Article Figures & Data
Figures
- Fig. 1.
Interaction of hyperforin and hypericin with OATP2B1. Inhibition of OATP2B1-mediated transport by hyperforin and hypericin was tested in MDCKII-OATP2B1 cells. The influence on cellular accumulation of estrone 3-sulfate was determined at low (0.005 μM; A and D) or high (50 μM; B and E) concentration to account for binding site A or B, respectively, or of bromosulfophthalein (C and F). The IC50 values were calculated fitting the data to a sigmoidal log(inhibitor)-normalized response curve, without constraining of top or bottom, or weighting. (G) Schematic of the molecular basis of competitive-counterflow experiments, in which substrates are reduced by the amount of another substrate in the steady state. Tested was the influence of hyperforin or hypericin on the cellular equilibrium of E1S in MDCKII-OATP2B1 cells compared with DMSO control (H). Atorvastatin and penicillin G were used as positive and negative controls, respectively. Data are presented as mean ± S.D. of n = 3 independent experiments, each performed in biologic triplicates. *P ≤ 0.05, one-way analysis of variance with Dunnett’s multiple comparisons test.
- Fig. 2.
Influence of OATP2B1 on hyperforin-induced PXR-mediated transactivation of CYP3A4. Luciferase activation was determined in HepG2 cells transfected with PXR-pEF6 in presence or absence of heterologously expressed OATP2B1 and treated with 0.1 μM hyperforin or DMSO (striped bars) (A). The influence of bromosulfophthalein on hyperforin-induced PXR activation was assessed in HepG2 cells transiently transfected with PXR-pEF6 and OATP2B1-pEF6 (B). Firefly luciferase activity was normalized to that of Renilla. Data are presented as mean ± S.D. of n = 3 independent experiments each performed in biologic triplicates. *P-value ≤ 0.05, two-way analysis of variance with Tukey’s multiple comparisons test.
- Fig. 3.
Interaction of hyperforin with OATP2B1-mediated transport of atorvastatin. The inhibitory effect of hyperforin on atorvastatin transport was determined in MDCKII-OATP2B1 cells, bromosulfophthalein, and estrone 3-sulfate served as control (A). The influence on transcellular fluxes was assessed on permeable supports using cultured Caco-2 cells. The unidirectional Papp-coefficients in (C) apical (a)-to-basal (b), or (D) b-to-a directions for atorvastatin were used to calculate (B) the efflux ratio [Papp (b–a)/Papp (a–b)]. Data are presented as mean ± S.D. of n = 3 independent experiments each performed in biologic triplicates, *P ≤ 0.05 one-way analysis of variance with Dunnett’s multiple comparisons test (A) or Student’s t test (B, C, D).
- Fig. 4.
Influence of commercially available St. John’s wort preparations on OATP2B1 function and transactivation of CYP3A4. Transport inhibition studies assessing binding site A were conducted in MDCKII-OATP2B1 cells. Formulations were tested in two concentrations representing a 1/100 or 1/1000 dilution of the respective formulation dissolved in 200 ml liquid (A). The influence of each formulation (1/100 dilution) on CYP3A4 activation was determined in cell-based reporter gene assays using (B) HepG2 or (C) HeLa cells as cellular models. Firefly luciferase was normalized to that of Renilla in each sample. Expression of HNF4α and OATP2B1 in HepG2 and HeLa cells was detected by Western blot analysis; actin served as control (D, E). Data are presented as mean ± S.D. of n = 3 independent experiments. *P-value ≤ 0.05 one-way analysis of variance followed by Dunnett’s multiple comparisons test; Vogel 1, Hyperimed; Vogel 2, Hyperiforce.
- Fig. 5.
Association of hyperforin or hypericin content with the observed effects in cell-based reporter gene assays, or transport inhibition studies. Luciferase activity was determined in transiently transfected HepG2 (A, D) or HeLa cells (B, E). Inhibition studies were conducted in MDCKII stably expressing OATP2B1 (C, F). Association of the observed effect with hyperforin or hypericin content was analyzed by linear regression (indicated by dotted line, shown with confidence interval), correlation was determined by calculating the Pearson coefficient. Data are presented as mean ± S.D. with results of n = 3 independent experiments performed in biologic triplicates. R, Pearson coefficient; open symbol indicated “Hyperiplant“, which was neither included in the linear regression nor the calculation of the Pearson coefficient.
Tables
- TABLE 1
Content of hypericin and hyperforin determined by HPLC-UV
Percentage of hypericin or hyperforin content given in the compendium was converted to content per 100 mg extract. For quantification by HPLC, a standard curve was used to calculate the concentration of hypericin or hyperforin, respectively. Data from quantification are from three independent experiments.
Formulation (Tradename) Hypericina (Declared Content) Hypericina (Quantified by HPLC) Hyperforinb (Declared Content) Hyperforinb (Quantified by HPLC) mg/100 mg mg/100 mg ± S.D. mg/100 mg mg/100 mg ± S.D. ARKOCAPSc 0.3 0.09 ± 0.04 — 0.24 ± 0.01 DEPRIVITAd 0.1–0.3 0.18 ± 0.01 — 1.04 ± 0.05 HAENSELER MENOPAUSEe 0.1–0.3 0.10 ± 0.01 — 0.34 ± 0.01 HYPERIPLANTf 0.1–0.3 0.15 ± 0.02 36 1.62 ± 0.03 JARSINg 0.1–0.3 0.20 ± 0.02 — 0.70 ± 0.02 REBALANCEh 0.1–0.3 0.08 ± 0.01 <0.2 0.02 ± 0.01 REMOTIVh 0.1–0.3 0.09 ± 0.01 <0.2 0.02 ± 0.01 SANDOZ HYPERICUMi 0.1–0.3 0.21 ± 0.02 — 0.85 ± 0.04 SOLEVITAd 0.1–0.3 0.16 ± 0.03 — 1.02 ± 0.07 VOGEL HYPERIMEDj 0.5–0.8 0.08 ± 0.01 — — — VOGEL HYPERIFORCEj 0.2–2.1 0.08 ± 0.01 — — — ↵a The content of hypericin corresponds to the sum of the content of hypericin and pseudohypericin.
↵b The content of hyperforin corresponds to the sum of the content of hyperforin and adhyperforin.
↵c Arko Diffusion SA, Les Avacias, Switzerland.
↵d Permamed AG, Therwil, Switzerland.
↵e Hänseler AG, Herisau, Switzerland.
↵f Schwabe Pharma AG, Küssnacht, Switzerland.
↵g Vifor SA, Bern, Switzerland.
↵h Zeller AG, Romanshorn, Switzerland.
↵i Sandoz Pharmaceuticals AG, Holzkirchen, Germany.
↵j Bioforce AG, Roggwil, Switzerland.
Data Supplement
- Supplemental Figures -
Supplemental Figure 1 - Counterflow experiments performed in MDCKII cells
Supplemental Figure 2 - Competitive counterflow experiment with hyperforin (A) or hypericin (B)
Supplemental Figure 3 - Chromatograms of a mixture of the references hypericin (TOCRIS) and pseudohypericin (Sigma-Aldrich), and of the 11 in Switzerland marketed St. John’s wort formulations
Supplemental Figure 4 - Chromatograms of the reference hyperforin (Sigma-Aldrich) and of the 11 in Switzerland marketed St. John’s wort formulations.
- Supplemental Figures -
