Study On The Analysis And Mechanism Of Anti-allergic And Antioxidant Substances From Mung Bean Sprouts

In this paper, the anti-allergic substance and its functional mechanism, the metabolic pathway of the active constituents and its interference by phytohormones MeJA during the mung bean (Vigna radiatus Linn.) germination process were clarified by the techniques of phytochemistry, analytical chemistry pharmacology, and molecular biology. For superoxide dismutases (SODs) and catalases (CATs) of mung bean, gene cloning and sequencing analysis were firstly made. The correlation between gene expression and antioxidant enzyme activity was initially investigated.The mung bean sprouts (0-168h) were tested for the anti-allergic activity by the in vitro hyaluronic acid enzyme inhibition experiments and in vivo relieving itching experiments by each12h. The sprouts after48h were formed to show the best activity. The optimal process conditions are80%ethanol solution as the extraction solvent, material-liquid ratio of1:10, extracting temperature of50℃and extracting duration of2h. For different extraction of mung bean sprouts, the ethyl acetate layer with the highest anti-allergic activity showed the effective anti-allergic extraction. The chemical constituents of effective anti-allergic extraction were analyzed by LC/MS.12phenolic acids and6flavonoids were identified with the concentration of isovitexin at45.75mg/g, isoquercitrin at193.52μg/g, kaempferol-3-O-rutinoside at925.95ng/g, and p-coumaric acid at205.89μg/g, respectively, determined by HPLC-QQQ/MS. The metabolites profiling of the polyphenols in the sprouting process of mung beans were performed using UPLC-Q-TOF-MS and PLS-DA.21polyphenols were identified accurately. The sprouts from different periods were clearly discriminated on PLS-DA score plots. The chemical markers assigned as8flavonoids, vitexin, isovitexin, rutin, kaempferol-3-O-rutinoside, isoquercitrin, genistein, daidzein, isorhamnetin and2phenolic acids, shikimic acid and caffeic acid, which were responsible for variations were identified by the loading plot of PLS-DA.8chemical markers of the flavonoids were quantified by the HPLC/DAD method. The established method showed good linearity, repeatability, intra-and inter-day precisions, accuracies and recovery. Then, the main metabolic and transformation pathways of the polyphenols during the sprouting process were discussed.The animal models of passive cutaneous anaphylaxis (PCA), mast cell stabilization activity and histamine estimation were applied to study anti-allergic activity of the active extraction, the model group showed significant deviation compared with the control group (p<0.01). The high and middle doses of the active extraction showed notable effectiveness about the above three models. The low dose of the extraction, increased the stability of mast cells and inhibited the release of histamine caused by systemic allergic reaction. The results of in vivo experiments showed that the active extract effectively relieved itching caused by histamine. At the same time, it eased swelling, redness and other phenomena by inhibiting passive cutaneous anaphylaxis and increasing mast cells stability.The anti-allergic function mechanism of isoquercitrin was elucidated by in vitro cell experiment. The method of Elisa was used to determine the level of histamine, TNF-α, interleukin (IL)-1β, interleukin (IL)-8and interleukin (IL)-6in the calcium ionophore A23187plusphorbol12-myristate13-acetate (PMA)-stimulated human basophilic KU812cells. The results showed that different concentration of (50μg/mL,25μg/mL,12.5μg/mL) reduced the level of histamine and proinflammatory cytokines, and showed a dose dependency. Western blot was used to analyze the effect of the active extract on the activation of mitogen-activated protein kinases (MAPKs) and NF-κB. The inhibitory effect on the pro-inflammatory cytokines was achieved by decreasing MAPKs subgroups extra cellular-regulated kinase (ERK) and activity NF-κB.MeJA was identified as the most important active constituents of mung bean sprouts in7different types of plant hormones (1.0mmol/L). MeJA intervention improved the antioxidant activity by increasing the synthesis of polyphenolic ingredient. The p-coumaric acid and isovitexin decreased by42.00%and85.85%, respectively after MeJA intervention. Kaempferol-3-O-rutinoside, daidzein, genistein, isoquercitrin and caffeic acid were improved by MeJA intervention. The results showed that MeJA improved the polyphenols metaboilic level and anti-allergic activity.The first complete gene cloning and sequencing analysis of mung beansuperoxide dismutases (SODs) and catalases (CATs) was carried out by the means of molecular biology. The sequences of SOD and CAT were submitted to GenBank and the registered numbers HQ259252, HQ259253, HQ260597, and HQ260598were obtained. VirSOD and VirCAT of the mung beans were clustered to the same subgroup through the construction of phylogenetic tree. Quantitative real time-polymerase chain reaction (qRT-PCR) assay was performed to assess the mRNA expression profiles of VirSOD and VirCAT during sprouting. The mRNA expression of VirSOD and VirCAT reached the highest level on the144h sample. The expression patterns of the2genes were related to the alteration activity of CAT and SOD. It could be concluded that the improved activity was due to VirSOD and VirCAT expression.