Structural characterization and immunomodulatory activity of a high-molecular-weight glucan-rich fraction from...

Industrial natural rubber serum (NRS), a major by-product of rubber processing, is an underexplored source of structurally diverse polysaccharides. In crude Hevea latex polysaccharides (HLPs) recovered from NRS, the coexistence of multiple molecular-weight populations limits fraction-specific structural interpretation and biological attribution. In the present study, a 10 kDa ultrafiltration strategy was applied to partition crude HLPs into structurally distinct polysaccharide fractions and to enrich the retained high-molecular-weight glucan-rich fraction, designated HLPs-R10K, for focused characterization. HLPs-R10K exhibited an average molecular weight of 285.5 kDa and was predominantly carbohydrate in nature with minimal residual protein. Monosaccharide composition, methylation analysis, and NMR spectroscopy indicated that the dominant structural population of HLPs-R10K was glucose-based and characterized mainly by (1 → 6)-linked glucosyl residues with minor (1 → 3)-related linkages. HPSEC-MALLS further showed that HLPs-R10K remained an enriched fraction rather than a completely homogeneous single polysaccharide species, although one high-molecular-weight population predominated. In RAW 264.7 macrophages, the dialyzed HLPs-R10K preparation enhanced nitric oxide production, induced moderate intracellular ROS generation, upregulated iNOS, COX-2, TNF-α, IL-1β, and IL-6 expression, and produced non-linear changes in ERK/JNK phosphorylation, whereas total p65 abundance was not significantly altered. These results support fraction-associated macrophage immunomodulatory responses. The PMAA peak-area ratios and multidimensional NMR data support a putative dominant structural motif; however, compound-specific response-factor-corrected linkage quantification and fully validated quantitative endotoxin determination were not available. Accordingly, the present structure-activity interpretation should be regarded as provisional. Collectively, these findings show that 10 kDa ultrafiltration is a useful fractionation step for resolving structurally distinct polysaccharide populations from industrial NRS and establish HLPs-R10K as a structurally enriched glucan-rich reference fraction for future structure-function studies and marker-guided standardization of Hevea latex polysaccharide preparations.