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17 pages, 2791 KiB  
Article
Enhancement of Doxorubicin Efficacy by Bacopaside II in Triple-Negative Breast Cancer Cells
by Sima Kianpour Rad, Kenny K. L. Yeo, Runhao Li, Fangmeinuo Wu, Saifei Liu, Saeed Nourmohammadi, William M. Murphy, Yoko Tomita, Timothy J. Price, Wendy V. Ingman, Amanda R. Townsend and Eric Smith
Biomolecules 2025, 15(1), 55; https://doi.org/10.3390/biom15010055 (registering DOI) - 3 Jan 2025
Viewed by 175
Abstract
Background: Triple-negative breast cancer (TNBC) is an aggressive subtype with limited treatment options and high resistance to chemotherapy. Doxorubicin is commonly used, but its efficacy is limited by variable sensitivity and resistance. Bacopaside II, a saponin compound, has shown anti-cancer potential. This study [...] Read more.
Background: Triple-negative breast cancer (TNBC) is an aggressive subtype with limited treatment options and high resistance to chemotherapy. Doxorubicin is commonly used, but its efficacy is limited by variable sensitivity and resistance. Bacopaside II, a saponin compound, has shown anti-cancer potential. This study evaluates the effects of doxorubicin and bacopaside II, both individually and in combination, across TNBC subtypes to explore mechanisms of resistance and enhanced drug efficacy. Methods: The growth-inhibitory effects of doxorubicin and bacopaside II were assessed in four TNBC cell lines. IC50 values were determined using dose–response assays, and doxorubicin accumulation was measured via spectral flow cytometry. ATP-binding cassette (ABC) transporter expression (ABCB1, ABCC1, ABCC3, and ABCG2) was analyzed for correlations with drug sensitivity. In silico docking assessed the binding affinity of bacopaside II to ABC transporters. A 3D culture model simulated drug-resistant TNBC, and combination effects were evaluated with live-cell imaging. Results: Doxorubicin sensitivity varied across TNBC molecular subtypes, correlating to intracellular accumulation. Bacopaside II inhibited growth across subtypes, inducing apoptosis in sensitive cells and necrosis in resistant cells. Bacopaside II increased doxorubicin accumulation, independent of P-glycoprotein (ABCB1), possibly through interactions with other ABC transporters. In drug-resistant 3D cultures, bacopaside II maintained efficacy and enhanced doxorubicin accumulation, counteracting ABC transporter-mediated resistance. The doxorubicin and bacopaside II combination showed synergistic growth inhibition. Conclusions: Bacopaside II enhances doxorubicin efficacy in TNBC by increasing drug accumulation and overcoming ABC transporter-mediated resistance, suggesting its potential as an adjuvant in TNBC treatment. These findings support further investigation of bacopaside II, particularly for resistant TNBC subtypes. Full article
(This article belongs to the Section Natural and Bio-derived Molecules)
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Figure 1
<p>Doxorubicin sensitivity, accumulation, and ABC transporter gene expression in TNBC cell lines. (<b>a</b>) TNBC cell lines (DU4475, MDA-MB-231, MDA-MB-453, and HCC1143) were treated with doxorubicin for five days, followed by crystal violet staining to assess growth inhibition. Data are presented as individual values with median, interquartile range (25th–75th percentiles), and whisker plots from at least six replicates, normalized to the vehicle control. (<b>b</b>) Doxorubicin accumulation was evaluated in TNBC cell lines after 24 h of treatment with increasing doxorubicin concentrations (0, 25, 50, and 100 nM). Intracellular doxorubicin levels were quantified via flow cytometry, with geometric mean fluorescence intensity (GMFI) representing doxorubicin accumulation. Data are shown as individual values with standard deviations (SD) from three independent experiments. (<b>c</b>) Correlation between doxorubicin IC50 values and intracellular GMFI across TNBC cell lines: DU4475 (blue), MDA-MB-231 (red), MDA-MB-453 (green), and HCC1143 (purple). Data points represent GMFI values from three separate experiments using 25 nM, 50 nM, and 100 nM doxorubicin (DOX). (<b>d</b>) Transcript expression levels of ABC transporters (<span class="html-italic">ABCB1</span>, <span class="html-italic">ABCC1</span>, <span class="html-italic">ABCC3</span>, and <span class="html-italic">ABCG2</span>) were measured in untreated TNBC cell lines using TaqMan Assays. Expression levels were normalized to the <span class="html-italic">HPRT1</span> reference gene using the 2<sup>−ΔCt</sup> method. Data represent the mean ± SD of three technical replicates across three independent cultures and were analyzed using ordinary one-way ANOVA with Holm–Sidak’s multiple comparisons test. Statistical significance is indicated as **** <span class="html-italic">p</span> &lt; 0.0001, ** <span class="html-italic">p</span> &lt; 0.01, * <span class="html-italic">p</span> &lt; 0.05, not significant (ns; <span class="html-italic">p</span> &gt; 0.05).</p>
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<p>Bacopaside II sensitivity and induction of cell death in TNBC cell lines. (<b>a</b>) TNBC cell lines (DU4475, MDA-MB-231, MDA-MB-453, and HCC1143) were treated with bacopaside II for five days, followed by crystal violet staining to assess growth inhibition. Data are presented as individual values with median, interquartile range (25th–75th percentiles), and whisker plots from at least six replicates, normalized to the vehicle control (2% methanol). (<b>b</b>) Kinetic live-cell imaging of HCC1143 cells showing the number of propidium iodide (PI)-positive cells (loss of membrane integrity; left panel) and caspase-3/7-positive cells (apoptosis; right panel) measured every 2 h over 48 h. Staurosporine (2.5 µM) was used as a positive control for apoptosis. Data represent the mean ± 95% confidence interval (CI) of up to six replicates. (<b>c</b>) Kinetic live-cell imaging of MDA-MB-231 cells showing the number of PI-positive cells (loss of membrane integrity; left panel) and caspase-3/7-positive cells (apoptosis; right panel) measured every 2 h over 48 h. Drozitumab (100 ng/mL) served as a positive control for apoptosis. Data represent the mean ± 95% CI of up to six replicates. (<b>d</b>) Western blot analysis of MLKL and phosphorylated MLKL (pMLKL) following 4 h of treatment with 0, 15, 20, and 30 µM bacopaside II. Band intensities were normalized to total protein levels.</p>
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<p>Effect of bacopaside II on doxorubicin accumulation. TNBC cell lines were co-treated with 25 nM doxorubicin and 0 µM, 1 µM, or 10 µM bacopaside II for 24 h. Intracellular doxorubicin levels in viable cells were quantified using flow cytometry. Data represent the mean ± SD of doxorubicin geometric mean fluorescence intensity (GMFI) from three individual experiments. Statistical significance was calculated using ordinary one-way ANOVA followed by Holm–Sidak’s multiple comparisons test. * <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, **** <span class="html-italic">p</span> &lt; 0.0001, ns—<span class="html-italic">p</span> &gt; 0.05.</p>
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<p>Bacopaside II overcomes multidrug resistance induced by 3D culture of MDA-MB-231 cells. (<b>a</b>) Transcript levels of stem cell markers <span class="html-italic">NANOG</span> and <span class="html-italic">OCT4</span> in MDA-MB-231 cells cultured as an adherent monolayer (2D), in scaffold-free 3D suspension for 4 weeks (3D), or after transitioning from 3D suspension back to adherent monolayer culture for 5 days (3D to 2D). Gene expression was normalized to the reference gene <span class="html-italic">HPRT1</span> and calculated relative to the 2D condition using the 2<sup>−ΔΔCt</sup> method. Data are presented as mean ± SD from three technical replicates across three independent cultures and analyzed using the Kruskal–Wallis test with Dunn’s multiple comparisons test. (<b>b</b>) Expression of ABC transporters <span class="html-italic">ABCB1</span>, <span class="html-italic">ABCC1</span>, <span class="html-italic">ABCC3</span>, and <span class="html-italic">ABCG2</span> in MDA-MB-231 cells cultured as a 2D monolayer or as a 3D suspension culture for 4 weeks. Data represent mean ± SD from three technical replicates across three independent cultures and were analyzed using the Mann–Whitney test. (<b>c</b>) Dose–response curves of 2D-cultured and 4-week 3D-cultured cells following 5 days of treatment with doxorubicin, paclitaxel, 5-fluorouracil, or bacopaside II. Data are presented as mean ± 95% CI from six replicates. (<b>d</b>) Intracellular doxorubicin accumulation, measured as geometric mean fluorescence intensity (GMFI) by flow cytometry, in 2D- and 3D-cultured cells after 24 h of treatment with 0, 25, 50, or 100 nM doxorubicin. Data represent mean ± SD from three individual experiments and were analyzed using a mixed-effects model with Geisser–Greenhouse correction and Sidak’s multiple comparisons test. (<b>e</b>) Doxorubicin GMFI in 3D-cultured cells following 24-h co-treatment with 0 µM (2% methanol vehicle control), 15 µM, or 30 µM bacopaside II and 0, 25, or 50 nM doxorubicin. Data represent mean ± SD from three individual experiments and were analyzed using a mixed-effects model with Geisser–Greenhouse correction and Sidak’s multiple comparisons test. Statistical significance is indicated as **** <span class="html-italic">p</span> &lt; 0.0001, *** <span class="html-italic">p</span> &lt; 0.001, ** <span class="html-italic">p</span> &lt; 0.01, ns—<span class="html-italic">p</span> &gt; 0.05.</p>
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<p>Synergistic inhibition of MDA-MB-231 cell proliferation by combined treatment with doxorubicin and bacopaside II. MDA-MB-231 cells were treated with the vehicle control (VC, 2% methanol), 10 µM bacopaside II (BII), 100 nM doxorubicin (DOX), or a combination of 10 µM bacopaside II and 100 nM doxorubicin (BII + DOX). Cell growth was monitored every 2 h over 72 h using kinetic live-cell imaging, with confluence measurements normalized to the 2-h time point. Data represent the mean ± SD of up to 12 replicates.</p>
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16 pages, 2652 KiB  
Article
Assessment of a Natural Phytobiotic Mixture as Feed Additive for Broiler Chicken: Studies on Animal Performance, Gut Health, and Antioxidant Status After Experimental Infection with Eimeria spp.
by Dimitrios Galamatis, Ioannis Panitsidis, Tilemachos Mantzios, Georgios Sioutas, Ioanna Stylianaki, Elias Papadopoulos, Jog Raj, Marko Vasiljević, Jasna Bošnjak-Neumüller, Damer Blake, Vasilios Tsiouris and Ilias Giannenas
Poultry 2025, 4(1), 4; https://doi.org/10.3390/poultry4010004 - 2 Jan 2025
Viewed by 218
Abstract
This study evaluated the effectiveness of phytobiotic supplementation in managing coccidiosis in broiler chickens, a disease that impacts productivity. Three hundred sixty Ross-308 one-day-old chicks were assigned to five treatment groups: control negative (CN), phyto negative (PN), control infected (CI), phyto infected (PI), [...] Read more.
This study evaluated the effectiveness of phytobiotic supplementation in managing coccidiosis in broiler chickens, a disease that impacts productivity. Three hundred sixty Ross-308 one-day-old chicks were assigned to five treatment groups: control negative (CN), phyto negative (PN), control infected (CI), phyto infected (PI), and salinomycin infected (SI). All diets were fed for the entire experiment duration. CN and CI groups were given a common diet, with CN remaining uninfected and CI exposed to Eimeria spp., while PN and PI groups received a phytobiotic supplement containing essential oils, saponins, and tannins (with PI challenged by Eimeria spp.), and SI received salinomycin post-infection. All infected groups were challenged on day 14 with Eimeria acervulina, E. maxima, and E. tenella. By day 21, PN had the highest body weight (744.9 g). Body weight gain (BWG) was highest in CN and PN from days 14–35, while CI consistently showed the lowest BWG. PI demonstrated significantly lower oocyst counts than CI, outperforming even SI by day 7, and showed milder intestinal lesions. A high anticoccidial index (ACI) of 188.45 was observed in PI, close to uninfected groups, while SI achieved a partially effective ACI of 136.91. Overall, PN and PI exhibited improved performance and intestinal health, highlighting the potential of phytobiotics in coccidiosis management for broilers. Full article
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<p>Left column: HE. Small intestine. (<b>a</b>) Villus shortening and surface and glandular epithelial hyperplasia, (<b>b</b>) villus blunting and fusion, surface and glandular epithelial hyperplasia, lymphoplasmacytic infiltrate in the lamina propria, (<b>c</b>) multiple apicomplexan coccidian forms in various developmental stages in the mucosa. Central column. (<b>d</b>–<b>f</b>) The immunohistochemical expression of Claudin-3 (on apical and basal regions and the pericellular borders of the epithelial cells) showed continuousity or differences in the spatial distribution, resulting in epithelial gaps. Right column. (<b>g</b>–<b>i</b>) CD3+ positive cells. Magnification 10×.</p>
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19 pages, 1622 KiB  
Article
Saponins Effect on Human Insulin Amyloid Aggregation
by Eleonora Mari, Silvia Vilasi, Paolo Moretti, Maria Rosalia Mangione, Giorgia Giorgini, Roberta Galeazzi and Maria Grazia Ortore
Biomolecules 2025, 15(1), 40; https://doi.org/10.3390/biom15010040 - 31 Dec 2024
Viewed by 333
Abstract
The misfolding and amyloid aggregation of proteins have been attracting scientific interest for a few decades, due to their link with several diseases, particularly neurodegenerative diseases. Proteins can assemble and result in insoluble aggregates that, together with intermediate oligomeric species, modify the extracellular [...] Read more.
The misfolding and amyloid aggregation of proteins have been attracting scientific interest for a few decades, due to their link with several diseases, particularly neurodegenerative diseases. Proteins can assemble and result in insoluble aggregates that, together with intermediate oligomeric species, modify the extracellular environment. Many efforts have been and are devoted to the search for cosolvents and cosolutes able to interfere with amyloid aggregation. In this work, we intensively study the effect of saponins, bioactive compounds, on human insulin aggregation. To monitor the kinetic of amyloid aggregation following secondary structure changes, we perform fluorescence and UV-Visible absorption spectroscopies, using Thioflavin T and Congo Red as amyloid specific probes, and Circular Dichroism. To study the overall structural features and size of aggregates, we perform Synchrotron Small-Angle X-ray Scattering and Dynamic Light Scattering experiments. The morphology of the aggregates was assessed by Atomic Force Microscopy. To deepen the understanding of the saponins interaction with insulin, a Molecular Dynamics investigation is performed, too. The reported data demonstrate that saponins interfere with the amyloid aggregation by inducing a strong inhibition on the formation of insulin fibrils, likely through specific interactions with insulin monomers. A dose-dependent effect is evident, and amyloid inhibition is already clear when saponins are just 0.01% w/w in solution. We suggest that saponins, which are natural metabolites present in a wide range of foods ranging from grains, pulses, and green leaves to sea stars and cucumbers, can be promising metabolites to inhibit human insulin aggregation. This basic research work can pave the way to further investigations concerning insulin amyloidosis, suggesting the use of saponins as amyloid inhibitors and/or stabilizing agents in solution. Full article
(This article belongs to the Collection Feature Papers in Molecular Structure and Dynamics)
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Graphical abstract

Graphical abstract
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<p>UV/Vis spectroscopy results of the effects of saponins on human insulin aggregation. To monitor the relative amount of <math display="inline"><semantics> <mi>β</mi> </semantics></math>-sheet structures in solution, the ratio between the intensity of the absorption peak due to Congo Red (CR) bound to fibrils and the one due to CR free in solution was calculated as a function of time. <math display="inline"><semantics> <mi>β</mi> </semantics></math>-sheet structures as a function of time during fibrillation: blue markers correspond to human insulin in solution in the absence of saponins, and purple markers correspond to human insulin in solution in the presence of 1 mg/mL of saponins. Error bars are estimated on the average of several replicas of the same experiment.</p>
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<p>In situ real-time ThT fluorescence assay for monitoring the aggregation kinetics of insulin incubated at 37 <math display="inline"><semantics> <mo>°</mo> </semantics></math>C under agitation in the absence and in the presence of increasing concentrations of saponins as indicated in the legend. Continuous lines are the theoretical fitting curves obtained by the analysis that provides the halftime featuring each kinetic pattern.</p>
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<p>Circular Dichroism (CD) curves of human insulin recorded after 48 h of aggregation kinetics at 37 <math display="inline"><semantics> <mo>°</mo> </semantics></math>C. The curves are related to the human insulin solution in the presence of increasing amounts of saponins as indicated in the legend.</p>
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<p>Percentages of <math display="inline"><semantics> <mi>α</mi> </semantics></math> and <math display="inline"><semantics> <mi>β</mi> </semantics></math> structure, as in the legend, provided by CD data analysis. Blue and cyan shades refer to <math display="inline"><semantics> <mi>α</mi> </semantics></math>-helices, which dramatically decrease in the absence of saponins. On the contrary, in the same conditions, <math display="inline"><semantics> <mi>β</mi> </semantics></math> structures arise, in shades from red to yellow.</p>
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<p>DLS results: particle size distribution of human insulin at 0.5 mg/mL, after 5 h of kinetics of aggregation in the presence (pink line) and in the absence (blue line) of saponins.</p>
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<p>SAXS results. Upper panels: SAXS spectra relative to human insulin (c = 0.5 mg/mL, pH 7.4) aggregation patterns at the beginning (0 h, in the legend, blue points), and after 6 (purple points) and 12 h (cyan points). On the left side, data concern insulin dissolved without saponins, and on the right side are data collected in the presence of 1 mg/mL saponins as shown in the legend. Bottom figure: structural parameters obtained by Guinier (Rg) and Guinier rod-like (Rc) analysis as a function of aggregation time. The empty cyan triangles represent the parameters achieved (Rg) for the curves obtained for the kinetics in the presence of saponins, while the orange (Rg) and the blue (Rc) triangles represent the parameters achieved for the kinetics in the absence of saponins.</p>
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<p>Atomic Force Microscopy images. Top and bottom images are two replicates of the same sample. Grey panel: insulin without saponin. Light blue panel: insulin with saponins (1 mg/mL). Powder pink panel: saponin without insulin (1 mg/mL).</p>
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<p>Chichipenoside B (in tube representation) and insulin (ribbons) molecular organization at the final steady state of the 200 ns MD simulation.</p>
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<p>Intermolecular distances between the four Chichipenoside B molecules (<b>a</b>) and insulin (<b>b</b>) along the 200 ns MD simulation.</p>
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<p>Visualization obtained from the 200 ns MD simulation of Chichipenoside B and human insulin, evidencing the residues playing a fundamental role in dimerization.</p>
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16 pages, 5763 KiB  
Article
Anti-Inflammatory Potential and Synergic Activities of Eclipta prostrata (L.) L. Leaf-Derived Ointment Formulation in Combination with the Non-Steroidal Anti-Inflammatory Drug Diclofenac in Suppressing Atopic Dermatitis (AD)
by Muhammad M. Poyil, Mohammed H. Karrar Alsharif, Mahmoud H. El-Bidawy, Salman Bin Dayel, Mohammed Sarosh Khan, Zainab Mohammed M. Omar, Alaaeldin Ahmed Mohamed, Reda M. Fayyad, Tarig Gasim Mohamed Alarabi, Hesham A. Khairy, Nasraddin Othman Bahakim, Mohamed A. Samhan and Abd El-Lateef Saeed Abd El-Lateef
Life 2025, 15(1), 35; https://doi.org/10.3390/life15010035 - 30 Dec 2024
Viewed by 293
Abstract
Atopic dermatitis (AD) or eczema is an important inflammatory chronic skin disease that brings many complications in its management and treatment. Although several chemical agents are used for treatment, the search for better anti-inflammatory and antibacterial agents of plant origin has been ongoing, [...] Read more.
Atopic dermatitis (AD) or eczema is an important inflammatory chronic skin disease that brings many complications in its management and treatment. Although several chemical agents are used for treatment, the search for better anti-inflammatory and antibacterial agents of plant origin has been ongoing, since natural compounds, it is commonly believed, are less dangerous than synthetic ones. Therefore, the present study explored a medicinal plant—Eclipta prostrata (L.) L.—for its anti-inflammatory activity alone and in combination with a non-steroidal anti-inflammatory drug (NSAID), diclofenac. The plant extract was used to make a cream formulation for treating atopic dermatitis and as an antibacterial agent against Staphylococcus aures, the major infectious agent associated with AD. The phytochemical analysis of the E. prostrata extract showed the presence of various phytochemicals, including flavonoids, Tannin, saponin, terpenoids, glycosides, phenol, alkaloids, quinone, and protein. The GC-MS profiling of methanolic E. prostrata extract was performed predicted the presence of twenty important phytochemicals, including 2-[5-(2-Hydroxypropyl) oxolan-2-yl]propanoic acid, dl-Menthol, dodecane, undecane, 4,7-dimethyl-, dodecane, 2,6,10-trimethyl-, decane, 2,3,5,8-tetramethyl-, cholest-5-en-3-ol, (3.alpha.)-, TMS derivative, cyclopropane carboxylic acid, 1-hydroxy-, (2,6-di-t-butyl-4-methylphenyl) ester, alpha.-farnesene, propanoic acid, 2-methyl-, 2-ethyl-1-propyl-1,3-propanediyl ester, diethyl phthalate, corticosterone, 2-methylpropionate, hentriacontan-13-ol, O-TMS, phthalic acid, 2,4-dimethylpent-3-yl dodecyl ester, hexasiloxane, 1,1,3,3,5,5,7,7,9,9,11,11-dodecamethyl-, acetic acid, 4-t-butyl-4-hydroxy-1,5-dimethyl-hex-2-ynyl ester, octadecane, 2-methyl- octacosane, 1-iodo-, nonacosane, and eicosyl isopropyl ether. Using an egg albumin denaturation inhibition assay, the anti-inflammatory activities of E. prostrata alone and in combination with diclofenac were investigated, and they showed 93% and 99% denaturation inhibition at 5 mg concentration of E. prostrata in alone and combination with diclofenac, respectively. Heat-induced haemolysis showed 2.5% and 2.4% of haemolysis at 5 mg of E. prostrata alone and in combination with diclofenac, respectively. An MTT assay performed using L929 cells proved that the extract has no cytotoxic effect. The plant extract displayed potential antibacterial activity against Staphylococcus aureus; the growth was inhibited at 1 mg/mL of E. prostrata extract. Thus, based on this evidence, the authors suggest that E. prostrata extract should be studied further for its anti-inflammatory and antibacterial activities and topical application in the treatment of atopic dermatitis. Full article
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<p>GC-MS chemical profiling of <span class="html-italic">E. prostrata</span> in methanolic fraction. TIC = tentatively identified compounds); min = minutes.</p>
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<p>Inhibition percentage of <span class="html-italic">E. prostrata</span> methanolic extract alone and in combination with diclofenac on egg albumin denaturation. EP = <span class="html-italic">E. prostrata,</span> EP + D = <span class="html-italic">E. prostrata</span> + diclofenac.</p>
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<p>Heat-inducing hemolysishaemolysis percentage of <span class="html-italic">E. prostrata</span> methanolic extract alone and in combination with diclofenac. EP = <span class="html-italic">E. prostrata,</span> EP + D = <span class="html-italic">E. prostrata</span> + diclofenac.</p>
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<p><span class="html-italic">E. prostrata</span> 15% methanolic extract cream effect on human skin prepared.</p>
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<p>Cell viability percentage of <span class="html-italic">E. prostrata</span> methanolic extract after treating L<sub>929</sub> cells using MTT assay. (<b>A</b>) Untreated L<sub>929</sub> cell morphology. (<b>B</b>) L<sub>929</sub> cell morphology after treatment with <span class="html-italic">E. prostrata</span>. (<b>C</b>) The graph represents the percentage of cell viability after treatment.</p>
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<p><span class="html-italic">E. prostrata</span> methanolic extract antibacterial activity against <span class="html-italic">S. aureus.</span> Two different concentrations exhibited inhibition zones around the well. Note: P—positive control (tetracycline) and v—vehicle control.</p>
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<p><span class="html-italic">E. prostrata</span> methanolic extract MIC was determined against <span class="html-italic">S. aureus</span>, and the growth inhibition was noted at 1 mg/mL.</p>
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25 pages, 8550 KiB  
Article
Potential Mechanisms and Effects of Dai Bai Jie Ethanol Extract in Preventing Acute Alcoholic Liver Injury
by Niantong Xia, Hongwei Xue, Yihang Li, Jia Liu, Yang Lou, Shuyang Li, Yutian Wang, Juan Lu and Xi Chen
Curr. Issues Mol. Biol. 2025, 47(1), 3; https://doi.org/10.3390/cimb47010003 - 24 Dec 2024
Viewed by 486
Abstract
This study investigated the protective effect of Dai Bai Jie (DBJ) extract against acute alcoholic liver injury (AALI) and elucidated its potential mechanism. The total saponin level in the DBJ extracts was measured using vanillin–chloroform acid colorimetry. To observe the preventive and protective [...] Read more.
This study investigated the protective effect of Dai Bai Jie (DBJ) extract against acute alcoholic liver injury (AALI) and elucidated its potential mechanism. The total saponin level in the DBJ extracts was measured using vanillin–chloroform acid colorimetry. To observe the preventive and protective effects of DBJ on AML-12 cells in an ethanol environment, the effective components of DBJ were identified. An alcohol-induced AALI mouse model was used to evaluate the efficacy of DBJ against AALI. For this purpose, alcohol dehydrogenase (ADH) and acetaldehyde dehydrogenase (ALDH) levels were assessed, liver function indices and oxidative and inflammatory markers were determined, and histopathological examinations were performed. Mechanistic investigations were conducted using RT-qPCR assays and immunohistochemical analysis to determine the protective effects of DBJ. The samples (DBJ-1, DBJ-2, and DBJ-3) were obtained by extracting DBJ with water, 50% ethanol, and 95% ethanol, yielding total saponin contents of 5.35%, 6.64%, and 11.83%, respectively. DBJ-3 was isolated and purified, and its components were identified by Ultra Performance Liquid Chromatography-Mass Spectrometry (UPLC-MS). DBJ-3 had the greatest effect on cell viability in an ethanol environment. Moreover, DBJ-3 reduced inflammatory infiltration, liver cell degeneration, and hemorrhage, while increasing ADH and ALDH levels in liver tissues. Additionally, DBJ-3 considerably decreased the serum alanine aminotransferase (ALT), aspartate aminotransferase (AST), total cholesterol (TC), and triglyceride (TG) levels. DBJ-3 reduced malondialdehyde (MDA), reactive oxygen species (ROS), and inflammatory factors, such as tumor necrosis factor (TNF-α), interleukin-1β (IL-1β), and interleukin 6 (IL-6), while increasing superoxide dismutase (SOD) and glutathione S-transferase (GST) activities. Furthermore, DBJ-3 significantly increased alcohol dehydrogenase 1b (ADH1B) and aldehyde dehydrogenase 2 (ALDH2) expression at the gene and protein levels within alcohol metabolism pathways and reduced the nuclear factor kappa-B (NF-κB) gene and protein levels. These findings suggest that DBJ-3 can prevent AALI by enhancing alcohol metabolism via the regulation of ADH1B and ALDH2 and the modulation of the NF-κB pathway to improve antioxidant and anti-inflammatory effects. Full article
(This article belongs to the Special Issue Biochemical Composition and Activity of Medicinal Plants and Food)
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<p>Standard curve of ginsenoside Re.</p>
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<p>Total ion chromatogram of DBJ-3 in negative ion mode. The arrow in <a href="#cimb-47-00003-f002" class="html-fig">Figure 2</a> indicates the detected saponin.</p>
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<p>The effects of different DBJ extracts on AML-12 cell survival were evaluated after 24 h. (<b>A</b>) DBJ-1; (<b>B</b>) DBJ-2; (<b>C</b>) DBJ-3. All the data are presented as the means ± SDs (<span class="html-italic">n</span> = 6).</p>
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<p>After 24 h, the survival rate of the AML-12 cells was determined with different concentrations of ethanol. All the data are presented as the means ±SDs (<span class="html-italic">n</span> = 6); ** <span class="html-italic">p</span> &lt; 0.01 and *** <span class="html-italic">p</span> &lt; 0.001 vs. the normal control group.</p>
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<p>Alterations in the survival rate of AML-12 cells. All the data are presented as the means ± SDs (<span class="html-italic">n</span> = 6); <sup>###</sup> <span class="html-italic">p</span> &lt; 0.001 vs. the NC group; ** <span class="html-italic">p</span> &lt; 0.01 and *** <span class="html-italic">p</span> &lt; 0.001 vs. the M group.</p>
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<p>Changes in the serum biochemical indices. The NC group (normal control, distilled water), M group (model, 56% alcohol at 15 mL/kg), S group (silymarin, 60 mg/kg), DBJ-3-L group (100 mg/kg DBJ-3), DBJ-3-M group (300 mg/kg DBJ-3), DBJ-3-H group (600 mg/kg DBJ-3). (<b>A</b>) ALT; (<b>B</b>) AST; (<b>C</b>) TC; (<b>D</b>) TG. The symbol corresponding to each group represents the data for that group, and all the data are presented as the means ± SDs (n = 6); <sup>##</sup> <span class="html-italic">p</span> &lt; 0.01 vs. the NC group; * <span class="html-italic">p</span> &lt; 0.05 and ** <span class="html-italic">p</span> &lt; 0.01 vs. the M group.</p>
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<p>Changes in ADH and ALDH levels in mouse liver tissue. (<b>A</b>) ADH; (<b>B</b>) ALDH. All the data are presented as the means ± SDs (<span class="html-italic">n</span> = 6); <sup>##</sup> <span class="html-italic">p</span> &lt; 0.01 vs. the NC group; * <span class="html-italic">p</span> &lt; 0.05 and ** <span class="html-italic">p</span> &lt; 0.01 vs. the M group.</p>
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<p>Histopathological sections from each group. (<b>A</b>) NC group; (<b>B</b>) M group; (<b>C</b>) S group; (<b>D</b>) DBJ-3-L group; (<b>E</b>) DBJ-3-M group; (<b>F</b>) DBJ-3-H group. The left image scale bar was 500 μm, and the right image scale bar was 100 μm in each group. Yellow arrows indicate cellular swelling and cytoplasmic looseness with light staining, blue arrows indicate lipid vacuolation in hepatocytes, and red arrows indicate a small amount of vascular congestion. The black arrows represent slight hydropic degeneration of many hepatocytes in the liver tissue.</p>
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<p>Changes in ROS, MDA, SOD, and GST levels in mouse liver tissues. (<b>A</b>) ROS; (<b>B</b>) MDA; (<b>C</b>) SOD; (<b>D</b>) GST. All the data are presented as the means ±SDs (<span class="html-italic">n</span> = 6); <sup>##</sup> <span class="html-italic">p</span> &lt; 0.01 vs. the NC group; * <span class="html-italic">p</span> &lt; 0.05 and ** <span class="html-italic">p</span> &lt; 0.01 vs. the M group.</p>
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<p>Changes in TNF-α, IL-1β, and IL-6 levels in mouse liver tissues. (<b>A</b>) TNF-α; (<b>B</b>) IL-6; (<b>C</b>) IL-1β. All the data are presented as the means ± SDs (<span class="html-italic">n</span> = 6); <sup>##</sup> <span class="html-italic">p</span> &lt; 0.01 vs. the NC group; * <span class="html-italic">p</span> &lt; 0.05 and ** <span class="html-italic">p</span> &lt; 0.01 vs. the M group.</p>
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<p>Expression levels of the <span class="html-italic">Adh1b</span> and <span class="html-italic">Aldh2</span> mRNAs. (<b>A</b>) <span class="html-italic">Adh1b</span>; (<b>B</b>) <span class="html-italic">Aldh2</span>. All the data are presented as the means ± SDs (<span class="html-italic">n</span> = 6); <sup>##</sup> <span class="html-italic">p</span> &lt; 0.01 vs. the NC group; ** <span class="html-italic">p</span> &lt; 0.01 vs. the M group.</p>
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<p>Immunohistochemical staining analysis of ADH1B and ALDH2 in the different groups. (<b>A</b>) Images illustrating the immunoreactivity of ADH1B and ALDH2; (<b>B</b>) ADH1B; (<b>C</b>) ALDH2. All the data are presented as the means ±SDs (<span class="html-italic">n</span> = 6); <sup>##</sup> <span class="html-italic">p</span> &lt; 0.01 vs. the NC group; ** <span class="html-italic">p</span> &lt; 0.01 vs. the M group.</p>
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<p><span class="html-italic">Nf-κb</span> mRNA expression. All the data are presented as the means ± SDs (<span class="html-italic">n</span> = 6); <sup>##</sup> <span class="html-italic">p</span> &lt; 0.01 vs. the NC group; ** <span class="html-italic">p</span> &lt; 0.01 vs. the M group.</p>
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<p>IHC analysis of NF-κB in the different groups. (<b>A</b>) Images illustrating the IHC of NF-κB; (<b>B</b>) NF-κB P65 protein expression. All the data are presented as the means ± SDs (<span class="html-italic">n</span> = 6); <sup>##</sup> <span class="html-italic">p</span> &lt; 0.01 vs. the NC group; * <span class="html-italic">p</span> &lt; 0.05 vs. the M group.</p>
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15 pages, 1572 KiB  
Article
Qualitative Phytochemical Profiling and In Vitro Antioxidant Potential Evaluation of South African Momordica Balsamina Linn Fruit Pulp
by Buang Matseke, Sipho Mapfumari and Mmamosheledi Mothibe
Life 2025, 15(1), 4; https://doi.org/10.3390/life15010004 - 24 Dec 2024
Viewed by 377
Abstract
Momordica balsamina Linn is a well-known African traditional herb due to its tremendous medicinal and nutritional properties. It is used worldwide for the treatment of different ailments and diseases. In the present study, the phytochemical and antioxidant activity of South African M. balsamina [...] Read more.
Momordica balsamina Linn is a well-known African traditional herb due to its tremendous medicinal and nutritional properties. It is used worldwide for the treatment of different ailments and diseases. In the present study, the phytochemical and antioxidant activity of South African M. balsamina fruit pulp extracts was evaluated. The fruit pulp extracts were obtained by using the serial exhaustive extraction procedure using the solvents hexane, DCM, acetone, and methanol. The resulting extracts were subjected to different standard colorimetric tests for phytochemical analysis. The presence of compounds with antioxidant activity was determined using dot plot and TLC. The DPPH radical scavenging assay, hydrogen peroxide activity, and reducing power assay, coupled with linear regression, were employed to determine the quantity of antioxidants and their IC50. The results for qualitative phytochemical screening have shown that the fruit pulp contains alkaloids, cardiac glycosides, saponins, phenolic compounds, tannins, flavonoids, terpenoids, and steroids. All the extracts revealed the presence of antioxidant activity in both dot plot and TLC. Acetone extracts (0.279 mg/mL) showed the lowest IC50 compared to the standards gallic acid (0.4 mg/mL) and diosgenin (0.42 mg/mL). These findings confirmed that M. balsamina is very rich in phytochemical compounds and has strong antioxidant potential; therefore, it could be a potential source of drugs which in the future may serve the production of synthetically improved therapeutic agents. Full article
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<p>Dot plot of <span class="html-italic">M. balsamina</span> extracts before spraying with DPPH (<b>left</b>) and after spraying with DPPH (<b>right</b>).</p>
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<p>Chromatograms of <span class="html-italic">M. balsamina</span> (<b>A</b>) hexane, (<b>B</b>) DCM, (<b>C</b>) acetone and (<b>D</b>) methanol extracts developed with different solvent systems. The chromatograms are visualised from left to right under (1). visible light, (2). UV short wave (214 nm), (3). UV long wave (314 nm), and (4). DPPH spray for antioxidants.</p>
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<p>Percentage DPPH radical scavenging activity of <span class="html-italic">M. balsamina</span> extracts of different concentrations.</p>
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<p>Percentage hydrogen peroxide radical scavenging activity of <span class="html-italic">M. balsamina</span> extracts of different concentrations.</p>
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<p>Percentage reducing power of <span class="html-italic">M. balsamina</span> extracts of different concentrations.</p>
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16 pages, 1191 KiB  
Article
Gigaspora roseae and Coriolopsis rigida Fungi Improve Performance of Quillaja saponaria Plants Grown in Sandy Substrate with Added Sewage Sludge
by Guillermo Pereira, Diyanira Castillo-Novales, Cristian Salazar, Cristian Atala and Cesar Arriagada-Escamilla
J. Fungi 2025, 11(1), 2; https://doi.org/10.3390/jof11010002 - 24 Dec 2024
Viewed by 279
Abstract
The use of living organisms to treat human by-products, such as residual sludge, has gained interest in the last years. Fungi have been used for bioremediation and improving plant performance in contaminated soils. We investigated the impact of the mycorrhizal fungus (MF) Gigaspora [...] Read more.
The use of living organisms to treat human by-products, such as residual sludge, has gained interest in the last years. Fungi have been used for bioremediation and improving plant performance in contaminated soils. We investigated the impact of the mycorrhizal fungus (MF) Gigaspora roseae and the saprophytic fungus (SF) Coriolopsis rigida on the survival and growth of Quillaja saponaria seedlings cultivated in a sandy substrate supplemented with residual sludge. Q. saponaria is a sclerophyllous tree endemic to Chile, known for its high content of saponins. We inoculated plants with the MF, the SF, and a combination of both (MF + SF). Following inoculation, varying doses of liquid residual sludge equivalent to 0, 75, and 100% of the substrate’s field capacity were applied. After 11 months, we found a positive influence of the utilized microorganisms on the growth of Q. saponaria. Particularly, inoculation with the SF resulted in higher plant growth, mycorrhizal colonization percentage, and higher enzymatic activity, especially after the application of the sludge. This increase was more evident with higher doses of the applied sludge. These results highlight the potential of combined microorganism and residual sludge application as a sustainable strategy for enhancing plant growth and reducing waste. Full article
(This article belongs to the Special Issue Fungi Activity on Remediation of Polluted Environments, 2nd Edition)
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<p>Growth performance of <span class="html-italic">Q. saponaria</span> plants inoculated with mycorrhizal and saprophytic fungi in sandy substrate. Height growth (<b>A</b>) and DAC (<b>B</b>) of <span class="html-italic">Q. saponaria</span> plants inoculated with the MF <span class="html-italic">G. roseae</span> and the SF <span class="html-italic">C. rigida</span>, and with both (MF + SF) in sandy substrate. Different letters denote significant differences (Tukey test <span class="html-italic">p</span> &lt; 0.05).</p>
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<p>Aerial and radicle biomass of <span class="html-italic">Q. Saponaria</span> plants inoculated with the MF <span class="html-italic">G. roseae</span> and SF <span class="html-italic">C. rigida</span>, or their combination (MF + SF), under increasing doses of residual sludge. Different letters denote significant differences (Tukey test <span class="html-italic">p</span> &lt; 0.05).</p>
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<p>Mycorrhization % in <span class="html-italic">Q. saponaria</span> plants inoculated with the MF <span class="html-italic">G. roseae</span> or with a combination of MF + SF (<span class="html-italic">G. roseae</span> and <span class="html-italic">C. rigida</span>) after the addition of increasing concentrations of waste sludge. Different letters denote significant differences (Tukey test <span class="html-italic">p</span> &lt; 0.05).</p>
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19 pages, 2461 KiB  
Article
Optimization of Breeding Tools in Quinoa (Chenopodium quinoa) and Identification of Suitable Breeding Material for NW Europe
by Tim Vleugels, Chris Van Waes, Ellen De Keyser and Gerda Cnops
Plants 2025, 14(1), 3; https://doi.org/10.3390/plants14010003 - 24 Dec 2024
Viewed by 328
Abstract
Quinoa (Chenopodium quinoa) cultivation has become increasingly popular in NW Europe but little is known about the performance of contract-free varieties in this region. In this study, we phenotyped 25 quinoa varieties on a single-plant basis in a field trial in [...] Read more.
Quinoa (Chenopodium quinoa) cultivation has become increasingly popular in NW Europe but little is known about the performance of contract-free varieties in this region. In this study, we phenotyped 25 quinoa varieties on a single-plant basis in a field trial in Belgium. In addition, we optimized breeding tools such as NIRS (near-infrared reflectance spectroscopy) to estimate the seed crude protein content and a multiplex PCR set to identify true F1 progeny from pair crosses. We identified 14 varieties with sufficiently early maturity, 17 varieties with plant height below 150 cm, 21 large-seeded varieties, four varieties with a crude protein content exceeding 15%, and two low-saponin varieties. A variety of seed colors and plant morphological traits was observed. Seed yield was not correlated with maturity, plant height or saponin content, but was negatively correlated with seed crude protein content. NIRS could accurately predict seed crude protein content with a determination coefficient of 0.94. Our multiplex SSR set could correctly identify the paternity in 77% to 97% of progeny, depending on the pair cross. In conclusion, our study identified various contract-free varieties that may be suitable for cultivation in NW Europe. In addition, our study provides valuable phenotypic information and breeding tools that breeders can harness for breeding efforts in NW European quinoa. Full article
(This article belongs to the Special Issue Genomics-Assisted Improvement of Quinoa)
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<p>Daily average temperatures (black line, right axis) and daily precipitation (gray bars, left axis) for the trial site during the growing season of 2021, obtained from the Royal Meteorological Institute (KMI) in the form of interpolated data from nearby weather stations.</p>
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<p>Example of the phenotypic diversity observed in panicle traits among our panel of varieties and plants: narrow (<b>A</b>), intermediate (<b>B</b>,<b>C</b>) to broad (<b>D</b>) panicle width; dense (<b>A</b>), intermediate (<b>B</b>,<b>C</b>) to loose (<b>D</b>) panicle density; and purple (<b>A</b>), red (<b>B</b>), yellow (<b>C</b>) or green (<b>D</b>) panicle color.</p>
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<p>Measured crude protein content and NIRS-estimated crude protein content for 178 quinoa seed samples used in the calibration, with indication of the 163 samples that were used in the final calibration (circles) and the 18 outlier samples (triangles) that were omitted.</p>
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26 pages, 4557 KiB  
Article
Ethanolic Extract of Averrhoa carambola Leaf Has an Anticancer Activity on Triple-Negative Breast Cancer Cells: An In Vitro Study
by Oscar F. Beas-Guzmán, Ariana Cabrera-Licona, Gustavo A. Hernández-Fuentes, Silvia G. Ceballos-Magaña, José Guzmán-Esquivel, Luis De-León-Zaragoza, Mario Ramírez-Flores, Janet Diaz-Martinez, Idalia Garza-Veloz, Margarita L. Martínez-Fierro, Iram P. Rodríguez-Sanchez, Gabriel Ceja-Espíritu, Carmen Meza-Robles, Víctor H. Cervantes-Kardasch and Iván Delgado-Enciso
Pharmaceutics 2025, 17(1), 2; https://doi.org/10.3390/pharmaceutics17010002 - 24 Dec 2024
Viewed by 328
Abstract
Background/Objectives: Averrhoa carambola, or star fruit, is a shrub known for its medicinal properties, especially due to bioactive metabolites identified in its roots and fruit with anti-cancer activity. However, the biological effects of its leaves remain unexplored. This study aimed to [...] Read more.
Background/Objectives: Averrhoa carambola, or star fruit, is a shrub known for its medicinal properties, especially due to bioactive metabolites identified in its roots and fruit with anti-cancer activity. However, the biological effects of its leaves remain unexplored. This study aimed to assess the effects of ethanolic extract from A. carambola leaves on triple-negative breast cancer (TNBC), an aggressive subtype lacking specific therapy. Methods: Phytochemical analysis and HPLC profile and additional cell line evaluation employing MDA-MB-231 were carried out. Results: Phytochemical screening revealed that the ethanolic extract was rich in flavonoids, saponins, and steroids, demonstrating an antioxidant capacity of 45%. 1H NMR analysis indicated the presence of flavonoids, terpenes, and glycoside-like compounds. Cell viability assays showed a concentration-dependent decrease in viability, with an IC50 value of 20.89 μg/mL at 48 h. Clonogenic assays indicated significant inhibition of replicative immortality, with only 2.63% survival at 15 μg/mL. Migration, assessed through a wound healing assay, was reduced to 3.06% at 100 μg/mL, with only 16.23% of cells remaining attached. An additive effect was observed when combining lower concentrations of the extract with doxorubicin, indicating potential synergy. Conclusions: These results suggest that the ethanolic extract of A. carambola leaves contains metabolites with anti-cancer activity against TNBC cells, supporting further research into their bioactive compounds and therapeutic potential. Full article
(This article belongs to the Special Issue Pharmaceutical Applications of Plant Extracts, 2nd Edition)
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<p>Chromatograms obtained at 290 nm from HPLC analysis. (<b>A</b>) Chromatogram of standards: gallic acid (GA, Rt 2.385 min), cinnamic acid (CA, Rt 30.795 min), anthrone (ANT, Rt 20.000 min), quercetin (Q, Rt 17.955 min), and 4-methylumbelliferone (4-ML, Rt 10.908 min). (<b>B</b>) Chromatogram of the ethanolic extract of <span class="html-italic">A. carambola</span> (500 ppm). (<b>C</b>) Chromatogram of the hydrolysate of the leaves of <span class="html-italic">A. carambola</span> (500 ppm). S: signal.</p>
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<p>Viability experiments employing ethanolic extract of <span class="html-italic">A. carambola</span> on MDA-MB-231 cells. (<b>A</b>) No changes in viability were observed in cervical cancer cell line TC-1 exposed to A. carambola extract in increasing concentrations. (<b>B</b>) A concentration-dependent effect was observed on MDA-MB-231 cell line exposed to the extract. (<b>C</b>) The ethanolic extract of <span class="html-italic">A. carambola</span> leaves had an experimental IC<sub>50</sub> of 20.83 μg/mL in triple-negative breast cancer cell line. (<b>D</b>) Morphological changes and detached cells were observed from the concentration of 25 μg/mL of ethanolic extract. Magnification 10×. The <span class="html-italic">p</span>-values correspond to significant differences compared to the control, DMEM-F12 medium with 0.1% DMSO, * <span class="html-italic">p</span> &lt; 0.05.</p>
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<p>Ethanolic extract of <span class="html-italic">A. carambola</span> leaves decreases replicative immortality of MDA-MB-231 cells. (<b>A</b>) Photographs depict the number of colonies formed after the exposition of each treatment. It is observed that a concentration-dependent effect completely inhibits cell survival. (<b>B</b>) The graph shows the percentage of survival treatment. The <span class="html-italic">p</span>-values correspond to significant differences compared to the control, only DMEM medium, * <span class="html-italic">p</span> &lt; 0.05.</p>
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<p>Ethanolic extract of <span class="html-italic">A. carambola</span> leaves interferes with MDA-MB-231 cell migration. (<b>A</b>) Images captured at 48 h of the wound area made in MDA-MB-231 cell monolayers. Magnification 4×. (<b>B</b>) The graph shows the changes in the open area; a concentration-dependent inhibitory effect can be observed at 48 h that was superior to the doxorubicin effect. Comparison to 48 h control, * <span class="html-italic">p</span> &lt; 0.05.</p>
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<p>The ethanolic extract of <span class="html-italic">A. carambola</span> leaves affects the cell adhesion of MDA-MB-231 cells. (<b>A</b>) The micrographs show the adhesive capacity of cells recovered after exposure to <span class="html-italic">A. carambola</span> extract and reseeded for 24 h. The adhesive capacity decreases as the concentration of the extract increases. Magnification 10×. (<b>B</b>) The graphs show the percentage of cells adhered to the monolayer after being treated with the extract for 48, showing a concentration-dependent decrease in adhesion. (<b>C</b>) The graph shows the percentage of cell death after 48 h of treatment. (<b>D</b>) The graph shows the percentage of adhesion of detached cells after treatment that were recovered and reseeded. The <span class="html-italic">p</span>-values correspond to significant changes compared to the control, * <span class="html-italic">p</span> &lt; 0.05.</p>
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<p>The combination of a low dose of doxorubicin and intermediate doses of <span class="html-italic">A. carambola</span> extract reduces the cell viability of MDA-MB-231 cells. The graph shows the reduction in cell viability induced by the different combinations after 48 h of treatment. An additive effect was observed between the 1/5 IC<sub>50</sub> dose of doxorubicin (DOX) and the three tested concentrations of the extract. <sup>a</sup> 0.4 μM DOX + 15 μg/mL extract vs. 15 μg/mL of the extract, <sup>b</sup> 0.4 μM DOX + 25 μg/mL vs. 25 μg/mL, <sup>c</sup> 0.4 μM DOX + 50 μg/mL vs. 50 μg/mL, <sup>d</sup> 2 μM DOX + 15 μg/mL vs. 15 μg/mL, <sup>e</sup> 2 μM DOX + 25 μg/mL vs. 25 μg/mL, <sup>f</sup> 2 μM DOX + 50 μg/mL vs. 50 μg/Ml, <sup>a’</sup> 0.4 μM DOX + 15 μg/mL vs. 0.4 μM DOX, <sup>b’</sup> 0.4 μM DOX + 25 μg/mL vs. 0.4 μM DOX <sup>c’</sup> 0.4 μM DOX + 50 μg/mL vs. 0.4 μM DOX, <sup>d’</sup> 2 μM DOX + 15 μg/mL vs. 2 μM DOX, <sup>e’</sup> 2 μM DOX + 25 μg/mL vs. 2 μM DOX, <sup>f’</sup> 2 μM DOX + 50 μg/mL vs. 2 μM DOX, * <span class="html-italic">p</span> &lt; 0.05.</p>
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14 pages, 3895 KiB  
Article
In-Depth LC-ESI/HRMS-Guided Phytochemical Analysis and Antioxidant Activity Analysis of Eco-Sustainable Extracts of Cynara cardunculus (Carciofo di Paestum PGI) Leaves
by Antonietta Cerulli, Roberta Cuozzo, Maria Paola Melis, Gabriele Serreli, Monica Deiana, Milena Masullo and Sonia Piacente
Plants 2024, 13(24), 3591; https://doi.org/10.3390/plants13243591 - 23 Dec 2024
Viewed by 267
Abstract
The Italian Carciofo di Paestum (C. scolymus) PGI, an artichoke variety from the Campania region, was investigated for its potential to reuse by-products for food supplements. EtOH:H2O 50:50 and 75:25 extracts of its leaves were analyzed for phenolic and [...] Read more.
The Italian Carciofo di Paestum (C. scolymus) PGI, an artichoke variety from the Campania region, was investigated for its potential to reuse by-products for food supplements. EtOH:H2O 50:50 and 75:25 extracts of its leaves were analyzed for phenolic and flavonoid content and antioxidant activity (TEAC: 1.90 and 1.81 mM of Trolox; DPPH IC50: 106.31 µg/mL and 128.21 µg/mL; FRAP: 1.68 and 1.58 mM FeSO₄/g extract). To further investigate the antioxidant potential, the ability of the two extracts to scavenge reactive species was assessed in Caco-2 cell cultures, showing a dose-dependent antioxidant capacity. To highlight metabolites responsible for the activity, LC-ESI/HRMSMS analysis was achieved, revealing 28 compounds (sesquiterpenes, megastigmanes, quinic acid and hydroxycinnamic acid derivatives, flavonoids, lignans, triterpenoid saponins, and polar fatty acids), of which structures were determined using 1D- and 2D-NMR analysis. In addition, quantitative determination of caffeoyl, dicaffeoyl, and quinic acid derivatives (CQAs) was performed through LC-ESI/QTrap/MS/MS, highlighting that the most abundant compound was 5-caffeoylquinic acid (6), with values of 9.310 and 7.603 mg/g extract in EtOH:H2O (75:25) and EtOH:H2O (50:50), respectively. The analysis showed that extracts were rich in bioactive compounds, suggesting their potential for development into antioxidant-based food supplements that may protect cells from oxidative stress and support overall wellness. Full article
(This article belongs to the Special Issue Bio-Active Compounds in Horticultural Plants)
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<p>Total phenolic and flavonoid content, TEAC, DPPH, and FRAP assays of EtOH:H<sub>2</sub>O (50:50) (blue) and EtOH:H<sub>2</sub>O (75:25) (orange) extracts of “Carciofo di Paestum” PGI leaves.</p>
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<p>Percentage of viable Caco-2 cells calculated relative to the control (0 μM, 100% viability) following 24 h of incubation with different concentrations of EtOH:H<sub>2</sub>O (50:50) (<b>A</b>) and EtOH:H<sub>2</sub>O (75:25) (<b>B</b>) extracts (0.1–50 μM). Data are presented as the mean ± SD from independent experiments (n = 16).</p>
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<p>ROS levels, detected via H<sub>2</sub>-DCF-DA fluorescence and expressed as % of the control samples (CTR) in Caco-2 cells following 2 h of incubation with varying concentrations of the EtOH:H<sub>2</sub>O (50:50) (<b>A</b>) and EtOH:H<sub>2</sub>O (75:25) (<b>B</b>) (2.5–50 μM) in co-incubation with TBH 2.5 mM. °°° = <span class="html-italic">p</span> &lt; 0.001 TBH vs. CTR; * = <span class="html-italic">p</span> &lt; 0.05 extracts vs. TBH; ** = <span class="html-italic">p</span> &lt; 0.01 extracts vs. TBH; *** = <span class="html-italic">p</span> &lt; 0.001 extracts vs. TBH (n = 12).</p>
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<p>LC-ESI/HRMS profile of EtOH:H<sub>2</sub>O (50:50) (<b>A</b>) and EtOH:H<sub>2</sub>O (75:25) (<b>B</b>) extracts of “Carciofo di Paestum” PGI leaves.</p>
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<p>Specialized metabolites isolated from EtOH:H<sub>2</sub>O (50:50) extract of “Carciofo di Paestum” PGI leaves.</p>
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19 pages, 3607 KiB  
Article
Mass Spectrometric Based Metabolomics of the Saudi Cultivar of Fenugreek (Trigonella foenum-graecum L.): A Combined GC-MS, Antimicrobial and Computational Approach
by Nujud A. M. Almuzaini, Abdel Moneim E. Sulieman, Naimah A. Alanazi, Riadh Badraoui and Emad M. Abdallah
Pharmaceuticals 2024, 17(12), 1733; https://doi.org/10.3390/ph17121733 - 21 Dec 2024
Viewed by 753
Abstract
Background and Objective: In Saudi Arabia, numerous plant species with promising medicinal properties are cultivated, widely traded, and commonly utilized in traditional medicine, including fenugreek (Trigonella foenum-graecum). This study aimed to comprehensively assess the phytochemical composition and antimicrobial potential of the [...] Read more.
Background and Objective: In Saudi Arabia, numerous plant species with promising medicinal properties are cultivated, widely traded, and commonly utilized in traditional medicine, including fenugreek (Trigonella foenum-graecum). This study aimed to comprehensively assess the phytochemical composition and antimicrobial potential of the Saudi cultivar of fenugreek using an integrative approach combining in vitro and in silico methodologies. Methods: A comprehensive investigation was conducted on the ethanol extract of fenugreek seeds, assessing its antibacterial, antifungal, properties. Computational modeling was employed to predict pharmacokinetic behavior and potential toxicity of the identified bioactive compounds. Results: Qalitative phytochemical analysis showed presence of alkaloids, tannins, saponins, glycosides, flavonoids, and steroids, while terpenoids were notably absent. GC-MS analysis of Trigonella foenum-graecum (fenugreek) seeds identified 25 bioactive compounds, with Ethyl methane sulfonate (12.41%) being the predominant component. Other key compounds included n-Hexadecanoic acid, 4-Butyl-2(4-nitrophenyl)-1,3-thiazole, and α-Tocopherol. In silico modeling of fenugreek phytochemicals supported their antibacterial, antioxidant, and neuroprotective potential, with compounds 21 and 24 showing strong binding to key targets like Tyrosyl-tRNA Synthetase (TyrRS) of Staphylococcus aureus (S. aureus), Aspartic proteinase from Candida albicans (C. albicans) and human peroxiredoxin 5. Pharmacokinetic analysis indicated good oral bioavailability, minimal CYP inhibition, and blood-brain barrier penetration, suggesting potential for treating neurodegenerative diseases. These bioactive compounds, including diosgenin and trigonelline, support fenugreek’s therapeutic promise and warrant further in vitro, in vivo, and clinical studies. Conclusion: The Saudi fenugreek cultivar is rich in bioactive compounds with good antibacterial potential. These findings establish a robust foundation for continued pharmacological research on the Saudi cultivar of T. foenum-graecum, highlighting its potential as a rich source of bioactive compounds with significant medicinal value. Full article
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<p>Chromatogram of the ethanol extract of fenugreek seeds.</p>
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<p>Tridimensional illustrations of the 3 targeted receptors 1JIJ (<b>A</b>–<b>A″</b>), 2QZW (<b>B</b>–<b>B″</b>) and 1H2D (<b>C</b>–<b>C″</b>) with the three predicted best compounds identified in the fenugreek seeds. 1JIJ complexed with compounds no. <b>21</b> (<b>A</b>), <b>24</b> (<b>A′</b>), and <b>15</b> (<b>A″</b>). 2QZW complexed with compounds no. <b>21</b> (<b>B</b>), <b>24</b> (<b>B′</b>), and <b>15</b> (<b>B″</b>). 1HD2 complexed with compounds no. <b>24</b> (<b>C</b>), <b>21</b> (<b>C′</b>), and <b>23</b> (<b>C″</b>).</p>
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<p>Illustration of the 2D diagrams of interactions of the 3 targeted receptors 1JIJ (<b>A</b>–<b>A″</b>), 2QZW (<b>B</b>–<b>B″</b>) and 1H2D (<b>C</b>–<b>C″</b>) with the three predicted best compounds identified in the fenugreek seeds. 1JIJ complexed with compounds no. <b>21</b> (<b>A</b>), <b>24</b> (<b>A′</b>), and <b>15</b> (<b>A″</b>). 2QZW complexed with compounds no. <b>21</b> (<b>B</b>), <b>24</b> (<b>B′</b>), and <b>15</b> (<b>B″</b>). 1HD2 complexed with compounds no. <b>24</b> (<b>C</b>), <b>21</b> (<b>C′</b>), and <b>23</b> (<b>C″</b>).</p>
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<p>Chemical structure of the best compounds (<b>15</b>, <b>21</b>, <b>23</b> and <b>24</b>) from the docking analysis.</p>
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<p>Boiled-egg model of the identified phytochemicals. The yellow and white areas correspond to the BBB (for blood-brain-barrier) permeation and GI (for gastro-intestinal) absorption, respectively.</p>
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9 pages, 593 KiB  
Article
Plant Cell Culture-Derived Saponin Adjuvant Enhances Immune Response Against a Stabilized Human Metapneumovirus Pre-Fusion Vaccine Candidate
by Maarten Swart, Jessica Allen, Brendan Reed, Ana Izquierdo Gil, Johan Verspuij, Sonja Schmit-Tillemans, Anish Chakkumkal, Mark Findeis, Angela V. Hafner, Chandresh Harjivan, Rebecca Kurnat, Harmjan Kuipers, Roland Zahn and Boerries Brandenburg
Vaccines 2024, 12(12), 1435; https://doi.org/10.3390/vaccines12121435 - 20 Dec 2024
Viewed by 576
Abstract
Human metapneumovirus (HMPV) is a significant respiratory pathogen, particularly in vulnerable populations. Background: No vaccine for the prevention of HMPV is currently licensed, although several subunit vaccines are in development. Saponin-based adjuvant systems (AS), including QS-21, have transformed the field of subunit vaccines [...] Read more.
Human metapneumovirus (HMPV) is a significant respiratory pathogen, particularly in vulnerable populations. Background: No vaccine for the prevention of HMPV is currently licensed, although several subunit vaccines are in development. Saponin-based adjuvant systems (AS), including QS-21, have transformed the field of subunit vaccines by dramatically increasing their potency and efficacy, leading to the development of several licensed vaccines. However, naturally sourced tree bark-extracted QS-21 faces supply and manufacturing challenges, hindering vaccine development. Objective: This study reports on an alternative plant cell culture system for the consistent production of highly pure QS-21. Method: We evaluated the efficacy of cultured plant cell (cpc)-produced QS-21 in a novel HMPV vaccine, formulating a recombinant pre-fusion stabilized HMPV F protein (preF) with cpcQS-21 and a synthetic toll-like receptor 4 (TLR4) agonist adjuvant formulation. Results: In mice, TLR4 agonist containing adjuvant formulations with plant cell-produced QS-21 performed equally to licensed adjuvant AS01 containing tree-bark-extracted QS-21 and demonstrated a significant increase in immunogenicity against HMPV preF compared to the unadjuvanted control. Conclusion: Our findings pave the way for a reliable, scalable, and sustainable source of pure QS-21, enabling the development of highly effective HMPV and other vaccines with significant public health impact. Full article
(This article belongs to the Section Vaccine Adjuvants)
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<p>Anti-F antibody titers following immunization. AS01 formulated with cpcQS-21 or beQS-21 elicited similar humoral immune responses in a prime-boost model of HMPV vaccination. Anti-F serum IgG response from BALB/c mice immunized with two doses (day 0 and 28) of 5 µg recombinant HMPV A2 PreF combined with AS01<sub>B</sub>, AS01-SPQX, or AS100-SPQX. Anti-F antibody titers were measured by ELISA on day 27 (<b>A</b>) or 42 (<b>B</b>). Concentrations of TLR4 agonist (MPLA for AS01<sub>B</sub> or PHAD<sup>®</sup> for SPQX) and (be or cpc) QS-21 are indicated. Log10 relative potency (RP) titers are compared with a reference serum pool. Red horizontal bars indicate the median response per group, and the dotted line indicates the lower limit of detection (LLOD). Open symbols indicate that the response is at or below the LLOD. AS01B and AS01-SPQX were compared across doses by a t-test. AS100-SPQX was compared with 5:5 μg AS01B and 5:5 μg AS01-SPQX by a <span class="html-italic">t</span>-test. (ns, not significant; ** <span class="html-italic">p</span> ≤ 0.01).</p>
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<p>IFN-γ-secretion by splenocytes stimulated with an HMPV A2 F peptide pool. HMPV vaccine formulations containing cpcQS-21 or beQS-21 induced HMPV-specific T cell-mediated immunity. Fourteen days following the second immunization (boost), IFN-γ secretion was assessed upon ex vivo stimulation with an F-pool for 18 h. The frequency of IFN-γ-secreting cells is depicted as the number of spot-forming units (SFU) per million splenocytes. Horizontal red bars denote group geometric means, and horizontal dashed lines indicate the lower limit of detection (LLOD) based on the 95th percentile of the background response. Open symbols indicate the response is at or below the LLOD. AS01B and AS01-SPQX adjuvant formulations were compared across doses by a Cochran–Mantel–Haenszel test. AS100-SPQX was compared with 5:5 μg AS01B and 5:5 μg AS01-SPQX with a Mann-Whitney U-test. No significant differences were found. ns, not significant.</p>
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23 pages, 4328 KiB  
Article
Herbal Extracts Mixed with Essential Oils: A Network Approach for Gastric and Intestinal Motility Disorders
by Roberta Budriesi, Ivan Corazza, Simone Roncioni, Roberta Scanferlato, Dalila De Luca, Carla Marzetti, Roberto Gotti, Nicola Rizzardi, Christian Bergamini, Matteo Micucci, Davide Roncarati and Laura Beatrice Mattioli
Nutrients 2024, 16(24), 4357; https://doi.org/10.3390/nu16244357 - 17 Dec 2024
Viewed by 578
Abstract
Background: Three herbal extracts (Asparagus racemosus Willd., Tabebuia avellanedae Lorentz, and Glycyrrhiza glabra L.) were mixed with three essential oils (Foeniculum vulgare Mill., Mentha piperita L., and Pimpinella anisum L.) to formulate a product (HEMEO) whose active compounds include saponins and [...] Read more.
Background: Three herbal extracts (Asparagus racemosus Willd., Tabebuia avellanedae Lorentz, and Glycyrrhiza glabra L.) were mixed with three essential oils (Foeniculum vulgare Mill., Mentha piperita L., and Pimpinella anisum L.) to formulate a product (HEMEO) whose active compounds include saponins and steroids in Asparagus racemosus, known for their anti-inflammatory properties; glycyrrhizin and flavonoids in Glycyrrhiza glabra, which exhibit gastroprotective and antispasmodic effects; menthol in Mentha piperita, contributing with antispasmodic and antimicrobial properties; and anethole and polyphenols in Pimpinella anisum, which modulate intestinal motility and offer antimicrobial activity. Objective: HEMEO was formulated for applications in intestinal motility disorders. Methods: HEMEO was evaluated for spontaneous and induced motility effects in isolated guinea pig ileum, colon, and stomach. Ex vivo experiments were conducted using LabChart software v7.0, and the product’s antibacterial action against Helicobacter pylori and its antioxidant effects were assessed through disc diffusion and FRAP assays. The presence of the volatile compounds in the formulation was confirmed by GC-MS analysis; the TPC of HEMEO, determined using the Folin–Ciocalteu method, was 9.925 ± 0.42 mg GAE/g. Conclusions: HEMEO showed a phenolic content correlated with its antioxidant potential and in addition inhibited H. pylori growth and demonstrated notable antioxidant properties, suggesting its role as a supportive agent in digestive processes and in managing motility disorders. Full article
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<p>Overview of experimental design of HEMEO.</p>
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<p>Plants pictures and chemical structure of key compounds found in the plant extracts and essential oils.</p>
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<p>Example of experimental original recording of the concentration–response curve of HEMEO on spontaneous longitudinal (Long) stomach basal contractility. (<b>a</b>) Spontaneous contraction (SC) signals for each concentration; (<b>b</b>) mean contraction amplitude (MCA) and spontaneous contraction variability (SCV), represented as error bars in the MCA plot and contraction percentage variation for the control (BSMA) for each considered condition; all the comparisons resulted in being statistically significant (<span class="html-italic">p</span> &lt; 0.05); (<b>c</b>) power spectral density (PSD) and percentage variations. Despite statistically significant differences between muscular tones at different HEMEO concentrations, the MCA variations are not physiologically significant (less than 20%). MF and HF bands values increase over 50% only for concentrations of 5.0 mg/mL and 10 mg/mL, suggesting a slight increase in mixing and pain.</p>
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<p>Example of experimental original recording of the concentration–response curve of HEMEO on spontaneous longitudinal (Long) ileum basal contractility. (<b>a</b>) Spontaneous contraction (SC) signals for each concentration; (<b>b</b>) mean contraction amplitude (MCA) and spontaneous contraction variability (SCV), represented as error bars in the MCA plot and contraction percentage variation for the control (BSMA) for each considered condition; not statistically significant differences (<span class="html-italic">p</span> &gt; 0.05) between MCAs at different concentrations are reported in the graph with a curly bracket. All the comparisons not reported are to be considered significant (<span class="html-italic">p</span> &lt; 0.05); (<b>c</b>) power spectral density (PSD) and percentage variations. Longitudinal tones present a biphasic behavior: it increases for 0.1 mg/mL concentrations and then decreases until a maximum difference of −60%. LF and HF present the same tend.</p>
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<p>Example of experimental original recording of the concentration–response curve of HEMEO on spontaneous longitudinal (Long) colon basal contractility. (<b>a</b>) Spontaneous contraction (SC) signals for each concentration; (<b>b</b>) mean contraction amplitude (MCA) and spontaneous contraction variability (SCV), represented as error bars in the MCA plot and contraction percentage variation for the control (BSMA) for each considered condition; all the comparisons reported resulted statistically significant (<span class="html-italic">p</span> &lt; 0.05); (<b>c</b>) power spectral density (PSD) and percentage variations. Longitudinal tones present a biphasic behavior: it increases for 0.1 mg/mL concentrations and then decreases until a maximum difference of −80%. LF and HF bands present the same biphasic trend. MF has a maximum increase of 300% at 0.5 mg/mL, then decreases after 5.0 mg/mL.</p>
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<p>Effect of HEMEO on histamine-induced contraction in isolated guinea pig ileum (<b>a</b>) and in spontaneously beating right atrium (<b>b</b>). Cumulative concentration–response curves were obtained before and after exposure to HEMEO (1 mg/mL) for 30 min. Each point is the mean ± SEM (<span class="html-italic">n</span> = 5–6). Where error bars are not shown, these are covered by the point itself.</p>
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<p>(<b>a</b>) The antioxidant power of the HEMEO solution was measured using the FRAP assay. (<b>b</b>) Comparison of 1 mg/mL HEMEO antioxidant power with the reference compound ascorbate.</p>
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<p>Schematic diagram showing the detail of the experimental design.</p>
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15 pages, 3359 KiB  
Article
Optimization and Standardization of the Extraction Method of Balanites aegyptiaca Del. Seeds (Zygophyllaceae) Used in the Formulation of an Antiparasitic Phytomedicine
by Mohamed Bonewendé Belemlilga, Salfo Ouedraogo, Gilchrist Abdoul Laurent Boly, Do Harouna Dao, Jonas Tiami Coulibaly, Jean Claude Romaric Pingdwindé Ouedraogo, Souleymane Compaoré, Sidiki Traore, Moumouni Koala, Estelle Noëla Hoho Youl, Lazare Belemnaba, Félix Bondo Kini, Aristide Traore, Séni Kouanda and Sylvin Ouedraogo
Pharmaceuticals 2024, 17(12), 1698; https://doi.org/10.3390/ph17121698 - 17 Dec 2024
Viewed by 322
Abstract
Background/Objectives: Balanites aegyptiaca Del. (Zygophyllaceae) is widely used in traditional medicine, both human and veterinary, throughout Africa for its many properties, including antiparasitic properties. This experimental study aims to optimize the extraction conditions of the seeds of Balanites aegyptiaca Del. Methods: Aqueous [...] Read more.
Background/Objectives: Balanites aegyptiaca Del. (Zygophyllaceae) is widely used in traditional medicine, both human and veterinary, throughout Africa for its many properties, including antiparasitic properties. This experimental study aims to optimize the extraction conditions of the seeds of Balanites aegyptiaca Del. Methods: Aqueous maceration was carried out with mass-to-volume ratios of 40%, 30%, 20%, 10% and 5% and extraction times of 6, 12, 24, 36 and 48 h. Extraction yields, phytochemical screening, saponins assay, antioxidant activities ABTS+ free radical scavenging activities, Ferric-reducing antioxidant power (FRAP) assay and antiparasitic tests on Heligmosomoides bakeri were used to compare the different extracts. Results: The pharmaco-chemical study generally showed that aqueous maceration gave the best results, with a mass/volume ratio of 10% after 12 h of maceration. The yield obtained was 28.03% with a saponins content of 13.81 mg/g. The antioxidant activities were 4.25 ± 0.17 µg/mL by the ABTS method and 0.739 µg/mL by the FRAP method. The larvicidal activity also showed that the 10% 12 h extract produced 100% larval mortality from 25 µg/mL. Conclusions: These data provide a basis for guiding the extraction process parameters in producing this antiparasitic phytomedicine. Full article
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<p>Almond powder (<b>a</b>) and lyophilized extract (<b>b</b>) of <span class="html-italic">Balanites aegyptiaca</span> seeds.</p>
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<p>The residual moisture content of different lyophilizate. ** <span class="html-italic">p</span> &lt; 0.01, **** <span class="html-italic">p</span> &lt; 0.0001 is considered significant compared with the other % macerates (two-way ANOVA followed by the “Tukey” multiple comparison test; ns is considered not significant). <span class="html-italic">n</span> = 3.</p>
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<p>Extraction yield histogram of aqueous macerates mass/volume ratio as a function of time (hours). *** <span class="html-italic">p</span> &lt; 0.01, **** <span class="html-italic">p</span> &lt; 0.0001 is considered significant compared with the other % macerates (two-way ANOVA followed by the “Tukey” multiple comparison test; ns is considered not significant). <span class="html-italic">n</span> = 3.</p>
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<p>Analytical TLC profile of different extracts. (<b>a</b>) The presence of saponins revealed by sulfuric anisaldehyde after heating the plate observed in visible light. (<b>b</b>) Presence of flavonoids (at 254 nm) detected by NEU reagent.</p>
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<p>Larvicidal effect of <span class="html-italic">B. aegyptiaca</span> extracts and the standard on <span class="html-italic">H. bakeri</span>.</p>
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<p>Whole plant (<b>a</b>), fruits (<b>b</b>) and isolated saponins (<b>c</b>,<b>d</b>) of <span class="html-italic">Balanites aegyptiaca</span>.</p>
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22 pages, 5864 KiB  
Article
The Effect of Seasonal and Annual Variation on the Quality of Polygonatum Cyrtonema Hua Rhizomes
by Weiting Pu, Yefei Yu, Xiaoxiao Shi, Ye Shao, Bihuan Ye, Youwu Chen, Qiyan Song, Jianjun Shen and Haibo Li
Plants 2024, 13(24), 3459; https://doi.org/10.3390/plants13243459 - 10 Dec 2024
Viewed by 404
Abstract
This study aims to reveal the interannual and seasonal variations in functional components in Polygonatum cyrtonema Hua. rhizomes and evaluate whether the variations significantly affect the quality of rhizomes as a traditional Chinese herbal medicine. The interannual and seasonal variations in total flavonoid [...] Read more.
This study aims to reveal the interannual and seasonal variations in functional components in Polygonatum cyrtonema Hua. rhizomes and evaluate whether the variations significantly affect the quality of rhizomes as a traditional Chinese herbal medicine. The interannual and seasonal variations in total flavonoid content and total saponin content were analyzed. The global dynamic variation in secondary metabolites in the rhizomes during a five-year growth period and in two traditional harvesting seasons were investigated based on metabolomics method. Results clearly showed that the functional components in P. cyrtonema rhizomes exhibited a significant increase in accumulation during the one- to four-year growth period and a significant decrease in accumulation during the four- to five-year growth period. The most active accumulation occurred during the three- to four-year growth period. Drastic variations in functional components occurred from spring to autumn. The significant interannual variation and drastic seasonal variation were strongly associated with the enrichment in some pathways related to the biosynthesis of secondary metabolites and the metabolisms of amino acids. The interannual and seasonal variations in functional components significantly affected the quality of P. cyrtonema rhizomes. The four-year-old rhizomes had the most superior quality due to their higher content of functional components and much more newly formed components. The rhizomes harvested in spring or autumn had unequal quality because of their significant differences in composition and content of functional components. Specifically, the rhizomes from spring contained more flavonoids, alkaloids, and phenolic acids, while those from autumn comprised more steroids. In conclusion, this study reveals that the interannual and seasonal variations in functional components can significantly affect the quality of P. cyrtonema rhizomes as a traditional Chinese herbal medicine. This study provides foundational insights and theoretical guidance for determining an optimal cultivation period to obtain medicinal rhizomes with superior quality. It also offers a strategy for harvesting medicinal rhizomes in two different seasons to achieve unequal quality. Full article
(This article belongs to the Section Phytochemistry)
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<p>Total flavonoid content and total saponin content in <span class="html-italic">P. cyrtonema</span> rhizomes. (<b>A</b>), Total flavonoid content in five age sections; (<b>B</b>), total flavonoid content in entire rhizomes in spring and autumn; (<b>C</b>), total saponin content in five age sections; (<b>D</b>), total saponins content in entire rhizomes in spring and autumn. The letters PS and PA refer to the rhizomes harvested in spring and autumn, respectively. The abbreviated ns represents no significant difference. ****, ***, **, and * represent statistically significant at <span class="html-italic">p</span> &lt; 0.0001, <span class="html-italic">p</span> &lt; 0.001, <span class="html-italic">p</span> &lt; 0.01, and <span class="html-italic">p</span> &lt; 0.05, respectively.</p>
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<p>Basic information on secondary metabolites detected from <span class="html-italic">P. cyrtonema</span> rhizomes. (<b>A</b>), Clustered heatmap of secondary metabolites in samples of one-year-old age sections (AS1 group), two-year-old age sections (AS2 group), three-year-old age sections (AS3 group), four-year-old age sections (AS4 group), and five-year-old age sections (AS5 group); (<b>B</b>), clustered heatmap of secondary metabolites in samples of spring rhizome (PS group) and autumn rhizome (PA group). The shades of color indicate the quantity of metabolites, with redder shades representing more and greener shades representing fewer.</p>
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<p>Quantity of differential accumulated secondary metabolites (DASMs) in five pairwise comparisons.</p>
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<p>The differential accumulated homoisoflavonoids in <span class="html-italic">P. cyrtonema</span> rhizomes. (<b>A</b>), In five age sections of rhizomes during the entire five-year growing period; (<b>B</b>), in entire rhizomes during the growing period from spring to autumn. The letters AS1, AS2, AS3, AS4, and AS5 refer to one-, two-, three-, four-, and five-year-old age sections, respectively.</p>
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<p>Interannual and seasonal variation in secondary metabolites in <span class="html-italic">P. cyrtonema</span> rhizomes. (<b>A</b>), From the one- to five-year growing periods; (<b>B</b>), during the spring–autumn growing period.</p>
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<p>Class and quantity of main differential accumulated secondary metabolites (DASMs) in <span class="html-italic">P. cyrtonema</span> rhizomes. (<b>A</b>), From the one- to five-year growing periods; (<b>B</b>), during the spring–autumn growing period.</p>
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<p>The top 20 significantly enriched KEGG pathways identified from AS4 vs. AS3, AS5 vs. AS4, and PS vs. PA. The color of dots in plots refers to higher or lower <span class="html-italic">p</span>-values, with bluer shades representing higher and redder shades representing lower. The size of a dot represents more or fewer secondary metabolites, with larger dots representing more and smaller dots representing fewer.</p>
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<p>Five-year-old <span class="html-italic">P. cyrtonema</span> rhizomes used in this study. (<b>A</b>), <span class="html-italic">P. cyrtonema</span> plant; (<b>B</b>), the age sections of rhizome used for interannual variation analysis; (<b>C</b>), the entire rhizomes harvested in spring and autumn used for seasonal variation analysis. The letters AS1, AS2, AS3, AS4, and AS5 refer to the one-, two-, three-, four-, and five-year-old age sections, respectively; The letters PS and PA refer to the rhizomes harvested in spring and autumn, respectively.</p>
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