Echinacea sanguinea and Echinacea pallida extracts stimulate glucuronidation and basolateral transfer of Bauer alkamides 8 and 10 and ketone 24 and inhibit P-glycoprotein transporter in Caco-2 cells

Abstract

The use of Echinacea as a medicinal herb is prominent in the United States, and many studies have assessed the effectiveness of Echinacea as an immunomodulator. We hypothesized that Bauer alkamides 8, 10, and 11 and ketone 24 were absorbed similarly either as pure compounds or from Echinacea sanguinea and Echinacea pallida ethanol extracts, and that these Echinacea extracts could inhibit the P-glycoprotein transporter in Caco-2 human intestinal epithelial cells. Using HPLC analysis, the permeation rate of Bauer alkamides by passive diffusion across Caco-2 cells corresponded with compound hydrophilicity (alkamide 8 > 10 > 11), independent of the plant extract matrix. Both Echinacea ethanol extracts stimulated apparent glucuronidation and basolateral efflux of glucuronides of alkamides 8 and 10 but not alkamide 11. Bauer ketone 24 was totally metabolized to more hydrophilic metabolites when administered as a single compound, but was also glucuronidated when present in Echinacea extracts. Bauer alkamides 8, 10, and 11 (175-230 µM) and ethanol extracts of E. sanguinea (1 mg/mL, containing ~ 90 µM total alkamides) and E. pallida (5 mg/mL, containing 285 µM total alkamides) decreased the efflux of the P-glycoprotein transporter probe calcein-AM from Caco-2 cells. These results suggest that other constituents in these Echinacea extracts facilitated the metabolism and efflux of alkamides and ketones, which might improve therapeutic benefits. Alkamides and Echinacea extracts might be useful in potentiating some chemotherapeutics, which are substrates for the P-glycoprotein transporter.

Fig. 1

The chemical structures of Bauer alkamides 8, 10, 11 and ketone 24.

Fig. 2

Transport of pure Bauer alkamides 8, 10 and 11 across Caco-2 cells. A. Concentration dependency of the transport of three alkamides. B. Non-saturable transport of three alkamides at 25 μM during 90 min incubation period with no difference between apical to basolateral (AP-BL) and basolateral to apical (BL-AP) direction at each time point. C. The effect of temperature on the transport of the three alkamides (37°C vs 4°C). Means bearing different letters were significantly different among three alkamides by ANOVA and Tukey’s multiple comparison (p < 0.05). Data are presented as the mean ± S.D. (n=6).

Fig. 2

Transport of pure Bauer alkamides 8, 10 and 11 across Caco-2 cells. A. Concentration dependency of the transport of three alkamides. B. Non-saturable transport of three alkamides at 25 μM during 90 min incubation period with no difference between apical to basolateral (AP-BL) and basolateral to apical (BL-AP) direction at each time point. C. The effect of temperature on the transport of the three alkamides (37°C vs 4°C). Means bearing different letters were significantly different among three alkamides by ANOVA and Tukey’s multiple comparison (p < 0.05). Data are presented as the mean ± S.D. (n=6).

Fig. 3

Glucuronidation of Bauer alkamides 8, 10, 11 and ketone 24 for pure compounds or from plant extracts by Caco-2 cells. Before: prior to deconjugation with β-glucuronidase; After: subsequent to deconjugation with β-glucuronidase. The concentration of three Bauer alkamides and ketone 24 was 25 μM both for pure compounds or as found in the two Echinacea extracts. A. Bauer alkamide 8. B. Bauer alkamide 10. C. Bauer alkamide 11. D. Bauer ketone 24. ^* Significantly different compared with the recovery relative to the initial amount before the enzyme treatment by two-sample t-test (p < 0.05). Data are presented as the mean ± S.D. (n=6).

Fig. 3

Glucuronidation of Bauer alkamides 8, 10, 11 and ketone 24 for pure compounds or from plant extracts by Caco-2 cells. Before: prior to deconjugation with β-glucuronidase; After: subsequent to deconjugation with β-glucuronidase. The concentration of three Bauer alkamides and ketone 24 was 25 μM both for pure compounds or as found in the two Echinacea extracts. A. Bauer alkamide 8. B. Bauer alkamide 10. C. Bauer alkamide 11. D. Bauer ketone 24. ^* Significantly different compared with the recovery relative to the initial amount before the enzyme treatment by two-sample t-test (p < 0.05). Data are presented as the mean ± S.D. (n=6).

Fig. 4

HPLC chromatograms of Bauer ketone 24 across Caco-2 cell monolayers as pure compound or from Echinacea species. A. Bauer ketone 24 standard at 100 μM (retention time 17.6 min). B. Bauer ketone 24 (100 μM) was not detected in the basolateral supernatant fluid, but two more hydrophilic metabolites were detected (retention time 13.3 and 15.4 min). C. Bauer ketone 24 was shown in the basolateral side after apically applied E. sanguinea containing 100 μM of ketone 24 (retention time 17.6 min). D. Bauer ketone 24 was shown in the basolateral side after apically applied E. pallida containing 100 μM of ketone 24 (retention time 17.6 min).

Fig. 4

HPLC chromatograms of Bauer ketone 24 across Caco-2 cell monolayers as pure compound or from Echinacea species. A. Bauer ketone 24 standard at 100 μM (retention time 17.6 min). B. Bauer ketone 24 (100 μM) was not detected in the basolateral supernatant fluid, but two more hydrophilic metabolites were detected (retention time 13.3 and 15.4 min). C. Bauer ketone 24 was shown in the basolateral side after apically applied E. sanguinea containing 100 μM of ketone 24 (retention time 17.6 min). D. Bauer ketone 24 was shown in the basolateral side after apically applied E. pallida containing 100 μM of ketone 24 (retention time 17.6 min).

Fig. 5

The effects of Bauer alkamides 8, 10, 11, ketone 24 on P-glycoprotein transporter activity. Control was 0.3% v/v DMSO in PBS. Verapamil was used as positive control at 10 μg/mL. ^{*, **} Significantly different compared with control by two-sample t-test (p < 0.05, 0.01, respectively). Data are presented as the mean ± S.D. (n=6). The ethanolic extract of E. sanguinea at 1 mg/mL contained 85 μM of alkamide 8, 2 μM of alkamide 10, and 0.7 μM of alkamide 11. The ethanolic extract of 5 mg/mL of E. pallida extract contained 215 μM of alkamide 8, 25 μM of alkamide 10, and 45 μM of alkamide 11.