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

Preparation of non-extractable polyphenols from Kumquat and its immunomodulatory effect in RAW 264.7 macrophage cells

以萃取游离多酚后的金柑果渣为材料,采用高压脉冲电场(PEF)进行处理,提取其中的结合多酚(NEPP)。在单因素(场强、脉冲数和液料比)实验基础上,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含量为1.6728 mg GAE/g DW(即每克金柑干重所含有的没食子酸当量);优化后的PEF提取条件为:脉冲数250次,场强11.8 kV/cm,液料比0.34∶1(L∶g)。在优化条件下,金柑NEPP含量(1.6382 mg GAE/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质量浓度为100 mg/L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。

Non-extractable polyphenol(NEPP) was extracted from the pomace of post-citrus polyphenols by high intensity pulsed electric field(PEF). On the basis of single-factor experiments(field intensity, pulse number and liquid-solid ratio), the extraction condition of NEPP in Kumquat was optimized by response surface method using NEPP content as response value. MTT assay, neutral red colorimetric assay, and Griess assay were used to test the growth, phagocytic activity, and NO release of RAW 264.7 macrophage cells after NEPP treatment to evaluate the immunomodulatory effect of NEPP. The results showed that the obtained regression model for the extraction process of Kumquat NEPP was significant with good fitting, which predicted the content of Kumquat NEPP was 1.6728 mg GAE/g DW(gallic acid equivalent per gram in dry weight of Kumquat). The optimized extraction conditions for Kumquat NEPP by PEF were obtained as follows: pulse number of 250, field intensity of 11.8 kV/cm, liquid-solid ratio of 0.34∶1(L∶g). Under the optimized conditions, the Kumquat NEPP content(1.6382 mg GAE/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 mg/L) showed no toxicity to RAW 264.7 macrophage cells, increased NO secretion and boomed phagocytic activity.