A rhamnogalacturonan I-enriched pectic polysaccharide from alfalfa and its protective effects against oxidative stress in Caenorhabditis elegans.

Alfalfa polysaccharides (APs) have attracted considerable interest for their reported antioxidant activities; however, chemically defined AP fractions remain insufficiently characterized, and their biological activities have rarely been evaluated in whole-organism oxidative-stress models. In this study, a major polysaccharide fraction (APs-4) was isolated from alfalfa by ultrasonic-enzymatic extraction method and purified through sequential ion-exchange and gel-filtration chromatography. APs-4 was characterized as a low-methoxyl pectic polysaccharide with a weight-average molecular weight (Mw) of 71.97 kDa and a dispersity (Đ) of 1.02. Monosaccharide composition, methylation analysis, and nuclear magnetic resonance spectroscopy supported a rhamnogalacturonan-I (RG-I)-enriched structural model, with estimated RG-I- and homogalacturonan (HG)-related contents of 80.2% and 8.0%, respectively. Its proposed backbone is predominantly composed of alternating →4)-α-galactopyranosyluronic acid (GalpA)-(1→ and →2,4)-α-rhamnopyranosyl (Rhap)-(1→ residues bearing arabinan and galactan-rich side chains. In a paraquat-induced oxidative-stress model of Caenorhabditis elegans (C. elegans), treatment with 0.5 mg/mL APs-4 extended the mean survival time from 4.57 h to 8.87 h. This protective effect was accompanied by increased activities of superoxide dismutase (SOD), catalase (CAT), and glutathione reductase (GR), elevated glutathione (GSH) levels, reduced malondialdehyde (MDA) levels, and upregulated transcription of stress-response genes including daf-16, skn-1, sir-2.1, and sod isoforms. These findings indicate a protective effect of APs-4 against paraquat-induced oxidative stress in C. elegans and provide correlative evidence linking treatment with changes in antioxidant indices and stress-related gene transcription. The present data do not establish causal pathway involvement or a direct structure-activity relationship, but they provide a basis for future pathway-specific genetic studies and comparative structural analyses.