Investigation Of Antioxidant Activity And Primary Antioxidant Mechanism Of Black Soybean Sprouts

Black soybean (Glycine max var) is the black seed of the soybean Glycine max (L.) merr,also known as the black bean. Black beans are rich in protein and mainly cultivated in theprovinces of Anhui, Dongbei, Henan, Hebei, Shandong and Shanxi for both food andmedicinal purposes. The Supplement to Compendium of Materia Medica states that blackbeans can be beneficial to sperm and bone marrow production, muscle strength, hair growth,and the immune system. Modern scientific research shows that black beans havehypolipidemic and antioxidant properties and can be used to beautify the skin.Antioxidant Activity: Anti-oxidizing capacities of extracts were measured in vitro usingthe DPPH scavenging test, OH· scavenging test, tyrosinase inhibition test and MTTcolorimetric assay which used to study the effects of the bean sprout extracts on human skinfibroblasts damnified by H2O2. Results show that radical scavenging rates at different shootlengths were all greater than90%(40mg/mL), while the tyrosinase inhibition capacity of theextracts reached90%. Extracts from sprouts grown to0.5cm had the highest capacity.Analysis of Gene Expression: Microarray and GO (gene ontology) term enrichmentanalysis were used to research the expressed genes related to antioxidant activity, and theresult was confirmed by qRT-PCR. Results indicated that: sprout of0.5cm in length has thehighest antioxidant activity; Microarray analysis of expressed genes between sprouts (0.5and5cm) showed that the most up-regulated genes in0.5cm sprout belonged to GO:0006979(response to oxidative stress), in which ten genes were significant highly expressed; Theresult of qRT-PCR analyses on up-regulation genes was consistent with the microarrayanalysis, indicating that the results were biologically reproducible and believable.Active Ingredient: Main active ingredients in black bean sprout were studied. The massconcentrations of proteins, polysaccharides and total flavonoid in water extracts of blacksoybean sprouts were measured by the Flint-phenol method, the phenol-sulfuric acid methodand AlCl3reagent color-developing method respectively. Results showed that black beansprouts contain much proteins, polysaccharides and little flavonoids. Proteins andpolysaccharides were separated using ammonium sulfate fractionation and alcoholprecipitation fractionation. Anti-oxidizing capacities of ingredients were measured byDPPH·scavenging test. The molecular weight distribution of proteins and polysaccharideswere measured by SDS polyacrylamide gel electrophoresis and multi-angle laser lightscattering, gel permeation chromatography (MALLS/GPC) respectively. Results showed thatproteins were the main antioxidant, and polysaccharides also have antioxidant activity, andproteins and polysaccharides with lower molecular weight have the strongest antioxidantactivity. Optimization of Extraction Using RSM: Response surface methodology (RSM) usinga central composite design (CCD) was employed to optimize the conditions for extraction ofantioxidants from black soybean (Glycine max var) sprouts. Three influencing factors:liquid-solid ratio, period of ultrasonic assisted extraction and extraction temperature wereinvestigated in the ultrasonic aqueous extraction. Then Response Surface Methodology (RSM)was applied to optimize the extraction process focused on DPPH radical-scavenging capacityof the antioxidants with respect to the above influencing factors. The best combination ofeach significant factor was determined by RSM design and optimum pretreatment conditionsfor maximum radical-scavenging capacity were established to be liquid-solid ratio of29.19:1,extraction time of32.13min, and extraction temperature of30°C. Under these conditions,67.60%of DPPH radical-scavenging capacity was observed experimentally, similar to thetheoretical prediction of66.36%.Safety and Stability: The safety of the extracts was determined using the red blood cell(RBC) test, chick chorioallantoic membrane (CAM) assay and human patch test. Hemolysisrate in all extracts were lower than10%, below the20%regulatory limit for the RBC test; TheCAM results show that the effect of sample groups on CAM was similar to normal saline(negative control) without irritation; No signs of allergic reactions were observed in thehuman patch tests; indating that black soybean sprout extract was safe. Results of stabilitystudy show that black soybean sprout extract was stable at room temperature, light, heat, cold,alternating hot and cold, or with pH stay within the range of6to10; The anticorrosion wasstable; The extract was safe, stable, active and can be added into gel, cream, emulsion andalcohol formula.