Hsu H J, Huang R F, Kao T H, Inbaraj B S, Chen B H
Department of Food Science, Fu Jen Catholic University, New Taipei City 242, Taiwan.
Nanotechnology. 2017 Mar 7;28(13):135103. doi: 10.1088/1361-6528/aa5e86.
Lycium barbarum L., a traditional Chinese herb widely used in Asian countries, has been demonstrated to be protective against chronic diseases such as age-related macular degeneration. The objectives of this study were to determine the carotenoid content in L. barbarum by high-performance liquid chromatography-mass spectrometry, followed by preparation of a carotenoid nanoemulsion to evaluate the mechanism of inhibition on HT-29 colon cancer cells. The highest extraction yield of carotenoids was attained by employing a solvent system of hexane-ethanol-acetone (1:1:1, v/v/v). Nine carotenoids, including neoxanthin (4.47 μg g), all-trans-zeaxanthin and its cis-isomers (1666.3 μg g), all-trans-β-cryptoxanthin (51.69 μg g), all-trans-β-carotene and its cis-isomers (20.11 μg g), were separated within 45 min and quantified using a YMC C column and a gradient mobile phase of methanol-water (9:1, v/v) (A) and methylene chloride (B). A highly stable carotenoid nanoemulsion composed of Capryol 90, TranscutolHP, Tween 80 and deionized water was prepared with a mean particle size of 15.1 nm. Characterization of zeaxanthin standard, blank nanoemulsion, carotenoid extract and carotenoid nanoemulsion by differential scanning calorimetry curves and Fourier transform infrared spectra revealed a good dispersion of zeaxanthin-dominated carotenoid extract with no significant chemical change after incorporation into nanoemulsion. The in vitro release kinetic study showed a higher release profile at pH 5.2 than at physiological pH 7.4, suggesting a rapid release of carotenoids in the acidic environment (pH 4.5-6.5) characteristic of tumors. Both the carotenoid nanoemulsion and the extract were effective at inhibiting growth of HT-29 colon cancer cells, with an IC of 4.5 and 4.9 μg ml, respectively. Also, both treatments could up-regulate p53 and p21 expression and down-regulate CDK2, CDK1, cyclin A and cyclin B expression and arrest the cell cycle at G/M. The study may form a basis for further exploration of L. barbarum nanoemulsion in cancer treatment.
枸杞是一种在亚洲国家广泛使用的传统中草药，已被证明对诸如年龄相关性黄斑变性等慢性疾病具有保护作用。本研究的目的是通过高效液相色谱 - 质谱法测定枸杞中的类胡萝卜素含量，随后制备类胡萝卜素纳米乳剂以评估其对HT - 29结肠癌细胞的抑制机制。采用己烷 - 乙醇 - 丙酮（1:1:1，v/v/v）的溶剂体系可获得最高的类胡萝卜素提取率。在45分钟内分离出9种类胡萝卜素，包括新黄质（4.47μg/g）、全反式玉米黄质及其顺式异构体（1666.3μg/g）、全反式β - 隐黄质（51.69μg/g）、全反式β - 胡萝卜素及其顺式异构体（20.11μg/g），并使用YMC C柱和甲醇 - 水（9:1，v/v）（A）与二氯甲烷（B）的梯度流动相进行定量。制备了一种由辛酸癸酸甘油三酯90、二乙二醇单乙基醚、吐温80和去离子水组成的高度稳定的类胡萝卜素纳米乳剂，其平均粒径为15.1nm。通过差示扫描量热曲线和傅里叶变换红外光谱对玉米黄质标准品、空白纳米乳剂、类胡萝卜素提取物和类胡萝卜素纳米乳剂进行表征，结果表明以玉米黄质为主的类胡萝卜素提取物分散良好，掺入纳米乳剂后无明显化学变化。体外释放动力学研究表明，在pH 5.2时的释放曲线高于生理pH 7.4时的释放曲线，这表明类胡萝卜素在肿瘤特征性的酸性环境（pH 4.5 - 6.5）中快速释放。类胡萝卜素纳米乳剂和提取物均能有效抑制HT - 29结肠癌细胞的生长，其IC50分别为4.5和4.9μg/ml。此外，两种处理均可上调p53和p21的表达，下调CDK2、CDK1、细胞周期蛋白A和细胞周期蛋白B的表达，并使细胞周期停滞在G/M期。该研究可能为进一步探索枸杞纳米乳剂在癌症治疗中的应用奠定基础。
