Compound | Molar Equivalents of GSH Depleted3-a | Distribution Coefficient | pKa | Log P | LD503-e | |||
---|---|---|---|---|---|---|---|---|
HRP/H2O23-a | Tyrosinase/O23-a | Microsomes3-b | pH 4.03-c | pH 7.43-d | ||||
mM | ||||||||
CA | 1.8 ± 0.2 | 1.0 ± 0.1 | 0.5 ± 0.1 | 14.0 ± 0.1 | 0.029 | 4.623-f | 1.24 | 7 |
CGA | 1.6 ± 0.1 | 1.1 ± 0.1 | 0.2 ± 0.1 | 1.1 ± 0.1 | 0.001 | 3.583-f 3-g | 0.60 | 23 |
DHCA | 1.9 ± 0.2 | 0.8 ± 0.1 | 0.6 ± 0.1 | 3.0 ± 0.4 | 0.003 | 4.363-f | 0.63 | 6 |
↵3-a HRP (0.1 μM)/H2O2 (50 μM) and a mixture of dihydroxycinnamic acids (50 μM) and GSH (200 μM) incubated in Tris/HCl buffer 0.1 M pH 7.4 at 37°C for 30 min. An aliquot of 25 μl DTNB (2 mg/ml) was added to 100 μl of the reaction mixture and the volume made up to 1 ml by Tris/HCl buffer 0.1 M, pH 8.94 containing 1 mM DETAPAC. Absorbance was monitored at 412 nm for both DHCA and CA and at 460 nm for CGA. Tyrosinase (20 units/ml) was used instead of HRP/H2O2. The data are the average of three separate measurements. Negligible GSH depletion (<0.1 molar equivalent) occurred in the absence of the enzyme.
↵3-b Glucose 6-phosphate (7.5 mM) was added to a mixture of the test compound (0.5 mM), NADP+ (0.5 mM), magnesium chloride (5 mM), microsomes (1 mg/ml), glucose-6-phosphate dehydrogenase (2.5 units/ml), and GSH (0.5 mM) in 1 ml Tris/HCl buffer (0.1 M, pH 7.4 containing DETAPAC 1 mM). The mixture was preincubated at 37°C from which 250-μl samples were taken at 60 min and added to 25 μl of trichloroacetic acid (30% w/v), vortexed, left for 5 min, and centrifuged to discard the protein pellet. An aliquot of 62.5 μl of DTNB (2 mg/ml) was added to 100 μl of the supernatant and the volume made up to 1 ml by Tris/HCl buffer (0.1 M, pH 8.9 containing DETAPAC 1 mM). Negligible GSH depletion (<0.1 molar equivalent) occurred in the absence of NADP+.
↵3-c Aliquots of 5 to 10 ml of octanol were added in time intervals of 30 min to the phosphate buffer (50 mM, pH 4.0 and 1 mM DETAPAC) containing dihydroxycinnamic acids (100 μM). The absorbance of the aqueous solution was monitored at 300 and 325 nm for CGA and CA, and 280 nm for DHCA before and after each 1-octanol addition. The values are mean of three measurements.
↵3-d Distribution coefficient value at pH 7.4 was calculated from P = D7.4/(1 − α) where P, α, and D7.4 are partition coefficient, degree of ionization at pH 7.4, and distribution coefficient at pH 7.4, respectively.
↵3-e Hepatocytes (10 ml) (106cells/ml) were preincubated at 37°C in Krebs-Henseleit buffer, pH 7.4, with CA, DHCA, and CGA at various concentration to determine the concentration at which the compound caused 50% death (LD50) (2 h) in the isolated rat hepatocytes. Cell viability was determined by trypan blue uptake. Similar experiments were undertaken for CGA and DHCA. Three separate experiments were carried out. Values shown are means.
↵3-f Martell and Smith (1977–1989).
↵3-g pKa of quinic acid, the sugar moiety of CGA.