Enzymatic Assisted Extraction Of Narirutin And Its Effect On Oral Tolerance Induced By OVA

Eggs are rich in high-quality protein,which is the major source of early dietary protein for infants.However,eggs contain a variety of allergens,which induce food allergies in infants,and affect the quality of life of allergic children.Ovalbumin(OVA)is the key allergen causing egg allergy,which is the most abundant protein in egg white.Oral tolerance is the state of local and systemic immune unresponsiveness that is induced by oral administration of innocuous antigen such as food proteins.Reports shown that food allergy happen to food proteins may occur if tolerance fails.Oral tolerance is a major way to prevent food allergy.Citrus peels are wasted,however researches show they were rich of biomaterials such as flavonoids.In particular,narirutin is dominant component in peels of Citrus.It has antioxidative,anti-cancer,anti-viral,and anti-inflammatory activities,but its effect on oral tolerance has yet to be reported.In this work,the extraction processing of narirutin was investigated by enzymatic dissociation and reflux extraction method,and its effects on oral tolerance induced in BALB/C mice by OVA.Details are shown as follows:1.Extraction processing of narirutin: Enzymatic dissociation and reflux extraction method was applied.(Reflux extraction method is based on our previous study): the concentration of ethanol was 60%,extraction solvent was 8 times of amount of material,extraction times were three times:1.5,1.0,and 0.5 h.Extraction fields of narirutin were detected by HPLC.The conditions of enzyme concentration,temperature,time,and pH were studied.As the results shown,when the concentration fo pectinase was 0.2 mg/mL,the temperature was set at 45℃,reaction time was set at 120 min,and pH was set at 4.5,the extraction fields of narirutin was 5.31 mg/g.Orthogonal test method was used to investigate the best conditions.As the results shown,when the concentration of pectinase was 0.3 mg/mL,enzyme reaction temperature was set as 45℃,reaction time was 120 min,pH was set as 5.0,the extraction field of narurutin is 5.83 mg/g.2.Effects on oral tolerance.The effects of narirutin on oral tolerance was applied by OVA induced oral tolerance model of BALB/c mice.The body weight of mice,body temperature,and diarrhoea score were studied,the concentrations of OVA-specific IgE,OVA-specific IgA,OVA-specific IgG1 and MCP-1 in serum and OVA-specific IgA in mice intestinal were measured by ELISA method.The expression of GATA3,Foxp3,STAT5,MHCⅡ,CXCR1,and CCR7 protein in colon tissue of mice was invested by Western Blot.As the results shown,narirutin helped the acquisition of oral tolerance.Hypothermia and allergic diarrhea with elevation of OVA-specific IgE,OVA-specific IgG1,OVA-specific IgA and MCP-1 were induced in serum of murine model of oral tolerance and narirutin treated oral tolerance.Analyses of protein expressing in colon showed the relative density of GATA3,MHCⅡ and CXCR1 decreased and Foxp3 and STAT5 expressing were increased.Additionally,the concentrations of mice serum MCP-1 have increased with narirutin treated oral tolerance groups and the relative density of CCR7 in the colon of mice have decreased.Therefore,Narirutin narirutin helped the acquisition of oral tolerance.