Goldenseal (Hydrastis canadensis) 43525850
Inhibitory Mechanisms of Human CYPs by Three Alkaloids Isolated from Traditional Chinese Herbs CSV JSON

The three purified herbal compounds tetrahydropalmatine (Tet), neferine and berberine (Ber) were exploredin vitro for basic inhibition mechanisms towards recombinant human CYP1A2, CYP2D6 and CYP3A4 meta- bolic activities. Phenacetin, dextromethorphan and testosterone, respectively, were used as CYP1A2, CYP2D6 and CYP3A4 substrates, and their metabolites were determined by validated HPLC methodologies. Positive inhibition controls were used. Mechanism-based (irreversible) inhibition was assessed by time-dependent and nicotinamide adenine dinucleotide phosphate-dependent and reversible inhibition by Lineweaver–Burk plot assessments. Inhibition mechanisms were also assessed by computerized interaction prediction by using the Discovery Studio CDOCKER software (Accelrys, San Diego, CA, USA). Tetrahydropalmatine showed a mechanism-based inhibition of both CYP1A2 and CYP2D6, and Ber of CYP2D6. Neferine and Ber both showed a nonmechanistic inhibition of CYP1A2. All compounds showed a similar and significant mechanism-based inhibition of CYP3A4. Tetrahydropalmatine and Ber demonstrated both reversible and irreversible inhi- bition of CYP2D6 and CYP3A4. Tetrahydropalmatine and Ber displayed H-bond and several Pi-bond connec- tions with specific amino acid residues of CYP1A2, CYP2D6 and CYP3A4, giving further knowledge to the identified reversible and irreversible herb–drug interactions. Tetrahydropalmatine and Ber should be considered for herb–drug interactions in clinical therapy until relevant clinical studies are available.

1 . 1A2 preincubation with NADPH (id=NPDI-EjO7dA)

In Vitro Enzyme Inhibition Experiment

Inhibition was detected.  Cutoff used — not specified

testosterone

Berberine 19012197

6beta-Hydroxytestosterone

  • CYP1A2 4312402

Recombinant expression system Baculovirus-insect cells
Cytochrome B5 No

Results

estimated from fig 2 
table 2

Compound measured Measurement Value Study sequence Additional information N replicates

Experimental Conditions

110-pmol CYP/mg

Commercially available

41273

400 μL

25 min

NADPH
NADPH regenerating system
P450 reductase

100 μM

0,5,15,30, and 45 min

NADPH with precipitant

75 μM

2 . 1A2 preincubation without NADPH (id=NPDI-MsX4aw)

In Vitro Enzyme Inhibition Experiment

Inhibition was detected.  Cutoff used — not specified

testosterone

Berberine 19012197

6beta-Hydroxytestosterone

  • CYP1A2 4312402

Recombinant expression system Baculovirus-insect cells
Cytochrome B5 No

Results

Estimated from fig 2 at 30 mins

Compound measured Measurement Value Study sequence Additional information N replicates

Experimental Conditions

110-pmol CYP/mg

Commercially available

41273

400 μL

25 min

NADPH
NADPH regenerating system
P450 reductase

100 μM

0,5,15,30, and 45 min

No NADPH with precipitant

75 μM

3 . 2D6 pre-incubation with NADPH (id=NPDI-ykfGxQ)

In Vitro Enzyme Inhibition Experiment

Inhibition was detected.  Cutoff used — not specified

testosterone

Berberine 19012197

6beta-Hydroxytestosterone

  • CYP2D6 4173631

Recombinant expression system Baculovirus-insect cells
Cytochrome B5 No

Results

Estimated from fig 2 at 30 mins
Table 2

Compound measured Measurement Value Study sequence Additional information N replicates

Experimental Conditions

100-pmol CYP/mg

Commercially available

73755

400 μL

25 min

NADPH
NADPH regenerating system
P450 reductase

100 μM

0,5,15,30, and 45 min

NADPH with precipitant

7.43-μM

4 . 2D6 preincubation without NADPH (id=NPDI-wjO31A)

In Vitro Enzyme Inhibition Experiment

Inhibition was detected.  Cutoff used — not specified

testosterone

Berberine 19012197

6beta-Hydroxytestosterone

  • CYP2D6 4173631

Recombinant expression system Baculovirus-insect cells
Cytochrome B5 No

Results

Estimate from Fig 2 at 30 mins

Compound measured Measurement Value Study sequence Additional information N replicates

Experimental Conditions

100-pmol CYP/mg

Commercially available

73755

400 μL

25 min

NADPH
NADPH regenerating system
P450 reductase

100 μM

0,5,15,30, and 45 min

No NADPH with precipitant

7.43-μM

5 . 3A4 preincubation with NADPH (id=NPDI-q61pww)

In Vitro Enzyme Inhibition Experiment

Inhibition was detected.  Cutoff used — not specified

testosterone

Berberine 19012197

6beta-Hydroxytestosterone

  • CYP3A4 4306811

Recombinant expression system Baculovirus-insect cells
Cytochrome B5 Co-expressed

Results

Results above from 30 min preincubation with NADPH (fig 2)
Table 2

Compound measured Measurement Value Study sequence Additional information N replicates

Experimental Conditions

141-pmol and 149-pmol

Commercially available

82636 and 38275

400 μL

10 min

NADPH
NADPH regenerating system
P450 reductase

100 μM

0,5,15,30, and 45 min

NADPH with precipitant

50.5 μM

6 . 3A4 preincubation without NADPH (id=NPDI-iBWK6Q)

In Vitro Enzyme Inhibition Experiment

Inhibition was detected.  Cutoff used — not specifed

testosterone

Berberine 19012197

6beta-Hydroxytestosterone

  • CYP3A4 4306811

Recombinant expression system Baculovirus-insect cells
Cytochrome B5 Co-expressed

Results

Results estimated above from 30 min of preincubation (Fig 2)

Compound measured Measurement Value Study sequence Additional information N replicates

Experimental Conditions

141-pmol and 149-pmol

Commercially available

82636 and 38275

400 μL

10 min

NADPH
NADPH regenerating system
P450 reductase

100 μM

0,5,15,30, and 45 min

No NADPH with precipitant

50.5 μM