Comparison of Antiplatelet Effects of Phenol Derivatives in Humans

Abstract

Flavonoids are associated with positive cardiovascular effects. However, due to their low bioavailability, metabolites are likely responsible for these properties. Recently, one of these metabolites, 4-methylcatechol, was described to be a very potent antiplatelet compound. This study aimed to compare its activity with its 22 close derivatives both of natural or synthetic origin in order to elucidate a potential structure-antiplatelet activity relationship. Blood from human volunteers was induced to aggregate by arachidonic acid (AA), collagen or thrombin, and plasma coagulation was also studied. Potential toxicity was tested on human erythrocytes as well as on a cancer cell line. Our results indicated that 17 out of the 22 compounds were very active at a concentration of 40 μM and, importantly, seven of them had an IC₅₀ on AA-triggered aggregation below 3 μM. The effects of the most active compounds were confirmed on collagen-triggered aggregation too. None of the tested compounds was toxic toward erythrocytes at 50 μM and four compounds partly inhibited proliferation of breast cancer cell line at 100 μM but not at 10 μM. Additionally, none of the compounds had a significant effect on blood coagulation or thrombin-triggered aggregation. This study hence reports four phenol derivatives (4-ethylcatechol, 4-fluorocatechol, 2-methoxy-4-ethylphenol and 3-methylcatechol) suitable for future in vivo testing.

Keywords: 4-methylcatechol; aggregation; catechol; flavonoid; platelet; whole blood.

Figure 1

Effect of Phenolic Compounds on AA-induced Aggregation: Results are presented as means with ±SD. * p ˂ 0.05 vs. DMSO; *** p ˂ 0.001 vs. DMSO; ⁺ p ˂ 0.05 vs. ASA; ⁺⁺⁺ p ˂ 0.001 vs. ASA; ^### p ˂ 0.001 vs. 4-methylcatechol.

Figure 2

Examples of Dose–response Curves in Arachidonic Acid-triggered Platelet Aggregation. Four compounds with different potencies were intentionally selected. Results show the summary of three independent experiments. Curves were fitted using a non-linear regression method.

Figure 3

Comparison of Antiplatelet Activity of All Compounds on Arachidonic Acid-platelet Aggregation. The direction of black arrow shows a more potent compound. Analysis was performed by comparing 95% confidence intervals of aggregation curves. Two-way grey arrows mean no statistical difference in the activity. (A): 4-allyl-1,2-dimethylcatechol, (B): 1,2-dimethylcatechol, (C): o-cresol, (D): 2,4-dimethoxytoluene, (E): o-toluidine, (F): 2-aminophenol, (G): 3-aminocatechol, (H): pyrocatechol, (I): 3-isopropylcatechol, (J): 4-nitrocatechol, (K): 4-aminocatechol, (L): 3-methylcatechol, (M): 4-methylcatechol, (N): 4-ethylcatechol, (O): 4-tert-butylcatechol, (P): 3-methoxycatechol, (Q): 3-fluorocatechol, (R): 4-fluorocatechol, (S): 4-chlorocatechol, (T): 2-methoxy-4-methylphenol, (U): 2-methoxy-4-ethylphenol, (V): 3,5-dichlorocatechol, (W): 4,5-dichlorocatechol.

Figure 4

Cytotoxicity of All the Compounds was Tested in MCF7/S0.5 Breast Cancer Cell Line. All compounds were tested in three different concentrations and results are compared to DMSO 0.1%, set to 100% viability. SDS 10% was used as a positive control. All compounds were tested in triplicates at least three times. Statistical significance was established using a one-way ANOVA assay with confidence interval of 95% (* p < 0.001). o-Toluidin was not tested.

Figure 5

Dose–response Curves of the Most Efficient and Safe Compounds on Collagen-induced Platelet Aggregation. 4,5-dichlorcatechol is not shown since it blocked cell proliferation at 100 μM.