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-alpha) 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.