Purification of Chinese yam polysaccharide with marked hydroxyl radical scavenging capacity: Mechanistic insights into gel permeation chromatography separation efficiency

Abstract

Hydroxyl radicals are the most aggressive reactive oxygen species and directly damage biomolecules under physiological conditions, making their elimination critical for alleviating oxidative stress. Chinese yam polysaccharides (CYPs) possess antioxidant potential, yet the contribution of individual fractions remains unclear. Therefore, four fractions (CYP20, CYP40, CYP60, and CYP80) were obtained by gradient ethanol precipitation. Their hydroxyl radical scavenging rates were 34.60%, 22.65%, 51.45%, and 22.88%, respectively. Among them, CYP60, with a molecular weight of 43.58 kDa and composed mainly of galactose and galacturonic acid, exhibited the strongest hydroxyl radical scavenging activity. During gel permeation chromatography (GPC) purification, CYP60 could not be effectively separated when ultrapure water was used as the mobile phase, whereas 0.1 mol/L NaNO₃ solution successfully yielded a purified polysaccharide with a narrow molecular weight distribution. Particle size, zeta potential, and rheological analyses revealed that electrostatic shielding disrupted hydrogen bonding networks, reduced aggregate size, and altered the aggregation state of the polysaccharide in solution. This study established an efficient purification strategy for CYP60. It also demonstrated that electrostatic interactions governing polysaccharide aggregation determine GPC separation efficiency, laying the groundwork for future fine structural studies.