金柑结合多酚的制备及其对RAW 264.7巨噬细胞免疫调节活性的研究

Preparation and Characterization of Non-extractable Polyphenols from Kumquat and Its Immunomodulatory Effect in RAW 264.7 Macrophage

摘要:

以萃取游离多酚后的金柑残渣为材料，采用高压脉冲电场（High intensity pulsed electric field，PEF）进行处理，提取其中的结合多酚（Non-extractable polyphenol，NEPP）。在单因素（场强、脉冲数和液料比等）实验考察的基础上，以金柑NEPP含量为响应值，通过响应面法对金柑NEPP提取条件进行优化；之后应用RAW 264.7巨噬细胞模型，分别采用MTT 法、中性红比色法以及Griess法，测定金柑NEPP处理后的RAW 264.7细胞生长情况、吞噬活性和释放NO能力，以评价其免疫活性。结果表明，金柑NEPP的提取工艺回归模型显著，拟合性良好，并预测金柑NEPP的最大理论得率为1.756 mg/g DW；优化后的PEF提取条件为：脉冲数450次，场强11.86 kv/cm，液料比0.341: 1 mL/mg。在优化条件下，金柑NEPP的得率（1.5 mg/g DW）接近预测值，表明优化后的PEF法提取工艺可用于金柑NEPP的提取。当金柑NEPP浓度为100 μg/mL时，对RAW 264.7巨噬细胞无毒性作用，可以显著地抑制巨噬细胞NO的分泌水平，并提高其吞噬活性

Abstract:

Non-extractable polyphenol (NEPP) was extracted from the residue of post-citrus polyphenols by high intensity pulsed electric field (PEF). On the basis of single-factor experimental investigation (field intensity, pulse number and liquid to solid ratio, etc.), MTT assay, neutral red colorimetric assay, and Griess assay were used to evaluate the effects of NEPP on the growth, phagocytic activity, and NO release of RAW 264.7 cells. The results showed that the obtained process regression model of kumquat NEPP was significant with great fitting, which predicted the maximum theoretical value of kumquat NEPP content was 1.756 mg/g DW. The optimized NEPP extraction condition of kumquat by PEF was shown as follows: pulse number 450 times, field intensity 11.86 kV/cm, liquid to solid ratio V (water): M (fruit residue) = 0.341 mL: 1mg. Following the optimized extraction method, the NEPP content of kumquat (1.5 mg/g DW) was close to the predicted value, indicating that the optimized PEF process was acceptable to the extraction of kumquat NEPP. Kumquat NEPP (100 g/mL) showed no toxicity to RAW 264.7 macrophages, increased NO secretion and boomed phagocytic activity.