Cardioprotective potential of oleuropein, hydroxytyrosol, oleocanthal and their combination: Unravelling complementary effects on acute myocardial infarction and metabolic syndrome

Abstract

Clinical studies have previously established the role of olive products in cardiovascular disease (CVD) prevention, whilst the identification of the responsible constituents for the beneficial effects is still pending. We sought to assess and compare the cardioprotective potential of oleuropein (OL), hydroxytyrosol (HT), oleocanthal (OC) and oleanolic Acid (OA), regarding Ischemia/Reperfusion Injury (IRI) and CVD risk factors alleviation. The scope of the study was to design a potent and safe combinatorial therapy for high-cardiovascular-risk patients on a bench-to-bedside approach. We evaluated the IRI-limiting potential of 6-weeks treatment with OL, HT, OC or OA at nutritional doses, in healthy and metabolic syndrome (MS)-burdened mice. Three combinatorial regimens were designed and the mixture with preponderant benefits (OL-HT-OC, Combo 2), including infarct sparing and antiglycemic potency, compared to the isolated compounds, was further investigated for its anti-atherosclerotic effects. In vivo experiments revealed that the combination regimen of Combo 2 presented the most favorable effects in limiting infarct size and hyperglycemia, which was selected to be further investigated in the clinical setting in Chronic Coronary Artery Syndrome (CCAS) patients. Cardiac function, inflammation markers and oxidative stress were assessed at baseline and after 4 weeks of treatment with the OL-HT-OC supplement in the clinical study. We found that OL, OC and OA significantly reduced infarct size in vivo compared to Controls. OL exhibited antihyperglycemic properties and OA attenuated hypercholesterolemia. OL-HT-OA, OL-HT-OC and OL-HT-OC-OA combination regimens were cardioprotective, whereas only OL-HT-OC mitigated hyperglycemia. Combo 2 cardioprotection was attributed to apoptosis suppression, enhanced antioxidant effects and upregulation of antioxidant enzymes. Additionally, it reduced atherosclerotic plaque extent in vivo. OL-HT-OC supplement ameliorated cardiac, vascular and endothelial function in the small-scale clinical study. Conclusively, OL-HT-OC combination therapy exerts potent cardioprotective, antihyperglycemic and anti-atherosclerotic properties in vivo, with remarkable and clinically translatable cardiovascular benefits in high-risk patients.

Keywords: Cardiometabolic syndrome; Hydroxytyrosol; Ischemia-reperfusion injury; Oleanolic acid; Oleocanthal; Oleuropein.

Graphical abstract

Fig. 1

Oleuropein, oleocanthal and oleanolic acid reduce infarct size in MS-burdened mice.Graphs ofA.Infarct/Risk Area Ratio % andB.Area at risk % of NS- and DMSO 5%-treated mice.C.Infarct/Risk Area Ratio % andD.Area at risk % of NS-, OL- and HT-treated mice.E.Infarct/Risk Area Ratio % andF.Area at risk % of DMSO 5%-, OC- and OA-treated mice. A, B Unpaired t-test. C, D, E, F One-way ANOVA, Tukey post hoc test. *p < 0.05, **p < 0.01, ***p < 0.005. All values are presented as Mean ± SEM (n = 5-8). DMSO: Dimethyl sulfoxide; HT: Hydroxytyrosol; NS: Normal saline; OA: Oleanolic Acid; OC: Oleocanthal; OL: Oleuropein.

Fig. 2

Oleuropein attenuates hyperglycemia and increases insulin levels in plasma.Graphs ofA.Fasting blood glucose of NS-, OL- and HT-treated mice.B.Fasting blood glucose of DMSO 5%-, OC- and OA-treated mice.C.Fasting insulin levels in plasma at the 14^thweek.D.Calculated HOMAIR index.E.Glucose curves for all treated groups and the ND group.F.Calculated Area Under Curve for each glucose curve, including the ND group. A, B Two-way ANOVA, Tukey post hoc test. C, D, F One-way ANOVA, Tukey post hoc test.⁺⁺p < 0.01 vs Veh (DMSO 5 %),^###p < 0.001 vs HT, *p < 0.05, **p < 0.01,^ꞎꞎp<0.01 vs ND,^ꞎꞎꞎꞎp<0.0001 vs ND. All values are presented as Mean ± SEM (n = 5-13). DMSO: Dimethyl sulfoxide; HT: Hydroxytyrosol; ND: Normal Diet; OA: Oleanolic Acid; OC: Oleocanthal; OL: Oleuropein.

Fig. 3

Oleanolicacid reduces total cholesterol but none of the treatments mitigates obesity.Graphs ofA.Total cholesterol and triglycerides levels of NS-, OL- and HT-treated mice (mg/dl).B.Total cholesterol and triglycerides levels of DMSO 5%-, OC- and OA-treated mice (mg/dl).C.Body weight of mice treated with NS, OL, HT.D.Body weight of mice treated with DMSO 5 %, OC, OA.E.Relative weight of epidydimal adipose tissue in mice treated with NS, OL, HT.F.Relative weight of epidydimal adipose tissue in mice treated with DMSO 5 %, OC, OA. A, B, C, D Two-way ANOVA, Tukey post hoc test. E, F One-way ANOVA, Tukey post hoc test. *p < 0.05, ***p < 0.001. All values are presented as Mean ± SEM (n = 5-14). HT: Hydroxytyrosol; ND: Normal Diet; OA: Oleanolic Acid; OC: Oleocanthal; OL: Oleuropein.

Fig. 4

Combinatorial treatments 1, 2 and 3 significantly reduce the infarct size in similar extent to the cardioprotective oleuropein, oleocanthal and oleanolic acid.Graphs ofA.Infarct/Risk Area Ratio % andB.Area at Risk % of mice treated with combinatorial treatments or DMSO 5 %.C.Infarct/Risk Area Ratio % andD.Area at Risk % of all mice treated with the isolated compounds or combinations. A, B, C, D One-way ANOVA, Tukey post hoc test. **p < 0.01, ***p < 0.001, ****p < 0.0001. All values are presented as Mean ± SEM (n = 6-11) Combo: Combinatorial therapy; DMSO: Dimethyl sulfoxide; HT: Hydroxytyrosol; ND: Normal Diet; OA: Oleanolic Acid; OC: Oleocanthal; OL: Oleuropein.

Fig. 5

Combo 2 (OL, HT, OC) reduces fasting glucose levels and increases insulin in plasma, with no significant effects on hypercholesterolemia and obesity.Graphs ofA.Fasting blood glucose of DMSO 5%-, Combo 1-, Combo 2- and Combo 3- treated mice.B.Fasting insulin level in plasma at the 14^thweek.C.Calculated HOMAIR index.D.Glucose curves.E.Calculated Area Under Curve for each glucose curve.F.Body weight of DMSO 5%-, Combo 1-, Combo 2- and Combo 3- treated mice.G.Relative adipose tissue weight.H.Total cholesterol and Triglycerides in plasma. A, F, H Two-way ANOVA, Tukey post hoc test. B, C, E, G One-way ANOVA, Tukey post hoc test.^##p < 0.01,^####p < 0.0001 vs Baseline,^$p < 0.05,^$$p < 0.01,^$$$p < 0.001,^$$$$p < 0.0001 vs 8 weeks,^ꞎꞎꞎꞎp<0.0001 vs ND, *p < 0.05, **p < 0.01. All values are presented as Mean ± SEM (n = 5-11). DMSO: dimethyl sulfoxide; Combo 1: OL, HT, OA; Combo2: OL, HT, OC; Combo 3: OL, HT, OC, OA.

Fig. 6

Oleuropein, Oleocanthal and Combo 2 reduce the apoptotic ratio of Bax/BcL-xL, while Oleocanthal and Combo 2 additionally upregulate Nrf2 levels and the antioxidant enzymes MnSOD and CAT.A.Representative blots at 2 h of reperfusion of CAT, GAPDH, Bax, BcL-xL, α-Actinin, p-Akt (S473), t-Akt, HO-1, p-GSK-3β (S9), t-GSK-3β and MnSOD.B.Representative blots at 2 h of reperfusion of Nrf2, gp91^phox, p47^phox, NOX-1 and β-tubulin. Relative densitometric graphs after normalization to total protein ofC.Bax/GAPDH.D.BcL-xL/GAPDH.E.Bax/BcL-xL.F.p-GSK-3β (S9)/t-GSK-3β.G.t-GSK-3β/α-Actinin.H.p-Akt (S473)/t-Akt.I.t-Akt/α-Actinin.J.MnSOD/a-Actinin.K.CAT/GAPDH.L.HO-1/α-Actinin.M.Nrf2/β-tubulin,N.NOX-1/β-tubulin,O.gp91^phox/β-tubulin,P.p47^phox/β-tubulin.C, M, N, O, P Kruskal-Wallis, Dunn's post hoc test. D, E, F, G, H, I, J, K, L, One-way ANOVA, Tukey post hoc test. *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001. All values are presented as Mean ± SEM (n = 6-8). Akt: Protein Kinase B; Bax: BCL2 Associated X; Bcl-xL: B-cell lymphoma-extra-large; CAT: Catalase; Combo 2: Combinatorial treatment with OL, HT, OC; DMSO: Dimethyl sulfoxide; GAPDH: Glyceraldehyde-3-phosphate dehydrogenase; HO-1: Heme oxygenase 1; HT: Hydroxytyrosol; MnSOD: Manganese-superoxide dismutase; ND: Normal Diet; NOX -1: NADPH oxidase 1, Nrf2: nuclear factor erythroid 2–related factor 2;OC: Oleocanthal; OL: Oleuropein.

Fig. 7

Hydroxytyrosol and Oleocanthal limit NETosis in vitro, while Hydroxytyrosol, Oleocanthal and Combo 2 significantly reduce atherosclerotic lesion extent and oxidative stress in vivo.Representative images ofA.andB.the effect of the isolated compounds and their combination on neutrophil MPO release, with or without PMA activation, under fluorescent microscope andCthe relative quantification of % netosis for each treatment.D.ApoE^−/−aortic sections stained with Oil Red O under brightfield microscope (scale is 150 μm) andE. Oil Red O (ORO) densitometry (% of total aortic area) in aortic rings of ApoE^−/−mice after 4-week treatment with OL, HT, OC or Combo 2.F.Representative images of aortic segments stained with DHE and DAPI to identify reactive oxygen species formation (scale is 75μΜ) andE.bar plots with relative fluorescent intensity quantification of DHE/DAPI for all experimental groups after 4-week treatment with OL, HT, OC or Combo 2. Bar plots ofH. MDA andI.ox-LDL andJ. total plasma cholesterol in the circulation of ApoE^−/−mice after the 4-week pretreatment. E, G, H, I, J One-way ANOVA, Tukey post hoc test. *p < 0.05, **p < 0.01, ***p < 0.001. All values are presented as Mean ± SEM (n = 5-6 for in vivo and n = 3 biological replicates for in vitro experiments). Combo 2: Combinatorial treatment with OL, HT, OC; DAPI: 49,6‐diamidino‐2‐phenylindole; DHE: dihydroethidium DMSO; Dimethyl sulfoxide; HT: Hydroxytyrosol; LDL: Low density lipoprotein; MDA: malondialdehyde OC: Oleocanthal; OL: Oleuropein; ORO: Oil Red O; TBARs: thiobarbituric acid reactive substances. (For interpretation of the references to colour in this figure legend, the reader is referred to the Web version of this article.)