Keywords: dl-praeruptorin A; d-praeruptorin A; Caco-2 monolayers; absorption; liver microsomes; metabolism; enzyme kinetics Qian-hu (Radix Peucedani), the dry roots of peucdanum praeruptorum Dunn, has been used for treatments of certain respiratory disorders such as cough and asthma, and pulmonary hypertension for centuries. The extract of Qian-hu is able to relax tracheal and pulmonary arterial preparations. dl-Praeruptorin A (dl-PA) and d-praeruptorin A (dPA) are two main bioactive constituents of the herb and dl-PA was also documented as the chemical marker for quality control of Qian-hu in China Pharmacopoeia. dPA is dextrorotatory enantiomer with two chiral centers, dl-PA is the racemate of dPA. Modern pharmacologic studies demonstrated that both dl-PA and dPA exhibited strong bioactivities, espeacially the cardiovascular protective effect. Recently, one literature reported that dPA showed more potent relaxation than its antipode l-praeruptoin A (lPA) on phenylephrine-induced contraction of rat isolated aortic rings with intact endothelium. However, there is no pharmacokinetics report on Qian-hu or its main bioactive constituents, thus the pharmacokinetic basis of the actions of the herb remains to be addressed. The present study aims to elucidate the intestinal transport mechanism as well as the hepatic metabolic stability of dl-PA and dPA using in vitro models, which will help to understand the in vivo fates of the mainconstituents of the herb and provide important information for a systemic pharmacokinetic evaluation of Qian-hu ongoing in our laboraboratory. In addition, the present study provides preliminary data on chiral discrimination, if any, in ADME properties by comparing in vitro absorption and metabolism of dl-PA and dPA . The bi-directional transports of dl-PA and d-PA were studied in the Caco-2 cell monolayer model and quantified by chiral-HPLC/UV. The identification of the metabolites which were generated in the caco-2 model was carried out by LC-MS/MS. For the metabolism part, it was investigated by incubation dl-PA and d-PA with human liver microsomes (HLMs) and rat liver microsomes (RLMs). For comparing the metabolic stability of dl-PA and dPA and speciesdifference, the generated metabolites were identified by LC-MS/MS and quantified by HPLC/UV. The bi-directional transport study showed that both dl-PA and dPA are mainly transported via transcellular pathway by passive diffusion with the Papp > 10-5 ; there is no stereoselectivity of dPA and lPA in racemate during the transport process. In addition, during the transport of dl-PA and dPA, their recoveries were low, esprecially for dl-PA (60-70%). Further study the bingding ratio of dl-PA, the results showed that 10% non-specific binding to TranswellTM membrane, other 10% irreversible binding to cellular components during transport were demonstrated to contribute to the loss with no stereoselectivity binding. In addition, two new metabolites, which have the same molecular wight but differernt retenteion time, were detected from the bi-directon transport studies of dl-PA not dPA on Caco-2 monlayer. They were identified to be the hydrolysis of dl-PA at 4′ position by LC-MS/MS. And further enzyme inhibition study of dl-PA on the Caco-2 cell monolayers showed that this metabolism was mediated by hCE-1. For the in vitro liver metabolism part, either dl-PA or dPA could produce phase I metabolites. 12 metabolites colud be detected when dl-PA was incubated with RLMs, and 9 metabolites formed with HLMs. Different with dl-PA, 12 metabolites colud be detected when dPA was incubated with RLMs, while 6 metabolites formed with HLMs. All the metabolites were identified by LC-MS/MS, and the result showed that all metabolisms occurred at the 3′ and/or 4′ position but not on the coumarin ring, with oxidation and hydrolysis the two predominant pathways. Enzyme kinetics of dPA in liver microsomes was studied. M1 which is the major metabolite of dPA in HLMs was isolated by wet chemistry method and silica gel column chromatography. A HPLC-MRM method was established for this study. Further more, the isoenzymes which were involved in the metabolism of dPA was identified by chemical inhibition, idiosyncratic antibody inhibition and recombinase screening. In conclusion, dl-PA and dPA could be readily transported across Caco-2 cell monolayer by passive diffusion. dl-PA could be metabolized by carboxylesterases in Caco-2 cells. dl-PA and dPA are subjected to extensive phase Ⅰ metabolism in liver microsomes. They separately exhibited the species differences and enantiomers metabolism differences in the same liver microsomes. Those results could provide important reference for further studying the in vivo pharmacokinetics of peucdanum praeruptorum Dunn or dl-PA and dPA.

關鍵詞：白花前胡甲素，白花前胡丙素，Caco-2 單層細胞模型，肝微粒體，代 謝，酶動力學 中藥前胡為傘形科植物白花前胡 Peucedanum praeruptorum Dunn 的乾燥 根，具有散風清熱，降氣化痰的作用，臨床上用於風熱咳嗽痰多，痰熱喘滿等癥。 白花前胡甲素（以下簡稱甲素）及白花前胡丙素（以下簡稱丙素）為前胡中主要 成份。甲素具有兩個手性中心，為消旋體，兩光學異構體均為順式構型，是中國 藥典規定的中藥前胡的品質控制的指標成分，丙素為其右旋異構體，為順式構 型。現代藥理研究表明，此兩種成份均具有較強的生物活性，尤以降壓、抗心衰、 抗心肌缺血等心血管活性為顯著。最近研究發現，在舒張血管活性方面右旋體的 作用強於其左旋對映異構體。然而，至今未見有關中藥前胡及其主要成分的藥動 學研究報導，並且對其發揮體內效應的機制尚不清楚。本研究通過研究前胡主要 成分白花前胡甲素和丙素的體外吸收及代謝機制，旨在預測其體內動態變化過程 及主要影響因素，為本研究組正在展開的前胡體內藥動學及藥效學系統評價提供 重要的實驗參考；此外，通過比較消旋體與右旋對映體的吸收及代謝差異，初步揭示甲素和丙素的結構上的區別可能導致的體內過程差異。 在 Caco-2 單層細胞模型上研究甲素及丙素的雙向（從腸腔側（AP 側）到 達基底側（BL 側）和從基底側外排到腸腔側）轉運及分子機制，採用手性 C18 柱對甲素和丙素進行 HPLC/UV 定量分析，LC-MS/MS 進行代謝產物的鑒定分 析；採用大鼠及人肝微粒體體外孵育法，比較研究甲素和丙素的肝臟代謝穩定性 及種屬差異。定量分析採用 HPLC/UV，代謝產物鑒定採用 LC-MS/MS. 透過實驗的結果表明，甲素及丙素的體外吸收均主要通過被動擴散，較易 透過（Papp > 10-5）Caco-2 細胞單層，其表觀滲透係數隨濃度升高而降低；甲素 的兩個手性對映體的透過無明顯立體選擇性。在 Caco-2 透過實驗中，甲素及丙 素的回收率均較低（大概的數值），並且甲素的回收率低於丙素。進一步對甲素 進行膜結合研究，發現約 10%的甲素與空白碳聚酯膜結合，另有約 10%的藥物 與甲醇處理過的細胞膜非特異結合，沒有發現甲素的左旋體和右旋體存在結合的 立體選擇性。此外，甲素在透過 Caco-2 細胞單層過程中發生水解，產生兩個代 謝產物（M1 和 M2），但單獨的丙素在透過 Caco-2 細胞單程過程中並未檢測到 任何代謝產物。利用 LC-MS/MS 進行定性分析，確定 M1 和 M2 為甲素在 4′位 酯鍵水解產生的同分異構體，通過觀察酯酶抑製劑對甲素在透過過程中代謝的影 響，明確了催化甲素生成 M1 和 M2 的酶為羧基酯酶。 通過體外溫孵實驗，發現白花前胡甲素和丙素在大鼠及人肝微粒體中均發 生較為強烈的一相代謝。甲素在大鼠肝微粒體中產生 12 個代謝產物，而在人肝 微粒體中產生了 9 個代謝產物；丙素在大鼠和人肝微粒體中則分別產生 12 個和 6 個代謝產物。對代謝產物進行 LC-MS/MS 定性分析，發現代謝均發生在甲素或丙素的 3′和/或 4′位側鏈上，代謝反應類型為水解和/或氧化，沒有發生骨架凱 琳內酯的開環或氧化。 同時，我們進行了 dPA 在人肝微粒體中酶動力學的相關研究。其主要代謝 產物 M1 通過濕化學法和矽膠柱色譜分離得到。進而我們建立了適合於酶動力學 研究的高效液相串聯多反應監測模式的分析方法。為了得到人肝微粒體中相應的 Km 和 Vmax 值，我們採用一系列的底物濃度在有 NADPH 系統參與下與人肝微粒體 共孵育 20 分鐘。參與白花前胡丙素代謝的異構酶的確定，我們通過化學抑制劑、 特異性的抗體和重組酶篩選來共同佐證。結果顯示：CYP 3A4、2C19 和 2B6 是參 與其代謝的主要酶亞型。 總之，白花前胡甲素和丙素均主要以被動擴散的形式快速通過 Caco-2 細胞 單層，且在吸收過程中白花前胡甲素在羧酸酯酶介導下發生了水解。此外，白花 前胡甲素和丙素的體外肝臟代謝存在種屬差異，而白花前胡甲素及丙素在同一種 屬的肝微粒體的代謝表現出立體差異。以上研究結果為進一步研究中藥前胡及主 要成分甲素和丙素的體內過程及影響因素提供了重要參考