by EPE, fenofibrate (Feno), or metformin (Metf) treatment daily for another 4-week HFD in HFD-fed mice. Finally, we harvested blood to analyze some tests on circulating glycemia and blood lipid levels. Western blotting analysis was performed on target gene expressions in peripheral tissues. The present findings indicated that EPE treatment reversed the HFD-induced increases in blood glucose, glycosylated HbA1C, and insulin levels. Our findings proved that treatment with EPE in HFD mice effectively controls hyperglycemia and hyperinsulinemia. Our results showed that EPE reduced blood lipid levels, including a reduction in blood triglyceride (TG), total cholesterol (TC), and free fatty acid (FFA); moreover, EPE reduced blood leptin levels and enhanced adiponectin concentrations. EPE treatment in HFD mice reduced BUN and creatinine in both blood and urine and lowered albumin levels in urine; moreover, EPE decreased circulating concentrations of inflammatory NLR family pyrin domain containing 3 (NLRP3) and kidney injury molecule-1 (KIM-1). These results indicated that EPE displayed antihyperglycemic and antihyperlipidemic activities but alleviated renal dysfunction in HFD mice. The histology examinations indicated that EPE treatment decreased adipose hypertrophy and hepatic ballooning, thus contributing to amelioration of lipid accumulation. EPE treatment decreased visceral fat amounts and led to improved systemic insulin resistance. For target gene expression levels, EPE enhanced AMP-activated protein kinase (AMPK) phosphorylation expressions both in livers and skeletal muscles and elevated the muscular membrane glucose transporter 4 (GLUT4) expressions. Treatment with EPE reduced hepatic glucose-6-phosphatase (G6Pase) and phosphoenolpyruvate carboxykinase (PEPCK) expressions to suppress glucose production in the livers and decreased phosphorylation of glycogen synthase kinase 3β (GSK3β) expressions to affect hepatic glycogen synthesis, thus convergently contributing to an antidiabetic effect and improving insulin resistance. The mechanism of the antihyperlipidemic activity of EPE involved a decrease in the hepatic phosphorylation of mammalian target of rapamycin complex C1 (mTORC1) and p70 S6 kinase 1 (S6K1) expressions to improve insulin resistance but also a reduction in hepatic sterol regulatory element binding protein (SREBP)-1c expressions, and suppression of ACC activity, thus resulting in the decreased fatty acid synthesis but elevated hepatic peroxisome proliferator-activated receptor (PPAR) α and SREBP-2 expressions, resulting in lowering TG and TC concentrations. Our results demonstrated that EPE improves insulin resistance and ameliorates hyperlipidemia in HFD mice. Full article
(This article belongs to the Section Molecular Pharmacology)
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Figure 1

Figure 1
<p>The fruits of <span class="html-italic">Phyllanthus emblica</span> L.</p>
Full article ">Figure 2
<p>Effects of ethyl acetate extract of <span class="html-italic">Phyllanthus emblica</span> L. (EPE) on the insulin (Ins)-stimulated expression levels of membrane glucose transporter type 4 (GLUT4), the ratio of phospho-5′-adenosine monophosphate kinase (p-AMPK) to total AMPK (t-AMPK), or phospho-Akt (p-Akt)/total Akt (t-Akt) in insulin-resistant C2C12 myotube cells induced by palmitate (Pal). The symbols “+++”, “###” and “***” represent <span class="html-italic">p</span> &lt; 0.001 as, respectively, compared to the value of the blank control, positive control (insulin) and negative control (insulin + palmitate) using analysis of variance (ANOVA) and with Dunnett’s tests. (<b>A</b>) Representative image, (<b>B</b>–<b>D</b>) quantification of the membrane GLUT4 expression levels, the ratio of p-AMPK to t-AMPK, or p-Akt/t-Akt expression levels. CON, the blank control; DMSO, a solvent control. All values are means ± SE (<span class="html-italic">n</span> = 3).</p>
Full article ">Figure 2 Cont.
<p>Effects of ethyl acetate extract of <span class="html-italic">Phyllanthus emblica</span> L. (EPE) on the insulin (Ins)-stimulated expression levels of membrane glucose transporter type 4 (GLUT4), the ratio of phospho-5′-adenosine monophosphate kinase (p-AMPK) to total AMPK (t-AMPK), or phospho-Akt (p-Akt)/total Akt (t-Akt) in insulin-resistant C2C12 myotube cells induced by palmitate (Pal). The symbols “+++”, “###” and “***” represent <span class="html-italic">p</span> &lt; 0.001 as, respectively, compared to the value of the blank control, positive control (insulin) and negative control (insulin + palmitate) using analysis of variance (ANOVA) and with Dunnett’s tests. (<b>A</b>) Representative image, (<b>B</b>–<b>D</b>) quantification of the membrane GLUT4 expression levels, the ratio of p-AMPK to t-AMPK, or p-Akt/t-Akt expression levels. CON, the blank control; DMSO, a solvent control. All values are means ± SE (<span class="html-italic">n</span> = 3).</p>
Full article ">Figure 2 Cont.
<p>Effects of ethyl acetate extract of <span class="html-italic">Phyllanthus emblica</span> L. (EPE) on the insulin (Ins)-stimulated expression levels of membrane glucose transporter type 4 (GLUT4), the ratio of phospho-5′-adenosine monophosphate kinase (p-AMPK) to total AMPK (t-AMPK), or phospho-Akt (p-Akt)/total Akt (t-Akt) in insulin-resistant C2C12 myotube cells induced by palmitate (Pal). The symbols “+++”, “###” and “***” represent <span class="html-italic">p</span> &lt; 0.001 as, respectively, compared to the value of the blank control, positive control (insulin) and negative control (insulin + palmitate) using analysis of variance (ANOVA) and with Dunnett’s tests. (<b>A</b>) Representative image, (<b>B</b>–<b>D</b>) quantification of the membrane GLUT4 expression levels, the ratio of p-AMPK to t-AMPK, or p-Akt/t-Akt expression levels. CON, the blank control; DMSO, a solvent control. All values are means ± SE (<span class="html-italic">n</span> = 3).</p>
Full article ">Figure 3
<p>Effects of ethyl acetate extract of <span class="html-italic">Phyllanthus emblica</span> L. (EPE) on (<b>A</b>) final body weight; (<b>B</b>) body weight gain over 4-week treatment; (<b>C</b>) absolute fat tissue weight; (<b>D</b>) absolute weights of spleen and pancreas; (<b>E</b>) blood glucose levels; (<b>F</b>) blood glycated hemoglobin (HbA1<sub>C</sub>) levels; (<b>G</b>) triglyceride levels; (<b>H</b>) total cholesterol levels; (<b>I</b>) insulin levels; (<b>J</b>) leptin levels; (<b>K</b>) adiponectin levels; (<b>L</b>) free fatty acid levels; (<b>M</b>) blood BUN levels; (<b>N</b>) plasma creatinine levels; (<b>O</b>) urine BUN levels; (<b>P</b>) urine creatinine levels; (<b>Q</b>) urine albumin levels; (<b>R</b>) blood kidney injury molecule-1 (KIM-1) levels; (<b>S</b>) blood CRP levels; and (<b>T</b>) blood NLRP3 levels in high-fat diet (HFD)-induced diabetic mice. <sup>#</sup> <span class="html-italic">p</span> &lt; 0.05, <sup>##</sup> <span class="html-italic">p</span> &lt; 0.01, and <sup>###</sup> <span class="html-italic">p</span> &lt; 0.001 compared with the control (CON) group; * <span class="html-italic">p</span> &lt; 0.05, ** <span class="html-italic">p</span> &lt; 0.01, and *** <span class="html-italic">p</span> &lt; 0.001 compared with the high-fat diet (HFD) plus vehicle (distilled water) (HF) group. All values are means ± SE (<span class="html-italic">n</span> = 8 per group). The extracts of <span class="html-italic">Phyllanthus emblica</span> L. (EPE), EPE1, 100, EPE2, 200, EPE3, 400 mg/kg body weight; Feno, fenofibrate (250 mg/kg body weight); Meft, metformin (300 mg/kg body weight). EWAT, epididymal white adipose tissue; RWAT, retroperioneal white adipose tissue; MWAT, mesenteric white adipose tissue.</p>
Full article ">Figure 3 Cont.
<p>Effects of ethyl acetate extract of <span class="html-italic">Phyllanthus emblica</span> L. (EPE) on (<b>A</b>) final body weight; (<b>B</b>) body weight gain over 4-week treatment; (<b>C</b>) absolute fat tissue weight; (<b>D</b>) absolute weights of spleen and pancreas; (<b>E</b>) blood glucose levels; (<b>F</b>) blood glycated hemoglobin (HbA1<sub>C</sub>) levels; (<b>G</b>) triglyceride levels; (<b>H</b>) total cholesterol levels; (<b>I</b>) insulin levels; (<b>J</b>) leptin levels; (<b>K</b>) adiponectin levels; (<b>L</b>) free fatty acid levels; (<b>M</b>) blood BUN levels; (<b>N</b>) plasma creatinine levels; (<b>O</b>) urine BUN levels; (<b>P</b>) urine creatinine levels; (<b>Q</b>) urine albumin levels; (<b>R</b>) blood kidney injury molecule-1 (KIM-1) levels; (<b>S</b>) blood CRP levels; and (<b>T</b>) blood NLRP3 levels in high-fat diet (HFD)-induced diabetic mice. <sup>#</sup> <span class="html-italic">p</span> &lt; 0.05, <sup>##</sup> <span class="html-italic">p</span> &lt; 0.01, and <sup>###</sup> <span class="html-italic">p</span> &lt; 0.001 compared with the control (CON) group; * <span class="html-italic">p</span> &lt; 0.05, ** <span class="html-italic">p</span> &lt; 0.01, and *** <span class="html-italic">p</span> &lt; 0.001 compared with the high-fat diet (HFD) plus vehicle (distilled water) (HF) group. All values are means ± SE (<span class="html-italic">n</span> = 8 per group). The extracts of <span class="html-italic">Phyllanthus emblica</span> L. (EPE), EPE1, 100, EPE2, 200, EPE3, 400 mg/kg body weight; Feno, fenofibrate (250 mg/kg body weight); Meft, metformin (300 mg/kg body weight). EWAT, epididymal white adipose tissue; RWAT, retroperioneal white adipose tissue; MWAT, mesenteric white adipose tissue.</p>
Full article ">Figure 3 Cont.
<p>Effects of ethyl acetate extract of <span class="html-italic">Phyllanthus emblica</span> L. (EPE) on (<b>A</b>) final body weight; (<b>B</b>) body weight gain over 4-week treatment; (<b>C</b>) absolute fat tissue weight; (<b>D</b>) absolute weights of spleen and pancreas; (<b>E</b>) blood glucose levels; (<b>F</b>) blood glycated hemoglobin (HbA1<sub>C</sub>) levels; (<b>G</b>) triglyceride levels; (<b>H</b>) total cholesterol levels; (<b>I</b>) insulin levels; (<b>J</b>) leptin levels; (<b>K</b>) adiponectin levels; (<b>L</b>) free fatty acid levels; (<b>M</b>) blood BUN levels; (<b>N</b>) plasma creatinine levels; (<b>O</b>) urine BUN levels; (<b>P</b>) urine creatinine levels; (<b>Q</b>) urine albumin levels; (<b>R</b>) blood kidney injury molecule-1 (KIM-1) levels; (<b>S</b>) blood CRP levels; and (<b>T</b>) blood NLRP3 levels in high-fat diet (HFD)-induced diabetic mice. <sup>#</sup> <span class="html-italic">p</span> &lt; 0.05, <sup>##</sup> <span class="html-italic">p</span> &lt; 0.01, and <sup>###</sup> <span class="html-italic">p</span> &lt; 0.001 compared with the control (CON) group; * <span class="html-italic">p</span> &lt; 0.05, ** <span class="html-italic">p</span> &lt; 0.01, and *** <span class="html-italic">p</span> &lt; 0.001 compared with the high-fat diet (HFD) plus vehicle (distilled water) (HF) group. All values are means ± SE (<span class="html-italic">n</span> = 8 per group). The extracts of <span class="html-italic">Phyllanthus emblica</span> L. (EPE), EPE1, 100, EPE2, 200, EPE3, 400 mg/kg body weight; Feno, fenofibrate (250 mg/kg body weight); Meft, metformin (300 mg/kg body weight). EWAT, epididymal white adipose tissue; RWAT, retroperioneal white adipose tissue; MWAT, mesenteric white adipose tissue.</p>
Full article ">Figure 3 Cont.
<p>Effects of ethyl acetate extract of <span class="html-italic">Phyllanthus emblica</span> L. (EPE) on (<b>A</b>) final body weight; (<b>B</b>) body weight gain over 4-week treatment; (<b>C</b>) absolute fat tissue weight; (<b>D</b>) absolute weights of spleen and pancreas; (<b>E</b>) blood glucose levels; (<b>F</b>) blood glycated hemoglobin (HbA1<sub>C</sub>) levels; (<b>G</b>) triglyceride levels; (<b>H</b>) total cholesterol levels; (<b>I</b>) insulin levels; (<b>J</b>) leptin levels; (<b>K</b>) adiponectin levels; (<b>L</b>) free fatty acid levels; (<b>M</b>) blood BUN levels; (<b>N</b>) plasma creatinine levels; (<b>O</b>) urine BUN levels; (<b>P</b>) urine creatinine levels; (<b>Q</b>) urine albumin levels; (<b>R</b>) blood kidney injury molecule-1 (KIM-1) levels; (<b>S</b>) blood CRP levels; and (<b>T</b>) blood NLRP3 levels in high-fat diet (HFD)-induced diabetic mice. <sup>#</sup> <span class="html-italic">p</span> &lt; 0.05, <sup>##</sup> <span class="html-italic">p</span> &lt; 0.01, and <sup>###</sup> <span class="html-italic">p</span> &lt; 0.001 compared with the control (CON) group; * <span class="html-italic">p</span> &lt; 0.05, ** <span class="html-italic">p</span> &lt; 0.01, and *** <span class="html-italic">p</span> &lt; 0.001 compared with the high-fat diet (HFD) plus vehicle (distilled water) (HF) group. All values are means ± SE (<span class="html-italic">n</span> = 8 per group). The extracts of <span class="html-italic">Phyllanthus emblica</span> L. (EPE), EPE1, 100, EPE2, 200, EPE3, 400 mg/kg body weight; Feno, fenofibrate (250 mg/kg body weight); Meft, metformin (300 mg/kg body weight). EWAT, epididymal white adipose tissue; RWAT, retroperioneal white adipose tissue; MWAT, mesenteric white adipose tissue.</p>
Full article ">Figure 3 Cont.
<p>Effects of ethyl acetate extract of <span class="html-italic">Phyllanthus emblica</span> L. (EPE) on (<b>A</b>) final body weight; (<b>B</b>) body weight gain over 4-week treatment; (<b>C</b>) absolute fat tissue weight; (<b>D</b>) absolute weights of spleen and pancreas; (<b>E</b>) blood glucose levels; (<b>F</b>) blood glycated hemoglobin (HbA1<sub>C</sub>) levels; (<b>G</b>) triglyceride levels; (<b>H</b>) total cholesterol levels; (<b>I</b>) insulin levels; (<b>J</b>) leptin levels; (<b>K</b>) adiponectin levels; (<b>L</b>) free fatty acid levels; (<b>M</b>) blood BUN levels; (<b>N</b>) plasma creatinine levels; (<b>O</b>) urine BUN levels; (<b>P</b>) urine creatinine levels; (<b>Q</b>) urine albumin levels; (<b>R</b>) blood kidney injury molecule-1 (KIM-1) levels; (<b>S</b>) blood CRP levels; and (<b>T</b>) blood NLRP3 levels in high-fat diet (HFD)-induced diabetic mice. <sup>#</sup> <span class="html-italic">p</span> &lt; 0.05, <sup>##</sup> <span class="html-italic">p</span> &lt; 0.01, and <sup>###</sup> <span class="html-italic">p</span> &lt; 0.001 compared with the control (CON) group; * <span class="html-italic">p</span> &lt; 0.05, ** <span class="html-italic">p</span> &lt; 0.01, and *** <span class="html-italic">p</span> &lt; 0.001 compared with the high-fat diet (HFD) plus vehicle (distilled water) (HF) group. All values are means ± SE (<span class="html-italic">n</span> = 8 per group). The extracts of <span class="html-italic">Phyllanthus emblica</span> L. (EPE), EPE1, 100, EPE2, 200, EPE3, 400 mg/kg body weight; Feno, fenofibrate (250 mg/kg body weight); Meft, metformin (300 mg/kg body weight). EWAT, epididymal white adipose tissue; RWAT, retroperioneal white adipose tissue; MWAT, mesenteric white adipose tissue.</p>
Full article ">Figure 3 Cont.
<p>Effects of ethyl acetate extract of <span class="html-italic">Phyllanthus emblica</span> L. (EPE) on (<b>A</b>) final body weight; (<b>B</b>) body weight gain over 4-week treatment; (<b>C</b>) absolute fat tissue weight; (<b>D</b>) absolute weights of spleen and pancreas; (<b>E</b>) blood glucose levels; (<b>F</b>) blood glycated hemoglobin (HbA1<sub>C</sub>) levels; (<b>G</b>) triglyceride levels; (<b>H</b>) total cholesterol levels; (<b>I</b>) insulin levels; (<b>J</b>) leptin levels; (<b>K</b>) adiponectin levels; (<b>L</b>) free fatty acid levels; (<b>M</b>) blood BUN levels; (<b>N</b>) plasma creatinine levels; (<b>O</b>) urine BUN levels; (<b>P</b>) urine creatinine levels; (<b>Q</b>) urine albumin levels; (<b>R</b>) blood kidney injury molecule-1 (KIM-1) levels; (<b>S</b>) blood CRP levels; and (<b>T</b>) blood NLRP3 levels in high-fat diet (HFD)-induced diabetic mice. <sup>#</sup> <span class="html-italic">p</span> &lt; 0.05, <sup>##</sup> <span class="html-italic">p</span> &lt; 0.01, and <sup>###</sup> <span class="html-italic">p</span> &lt; 0.001 compared with the control (CON) group; * <span class="html-italic">p</span> &lt; 0.05, ** <span class="html-italic">p</span> &lt; 0.01, and *** <span class="html-italic">p</span> &lt; 0.001 compared with the high-fat diet (HFD) plus vehicle (distilled water) (HF) group. All values are means ± SE (<span class="html-italic">n</span> = 8 per group). The extracts of <span class="html-italic">Phyllanthus emblica</span> L. (EPE), EPE1, 100, EPE2, 200, EPE3, 400 mg/kg body weight; Feno, fenofibrate (250 mg/kg body weight); Meft, metformin (300 mg/kg body weight). EWAT, epididymal white adipose tissue; RWAT, retroperioneal white adipose tissue; MWAT, mesenteric white adipose tissue.</p>
Full article ">Figure 3 Cont.
<p>Effects of ethyl acetate extract of <span class="html-italic">Phyllanthus emblica</span> L. (EPE) on (<b>A</b>) final body weight; (<b>B</b>) body weight gain over 4-week treatment; (<b>C</b>) absolute fat tissue weight; (<b>D</b>) absolute weights of spleen and pancreas; (<b>E</b>) blood glucose levels; (<b>F</b>) blood glycated hemoglobin (HbA1<sub>C</sub>) levels; (<b>G</b>) triglyceride levels; (<b>H</b>) total cholesterol levels; (<b>I</b>) insulin levels; (<b>J</b>) leptin levels; (<b>K</b>) adiponectin levels; (<b>L</b>) free fatty acid levels; (<b>M</b>) blood BUN levels; (<b>N</b>) plasma creatinine levels; (<b>O</b>) urine BUN levels; (<b>P</b>) urine creatinine levels; (<b>Q</b>) urine albumin levels; (<b>R</b>) blood kidney injury molecule-1 (KIM-1) levels; (<b>S</b>) blood CRP levels; and (<b>T</b>) blood NLRP3 levels in high-fat diet (HFD)-induced diabetic mice. <sup>#</sup> <span class="html-italic">p</span> &lt; 0.05, <sup>##</sup> <span class="html-italic">p</span> &lt; 0.01, and <sup>###</sup> <span class="html-italic">p</span> &lt; 0.001 compared with the control (CON) group; * <span class="html-italic">p</span> &lt; 0.05, ** <span class="html-italic">p</span> &lt; 0.01, and *** <span class="html-italic">p</span> &lt; 0.001 compared with the high-fat diet (HFD) plus vehicle (distilled water) (HF) group. All values are means ± SE (<span class="html-italic">n</span> = 8 per group). The extracts of <span class="html-italic">Phyllanthus emblica</span> L. (EPE), EPE1, 100, EPE2, 200, EPE3, 400 mg/kg body weight; Feno, fenofibrate (250 mg/kg body weight); Meft, metformin (300 mg/kg body weight). EWAT, epididymal white adipose tissue; RWAT, retroperioneal white adipose tissue; MWAT, mesenteric white adipose tissue.</p>
Full article ">Figure 3 Cont.
<p>Effects of ethyl acetate extract of <span class="html-italic">Phyllanthus emblica</span> L. (EPE) on (<b>A</b>) final body weight; (<b>B</b>) body weight gain over 4-week treatment; (<b>C</b>) absolute fat tissue weight; (<b>D</b>) absolute weights of spleen and pancreas; (<b>E</b>) blood glucose levels; (<b>F</b>) blood glycated hemoglobin (HbA1<sub>C</sub>) levels; (<b>G</b>) triglyceride levels; (<b>H</b>) total cholesterol levels; (<b>I</b>) insulin levels; (<b>J</b>) leptin levels; (<b>K</b>) adiponectin levels; (<b>L</b>) free fatty acid levels; (<b>M</b>) blood BUN levels; (<b>N</b>) plasma creatinine levels; (<b>O</b>) urine BUN levels; (<b>P</b>) urine creatinine levels; (<b>Q</b>) urine albumin levels; (<b>R</b>) blood kidney injury molecule-1 (KIM-1) levels; (<b>S</b>) blood CRP levels; and (<b>T</b>) blood NLRP3 levels in high-fat diet (HFD)-induced diabetic mice. <sup>#</sup> <span class="html-italic">p</span> &lt; 0.05, <sup>##</sup> <span class="html-italic">p</span> &lt; 0.01, and <sup>###</sup> <span class="html-italic">p</span> &lt; 0.001 compared with the control (CON) group; * <span class="html-italic">p</span> &lt; 0.05, ** <span class="html-italic">p</span> &lt; 0.01, and *** <span class="html-italic">p</span> &lt; 0.001 compared with the high-fat diet (HFD) plus vehicle (distilled water) (HF) group. All values are means ± SE (<span class="html-italic">n</span> = 8 per group). The extracts of <span class="html-italic">Phyllanthus emblica</span> L. (EPE), EPE1, 100, EPE2, 200, EPE3, 400 mg/kg body weight; Feno, fenofibrate (250 mg/kg body weight); Meft, metformin (300 mg/kg body weight). EWAT, epididymal white adipose tissue; RWAT, retroperioneal white adipose tissue; MWAT, mesenteric white adipose tissue.</p>
Full article ">Figure 3 Cont.
<p>Effects of ethyl acetate extract of <span class="html-italic">Phyllanthus emblica</span> L. (EPE) on (<b>A</b>) final body weight; (<b>B</b>) body weight gain over 4-week treatment; (<b>C</b>) absolute fat tissue weight; (<b>D</b>) absolute weights of spleen and pancreas; (<b>E</b>) blood glucose levels; (<b>F</b>) blood glycated hemoglobin (HbA1<sub>C</sub>) levels; (<b>G</b>) triglyceride levels; (<b>H</b>) total cholesterol levels; (<b>I</b>) insulin levels; (<b>J</b>) leptin levels; (<b>K</b>) adiponectin levels; (<b>L</b>) free fatty acid levels; (<b>M</b>) blood BUN levels; (<b>N</b>) plasma creatinine levels; (<b>O</b>) urine BUN levels; (<b>P</b>) urine creatinine levels; (<b>Q</b>) urine albumin levels; (<b>R</b>) blood kidney injury molecule-1 (KIM-1) levels; (<b>S</b>) blood CRP levels; and (<b>T</b>) blood NLRP3 levels in high-fat diet (HFD)-induced diabetic mice. <sup>#</sup> <span class="html-italic">p</span> &lt; 0.05, <sup>##</sup> <span class="html-italic">p</span> &lt; 0.01, and <sup>###</sup> <span class="html-italic">p</span> &lt; 0.001 compared with the control (CON) group; * <span class="html-italic">p</span> &lt; 0.05, ** <span class="html-italic">p</span> &lt; 0.01, and *** <span class="html-italic">p</span> &lt; 0.001 compared with the high-fat diet (HFD) plus vehicle (distilled water) (HF) group. All values are means ± SE (<span class="html-italic">n</span> = 8 per group). The extracts of <span class="html-italic">Phyllanthus emblica</span> L. (EPE), EPE1, 100, EPE2, 200, EPE3, 400 mg/kg body weight; Feno, fenofibrate (250 mg/kg body weight); Meft, metformin (300 mg/kg body weight). EWAT, epididymal white adipose tissue; RWAT, retroperioneal white adipose tissue; MWAT, mesenteric white adipose tissue.</p>
Full article ">Figure 3 Cont.
<p>Effects of ethyl acetate extract of <span class="html-italic">Phyllanthus emblica</span> L. (EPE) on (<b>A</b>) final body weight; (<b>B</b>) body weight gain over 4-week treatment; (<b>C</b>) absolute fat tissue weight; (<b>D</b>) absolute weights of spleen and pancreas; (<b>E</b>) blood glucose levels; (<b>F</b>) blood glycated hemoglobin (HbA1<sub>C</sub>) levels; (<b>G</b>) triglyceride levels; (<b>H</b>) total cholesterol levels; (<b>I</b>) insulin levels; (<b>J</b>) leptin levels; (<b>K</b>) adiponectin levels; (<b>L</b>) free fatty acid levels; (<b>M</b>) blood BUN levels; (<b>N</b>) plasma creatinine levels; (<b>O</b>) urine BUN levels; (<b>P</b>) urine creatinine levels; (<b>Q</b>) urine albumin levels; (<b>R</b>) blood kidney injury molecule-1 (KIM-1) levels; (<b>S</b>) blood CRP levels; and (<b>T</b>) blood NLRP3 levels in high-fat diet (HFD)-induced diabetic mice. <sup>#</sup> <span class="html-italic">p</span> &lt; 0.05, <sup>##</sup> <span class="html-italic">p</span> &lt; 0.01, and <sup>###</sup> <span class="html-italic">p</span> &lt; 0.001 compared with the control (CON) group; * <span class="html-italic">p</span> &lt; 0.05, ** <span class="html-italic">p</span> &lt; 0.01, and *** <span class="html-italic">p</span> &lt; 0.001 compared with the high-fat diet (HFD) plus vehicle (distilled water) (HF) group. All values are means ± SE (<span class="html-italic">n</span> = 8 per group). The extracts of <span class="html-italic">Phyllanthus emblica</span> L. (EPE), EPE1, 100, EPE2, 200, EPE3, 400 mg/kg body weight; Feno, fenofibrate (250 mg/kg body weight); Meft, metformin (300 mg/kg body weight). EWAT, epididymal white adipose tissue; RWAT, retroperioneal white adipose tissue; MWAT, mesenteric white adipose tissue.</p>
Full article ">Figure 4
<p>Histology examinations on (<b>A</b>) liver tissues (at 400×) and (<b>B</b>) white adipose tissues of mice (at 400×) in the control (CON), high-fat diet (HFD) plus vehicle (distilled water) (HF), HFD + EPE1, HFD + EPE2, HFD + EPE3, HFD + fenofibrate (Feno), or HFD + metformin (Metf) groups (<span class="html-italic">n</span> = 8 per group) by hematoxylin and eosin staining. The ethyl acetate extract of <span class="html-italic">Phyllanthus emblica</span> L. (EPE), EPE1, 100, EPE2, 200, EPE3, 400 mg/kg body weight; Feno, fenofibrate (250 mg/kg body weight); Metf, metformin (300 mg/kg body weight).</p>
Full article ">Figure 5
<p>The expression levels of (<b>A</b>,<b>B</b>) membrane GLUT4, p-AMPK (Thr<sup>172</sup>)/t-AMPK, (<b>C</b>,<b>D</b>) PEPCK, G6Pase, p-GSK3β/t-GSK3β, (<b>E</b>,<b>F</b>) p-mTORC1/t-mTORC1, p-p70S6K1/t-p70S6K1, p-ACC/t-ACC, (<b>G</b>–<b>K</b>) PPARα, FAS, PPARγ, SREBP1<sub>C</sub>, SREBP2 and (<b>L</b>,<b>M</b>) p-Akt (Ser<sup>473</sup>)/t-Akt (Ser<sup>473</sup>), and p-FoxO1 (Ser<sup>256</sup>)/t-FoxO1 (Ser<sup>256</sup>) in the skeletal muscles, livers, or adipose tissues of high-fat diet (HFD)-induced diabetic mice by oral gavage ethyl acetate extract of <span class="html-italic">Phyllanthus emblica</span> L. (EPE). (<b>A</b>,<b>C</b>,<b>E</b>,<b>G</b>,<b>J</b>,<b>L</b>) representative image; (<b>B</b>,<b>D</b>,<b>F</b>,<b>H</b>,<b>I</b>,<b>K</b>,<b>M</b>) quantification of the p-AMPK to t-AMPK, p-GSK3β to t-GSK3β, p-mTORC1 to t-mTORC1, p-p70S6K1 to t-p70S6K1, p-ACC to t-ACC p-Akt (Ser<sup>473</sup>) to t- Akt (Ser<sup>473</sup>), and p-FoxO1 (Ser<sup>256</sup>) to t-FoxO1 (Ser<sup>256</sup>). Protein was separated by 12% SDS-PAGE detected by Western blot. <sup>###</sup> <span class="html-italic">p</span> &lt; 0.001 compared with the control (CON) group; * <span class="html-italic">p</span> &lt; 0.05, ** <span class="html-italic">p</span> &lt; 0.01, *** <span class="html-italic">p</span> &lt; 0.001 compared with the high-fat diet (HFD) plus vehicle (distilled water) (HF) group. All values are means ± SE (<span class="html-italic">n</span> = 8 per group). EPE1, EPE2, and EPE3, ethyl acetate extract of <span class="html-italic">Phyllanthus emblica</span> L. (EPE) (EPE1, EPE2, and EPE3, 100, 200, and 400 mg/kg body weight, resp.); fenofibrate (Feno; 250 mg/kg body weight); metformin (Metf, 300 mg/kg body weight).</p>
Full article ">Figure 5 Cont.
<p>The expression levels of (<b>A</b>,<b>B</b>) membrane GLUT4, p-AMPK (Thr<sup>172</sup>)/t-AMPK, (<b>C</b>,<b>D</b>) PEPCK, G6Pase, p-GSK3β/t-GSK3β, (<b>E</b>,<b>F</b>) p-mTORC1/t-mTORC1, p-p70S6K1/t-p70S6K1, p-ACC/t-ACC, (<b>G</b>–<b>K</b>) PPARα, FAS, PPARγ, SREBP1<sub>C</sub>, SREBP2 and (<b>L</b>,<b>M</b>) p-Akt (Ser<sup>473</sup>)/t-Akt (Ser<sup>473</sup>), and p-FoxO1 (Ser<sup>256</sup>)/t-FoxO1 (Ser<sup>256</sup>) in the skeletal muscles, livers, or adipose tissues of high-fat diet (HFD)-induced diabetic mice by oral gavage ethyl acetate extract of <span class="html-italic">Phyllanthus emblica</span> L. (EPE). (<b>A</b>,<b>C</b>,<b>E</b>,<b>G</b>,<b>J</b>,<b>L</b>) representative image; (<b>B</b>,<b>D</b>,<b>F</b>,<b>H</b>,<b>I</b>,<b>K</b>,<b>M</b>) quantification of the p-AMPK to t-AMPK, p-GSK3β to t-GSK3β, p-mTORC1 to t-mTORC1, p-p70S6K1 to t-p70S6K1, p-ACC to t-ACC p-Akt (Ser<sup>473</sup>) to t- Akt (Ser<sup>473</sup>), and p-FoxO1 (Ser<sup>256</sup>) to t-FoxO1 (Ser<sup>256</sup>). Protein was separated by 12% SDS-PAGE detected by Western blot. <sup>###</sup> <span class="html-italic">p</span> &lt; 0.001 compared with the control (CON) group; * <span class="html-italic">p</span> &lt; 0.05, ** <span class="html-italic">p</span> &lt; 0.01, *** <span class="html-italic">p</span> &lt; 0.001 compared with the high-fat diet (HFD) plus vehicle (distilled water) (HF) group. All values are means ± SE (<span class="html-italic">n</span> = 8 per group). EPE1, EPE2, and EPE3, ethyl acetate extract of <span class="html-italic">Phyllanthus emblica</span> L. (EPE) (EPE1, EPE2, and EPE3, 100, 200, and 400 mg/kg body weight, resp.); fenofibrate (Feno; 250 mg/kg body weight); metformin (Metf, 300 mg/kg body weight).</p>
Full article ">Figure 5 Cont.
<p>The expression levels of (<b>A</b>,<b>B</b>) membrane GLUT4, p-AMPK (Thr<sup>172</sup>)/t-AMPK, (<b>C</b>,<b>D</b>) PEPCK, G6Pase, p-GSK3β/t-GSK3β, (<b>E</b>,<b>F</b>) p-mTORC1/t-mTORC1, p-p70S6K1/t-p70S6K1, p-ACC/t-ACC, (<b>G</b>–<b>K</b>) PPARα, FAS, PPARγ, SREBP1<sub>C</sub>, SREBP2 and (<b>L</b>,<b>M</b>) p-Akt (Ser<sup>473</sup>)/t-Akt (Ser<sup>473</sup>), and p-FoxO1 (Ser<sup>256</sup>)/t-FoxO1 (Ser<sup>256</sup>) in the skeletal muscles, livers, or adipose tissues of high-fat diet (HFD)-induced diabetic mice by oral gavage ethyl acetate extract of <span class="html-italic">Phyllanthus emblica</span> L. (EPE). (<b>A</b>,<b>C</b>,<b>E</b>,<b>G</b>,<b>J</b>,<b>L</b>) representative image; (<b>B</b>,<b>D</b>,<b>F</b>,<b>H</b>,<b>I</b>,<b>K</b>,<b>M</b>) quantification of the p-AMPK to t-AMPK, p-GSK3β to t-GSK3β, p-mTORC1 to t-mTORC1, p-p70S6K1 to t-p70S6K1, p-ACC to t-ACC p-Akt (Ser<sup>473</sup>) to t- Akt (Ser<sup>473</sup>), and p-FoxO1 (Ser<sup>256</sup>) to t-FoxO1 (Ser<sup>256</sup>). Protein was separated by 12% SDS-PAGE detected by Western blot. <sup>###</sup> <span class="html-italic">p</span> &lt; 0.001 compared with the control (CON) group; * <span class="html-italic">p</span> &lt; 0.05, ** <span class="html-italic">p</span> &lt; 0.01, *** <span class="html-italic">p</span> &lt; 0.001 compared with the high-fat diet (HFD) plus vehicle (distilled water) (HF) group. All values are means ± SE (<span class="html-italic">n</span> = 8 per group). EPE1, EPE2, and EPE3, ethyl acetate extract of <span class="html-italic">Phyllanthus emblica</span> L. (EPE) (EPE1, EPE2, and EPE3, 100, 200, and 400 mg/kg body weight, resp.); fenofibrate (Feno; 250 mg/kg body weight); metformin (Metf, 300 mg/kg body weight).</p>
Full article ">Figure 5 Cont.
<p>The expression levels of (<b>A</b>,<b>B</b>) membrane GLUT4, p-AMPK (Thr<sup>172</sup>)/t-AMPK, (<b>C</b>,<b>D</b>) PEPCK, G6Pase, p-GSK3β/t-GSK3β, (<b>E</b>,<b>F</b>) p-mTORC1/t-mTORC1, p-p70S6K1/t-p70S6K1, p-ACC/t-ACC, (<b>G</b>–<b>K</b>) PPARα, FAS, PPARγ, SREBP1<sub>C</sub>, SREBP2 and (<b>L</b>,<b>M</b>) p-Akt (Ser<sup>473</sup>)/t-Akt (Ser<sup>473</sup>), and p-FoxO1 (Ser<sup>256</sup>)/t-FoxO1 (Ser<sup>256</sup>) in the skeletal muscles, livers, or adipose tissues of high-fat diet (HFD)-induced diabetic mice by oral gavage ethyl acetate extract of <span class="html-italic">Phyllanthus emblica</span> L. (EPE). (<b>A</b>,<b>C</b>,<b>E</b>,<b>G</b>,<b>J</b>,<b>L</b>) representative image; (<b>B</b>,<b>D</b>,<b>F</b>,<b>H</b>,<b>I</b>,<b>K</b>,<b>M</b>) quantification of the p-AMPK to t-AMPK, p-GSK3β to t-GSK3β, p-mTORC1 to t-mTORC1, p-p70S6K1 to t-p70S6K1, p-ACC to t-ACC p-Akt (Ser<sup>473</sup>) to t- Akt (Ser<sup>473</sup>), and p-FoxO1 (Ser<sup>256</sup>) to t-FoxO1 (Ser<sup>256</sup>). Protein was separated by 12% SDS-PAGE detected by Western blot. <sup>###</sup> <span class="html-italic">p</span> &lt; 0.001 compared with the control (CON) group; * <span class="html-italic">p</span> &lt; 0.05, ** <span class="html-italic">p</span> &lt; 0.01, *** <span class="html-italic">p</span> &lt; 0.001 compared with the high-fat diet (HFD) plus vehicle (distilled water) (HF) group. All values are means ± SE (<span class="html-italic">n</span> = 8 per group). EPE1, EPE2, and EPE3, ethyl acetate extract of <span class="html-italic">Phyllanthus emblica</span> L. (EPE) (EPE1, EPE2, and EPE3, 100, 200, and 400 mg/kg body weight, resp.); fenofibrate (Feno; 250 mg/kg body weight); metformin (Metf, 300 mg/kg body weight).</p>
Full article ">Figure 5 Cont.
<p>The expression levels of (<b>A</b>,<b>B</b>) membrane GLUT4, p-AMPK (Thr<sup>172</sup>)/t-AMPK, (<b>C</b>,<b>D</b>) PEPCK, G6Pase, p-GSK3β/t-GSK3β, (<b>E</b>,<b>F</b>) p-mTORC1/t-mTORC1, p-p70S6K1/t-p70S6K1, p-ACC/t-ACC, (<b>G</b>–<b>K</b>) PPARα, FAS, PPARγ, SREBP1<sub>C</sub>, SREBP2 and (<b>L</b>,<b>M</b>) p-Akt (Ser<sup>473</sup>)/t-Akt (Ser<sup>473</sup>), and p-FoxO1 (Ser<sup>256</sup>)/t-FoxO1 (Ser<sup>256</sup>) in the skeletal muscles, livers, or adipose tissues of high-fat diet (HFD)-induced diabetic mice by oral gavage ethyl acetate extract of <span class="html-italic">Phyllanthus emblica</span> L. (EPE). (<b>A</b>,<b>C</b>,<b>E</b>,<b>G</b>,<b>J</b>,<b>L</b>) representative image; (<b>B</b>,<b>D</b>,<b>F</b>,<b>H</b>,<b>I</b>,<b>K</b>,<b>M</b>) quantification of the p-AMPK to t-AMPK, p-GSK3β to t-GSK3β, p-mTORC1 to t-mTORC1, p-p70S6K1 to t-p70S6K1, p-ACC to t-ACC p-Akt (Ser<sup>473</sup>) to t- Akt (Ser<sup>473</sup>), and p-FoxO1 (Ser<sup>256</sup>) to t-FoxO1 (Ser<sup>256</sup>). Protein was separated by 12% SDS-PAGE detected by Western blot. <sup>###</sup> <span class="html-italic">p</span> &lt; 0.001 compared with the control (CON) group; * <span class="html-italic">p</span> &lt; 0.05, ** <span class="html-italic">p</span> &lt; 0.01, *** <span class="html-italic">p</span> &lt; 0.001 compared with the high-fat diet (HFD) plus vehicle (distilled water) (HF) group. All values are means ± SE (<span class="html-italic">n</span> = 8 per group). EPE1, EPE2, and EPE3, ethyl acetate extract of <span class="html-italic">Phyllanthus emblica</span> L. (EPE) (EPE1, EPE2, and EPE3, 100, 200, and 400 mg/kg body weight, resp.); fenofibrate (Feno; 250 mg/kg body weight); metformin (Metf, 300 mg/kg body weight).</p>
Full article ">Figure 5 Cont.
<p>The expression levels of (<b>A</b>,<b>B</b>) membrane GLUT4, p-AMPK (Thr<sup>172</sup>)/t-AMPK, (<b>C</b>,<b>D</b>) PEPCK, G6Pase, p-GSK3β/t-GSK3β, (<b>E</b>,<b>F</b>) p-mTORC1/t-mTORC1, p-p70S6K1/t-p70S6K1, p-ACC/t-ACC, (<b>G</b>–<b>K</b>) PPARα, FAS, PPARγ, SREBP1<sub>C</sub>, SREBP2 and (<b>L</b>,<b>M</b>) p-Akt (Ser<sup>473</sup>)/t-Akt (Ser<sup>473</sup>), and p-FoxO1 (Ser<sup>256</sup>)/t-FoxO1 (Ser<sup>256</sup>) in the skeletal muscles, livers, or adipose tissues of high-fat diet (HFD)-induced diabetic mice by oral gavage ethyl acetate extract of <span class="html-italic">Phyllanthus emblica</span> L. (EPE). (<b>A</b>,<b>C</b>,<b>E</b>,<b>G</b>,<b>J</b>,<b>L</b>) representative image; (<b>B</b>,<b>D</b>,<b>F</b>,<b>H</b>,<b>I</b>,<b>K</b>,<b>M</b>) quantification of the p-AMPK to t-AMPK, p-GSK3β to t-GSK3β, p-mTORC1 to t-mTORC1, p-p70S6K1 to t-p70S6K1, p-ACC to t-ACC p-Akt (Ser<sup>473</sup>) to t- Akt (Ser<sup>473</sup>), and p-FoxO1 (Ser<sup>256</sup>) to t-FoxO1 (Ser<sup>256</sup>). Protein was separated by 12% SDS-PAGE detected by Western blot. <sup>###</sup> <span class="html-italic">p</span> &lt; 0.001 compared with the control (CON) group; * <span class="html-italic">p</span> &lt; 0.05, ** <span class="html-italic">p</span> &lt; 0.01, *** <span class="html-italic">p</span> &lt; 0.001 compared with the high-fat diet (HFD) plus vehicle (distilled water) (HF) group. All values are means ± SE (<span class="html-italic">n</span> = 8 per group). EPE1, EPE2, and EPE3, ethyl acetate extract of <span class="html-italic">Phyllanthus emblica</span> L. (EPE) (EPE1, EPE2, and EPE3, 100, 200, and 400 mg/kg body weight, resp.); fenofibrate (Feno; 250 mg/kg body weight); metformin (Metf, 300 mg/kg body weight).</p>
Full article ">Figure 5 Cont.
<p>The expression levels of (<b>A</b>,<b>B</b>) membrane GLUT4, p-AMPK (Thr<sup>172</sup>)/t-AMPK, (<b>C</b>,<b>D</b>) PEPCK, G6Pase, p-GSK3β/t-GSK3β, (<b>E</b>,<b>F</b>) p-mTORC1/t-mTORC1, p-p70S6K1/t-p70S6K1, p-ACC/t-ACC, (<b>G</b>–<b>K</b>) PPARα, FAS, PPARγ, SREBP1<sub>C</sub>, SREBP2 and (<b>L</b>,<b>M</b>) p-Akt (Ser<sup>473</sup>)/t-Akt (Ser<sup>473</sup>), and p-FoxO1 (Ser<sup>256</sup>)/t-FoxO1 (Ser<sup>256</sup>) in the skeletal muscles, livers, or adipose tissues of high-fat diet (HFD)-induced diabetic mice by oral gavage ethyl acetate extract of <span class="html-italic">Phyllanthus emblica</span> L. (EPE). (<b>A</b>,<b>C</b>,<b>E</b>,<b>G</b>,<b>J</b>,<b>L</b>) representative image; (<b>B</b>,<b>D</b>,<b>F</b>,<b>H</b>,<b>I</b>,<b>K</b>,<b>M</b>) quantification of the p-AMPK to t-AMPK, p-GSK3β to t-GSK3β, p-mTORC1 to t-mTORC1, p-p70S6K1 to t-p70S6K1, p-ACC to t-ACC p-Akt (Ser<sup>473</sup>) to t- Akt (Ser<sup>473</sup>), and p-FoxO1 (Ser<sup>256</sup>) to t-FoxO1 (Ser<sup>256</sup>). Protein was separated by 12% SDS-PAGE detected by Western blot. <sup>###</sup> <span class="html-italic">p</span> &lt; 0.001 compared with the control (CON) group; * <span class="html-italic">p</span> &lt; 0.05, ** <span class="html-italic">p</span> &lt; 0.01, *** <span class="html-italic">p</span> &lt; 0.001 compared with the high-fat diet (HFD) plus vehicle (distilled water) (HF) group. All values are means ± SE (<span class="html-italic">n</span> = 8 per group). EPE1, EPE2, and EPE3, ethyl acetate extract of <span class="html-italic">Phyllanthus emblica</span> L. (EPE) (EPE1, EPE2, and EPE3, 100, 200, and 400 mg/kg body weight, resp.); fenofibrate (Feno; 250 mg/kg body weight); metformin (Metf, 300 mg/kg body weight).</p>
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<p>Effects of seven fractions of EPE (EA) on expression levels of phospho-AMPK/total-AMPK in C2C12 myoblasts cells by Western blotting analyses. C2C12 skeletal myoblast cells were treated with seven fractions and equal amounts of lysates were resolved by SDS-PAGE and blotted for phospho-AMPK (Thr<sup>172</sup>). (<b>A</b>,<b>B</b>) Effects of 7 fractions of ethyl acetate extract of <span class="html-italic">Phyllanthus emblica</span> L. (EtOAc soluble fractions) (EA-1, EA-2, EA-3, EA-A, EA-5, EA-6, and EA-7) on expression levels of phospho-AMPK (Thr<sup>172</sup>) in C2C12 myoblasts cells by Western blotting analysis. C2C12 skeletal myoblast cells were treated with 7 fractions as described in the experimental procedures and equal amounts of lysates were resolved by SDS-PAGE and blotted for phospho-AMPK (Thr<sup>172</sup>). (<b>A</b>) Representative blots for 7 fractions in C2C12 myoblasts cells; (<b>B</b>) quantification of the expression levels of the ratio of phospho-AMPK to total-AMPK. All values are means ± S.E. <sup>+++</sup> <span class="html-italic">p</span> &lt; 0.001 compared with the control group; <sup>###</sup> <span class="html-italic">p</span> &lt; 0.001 compared with the insulin group ** <span class="html-italic">p</span> &lt; 0.01, *** <span class="html-italic">p</span> &lt; 0.001 compared with the palmitate+ insulin group. (<b>C</b>,<b>D</b>) High-performance liquid chromatography analysis of (<b>C</b>) 2500 ppm ethyl acetate of <span class="html-italic">Phyllanthus emblica</span> L. (EPE), (<b>D</b>) 10.3 mg/5 mL EA-4 of ethyl acetate of <span class="html-italic">Phyllanthus emblica</span> L. [<a href="#B55-cimb-46-00623" class="html-bibr">55</a>].</p>
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<p>Effects of seven fractions of EPE (EA) on expression levels of phospho-AMPK/total-AMPK in C2C12 myoblasts cells by Western blotting analyses. C2C12 skeletal myoblast cells were treated with seven fractions and equal amounts of lysates were resolved by SDS-PAGE and blotted for phospho-AMPK (Thr<sup>172</sup>). (<b>A</b>,<b>B</b>) Effects of 7 fractions of ethyl acetate extract of <span class="html-italic">Phyllanthus emblica</span> L. (EtOAc soluble fractions) (EA-1, EA-2, EA-3, EA-A, EA-5, EA-6, and EA-7) on expression levels of phospho-AMPK (Thr<sup>172</sup>) in C2C12 myoblasts cells by Western blotting analysis. C2C12 skeletal myoblast cells were treated with 7 fractions as described in the experimental procedures and equal amounts of lysates were resolved by SDS-PAGE and blotted for phospho-AMPK (Thr<sup>172</sup>). (<b>A</b>) Representative blots for 7 fractions in C2C12 myoblasts cells; (<b>B</b>) quantification of the expression levels of the ratio of phospho-AMPK to total-AMPK. All values are means ± S.E. <sup>+++</sup> <span class="html-italic">p</span> &lt; 0.001 compared with the control group; <sup>###</sup> <span class="html-italic">p</span> &lt; 0.001 compared with the insulin group ** <span class="html-italic">p</span> &lt; 0.01, *** <span class="html-italic">p</span> &lt; 0.001 compared with the palmitate+ insulin group. (<b>C</b>,<b>D</b>) High-performance liquid chromatography analysis of (<b>C</b>) 2500 ppm ethyl acetate of <span class="html-italic">Phyllanthus emblica</span> L. (EPE), (<b>D</b>) 10.3 mg/5 mL EA-4 of ethyl acetate of <span class="html-italic">Phyllanthus emblica</span> L. [<a href="#B55-cimb-46-00623" class="html-bibr">55</a>].</p>
Full article ">Figure 6 Cont.
<p>Effects of seven fractions of EPE (EA) on expression levels of phospho-AMPK/total-AMPK in C2C12 myoblasts cells by Western blotting analyses. C2C12 skeletal myoblast cells were treated with seven fractions and equal amounts of lysates were resolved by SDS-PAGE and blotted for phospho-AMPK (Thr<sup>172</sup>). (<b>A</b>,<b>B</b>) Effects of 7 fractions of ethyl acetate extract of <span class="html-italic">Phyllanthus emblica</span> L. (EtOAc soluble fractions) (EA-1, EA-2, EA-3, EA-A, EA-5, EA-6, and EA-7) on expression levels of phospho-AMPK (Thr<sup>172</sup>) in C2C12 myoblasts cells by Western blotting analysis. C2C12 skeletal myoblast cells were treated with 7 fractions as described in the experimental procedures and equal amounts of lysates were resolved by SDS-PAGE and blotted for phospho-AMPK (Thr<sup>172</sup>). (<b>A</b>) Representative blots for 7 fractions in C2C12 myoblasts cells; (<b>B</b>) quantification of the expression levels of the ratio of phospho-AMPK to total-AMPK. All values are means ± S.E. <sup>+++</sup> <span class="html-italic">p</span> &lt; 0.001 compared with the control group; <sup>###</sup> <span class="html-italic">p</span> &lt; 0.001 compared with the insulin group ** <span class="html-italic">p</span> &lt; 0.01, *** <span class="html-italic">p</span> &lt; 0.001 compared with the palmitate+ insulin group. (<b>C</b>,<b>D</b>) High-performance liquid chromatography analysis of (<b>C</b>) 2500 ppm ethyl acetate of <span class="html-italic">Phyllanthus emblica</span> L. (EPE), (<b>D</b>) 10.3 mg/5 mL EA-4 of ethyl acetate of <span class="html-italic">Phyllanthus emblica</span> L. [<a href="#B55-cimb-46-00623" class="html-bibr">55</a>].</p>
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<p>The graphic abstract of ethyl acetate extract of <span class="html-italic">Phyllanthus emblica</span> L. (EPE) in high-fat diet (HFD)-induced diabetic mice.</p>
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17 pages, 15759 KiB  
Article
Full-Scale Fire Experiment on Mezzanine Racks in Logistics Facilities
by Byeongheun Lee, Nam Jeon and Jeongki Min
Fire 2024, 7(9), 326; https://doi.org/10.3390/fire7090326 - 20 Sep 2024
Viewed by 303
Abstract
The increased demand for contactless services has facilitated a rapid increase in logistics facilities. There are shorter distances between the shelf racks used in mezzansine racks in such facilities compared to standard racks and can store various items; however, research on fire safety [...] Read more.
The increased demand for contactless services has facilitated a rapid increase in logistics facilities. There are shorter distances between the shelf racks used in mezzansine racks in such facilities compared to standard racks and can store various items; however, research on fire safety related to this remains insufficient. In this study, we visited four logistics facilities with mezzanine racks and one logistics facility using shelf racks to investigate their fundamental characteristics. Considering the characteristics of logistics facilities that store various combustibles, a fire test was conducted using unit shelf racks with packaging materials, boxes, and expandable polystyrene (EPS). Shelf racks loaded with corrugated fiberboard, cardboard boxes, and EPS exhibited the highest fire risk and were set as combustibles inside the rack. Before the experiment, the radiative heat flux was measured by considering the spacing distances of mezzanine racks observed on-site. The most frequently measured range was 43.7–67.3 kW/m2 at 1.0–1.5 m. After beginning the fire experiment, when simulating mezzanine racks with aisle widths of 1.2–2.0 m, fire owing to radiative heat occurred within 5 min in the separated shelf racks. Based on the results, we estimate that the minimum separation distance required to prevent radiant heat-based fires between shelving racks inside a mezzanine is 3.2 m. These findings are expected to be utilized in fire prevention by increasing the understanding of the spread of fire in shelf racks. Full article
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<p>Mezzanine floor racking system.</p>
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<p>The parts of a shelf rack.</p>
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<p>Detailed conditions and the actual shelf rack used in the experiment.</p>
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<p>Cardboard box. (<b>a</b>) Immediately after ignition; (<b>b</b>) 5 min after ignition; (<b>c</b>) 13 min after ignition.</p>
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<p>EPS. (<b>a</b>) Immediately after ignition; (<b>b</b>) 1 min after ignition; (<b>c</b>) 5 min after ignition.</p>
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<p>Box + EPS. (<b>a</b>) Box + EPS; (<b>b</b>) 2 min 30 s after ignition; (<b>c</b>) 4 min after ignition.</p>
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<p>Combustibles placed on a single shelf rack.</p>
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<p>Single shelf rack fire experiment. (<b>a</b>) Immediately after ignition; (<b>b</b>) 4 min 30 s after ignition; (<b>c</b>) 7 min after ignition.</p>
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<p>Combustion behavior of corrugated fiberboard + EPS specimen. (<b>a</b>) Immediately after ignition; (<b>b</b>) 6 min after ignition; (<b>c</b>) 7 min after ignition; (<b>d</b>) 8 min after ignition. (<b>e</b>) Time of ignition (immediately after ignition); (<b>f</b>) peak time (8 min after ignition).</p>
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<p>Combustion behavior of corrugated fiberboard + cardboard box specimen (<b>a</b>) 30 s after ignition; (<b>b</b>) 2 min after ignition; (<b>c</b>) 3 min after ignition; (<b>d</b>) 6 min after ignition. (<b>e</b>) Time of ignition (immediately after ignition). (<b>f</b>) Peak time (6 min after ignition).</p>
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<p>Combustion behavior of corrugated fiberboard + cardboard box + EPS specimen (<b>a</b>) 15 s after ignition; (<b>b</b>) 1 min 30 s after ignition; (<b>c</b>) 3 min after ignition; (<b>d</b>) 6 min after ignition. (<b>e</b>) Time of ignition (immediately after ignition); (<b>f</b>) peak time (6 min after ignition).</p>
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<p>Comparison of shelf rack temperatures over time.</p>
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<p>Test setup simulating mezzanine racks.</p>
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<p>Position of heat flux sensors.</p>
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<p>Combustion behavior over time. (<b>a</b>) Immediately after ignition; (<b>b</b>) 2 min 30 s after ignition; (<b>c</b>) 4 min after ignition; (<b>d</b>) 7 min 40 s after ignition.</p>
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<p>Heat flux on surface distance.</p>
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<p>Formula derivation through regression analysis.</p>
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<p>Position of thermocouples on opposite shelf rack.</p>
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<p>Temperature measurement results from thermocouples: (<b>a</b>) ch.1; (<b>b</b>) ch.2; (<b>c</b>) ch.3; (<b>d</b>) ch.4.</p>
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<p>Fire experiment with shelf racks spaced 1.2 m apart. (<b>a</b>) Immediately after ignition; (<b>b</b>) vertical fire spread; (<b>c</b>) ignition on opposite shelf rack (05:10).</p>
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<p>Fire experiment with shelf racks spaced 1.5 m apart. (<b>a</b>) Immediately after ignition; (<b>b</b>) vertical fire spread; (<b>c</b>) ignition on opposite shelf rack (04:30).</p>
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<p>Fire experiment with shelf racks spaced 2.0 m apart. (<b>a</b>) Immediately after ignition; (<b>b</b>) vertical fire spread; (<b>c</b>) ignition on opposite shelf rack (05:15).</p>
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<p>Estimated radiative heat flux based on spacing distance.</p>
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20 pages, 3351 KiB  
Article
Potential Bioactivities, Chemical Composition, and Conformation Studies of Exopolysaccharide-Derived Aspergillus sp. Strain GAD7
by Mohamed I. A. Ibrahim, Hassan A. H. Ibrahim, Tatsuki Haga, Atsuhiko Ishida, Tatsuo Nehira, Koichi Matsuo and Ahmed M. Gad
J. Fungi 2024, 10(9), 659; https://doi.org/10.3390/jof10090659 - 19 Sep 2024
Viewed by 614
Abstract
This research identified a marine fungal isolate, Aspergillus sp. strain GAD7, which produces an acidic and sulfated extracellular polysaccharide (EPS) with notable anticoagulant and antioxidant properties. Six fungal strains from the Egyptian Mediterranean Sea were screened for EPS production, with Aspergillus sp. strain [...] Read more.
This research identified a marine fungal isolate, Aspergillus sp. strain GAD7, which produces an acidic and sulfated extracellular polysaccharide (EPS) with notable anticoagulant and antioxidant properties. Six fungal strains from the Egyptian Mediterranean Sea were screened for EPS production, with Aspergillus sp. strain GAD7 (EPS-AG7) being the most potent, yielding ~5.19 ± 0.017 g/L. EPS-AG7 was characterized using UV-Vis and FTIR analyses, revealing high carbohydrate (87.5%) and sulfate (24%) contents. HPLC and GC-MS analyses determined that EPS-AG7 is a heterogeneous acidic polysaccharide with an average molecular weight (Mw¯) of ~7.34 × 103 Da, composed of mannose, glucose, arabinose, galacturonic acid, galactose, and lyxose in a molar ratio of 6.6:3.9:1.8:1.3:1.1:1.0, linked through α- and β-glycosidic linkages as confirmed by NMR analysis. EPS-AG7 adopted a triple helix-like conformation, as evidenced by UV-Vis (Congo Red experiment) and circular dichroism (CD) studies. This helical arrangement demonstrated stability under various experimental conditions, including concentration, ionic strength, temperature, and lipid interactions. EPS-AG7 exhibited significant anticoagulant activity, doubling blood coagulation time at a concentration of 3.0 mg/mL, and showed significant antioxidant activity, with scavenging activities reaching up to 85.90% and 58.64% in DPPH and ABTS+ assays at 5.0 mg/mL, and EC50 values of 1.40 mg/mL and 3.80 mg/mL, respectively. These findings highlight the potential of EPS-AG7 for therapeutic applications due to its potent biological activities. Full article
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<p>Sampling sites for marine algae samples and the corresponding fungi isolates along the Abu-Qir shore in Egypt during September 2022.</p>
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<p>(<b>a</b>) <span class="html-italic">Mycelium</span> dry weights and EPS yields of fungal isolates, (<b>b</b>) macrographs showing macroscopic features on SD agar (<b>b</b>-<b>left</b>) and microscopy details of <span class="html-italic">Aspergillus</span> sp. strain GAD7 (<b>b-right</b>), and (<b>c</b>) phylogenetic tree of <span class="html-italic">Aspergillus</span> sp. strain GAD7.</p>
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<p>(<b>a</b>) FTIR spectrum, and (<b>b</b>) SEC chromatogram of the EPS-AG7 produced by <span class="html-italic">Aspergillus</span> sp. GAD7.</p>
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<p>(<b>a</b>) Plot of <math display="inline"><semantics> <mrow> <msub> <mrow> <mi>λ</mi> </mrow> <mrow> <mi>m</mi> <mi>a</mi> <mi>x</mi> </mrow> </msub> </mrow> </semantics></math> of CR-EPS against [NaOH] using UV-Vis, (<b>b</b>) VUV-CD spectrum of EPS-AG7 by <span class="html-italic">Aspergillus</span> sp. GAD7 (2.5 <span class="html-italic">w</span>/<span class="html-italic">v</span>% in DW, 25 °C); (<b>c</b>) VUV-CD concentration dependent of EPS-AG7 (0.5–6.0 <span class="html-italic">w</span>/<span class="html-italic">v</span>% in DW); (<b>d</b>) VUV-CD spectra in absence and presence of NaF (50 mmol/L); and (<b>e</b>) VUV-CD temperature-dependent spectra of EPS-AG7 (6.0 <span class="html-italic">w</span>/<span class="html-italic">v</span>%, 20 °C–65 °C in DW).</p>
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<p>VUV-CD spectra of EPS-AG7/lipid at different weight ratios (<span class="html-italic">w</span>/<span class="html-italic">w</span>%): (<b>a</b>) EPS-AG7/DOPE; (<b>b</b>) EPS-AG7/DOPS; and (<b>c</b>) EPS-AG7/DOPC, in Tris-HCl buffer of pH 7.6 at 25 °C, and EPS-AG7 concentration of 2.5 <span class="html-italic">w</span>/<span class="html-italic">v</span>%.</p>
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<p>DPPH and ABTS<sup>+</sup> free radicals scavenging activity (%) of (<b>a</b>) EPS-AG7 at a final concentration range of 0.25–5.0 mg/mL, versus (<b>b</b>) ascorbic acid at a final concentration range of 0.005–1.0 mmol/L (0.88–176.12 µg/mL).</p>
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16 pages, 3227 KiB  
Article
Euglena gracilis Enhances Innate and Adaptive Immunity through Specific Expression of Dectin-1 in CP-Induced Immunosuppressed Mice
by Hwan Hee Lee, Ji-Yeon Seong, Hyojeung Kang and Hyosun Cho
Nutrients 2024, 16(18), 3158; https://doi.org/10.3390/nu16183158 - 18 Sep 2024
Viewed by 549
Abstract
Background: Euglena gracilis (E. gracilis), a species of unicellular algae, can accumulate large amounts of β-1,3-glucan paramylon, a polysaccharide, in its cytoplasm and has recently attracted interest as a bioproduct due to its various health benefits. In this study, the immune-enhancing [...] Read more.
Background: Euglena gracilis (E. gracilis), a species of unicellular algae, can accumulate large amounts of β-1,3-glucan paramylon, a polysaccharide, in its cytoplasm and has recently attracted interest as a bioproduct due to its various health benefits. In this study, the immune-enhancing effect of E. gracilis powder (EP) was investigated in vitro and in vivo. Methods: In vitro, the production of NO and cytokines and the mechanism of the signaling pathway of β-1,3-glucan were identified in RAW264.7 cells. In vivo, cyclophosphamide-induced (CP-induced) immunosuppressed C57BL/6 female mice were orally administered with three different concentrations (100, 300, and 600 mg/kg) of EP daily. After 14 days, the organs and whole blood were collected from each animal for further study. Results: The weight loss of CP-treated mice was reversed by treatment with EP to levels comparable to those of control mice. In addition, the frequencies of NK1.1+, CD3+, CD4+, CD8+, and B220+ in immune cells isolated from the spleen were increased by EP treatment compared with water or RG. The secretion of TNF-α, IFN-γ, and IL-12 from splenocytes was also increased by EP treatment, as was the level of IgM in the serum of the mice. Finally, EP treatment specifically upregulated the expression of dectin-1 in the liver of CP-treated mice. Conclusions: E. gracilis could be a good candidate for a natural immune stimulator in the innate and adaptive response by secreting TNF-α, IFN-γ, and IL-12 through stimulating dectin-1 expression on the surface of immune cells. Full article
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<p>Production of NO metabolites by β-1,3-glucan in RAW264.7 cells. Cells were incubated with a series of concentrations of β-1,3-glucan (0, 50, 100, 250, 500, and 1000 μg/mL) or LPS (1 μg/mL) for 24 h. After the incubation, the (<b>A</b>) cytotoxicity by CCK-8 assay and (<b>B</b>) NO production in the supernatants collected from the cells by a nitrite/nitrate assay kit were determined. All data are presented as means ± SD, and experiments were performed at least three times. * <span class="html-italic">p</span> &lt; 0.05 vs. untreated; ns: not significant.</p>
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<p>Effect of β-1,3-glucan on TNF-α production and the activation-related proteins in RAW264.7 cells. Cells were incubated with different concentrations (0, 50, 100, 250, and 500 μg/mL) of β-1,3-glucan or LPS for 24 h. The supernatants and cells were then harvested separately for each experiment. (<b>A</b>) TNF-α by ELISA assay and the expression of (<b>B</b>) TLR-6, (<b>C</b>) Dectin-1, (<b>D</b>) pERK, and (<b>E</b>) pNF-κB by Western blot analysis were identified. All data are presented as means ± SD, and experiments were performed at least three times. * <span class="html-italic">p</span> &lt; 0.05 vs. untreated; ns: not significant.</p>
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<p>Change in body weight and liver, spleen, and thymus weights of the mice after oral administration of EP. Mice were injected intraperitoneally with CP on days 1 and 4 after starting oral administration of water, RG (10 mg/kg), and three concentrations (100, 300, and 600 mg/kg) of EP, except for the group receiving water only (NC). All specimens were administered orally to the mice for a total of 14 days. (<b>A</b>–<b>C</b>) Body weight of mice on days 6, 8, and 14 and the weight of (<b>D</b>) liver, (<b>E</b>) spleen, and (<b>F</b>) thymus isolated from each animal after 14 days of oral administration. All data are presented as means ± SD (n = 8) and analyzed by one-way ANOVA to compare differences between groups. The different letters in the graph indicate a significance between groups, and the <span class="html-italic">p</span>-value is less than 0.05.</p>
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<p>Effect of EP on peripheral blood cells of immunosuppressed mice. Whole blood was collected from mice after oral administration of each sample for 14 days. (<b>A</b>) White blood cells (WBC, ×10<sup>6</sup> cells/μL), (<b>B</b>) neutrophils (NEU, %), (<b>C</b>) lymphocytes (LYM, %), (<b>D</b>) red blood cells (RBC, ×10<sup>6</sup> cells/μL), (<b>E</b>) red blood cell distribution width (RDW, %), and (<b>F</b>) platelets (PLT, ×10<sup>3</sup> cells/μL). All data are presented as means ± SD (n = 8) and analyzed with one-way ANOVA to compare differences between groups. The different letters in the graph indicate a significance between groups, and the <span class="html-italic">p</span>-value is less than 0.05.</p>
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<p>Effect of EP on splenocytes of immunosuppressed mice. (<b>A</b>) Splenocyte proliferation by CCK-8 assay and the frequencies of (<b>B</b>) NK1.1<sup>+</sup>, (<b>C</b>) CD3<sup>+</sup>, (<b>D</b>) CD4<sup>+</sup>, (<b>E</b>) CD8<sup>+</sup>, and (<b>F</b>) B220<sup>+</sup> cells by flow cytometry were determined. All results are expressed as means ± SD (n = 8). Data were analyzed by one-way ANOVA to compare differences between groups. The different letters in the graph indicate a significance between groups, and the <span class="html-italic">p</span>-value is less than 0.05.</p>
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<p>Effect of EP on the production of cytokines and antibodies. Splenocytes isolated from mice were incubated with ConA (10 μg/mL) for 72 h, and the cell-free supernatants were then collected, and mouse serum was separated from whole blood. The levels of cytokines and antibodies were analyzed by ELISA assay kits. The production of (<b>A</b>) TNF-α, (<b>B</b>) IFN-γ, (<b>C</b>) IL-12, and (<b>D</b>) IgM in the cell-free supernatants and the levels of (<b>E</b>) IgM and (<b>F</b>) IgG in the serum were determined. Data were analyzed by one-way ANOVA to compare differences between groups. The different letters in the graph indicate a significance between groups, and the <span class="html-italic">p</span>-value is less than 0.05.</p>
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<p>Effect of EP on dectin-1 expression in mouse liver. The liver tissue of mice was collected after 14 days of specimen treatment. The analysis of dectin-1 expression in the liver tissue of mice was performed by immunohistochemistry (IHC) staining. The picture was visualized by light microscopy (40× and 200×). Dectin-1 expression is indicated by black dots, shown by red arrows. The experiment was performed at least three times on different liver tissues from each group, and the upper picture was a representative of all results.</p>
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23 pages, 11085 KiB  
Article
Exopolysaccharides from the Green Microalga Strain Coelastrella sp. BGV—Isolation, Characterization, and Assessment of Anticancer Potential
by Tanya Toshkova-Yotova, Inna Sulikovska, Vera Djeliova, Zdravka Petrova, Manol Ognyanov, Petko Denev, Reneta Toshkova and Ani Georgieva
Curr. Issues Mol. Biol. 2024, 46(9), 10312-10334; https://doi.org/10.3390/cimb46090614 - 16 Sep 2024
Viewed by 459
Abstract
Algal metabolites have been extensively studied as potential anticancer therapeutics. Among them, polysaccharides have attracted much attention because of their beneficial biological effects and safety. In the present research, the chemical characteristics, antitumor, and proapoptotic activities of extracellular polysaccharides (EPS) isolated from a [...] Read more.
Algal metabolites have been extensively studied as potential anticancer therapeutics. Among them, polysaccharides have attracted much attention because of their beneficial biological effects and safety. In the present research, the chemical characteristics, antitumor, and proapoptotic activities of extracellular polysaccharides (EPS) isolated from a new Bulgarian strain of the green microalga Coelastrella sp. BGV were investigated. A fast and convenient method of precipitation with cold ethanol was used to isolate EPS from the culture medium. The chemical characteristics of the isolated EPS were examined by colorimetric and spectrophotometric analyses, HPSEC-RID and HPLC-UV chromatography, and FT-IR spectroscopy. The results showed that the isolated EPS sample consists of three carbohydrate fractions with different molecular weights (11.5 × 104 Da, 30.7 × 104 Da, and 72.4 × 104 Da, respectively) and contains 7.14 (w/w%) protein. HPLC-UV analysis revealed the presence of galactose and fucose. The total uronic acid content in the sample was 4.5 (w/w%). The IR-FT spectrum of EPS revealed the presence of various functional groups typical of a polysaccharide (or proteoglycan) composed primarily of neutral sugars. The anticancer potential of the obtained EPS was assessed using cell lines with cancerous and non-cancerous origins as in vitro experimental models. The results of the performed MTT assay showed that EPS reduced the viability of the cervical and mammary carcinoma cell lines HeLa and MCF-7, while the control non-cancer cell lines BALB/3T3 and HaCaT were less affected. The HeLa cell line showed the highest sensitivity to the effects of EPS and was therefore used for further studies of its anticancer potential. The ability of EPS to inhibit cancer cell migration was demonstrated by wound-healing (scratch) assay. The cell cycle FACS analysis indicated that the EPS treatment induced significant increases in the sub G1 cell population and decreases of the percentages of cells in the G1, S, and G2-M phases, compared to the control. The fluorescent microscopy studies performed using three different staining methods in combination with Annexin V-FITC flow cytometric analysis clearly demonstrate the ability of EPS to induce cancer cell death via the apoptosis pathway. Moreover, an altered pattern and intensity of the immunocytochemical staining for the apoptosis- and proliferation-related proteins p53, bcl2, and Ki67 was detected in EPS-treated HeLa cancer cells as compared to the untreated controls. The obtained results characterize the new local strain of green microalgae Coelastrella sp. BGV as a producer of EPS with selective antitumor activity and provide an opportunity for further studies of its pharmacological and biotechnological potential. Full article
(This article belongs to the Special Issue Phytochemicals in Cancer Chemoprevention and Treatment)
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Figure 1
<p>An HPLC-UV chromatogram of PMP (1-phenyl-3-methyl-5-pyrazolone)—monosaccharide derivatives obtained from EPS from <span class="html-italic">Coelastrella</span> sp. BGV.</p>
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<p>HPSEC elution profile of EPS from <span class="html-italic">Coelastrella</span> sp. BGV. (<b>A</b>) exopolysaccharide; (<b>B</b>) pullulan standards. Molecular weights of the standards used—from left to right: 78.8 × 10<sup>4</sup>, 40.4 × 10<sup>4</sup>, 21.2 × 10<sup>4</sup>, 11.2 × 10<sup>4</sup>, 4.73 × 10<sup>4</sup>, 2.28 × 10<sup>4</sup>, 1.18 × 10<sup>4</sup>, 0.59 × 10<sup>4</sup> Da.</p>
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<p>FT-IR absorption spectrum of the EPS from <span class="html-italic">Coelastrella</span> sp. BGV over the range of 4000–400 cm<sup>−1</sup>.</p>
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<p>Effect of extracellular polysaccharide from Coelastrella sp. BGV on the viability of HeLa, MCF-7, HaCaT and BALB/3T3 cells assessed by an MTT test at 24 h and 48 h. Untreated cells and cultivated cells treated with the antitumor drug Doxorubicin (Dox; 2.5 μg/mL) were used as negative and positive controls, respectively. The data are expressed as the mean ± SD of five samples from each treatment group. * <span class="html-italic">p</span> &lt; 0.05, ** <span class="html-italic">p</span> &lt; 0.01, and *** <span class="html-italic">p</span> &lt; 0.001 indicate significant differences compared to the negative control.</p>
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<p>Effect of Coelastrella sp. BGV EPS treatment on the migration of HeLa cervical carcinoma cells evaluated by wound-healing assay. (<b>Upper panel</b>) Light microscopy images of untreated cell cultures and cell cultures treated with EPS (250 μg/mL); (<b>Lower panel</b>) Quantification of the EPS effect on the migration potential of the cancer cells. Data are presented as Mean ± SD; *** <span class="html-italic">p</span> &lt; 0.001 compared to the untreated control.</p>
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<p>Morphological changes of the HeLa tumor cells cultured in the presence of EPS (500 μg/mL) for 24 h followed by AO/EB (<b>Upper row</b>), DAPI (<b>middle row),</b> and Annexin V (<b>Lower row</b>) staining. Fluorescence micrographs of: (<b>a</b>,<b>d</b>,<b>g</b>)—untreated HeLa cells; (<b>b</b>,<b>e</b>,<b>h</b>)—cells after incubation with 500 µg/mL of EPS; (<b>c</b>,<b>f</b>,<b>i</b>)—cells after incubation with 2.5 µg/mL Dox; Scale bar = 20 µm.</p>
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<p>Proapoptotic effect of <span class="html-italic">Coelastrella</span> sp. BGV EPS on HeLa cervical carcinoma cells as evaluated by FACS analysis. (<b>Upper panel</b>) Representative histogram of control untreated cells and cells treated with 500 µg/mL EPS for 24 h. (<b>Lower panel</b>) Bar graph showing the percentages of the live, early and late apoptotic, and necrotic cells. The data are expressed as mean ± SD from three independent experiments; *** <span class="html-italic">p</span> &lt; 0.001 indicates significant difference as compared to the negative control.</p>
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<p>Effect of Coelastrella sp BGV EPS treatment on the cell cycle progression of HeLa cervical carcinoma cells. (<b>Upper panel</b>) Representative histogram of control untreated cells and cells treated with 500 µg/mL EPS for 24 h. (<b>Lower panel</b>) Bar graph representing the distribution of the cells in the different cell cycle phases. The data are expressed as mean ± SD from three independent experiments; *** <span class="html-italic">p</span> &lt; 0.001 indicate significant difference as compared to the negative control.</p>
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<p>Immunocytochemical analysis of the EPS effects on the expression and intracellular localization of the p53, bcl2, and Ki67 proteins in HeLa carcinoma cells. (<b>a</b>,<b>d</b>,<b>g</b>) untreated control cells; (<b>b</b>,<b>e</b>,<b>h</b>) cells treated with EPS (500 μg/mL); (<b>c</b>,<b>f</b>,<b>i</b>) cells treated with Dox (2.5 μg/mL); (<b>a</b>–<b>c</b>) p53 immunostaining; (<b>d</b>–<b>f</b>) bcl2 immunostaining; (<b>g</b>–<b>i</b>) Ki67 immunostaining.</p>
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24 pages, 11508 KiB  
Article
Discovery and Optimization of Ergosterol Peroxide Derivatives as Novel Glutaminase 1 Inhibitors for the Treatment of Triple-Negative Breast Cancer
by Ran Luo, Haoyi Zhao, Siqi Deng, Jiale Wu, Haijun Wang, Xiaoshan Guo, Cuicui Han, Wenkang Ren, Yinglong Han, Jianwen Zhou, Yu Lin and Ming Bu
Molecules 2024, 29(18), 4375; https://doi.org/10.3390/molecules29184375 - 14 Sep 2024
Viewed by 623
Abstract
In this study, novel ergosterol peroxide (EP) derivatives were synthesized and evaluated to assess their antiproliferative activity against four human cancer cell lines (A549, HepG2, MCF-7, and MDA-MB-231). Compound 3g exhibited the most potent antiproliferative activity, with an IC50 value of 3.20 [...] Read more.
In this study, novel ergosterol peroxide (EP) derivatives were synthesized and evaluated to assess their antiproliferative activity against four human cancer cell lines (A549, HepG2, MCF-7, and MDA-MB-231). Compound 3g exhibited the most potent antiproliferative activity, with an IC50 value of 3.20 µM against MDA-MB-231. This value was 5.4-fold higher than that of the parental EP. Bioassay optimization further identified 3g as a novel glutaminase 1 (GLS1) inhibitor (IC50 = 3.77 µM). In MDA-MB-231 cells, 3g reduced the cellular glutamate levels by blocking the glutamine hydrolysis pathway, which triggered reactive oxygen species production and induced caspase-dependent apoptosis. Molecular docking indicated that 3g interacts with the reaction site of the variable binding pocket by forming multiple interactions with GLS1. In a mouse model of breast cancer, 3g showed remarkable therapeutic effects at a dose of 50 mg/kg, with no apparent toxicity. Based on these results, 3g could be further evaluated as a novel GLS1 inhibitor for triple-negative breast cancer (TNBC) therapy. Full article
(This article belongs to the Special Issue Bioactivity of Natural Compounds: From Plants to Humans)
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<p>Design of GLS1 inhibitors based on the EP and BPTES binding groups.</p>
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<p>The inhibitory effect of compound <b>3g</b> on GLS1 activity in MDA-MB-231 cells. (<b>A</b>) A GLS1 inhibitor screening kit was utilized to detect the GLS1 levels of EP, <b>3g</b>, and BPTES. (<b>B</b>) After treating MDA-MB-231 cells with different concentrations of <b>3g</b> for 48 h, the expression of the GLS1 protein was detected by Western blot. (<b>C</b>) Quantitative analysis. The data are expressed as the mean ± SD (<span class="html-italic">n</span> = 3), *** <span class="html-italic">p</span> &lt; 0.001, compared with the control group.</p>
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<p>Compound <b>3g</b> inhibited the proliferation of MDA-MB-231 cells. (<b>A</b>) Compound <b>3g</b> inhibited the colony formation of MDA-MB-231 cells. (<b>B</b>) Clonogenic suppression expressed as a percentage relative to the vehicle-treated controls. Data represent the mean ± SD (<span class="html-italic">n</span> = 3), ** <span class="html-italic">p</span> &lt; 0.01, *** <span class="html-italic">p</span> &lt; 0.001, compared with the control group.</p>
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<p>Compound <b>3g</b> induced apoptosis of MDA-MB-231 cells. (<b>A</b>) MDA-MB-231 cells were treated with different concentrations of <b>3g</b> for 48 h; then, the cells were fixed and stained with Annexin V-FITC/PI and analyzed via flow cytometry. Annexin V-FITC and PI data are expressed as percentages (%) for each quadrant. (<b>B</b>) The apoptosis rate was quantitatively detected. (<b>C</b>) Western blot analysis. MDA-MB-MB-231 cells were treated with different concentrations of <b>3g</b> for 48 h, and the protein expressions of Bcl-2, Bax, Cyt C, caspase-9, cleaved caspase-9, caspase-3, and cleaved caspase-3 were detected via Western blot. (<b>D</b>) Quantitative analysis. Data represent the means ± SD (<span class="html-italic">n</span> = 3), ** <span class="html-italic">p</span> &lt; 0.01, *** <span class="html-italic">p</span> &lt; 0.001, compared with the control group.</p>
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<p>The effect of compound <b>3g</b> and EP on the glutamate levels in MDA-MB-231 cells. MDA-MB-231 cells were treated with different concentrations of <b>3g</b> (2, 4, and 8 μM) and EP for 48 h. The changes in the glutamate levels of MDA-MB-231 cells were detected by using a glutamate kit. Data represent the means ± SD (<span class="html-italic">n</span> = 3), ** <span class="html-italic">p</span> &lt; 0.01, *** <span class="html-italic">p</span> &lt; 0.001, compared with the control group.</p>
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<p>Compound <b>3g</b> induced an increase in the ROS levels in MDA-MB-231 cells. MDA-MB-231 cells were subjected to treatment with <b>3g</b> and EP at different concentrations for 48 h. (<b>A</b>) Fluorescence microscopy image of intracellular ROS production in MDA-MB-231 cells stained with DCFH-DA (green). (<b>B</b>) Quantification of ROS levels by flow cytometry. (<b>C</b>) Quantitative analysis. Data represent the means ± SD (<span class="html-italic">n</span> = 3), ** <span class="html-italic">p</span> &lt; 0.01, *** <span class="html-italic">p</span> &lt; 0.001, compared with the control group.</p>
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<p>The eutectic structure of compound <b>3g</b> with GLS1 (PDB ID: 3UO9). (<b>A</b>) Modeled and enlarged close-up of the surface mosaic of the GLS1 tetramer and <b>3g</b> binding. (<b>B</b>) Close-up of the <b>3g</b> interactions in the GLS1 allosteric binding pocket. Here, <b>3g</b> is rendered as a rod and colored according to the atom type. Green denotes carbon, blue denotes nitrogen, and red denotes oxygen. The key residual atoms in GLS1 that interacted with the compound are denoted in cyan. The red dashed lines indicate hydrogen bonds, and the numbers are the hydrogen bond lengths.</p>
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<p>Compound <b>3g</b> inhibited the growth of 4T1 cells in vivo. (<b>A</b>) Tumor images of 4T1 tumor-bearing mice treated with <b>3g</b> or BPTES; and (<b>B</b>) tumor HE staining. Scale = 50 μm. (<b>C</b>) Changes in the tumor volume; (<b>D</b>) tumor weight; and (<b>E</b>) body weight of 4T1 tumor-bearing mice. Data represent the mean ± SD (<span class="html-italic">n</span> = 6), * <span class="html-italic">p</span> &lt; 0.05, ** <span class="html-italic">p</span> &lt; 0.01, *** <span class="html-italic">p</span> &lt; 0.001, compared with the control group. Scale = 50 μm.</p>
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<p>The effect of compound <b>3g</b> on organ damage in model mice. The hearts, livers, spleens, lungs, and kidneys of the mice were harvested and sectioned for HE staining. Scale bars = 50 μm.</p>
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<p>Synthesis of EP derivatives (<b>1a</b>–<b>h</b>, <b>2a</b>–<b>h</b>, <b>3a</b>–<b>h</b>, and <b>4a</b>–<b>h</b>). Reagents and conditions: (i) Et<sub>3</sub>N, CH<sub>2</sub>Cl<sub>2</sub>, SA (A), MA (B), GA (C), or PA (D), reflux, 24 h, 76–85%; and (ii) R<sub>2</sub>-H, HOBT·H<sub>2</sub>O, EDCI·HCl, pyridine, DMF, room temperature, 12–48 h, 77–88%.</p>
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20 pages, 2715 KiB  
Article
Characterization of Extractable and Non-Extractable Phenols and Betalains in Berrycactus (Myrtillocactus geometrizans) and Its Chemoprotective Effect in Early Stage of Colon Cancer In Vivo
by Rosa Iris Godínez-Santillán, Aarón Kuri-García, Iza Fernanda Ramírez-Pérez, María Guadalupe Herrera-Hernández, Santiaga Marisela Ahumada-Solórzano, Salvador Horacio Guzmán-Maldonado and Haydé Azeneth Vergara-Castañeda
Antioxidants 2024, 13(9), 1112; https://doi.org/10.3390/antiox13091112 - 14 Sep 2024
Viewed by 480
Abstract
This research identified the bioactive compounds and antioxidant capacity of the extractable (EP) and non-extractable (NEP) polyphenol fractions of berrycactus (BC). Additionally, the effects of BC and its residue (BCR) on preventing AOM/DSS-induced early colon carcinogenesis were evaluated in vivo. Male Sprague Dawley [...] Read more.
This research identified the bioactive compounds and antioxidant capacity of the extractable (EP) and non-extractable (NEP) polyphenol fractions of berrycactus (BC). Additionally, the effects of BC and its residue (BCR) on preventing AOM/DSS-induced early colon carcinogenesis were evaluated in vivo. Male Sprague Dawley rats were randomly assigned to six groups (n = 12/group): healthy control (C), AOM/DSS, BC, BCR, BC+AOM/DSS, and BCR+AOM/DSS. NEP was obtained through acid hydrolysis using H2SO4 and HCl (1 M or 4 M). The HCl-NEP fraction exhibited the highest total phenolic and flavonoid content, while condensed tannins were more abundant in the H2SO4-NEP fraction. A total of 33 polyphenols were identified by UPLC-QTOF-MSE in both EP and NEP, some of which were novel to BC. Both NEP hydrolysates demonstrated significant total antioxidant capacity (TEAC), with HCl-NEP exhibiting the highest ORAC values. The BC+AOM/DSS and BCR+AOM/DSS groups exhibited fewer aberrant crypt foci (p < 0.05), reduced colonic epithelial injury, and presented lower fecal β-glucuronidase activity, when compared to AOM/DSS group. No differences in butyric acid concentrations were observed between groups. This study presents novel bioactive compounds in EP and NEP from BC that contribute to chemopreventive effects in early colon carcinogenesis, while reducing fecal β-glucuronidase activity and preserving colonic mucosal integrity. Full article
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Figure 1
<p>In vivo experimental design. Evaluation of the chemoprotective effect of BC or BCR in AOM/DSS-induced colorectal cancer Sprague Dawley rats. C−: negative control (saline solution 0.9% <span class="html-italic">v</span>/<span class="html-italic">v</span> injection); BC: berrycactus (5 g/kg BW); BCR: berrycactus residue (5 g/kg BW); AOM: azoxymethane (10 mg/kg BW, via intraperitoneal); DSS: dextran sulfate sodium (2%, via oral, daily for 7 days in drinking water ad libitum). BC and BCR were administered orally mixed with diet pellets. All mice consumed basal diet (Rodent Lab diet) ad libitum.</p>
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<p>Normal and aberrant crypt foci of the distal colon with methylene blue staining. (<b>A</b>) Number of ACF in the distal colon (1 cm<sup>2</sup>). (<b>B</b>) Multiplicity counted by number of crypts per focus in the distal colon. Results are expressed as mean of n = 12 rats ± SD. Different letters per column indicate a significant difference <span class="html-italic">p</span> = 0.05, Tukey test. C-: negative; AOM: azoxymethane; DSS: Dextran sulfate sodium; BC: berrycactus; BCR: berrycactus residue.</p>
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<p>Histopathologic analysis of preneoplastic colonic lesions caused by AOM/DSS and treated with G and R. (<b>A</b>) Incidence of inflammation and premalignant damage in the colon; (<b>B</b>) Hematoxylin–eosin-stained histopathologic analyzes of colon lesions (40×). Orange arrows indicate aberrant crypt foci. AOM: azoxymethane; DSS: dextran sulfate sodium; BC: berrycactus; BCR: berrycactus residue.</p>
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<p>β-glucoronidase activity in experimental groups. Results are expressed as mean of two independent experiments by duplicate ± SD. Different letters per column indicate a significant difference <span class="html-italic">p</span> = 0.05, Tukey test. AOM: azoxymethane; DSS: dextran sulfate sodium; BC: berrycactus; BCR: berrycactus residue.</p>
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<p>Short-chain fatty acids production in feces of experimental groups. Results are expressed as mean of two independent experiments by duplicate ± SD. Different uppercase letters indicate a significant difference between experimental groups, and different lowercase letters indicate a significant difference between times (<span class="html-italic">p</span> = 0.05), Tukey test. C−: negative; AOM: azoxymethane; DSS: dextran sulfate sodium; BC: garambullo; BCR: berrycactus residue.</p>
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14 pages, 9815 KiB  
Article
Resin Acid Copper Salt, an Interesting Chemical Pesticide, Controls Rice Bacterial Leaf Blight by Regulating Bacterial Biofilm, Motility, and Extracellular Enzymes
by Lihong Shi, Xiang Zhou and Puying Qi
Molecules 2024, 29(18), 4297; https://doi.org/10.3390/molecules29184297 - 11 Sep 2024
Viewed by 461
Abstract
Bacterial virulence plays an important role in infection. Antibacterial virulence factors are effective for preventing crop bacterial diseases. Resin acid copper salt as an effective inhibitor exhibited excellent anti-Xanthomonas oryzae pv. oryzae (Xoo) activity with an EC50 of 50.0 [...] Read more.
Bacterial virulence plays an important role in infection. Antibacterial virulence factors are effective for preventing crop bacterial diseases. Resin acid copper salt as an effective inhibitor exhibited excellent anti-Xanthomonas oryzae pv. oryzae (Xoo) activity with an EC50 of 50.0 μg mL−1. Resin acid copper salt (RACS) can reduce extracellular polysaccharides’ (EPS’s) biosynthesis by down-regulating gumB relative expression. RACS can also effectively inhibit the bio-mass of Xoo biofilm. It can reduce the activity of Xoo extracellular amylase at a concentration of 100 μg mL−1. Meanwhile, the results of virtual computing suggested that RACS is an enzyme inhibitor. RACS displayed good curative activity with a control effect of 38.5%. Furthermore, the result of the phytotoxicity assessment revealed that RACS exhibited slight toxicity compared with the control at a concentration of 200 μg mL−1. The curative effect was increased to 45.0% using an additional antimicrobial agent like orange peel essential oil. RACS markedly inhibited bacterial pathogenicity at a concentration of 100 μg mL−1 in vivo. Full article
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Graphical abstract

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<p>The chemical structure of RACS.</p>
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<p>The quantitative assay of crystal violet staining <span class="html-italic">Xoo</span> biofilm. (<b>A</b>) The inhibition rate of RACS regulated biofilm production at doses of the control value (0), 12.5, 25.0, 50.0, and 100 μg mL<sup>−1</sup>. (<b>B</b>) The relative normalized expression of the <span class="html-italic">gumB</span> gene at doses of the control value, 50.0, and 100 μg mL<sup>−1</sup>. Lowercase letters above the histogram indicate significant differences (<span class="html-italic">p</span> &lt; 0.5). (* <span class="html-italic">p</span> &lt; 0.05).</p>
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<p>Analysis of Xoo biofilm formation using acridine orange staining. (<b>A</b>–<b>E</b>) Acridine orange staining <span class="html-italic">Xoo</span> biofilms. (<b>F</b>) Inhibitory rate of <span class="html-italic">Xoo</span> biofilm production. Lowercase letters above dots indicate significant differences (<span class="html-italic">p</span> &lt; 0.5).</p>
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<p>The analysis of <span class="html-italic">Xoo</span> motility. (<b>A</b>–<b>E</b>) RACS suppressed the <span class="html-italic">Xoo</span> motility at doses of the control value (0), 12.5, 25.0, 50.0, and 100 μg mL<sup>−1</sup>. (<b>F</b>) The inhibitory ratio of <span class="html-italic">Xoo</span> motility. The scale plate was 10 mm. Lowercase letters above the dots indicate significant differences (<span class="html-italic">p</span> &lt; 0.5).</p>
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<p>The activity of <span class="html-italic">Xo</span> extracellular amylase at concentrations of the control value (0) and 100 μg mL<sup>−1</sup> (<b>A</b>,<b>B</b>). (<b>C</b>) The results of RACS as an enzyme inhibitor by virtual computing. Scale bar = 1 cm.</p>
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<p>The control effect and phytotoxicity evaluation of RACS against <span class="html-italic">Xoo</span> at a dose of 200 μg mL<sup>−1</sup> in vivo. (<b>A</b>) Control. (<b>B</b>) RACS curative activity. (<b>C</b>) Phytotoxicity evaluation. The white arrow indicates the lesion length.</p>
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<p>The multi-task graph attention (MGA) framework of the ADMET Evaluation.</p>
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<p>The results of the ADMET Evaluation. A green filled circle indicates good results, a red filled circle indicates bad results, and a yellow filled circle indicates general results.</p>
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<p>The curative effect evaluation of RACS containing 0.3% OPEO against <span class="html-italic">Xoo</span> at a dose of 200 μg mL<sup>−1</sup> in vivo. (<b>A</b>) The contact angle of water. (<b>B</b>) The contact angle of RACS + 0.3% OPEO. (<b>C</b>) The curative effect evaluation of the control. (<b>D</b>) The curative effect evaluation of RACS + 0.3% OPEO.</p>
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<p>The influence of RACS on bacterial pathogenicity on rice plant at the control value (<b>A</b>) and 100 μg/mL (<b>B</b>). (<b>C</b>) The length of lesions.</p>
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20 pages, 2279 KiB  
Article
Halotolerant Endophytic Bacteria Priestia flexa 7BS3110 with Hg2+ Tolerance Isolated from Avicennia germinans in a Caribbean Mangrove from Colombia
by Zamira E. Soto-Varela, Christian J. Orozco-Sánchez, Hernando José Bolívar-Anillo, José M. Martínez, Nuria Rodríguez, Natalia Consuegra-Padilla, Alfredo Robledo-Meza and Ricardo Amils
Microorganisms 2024, 12(9), 1857; https://doi.org/10.3390/microorganisms12091857 - 7 Sep 2024
Viewed by 633
Abstract
The mangrove ecosystems of the Department of Atlántico (Colombian Caribbean) are seriously threatened by problems of hypersalinization and contamination, especially by heavy metals from the Magdalena River. The mangrove plants have developed various mechanisms to adapt to these stressful conditions, as well as [...] Read more.
The mangrove ecosystems of the Department of Atlántico (Colombian Caribbean) are seriously threatened by problems of hypersalinization and contamination, especially by heavy metals from the Magdalena River. The mangrove plants have developed various mechanisms to adapt to these stressful conditions, as well as the associated microbial populations that favor their growth. In the present work, the tolerance and detoxification capacity to heavy metals, especially to mercury, of a halotolerant endophytic bacterium isolated from the species Avicennia germinans located in the Balboa Swamp in the Department of Atlántico was characterized. Diverse microorganisms were isolated from superficially sterilized A. germinans leaves. Tolerance to NaCl was evaluated for each of the obtained isolates, and the most resistant was selected to assess its tolerance to Pb2+, Cu2+, Hg2+, Cr3+, Co2+, Ni2+, Zn2+, and Cd2+, many of which have been detected in high concentrations in the area of study. According to the ANI and AAI percentages, the most halotolerant strain was identified as Priestia flexa, named P. flexa 7BS3110, which was able to tolerate up to 12.5% (w/v) NaCl and presented a minimum inhibitory concentrations (MICs) of 0.25 mM for Hg, 10 mM for Pb, and 15 mM for Cr3+. The annotation of the P. flexa 7BS3110 genome revealed the presence of protein sequences associated with exopolysaccharide (EPS) production, thiol biosynthesis, specific proteins for chrome efflux, non-specific proteins for lead efflux, and processes associated with sulfur and iron homeostasis. Scanning electron microscopy (SEM) analysis showed morphological cellular changes and the transmission electron microscopy (TEM) showed an electrodense extracellular layer when exposed to 0.25 mM Hg2+. Due to the high tolerance of P. flexa 7BS3110 to Hg2+ and NaCl, its ability to grow when exposed to both stressors was tested, and it was able to thrive in the presence of 5% (w/v) NaCl and 0.25 mM of Hg2+. In addition, it was able to remove 98% of Hg2+ from the medium when exposed to a concentration of 14 mg/L of this metalloid. P. flexa 7BS3110 has the potential to bioremediate Hg2+ halophilic contaminated ecosystems. Full article
(This article belongs to the Special Issue Halophilic Microorganisms, 2nd Edition)
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<p>Location and evolution of the Atlántico department coastline, Caribbean coast of Colombia and Balboa Swamp. (<b>A</b>) Colombia map with sampled region marked with red; (<b>B</b>) Balboa swamp in the Colombian Caribbean coastal region; (<b>C</b>) Magdalena river delta depicted in the early 1800s; (<b>D</b>) Magdalena river delta today.</p>
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<p>Effect of Hg<sup>2+</sup> on cell morphology and growth of <span class="html-italic">P. flexa</span> 7BS3110 (<b>A</b>) Controls at different concentrations of NaCl without Hg<sup>2+</sup>. (<b>B</b>) Growth in the presence of 0.25 mM Hg<sup>2+</sup> at different concentrations of NaCl. (<b>C</b>) SEM image of cells in culture without Hg<sup>2+</sup>. (<b>D</b>) SEM image of cells with Hg<sup>2+</sup> (0.25 mM) at 2 days. (<b>E</b>) SEM image of cells with Hg<sup>2+</sup> at 7 days.</p>
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<p>Reduction and extracellular sequestration of Hg<sup>2+</sup> by <span class="html-italic">P. flexa 7BS3110</span>. (<b>A</b>) Hg<sup>2+</sup> concentration in solution at different incubation times. (<b>B</b>) SEM image showing the presence of EPS. (<b>C</b>) TEM image of cells grown without Hg<sup>2+</sup>. (<b>D</b>) TEM images of cells grown in the presence of 0.25 mM Hg<sup>2+</sup>. (<b>E</b>) TEM image of cells grown in the absence of Hg<sup>2+</sup>. (<b>F</b>) TEM images of cells grown in the presence of 0.25 mM Hg<sup>2+</sup>.</p>
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17 pages, 6491 KiB  
Article
Identification and Characterization of a Highly Active Hyaluronan Lyase from Enterobacter asburiae
by Linjing Zhang, Jiayu Jiang, Wei Liu, Lianlong Wang, Zhiyuan Yao, Heng Li, Jinsong Gong, Chuanli Kang, Lei Liu, Zhenghong Xu and Jinsong Shi
Mar. Drugs 2024, 22(9), 399; https://doi.org/10.3390/md22090399 - 31 Aug 2024
Viewed by 748
Abstract
Hyaluronic acid (HA) is a well-known functional marine polysaccharide. The utilization and derivative development of HA are of great interest. Hyaluronan lyase has wide application prospects in the production of HA oligosaccharides and lower molecular weight HA. In this study, a strain of [...] Read more.
Hyaluronic acid (HA) is a well-known functional marine polysaccharide. The utilization and derivative development of HA are of great interest. Hyaluronan lyase has wide application prospects in the production of HA oligosaccharides and lower molecular weight HA. In this study, a strain of Enterobacter asburiae CGJ001 with high hyaluronan lyase activity was screened from industrial wastewater. This strain exhibited an impressive enzyme activity of 40,576 U/mL after being incubated for 14 h. Whole genome sequencing analysis revealed that E. asburiae CGJ001 contained a cluster of genes involved in HA degradation, transport, and metabolism. A newly identified enzyme responsible for glycosaminoglycan degradation was designated as HylEP0006. A strain of E. coli BL21(DE3)/pET-22b(+)-hylEP0006 was successfully constructed. HylEP0006 exhibited optimal degradation at 40 °C and pH 7.0, showing a high activity of 950,168.3 U/mg. HylEP0006 showed specific activity against HA. The minimum degradation fragment of HylEP0006 was hyaluronan tetrasaccharides, and HylEP0006 could efficiently degrade HA into unsaturated disaccharides (HA2), with HA2 as the final product. These characteristics indicate that HylEP0006 has a potential application prospect for the extraction and utilization of hyaluronic acid. Full article
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<p>Typical time course of the growth of <span class="html-italic">Enterobacter</span> sp. CGJ001 in a shake flask. Enzyme activity (red, U/mL) and cell growth density (black, OD<sub>600</sub> values) were measured regularly. Values represent the mean of three replicates ± SD.</p>
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<p>The phylogenetic tree of strain <span class="html-italic">Enterobacter</span> sp. CGJ001 was constructed based on the analysis of 16S rDNA sequences, and the phylogenetic tree was generated by MEGA X using the neighbor–joining method.</p>
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<p>Whole-genome analysis of <span class="html-italic">E. asburiae</span> CGJ001: (<b>a</b>) Chromosome genome pattern of <span class="html-italic">E. asburiae</span> CGJ001. The circle diagram shows seven kinds of information, from outside to inside: The first circle is the genomic position information, the second circle is the GC content information, the third circle is the coding genes on the plus strand (red marks), the fourth circle is the coding genes on the minus strand (green marks), the fifth circle is the ncRNA information on the plus strand (blue marks), and the sixth circle is the ncRNA information on the minus strand (purple marks). The seventh circle is marked with information on long genomic repeats (orange marks). (<b>b</b>) KEGG metabolic pathway secondary classification <span class="html-italic">of E. asburiae</span> CGJ001. KEGG classified the biological metabolic pathways into 6 categories, and each category was systematically divided into secondary classifications. The number of genes in each metabolic pathway in the secondary classification was counted. (<b>c</b>) COG functional classification statistics <span class="html-italic">of E. asburiae</span> CGJ001. (<b>d</b>) GO functional classification statistics <span class="html-italic">of E. asburiae</span> CGJ001.</p>
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<p>The analysis of plasmid A: (<b>a</b>) Plasmid A genome pattern of <span class="html-italic">E. asburiae</span> CGJ001. (<b>b</b>) The PUL<sub>HA</sub> was anticipated in plasmid A of strain <span class="html-italic">E. asburiae</span> CGJ001.</p>
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<p>The expression of <span class="html-italic">hylEP0006</span>: (<b>a</b>) Colony PCR validation of the recombinant strain. Lane M: DNA marker; 1–10: PCR amplification bands of positive clones (the DNA fragment size of <span class="html-italic">hylEP0006</span>: 2379 bp). (<b>b</b>) SDS-PAGE of purified recombinant HylEP0006. Lane M, unstained protein molecular weight marker; lane <span class="html-italic">Hyl</span>, purified HylEP0006.</p>
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<p>The effects of temperature and pH on the enzymatic activity of HylEP0006: (<b>a</b>) Effect of temperature. The enzymatic activity of HylEP0006 was measured at 25–50 °C. HylEP0006 had the highest specific activity at 40 °C, equivalent to 100%. (<b>b</b>) Thermal stability of HylEP0006. The cells were incubated at different temperatures (30–50 °C) for 2 h, and the residual activity of HylEP0006 was determined at 40 °C. The initial specific activities of HylEP0006 were all set to be 100%. (<b>c</b>) Effect of pH. The enzymatic activity of HylEP0006 was assessed in a 50 mM buffer solution, comprising Na<sub>2</sub>HPO<sub>4</sub>-Citrate buffer (pH 3.0–5.0), NaH<sub>2</sub>PO<sub>4</sub>-Na<sub>2</sub>HPO<sub>4</sub> buffer (pH 6.0–8.0), and Glycine-NaOH buffer (pH 9.0–10.0). In NaH<sub>2</sub>PO<sub>4</sub>-Na<sub>2</sub>HPO<sub>4</sub> buffer (pH 7.0), HylEP0006 exhibited its maximum specific activity, which was recorded as being equivalent to 100%. (<b>d</b>) pH stability of HylEP0006. The residual activity of HylEP0006 was assessed at a temperature of 40 °C by subjecting it to incubation in the aforementioned buffer (with pH ranging from 3.0 to 9.0) for 2 h, maintaining the incubation temperature at 30 °C. The initial specific activities of HylEP0006 were all set to be 100%. Values represent the mean of three replicates ± SD.</p>
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<p>Enzymatic kinetic study of HylEP0006: (<b>a</b>) HylEP0006 degradation process of different concentrations of substrate figure. Values represent the mean of three replicates ± SD. (<b>b</b>) Lineweaver–Burk double reciprocal plot of HylEP0006.</p>
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<p>The analysis of HA digested by HylEP0006: (<b>a</b>) The final product of HA digested with HylEP0006 at 40 °C was analyzed by HPLC using a YMC-Pack Polyamine II column. (<b>b</b>) Analysis of the end product resulting from the digestion of HA with HylEP0006 using electrospray ionization mass spectrometry (ESI-MS).</p>
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<p>The analysis of HA4 degraded products by HylEP0006: (<b>a</b>) The product of HA4 degraded with HylEP0006 at 40 °C was analyzed by HPLC. (<b>b</b>) Scheme diagram of the cleavage of oligosaccharide HA4 substrates by HylEP0006.</p>
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<p>The analysis of HA10 degraded products by HylEP0006: (<b>a</b>) The product of HA10 degraded with HylEP0006 at 40 °C was analyzed by HPLC using a YMC-Pack Polyamine II column. (<b>b</b>) The oligosaccharide content of each product component during the initial 6 h degradation period of HA10 at 40 °C was analyzed by HPLC. Values represent the mean of three replicates ± SD. (<b>c</b>) Scheme diagram of the cleavage of oligosaccharide HA10 substrates by HylEP0006.</p>
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12 pages, 2895 KiB  
Article
Inhibitory Effects of Lactobionic Acid on Biofilm Formation and Virulence of Staphylococcus aureus
by Shimo Kang, Yahui Yang, Wanwan Hou and Yan Zheng
Foods 2024, 13(17), 2781; https://doi.org/10.3390/foods13172781 - 31 Aug 2024
Viewed by 598
Abstract
Staphylococcus aureus biofilm is a common bio-contaminant source that leads to food cross-contamination and foodborne disease outbreaks. Hence, there is a need for searching novel antibiofilm agents with potential anti-virulence properties to control S. aureus contamination and infections in food systems. In this [...] Read more.
Staphylococcus aureus biofilm is a common bio-contaminant source that leads to food cross-contamination and foodborne disease outbreaks. Hence, there is a need for searching novel antibiofilm agents with potential anti-virulence properties to control S. aureus contamination and infections in food systems. In this study, the antibiofilm effects of lactobionic acid (LBA) against S. aureus and its influence on virulence were explored. The minimum inhibition concentration of LBA on S. aureus was 8 mg/mL. Viable count and crystal violet assays revealed that LBA inhibited and inactivated S. aureus biofilms. Microscopic observations further confirmed the antibiofilm activity of LBA on S. aureus that disrupted the biofilm architecture and inactivated the viable cells in biofilms. Moreover, LBA decreased the release of extracellular DNA (eDNA) and extracellular polysaccharide (EPS) in S. aureus biofilms. LBA suppressed biofilm formation by intervening metabolic activity and reduced virulence secretion by repressing the hemolytic activity of S. aureus. Furthermore, LBA altered the expressions of biofilm- and virulence-related genes in S. aureus, further confirming that LBA suppressed biofilm formation and reduced the virulence secretion of S. aureus. The results suggest that LBA might be useful in preventing and controlling biofilm formation and the virulence of S. aureus to ensure food safety. Full article
(This article belongs to the Special Issue Foodborne Pathogenic Bacteria: Prevalence and Control—Volume II)
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<p>Effect of LBA on <span class="html-italic">S. aureus</span> biofilm formation (<b>A</b>) and the viable cells in biofilms (<b>B</b>). ** <span class="html-italic">p</span> &lt; 0.01, compared with control.</p>
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<p>Microscopic visualization of <span class="html-italic">S. aureus</span> biofilms treated with diverse LBA concentrations. (<b>A1</b>–<b>A4</b>) SEM images at 20,000× magnification. (<b>B1</b>–<b>B4</b>) CLMS images at 20× magnification.</p>
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<p>Effect of LBA on the release of EPS (<b>A</b>) and eDNA (<b>B</b>) of <span class="html-italic">S. aureus</span> biofilm cells. * <span class="html-italic">p</span> &lt; 0.05, ** <span class="html-italic">p</span> &lt; 0.01, compared with control.</p>
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<p>Effect of LBA on the biofilm metabolic activity (<b>A</b>) and hemolysis activity (<b>B</b>) of <span class="html-italic">S. aureus</span>. ** <span class="html-italic">p</span> &lt; 0.01, compared with control.</p>
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<p>Expression of biofilm formation and virulence-related genes in <span class="html-italic">S. aureus</span> after treatment with 1 × MIC LBA by RT-qPCR. The expression level of 16s rRNA was used to normalize the expressions of the target genes. * <span class="html-italic">p</span> &lt; 0.05, ** <span class="html-italic">p</span> &lt; 0.01, compared with control.</p>
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15 pages, 2249 KiB  
Article
Exploring the Volatile Profile of Vanilla planifolia after Fermentation at Low Temperature with Bacillus Isolates
by Thabani-Sydney Manyatsi, Yu-Hsin Lin, Pin-Hui Sung and Ying-Tzy Jou
Foods 2024, 13(17), 2777; https://doi.org/10.3390/foods13172777 - 30 Aug 2024
Viewed by 656
Abstract
Vanilla planifolia is grown as a high-value orchid spice for its odor and savor attributes that increase due to the curing process associated with microbial colonization. This tends to influence the aromatic properties of vanilla. Hence, 11 Bacillus sp. strains were isolated from [...] Read more.
Vanilla planifolia is grown as a high-value orchid spice for its odor and savor attributes that increase due to the curing process associated with microbial colonization. This tends to influence the aromatic properties of vanilla. Hence, 11 Bacillus sp. strains were isolated from V. planifolia and identified with 16S rRNA gene sequencing. The liquid culture (1 mL of 107 CFU mL−1) of selected Bacillus vallismortis NR_104873.1:11-1518, Bacillus velezensis ZN-S10, and Bacillus tropicus KhEp-2 effectively fermented green-blanched vanilla pods kept at 10 °C during the sweating stage. GC-MS analysis showed that the methanol extract of non-coated, and B. vallismortis treated vanilla detected three (3) volatile compounds, whereas seven (7) components were obtained in B. tropicus and B. velezensis treatment. 4H-pyran-4-one, 2,3-dihydro-3,5-dihydroxy-6-methyl was found in B. velezensis ZN-S10, B. tropicus KhEp-2, and B. vallismortis while it was not present in the control samples. This ketone compound suggested a Maillard reaction resulting in brown-increased aroma pods. Linoleic acid and Hexadecanoic acid ethyl esters were detected only in ZN-S10 strain-coated vanilla. A novel 3-Deoxy-d-mannoic lactone was detected only in B. vallismortis-treated vanilla characterized as a new compound in V. planifolia which suggested that the new compound can be altered with the coating of bacteria in vanilla during fermentation. Thus, the Bacillus strains improved the volatile profile and exhibited a new aroma and flavor profile of vanilla owing to bacteria fermentation during the curing process. Full article
(This article belongs to the Section Food Biotechnology)
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<p>Vanilla plants in a greenhouse (<b>left</b>) in Pingtung, Taiwan, being harvested as green matured vanilla pods (<b>right</b>) used as samples for this study.</p>
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<p>Morphological characteristics of isolated <span class="html-italic">Bacillus</span> strains from <span class="html-italic">Vanilla planifolia</span> that were cultured and grown on modified MRS agar medium plates and hemolysis agar plates of the <span class="html-italic">Bacillus</span> sp. strains isolated from <span class="html-italic">Vanilla planifolia</span> cultured at 37 °C on blood agar media for 48 h. The strains presented include <span class="html-italic">Bacillus tropicus</span> KhEp-2 (<b>A</b>,<b>B</b>), <span class="html-italic">Bacillus velezensis</span> ZN-S10 (<b>C</b>,<b>D</b>), and <span class="html-italic">Bacillus vallismortis</span> NR_104873.1:11-1518 (<b>E</b>,<b>F</b>) colonies on MRS and blood agar medium, respectively.</p>
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<p>The neighbor-joining phylogenetic tree of the relationship among 11 <span class="html-italic">Bacillus</span> sp. strains isolated from vanilla beans (<span class="html-italic">V. planifolia</span>). The tree analyzed 11 nucleotide sequences evolutionarily using MEGA11 software (version 11.0.13). A pairwise deletion option on 1514 total positions was employed in the final dataset. Bar distance scale = 0.10.</p>
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<p>Gas chromatography of (<b>A</b>) non-bacteria-treated vanilla (control); (<b>B</b>) <span class="html-italic">Bacillus tropicus</span> KhEp-2-treated vanilla (<b>C</b>); <span class="html-italic">Bacillus velezensis</span> ZN-S10 treatment and (<b>D</b>) <span class="html-italic">Bacillus vallismortis</span> NR_104873.1:11-1518-coated vanilla beans, analyzed as methanol extract.</p>
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