矢车菊素-3-O-葡萄糖苷及其月桂酸酶法酰化产物对Caco-2细胞胆固醇摄取的作用比较

Comparison of The Effects of Black Rice Anthocyanin Monomer C3G of and Its Laurate Acylation Product C3G-C12 on Cholesterol Uptake in Caco-2 Cells

摘要:

为了探讨黑米花青素矢车菊素-3-O-葡萄糖苷（C3G）及其月桂酸酶法酰化产物矢车菊素-3-（6″-月桂酰）葡萄糖（C3G-C12）对Caco-2细胞胆固醇吸收转运的影响及其可能作用机制。该研究通过半制备液相色谱（Semi-HPLC）从黑米花青素中提取C3G，采用液相色谱质谱联用（LC-MS/MS）鉴定该单体。以C3G为原料，用月桂酸和Novozymes 435酶对其进行酶法酰基化。进一步通过Semi-HPLC分离酰化产物，LC-MS/MS和傅里叶红外光谱（FTIR）验证该单体。建立21天Caco-2肠道上皮细胞模型，分别用质量浓度为50、100、150 μg/mL C3G和C3G-C12及20 μmol·L-1胆固醇吸收抑制剂依折麦布孵育开展转运试验，利用高效液相色谱（HPLC）检测细胞胆固醇吸收转运量，并用荧光定量PCR（qPCR）检测NPC1L1 mRNA的表达。结果表明，Caco-2细胞胆固醇转运可被二者呈浓度依赖性抑制。150 μg/mL质量浓度的C3G和C3G-C12可分别使胆固醇吸收下降46.21%和56.36%，引起56.93%和59.76%的NPC1L1 mRNA表达下降。综上所述，C3G-C12较C3G更有效抑制胆固醇吸收，这可能与其抑制NPC1L1 mRNA的表达有关。该研究为黑米花青素酶法酰基化修饰及其在调节肠道胆固醇代谢的作用提供了理论基础。

Abstract:

The effects of the black rice anthocyanidin monomer cyanidin-3-O-glucoside (C3G) and its lauric acid enzymatic acylation product cyanidin-3-(6″-lauroyl) glucose (C3G-C12) on cholesterol absorption and transport in Caco-2 cells, and its possible mechanism of action, were investigated. In this study, C3G was extracted from black rice anthocyanidins by semi-preparative liquid chromatography (Semi-HPLC) and the monomer was identified by liquid chromatography-mass spectrometry (LC-MS/MS). C3G was used as raw material and enzymatically acylated with lauric acid and Novozymes 435. The acylated products were further separated by Semi-HPLC, and the monomer was verified by LC-MS/MS and Fourier transform infrared spectroscopy (FTIR). A 21-day Caco-2 intestinal epithelial cell model was established. The cells were incubated with mass concentrations of 50, 100, and 150 μg/mL C3G and C3G-C12 and 20 μmol·L-1 cholesterol absorption inhibitor Ezetimibe to carry out transport experiments. High performance liquid chromatography (HPLC) was used to detect the cellular cholesterol absorption, and fluorescence quantitative PCR (qPCR) was used to detect the expression of NPC1L1 mRNA. The results showed that the cholesterol transport of Caco-2 cells could be inhibited by the both in a concentration-dependent manner. C3G and C3G-C12 at a mass concentration of 150 μg/mL reduced cholesterol absorption by 46.21% and 56.36%, respectively, and cause a 56.93% and 59.76% decrease in NPC1L1 mRNA expression. In summary, C3G-C12 was more effective in inhibiting cholesterol absorption than C3G, which might be related to its inhibition of NPC1L1 mRNA expression. This study provided a theoretical basis for the enzymatic acylation modification of black rice anthocyanins and their role in regulating intestinal cholesterol metabolism.