In vitro inhibitory effects of dihydromyricetin on human liver cytochrome P450 enzymes

Figures & data

Table 1. Isoforms tested, marker reactions, incubation conditions and K_m used in the inhibition study.

CYPs	Marker reactions	Substrate concentration (μM)	Protein concentration (mg/mL)	Incubation time (min)	Estimated Km (μM)
1A2	Phenacetin O-deethylation	40	0.2	30	48
3A4	Testosterone 6β-hydroxylation	50	0.5	10	53
2A6	Coumarin 7-hydroxylation	1.0	0.1	10	1.5
2E1	Chlorzoxazone 6-hydroxylation	120	0.4	30	126
2D6	Dextromethorphan O-demethylation	25	0.25	20	4.8
2C9	diclofenac 4'-hydroxylation	10	0.3	10	13
2C19	S-Mephenytoin 4-hydroxylation	100	0.2	40	105
2C8	Paclitaxel 6α-hydroxylation	10	0.5	30	16

Figure 1. The chemical structure of DHM.

Figure 2. Inhibition of DHM on CYP450 enzymes in pooled HLMs. All data represent mean ± SD of the triplicate incubations. *p < 0.05, significantly different from the negative control. Negative control: incubation systems without DHM; DHM: incubation systems with DHM (100 μM); positive control: incubation systems with their corresponding positive inhibitors (10 μM furafylline for CYP1A2, 1 μM ketoconazole for CYP3A4, 10 μM tranylcypromine for CYP2A6, 50 μM clomethiazole for CYP2E1, 10 μM quinidine for CYP2D6, 10 μM sulphaphenazole for CYP2C9, 50 μM tranylcypromine for CYP2C19, 5 μM montelukast for CYP2C8).

Figure 3. Inhibitory effects of DHM on the activities of CYP3A4, CYP2E1 and CYP2D6.

Figure 4. Lineweaver–Burk plots (A) and the secondary plot for Ki (B) of inhibition of DHM on CYP3A4 catalyzed reactions (testosterone 6β-hydroxylation) in pooled HLM. Data are obtained from a 30 min incubation with testosterone (20–100 μM) in the absence or presence of DHM (0–30 μM). The data represent the mean of the incubations (performed in triplicate).

Figure 5. Lineweaver–Burk plots (A) and the secondary plot for Ki (B) of inhibition of DHM on CYP2E1 catalyzed reactions (chlorzoxazone 6-hydroxylation) in pooled HLM. Data are obtained from a 30 min incubation with chlorzoxazone (20–200 μM) in the absence or presence of DHM (0–50 μM). All data represent the mean of the incubations (performed in triplicate).

Figure 6. Lineweaver–Burk plots (A) and the secondary plot for Ki (B) of inhibition of DHM on CYP2D6 catalyzed reactions (dextromethorphan O-demethylation) in pooled HLM. Data are obtained from a 30 min incubation with dextromethorphan (5–50 μM) in the absence or presence of DHM (0–50 μM). All data represent the mean of the incubations (performed in triplicate).

Figure 7. Time-dependent inhibition investigations of CYP3A4, 2E1 and 2D6 catalyzed reactions by DHM (20 μM). All data represent the mean of the incubations (performed in triplicate).

Figure 8. Time (0–30 min) and concentration (0–50 μM)-inactivation of microsomal CYP3A4 activity by DHM in the presence of NADPH. The initial rate constant of inactivation of CYP3A4 by each concentration (K_obs) was determined through linear regression analysis of the natural logarithm of the percentage of remaining activity versus pre-incubation time (A). The K_I and K_inact values were determined through non-linear analysis of the K_obs versus the DHM concentration (B).