Separation、purification And Structure Identification Of Phenolics In Brown Rice

Rice is the second production crop in the world,and it is the staple food for above half of the population.Research on brown rice polyphenol mainly concentrate on colored rice polyphenol,the research about free,enzyme and bound polyphenol of colourless rice in China,including representative early indica rice,late indica rice,and japonica rice varieties had not been reported yet.This work selected 5 brown rice varieties from the top 10 Chinese brown rice varieties of planting area in 2009²012,they were early indica rice（Zhongjiazao 17）,late indica rice（Tianyouhuazhan,Wuyou 308）,japonica rice（Longjing25,Longjing26）,respectively.Moreover,2brown rice varieties（Huanghuazhan,Fenghuazhan）were selected from the top 5brown rice varieties of planting area of Jiangxi province in 2013.The free,enzyme and bound polyphenol of different rice varieties was studied.The main contents and results are as follows:1.The polyphenol content,flavonoids content and antioxidant activity of different rice varieties were studied.It was found that the iron reducing power,ABTS radical scavenging activity,polyphenol content and flavonoids content of total polyphenols in brown rice was 1499.7¹676.4μg GAE/g DW,365.2⁵68.2μg CE/g DW,661.1⁷23.1μmol Fe²⁺/100 g DW and 598.8⁷23.6 mg troxlox/100 g DW,respectively.The free flavonoids content acconted for 63.7%of total flavonoids content,the bound flavonoids content and the enzyme flavonoids content was 32.0%and 4.2%,respectively.The percentage of free,enzyme and bound polyphenol content were very close,with vuales at35.2%,38.1%and 26.7%,respectively.There was significant difference（P<0.05）in the polyphenol content,flavonoids content and antioxidant activity of different rice varieties.The flavonoids content of different cultivars showed greatest difference.Moreover the highest flavonoids content（Longjing 26）was 55.6%higher than the lowest flavonoids content（Tianyouhuazhan）,while the highest polyphenol content（Wuyou 308）was just 11.8%higher than the lowest（Longjing25）.2.Liquid-liquid extraction,macroporous resin and C₁₈8 solid extraction were used to purify the brown rice crude extract.The phenol recovery and antioxidation of different purification methods were studied,then analysised 9 phenolic acid and total phenolic acid recovery of 3 purification methods through High Performance Liquid Chromatography（HPLC）.The polyphenol recovery is respectively 20.51%,53.45%and 87.87%,the total phenol acid recovery is respectively 56.84%,67.35 and 68.99%,polyphenols purity respectively increased from 19.23%to 40.72%,46.84%and78.71%.The optimal purification method was C₁₈8 solid phase extraction method,polyphenols recovery difference of 3 purification methods were primarily caused by the extracting efficiency of non-phenolic acids.The article compared 9 phenolic acids recovery rate of 3 purification methods（p-hydroxy benzoic acid,chlorogenic acid,vanillic acid,caffeic acid,2,4-dihydroxy benzoic acid,syringic acid,coumaric acid,ferulic acid,sinapic acid）,the phenolic acid recovery rate had significant difference between different purification methods（P<0.05）.So the optimal purification method of one single phenolic acid should be choosed by the recovery of target phenolic acid not just according to the total polyphenol recovery or total phenolic acid recovery.3.Taking gradient elution of brown rice polyphenol by using C₁₈ solid phase extraction column,the elution phase was respectively 0%,10%,15%,20%,25%,30%,50%,70%,100%ethanol solution,polyphenols extraction yield and oxidation resistance of eluent were studied,and the liquid chromatogram of different eluents was investigate by high performance liquid chromatography（HPLC）.25%ethanol gradient eluent has the maximum extraction ratio and oxidative stability,it also has the largest range of peak time in all in gradient eluent.The polyphenol content has a positive correlation between ferric reducing ability and DPPH free radical scavenging capacity（P<0.01）.4.The free,enzyme and bound brown rice polyphenols were identified using HPLC/ESI/MS/MS,we detected 9 phenol acids,11 glycoside phenol acids,2 phenol acid esters,4 phenol acid amines and 20 flavonoids in free phenol;But we detected a lot of amino acids in zymolytic phenol;We detected 20 phenol acids in bound phenol.The Hydroxbenzoic acid,Gentisic acid and Feruloyl O-feruloyl O-anhydroglucoside were first detected in brown rice.The polyphenol content（quantified by HPLC）in free polyphenols of brown rice is 45.39μg/g,of which phenolic acid,phenolic acid class（glycoside phenol acid,phenol acid esters and phenol acid amines）and flavonoids content is respectively 9.23μg/g,19.55μg/g（16.27,0.33 and 2.95μg/g）and 16.62μg/g.Taking quantitative analysis of previous identified phenolic acid class and flavonoid substances.Phenolic acid class and flavonoids were important composition of free polyphenols,they accounted for 79.7%of free polyphenol content.Phenolic acid class has a stable proportion（36%）in different varieties,the proportion of phenolic acids,phenolic acid amines and flavonoids changes with species.The highest content of phenolic acids is ferulic acid,followed by vanillic acid,the highest content of phenolic acids class is ferulic acid glycosides,the highest content of flavonoids is Luteolin C-Glycoside C-Glycoside.The bound polyphenols of brown rice were all phenolic acids.The average polyphenol content（quantified by HPLC）of phenolic acid monomer and phenolic acid dimer is 251.22μg/g,33.19μg/g,the average polyphenol content（quantified by HPLC）in bound polyphenols of brown rice is 284.41μg/g.The highest content of phenolic acids is ferulic acid,followed by p-coumaric acid...