灵芝多糖的超声辅助提取工艺优化及抗氧化活性分析

Optimization of ultrasonic-assisted extraction process and antioxidant activity analysis of Ganoderma lucidum polysaccharides

摘要:

为实现灵芝多糖（Ganoderma licidum Polysaccharides，GLP）的高效利用，该研究采用单因素及响应面法对灵芝子实体多糖超声辅助提取工艺进行优化，测定GLP的体外抗氧化能力，在此基础上建立H2O2诱导的HepG2细胞氧化损伤模型，研究其对细胞氧化应激的保护作用。结果表明，GLP最优提取工艺为：温度88 ℃，水提时间2.5 h，液料比41:1（mL/g），超声时间40 min，多糖得率为3.05%；1 mg/mL质量浓度下的GLP对羟基（Oxhydryl, ·OH）、1,1-二苯基-2-三硝基苯肼（1,1-Diphenyl-2-Trinitrophenylhydrazine, DPPH）、2,2'-连氮基-双-（3-乙基苯并二氢噻唑啉-6-磺酸）(2,2'-Azidobis (3-Ethylbenzo Dihydrothiazoline-6-Sulfonic), ABTS）分别可达79.49%、70.55%、79.61%；细胞试验结果表明，质量浓度为8 μg/mL的GLP可使细胞存活率恢复到将近100%，与模型组相比，GLP各剂量组的超氧化物歧化酶（SOD）提高28.44%~61.61%，谷胱甘肽过氧化物酶（GSH-Px）提高58.61%~101.75%，丙二醛（MDA）降低14.28%~32.89%，减少活性氧（Reactive Oxygen Species, ROS）累积，显著下调Nrf2，HO-1、上调Keap1基因表达（P<0.01），通过激活keap1/Nrf2信号通路，缓解细胞氧化应激损伤。该研究可为GLP提取及抗氧化功能产品的开发利用提供参考依据。

Abstract:

To achieve efficient utilization of Ganoderma lucidum Polysaccharides (GLP), the extraction process of polysaccharides from Ganoderma lucidum fruiting bodies was optimized through single-factor and response surface methodology. The in vitro antioxidant capacity of GLP was subsequently determined. Based on these findings, an oxidative damage model was established using H2O2-induced HepG2 cells to investigate the protective effects of GLP against cellular oxidative stress. The results demonstrated that the optimal extraction conditions were determined to be: extraction temperature of 88℃, water extraction time of 2.5 hours, liquid-to-solid ratio of 41:1 (mL/g), and ultrasonic treatment duration of 40 minutes, yielding a polysaccharide yield rate of 3.05%. At a concentration of 1 mg/mL, GLP exhibited remarkable scavenging activities against Oxhydryl (·OH), 1,1-Diphenyl-2-Trinitrophenylhydrazine (DPPH), and 2,2'-Azidobis (3-Ethylbenzo Dihydrothiazoline-6-Sulfonic), (ABTS), with scavenging rates reaching 79.49%, 70.55%, and 79.61%, respectively. Cellular experiments revealed that GLP at a concentration of 8 μg/mL was able to restore cell viability to nearly 100%. Compared with the model group, all GLP treatment groups showed significant increases in Superoxide Dismutase (SOD) by 28.44% to 61.61% and Glutathione Peroxidase (GSH-Px) by 58.61% to 101.75%, while Malondialdehyde (MDA) levels were reduced by 14.28% to 32.89%. Furthermore, GLP treatment was found to effectively reduce Reactive Oxygen Species (ROS) accumulation and significantly downregulate the expression of Nrf2 and HO-1 genes while upregulating Keap1 gene expression (P<0.01). The protective effects against oxidative stress were mediated through the activation of the Keap1/Nrf2 signaling pathway. This study provides valuable reference for the development of GLP extraction methods and antioxidant functional products.