Identification of novel anti-inflammatory peptides from bee pollen (Apis mellifera) hydrolysate in lipopolysaccharide-stimulated RAW264.7 macrophages

Bee pollen protein was hydrolyzed using the commercial Alcalase, Flavourzyme and Neutrase enzymes. The Neutrase hydrolysate formed from a 1:1 (v/v) enzyme/substrate ratio (NH1) showed the highest nitric oxide (NO) radical scavenging activity. The NH1 was further separated into five fractions based on molecular weight (MW1–5) and MW1, the smallest weight fraction (< 0.65 kDa), possessed the highest NO inhibitory activity. The effects of NH1 on the production of NO were assessed by incubating with lipopolysaccharide-stimulated RAW264.7 macrophage cells. NO levels from the culture supernatants were determined by the Griess reaction. The results showed that MW1 suppressed the production of pro-inflammatory cytokines including cyclooxygenase-2 (COX-2), inducible nitric oxide synthase (iNOS), tumor interleukin-6 (IL-6) and necrosis factor transcript expression (TNF-α) in RAW264.7 macrophage cells. Thus, the MW1 fraction was fractionated using reversed-phase high-performance liquid chromatography into six principal fractions (H1–6), where H2, H3 and H4 showed strong NO inhibitory activity. Seven peptide sequences were obtained by quadrupole time-of-flight mass spectrometry, three of which displayed potent anti-inflammatory activity and may be useful ingredients in functional food and pharmaceutical drugs.