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Fig. 1. Chemical structures. Structure of oleuropein (IUPAC name: methyl (4S,5E,6S)-4-[2-[2-(3,4-dihydroxyphenyl)ethoxy]-2-oxoethyl]-5-ethylidene-6-[(2S,3R,4S,5S,6R)-3,4,5-trihydroxy-6-(hydroxymethyl)oxan-2-yl]oxy-4H-pyran-3-carboxylate) and “oleuropein aglycone” (IUPAC name: methyl (4S,5E,6R)-4-[2-[2-(3,4-dihydroxyphenyl)ethoxy]-2-oxoethyl]-5-ethylidene-6-hydroxy-4H-pyran-3-carboxylate)
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Fig. 2. Stability of OLE and interaction with human salivary α-amylase. a Enzymatic hydrolysis of oleuropein as assessed using HPLC with diode array detection (see “Materials and methods”). Oleuropein was incubated at 37 °C with porcine pancreatin (79 μg protein/ml) (dark grey bars), a protein extract from rat intestine (124 μg protein/ml) (grey bars), or “hesperidinase” from A. niger (1.3 μg protein/ml) (light grey bars), in 0.05 M sodium phosphate buffer pH 6. Black bars indicate oleuropein with no added enzymes. Under similar conditions, quercetin-3-O-glucoside (100 μM), as control, was rapidly hydrolysed by the rat intestinal preparation (~ 30% remaining at 3 h, none detectable at 6 h) and by hesperidinase (~ 40% remaining after 6 h, none detectable at 70 h). Quercetin-3-O-glucoside was unaffected by pancreatin. b Inhibition of human salivary α-amylase using either amylose (black triangle) or amylopectin (black circle) as substrate, and measuring product using 2,4-dinitroasalicylic acid. c Effect of combining oleuropein with acarbose on inhibition of human salivary α-amylase. Black bars show OLE alone, dark grey bars show acarbose and the light grey bars show a combination: Treatment number 1: OLE (oleuropein, 0.8 mg/ml), acarbose, 2.5 µM; 2: OLE (oleuropein, 0.6 mg/ml), acarbose, 1.88 µM; 3: OLE (oleuropein, 0.4 mg/ml), acarbose, 1.25 µM. *p ≤ 0.05, **p ≤ 0.01
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Fig. 3. Inhibition of α-glucosidases by OLE. a Inhibition of rat intestinal maltase activity, using maltose (4 mM) as substrate, and quantifying glucose produced using a hexokinase-linked assay. b Inhibition of crude and purified rat intestinal maltase by OLE. Crude rat intestinal preparation (grey bars) and 2300-fold purified preparation of rat maltase (black bars) were incubated with maltose and inhibitor as described in the experimental section. The IC50 values for OLE inhibition were not significantly different (0.46 ± 0.14 and 0.40 ± 0.26 mg/ml of oleuropein respectively). c, d Inhibition of human maltase and sucrase activity using Caco-2/TC7 cells as the enzyme source. Glucose was quantified using a hexokinase-linked assay after the removal of interfering compounds by SPE. The IC50 values for maltase and sucrase were 1.28 ± 0.4 and 3.2 ± 1.0 mg/mL oleuropein, respectively
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Fig. 4. Effect of OLE on sugar transport. a Concentration dependence of OLE on transport of [14C(U)]-glucose (5 mM) across differentiated Caco-2/TC7 cell monolayers. ***p ≤ 0.001, compared to control with no OLE (n = 3 separate experiment with 6 replicates each). Error bars represent SD. b Effect of OLE (0.4 mg oleuropein /ml) on transport of [14C(U)]-glucose (5 mM) across differentiated Caco-2/TC7 cell monolayers at different apical [14C(U)]-glucose concentrations (n = 3 biological replicates with n = 6 wells per biological replicate; black bars). Error bars represent SD. **p ≤ 0.01, ***p ≤ 0.001 compared to control (grey bars) with no OLE. c Effect of OLE on glucose uptake by Xenopus oocytes expressing GLUT2. Two days post-cRNA microinjection, oocytes were incubated in 0.1 mM [14C(U)]-glucose with OLE for 5 min. Each data point represents the mean ± SEM of twelve replicates; IC50 = 0.012 ± 0.001 mg/ml. *p ≤ 0.05. d Effect of OLE on glucose uptake by Xenopus oocytes expressing GLUT5. One day post-cRNA microinjection, oocytes were incubated in 0.1 mM [14C(U)]-fructose with OLE for 5 min. Each data point represents the mean ± SEM of six replicates (18 oocytes)
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Fig. 5. Effect of OLE on sucrose hydrolysis. a Representative trace of separation of glucose, fructose and sucrose at 5 μM (0.04 μl loaded; 0.2 pmol each loaded onto the column) on a Carbopac PA20 column at 0.008 ml/min by HPAE-PAD on an ICS-4000 system. b, c Standard curves used for quantification of glucose and fructose respectively in the presence of final concentrations of 0 (black square), 0.04 (black circle), 0.12 (black triangle) or 0.24 (cross symbol) mg oleuropein/mL. d Transport experiments in Caco-2/TC7 cells. After incubation with 1, 5 or 25 mM sucrose (60 min) in the apical compartment, apical fructose (black), apical glucose (grey) and basolateral glucose (white) were quantified by HPAE-PAD. Basolateral fructose concentration was < 5 µM within the timeframe of the experiment. e Concentration-dependence of OLE on sucrose hydrolysis and glucose transport by differentiated Caco-2/TC7 cell monolayers after incubation with 5 mM sucrose (60 min). Apical glucose (black square), basolateral glucose (black square) and apical fructose (black triangle) were quantified. Results are means ± SEM, transport experiments were performed with n = 3 biological replicates with n = 6 wells per biological replicate. ***p ≤ 0.001, **p ≤ 0.01, *p ≤ 0.05
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Fig. 6. Effect of OLE or olives on postprandial blood glucose area under the curve during consumption of carbohydrates. Time dependence of blood glucose after consumption of control (black circle; with solid line) and test (black triangle; with dotted line) meals. a Study 1: double-blinded, randomised, crossover, placebo controlled in 24 healthy volunteers consuming bread (109 g containing 50 g carbohydrate) with OLE in capsules (500 mg, equivalent to 100 mg oleuropein). b Study 2: double-blinded, randomised, crossover, placebo controlled in 24 healthy volunteers consuming white bread (109 g containing 50 g carbohydrate) with OLE in capsules (2 × 500 mg, equivalent to 200 mg oleuropein). c Study 3: randomised, crossover, controlled study in 16 healthy volunteers consuming white bread (109 g containing 50 g carbohydrate) with 200 ml water with and without olives (100 g Kalamata olives containing 35 g oleuropein). d Study 4: randomised, crossover, controlled study on 10 healthy volunteers consuming white bread (109 g containing 50 g carbohydrate) with 200 ml water (control) or containing 125 mg dissolved OLE (50 mg oleuropein). e Study 5: randomised, crossover, controlled study in 10 volunteers consuming wholemeal bread (132 g containing 50 g carbohydrate) with 200 ml water (control) or containing 125 mg dissolved OLE (50 mg oleuropein). For additional details, see Table 2
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Fig. 7. Effect of OLE on postprandial blood glucose area under the curve during consumption of sugars. Time dependence of blood glucose after consumption of control (black circle; with solid line) and test (black triangle; with dotted line) meals. a Study 6: randomised, crossover, controlled study on 10 healthy volunteers consuming glucose (50 g) with 200 ml water (control) or containing 125 mg dissolved OLE (50 mg oleuropein). b Study 7: randomised, crossover, controlled study on 10 healthy volunteers consuming sucrose (50 g) with 200 ml water (control) or containing 125 mg dissolved OLE (50 mg oleuropein). c Study 8: randomised, crossover, controlled study on 10 healthy volunteers consuming sucrose (25 g) with 250 ml water (control) or containing 400 mg dissolved OLE (160 mg oleuropein). For additional details, see Table 3
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Fig. 8. Inter-individual differences in responses to sucrose and oleuropein. Randomised, crossover, controlled study on 10 healthy volunteers consuming sucrose (25 g) with 250 ml water (control) (a) or 250 ml water containing 400 mg dissolved OLE (160 mg oleuropein) (b), indicating changes in IAUC for each volunteer by linked data points from control to treatment, with mean of all data shown as dotted line
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Fig. 9. Summary of IAUC and peak blood glucose from intervention studies. Studies are numbered 1–9, and full details are given in Table 2
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Fig. 10. Mechanism of action of oleuropein on sucrose hydrolysis and subsequent sugar transport. Digestion of starch, maltose and sucrose in the gut lumen and transport of glucose across the intestinal barrier. The arrows indicate the sites where oleuropein exhibits inhibition according to our data
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