Intestinal absorption mechanisms of prenylated flavonoids present in the heat-processed Epimedium koreanum Nakai (Yin Yanghuo)

Abstract

Purpose: The purpose is to determine absorption mechanism of five bioactive prenylated flavonoids (baohuoside I, icariin, epimedine A, B, and C) present in heat-processed Epimedium koreanum Nakai (Yin Yanghuo).

Methods: Transport of five prenylated flavonoids present in heat-processed herbs were studied in the human intestinal Caco-2 model and the perfused rat intestinal model.

Results: In the perfused rat intestinal model, prenylated flavonoids with a monoglucosidic bond (e.g., icariin) was rapidly hydrolyzed into corresponding metabolites (e.g., baohuoside I). In the Caco-2 model, apical to basolateral permeability of a monoglycoside baohuoside I (1.46 x 10(-6) cm/sec) was more than 2 folds greater than four prenylated flavonoids with 2 or more sugar moieties (<0.6 x 10(-6) cm/sec). The slow apical to basolateral transport of baohuoside I was the result of efflux. This efflux was carrier-mediated and active since its transport was vectorial, concentration- and temperature-dependent with activation energies greater than 15 kcal/mol. Efflux of baohuoside I was significantly suppressed by inhibitors of BCRP and MRP2, whereas efflux of icariin was significantly inhibited only by p-glycoprotein inhibitor verapamil. Because YHH is often heat-processed for better efficacy, we determined and found the optimal condition for increasing contents of more bioavailable flavonoids (i.e., baohuoside I) to be 160-170 degrees C for 5-7 min.

Conclusions: Poor bioavailability of prenylated flavonoids results from their poor intrinsic permeation and transporter-mediated efflux. Heat processing parameters may be optimized to preserve the herb's bioavailable flavonoids, which help retain and improve its efficacy during processing.

Fig. 1

Chemical structures of five prenylated flavonoids isolated from Epimedium koreamum Nakai or Yin Yanghuo: icariin, epimedin A, epimedin B, epimedin C, and baohuoside I. The symbol “glc” refers to glucose, “rha” to rhamnose, and “xyl” to xylose.

Fig. 2

UPLC elution profiles of four 7-O-glucose-prenylflavonoids and their intestinal metabolites. In Fig. 2a. the UPLC profile represents a perfusate that contains 10μM each of epimedin A, epimedin B, epimedin C and icariin, which were labeled in the UPLC profile as peak number 1, 2, 3, and 4, respectively. In Fig. 2b. the profile is a representative perfusate samples after the perfusate present in Fig. 2a has undergone 2 hr single-pass rat intestinal perfusion as described in the method. The rat intestinal perfusate sample has 4 corresponding metabolites of 1, 2, 3 and 4, and only baohuoside I (peak number 8) is identified whereas the other three peaks are unidentified glucosidase metabolites of 1, 2 and 3. Testosterone was used as an internal standard (IS) in both panels.

Fig. 3

Permeabilities of baohuoside I at different concentrations (from 5μM to 40μM). The experiments were performed at 37°C. Five samples were taken time at 0, 2, 3, 4, 5 hr after the start of the experiments. The amounts transported were then plotted against sampling time (2, 3, 4, and 5 hr), which always generated linear curves, the slopes of which are rates of drug transport. The rates of transport were then used to calculate permeabilities using equation (1) and the calculated permeabilities are plotted here as bars. Each bar represents the average of three determinations and the error bars are the standard deviation of the means. The “*” symbol indicates a statistically significant difference between permeability at 5μM (control) and that at a higher concentration. The number of “*” symbol indicates the level of significance with ***p<0.001, **p<0.01 and *p<0.05. One-way ANOVA with Tamhane’s post hoc was used to analyze the data statistically.

Fig. 4

Permeabilities of baohuoside I (20μM) at different temperatures. The experiments were performed at 37°C. Permeabilities plotted here are similarly generated as described in Fig. 3. Each bar represents the average of three determinations and the error bars are the standard deviation of the means. The “*” symbol indicates that absorptive permeability (P_AB) and secretory permeabilities (P_BA) values at this concentration are significantly different from each other (p<0.01). One-way ANOVA with Tamhane’s post hoc was used to analyze the data statistically.

Fig. 5

Effects of various potential inhibitors on permeabilities of baohuoside I (20μM). The experiments were performed at 37°C. The inhibitors were present at the donor side of the cell monolayers at the stated concentration (50 μM for MK-571, 25 μM for dipyridamole, and 20 μM for verapamil). Permeabilities plotted here are similarly generated as described in Fig. 3. The “*” symbol indicates that P_AB and P_BA are significantly different (p<0.05) from each other. The symbol “α” indicates that the absorptive permeability (P_AB) of baohuoside I in the presence of an inhibitor is significantly different (p<0.05) from that of the control, whereas the symbol “β” indicates the same for its secretory permeabilities (P_BA). One-way ANOVA with Tamhane’s post hoc was used to analyze the data statistically.

Fig. 6

Effects of various potential inhibitors on permeabilities of icariin (20μM). Permeabilities plotted here are similarly generated as described in Fig. 3. Absorptive permeability is expressed as P_AB and secretory permeability is expressed as P_BA. The experiments were performed at 37°C. The inhibitors were present at the donor side of the cell monolayers at the stated concentration (50 μM for MK-571, 25 μM for dipyridamole, and 20 μM for verapamil). The “*” symbol indicates that P_AB and P_BA are significantly different (p<0.01) from each other. The symbol “α” indicates that the absorptive permeability (P_AB) of icariin in the presence of an inhibitor is significantly different (p<0.05) from that of the control, whereas the symbol “β” indicates the same for its secretory permeabilities P_BA. One-way ANOVA with Tamhane’s post hoc was used to analyze the data statistically.

Fig. 7

Effect of concentration on the cellular accumulation of baohuoside I at 37°C. The accumulation was measured after 5 hr incubation of cell monolayers with baohuoside I. Each data point is the average of three determinations, and the error bars represent the standard deviation of the mean. The “*” symbol indicated statistically significant difference between loading side. One-way ANOVA with Tamhane’s post hoc was used to analyze the data statistically.

Fig. 8

Effects of processing temperature and time on the prenylated flavonoid content in Yin Yanghuo (Epimedium koreamum Nakai). The drug powder was heated at different temperature (from 160°C to 210°C)and/or for different length of time (from 0–30 min) and the contents of icariin (a), epimedin A (b), epimedin B (c), epimedin C (d), and baohuoside I (e) were analyzed using HPLC and plotted against the duration of the heating.