Mechanism Of Enhancement Of Antioxidant Activity Of Oat Flour Afterenzymatic Hydrolysis

Oat(Avena sativa L. and Avenanuda L.) is regarded as a kind of nutritional and functional food. Polyphenols are one of the main biological components in oat, they can scavenge free radicals, also have proliferation inhibition effect on some tumor cells. Our early study found that the antioxidant activity of oat whole flour would be enhanced by α-amylasetreatment. We speculated that this phenomenon was due to the increase of polyphenol content during enzymatic hydrolysis. In order to further study the mechanism, oat flour and oat isolates(starch, oat protein isolates(OPI), bran) were treated withα-amylase, papain, cellulase, respectively. UK, a kind of starch hydrolysate, was detected in large amounts after starch hydrolysis. In order to study the properties of UK, thin-layer chromatagraphy was used for the isolation and purification of UK. The antioxidant and anti-tumor activity, structure identification, producing conditions of UK were investigated. The main results are as follows:(1) Oat whole flour treated with enzymes. Compared with heating treatment(control), enzyme(α-amylase, papain and cellulase) treatment significantly increased extractable polyphenol content and phenolic compounds in oat flour. After treated by α-amylase, avenanthramide 2f increased most(7.49 vs 17.27 μg/g), followed by ferulic acid, caffeic acid, vanillin, gallic acid, p-coumaric acid, avenanthramide 2p, 2c and p-hydroxybenzaldehyde(0.15 vs 0.32 μg/g). After treated by papain, avenanthramide 2f also increased most(5.95 vs 9.31 μg/g), followed by ferulic acid, avenanthramide 2p, gallic acid and p-coumaric acid(0.46 vs 0.60 μg/g). After treated by cellulase, ferulic acid increased most(0.28 vs 6.90 μg/g), followed by avenanthramide 2f, p-coumaric acid, caffeic acid, vanillin, gallic acid and p-hydroxybenzaldehyde(0.31 vs 0.42 μg/g). These three enzyme treatments significantly enhanced ABTS, DPPH radical scavenging capacity, ferric reducing antioxidant power and protection against AAPH induced protein oxidative damage of phenolic extracts.(2) Oat isolates treated with enzymes. Compared with heating treatment, the extractable polyphenol contents of oat isolates(starch, OPI, bran) significantly incresed after treated by α-amylase, papain and cellulase. So did phenolic compounds. Types and total content of phenolic compounds in oat bran extract increased most after treated by cellulase, containing p-coumaric acid, ferulic acid, avenanthramide 2f, caffeic acid and vanillin, especially ferulic acid increaed up to 756%(12.23 vs 124.03 μg/g). Phenolic compounds containing ferulic acid, avenanthramide 2p and 2f significantly incresead after treated by papain, and gallic acid, ferulic acid, avenanthramide 2f and vanillin after treated by α-amylase. The types of increased phenolic compounds in oat isolates were less than those in oat flour. Phenolic compounds increased most in oat isolates were in accordance with those in oat flour after enzyme treatments. It is worth mentioning that UK, a kind of non-phenolic antioxidant, was detected in large amounts after starch hydrolysis. These three enzyme treatments significantly increased the total antioxidant activity of oat isolates extracts compared to heating treatments.(3) Purification and antioxidant activity of UK. In order to further study the UK’s contribution to antioxidant activity of starch hydrolysates, thin-layer chromatography was used for isolation and purification of UK. The optimized developing solvent was as follows: Toluene: Ethyl acetate: Acetic acid = 5:3:2(v/v/v). UK showed good ABTS, DPPH radical scavenging capacity, ferric reducing antioxidant power and protection protein from oxidative damage, and these antioxidant activities were in a dose-dependent manner.(4) Anti-tumor activity and tumor cell proliferation inhibition mechanism of UK. UK inhibited cell viability of HepG-2 and K562 in a dose-dependent manner. UK had no effect on cell cycle of tumor cells HepG-2 and K562. UK promoted apoptosis of HepG-2 and K562, especially the early apoptosis of HepG-2, suggesting that UK was capable of inducing apoptosis in tumor cells. UK up-regulated apoptosis-related genes(FasL, Caspase-3, 6, 7, 8, 9, 10) mRNA expression levels of HepG-2 and K562 cells, indicating that UK mediated through Fas pathway to induce apoptosis of tumor cells. UK treatment had no effect on the STAT signaling pathway of HepG-2 cells, but activated STAT signaling pathway of K562 cells. UK treatment did not affect the expression amounts of MMP-9 in HepG-2 cells, but reduced the expression amounts of MMP-2 in HepG-2 cells, indicating UK could block tumor cell invasion and migration by down-regulating the expression of MMP-2.(5) Structure identification of UK. Identified by ultraviolet-visible absorption spectrum, Fourier infrared spectrum and nuclear magnetic resonance spectrum, UK was a mixture composed of glycerol-monopalmitate, glycerol-monolinoleate, and 9E, 11 Econjugated glycerol monolinoleate. As the main ingredient in UK, the proportion of glycerol-monopalmitate was 60%-80%. Both of glycerol-monolinoleate and 9E, 11E-conjugated glycerol monolinoleate accounted for about 10%-15%.(6) Releasing conditions of UK. The effects of treatment, extraction solvent, micro-surface morphology and degree of hydrolysis(DE value) on the release of UK were as follows: both of enzymatic treatment and the extraction solvent containing HCl significantly promote the release of UK, but enzymatic treatment was the main factor. The content of UK was positively correlated with DE value, both of DE value and the content of UK increased with treatment time prolonged. The surface of starch granules showed obvious holes after enzymatic treatment, and the holes linked together to form ‘ditch’ with treating duration. The results indicated that UK formed inclusion complexes with amylose in starch granule and could be released after starch hydrolysis.