Oleanolic acid (O), a naturally occurring pentacyclic triterpenoid, is a biologically active marker compound commonly present in Chinese herbs, such as Fructus Ligustri Lucidi, Fructus Forsythiae and Radix Ginseng etc. Oleanolic acid has been successfully used as an OTC oral drug to treat human liver disorders in China due to its hepatoprotective effect. In addition, it has been shown to exhibit anti-inflammatory, antitumor and anti-hyperlipidemic activities. Up to now, the commonly used formulations of OA in the market are tablets, capsules, and pills. However, being hydrophobic, O exhibits poor aqueous solubility. During in-vitro dissolution study, less than 1 mg/ml OA were dissolved from raw solid form into an aqueous buffer (pH 1 or 7) after 2 hours. Furthermore, given the low Caco-2 cell permeability and extensive hepatic microsomal metabolism, the low oral bioavailability of oleanolic acid may be due to a poor gastrointestinal absorption and extensive hepatic first-pass metabolism. In order to modify the in vitro dissolution of oleanolic acid, two approaches are utilized in this study. One is to prepare oleanolic acid solvates through recrystallization method, while the other is to prepare its self-emulsifying nanoemulsion system (SNEDDS). Furthermore, the permeability of OA raw material and its nanoemulsion were determined on the Caco-2 cell monolayer model. The solid state properties of oleanolic acid recrystallized from a variety of solvents were investigated. Glassy materials were prepared with dichloromethane and chloroform solvents in both cold crystallization and solvent evaporation. Crystalline forms of OA, including a methanol solvate, an ethanol solvate and an acetone non-solvate, were prepared by dissolving its raw material into different organic solvents and evaporating organic solvents at room temperature in a dark place for several weeks. The filtered crystals were carefully dried and stored under ambient conditions and then characterized. Upon desolvation, both the methanol and ethanol solvates were found to undergo phase transformation to a crystalline phase similar to OA-Acetone around 190-195°C. The PXRD patterns of commercial pharmaceutical grade OA and the OA-Methanol were similar, so the commercial form is probably desolvated oleanolic acid methanol solvate. Specific surface area of the three crystalline materials was determined by BET nitrogen adsorption. HPLC was utilized to quantify the in vitro dissolution of OA raw material (OA-RW), solvates, non-solvate and commercial available tablet. Dissolution studies showed that ~89.5% of OA could be released from the raw material in 1% SDS (pH 7.0) after 24 hours, which is higher than those of the crystalline forms (<70%). Both of the initial and intrinsic dissolution rates of OA-RW were obviously faster than those of the other forms which may due to its higher specific surface area. For the other crystalline forms, methanol and ethanol solvates showed higher intrinsic dissolution rates than the acetone non-solvate because of the favorable reduction in free energy through the mixing of methanol/ethanol with water. Meanwhile, a self-nanoemulsified drug delivery system (SNEDDS) of oleanolic acid (OA) for oral delivery is also prepared in this study. Solubility of OA under different systems was determined for excipient selection purpose. Three formulations, where OA was fixed at the concentration of 20mg/g, were prepared utilizing Sefsol 218 as oil phase, Cremophor EL and Labrasol as primary surfactants, and Transcutol P as cosurfactant. Pseudo-ternary phase diagrams were constructed to identify self-emulsification regions for rational design of SNEDDS formulations. Sefsol 218 was found to provide the highest solubility among all medium-chained oils screened. Efficient self-emulsification was observed for the systems composing of Cremophor EL and Labrasol. The surfactant to cosurfactant ratio greatly affected the droplet size of the nanoemulsion. Based on the results in dissolution profiles, stability data and particle size distribution, three optimized formulations were selected: Sefsol 218:Cremophor EL:Labrasol:Transcutol P (50%:17.5%:17.5%:15%, w/w), Sefsol 218:Cremophor EL: Labrasol: Transcutol P (50:20:20:10%, w/w), and Sefsol 218:Cremophor EL:Labrasol (50:25:25% w/w). More than 90% and 60% 0A could be rapidly released from the nanoemulsions in water and simulated gastric fluid within 10min, respectively. In contrast, none of the OA could be detected in dissolution medium from the OA raw material and its commercial available tablet, where the equilibrated concentration was below the detection limit of HPLC (1 g/mI). Three formulations were stable at room and subambient temperature (e.g. 4°C) for at least six months. However, they are sensitive at higher temperature (e.g. 40°C). These results suggested the potential use of SNEDDS to improve solubility and dissolution for poorly water soluble triterpenoids such as OA. The Caco-2 cell monolayer, the well studied model for assessing drug absorption, was used to evaluate the permeation rates of oleanolic acid in both apical to basolateral and basolateral to apical directions. Papp values of oleanolic acid or its SNEDDS formulation (4 Mg/ml OA) were all lower than 1×106 cm/s, from both directions, suggesting that poor intestinal permeability maybe the rate limiting step for its oral absorption. Above results indicate that slower dissolution rate of OA can be engineered by the formation of O crystalline materials, which can be potentially useful for OA sustained release products. And because that O could be rapidly and completely released from its nanoemulsion system, it was suggested that SEDDS of OA could be developed into immediate-release products. However, due to the limited intestinal permeability of OA, more in vivo animal work needs to be performed to verify whether the oral bioavailability of 0A could be enhanced by formulated as SNEDDS.

齊墩果酸是天然五環三萜類化合物，廣泛分佈於中藥植物中如女貞子、連翘 和人參等。齊墩果酸具有消炎、抗腫瘤和抗血脂過高等多方面的臨床藥理作用，在中國己作為常用保肝 OTC 藥物洽療急性化學性肝損傷和慢性肝硬化、肝纖維化。目前市售劑型主要是齊墩果酸片、膠亵和丸劑等。 齊墩果酸難溶于水，原料藥粉末在酸性(pH 1)及中性水溶液(pH ⑦中溶出 2 小時後，溶解度仍然低於 HIPLC 方法的檢測限 1ug/ml，並且其在 Caco-2 單細胞 層中渗透率較低和肝微粒體中代謝嚴重，因此其在體内的絕對生物利用度極低的 原因可能歸咎於溶解度差、胃腸道吸收低及大量的肝首過效應。 在吸收過程中，藥物的溶出速率通常為其吸收的限速階段。因此，本文通過 制備齊墩果酸的溶劑化晶體及納米乳液來改變其在水中的溶解度和溶出速率同 時，還進一步考察了齊墩果酸原料藥及其納米乳液在 Caco-2 單細胞層中的透過 情況。 首先本文考察了使用不同溶劑製備的齊墩果酸重結晶物的固態化學性質。採 用冷析結晶和溶劑揮發法在二氯甲烷和氯仿中製備得到了齊墩果酸的玻璃様物 質。齊墩果酸溶解在不同有機溶劑中後，過濾並置於室溫避光數周至溶劑揮幹，得到其甲醇、乙醇溶劑化物及丙酮非溶劑化物，室溫下保存並進行理化性質考察。齊墩果酸甲醇和乙醇溶劑化物在 190-195°C 去溶劑化後發生了相轉化，得到 與丙酮非溶劑化物類似的結晶相。採用粉末 叉 射線衍射法分析表明，市售齊墩 果酸原料藥粉末和其甲醇溶劑化物的 PXRD 圖譜相似，說明原料藥粉末可能是去溶劑化的甲醇溶劑化物。利用 BET 氮吸附法測定重結晶樣品的比表面積，並 以 HPLC 為檢測方法考染和比毀丁齊墩果酸原料藥粉末 重結晶物及市售普通片 劑的能外浴出/度。在1%SDs 水浴液中，齊墩果酸可以從其原料樊和重結晶粉末中級慢溶出，24小時内原料藥的浴出!度為 89.5%，而其甲醇、乙醇和丙酮的重結弱的浴出度都低於70%。而且其原料樊的初始浴出速率和特性溶出速率最高，這可能與原料藥粉末具有較高的比表面積有關。雖然丙酮結晶性粉末比表面積高於 甲醇及乙醇結晶產物，但由於甲醇利乙醇與水混合過程中自由能降低，故兩種重 結晶粉末相對於丙酮重結晶產物表現出更高的特性溶出速率。 同時，本文還研究製備了齊墩果酸的自乳化口服納米乳液。通過不同系統中 齊墩果酸溶解度及自乳化效率考缭，筋選出合適的輔料，選用處方量的齊墩果酸 20mg/g, Sefsol 218 為油相，Cremophor EL 和 Labrasol 的混合物為表面活性劑 P 為助表面活性劑。給製偽三元相圖確定了 自乳化的存在區域，合理設計了白乳化納米乳液處方。在所選取的油相中，Sefsol 218 對齊墩果酸溶解性最好。表面活性劑 Cremophor EL 與 Labrasol 混合使用，得到了分散快速、乳滴 均勻的自納米乳體系。表面活性劑 Cremophor EL/Labrasol 與助表面 性劑 Transcutol P 的比例對納米乳液粒徑影響較大。通過溶出度、穩定性和粒徑的考 察，最終得到三個比例不同的理想處方分別為 Sefsol 218:Cremophor EL:Labrasol:Transcutol P (50%:17.5%:17.5%:15%, w/w), Sefsol 218:Cremophor EL:Labrasol: Transcutol P (50:20:20:10% w/w) fIl Sefsol 218:Cremophor EL: Labrasol ($0-25:25%% w/w)。齊墩果酸在純水及模擬胃液中 10 分鐘之內即可從其納米乳液中快速釋放，溶出度分別達到 90%和 60%以上，而原料藥粉末與市售片劑的溶出濃度則均低於 HIPLC 方法的最低檢測限 （1/g/ml)，即幾乎不能溶出。影響因 素實驗表明製劑對熱不穩定，在常溫和低溫 下(4°C)可保證穩定至少 6 個月。自 乳化釋藥系統可應用與三萜類化合物如齊墩果酸以提高其溶解度和溶出速率。 Caco-2細胞模型作為藥物吸收研究的一種成热而快速的工具，用於測定齊 墩果酸經 Caco-2 細胞單層轉運過程中的表觀渗透係數 Papp值以考察其透膜性。 結果表面，含4ug/ml OA 的原料藥及其納米乳液製劑經 AP 端到 BL端和從BL 端到 AP端的Ppo值都低於 1X10° cmls， 表示齊城果酸在小腸中的低透通率可能是其口服吸收的限速步驟。綜上所述，齊墩果酸可通過不同的晶型改造達到綴釋的效果，而納米乳液的製備則迅速並顯著地提高了齊墩果酸在純水中的溶出度，因此可作為一種速釋的 給藥方式來改善齊墩果酸的口服吸收。