橙皮素磷酸酯衍生物生物可利用率之研究

Abstract

橙皮素 (hesperetin, Hst) 為類黃酮化合物，廣泛存在於柑橘屬果實之植物次級代謝物，具有預防心血管疾病、提升免疫力、抗癌症等生理活性。然而Hst的水溶性不佳，生物可利用率 (bioavaiability) 極低，使其在食品與藥品應用上受到許多限制。本研究室先前篩選出Bacillus subtilis BCRC 80517菌株中具有類黃酮磷酸酯合成酶 (flavonoid phosphate synthetase) 可將類黃酮磷酸酯化形成橙皮素磷酸酯衍生物 (hesperetin phosphate derivatives, HstPs)，hesperetin 7-O-phosphate (Hst7P) 與hesperetin 3'-O-phosphate (Hst3'P) 。在製藥工業上常利用磷酸化手段合成磷酸酯前驅藥物 (phosphate ester prodrug) 進而提升生物可利用率。本研究的主要目的在於利用Caco-2 細胞與藥物動力學試驗評估HstPs之生物可利用率並和Hst及市售橙皮素衍生物產品如橙皮苷 (hesperidin, Hsd) 與葡糖基橙皮苷 (α glucosyl hesperidin, G-Hsd) 比較。HstPs與Hst的基本物化性質如溶解度與安定性的結果顯示Hst7P 和Hst3'P於橙皮素之相對溶解度分別為 1446與1544倍；HstPs與Hst在240分鐘模擬胃液 (simulated gastric fluid, SGF) 與模擬腸液 (simulated intestinal fluid, SIF) 反應下的安定性良好，顯示橙皮素磷酸酯可以原始型態順利達腸道被吸收。在HBSS緩衝溶液之穿透試驗，比較50 μM Hst7P、Hst3'P、Hst、Hsd與G-Hsd 五者之穿透比率與表觀穿透係數 (Apparent permeability coefficient, Papp )，結果顯示Hst7P與Hst3'P的Papp各為9.27×10−6 與10.67×10−6 (cm/sec)，遠高於Hst (4.87×10−6cm/sec)、Hsd (0.12×10−6cm/sec) 與G-Hsd (0.22×10−6cm/sec)，顯示化合物溶解程度與分子大小影響穿透程度。此外本研究比較Caco-2 細胞鹼性磷酸酶 (alkaline phosphatase, ALP) 對 fosphenytoin (phosphate ester prodrug之控制組) 和HstPs之水解效率，結果顯示與fosphenytoin 相比ALP對HstPs有較佳的水解效率。最後由藥物動力學體內試驗的結果顯示HstPs可大幅增加口服後血漿內Hst濃度，吸收率較佳。綜合而論，HstPs如Hst7P 和Hst3'P相較Hst及市售產品如Hsd與G-Hsd具有較高的生物可利用率，未來在保健食品應用上深具潛力，可作為目前市售產品的有利競爭者。

Hesperetin (Hst) is one of the secondary metabolite in citurs fruit, which has a number of pharmacological and biological activities, such as ameliorating cardiac inflammation, increasing intact immunity systems, activities of anti-cancer; however, the application of Hst has been restricted because of low water solubility and poor bioavailability. Our previous study had revealed two phosphorylated hesperetin (HstPs), e.g. hesperetin 7-O-phosphate (Hst7P) and hesperetin 3'-O-phosphate (Hst3'P), derived by Bacillus subtilis BCRC 80517 bioconversion with Hst. On the basis of phosphate prodrug, Hst7P and Hst3'P were supposed to be absorbed more effective in intestinal in contrast to Hst. This study was aimed to explore the bioavailability of HstPs, Hst and its derivatives, including hesperidin (Hsd) and alpha glucosyl hesperidin (G-Hsd), through in vitro Caco-2 cells monolayer assay and in vivo pharmacokinetic study. First, we revealed that HstPs displayed 1446 and 1544 times higher in aqueous solubility than Hst. In addition, either HstPs or Hst were stable in both simulated gastric fluid (SGF) and simulated intestinal fluid (SIF) during 240-min incubation. In in vitro transport study, HstPs exhibited higher permeation rate and apparent permeability rate (Papp). The results showed that the Papp values of Hst7P and Hst3’P were 9.27×10−6 and 10.67× 10−6 (×10-6cm/sec), respectively, higher than Hst (4.87×10−6cm/sec), Hsd (0.12×10−6cm/sec), and G-Hsd (0.22×10−6cm/sec), indicated that HstPs enhanced in vitro intestinal permeability of Hst and its derivatives. The results’ of dephosphorylation assay indicated the compounds could be hydrolyzed effectively by the membrane-associated alkaline phosphatase (ALP). In addition, the clearance half-life (T1/2) of Hst7P and Hst3'P were significantly lower than fosphenytoin. Moreover, HstPs greatly increased plasma exposure to Hst after oral administration in rats. Based on the phosphate prodrug concept, HstPs could be absorbed more effective and faster. Our finding indicated that HstPs have higher bioavailability than Hst, Hsd, and G-Hsd.