Development and Characterization of Self-emulsifying Drug Delivery Systems (SEDDS) of Angelica oil and Zedoary Turmeric oil by Zhao Yi Thesis Supervisor: Dr. Ying ZHENG Chinese Medicinal Sciences Radix Angelicae Sinensis (Danggui in Chinese, DG), the dried rhizome of Angelica Sinensis (Oliv.) Diels of Umbelliferae herb, grows widely in Gansu province. Because that it could nourish blood, regulate menstruation and relieve pain, so it was praised as "ten prescriptions with nine danggui in" in Chinese medicine. At the present, more than 70 compounds have been isolated and identified from DG, and nearly half of them belong to the essential oil. In Chinese pharmacopoeia (2005), ferulaic acid, which is a hydrophilic compound, is used as the quality control component for DG. Although the essential oil is not regarded as the quality control components for DG, many pharmacological and clinical studies indicated that coniferyl ferulate (CF), E/Z-ligustilide (E/Z-lig) and E/Z-butylidenephthalide (E/Z-bp) are considered as the main bioactive components in DG oil, which could inhibit platelet aggregation, relax uterus and tracheal muscle, prevent gynecological disease and treat menstrual disorders etc. Among of them, Z-ligustilide has been paid much attention because of its relatively high content in DG oil (more than 30%) as well as its well established preventive and therapeutic effect on cardiovascular and cerebrovascular diseases. Curcuma zedoaria Rosc. (Ezhu in Chinese) was the dried rhizome of Curcumа phaeocaulis Val., Curcuma kwangsiensis S. G. Lee et C. F. Liang, Curcuma. Wenyujin. Y. H. chen et C. Ling. Ezhu could guide "qi" downward, promote blood flow, remove food retention and relieve pain. The essential oil of Ezhu, also named as Zedoary Turmeric Oil (Ezhu oil), is extracted from the herb and consisted of many bioactive components. In Chinese pharmacopeia (2005), germacrone is utilized as the quality control component in Ezhu oil. Pharmacological and clinical studies indicated that the main six active components in essential oil are neocurdione (NCD), curdione (CD), germacrone (GM), curzerene (CZ), furanodiene (FD) and B-elemene (B-Е), which have anti-tumor, relieve pain, promote blood flow, remove the necrotic tissue and strengthen immune capability of the organism. Based on the previous research of Danggui and Ezhu oil, the objectives of the present study are to develop and characterize the self-emulsifying drug delivery systems (SEDDS) for above two essential oils. Moreover, pharmacokinetics study of SEDDS of Ezhu oil was also conducted in rat. The content of each chapter is organized as follows: 1. Isolation and purification of Z-ligustilide from Danggui oil The volatile component Z-ligustilide was purified from total essential oil of Danggui by middle-pressure column chromatography and identified by GC-MS with the purity of ~98%. The prepared Z-ligustilide was used as the standard compound in the following studies. 2. Development of SEDDS of Danggui and Ezhu oil Pseudo-ternary phase diagrams were constructed to identify the efficient self-emulsification regions. Each preparation was characterized in terms of emulsification time, droplet size and zeta potential after dispersion into water to determine the optimal formulation, which was further subjected to dissolution and stability evaluation. DG oil could serve as a partial oil phase with the aid of the second oil phase and the combined use of the surfactants produced better self-emulsifying microemulsions. Increasing the surfactant concentration reduced the droplet size but increased the emulsification time, while the reverse effect was observed by increasing the cosurfactant concentration. The optimized formulation consisted of DG oil, ethyl oleate, Tween 80, Labrasol and Transcutol (16:24:38.5:16.5:5, w/w) with droplet size of 56.2+4.2 nm and -potential of -42.4±3.7 mV. Similar results were also obtained for SEDDS of Ezhu oil. The optimized formulation consisted of Ezhu oil, ethyl oleate, Tween 80 and Transcuto! (30.8:7.7:40.5, w/w) with droplet size of 68.3±1.6 nm and 4-potential of -41.2±1.3 mV. The emulsification times for above two developed SEDDS were all below 1 min. 3. Characterization of SEDDS of Danggui and Ezhu oil The active components (e.g. E/Z-lig and E/Z-bp for DG oil; NCD, CD, GM, CZ, FZ and BE in Ezhu oil) could be rapidly released (> 85%) from the optimized SEDDS in aqueous media (e.g. water, PBS buffer or 0.1N HCl) and remained stable in intact SEDDS for at least 12 months (DG SEDDS) or 9 months (EZhu SEDDS) at room temperature. 4. Pharmacokinetics study of SEDDS of Ezhu oil Formulated SEDDS of Ezhu oil and original Ezhu oil were orally administrated to SD rat. The concentrations of the germacrone (GM) in rat plasma were quantified by HPLC-DAD. The results showed that compared with Ezhu oil, the bioavailability of developed SEDDS of Ezhu oil increased to 175.38%. Moreover, the Cmax significantly increased to about 2 times with a shorter time to reach the peak concentration (p<0.05). Keywords: Radix Angelicae Sinensis (Danggui); Curcuma Zedoaria Rosc. (Ezhu); Essential oil; Zedoary Turmeric Oil; Self-emulsifying drug delivery systems (SEDDS); Microemulsion; Pseudo-ternary phase diagrams; Droplet size; Zeta potential, Dissolution; Stability; Bioavailability XIV

當歸(Radix Angelicae Sinensis)是傘形科植物當歸(Angelica sinensis (Oliv.) Diels)的乾燥根,主產於我國甘肅岷縣和宕昌,其他地區亦有栽培。當歸性溫, 味甘辛,能補血和血,調經止痛,潤燥滑腸。中醫因其應用廣泛對其有“十方九歸 之稱。現階段已經從當歸中分離出70餘種化合物,其中主要為揮發油和水溶性 物質兩部分。2005 版中國藥典以水溶性成分阿魏酸的含量為當歸的質控標準。 當歸揮發油為當歸藥效的重要來源,而其中 Z/E-薬本内酯,Z/E-丁烯基苯酞,阿 魏酸松柏酯是揮發油中的主要有效成分,其中又以Z- 本內酯於揮發油中含量最 高,超過30%。它們主要用於防治心腦血管方面的疾病及女性婦科疾病方面的 治療。 義術(Curcuma zedoaria Rosc.)為薑科植物蓬莪術(Curcuma phaeocaulis Val.)、溫郁金(Curcuma. Wenyujin. Y. H. chen et C. Ling)或廣西莪術(Curcuma kwangsiensis S. G. Lee et C. F. Liang)的根莖。性溫,味苦辛。功能為行氣、破血、 消積、止痛。主治心腹脹痛、症痕、積聚、宿食不消、婦女血瘀經閉、跌打損 傷作痛。其中主要有效成分存在於其揮發油中,包括新莪術二酮,我術二酮, 吉馬酮,莪術烯, 二烯,β-欖香烯等,具有消炎、抗腫瘤、止痛、活血化 瘀、增強機體免疫能力的功能。2005 版中國藥典以吉馬酮含量作為我術揮發油 質控標準。 澳门大学硕士学位论文 本課題對當歸和我術揮發油進行了自乳化製劑處方設計、製備、質量標準、 穩定性、體外釋藥速率的研究,並對我術揮發油進行了體內藥物動力學的研究, 為當歸揮發油、莪術揮發油新劑型研究奠定了一定基礎。 其主要內容及結果如下: 1. 當歸揮發油中Z-藁本內酯的提取分離 通過中壓柱色譜法從當歸揮發油中分離純化得到Z- 本內酯,運用 GC-MS 結合文獻分析鑒定其為Z-藁本內酯。結果顯示用經典矽膠柱快速分離純化得到 的Z- 本內酯,通過氣相色譜-質譜法(GC-MS)檢測其含量≥98%,可用作 Z本内酯的標準品。 2. 當歸揮發油、莪術揮發油自乳化製劑的處方設計及製備 根據當歸揮發油、溫莪術提取得到的我術揮發油在不同輔料中的溶解度, 對其自乳化製劑進行了三批次重復的處方篩選,考察了油相、表面活性劑和助 表面活性劑對藥物油的溶解能力,再根據較好的處方設計偽三元相圖,通過自 乳化效果和乳滴粒徑、乳化速率、電位元,乳滴外觀形態對不同處方及處方中 各組分的比例進行考察,對較適宜的處方進行微調,並考察了得到的較優處方 的外部影響因素:溫度,溶液的流動形態,pH,抗稀釋能力,得到確定最佳處 方比例為:當歸油:油酸乙酯:Tween 80: Labrasol: Transcutol=16:24:38.5: 16.5:5:莪術油:油酸乙酯:Tween 80: Transcutol=30.8: 7.7:40.5:21. 3. 當歸揮發油、莪術揮發油自乳化製劑的質量評價 通過乳化速率、粒徑、電位、微乳形態對製劑進行了質量評價,並考察了 製劑於不同溶出介質中的溶出度,採用加速穩定性以預測長期穩定性結果並考 察室溫長期穩定性。結果顯示,當歸油 SEDDS 的相對自乳化時間≤30s;粒徑為 56.2±4.2 nm; PDI為 0.093±0.009: Zeta 電位為-42.4±3.7 mV;微乳形態形態呈 圓球形,大小均勻;其中質控成分 E/Z-lig及 E/Z-bp 溶出穩定,於不同介質中溶 出度均超過85%,同原藥物油在水性介質中無溶出相比較,大大提高了藥物的 溶出度;其加速穩定性結果顯示該製劑應避光保存,高溫60℃及低溫4℃加速 考察10天對 Z-lig 及E/Z-bp 無影響,外觀無晶體析出,顏色無變化,粒徑及乳 化速率無明顯變化。我術油 SEDDS 的相對自乳化時間 ≤1 min;粒徑為68.3±1.6 nm; PDI 為0.101±0.012: Zeta 電位為-41.2±1.7 mV;微乳形態形態呈圓球形, 大小均勻;其中質控成分 NCD, CD, GM, CZ, FZ, BE 溶出穩定,於不同介質中溶 X 澳门大学硕士学位论文 出度均超過88%,同原藥物油在水性介質中無溶出相比較,大大提高了藥物的 溶出度;之後採用留樣觀察法考察了當歸揮發油、莪術揮發油自乳化製劑的穩 定性,結果表明這2種自乳化製劑在12個月及9個月的留樣觀察中質量穩定。 4. 莪術揮發油自乳化製劑在大鼠體內的藥動學研究 採用高效液相色譜法以莪術油中吉馬酮作為指標考察了莪術油SEDDS 製劑 在大鼠體內的藥物動力學過程,並與莪術油原藥進行了比較。結果顯示製備成 自乳化製劑後藥物的口服生物利用度與原藥比提高為173.79% 對製劑同原藥的 藥動學參數進行統計分析,藥時曲線下面積 AUC、藥時曲線下面積一階矩 AUMC、平均滯留時間 MRT、統計矩消除半衰期 1/2z、達峰時間 Tax、最大血 藥濃度 Cmax均有顯著性差異,說明將莪術油製備為SEDDS 製劑,能顯著改善藥 物的生物利用度,提高莪油在生物體內的吸收。 關鍵字:當歸; 術;揮發油;自乳化釋藥系統;微乳:偽三元相圖;粒徑; 電位;溶出度;穩定性;藥動學;生物利用度 XI