Bioactive constituent profiling of brown sugar and functional evaluation of an isolated oligosaccharide with iron absorption-promoting and anti-anemia effects.

Brown sugar is a widely consumed non-centrifugal cane sugar with a complex chemical composition, but the constituents responsible for its potential effects on iron homeostasis remain poorly defined. Here, non-targeted UPLC-Q-Exactive MS/MS profiling identified 84 compounds beyond sucrose and glucose, among which 49 were bioactive constituents, including the cyclic peptide cyclo(Pro-Leu), flavonoids, phenolic acids, and itaconic acid. An oligosaccharide present at a relatively high abundance (3.31%, w/w) was isolated through sucrose hydrolysis, macroporous resin adsorption, and 300 Da membrane separation, reaching a purity of over 98%. Combined UPLC-Q-TOF-MS, FTIR, NMR, and differential scanning calorimetry analyses identified it as raffinose. In mice with iron-deficiency anemia, intragastric administration of the purified oligosaccharide restored red blood cell counts, hemoglobin, hematocrit, and serum ferritin, normalized the spleen index and red pulp architecture, and did not affect fasting blood glucose. The purified oligosaccharide also restored splenic ferroportin protein without changing its mRNA level. Molecular docking suggested that it occupies the hepcidin-binding cavity of ferroportin (-8.3 kcal mol-1), which may competitively prevent hepcidin-mediated ferroportin degradation and thus sustain splenic iron recycling. It also remained largely intact after simulated gastrointestinal digestion. In Ussing chamber experiments, it enhanced intestinal iron transport and was itself transported across the epithelium, pointing to a co-transport process supported by a stable 3 : 1 oligosaccharide-iron complex (ipTM = 0.87). These results suggest that this oligosaccharide, as a functional food ingredient, may support iron homeostasis by coupling ferroportin-mediated splenic iron recycling with chelation-assisted intestinal iron absorption.