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* Department of Human Physiology and Pharmacology,
Department of Chemistry, University of Rome "La Sapienza," Rome, Italy
3To whom correspondence should be addressed. E-mail: giuseppina.togna{at}uniroma1.i.
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ABSTRACT |
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 TOP ABSTRACT MATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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KEY WORDS: • isochromans • antioxidant • platelet aggregation • olive oil • cardiovascular diseases
Interest in antioxidants for the treatment of human disease and in the role of dietary antioxidants for disease prevention has been sustained for at least two decades. It has been suggested that the consumption of certain foods including fruits, vegetables and red wine may be beneficial in the prevention of cardiovascular diseases (1–3). Several in vitro studies have shown that this beneficial effect may be explained in part by the presence of polyphenols. In fact, polyphenols benefit the cardiovascular system because of their antioxidant and free radical-scavenging properties, which decrease LDL oxidation (4,5) and diminish platelet aggregation (6–8). Furthermore, the ability of polyphenols to induce endothelium-dependent vasorelaxation by enhanced nitric oxide synthesis (3,9,10) or by enhanced biological activity (11–13) has also been demonstrated. This pharmacologic effect is important because of the vasorelaxant and antiaggregant properties of this endothelial mediator.
The effects of certain red wine polyphenolic compounds, such as resveratrol (4,14–17) and quercetin (8,18,19), have been extensively investigated to determine the relationship between dietary phenolic compounds and decreased risk of cardiovascular diseases. However, the effects of polyphenolic compounds contained in other foods, such as olive oil, have received less attention and little information exists regarding its phenol fraction biological activities.
We have shown that 2-(3',4'-dihydroxyphenyl)ethanol (also known as hydroxytyrosol), a simple polyphenol found in olives (20,21) and olive oil (22), can react with aldehydes and ketones under very mild conditions to produce other polyphenolic compounds such as 6,7-dihydroxyisochromans (23). Because of the presence of many carbonyl compounds (24–26) we have been able to demonstrate the occurrence of this reaction in olive oil by preparing isochromans in this medium (27). We have shown that two of the synthesized isochromans are naturally present in this matrix (28). After a preliminary screening test for the biological activity of all the synthesized isochromans, we evaluated the antiplatelet activity and antioxidant power of the two isochromans found in extra-virgin olive oil.
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MATERIALS AND METHODS |
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TOPABSTRACTMATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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The synthesized compounds were initially tested for the ability to inhibit sodium arachidonate (SA)-induced aggregation of human platelets. Platelet aggregation was studied in a light-transmission aggregometer using Born’s turbidimetric method (29). Blood from healthy drug-free volunteers was collected in 130 mmol/L of sodium citrate (9:1). Platelet-rich plasma (PRP) was obtained by centrifugation at 200 x g for 20 min and platelet count was adjusted to 2·108 platelets/L PRP by adding platelet-poor plasma (blood centrifuged at 2000 x g for 10 min).
All compounds, solubilized in Tris buffer (pH = 7.8), were incubated in PRP samples for different times (3 and 15 min) before the addition of SA, a platelet agonist, at 130 mg/L of PRP. Each compound was first assayed at a concentration of 50 µmol/L, then reassayed at concentrations decreasing or increasing by 5 µmol/L each time. This allowed the identification of the minimal concentration required to completely inhibit platelet aggregation (MIC) for each compound. The MIC was then confirmed in subsequent tests by using three PRP samples. On the basis of the MIC, a scale of antiaggregant activity was constructed with the following cut-off values: high, 
10 µmol/L; intermediate >10–20 µmol/L; and low, 
30 µmol/L. We then investigated the two isochromans found in olive oil showing high antiaggregant activity in the screening test.
Platelet aggregation studies and thromboxane release.
The experiments were performed on compounds L116 and L137 using agonists that induce platelet aggregation involving (SA and collagen) or not involving (ADP) reactive oxygen species (ROS) production (30–32). Compounds were assayed at final concentrations ranging from 0.1 to 20 µmol/L. After a 15-min incubation, PRP samples were stimulated with aggregating concentrations of platelet agonists: SA (130 mg/L PRP), collagen (2 mg/L) and ADP (2 mg/L); and the platelet response was recorded for 10 min. After addition of 20 µmol/L of indomethacin, PRP was centrifuged (5 min at 2000 x g) and the amount of thromboxane (TX) B2, the stable breakdown product of TXA2 released by platelets, was measured in the supernatant by RIA (33).
Release of tritiated arachidonic acid.
The effects of L116 and L137 on tritiated arachidonic acid ([3H]AA) release by prelabeled platelets were investigated using the method of Leoncini and Signorello (34). Briefly, platelets obtained by centrifuging PRP were resuspended in pH 7.4 Tyrode’s-Hepes buffer containing 2 g/L of bovine serum albumin (BSA) and incubated with 37 MBq/L [3H]AA for 1 h at 37°C. Platelets were then washed twice to remove any remaining free arachidonate using Tyrode’s-Hepes buffer BSA-free and resuspended at a concentration of 3·1011 platelet/L with Tyrode’s-Hepes buffer with BSA. Aliquots of 500 µL containing labeled platelets were preincubated for 10 min at 37°C, then test compound (50 to 200 µmol/L) was added 20 min prior to thrombin (500 U/L) or collagen (5 mg/L) stimulation. Reactions were stopped at 5 min by adding 50 µL of a cold block mix (5 mmol/L of EGTA, 5 mmol/L of theophylline and 0.2 mg/L of prostaglandin E1) and the samples centrifuged (2000 x g for 10 min). The [3H]AA released into the supernatant was determined by liquid scintillation counting of 450 µL aliquots.
Radical-scavenging capacity (DPPH test).
The free radical-scavenging capacities of L116 and L137 were determined by the 1,1-diphenyl-2-picryl-hydrazyl (DPPH) test (35). An aliquot (0.1 mL) of methanol solution containing varying concentrations (5–100 µmol/L) of L116 and L137 was mixed with 3.9 mL of a 100 µmol/L DPPH methanol solution. Hydroxytyrosol, quercetin and resveratrol (2–200 µmol/L) were used as reference compounds. Absorbance at 515 nm was measured until the reaction reached a steady state (UV-160A spectrophotometer; Shimadzu, Kyoto, Japan). Measurements were performed in duplicate.
Antioxidant activity of the compounds was expressed as concentration of antioxidant needed to decrease initial DPPH concentration by 50% (EC50) and antiradical efficiency (AE = 1/EC50). The EC50 was calculated by plotting the percentage of DPPH remaining at steady state compared with mol antioxidant/mol DPPH (35).
Statistical analysis.
Continuous variables are presented as mean ± SD. The EC50 data were tested by one-way ANOVA and the Duncan’s test was used for post hoc comparison. Other data were tested by two-way ANOVA (treatment x concentration) with post hoc Dunnett’s test. Significant interactions indicated dose-dependent effects. SA and collagen data were analyzed separately. When the model showed a lack of homogeneity of the variances, the effects were subsequently verified by nonparametric as well as by parametric statistical testing on log-transformed data. If no substantial differences were revealed between the models, those shown pertain to the initial model. All statistical analyses were performed with STATISTICA (Cambridge, MA) and the P-value for significant difference was 0.05.
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RESULTS |
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TOPABSTRACTMATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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All of the compounds tested inhibited human platelet aggregation by SA at both times tested (Table 1). After 15 min incubation, compounds L137 and L116 exhibited high (10 µmol/L), compounds L112, L64, C216 and C 218 intermediate (>10–20 µmol/L) and L65 and C235 low (30 µmol/L) antiaggregating activity (Table 1). Because L116 and L137 are the isochromans in extra-virgin olive oil (28), these two compounds were chosen for further testing.
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Both L116 and L137 dose-dependently inhibited human platelet aggregation in response to SA or collagen, starting from a concentration of 1 µmol/L (P < 0.001) (Table 2). L116 and L137 also dose-dependently inhibited platelet thromboxane production (P < 0.001) (Table 2). On the contrary, at all concentrations tested these compounds did not affect the platelet reactivity to ADP in terms of aggregating response and TXB2 production (data not shown).
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L116 and L137 reduced [3H]AA release induced by thrombin (P < 0.001) and collagen (P < 0.001) at 100 µmol/L (Fig. 2). The inhibiting effect of L137 on the [3H]AA release induced by collagen was greater than that induced by thrombin (P < 0.001). A similar pattern was seen with L116 although the effect was not significant (P = 0.07).
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The antioxidant power (EC50) of the compounds examined differed (P < 0.001) (Table 3). L116 had a greater antioxidant power than L137 (P = 0.02) and resveratrol (P < 0.001), but did not differ from hydroxytyrosol or quercetin. Furthermore, L137 was more active than resveratrol (P < 0.001), but exhibited a lower antioxidant activity compared with hydroxytyrosol (P = 0.001) or quercetin (P = 0.007). Kinetic behavior (time to reach steady state) also differed among the tested compounds, being slow for hydroxytyrosol, quercetin and resveratrol (1–3 h) and fast for L116 and L137 (1–5 min).
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DISCUSSION |
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TOPABSTRACTMATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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A lipophilic and planar substituent, like the phenyl in L137, may enhance the activity of a compound by favoring entrance into the cell. On the contrary, a hydrophilic as in C235 (hydroxy-phenyl), or a nonplanar substituent as in L65 and L64 (propyl or isopropyl), could hinder this fundamental step. Furthermore, the greater activity of L116 compared with C218 and C235 may be related to the easy formation of an intramolecular hydrogen bond between the hydroxyl and methoxy groups.
Because L116 and L137 are the isochromans recently found in extra-virgin olive oil (28), a major component of the Mediterranean diet, we restricted our examination to these compounds. L116 and L137 inhibited the platelet response to SA and collagen, but not to ADP.
It has been reported that production of oxygen radicals appears to be more important during the initial phases of platelet activation when induced by SA and collagen than by the other agonists, such as ADP (30–32). This suggests that the capacity of L116 and L137 to interfere with platelet function is related to their radical-scavenging activity. The lack of effect when using ADP supports this hypothesis.
It is interesting to compare the platelet antiaggregating effects of L116 and L137 with the reported activity of the parent compound, hydroxytyrosol. For hydroxytyrosol, previous studies report a 50% inhibiting concentration (IC50) on platelet aggregation induced by collagen of 67 µmol/L (36,37). Surprisingly, 400 µmol/L of hydroxytyrosol was required to completely inhibit collagen-induced aggregation (36). On the basis of these data, L116 and L137 appear to be more active than hydroxytyrosol on collagen-induced platelet aggregation. Unlike hydroxytyrosol (IC50 = 27 µmol/L) (36), L116 and L137 did not modify the platelet response to ADP up to 30 µmol/L.
The inhibiting effect of L116 and L137 on collagen-induced platelet aggregation was comparable to that reported for quercetin (8) and resveratrol (14). Moreover, in terms of platelet response to SA, L116 and L137 were similar to quercetin (38), and considerably more active than resveratrol (17). Furthermore, resveratrol and quercetin required concentrations much higher (IC50 > 100 µmol/L) than those assayed in our experiments for L116 and L137 to inhibit platelet aggregation by ADP (19,38,39).
L116 and L137 inhibited arachidonic acid mobilization from platelet membrane phospholipids induced by thrombin and to a greater degree by collagen, suggesting a direct inhibition of phospholipase A2 (PLA2) by these isochromans. Thrombin induces release of arachidonic acid from membrane phospholipids by directly stimulating PLA2 without (unlike collagen) ROS production (40,41). Therefore, the greater level of inhibition exhibited when using collagen could be the result of an additional indirect effect of the compounds on PLA2 mediated by their scavenging activity (30,42,43).
Because it has already been ascertained that free radicals are involved in the pathology of vascular disease, these findings indicate that nutraceuticals other than the often studied quercetin or resveratrol also possess antioxidant activity that could contribute to the beneficial effects of the Mediterranean diet in the prevention of vascular disease.
The quantities of these two hydroxy-isochromans in samples of extra-virgin olive oil are very low and extremely variable, ranging from 20 to 390 ng/kg for compound L116 and 8 to 1400 ng/kg for compound L137 (28); this range is the result of variable amounts of hydroxytyrosol and carbonyl compounds related to different olive cultivars and harvest times. Thus, the level of these isochromans in olive oil may be too low to produce significant antiplatelet effects in vivo, even if we cannot exclude the possibility that other isochromans among those we have prepared could be present in olive oil. In fact, carbonylic compounds are numerous in this matrix (24–26) and nearly all can react with hydroxytyrosol to produce isochromans. The time required for the formation of isochromans (27) is very long (one month or more), so larger amounts of these compounds could be present in aged extra-virgin olive oils. Furthermore, the total daily intake of olive oil phenols in the Mediterranean region is estimated to be 10–20 mg (44) and therefore a synergic effect of phenolic oil constituents demonstrated for other phenolic compounds (8) cannot be excluded.
Considering the simplicity of synthesis methodology (23) and the good antiplatelet activity exerted by L116 and L137, these isochromans could also represent positive factors contributing to the production of safe nutritional products and additives with useful antioxidant properties.
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FOOTNOTES |
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2 This study received financial support from Italian Ministero dell’Universitá e della Ricerca and Consiglio Nazionale della Ricerche.
4 Abbreviations used: AE, antiradical efficiency; BSA, bovine serum albumin; DPPH, 1,1-diphenyl-2-picryl-hydrazyl; EC50, 50% efficient concentration (concentration required to scavenger DPPH radicals by 50%); [3H]AA, tritiated arachidonic acid; IC50, 50% inhibiting concentration; MIC, minimal inhibiting concentration; PLA2, phospholipase A2; PRP, platelet-rich plasma; TXB2, thromboxane B2; ROS, reactive oxygen species; SA, sodium arachidonate.
Manuscript received 27 February 2003. Initial review completed 1 April 2003. Revision accepted 30 May 2003.
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LITERATURE CITED |
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TOPABSTRACTMATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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1. Howard, B. V. & Kritchevsky, D. (1999) Phytochemicals and cardiovascular disease. Circulation 95:2591-2593.
2. Visioli, F., Borsani, L. & Galli, C. (2000) Diet and prevention of coronary heart disease: the potential role of phytochemicals. Cardiovasc. Res. 47:419-425.
3. Diebolt, M., Bucher, B. & Andriantsitohaina, R. (2001) Wine polyphenols decrease blood pressure, improve NO vasodilatation, and induce gene expression. Hypertension 38:159-165.
4. Frankel, E. N., Waterhouse, A. L. & Kinsella, J. E. (1993) Inhibition of human LDL oxidation by resveratrol. Lancet 341:1103-1104.[Medline]
5. de Rijke, Y. B., Demacker, P.N.M., Assen, N. A., Sloots, L. M., Katan, M. B. & Stalenhoef, A.F.H. (1999) Red wine consumption and oxidation of low-density lipoproteins. Lancet 345:325-326.
6. Pace-Asciak, C. R., Rounova, O., Hahn, S. E., Diamandis, E. P. & Goldberg, D. M. (1996) Wines and grape juices as modulators of platelet aggregation in healthy human subjects. Clin. Chim. Acta. 246:163-182.[Medline]
7. Ruf, J. C. (1999) Wine and polyphenols related to platelet aggregation and atherothrombosis. Drug Exp. Clin. Res. 25:125-131.
8. Pignatelli, P., Pulcinelli, F. M., Celestini, A., Lenti, L., Ghiselli, A., Gazzanica, P. P. & Violi, F. (2000) The flavonoids quercetin and catechin synergistically inhibit platelet function by antagonizing the intracellular production of hydrogen peroxide. Am. J. Clin. Nutr. 72:1150-1155.
9. Andriambeloson, E., Stoclet, J. C. & Andriantsitohaina, R. (1999) Mechanism of endothelial nitric oxide-dependent vasorelaxation induced by wine polyphenols in rat thoracic aorta. J. Cardiovasc. Pharmacol. 33:248-254.[Medline]
10. Lemos, V. S., Freitas, M. R., Muller, B., Lino, Y. D., Queiroga, C.E.G. & Cortes, S. F. (1999) Dioclein, a new nitric oxide- and endothelium-dependent vasodilator flavonoid. Eur. J. Pharmacol. 386:41-46.[Medline]
11. Fitzpatrick, D. F., Hirschfield, S. L., Ricci, T., Jansen, P. & Coffey, R. G. (1995) Endothelium-dependent vasorelaxation caused by various plant extracts. J. Cardiovasc. Pharmacol. 26:90-95.[Medline]
12. Carr, A. & Frei, B. (2000) The role of natural antioxidants in preserving the biological activity of endothelium-derived nitric oxide. Free Radical Biol. Med. 28:1806-1814.[Medline]
13. Duarte, J., Perez-Palencia, R., Vargas, F., Ocete, M. A., Perez-Vizcaino, F., Zarzuelo, A. & Tamargo, J. (2001) Antihypertensive effects of the flavonoid quercetin in spontaneously hypertensive rats. Br. J. Pharmacol. 133:117-124.[Medline]
14. Bertelli, A. A., Giovannini, L., Bernini, W., Migliori, M., Fregoni, M., Bavaresco, L. & Bertelli, A. (1996) Antiplatelet activity of cis-resveratrol. Drug Exp. Clin. Res. 22:61-63.
15. Rotondo, S., Rajtar, G., Manarini, S., Celardo, A., Rotillo, D., de Gaetano, G., Evangelista, V. & Cerletti, C. (1998) Effect of trans-resveratrol, a natural polyphenolic compound, on human polymorphonuclear leukocyte function. Br. J. Pharmacol. 123:1691-1699.[Medline]
16. Dobrydneva, Y., Williams, R. L. & Blackmore, P. F. (1999) Trans-resveratrol inhibits calcium influx in thrombin-stimulated human platelets. Br. J. Pharmacol. 128:149-157.[Medline]
17. Fremont, L. (2000) Biological effects of resveratrol. Life Sci 66:663-673.[Medline]
18. Janssen, K., Mensink, R. P., Cox, F. J., Harryvan, J. L., Hovenier, R., Hollman, P. C. & Katan, M. B. (1998) Effects of the flavonoids quercetin and apigenin on hemostasis in healthy volunteers: results from an in vitro and a dietary supplement study. Am. J. Clin. Nutr. 67:255-262.[Abstract]
19. Pace-Asciak, C. R., Hahn, S., Diamandis, E. P., Soleas, G. & Goldberg, D. M. (1995) The red wine phenolics trans-resveratrol and quercetin block human platelet aggregation and eicosanoid synthesis: implications for protection against coronary heart disease. Clin. Chim. Acta. 235:207-219.[Medline]
20. Romani, A., Mulinacci, N., Pinelli, P., Vincieri, F. F. & Cimato, A. (1999) Polyphenolic content in five tuscany cultivars of Olea europaea L. J. Agric. Food Chem. 47:964-967.[Medline]
21. Bianco, A. & Uccella, N. (2000) Biophenolic components of olives. Food Res. Int. 33:475-485.
22. Montedoro, G., Servili, M., Baldioli, M. & Miniati, E. (1992) Simple and hydrolyzable phenolic compounds in virgin olive oil: their extraction separation and quantitative and semi-quantitative evaluation by HPLC. J. Agric. Food Chem. 40:1571-1576.
23. Guiso, M., Marra, C. & Cavarischia, C. (2001) Isochromans from 2-(3', 4'-dihydroxy) phenylethanol. Tetrahedron Lett 42:6531-6534.
24. Servili, M., Selvaggini, R., Taticchi, A. & Montedoro, G. (2001) Headspace composition of virgin olive oil evaluated by solid phase microextraction: relationships with the oil sensory characteristics. Special Publication - Royal Society of Chemistry 274(Food Flavors and Chemistry):236-247.
25. Cartoni, G. P., Coccioli, F., Jasionowska, R. & Ramirez, D. (2000) HPLC analysis of the benzoic and cinnamic acids in edible vegetable oils. It. J. Food Sci. 12:163-167.
26. Kubo, I. & Kinst-Hori, I. (1999) Tyrosinase inhibitory activity of the olive oil flavor compounds. J. Agric. Food Chem. 47(11):4574-4578.[Medline]
27. Guiso, M., Marra, C., Togna, G. I. & Coccioli, F. (2001) "L’Olea Europaea: un’occasione d’incontro tra ricerca scientifica e alimenti" XXVII Convegno Nazionale della Divisione di Chimica Organica. P146, Trieste :3-7 settembre 2001.
28. Bianco, A., Coccioli, F., Guiso, M. & Marra, C. (2001) The occurrence in olive oil of a new class of phenolic compounds: hydroxy-isochromans. Food Chem 77:405-411.
29. Born, G.V.R. (1962) Aggregation of blood platelets adenosine diphosphatase and its reversal. Nature 194:927-929.[Medline]
30. Iuliano, L., Praticò, D., Bonavita, M. S. & Violi, F. (1992) Involvement of phospholipase A2 in H2O2-dependent platelet aggregation. Platelets 2:87-90.
31. Praticò, D., Iuliano, L., Pulcinelli, F. M., Bonavita, M. S., Gazzaniga, P. P. & Violi, F. (1992) Hydrogen peroxide triggers activation of human platelets selectively exposed to nonaggregating concentrations of arachidonic acid and collagen. J. Lab. Clin. Med. 119:364-370.[Medline]
32. Caccese, D., Praticò, D., Ghiselli, A., Natoli, S., Pignatelli, P., Sanguigni, V., Iuliano, L. & Violi, F. (2000) Superoxide anion and hydroxyl radical release by collagen-induced platelet aggregation. Role of arachidonic acid metabolism. Thromb. Haemost. 83:485-490.[Medline]
33. Patrignani, P., Filabozzi, P. & Patrono, C. (1982) Selective cumulative inhibition of platelet thromboxane production by low-dose aspirin in healthy subjects. J. Clin. Invest. 69:1366-1372.
34. Leoncini, G. & Signorello, M. G. (1999) N-ethylmaleimide-stimulated arachidonic acid release in human platelets. Biochem. Pharmacol. 57:785-791.[Medline]
35. Parejo, I., Codina, C., Petrakis, C. & Kefalas, P. (2000) Evaluation of scavenging activity assessed by Co(II)/EDTA-induced luminol chemiluminescence and DPPH (2, 2-diphenyl-1-picrylhydrazyl) free radical assay. J. Pharmacol. Toxicol. Method 44:507-512.[Medline]
36. Petroni, A., Blasevich, M., Salami, M., Papini, N., Montedoro, G. F. & Galli, C. (1995) Inhibition of platelet aggregation and eicosanoid production by phenolic components of olive oil. Thromb. Res. 78:151-160.[Medline]
37. Petroni, A., Blasevich, M., Salami, M., Servili, M., Montedoro, G. F. & Galli, C. (1994) A phenolic antioxidant extracted from olive oil inhibits platelet aggregation and arachidonic acid metabolism in vitro. World Rev. Nutr. Diet. 75:169-172.[Medline]
38. Tzeng, S. H., Ko, W. C., Ko, F. N. & Teng, C. M. (1991) Inhibition of platelet aggregation by some flavonoids. Thromb. Res. 64:91-100.[Medline]
39. Beretz, A., Cazenave, J- & P. & Anton, R. (1982) Inhibition of aggregation and secretion of human platelets by quercetin and other flavonoids: structure-activity relationships. Agents Actions 12:382-387.[Medline]
40. Kramer, R. M., Roberts, E. F., Manetta, J. V., Hylsop, P. A. & Jakubowski, J. A. (1993) Thrombin-induced phosphorylation and activation of Ca++-sensitive cytosolic phospholipase A2 in human platelets. J. Biol. Chem. 268:26796-26804.
41. Pignatelli, P., Pulcinelli, F. M., Lenti, L., Gazzaniga, P. P. & Violi, F. (1998) Hydrogen peroxide is involved in collagen-induced platelet activation. Blood 91:484-490.
42. Hashizume, T., Yamaguchi, H., Kawamoto, A., Tamura, A., Sato, T. & Fujii, T. (1991) Lipid peroxide makes rabbit platelet hyperaggregable to agonists through phospholipase A2 activation. Arch. Biochem. Biophys. 289:47-52.[Medline]
43. Iuliano, L., Pedersen, J. Z., Praticò, D., Rotilio, G. & Violi, F. (1994) Role of hydroxyl radicals in the activation of human platelets. Eur. J. Biochem. 221:695-704.[Medline]
44. Visioli, F., Bellomo, G., Montedoro, G. & Galli, C. (1995) Low density lipoprotein oxidation is inhibited in vitro by olive oil constituents. Atherosclerosis 117:25-32.[Medline]
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