Studies On Separation And Identification, Digestion And Absorption, Anti-Inflammatory Activity In Vitro Of Sialoglycoproteins In Edible Bird’s Nest

Edible bird’s nest (EBN) is rich in sialoglycoproteins (SGP) with lots of nutritionalbenefits, such as promoting cell growth, inhibiting blood coagulation, protecting againstinfluenza virus, improving bone strength and scavenging free radicals. To date, there are manyresearches about nutritional composition analysis and authenticity identification of EBN, butshort of researches about SGP. Therefore, in this research, superior White EBN of Malaysiawas used as experiment materials to get down to some research about purification andidentification of active substance, digestion and absorption in vitro, anti-inflammatory activity.The conclusion is as follows:The distribution of α2,3and α2,6sialoglycoprotein (SGP) in EBN extraction (EBNE)was analyzed by lectin blotting. Superdex75and Q Sepharose Fast Flow (QFF) werecombined to enrich main SGPs in EBNE. For purification and identification of α2,3SGP andα2,6SGP, MAA-Sepharose4B, SNA-Sepharose4B lectin affinity chromatography andMALDI TOF-TOF/MS were employed successively. The results suggested EBNE was rich inSGPs; and the main SGPs were distributed in30~60kDa, which contained terminal α2,3andα2,6sialic acid linkage and were successivelyenriched bytwo chromatography columns.43kDa α2,3SGP,43kDa α2,6SGP and50kDa α2,6SGP were further purified by affinitychromatography eluted with0.5M respectively. In addition, MALDI TOF-TOF/MS analysisshowed that, three purified SGPs all belonged to acidic mammalian chitinase-like proteins andacidic mammalian chitinase proteins.Caco-2cell monolayer model combined with digestion in vitro was employed in order toresearch for the effects of Maillard reaction in crystal sugar containing EBN process ondigestion and absorption. The results showed that EBNE degraded obviously in simulatedgastric fluid (SGF). The Maillard reaction reduced the lectin affinity of α2,3SGP and α2,6SGP in EBNE and also increased the resistance to pepsin digestion of EBNE. In addition, theanalysis of TEER change of Caco-2monolayer treated with200μg/mL DEBNE andDMEBNE suggested that glycoproteins in EBN could transport into basolateral side by the pathway of paracellular transport. DMEBNE was absorbed significantly in5~30min (P<0.05)and even highly significantly in60min (P<0.01). However, the Maillard reaction improvedthe intestinal absorption of EBNE through paracellular transport and the mechanism neededfurther research.The LPS-induced RAW264.7inflammatory cell model was used to study the influenceof Maillard reaction and different terminal sialic acid residues (including α2,3linkage and α2,6linkage) of SGP on inflammatory response. The MTT assay proved that glycoproteins inEBN could inhibit inflammatory cell proliferation and the inhibiting effect of SGP was higherthan that of the crude extraction. At the concentration0.01~150μg/mL, the inhibitinginflammatory cell proliferation activity of α2,3SGP was higher than α2,6SGP. At the sametime, the results of NO generation suggested that the NO inhibition rate of α2,3SGP was upto59.37%(P=0.002) while that of α2,6SGP was up to54.85%(P=0.005), which indicatedthe anti-inflammatory activity of SGP in EBN was highly significant (P<0.01) and wassignificantly higher (P<0.05) than EBNE, DEBNE and DMEBNE at1μg/mL and10μg/mLanalyzed by SPSS. In addition, at the concentration of0.01~10μg/mL, the anti-inflammatoryactivity of α2,3SGP was also more obvious than α2,6SGP in EBN, but without significantdifference (P>0.05). This conclusion lays the foundation of the study on SGPstructure-activity relationship in EBN, and also provides a scientific basis for evaluatingnutrition activity of EBN.