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

γH2AX staining patterns observed in EC and VSMC from patients with AAA.

<p><b>A</b>) Aortic aneurysmatic wall derived EC stained with anti-CD31 (green) and anti-γH2AX (red) mAbs. <b>B</b>) Aortic aneurysmatic wall derived VSMC stained with anti-α-smooth muscle actin (green) and anti-γH2AX (red) mAbs. Focal staining for γH2AX is evident (A, B). Nuclei stained with DAPI (A, B). Magnification, 100× (A, B). <b>C</b>) γH2AX-positive foci were significantly more abundant in EC and VSMC from AAA wall compared to controls (p = 0.006 and p = 0.010 respectively).</p

Telomere length and oxidative DNA damage in lymphocytes from each individual AAA patient and control.

<p><b>A</b>) Telomere length of peripheral blood lymphocytes from 23 patients with abdominal aortic aneurysms (AAA), and 34 healthy donors. Bars represent the mean. <b>B</b>) Lymphocytes interphase nuclei from an AAA patient hybridized with Cy3-PNA telomeric probe (red signals). <b>C</b>) Oxidative DNA damage in peripheral blood lymphocytes from 19 patients with AAA, and 23 healthy donors. <b>D</b>) A representative immunostaining of anti-8-oxo-dG (green) of blood lymphocytes from an AAA patient. Arrows show nuclei intensively staining for 8-oxo-dG (green). <b>D</b>) Immunostaining of anti-8-oxo-dG (green) of blood lymphocytes from an healthy donor. Arrow shows nucleus with several small positive regions.</p

Telomere length of EC, VSMC, blood lymphocytes and epidermal cells from patients with AAA and controls.

<p><b>A</b>) Telomeres length in EC from 21 AAA patients and in EC from 20 normal aorta. <b>B</b>) Telomeres length in VSMC from 21 AAA patients and in VSMC from 20 normal aorta. <b>C</b>) Telomeres length in epidermal cells from patients with 11 AAA and in these same cells from 6 controls. <b>D</b>) Telomeres length in peripheral blood lymphocytes in 23 AAA patients and in 34 controls.</p

Median telomere fluorescence.

<p>A minimum of 20 nuclei were scanned for every sample and the mean value of the fluorescence ratios of all cells analyzed was calculated.</p

Telomere length of EC and VSMC from patients with AAA measured using Q-FISH and immunofluorescence.

<p><b>A</b>) Aortic aneurysmatic wall derived EC stained with anti-CD31 mAb (green). The inset shows the nuclei analyzed by Q-FISH. <b>B</b>) EC interphase nuclei hybridized with Cy3-PNA telomeric probe (red signals). <b>C</b>) Aortic aneurysmatic wall derived VSMC stained with anti-α-smooth muscle actin mAb (green). <b>D</b>) VSMC nuclei hybridized with Cy3-PNA telomeric probe (red signals). DAPI was used to label nuclei.</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

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

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