人參.GinsengGinseng Roots Extract: Ginseng Roots Extract was refined from the underground pan (Root and Fibrous root) of ginseng, which belongs to the araliaceae plants. It was a yellow-black powder. The water solubility was 100%. The ginseng root extract not only contained the 29 monosomic saponins of ginseng root, but also contained ginseng polysaccharides, ginseng proteins, glucoproteins, amino acids, ginseng volatile oils, organic germanium and trace elements, etc. Ginseng Leaves Extract: Ginseng Leaves Extract was refined from the stein and leaf of ginseng, which belongs to the araliaceae plants. It was a white powder and mainly contained eighteen ginseng monosomic saponins. It could be solubilized in 80¡æ water and ethanol. In Vitro Effect of Standardized Ginseng Extracts and Individual Ginsenosides on the Catalytic Activity of Human CYP1A1, CYP1A2, and CYP1B1: Ginseng extract has been reported to decrease the incidence of 7,12-dimethylbenz[a]anthracene (DMBA)-initiated tumorigenesis in mice. A potential mechanism for this effect by ginseng is inhibition of DMBA-bioactivating cytochrome P450 (P450) enzymes. In the present in vitro study, we examined the effect of a standardized Panax ginseng (or Asian ginseng) extract (G115), a standardized Panax quinquefolius (or North American ginseng) extract (NAGE), and individual ginsenosides (Rb1, Rb2, Rc, Rd, Re, Rf, and Rg1) on CYP1 catalytic activities, as assessed by 7-ethoxyresorufin O-dealkylation. G115 and NAGE decreased human recombinant CYP1A1, CYP1A2, and CYP1B1 activities in a concentration-dependent manner. Except for the competitive inhibition of CYP1A1 by G115, the mode of inhibition was the mixed-type in the other cases. A striking finding was that NAGE was 45-fold more potent than G115 in inhibiting CYP1A2. Compared with G115, NAGE also preferentially inhibited 7-ethoxyresorufin O-dealkylation activity in human liver microsomes. Rb1, Rb2, Rc, Rd, Re, Rf, and Rg1, either individually or as a mixture and at the levels reflecting those found in an inhibitory concentration (100 µg/ml) of NAGE or G115, did not influence CYP1 activities. However, at a higher ginsenoside concentration (50 µg/ml), Rb1, Rb2, Rc, Rd, and Rf inhibited these activities. Overall, our in vitro findings indicate that standardized NAGE and G115 extracts, which were not treated with calf serum or subjected to acid hydrolysis, inhibited CYP1 catalytic activity in an enzyme-selective and extract-specific manner, but the effects were not due to Rb1, Rb2, Rc, Rd, Re, Rf, or Rg1.
Fig. 1. Content of individual ginsenosides in G115 and NAGE. The content (% w/w) of Rb1,Rb2,Rc,Rd,Re,Rf,Rg1,and Rg2 in the standardized G115 and NAGE extracts were quantified by HPLC, and the data were provided by our suppliers of G115 (Pharmaton S.A.) and NAGE (Canadian Phytopharmaceuticals Corp.). 7-Ethoxyresorufin O-Dealkylation Assay: 7-Ethoxyresorufin O-dealkylation activity was determined by a modification of a continuous spectrofluorometric assay. Briefly, each standard 2-ml incubation contained 100 mM potassium phosphate buffer, pH 7.4, 5 mM MgCl2, 1.5 mM EDTA, 0.2 µM 7-ethoxyresorufin (unless indicated otherwise), human recombinant P450 enzyme (1 pmol of CYP1A1, 3 pmol of CYP1A2, or 2.5 pmol of CYP1B1) or human liver microsomes (75 pmol of total microsomal P450), and 0.25 mM NADPH. Reaction was performed at 37°C and initiated by the addition of NADPH. Fluorescence was recorded every 30 s for 3 min using a Shimadzu model RF-540 spectrofluorometer. The excitation wavelength was set at 530 nm (5-nm slit width), and the emission wavelength was set at 582 nm (5-nm slit width). Calibration curves were constructed by determining the fluorescence in incubations containing known amounts of the authentic resorufin metabolite. Samples containing the authentic standard were processed in the same manner as the unknown samples but in the presence of heat-inactivated human liver microsomes or control insect cell microsomes.
Fig. 2. Concentration-dependent effect of G115 and NAGE on the catalytic activity of CYP1A1, CYP1A2, and CYP1B1.7-Ethoxyresorufin O-dealkylation assay (0.2 µM substrate concentration) was performed with human recombinant CYP1A1 (A), CYP1A2 (B), and CYP1B1 (C), and varying concentrations of G115 and NAGE. Control incubations contained the vehicle (100 mM potassium phosphate, pH 7.4). Shown are mean ± S.E.M. percentages of control activity for three independent experiments. Control enzyme activity (mean ± S.E.M.) for CYP1A1, CYP1A2, and CYP1B1 was 62 ± 1, 5.1 ± 0.3, and 14 ± 0.4 nmol/min/nmol of P450, respectively.
Fig. 3. Lineweaver-Burk plots for the inhibition of CYP1A1, CYP1A2, and CYP1B1 by G115. 7-Ethoxyresorufin O-dealkylation assay was performed with human recombinant CYP1A1 (A), CYP1A2 (B), or CYP1B1 (C) at multiple concentrations of 7-ethoxyresorufin (0.025-0.2 µM for CYP1A1 and CYP1B1; 0.1-0.8 µM for CYP1A2) and G115 (125-500 µg/ml for CYP1A1; 500-1500 µg/ml for CYP1A2; 75-225 µg/ml for CYP1B1). The plots were generated by nonlinear regression analysis of the experimental data, as described under Materials and Methods. Results are expressed as mean ± S.E.M. of reciprocal enzyme activity for three independent experiments. Fig. 4. Lineweaver-Burk plots for the inhibition of CYP1A1, CYP1A2, and CYP1B1 by NAGE. 7-Ethoxyresorufin O-dealkylation assay was performed with human recombinant CYP1A1 (A), CYP1A2 (B), or CYP1B1 (C) at multiple concentrations of 7-ethoxyresorufin (0.025-0.2 µM for CYP1A1 and CYP1B1; 0.1-0.8 µM for CYP1A2) and NAGE (125-500 µg/ml for CYP1A1; 5-20 µg/ml for CYP1A2; 25-100 µg/ml for CYP1B1). The plots were generated by nonlinear regression analysis of the experimental data, as described under Materials and Methods. Results are expressed as mean ± S.E.M. of reciprocal enzyme activity for three independent experiments.
Fig. 5. Effect of G115 and NAGE on 7-ethoxyresorufin O-dealkylation activity in individual human liver microsomes.7-Ethoxyresorufin O-dealkylation activity was determined in a panel of four individual human liver microsome samples (denoted as HG23, HG30, HG56, and HG89) in the presence of G115 or NAGE (each at 60 µg/ml). Control incubations contained the vehicle (100 mM potassium phosphate, pH 7.4). Results are expressed as mean ± S.E.M. of enzyme activity for three independent experiments. *, significantly different from the control, p < 0.05. |