Goldenseal (Hydrastis canadensis)
The Effect of Oxidation on Berberine-Mediated CYP1 Inhibition: Oxidation Behavior and Metabolite-Mediated Inhibition
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The protoberberine alkaloid berberine carries methylenedioxy moiety and exerts a variety of pharmacological effects, such as anti- inflammation and lipid-lowering effects. Berberine causes potent CYP1B1 inhibition, whereas CYP1A2 shows resistance to the inhibition. To reveal the influence of oxidative metabolism on CYP1 inhibition by berberine, berberine oxidation and the metabolite-mediated inhibition were determined. After NADPH-fortified preincubation of berberine with P450, the inhibition of CYP1A1 and CYP1B1 variants (CYP1B1.1, CYP1B1.3, and CYP1B1.4) by berberine was not enhanced, and CYP1A2 remained resistant. Demethyleneberberine was identified as the most abundant metabolite of CYP1A1- and CYP1B1-catalyzed oxidations, and thalifendine was generated at a relatively low rate. CYP1A1-catalyzed berberine oxidation had the highest maximal velocity (Vmax) and exhibited positive cooperativity, suggesting the assistance of substrate binding when the first substrate was present. In contrast, the demethylenation by CYP1B1 showed the property of substrate inhibition. CYP1B1-catalyzed berberine oxidation had low Km values, but it had Vmax values less than 8% of those of CYP1A1. The dissociation constants generated from the binding spectrum and fluorescence quenching suggested that the low Km values of CYP1B1-catalyzed oxidation might include more than the rate constants describing berberine binding. The natural protoberberine/berberine fmetabolites with methylenedioxy ring-opening (palmatine, jatrorrhizine, and demethyleneberberine) and the demethylation (thalifendine and berberrubine) caused weak CYP1 inhibition. These results demonstrated that berberine was not efficiently oxidized by CYP1B1, and metabolism-dependent irreversible inactivation was minimal. Metabolites of berberine caused a relatively weak inhibition of CYP1.
1 . CYP1A1 preincubation with NADPH (id=NPDI-5sDXyA)
In Vitro Enzyme Inhibition Experiment
Negligible Inhibition was detected. Cutoff used — not specified
- CYP1A1 4173297
Recombinant expression system
E.coli
Cytochrome B5 Not available
Results
Table 1
| Sample | Compound measured | Value | Measurement | Study sequence | Additional information | N replicates |
|---|
Experimental Conditions
Commercially available
MgCl2
NADPH
P450 reductase
65.5 mM (0.5 mL)
30 min
NADPH with precipitant
2 . CYP1A2 preincubation w/o NADPH (id=NPDI-ZdbG7w)
In Vitro Enzyme Inhibition Experiment
Negligible Inhibition was detected. Cutoff used — not specified
- CYP1A2 4312402
Recombinant expression system
E.coli
Cytochrome B5 Not available
Results
| Sample | Compound measured | Value | Measurement | Study sequence | Additional information | N replicates |
|---|
Experimental Conditions
Commercially available
MgCl2
NADPH
P450 reductase
2 μM
65.5 mM (0.5 mL)
30 min
No NADPH with precipitant
3 . 1A2 pre-incubation with NADPH (id=NPDI-jjh4MA)
In Vitro Enzyme Inhibition Experiment
Negligible Inhibition was detected. Cutoff used — not specified
- CYP1A2 4312402
Recombinant expression system
E.coli
Cytochrome B5 Not available
Results
| Sample | Compound measured | Value | Measurement | Study sequence | Additional information | N replicates |
|---|
Experimental Conditions
Commercially available
MgCl2
NADPH
P450 reductase
65.5 mM (0.5 mL)
30 min
NADPH with precipitant
4 . Pre-incubation w/o NADPH (id=NPDI-NAiMNw)
In Vitro Enzyme Inhibition Experiment
Negligible Inhibition was detected. Cutoff used — not specified
- CYP1A1 4173297
Recombinant expression system
E.coli
Cytochrome B5 Not available
Results
| Sample | Compound measured | Value | Measurement | Study sequence | Additional information | N replicates |
|---|
Experimental Conditions
Commercially available
MgCl2
NADPH
P450 reductase
65.5 mM (0.5 mL)
30 min
No NADPH with precipitant