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標題: 人參微脂體減緩過氧化氫對細胞氧化傷害之評估與黃連及小檗鹼微脂體抗人類肝癌細胞之效能分析
Reduction of hydrogen peroxide-induced oxidative damage by ginseng liposomal nanovesicles; anticancer effects on human hepatocellular carcinoma by huanglian and berberine liposomal nanovesicles
作者: Yu, Ming-Chiang
關鍵字: liposome;微脂體;ginseng;huanglian;ginseng liposome;berberine liposome;huanglian liposome;黃連;人參;人參微脂體;小檗鹼微脂體;黃連微脂體
出版社: 食品暨應用生物科技學系所
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人參(ginseng)屬五加科(araliaceae)植物人參(Panax ginseng C.A. Mey.)之乾燥根莖,而黃連(huanglian)為毛茛科植物黃連(Coptis chinensis Franch.)之乾燥根莖,都是中國數千年的傳統中藥材。微脂體(liposome)因可包裹疏水性與親水性的藥物,為良好的藥物傳輸載體。
本研究目的第一部分是以微脂體包裹人參萃液,製備成人參微脂體,並評估其物性、化性、細胞安全性及抗氧化之能力。四種人參微脂體(PEG0.0、0.5、1.5與3.0)以15000 psi均質5分鐘可使粒徑下降至140~150 nm,而其中PEG3.0-liposome清除DPPH自由基能力(16%)與包裹人參皂苷含量最佳。在細胞安全性結果發現四種微脂體在濃度50、100與200 nmol/mL對L929細胞沒有抑制生長的現象,而L929在細胞量4×104與6×104 cells/mL分别以250及350 μM H2O2傷害後,存活率約為76%,但若加入50、100與200 nmol/mL濃度人參微脂體,相對於控制組(不加人參微脂體),都有減緩H2O2對L929細胞的氧化傷害,其中以100 nmol/mL的PEG3.0-liposome最具功效。
本研究第二部分以微脂體包裹小檗鹼與黃連萃液以形成小檗鹼與黃連微脂體,冀望經由製成微脂體新劑型後可以提高對肝癌細胞的殺害能力並降低對正常細胞的副作用。黃連粉萃出的小檗鹼量是未磨粉黃連塊的6.1倍,此外再以濃縮方式提高黃連水萃液的小檗鹼量至未磨粉黃連塊的26.4倍。因標準品小檗鹼對水的最大溶解度為1.5 mg/mL,嘗試用酸與鹼調整pH值以增加小檗鹼在水的溶解度,但是並無顯著差異。接著以微脂體包裹黃連水萃液與小檗鹼水溶液形成黃連與小檗鹼微脂體,經25000 psi均質20分鐘,黃連與小檗鹼微脂體粒徑約為140~150 nm,而包裹的小檗鹼含量分別為160.0 μg/mL與25.6 μg/mL。以小檗鹼水溶液與黃連水萃液對HepG2細胞做生長抑制試驗,其所相對應IC50值分别為48.0 μg/mL與39.0 μg/mL;而以小檗鹼微脂體與黃連微脂體對HepG2細胞做生長抑制試驗,其所相對應的IC50值則分别為1.3 μg/mL與9.0 μg/mL。故小檗鹼與黃連水萃液在經微脂體包裹後,其抗肝癌細胞生長的效率分別提升36.6與4.3倍,故本實驗證明微脂體可有效提升小檗鹼溶液與黃連水萃液的抗肝癌細胞生長的效果。

Ginseng and huanglian are traditional Chinese medicines and have been used for thousand years. The former is the root of Panax ginseng C.A. Mey, and the latter is the stem of Coptis chinensis Franch. Liposome is an excellent drug delivery vehicle which can encapsulate both hydrophilic and hydrophobic drugs, and has been used from the 1960s. Therefore, in this study, the extracts of ginseng and huanglian were encapsulated inside liposomes with a purpose to enhance their therapeutic effects.

The first part of the project is to develop ginseng liposomes to improve the therapeutic effects of ginseng against the H2O2-induced oxidative damage to L929 cells. After determining the amounts of insenosides by HPLC, ginseng extract was encapsulated with various ratios of PEG (0, 0.5, 1.5, and 3 mol%). After homogenization at 15000 psi for 5 min, the sizes of those 4 batches of ginseng liposomes were all around 140-150 nm. Among them, PEG3.0 ginseng liposome had the highest scavenging ability of DPPH radicals (16%) and encapsulation efficiency of ginsenosides. In the cytotoxicity evaluation, different concentrations (50, 100, and 200 nmol/mL) of 4 batches of ginseng liposomes showed no inhibition on the growth of L929 cells. The survival rate of the L929 cells was about 76% after the H2O2 treatment. While ginseng liposomes were added to the H2O2-treated cells, the survival rates of the L929 cell had significant increase (5-50%), compared to the control group without the addition of ginseng liposomes. The increase of survival rates following the increase of PEG mol% on the liposomal surface. In addition, 100 nmol/mL of PEG3.0 ginseng liposome had the best efficiency in treating the oxidatively damaged L929 cells, and could also increase about 50% of cell survival rate.

The second section of this study is to develop berberine and huanglian liposomes to improve the anti-hepatoma effects of huanglian and berberine. First, huanglian power and stalk were extracted in distilled water, and the extract of huanglian power had higher berberine concentration than the stalk. Furthermore, huanglian extract was concentrated by rotary evaporator to increase its berberine concentration. On the other hand, to increase the solubility of berberine standard (1.5 mg/mL in water), the pH values of berberine solution were modified with acids or bases. However, no significant improvement of berberine solubility was observed. After huanglian and berberine liposomes were homogenized at 25000 psi for 20 min, the average sizes of these batches of liposomes were 140-150 nm. Moreover, the encapsulated berberine concentrations of huanglian and berberine liposomes were 160.0 and 25.6 μg/mL, respectively. At the therapeutic test, the HepG2 cells were treated with either unencaosulated free form drugs (huanglian extract and berberine solution) or liposomal drugs (huanglian and berberine liposomes). In the study of HepG2 cells, the IC50 values of free drugs were 39.0 μg/mL for huanglian extract and 48.0 μg/mL for berberine solution; and the IC50 values of liposomal drugs were 9.0 μg/mL for huanglian liposomes and 1.3 μg/mL for berberine liposomes. The IC50 values of liposomal drugs were lower than the free drugs for the growth inhibition of hepatoma cells. Therefore, liposome could be a good drug carrier to enhance the anti-hepatoma effects of huanglian and berberine.
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