Puerarin and conjugate bases as radical scavengers and antioxidants:: Molecular mechanism and synergism with β-carotene

The 4'-hydroxyl group of puerarin, a C-glycoside of the isoflavonoid daidzein, was shown, using 2,2'-azino-bis(3-ethylbenzthiazoline-6-sulfonic acid) radical cation and stopped-flow spectroscopy and by comparison with the 7-propylpuerarin (A ring derivative) and 4'-propylpuerarin (B ring derivative), to be a more efficient radical scavenger as compared to the 7-hydroxyl group by a factor of 2, a difference increasing upon deprotonation. The difference in radical scavenging agreed with the oxidation potentials (cyclic voltammetry in acetonitrile, 0.1 M Bu4NBF4 at 25 degrees C): E/mV = 862 +/- 3 for puerarin, 905 +/- 10 for 7-propylpuerarin, and 1064 +/- 2 for 4'-propylpuerarin relative to ferrocene/ferricenium. In aqueous solution, the reduction potential was shown to decrease for increasing pH, and deprotonation of the 4'-hydroxyl group increased radical scavenging more than deprotonation of the 7-hydroxyl group. The 7-hydroxyl was found to be more acidic (pK(a1) = 7.20 +/- 0.01 in puerarin and pK(a) = 7.23 +/- 0.01 in 4'-propylpuerarin) than the 4'-hydroxyl group (pK(a2) = 9.84 +/- 0.08 in puerarin and pK(a) = 9.51 +/- 0.02 in 7-propylpuerarin); aqueous solution, ionic strength of 0.1, and 25 degrees C. In phosphatidyl choline liposome of pH 7.4, puerarin and beta-carotene each showed a modest antioxidant activity measured as prolongation of the lag phase for formation of conjugate dienes and using the water-soluble radical initiator APPH with effects of puerarin and beta-carotene being additive. For the lipophilic initiator AMVN, the antioxidative effect decreased for puerarin and increased for beta-carotene as compared to APPH and showed a clear synergism. A regeneration of beta-carotene, effective in the liposome lipid phase as antioxidant, from the cation radical by deprotonated forms of puerarin was demonstrated in 9:1 chloroform/methanol using laser flash photolysis with k(2) = 2.7 x 10(4) L mol(-1) s(-1) for the bimolecular process between the cation radical and the puerarin dianion.