Danshen (Salvia miltiorrhiza) is a commonly used traditional Chinese medicine for the treatment of cardiovascular and cerebrovascular diseases. In China, numerous pharmaceutical products of Danshen are commercially available, which include tablets, capsules, granules, injectables and oral liquids etc. In the Chinese Pharmacopeia (2005), only salvianolic acid B and tanshinone IIA were determined by HPLC method as quality control markers. And no dissolution and stability tests were required. The objective of this study was to apply the improved quality evaluation method to evaluate the quality of Danshen products. Considering both hydrophilic and lipophilic active components, danshensu, protocatechuic aldehyde, salvianolic acid в, cryptotanshinone and tanshinone IIA were selected as five markers. A HPLC method was developed to simultaneously determine their contents, which were used to compare the products from different companies, and different batches from the same company. Similarity factor method was used to evaluate the similarities of in-vitro dissolution profiles. Moreover, accelerated and long-term stability tests were performed to predict the shelf-life of Danshen products. Results showed that all of the Danshen products passed the content requirements of Chinese pharmacopeia. However, the content variations of the hydrophilic components were much higher than those of the lipophilic components. In dissolution tests, most of the hydrophilic components were dissolved completely while all of the lipophilic components could not be detected. The f2 values showed that there were no similar dissolution profiles among different brands of Danshen products. Contents of the five markers decreased by the first-order kinetic processes in the accelerated and long-term stability tests. The predicted shelf-life of Danshen products were shorter than those labeled on the products. It was suggested that more markers with defined content range instead of lower content limit are required for better control of Danshen products. Dissolution and stability tests need to be added into the quality control criteria in the Chinese Pharmacopeia. Among the components isolated from Danshen, Tanshinone (TanIIA) is a major lipophilic and thermosensitive bioactive diterpenoid for the treatment of myocardial infarction and myocardial ischemia. However, TanIIA has very poor oral absorption (<5%), which may be due to its poor dissolution rate, low permeability across the intestinal membrane, intensive first-pass metabolism and interaction with efflux transporters. In this study, TanIIA was formulated with Flouric68 (F68), PEG4000 and polyvinylpyrrolidone (PVP40) to prepare solid dispersions by Spray-Freezing Drying (SFD) technology for the inhancement of its aqueous dissolution profile. Powder X-ray diffraction (PXRD) patterns showed that the intensities of characteristic peaks of TanlIA dramatically decreased in TanIIA/F68 and TanIIA/PVP SFD products compared to the unprocessed TanIIA. Scanning electron microscopy (SEM) micrographs also displayed that SFD products appeared lacking of crystallinity while TanIIA was styloid crystals. Fourier transform infrared spectroscopy (FTIR) spectrum revealed that the interactions between TanIIA and excipients were the evidence for the changed PXRD and SEM profiles. Dissolution results exhibited that SFD products with TanIIA/F68 (1:9,w/w) has a higher dissolution rate to about 70%. In an animal study which compared oral absorption of SFD products of TanIIA/F68-1:9 to unprocessed TanIIA group (n=4), Cmax of SFD products was significantly increased to 2.4- and 1.4-fold for unchanged and total TanIIA after enzyme hydrolysis, respectively (p < 0.05). However, AUC and MRT did not significantly increase (p > 0.05) compared to those of the control. In conclusion, SFD technology is feasible for preparing TanIIA SFD products with amorphous structure, larger surface area and higher dissolution rate. However, the pharmacokinetic profiles indicated the vi dissolution process might not be the rate-limiting step for the absorption of TanIIA in vivo. Keywords: Danshen; Quality Evaluation; Dissolution; Stability; Tanshinone IIA (TanIIA); Spray-Freezing Drying (SFD) Technology; Characterization; Absorption; Pharmacokinetics vii

澳門大學碩士學位論文 摘要 丹參制劑的質量评价及其脂溶性成分丹參酮 IIA 的剂型改良 張 導師:鄭穎博士 澳門大學 中華醫藥研究院 丹參為唇形科植物 Salvia miltiorrhiza Bge. 的乾燥根及根莖,具有祛瘀止痛、 活血通經及清心除煩等功效,近年來多用於治療冠心病、心絞痛等心腦血管系統 疾病的治療。作為傳統常用中藥,丹參已經被開發成各種復方成藥制劑,以它作 為主藥並被 05 版中國藥典收載的有復方丹參片和復方丹參滴丸等,而丹七片收 載於部頒標準。國內多個廠家均可生產。 現行 2005 版中國藥典一部標準中只規定了復方丹參片中兩種指標成分(丹 酚酸 B、丹參酮 IIA)的含量下限,在體外溶出和穩定性考察方面沒作要求。而 收載於部頒標準的丹七片未要求對指標成分進行含量測定。之前有提高復方丹參 制劑質控標準的報道,但多側重改進高效液相分析方法同時測定多指標成分的含 量,如同時測定水溶性和脂溶性成分,以及三七皂苷的含量,對其相同制劑不同 廠家及同廠家不同批次間的含量差異、體外溶出度及儲存穩定性考察未見報道。 本研究第一部份選取了5個活性指標成分,分別為丹參素、原兒茶醛、丹酚酸 B、 隱丹參酮和丹參酮 ILA,對市面上4個廠家的復方丹參片、2個廠家丹七片和1 個廠家復方丹參滴丸進行了含量、溶出度及穩定性測定。以含量柱形圖比較含 ii 澳門大學碩士學位論文 量、以相似因子法評價溶出曲線、用動力學過程描述指標成分含量的變化規律並 推算有效期。實驗結果表明,6個廠家的復方丹參市售品中指標成分的含量均超 出藥典規定下限。4個廠家復方丹參片的水溶性成分含量相差1.6~7.6倍,脂溶 性成分含量相差1.4倍;2個廠家丹七片中水溶性成分含量相差2.3~8.5倍;復方 丹參滴丸中指標成分含量批間 RSD <10%。復方丹參片的水溶性成分溶出完全、 脂溶性成分未見溶出;丹七片的水溶性成分溶出不完全;復方丹參滴丸中只有原 兒茶醛溶出不完全。相似因子結果表明,各廠家相同制劑的指標成分間無相似溶 出行為。各廠家制劑的指標成分降解均符合一級動力學 (≥0.9),預測的有效 期均小於1年,但出現這種情況的原因仍需深入探討。從實驗結果可知,現行的 復方丹參制劑質量標準應適當增加指標成分數量、增加指標成分含量上限規定、 增加溶出度測定和穩定性考察項目。 根據第一部份的溶出度實驗結果發現,復方丹參制劑中的丹參脂溶性成分, 如丹參酮 IIA 未見檢出。有關研究表明,丹參酮 IIA 具有非常低的口服吸收生物 利用度(<5%),而導致其低口服生物利用度的原因可能是其较差的水溶性和溶 出度,较低的細胞膜透過性,与细胞膜上反轉運蛋白的相互作用及较强的 II 相 首过代谢等。本研究第二部份選取丹參中脂溶性成分丹參酮 IIA 作為研究對象代 表,利用近年來新興的噴霧冷凍幹燥技術(SFD技術)制備丹參酮 IIA 固體分散 體以提高它的體外溶出度,並且考察其在大鼠體內的口服吸收。 这部分采用正丁醇-乙醇-水作為混合溶劑系統將丹參酮 IIA 和泊洛沙姆188 (F68)、聚乙二醇4000(PEG4000)及聚乙烯吡咯烷酮 40 (PVP40)以1:1和1:9 的质量比混合,通過噴霧冷凍幹燥技術制備固體分散體。並用粉末X射線衍射、 冷場掃描電鏡、紅外光譜、溶出實驗和比表面積測定等實驗考察了噴霧冷凍幹燥 澳門大學碩士學位論文 產品的體外物理化學性質,並选择其中一个处方進行了大鼠體內的药代動力學的 研究。實驗結果表明,運用三種辅料均可制备出蓬鬆多孔狀的無定形丹參酮 IIA 固體分散體,並使丹參酮 IIA 水中溶解度有顯著提高。但F68 和PVP40 作輔料 得到的產品較 PEG4000 制得的產品密度更低、溶出度更高。粉末X射線測試結 果中丹參酮 IIA 晶體的最強特征峰強度顯著降低,某些角度的相關峰消失,表明 產品中的丹參酮 IIA 晶體已大部分轉化成無定形非晶體的結構。在冷場掃描電鏡 下可觀察到,丹參酮 IIA 原藥為柱狀結晶,經噴霧冷凍幹燥後的產品大部分轉變 為無定形呈疏松狀分佈。紅外光譜的結果也印證了粉末 X 射線和掃描電鏡的結 果,輔料分子和丹參酮 IIA 之間差生了氫鍵作用。體外溶出實驗實驗表明,丹參 酮 IIA 混合泊洛沙姆188 (1:9) 的噴霧冷凍幹燥產品可使丹參酮 IIA 的溶出度提 高到約70%,而用 PVP或PEG4000 同樣比例作輔料得到的產品溶出度也提高到 約35%和20%。將丹參酮 IIA 混合 F68 的1:9比例噴霧冷凍乾燥產品(SFD TanIIA/F68-1:9 product)進行為期3個月的加速穩定性試驗,發現此產品只能在 1個月內保持穩定 第2、3個月的含量和溶出度已經有顯著性的下降(P<0.05)。 將丹參酮 IIA 混合混合泊洛沙姆 188 (1:9) 的噴霧冷凍幹燥產品進行大鼠體內實 驗,結果表明丹參酮 IIA 在大鼠體內只以很低濃度的原型存在,而主要以葡萄糖 醛酸結合物的形式存在。丹參酮 IIA 固體分散體組的原型及葡萄糖醛酸結合物的 Cmax 比對照組分別高出2.4和1.4倍 (P<0.05),但AUC 沒有統計學上的顯著性 差異 (P>0.05)。表明溶出步驟可能不是丹參酮 IIA 生物利用度低的限速步骤, 其他的限制其口服吸收的機理還需進一步深入探討。 關鍵詞:丹參制劑;含量;溶出度;穩定性;丹參酮 IIA;固體分散體;噴霧冷 凍幹燥;口服吸收 iv