Purpose: Herba Epimedit is a Traditional Chinese Medicine used to treat cardiovascular diseases and other chronic illness, such as infertility, amnesia, neurasthenia, impotence and senile functional diseases in China for thousands of years. The major active constituents of Herba Epimedit are flavonoids and more than 60 kinds of flavonoids have been identified. Among them, Epimedin A, Epimedin B, Epimedin C, Icariin and Icariside II are considered as the major bioactive flavonoids in Herba Epimedit. Although in vitro and in vivo studies have demonstrated that these flavonoids have many beneficial effects, especially potential biological activity against osteoporosis, the extent to which flavonoids are absorbed and the mechanisms involved are not known. This study aimed to investigate the intestinal transport mechanisms of Epimedin A, Epimedin B, Epimedin C, Icarin and Icariside II in different dosing formulations, by a well validated Caco-2 monolayer model in vitro. In addition, the impact of efflux transporters including P-glycoprotein and multidrug-resistance associated proteins on these flavonoids absorption was also studied. Methods: The transport of the five flavonoids from both apical to basolateral (AP-BL) and basolateral to apical (BL-AP) directions, in the form of individual pure compounds, pure compounds mixture and total flavonoids extract, were studied in the Caco-2 cell monolayer. The bi-directional transport studies were performed in the presence or absence of EGTA which disrupts tight junctions by its Ca?* chelating effect, in the presence or absence of cyclosporine, ketoconazole or verapamil which is inhibitor of the P-glycoprotein (P-gp), and in the presence or absence of MK571 which is inhibitor of the multidrug-resistance associated protein (MRP). In addition, the expression and localization of P-gp and MRP2 on Caco-2 cell monolayer were examined by western blot and fluorescent microscopy. Results: In the form of individual pure compounds, no flavonoids could be detected from both directions for Epimedin A, Epimedin B and Epimedin C. A small Papp value (0.89#0.07×106 cm/s) was obtained for the secretion transport of Icarin from basolateral to apical side (BL-AP) and no Icariin could be detected for the absorption transport (apical to basolateral side, AP-BL). On the other hand, Icariside II has a high Papp values (5.66#0.40×10% cm/s) from BL to AP, but no Icariside II could be detected from AP to BL. In the presence of EGTA; Papp values of Epimedin A, Epimedin B, Epimedin C and Icarin markedly increased to 106 cm/sec in both directions. Furthermore, the Papp value of Icariin from the BL to AP significantly decreased (P<0.05) in the presence of MK571 but did not affected by cyclosporine. On the other hand, in the case of Icariside II, still no flavonoid could be detected from the AP to BL direction while the Papp value significantly decreased (P<0.05) from the BL to AP direction in the presence of EGTA. However, the Papp values of Icariside II from AP to BL significantly increased to 0.68‡0.28×10% cm/s, 0.6410.07×106 cm/s and 2.561028×10 cm/s in the presence of 50 M varapamil, 50 M ketoconazole or 10 MM cyslosporin, respectively, while the Papp value decreased significantly (P<0.05) from BL to AP. Moreover, Papp values of Icariside II from AP to BL increased to 1.120.06×10 cm/s and 0.9010.13x10°° cm/s in the presence of 50 MM MK571 while the Papp value from BL to AP decreased significantly (P<0.05). For Epimedin A, Epimedin B and Epimedin C, no flavonoid could be detected in both directions neither in the form of pure individual compounds, pure compound mixture nor total flavonoids extracts. For Icarin, the Papp values from BL to AP decreased significantly (P<0.05) either in the mixture or extract form while still no flavonoid could be detected from AP to BL in any forms. In the case of Icariside II, Papp values from AP to BL increased to more than 10-6 cm/sec while the Papp values from BL to AP did not significantly changed in the mixture or extract form. In addition, P-gp and MRP2 were expressed on Caco-2 cell monolayer validated by western blot and fluorescent microscopy. Conclusion: The above results suggest that the absorption and secretion transport of Epimedin A, Epimedin B and Epimedin C and Icarin are mainly via paracellular pathway. Meanwhile, MRP efflux systems maybe involved in the secretion transport of Icariin. It is expected these four flavonoids are poorly absorbed in vivo. For Icariside II, its transport involves interaction with the P-gp and MRP efflux systems but not via the paracellular pathway. The results that Icariside II would be detected in the form of pure compound mixture and total flavonoids extract but not in the form of individual pure compound suggested that the co-occurring of Icariside I and Icarin or other flavonoids may saturate the efflux transporters, and/or these efflux transporters maybe partly inhibited in the mixture or extract form.

目的：淫羊歡 (Hlerba Epimedii) 是中國歷史悠久的傳統中藥，可用以治療心血管疾病和其他慢性疾如不育、健忘症、神经衰弱症、阳娄、老年功能性疾病。淫羊藿的主要成份是黃酮類。60多種的黄酮頻己被鑑定，其中，朝花定 朝藿定 B 朝藿定 C、淫羊蒮苷和寶歡苷工被認為主要的生物活性的黄酮類。雖然體外和體内研究提出這些黃酮類有益健康，特別是有對抗骨质疏松症的潛在活性，但是這些黄酮類的吸收程度和沙及的機制選不知道。本研究的目的在於以經過驗證的 Caco-2 細胞單層體外模型作為朝藿定 A、朝藿定 B、朝藿定 C、淫羊藿苷和寶藿苷 1在不同的劑量處方中腸道轉運機理。此外，也研究了外排轉運體包括 P-醣蛋白和多藥耐藥相關蛋白對這些黃酮吸收的影響。 方法：以 Caco-2 單細胞層來研究五種黃酮類從頂側—腸腔側（AP 側）到達底側 (BL側）和從BL 側外排到 AP 側的轉運。黃酮類以三種形式在 Caco-2 單細胞層進行研究，分別是純的單體化合物、五種純的單體化合物混合物和總黃酮提取物。雙向轉運實驗在以下化合物的存在和不存在下進行：可與鈣離子螯合而破壞 细胞之间緊密連接的 EGTA; P-醣蛋白抑制劑酮康唑、环孢素或维拉帕米：多藥耐藥相關蛋白抑制劑MK571。此外，P-醣蛋白和多藥耐藥相關蛋白在 Caco-2 單 細胞層的表達和定位分別以免疫印迹杂交法和萤光顯微鏡來檢測。 結果：在純單體的形式下，朝藿定 A、朝藿定 B和朝藿定C 在兩個轉運方向都測不到；溼羊藿苷在 BL 側到 AP 側（BL-AP）的方向測得小的表觀渗透系數 0.89‡0.07×106cm/3），而在 AP 側到BI側CAP-BL）則測不到。另一方面，寶藿苷！在 BL-AP 測得高的表觀渗透系數(5.66士0.40×10% cm/s)，但在 AP-BL 則測不到。在EGTA 的存在下，朝藿定 A、朝藿定 B、朝藿定 C和淫羊藿苷在兩個 轉運方向的表觀渗透系牧都增加至大約 10°cm/sec。而且，在MKST1 的存在下，淫羊歡苷在 BL-AP 側的表觀渗透系收明顯下降(P-0.05）但此表觀渗透系牧不受环孢素的影響。另一方面，在 EGTA 的存在下，地街有1 在 AP-BL 的方向依然 測不到而 BL-AP 則的表親渗透系蚊就明顯下降了（P<0.05）。然而，饮街苷工的 AP- BL 的表觀渗透系數在 50 wM 维拉帕水的存在下增加為 0.68士0.28×106cp/s;在50wM 酮康唑的存在下增加為0.64‡0.07x106cm/s；而在 10MM环孢素的存在下增加為 2.56+028×10°om/s：而在 BL-AP 的表觀渗透系數就明顯下降 (P<0.01)。此外，寶霍苷1的 AP-BL 的表觀渗透系數在 50 wM MK 571 的存在下 增加為 1.12‡0.06×106or/s，而在 BL-AP 表觀渗透系數就明顯下降(MK571, P<0.05) o 無論在純的單體化合物、 五種純的單體化合物混合物和總黃酮提取物的形式下，朝藿定 A、朝藿定 B和朝藿定C 在兩個轉運方向都測不到。與純的單體化合物 比較，淫羊藿苷在混合物或提取物的形式中 BL-AP 側方向的表觀渗透系敷明顯降低 （P-0.05)，在AP- BL則依然測不到；而寶霍苷1在 AP-BL 的表觀渗透系數增加至大於 106cm/sec，而在 BL -AP 的表觀渗透系數就沒有明顯增加。 此外，Caco-2 細胞單層上的 尸-醣蛋白和多藥耐藥相關蛋白的表達以免疫印迹杂交法和萤光顯微鏡來証實。 結論：以上的結果提出朝藿定 A、朝藿定 B和朝藿定C和淫羊藿苷的吸收轉運和外排轉運主要透過细胞旁路通路。同時，淫羊藿苷的外排轉運可能涉及多藥耐藥相關蛋白外排系統。我們預計這四種黄酮類在體內吸收不良。而寶藿苷1的轉運涉及外排系統 尸-醣蛋白和多藥耐藥相關蛋白的相互作用而非經由细胞旁路通路。寶藿苷工在純單體化合物混合物和總黃酮提取物中測到而在純單體化合物中 測不到的結果提出寶藿苷1與淫羊藿苷或其他黃酮類同時存在可能會使P-醣蛋白 和多藥耐藥相關蛋白飽和：另一可能是在混合物或總黃酮提取物中同時存在的其他黃酮類對卩-醣蛋白和多藥耐藥相關蛋白有某程度上的抽制作用。