碱法提取蝉蛹蛋白及其免疫调节功能研究

Study on extraction of protein from Cryptotympana atrata by alkali method and its immunomodulatory function

中文关键词:  蝉蛹蛋白  制备工艺  免疫调节

英文关键词:Cryptotympana atrata protein, process of preparation  immunomodulatory

中文摘要:

      目的 研究蝉蛹蛋白(Cryptotympana atrata protein, CA-P)的制备工艺及其免疫调节活性。方法 采用单因素法对影响CA-P提取的溶剂、料液比、氢氧化钠浓度、提取温度、提取时间、提取次数及沉降等电点进行优化。采用环磷酰胺所致小鼠免疫低下模型评价CA-P的免疫调节活性, 实验动物分为正常组、模型组、阳性对照组(香菇多糖250 mg/kg·d)、CA-P低、中和高剂量组[125、250和500 mg/(kg·d)]。以小鼠体重、免疫器官指数、脾淋巴细胞增殖率、自然杀伤细胞(natural killer cell, NK)活性、血清中免疫球蛋和细胞因子为指标, 对CA-P的体内免疫调节活性进行评价。结果 CA-P的最佳提取工艺为碱提[0.1 mol/L的NaOH溶液、料液比1:20 (g:mL)、在60℃条件下搅拌提取6 h], 酸沉(沉淀pH 4.0)。3个剂量的CA-P均对免疫低下小鼠的体重下降有改善作用, 可显著提高小鼠脾脏和胸腺指数, 有效促进小鼠脾淋巴细胞增殖, 增强NK活性。此外, CA-P干预组小鼠血清中免疫球蛋白G、免疫球蛋白A、免疫球蛋白M、细胞因子γ干扰素、肿瘤坏死因子α、白细胞介-2、白细胞介素-4和白细胞介素-10的分泌水平均有所提高。结论 采用碱提酸沉法可有效提取CA-P, 且提取物具有良好的免疫增强活性。

英文摘要:

      Objective To investigate the preparation process and immunomodulatory activity of Cryptotympana atrata protein (CA-P). Methods The single-factor method was employed to optimize the types of solvent, solid-liquid ratio, NaOH concentration, extraction temperature, extraction time, extraction cycles, and sedimentation isoelectric point that influenced the extraction of CA-P. The immunomodulatory activity of CA-P was evaluated using a mouse model of immunosuppression induced by cyclophosphamide. The experimental animals were divided into a normal group, model group, positive control group (lentinan 250 mg/kg·d), low-dose group (125 mg/kg·d), medium-dose group (250 mg/kg·d), and high-dose group (500 mg/kg·d). Mouse body weight, immune organ index, splenic lymphocyte proliferation rate, natural killer cell (NK) activity, serum immunoglobulins, and cytokines were used as indicators for evaluating the immunomodulatory activity of CA-P in vivo. Results The optimal extraction process for the CA-P involved alkaline extraction using a 0.1 mol/L NaOH solution, with a solid-liquid ratio of 1:20 (g:mL), at a stirring temperature of 60℃ for 6 h, followed by acid precipitation at pH 4.0. Three doses of CA-P had an improvement effect on weight loss in immunocompromised mice, significantly increasing spleen and thymus indices, effectively promoting spleen lymphocyte proliferation, and enhancing NK activity. In addition, the serum levels of immunoglobulin G, immunoglobulin A, and immunoglobulin M, interferon-γ, tumor necrosis factor α, the secretion levels of interleukin-2, interleukin-4, and interleukin-10 in the CA-P intervention group mice had all been increased. Conclusion CA-P can be effectively extracted by alkali extraction and acid precipitation, and the extract has good immunoenhancement activity.