梨果仙人掌多糖提取优化、结构表征及抗氧化活性研究

Extraction Process Optimization, Structural Characterization, and Antioxidant Activity Evaluation of Polysaccharides from Opuntia ficus-indica (L.) Mill.

摘要:

【目的】优化梨果仙人掌多糖的提取工艺，在降低蒽醌残留的同时提高多糖得率，并对所得多糖进行结构表征及抗氧化活性评价。【方法】采用热水浸提法，通过单因素及正交试验优化提取参数；利用乙醇多次洗涤沉淀以去除蒽醌类成分；利用紫外光谱（UV）、傅里叶红外光谱（FT-IR）、高效凝胶渗透色谱（HPGPC）等对其结构及单糖组成进行表征；通过测定DPPH自由基清除率及氧自由基吸收能力（ORAC）评价其体外抗氧化活性；建立H₂O₂诱导的HepaRG细胞氧化应激模型，检测细胞活力，并测定细胞内活性氧（ROS）水平及谷胱甘肽过氧化物酶（GSH-Px）、丙二醛（MDA）、过氧化氢酶（CAT）和超氧化物歧化酶（SOD）水平。【结果】最优提取条件为提取温度70 ℃、提取时间2.5 h、料液比1 g∶30 mL。结构表征显示，所得多糖重均分子量主要分布于722.3×10³、50.6×10³和1.6×10³，由甘露糖、葡萄糖醛酸、鼠李糖、半乳糖醛酸、葡萄糖、半乳糖、木糖、阿拉伯糖、岩藻糖9种单糖组成。该多糖对DPPH自由基的清除率为96.27%，ORAC值为0.66 mmol/L。在细胞模型中，梨果仙人掌多糖（2.50、5.00 mg/mL）可显著提升HepaRG细胞内CAT、SOD、GSH-Px活性，降低MDA和ROS水平。【结论】优化后的提取工艺可有效提高多糖得率，所得多糖为高纯度杂多糖。该多糖在体外及细胞水平均表现出显著的抗氧化活性，其作用机制与调控氧化应激相关生物标志物有关，可作为功能性食品领域的天然抗氧化剂。

Abstract:

[Objective] This study aims to optimize the extraction process of polysaccharides from Opuntia ficus-indica (L.) Mill., improve the polysaccharide yield while reducing anthraquinone residue, and characterize the structure and evaluate the antioxidant activity of the obtained polysaccharides. [Method] The hot water extraction parameters were optimized by single factor and orthogonal tests. The anthraquinone residue was reduced by repeated washing with ethanol. The polysaccharide structure and composition were characterized by ultraviolet (UV) spectroscopy, Fourier transform infrared (FT-IR) spectroscopy, and high performance gel permeation chromatography (HPGPC). The DPPH radical scavenging rate and oxygen radical absorbance capacity (ORAC) were measured to evaluate the in vitro antioxidant capacity. A HepaRG cell model of H₂O₂-induced oxidative stress was established, and the cell viability, intracellular reactive oxygen species (ROS) level, and the levels of glutathione peroxidase (GSH-Px), malondialdehyde (MDA), catalase (CAT), and superoxide dismutase (SOD) were determined. [Result] The optimal extraction conditions were extraction with a solid-to-liquid ratio of 1 g∶30 mL at 70 ℃ for 2.5 h. Structural characterization revealed three main weight-average molecular weights: 722.3×10³, 50.6×10³, and 1.6×10³. The polysaccharides consisted of 9 monosaccharides: mannose, glucuronic acid, rhamnose, galacturonic acid, glucose, galactose, xylose, arabinose, and fucose. The polysaccharides exerted the DPPH scavenging rate of 96.27% and the ORAC value of 0.66 mmol/L. The cell experiment showed that the polysaccharides (2.50 mg/mL and 5.00 mg/mL) significantly enhanced the activities of CAT, SOD and GSH-Px, while lowering the levels of MDA and ROS in the model HepaRG cells. [Conclusion]The optimized extraction process significantly improves the polysaccharide yield, and the obtained polysaccharides are high-purity heteropolysaccharides. Both in vitro and cell experiments demonstrate that the polysaccharides exhibit significant antioxidant activity, and its mechanism of action is associated with the regulation of oxidative stress-related biomarkers, highlighting its potential as a natural antioxidant for functional food applications.