Simultaneous ultrasound-assisted water extraction and β-cyclodextrin encapsulation of polyphenols from <i>Mangifera indica</i> stem bark in counteracting TNFα-induced endothelial dysfunction

<div><p>This study proposes an alternative technique to prevent heat degradation induced by classic procedures of bioactive compound extraction, comparing classical maceration/decoction in hot water of polyphenols from Mango (<i>Mangifera indica</i> L.) (MI) with ultrasound-assisted extraction (UAE) in a water solution of β-cyclodextrin (β-CD) at room temperature and testing their biological activity on TNFα-induced endothelial dysfunction. Both extracts counteracted TNFα effects on EAhy926 cells, down-modulating interleukin-6, interleukin-8, cyclooxygenase-2 and intracellular adhesion molecule-1, while increasing endothelial nitric oxide synthase levels. β-CD extract showed higher efficacy in improving endothelial function. These effects were abolished after pre-treatment with the oestrogen receptor inhibitor ICI1182,780. Moreover, the β-CD extract induced Akt activation and completely abolished the TNFα-induced p38MAPK phosphorylation. UAE and β-CD encapsulation provide an efficient extraction protocol that increases polyphenol bioavailability. Polyphenols from MI play a protective role on endothelial cells and may be further considered as oestrogen-like molecules with vascular protective properties.</p></div

Free IS levels, CD14⁺CD16⁺ monocytes and eGFR in study populations.

<p>(A) IS serum levels in AAA patients versus age- and e-GFR-matched control subjects, **p<0.01; (B) scattergram and regression line representing the relationship of CD14⁺CD16⁺monocyte percentages with IS serum levels (p = 0,014; r = 0,34); (C) and (D): scattergram and regression line representing the relationship between IS concentrations and eGFR in control subjects (empty circle, p = 0,02; r = -0,55) and AAA patients (grey triangle, p = 0,2; r = -0,17) respectively.</p

Protein expression in IS treated MdM.

<p>Western Blot analysis of total cell lysates for (A) AhR, (B) AhRR, (C) CYPOR, (D) Nrf2, (E) HO1,(F) PPARγ, (G) TGF-β, (H) TIMP-1, (I) COX2; values are normalized to GAPDH expression. (J) Gelatinolic activity of MMP-9. (K) IL-10 and (L) CCL2 content in IS-treated MdM conditioned medium. * p <0.05; ** p <0.01; *** p <0.001 vs. CTR.</p

Gene expression in IS-treated M/2M.

<p>RT-PCR for (A) AhR, (B) AhRR, (C) Nrf2, (D) HO1, (E) IL-6 and (F) CCL2. Results were plotted as mRNA fold induction in IS-treated versus Control cells; * p <0.05; ** p <0.01; ***p<0,001 vs. control cells.</p

Proposed scheme of IS effects on monocyte differentiation.

<p>Mild IS concentrations (1, 10, 20 μM) potentiate the detoxifying and anti-oxidant pathways of AhR and Nrf2 in monocytes (THP-1 cells), resulting in CD163 and HO1 overexpression and monocyte activation. Macrophages derived from IS-treated monocytes (IS-treated MdM) retain an upregulation of the AhR activity and features of a profibrotic, low inflammatory phenotype.</p

Effects of IS on THP-1 monocytes.

<p>Effect of 72 h treatment with 1, 10, 20 μM IS ±10 μM CH-223191 pretreatment (dashed bars) on (A) CD163 expression; § p< 0,05; **p< 0,01 vs. CTR; ***p<0,001 vs. CTR; (B) Proliferation rate expressed as % of cells in first (grey bars) and second (white bars) generation as evaluated by flow cytometry using the CFSE-DA probe; *p<0.05 vs. IS1; **p<0.01 vs. CTR; #p<0.01 vs. the corresponding IS alone concentrations; (C) Chemotaxis during 3 hours of incubation in a Boyden chamber after a 48 h IS treatment; CCL2 (10 ng/ml) and PBS were used as chemoattractant and negative control respectively. Values are expressed by means of cell number/field on a polycarbonate polyvinylpyrrolidone-free filter (5-nm pore size). *p<0.05; § p<0.05; #p<0.05 vs 20μM IS. (D) CCR2 membrane expression in cells after 72 h IS treatment evaluated by flow cytometry. (E) CCR2 and (F) CCL2 gene expression in cells after 24 h IS treatment as revealed by RT-PCR; **p< 0,01; ***p<0,001 vs. CTR. Results are plotted as mRNA fold induction (mean ± SEM) versus control cells.(G) Immunofluorescence localization of AhR (green) and AhRR (blue) after a 72 hour IS treatment; nuclei are counterstained with Propidium Iodide (red). (H) Protein expression of CYPOR (cytochrome p450 reductase); * p <0.05; ** p <0.01 vs. CTR.</p