| Anthocyanins are a kind of water-soluble natural pigments categorized as flavonoids,widely found in natural plants.Anthocyanins are an antioxidant that removes free radical damage,enhances vascular elasticity,improves circulatory system,protects liver and eyes,prevents radiation and cancer.The structural units of anthocyanins are 3,5,7-trihydroxy-2-phenylbenzopyran.Due to the presence of many phenolic hydroxyl groups on the C6-C3-C6 skeleton,anthocyanins are susceptible to temperature,light,pH,oxygen,ascorbic acid,enzymes,metal ions,etc.,which will make it appear fading,discoloration,precipitation and so on.This led to the poor stability of anthocyanins,which largely restricted the application of anthocyanins.At present,the development of high quality and cheap processing raw materials and improving the stability of anthocyanins have become a hot spot in the research of anthocyanins.In order to explore the basic components of anthocyanins from Vitis amurensis Rupr,to obtain relatively stable anthocyanin products in the application,and to expand the market of anthocyanin raw materials,this paper chose a representative Vitis amurensis Rupr cv.‘Beibinghong' as raw materials,and the anthocyanins of Vitis amurensis Rupr were extracted by non thermal processing technology-high voltage pulsed electric field with the least structural damage and good extraction effect.At the same time,the anthocyanins from Vitis amurensis Rupr were purified.We used semi-preparative liquid phase to obtain the anthocyanin monomers from Vitis amurensis Rupr.Light and thermal stabilities of anthocyanin were improved by using high hydrostatic pressure assisted organic acids to stabilize the anthocyanin of Vitis amurensis rupr.Stable treatment changed the structure of anthocyanins of Vitis amurensis rupr,we studied the acute toxicity,in vitro and in vivo antioxidant activity of its physiological function.The conclusions are as follows:1.The process parameters of high voltage pulsed electric field extraction of anthocyanins of Vitis amurensis Rupr were determined.In the four methods of high voltage pulsed electric field,high hydrostatic pressure,ultrasonic and heating oscillations,the high voltage pulsed electric field method and high hydrostatic pressure method had high extraction efficiency,which are non-thermal extraction,little damage to active components,short processing time and large processing capacity.The extraction effect of the pulsed electric field method is superior to that of the high hydrostatic pressure method.The optimum process parameters of extraction of Vitis amurensis Rupr with high voltage pulsed electric field was the ratio of liquid to material 8.5:1 w/w,the electric field intensity was 15 kv/cm,the pulse numberwas 4,then the total anthocyanins content was 163.15 ± 2.54 mg/100g·FW.2.The technological parameters for the preparation of anthocyanin monomers of Vitis amurensis Rupr were determined.After purification by D101 macroporous resin,the anthocyanin purification samples of Vitis amurensis Rupr were obtained with total anthocyanins content of ? 30%.Anthocyanins were fractionated from 2 mL of the concentrated colorant solution by using a recycling preparative HPLC system(Waters 2545,Waters 2489 UV/Vis Detector,USA)that was equipped with a Waters fraction collector III.SunFire Prep C18 column(10 mm × 250 mm,5 ?m,USA)was used for separation with the mobile phase consisting of 5%formic acid aqueous(A)and methanol(B)as the mobile phase at a flow rate of 2 mL/min with UV detection at 530 nm.The gradient program was as follow: 0-5 min,8-10% B;5-50 min,10% B;50-55 min,10-15% B;55-60 min,15-20% B;60-80 min,20-25% B;80-100 min,25-35% B;100-105 min,35-70% B.Delphinidin-3-O-glucoside,cyanidin-3-O-glucoside,malvidin-3,5-O-diglucoside,petunidin-3-O-glucoside,peonidin-3-O-glucoside,and malvidin-3-O-glucoside six kinds of anthocyanin monomers were obtained,and each anthocyanin monomer content ? 95%.3.The Vitis amurensis Rupr varieties were determined and the structures of anthocyanins of Vitis amurensis Rupr cv.Beibinghong were identified.The contents of total anthocyanins in the five kinds of Vitis amurensis Rupr cv.Beibinghong,Gongniang NO.1,Shuanghong,Shuangyou and Zuoyouhong were from 67.80-398.77 mg/100g·FW,there were significant difference between species.The acid hydrolyzate of Vitis amurensis Rupr contained five kinds of anthocyanin aglycans,including delphinidin,cyanidin,petunidin,peonidin and malvidin,the content of aglycones were significant difference.There were significant differences in the anthocyanins of the five kinds of Vitis amurensis Rupr after purification,among which Beibinghong contained the most abundant anthocyanins,and the content is rich(166.65 ± 4.63 mg/100g·FW).So we chose the Vitis amurensis Rupr cv.‘Beibinghong' as raw material for identification and anthocyanin stabilization treatment.The components of anthocyanins were analyzed by UV-Vis spectroscopy,FTIR spectra,HPLC,mass spectrometry and NMR,including delphinidin-3-O-glucoside,cyanidin-3-O-glucoside,malvidin-3,5-Odiglucoside,petunidin-3-O-glucoside,peonidin-3-O-glucoside,malvidin-3-O-glucoside,cyanidin,petunidin,delphinidin-3-O-(6-O-acetyl)glucoside,peonidin,malvidin and delphinidin-3-O-(6-O-p-coumaroyl)glucoside.Among them,a kind of anthocyanin rich in a variety of Vitis amurensis Rupr was analyzed by NMR spectroscopy for the first time,and it was identified as malvidin-3,5-O-diglucoside.This work will be a step forward in predecessors,and lay the foundation for the research and application of anthocyanin monomers of Vitis amurensisRupr.4.The stabilization process of high hydrostatic pressure assisted organic acids bonded anthocyanins of Vitis amurensis Rupr was optimized.The optimum conditions for the treatment of anthocyanins from Vitis amurensis Rupr by high hydrostatic pressure assisted organic acids were optimized.The complex organic acids(ferulic acid: D-gluconic acid:caffeic acid: vanillic acid = 1.5:2.5:2.5:0.5,w/w/w/w,0.7 mg/mL)and anthocyanins(0.1 mg/mL)were reacted at 300 MPa pressure for 2 min,which can achieve the best stable effect.The absorbance of anthocyanins treated by high hydrostatic pressure was 0.781 ± 0.005,retention rate was 42.26%,and the retention rate was 12% higher than that of anthocyanins treated by traditional method.5.The stabilization effect of high hydrostatic pressure assisted organic acids bonded anthocyanins was studied.The stability of high hydrostatic pressure assisted organic acids bonded anthocyanins was better,and processing time was short,which was high efficiency,compared with the traditional method has wider application prospects.The absorbance value of high hydrostatic pressure assisted complex organic acids bonded anthocyanins of Vitis amurensis Rupr was 0.433 ± 0.008 after 20 days of daylight treatment,which was 44% higher than that of untreated anthocyanins.The retention rate of high hydrostatic pressure assisted complex organic acids bonded anthocyanins of Vitis amurensis Rupr was 56.02% after 20 days of daylight treatment,which was about 10% higher than that of untreated anthocyanins.The absorbance of high hydrostatic pressure assisted complex organic acids bonded anthocyanins of Vitis amurensis Rupr was 0.568 ± 0.005 at 100 °C for 2 hours,which was 81% higher than the absorbance of untreated anthocyanins.The retention rate of high hydrostatic pressure assisted complex organic acids bonded anthocyanins of Vitis amurensis Rupr was about 73.48% at100 °C for 2 hours,which was about 13% higher than the absorbance of untreated anthocyanins.The results showed that the color of high hydrostatic pressure assisted organic acids bonded anthocyanins had better color stability under exposure and heat exposure.6.The structures of anthocyanins by high hydrostatic pressure assisted organic acids bonded were analyzed.UV-Vis spectroscopy,HPLC,FTIR spectra and mass spectrometry showed that the contrast between the anthocyanin of Vitis amurensis Rupr treated by high hydrostatic pressure assisted complex organic acids and the untreated anthocyanins were changed.The maximum absorption wavelength of the anthocyanin treated by high hydrostatic pressure shifted ??max = 11,the new material peaks appeared in the liquid chromatogram,the new characteristic peaks appeared in the infrared spectrum,and the new ion peaks appeared in the mass spectrum: 411,421,425,433,435,439,443,477,611,625,627,639,and 669.Thesephenomena showed that the high hydrostatic pressure promoted the intermolecular or intramolecular effect between anthocyanins of Vitis amurensis Rupr and organic acids in a short time,or promoted the formation of hydrogen bonds and hydrophobic bonds,so that anthocyanins were properly modified to improve light and thermal stabilities.7.The stabilization mechanism of high hydrostatic pressure assisted organic acids bonded anthocyanins was studied.According to the results of structural analysis,the stabilization mechanism of high hydrostatic pressure assisted complex organic acids bonded anthocyanins was deduced: under the condition of high hydrostatic pressure,anthocyanins of Vitis amurensis Rupr react with ferulic acid,caffeic acid and vanillic acid,and then to form new anthocyanin derivatives after deacrylic acid,decarboxylation and dehydration.These newly formed anthocyanin derivatives are not easy to be affected by the factors such as light and heat,so that anthocyanins of Vitis amurensis Rupr in a stable state,which may be the main reasons to improve the stability of anthocyanins of Vitis amurensis Rupr.High hydrostatic pressure promoted anthocyanin to form stable anthocyanin derivatives in a short time,which improved the stability of anthocyanins of Vitis amurensis Rupr.This study clarified the mechanism of protection of anthocyanins by organic acids in the literature.8.The acute toxicity and antioxidant activity of high hydrostatic pressure assisted organic acids bonded anthocyanins were evaluated.The acute oral toxicity of high hydrostatic pressure assisted complex organic acids bonded anthocyanins in the 2000 mg/(kg.d)test range was non-toxic.High hydrostatic pressure assisted complex organic acids bonded anthocyanins had better antioxidant activity in vitro.When the concentration of high hydrostatic pressure assisted complex organic acids bonded anthocyanins was 20 ?g/mL,the power of DPPH,ABTS free radical scavenging was > 90%,which was 2-3 times higher than the untreated anthocyanins and ascorbic acid(VC).The antioxidant activity in mice showed that the T-SOD and GSH-Px activity in serum,liver,kidney,heart and brain of mice fed with the high hydrostatic pressure assisted complex organic acids bonded anthocyanins were significantly higher than that in the model group(p < 0.01),and the content of MDA was significantly lower than that of model group(p < 0.01).The results showed that the high hydrostatic pressure assisted complex organic acids bonded anthocyanins had high antioxidant activity in vivo,better than untreated anthocyanins. |
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