本文采用超声波辅助水提、乙醇沉淀、DEAE-52纤维素阴离子交换层析法分离纯化得到从树蝴蝶原料中提取的中性多糖LKY-I。以不同浓度的多糖为原料，研究了LKY-I的体外免疫调节活性。结果表明，LKY-I在125~500 μg/mL浓度范围内对RAW 264.7细胞无明显毒性。LKY-I可显著增强巨噬细胞吞噬中性红的能力，当多糖浓度为500 μg/mL时，吸光度值可达到1.39，显著高于阳性对照组LPS。LKY-I可促进巨噬细胞RAW 264.7分泌NO，最大分泌量可达55.42±1.79 μM；与正常对照组相比，LKY-I促进巨噬细胞分泌IL-6、TNF-α细胞因子，分泌量分别增加了102.3%和289.5%。在125~500 μg/mL浓度范围内，LKY-I可显著提高iNOS、IL-6及TNF-α基因的表达。LKY-I协同LPS和ConA能显著促进小鼠脾淋巴细胞的增殖。研究结果表明，LKY-I多糖具有良好的体外免疫调节活性，可以作为一种有益健康的成分应用到食品工业中。

The aim of this study was to determine the immunomodulatory activities of the neutral polysaccharide LKY-I extracted from Lobaria kurokawae Yoshim. The polysaccharide LKY-I was isolated sequentially using ultrasonic-assisted water extraction, ethanol precipitation, diethylaminoethyl cellulose (DEAE-52) ion-exchange chromatography, and gel permeation chromatography. We investigated the immunomodulatory activities using different concentrations of LKY-I. The polysaccharide LKY-I at a concentration range of 31.25–500 μg/mL had no obvious in vitro toxic effects in macrophages (RAW 264.7). The phagocytic ability of macrophages treated with 500 μg/mL LKY-I was higher than that of the macrophages in the positive control group treated with lipopolysaccharide (LPS); absorbance, 1.39. In addition, LKY-I induced the production of nitric oxide (NO) up to 55.42 ± 1.79 μM. Compared with the control group, the LKY-I group showed an increase in the secretion of tumor necrosis factor α (TNF-α) and interleukin 6 (IL-6) by 102.3% and 289.5%, respectively. Furthermore, LKY-I at a dose of 125–500 μg/mL showed a marked increase in the mRNA expression levels of inducible nitric oxide synthase (iNOS), TNF-α, and interleukin 6 (IL-6). LKY-I in combination with LPS or concanavalin A (ConA) significantly promoted the proliferation of lymphocytes. Thus, our findings suggest that LKY-I has immunomodulatory effects and can be used as a health-promoting food supplement.

广东省自然科学基金项目（2014A030313242）