0.0 0.2 0.4 0.6 0.8 1.0 7-Ethoxy resorufin O-deethy lation nmol/min/mg protein
0.0 0.5 1.0 1.5
Methanol extract g/kg
0.0 0.2 0.4 0.6 0.8 1.0 7-Pentoxy resorufin O-dealky lati on nmol/min/mg protein
0.0 0.1 0.2 0.3 0.4
∗ ∗ ∗
∗
GST
Figure 9. Immunoblot analysis of liver microsomal cytochrome P450 and cytosol protein levels from control（C） and S. flavanscens methanol extract（ME）-treated mice. Electrophoresis and immunodetection were carried out as described in the section of Materials and Methods. Blots are shown in the right panel. The relative band density compared to control is shown in the left panel.
CYP3A
Figure 10. Dose-respone of effects of powdered concentrate of S. flavenscens on 7-ethoxyresorufin O-deethylation and 7-pentoxyresorufin O-dealkylation activities in rat liver. Rats were treated with increasing daily dosage of powdered concentrate through gastrogavage for 3 days.
Microsomal 7-ethoxyresorufin O-deethylation and 7-pentoxyresorufin O-dealkylation activities were determined as described in the Materials and Methods. Date represent mean ± S.E.M. of 3 rats in control and treated groups. ＊Asterisks represent values significantly different from the control values, p＜0.05
Powdered concentrate g/kg
0 1 2 3
7-Ethoxyresorufin O-deethylation nmol/min/mg protein 0.0 0.2 0.4 0.6 0.8
∗ ∗
Powdered concentrate g/kg
0.0 0.5 1.0 1.5 2.0 2.5 3.0 7-Pentoxyresorufin O-dealkylation nmol/min/mg protein
0.0 0.1 0.2 0.3 0.4 0.5
0.6
∗
CYP1A
Figure 11. Immunoblot analysis of liver microsomal cytochrome P450 and cytosol protein levels from control（C） and S. flavanscens powdered concentrate（PC）
-treated rats. Electrophoresis and immunodetection were carried out as described in the section of Materials and Methods. Blots are shown in the right panel. The relative band density compared to control is shown in the left panel.
Figure 12. The linear relationship between theophylline concentration and the peak area obtained from HPLC analysis.
Area
0 100 200 300 400 500 600 700
Theophylline, μg/ml
0 1 2 3 4 5
Figure 13. Mean unbound theophylline concentrate-time curves in rat blood after intravenous treatment with 2mg/kg theophylline.
(A)Control rat, the solid lines show the result of linear regression; (B) Effects of pre-treatment with S. flavanscens on unbound theophylline concentration. Rats were pre-treated with water (○) or S. flavanscens powdered concentrate(●) as descried Table6.
Time, min
0 100 200 300 400
Theophylline, μg/ml
0.1 1 10
(A)
Time (min)
0 100 200 300
log Theophylline ug/ml
0.1 1 10
control
S. flavescens (B)
Table 1. Effects of powdered concentrate of S. flavescens on the component conterts and activites of cytochrome P450-dependent monooxygenase in mouse liver
Assay Control Powdered concentrate
Body weight (g) 18.8 ± 0.6 17.9 ± 0.3
NADPH-Cytochrome P450 reductase (nmol/min/mg protein)
154 ± 6 178 ± 8*
7-Ethoxyresorufin O-deethylation (nmol/min/mg protein)
0.58 ± 0.04 1.12 ± 0.07*
Mice were treated with 3 g/kg/day powdered concentrate for three days. Results represent mean ± S.E.M. of 5 mice for control and treatment groups. *Asterisks represent values significantly different from the control values, p＜0.05.
Table 2. Effects of S. flavescens extracts on cytochrome P450-dependent monooxygenase components and activities in mouse liver
Assay Control Aqueous extract Methanol extract
Body weight (g) 19.7 ± 0.5 19.1 ± 0.4 19.0 ± 0.3
NADPH-Cytochrome P450 reductase (nmol/min/mg protein)
199 ± 6 235 ± 7* 239 ± 11*
7-Ethoxyresorufin O-deethylation (nmol/min/mg protein)
0.63 ± 0.03 1.40 ± 0.07* 1.51 ± 0.04*
7-Pentoxyresorufin O-dealkylation (pmol/min/mg protein)
99 ± 5 318 ± 19* 379 ± 21*
Coumarin hydroxylation (pmol/min/mg protein)
53 ± 6 84 ± 7* 107 ± 11*
Nifedipine oxidation (nmol/min/mg protein)
2.17 ± 0.14 3.08 ± 0.13* 2.43 ± 0.12
Nitrosodimethylamine N-demethylation (nmol/min/mg protein)
2.46 ± 0.18 2.76 ± 0.08 2.41 ± 0.06
Tolbutamide hydroxylation (pmol/min/mg protein)
34.8 ± 3.8 34.3 ± 2.2 28.1 ± 4.1
Mice were treated with 1 g/kg/day of the aqueous or methanol extract for 3 days. Control mice received the same volume of water without any extract. Data are presented as the mean ± SEM of five, five, and six mice for the control, aqueous extract-, and methanol extract-treated groups, respectively. *Asterisks represent values significantly different from the control values, p＜0.05.
Table 3. Effects of the powdered concentrate and extracts of S. flavescens on UDP-glucuronosyltransferase, glutathione S-transferase, and NAD(P)H-quinone oxidoreductase activities in mouse liver
Assay Control
(n = 5)
Powdered concentrate
(n = 5)
Aqueous extract (n = 5)
Methanol extract (n = 6) UDP-Glucuronosyltransferase
(nmol/min/mg protein)
20.2 ± 2.5 23.4 ± 8.9 22.9 ± 6.3 28.2 ± 8.7
Glutathione S-transferase (μmol/min/mg protein)
2.81 ± 0.18 4.36 ± 0.37* 4.38 ± 0.45* 4.02 ± 0.10*
NAD(P)H-Quinone oxidoreductase (nmol/min/mg protein)
85 ± 6 96 ± 9 96 ± 17 107 ± 6*
Mice were treated with 3 g/kg/day of the powdered concentrate, 1 g/kg/day of the aqueous extract, or 1 g/kg/day of the methanol extract for 3 days. Data are presented as the mean ± SEM. The number (n) of mice in each group is shown in parentheses.
*Asterisks represent values significantly different from the control values, p＜0.05.
Table 4. Effects of the powdered concentrate and extracts of S. flavescens on drug-metabolizing enzymes in mouse kidney
Assay Control
(n = 5)
Mice were treated with 3 g/kg/day of the powdered concentrate, 1 g/kg/day of the aqueous extract, or 1 g/kg/day of the methanol extract for 3 days. Data are presented as the mean ± SEM. The number (n) of mice in each group is shown in parentheses. n.d.: not detectable.
Table 5. Effects of powdered concentrate of S. flavescens on the component conterts and activites of cytochrome P450-dependent monooxygenase in rat liver
Assay Control Powdered concentrate
Body weight (g) 193 ± 10 194 ± 8
NADPH-Cytochrome P450 reductase (nmol/min/mg protein)
145 ± 17 167 ± 8
7-Ethoxyresorufin O-deethylation (nmol/min/mg protein)
0.307 ± 0.019 0.709 ± 0.032*
Rats were treated with 3 g/kg powdered concentrate for three days. Results represent mean ± S.E.M. of 3 rats for control and treatment groups. *Asterisks represent values significantly different from the control values, p＜0.05.
Table 6. Effects of powdered concentrate of S. flavescens on NAD(P)H-Quinone
oxidoreductase, UDP-glucuronosyltransferase, and glutathione S-transferase activites in rat liver
Assay Control Powdered concentrate
UDP-Glucuronosyltransferase 20.7 ± 6.3 28.0 ± 6.6 (nmol/min/mg protein)
NAD(P)H-Quinone oxidoreductase (nmol/min/mg protein)
252 ± 18 210 ± 34
Glutathione S-transferase (μmol/min/mg protein)
1.57 ± 0.05 2.24 ± 0.14*
Rats were treated with 3 g/kg powdered concentrate for three days. Results represent mean ± S.E.M. of 3 rats for control and treatment groups. *Asterisks represent values significantly different from the control values, p＜0.05.
Table 7. Effects of powdered concentrate of S. flavescens on drug-metabolizing enzymes in rat kidney
Assay Control Powdered concentrate
Kidney weight (g) 1.91 ± 0.15 1.81 ± 0.10 Microsomal protein
(mg/g)
17.5 ± 2.1 18.5 ± 1.4
Cytochrome P450 (pmol/mg protein)
0.037 ± 0.010 0.045 ± 0.005
Cytochrome b5 0.064 ± 0.006 0.069 ± 0.002 (nmol/mg protein)
NADPH-Cytochrome P450 reductase (nmol/min/mg protein)
27.4 ± 2.0 30.4 ± 1.2
7-Ethoxyresorufin O-deethylation (nmol/min/mg protein)
n.d. n.d.
7-Pentoxyresorufin O-dealkylation (pmol/min/mg protein)
n.d. n.d.
NAD(P)H-Quinone oxidoreductase (nmol/min/mg protein)
24.3 ± 2.2 23.5 ± 0.7
Glutathione S-transferase (μmol/min/mg protein)
0.110 ± 0.009 0.117 ± 0.003
Rats were treated with 3 g/kg powdered concentrate for three days.
Results represent mean ± S.E.M. of 3 rats for control and treatment groups. n.d.: not detectable.
Table 8. Effects of powdered concentrate of S. flavescens on drug-metabolizing enzymes in rat lung
Assay Control Powdered concentrate
lung weight (g) 0.97 ± 0.14 1.11 ± 0.03 Microsomal protein
(mg/g)
8.98 ± 1.54 10.6 ± 1.3
Cytochrome P450 (pmol/mg protein)
0.048 ± 0.029 0.090 ± 0.014
Cytochrome b5 0.040 ± 0.002 0.043 ± 0.002 (nmol/mg protein)
NADPH-Cytochrome P450 reductase (nmol/min/mg protein)
30.6 ± 2.9 27.6 ± 1.5
7-Ethoxyresorufin O-deethylation (nmol/min/mg protein)
n.d. n.d.
7-Pentoxyresorufin O-dealkylation (pmol/min/mg protein)
n.d. n.d.
NAD(P)H-Quinone oxidoreductase (nmol/min/mg protein)
106 ± 16 115 ± 5
Glutathione S-transferase (μmol/min/mg protein)
0.073 ± 0.005 0.079 ± 0.004
Rats were treated with 3 g/kg powdered concentrate for three days.
Results represent mean ± S.E.M. of 3 rats for control and treatment groups. n.d.: not detectable.
Table 9. Effects of powdered concentrate of S. flavescens on serum enzyme
Assay Control Powdered concentrate
AST (unit/l) 382 ± 134 314 ± 83 ALT (unit/l) 70 ± 33 59 ± 15 BUN（mg/dl） 15.7 ± 1.8 15.6 ± 1.9 CRE（mg/dl） 0.9 ± 0.2 0.8 ± 0.1 Rats were treated with 3g/kg/day powdered concentrate of S. flavescens for three days.
Data are presented as the mean ± SEM of three and five rats for the control and treatment groups, respectively.
Table 10. Intraday precision and accuracy of theophylline (n=3)
conc.(ug/ml) mean S.D. C.V.
早 1.08 1.09 1.09 1.09 0.01 0.62 1
晩 1.08 1.08 1.08 1.08 0.00 0.30 早 1.97 2.00 2.00 1.99 0.02 0.98 2
晩 1.98 1.99 1.98 1.99 0.00 0.22 早 3.03 3.03 3.04 3.03 0.00 0.14 3
晩 3.03 3.01 3.02 3.02 0.01 0.29
Table 11. Interday precision and accuracy of theophylline (n=3)
day mean S.D. C.V.
conc.
(ug/ml) 1 2 3
1 1.02 1.07 1.07 1.00 1.05 1.02 1.10 1.08 1.08 1.05 0.03 3.21 2 1.98 1.97 2.02 1.96 1.93 1.91 1.94 1.97 1.98 1.96 0.03 1.60 3 3.05 3.03 3.06 3.07 2.96 2.96 3.04 3.02 3.04 3.02 0.04 1.38
Table 12. Pharmacokinetic parameters of interaction between theophylline（2 mg/kg, i.v.）
and S. flavescens powdered concentrate in rat blood.（two-compartment model）
Pharmacokinetic parameters
theophylline theophylline＋ powdered concentrate
*Asterisks represent values significantly different from the control values, p＜0.05.
Table 13. Summary of in vivo effects of S. flavescens powdered concentrate, aqueous extract, methanol extract on drug-metabolizing enzymes activities in mouse liver
Phase I Phase II
CYP1A CYP2B CYP2A6 CYP3A CYP2E1 CYP2C UGT GST NQO Powdered
concentrate
↑ ↑ ↑ ↑ － ↑ － ↑ －
Aqueous extract
↑ ↑ ↑ ↑ － － － ↑
Methanol extract
↑ ↑ ↑ － － － － ↑ ↑
Mice were treated with powdered concentrate and extracts.