| Most of the flavonoids are found as a form of glycosides in plants,which have large molecular weight and poor membrane permeability,and are difficult to be directly absorbed by human.Reported studies shown that flavonoids have pharmacologically activities because of its aglycones.At present,mostly reported preparation of flavonoid aglycones are enzymatic hydrolysis,alkaline hydrolysis,strong acid hydrolysis,etc.,however,these reported methods have many defects such as poor solubility,low conversion rate and a large amount of solvent are needed.Natural deep eutectic solvents(NADES),a safety green ionic liquid,have been widely used to the extraction and separation of flavonoids from plants in recent years,due to its have higher flavonoids solubility than other solvents.In this manuscript,we first discovered that flavonoids had higher solubility in acidic NADES than other solvents.In the meantime,it can be used to prepare flavonoid aglycones by decomposing glycosidic bonds under the heating condition.Based on the discovery of acidic NADES can dissolve and degrade flavonoid glycosides(isoflavones and anthocyanins).The following work has been done in this article:(1)the effect of the NADES in the dissolution and degradation of soybean isoflavones(genistin)were demonstrated.The mechanism of NADES intermolecular hydrogen bonding was also discussed,and further applied to the dissolution and aglycon conversion of soybean isoflavone extracts.(2)The anthocyanins in blueberry pomace were efficiently extracted and converted into anthocyanin monomers by using NADES as the solvent.Combing with HPLC,we established a novel method which could analysis of anthocyanin content and anthocyanin monomer ratio at the same time.The major work and conclusions of present paper are descripted as follows:taken the formation of aglycones by using genistin in soy isoflavones as a representation,6 NADES systems were investigated.The results showed that the choline chloride-malic acid(ChApp)system was the best,which can efficiently transform genistin to its corresponding aglycones,and with sufficiently genistin solubility as well.Hence,this study mainly focus on the effects of kinds of water ratio and various temperature on the conversion of genistein by using ChApp system as the solvent.The experimental results showed that,including:(1)when the water ratio is constant and the temperature is above 60 ℃,the genistin is converted to genistein.Additionally,the conversion rate is increasing when the system temperature is rising;(2)when the system temperature is settled,the conversion rate decreased with the ChApp system water ratio increasing from 5%to 40%.Therefore,the ChApp system with 10%water and the temperature at 90 ℃ was chosen as the best condition of aglycon conversion.Meanwhile,the hydrolysis temperature and incubation time were also optimized when we using the crude extract of soybean isoflavones instead of standard genistein sample,and the 3.5 h and 90 ℃ were proven to the best reaction time and temperature.The crude extract of soybean isoflavones was handled under this optimal condition.Followed by purification of it by using X-5 macroporous resin,and the optimal enrichment conditions were described as follows:the loading concentration was 7.8 mg/mL,and the adsorption flow rate was 1.5 BV/h,70%ethanol as the eluent,the elution flow rate is 1 BV/h.At this time,the recovery of isoflavones reached 76%,the content of soy isoflavones increased from 25%to 49%,and the aglycon content increased from 12.42%to 40.02%.In this research,in order to explore the interaction mechanism between soybean isoflavone-genistin and NADES/H2O,we studied the influence of water content and system temperature on the dissolution of genistin with the aid of ChApp system as solvent.Simultaneously,’H-NMR and molecular dynamics were also used as the complement and verification.The results indicated that the concentration of genistin increased with the decrease of water content(10%,30%,50%and 70%)at the same temperature.At the same water ratio of the system,oncentration of genistin is higher at 40 ℃ than 30 ℃.1H-NMR results indicate that the chemical shift moves to the high field,the number of hydrogen bonds increases and the solubility of genistin increases with the water content of system decreased.Molecular dynamics simulation shows that a large amount of hydrogen bonds are formed between choline chloride and malic acid.Hydrogen bonds can also been found between genistin and ChApp.However,the number of hydrogen bonds between choline chloride and malic acid in the system decreases and the solubility of genistin decreases with the water content increases.Therefore,the system which have low water content benefit the dissolution of genistin.Usually,blueberry anthocyanins have five anthocyanins and in combination with three sugar ligands including glucose,arabinose and galactose.Therefore,it’s difficult to separate and detect them by liquid chromatography.Based on the above research,this paper proposed to extract anthocyanin from blueberry pomace through NADES and convert it into anthocyanin monomer.Combined with HPLC,a method was established to simultaneously analyze the total content of anthocyanin and the proportion of anthocyanin monomer in blueberry anthocyanin.By optimizing the extraction conditions:the choline chloride-citric acid(ChCa)system with water(20%)is selected as the extractant,the solid-liquid ratio is 1:30,water bath at 55℃ for 20 min,and the total anthocyanin extraction amount reaches 40.42 mg/g DW(twice than water as the extractant).At the same time,by optimizing the temperature and reaction time of the system,15 min blueberry anthocyanins were converted into corresponding aglycons at 100 0C.Using this condition in the ChCa system,the anthocyanins in the blueberry pomace were hydrolyzed and obtained five aglycones.And followed by HPLC to completely separate these aglycones.In addition,the ratio of aglycones obtained after hydrolysis is similar to unhydrolyzed anthocyanin.Therefore,the anthocyanins in blueberry pomace can be extracted by ChCa system and converted into anthocyanin monomers.Followed by combining with HPLC,a novel method for analysis of blueberry anthocyanin content and anthocyanin monomer ratio is established.The accuracy and convenience of this method was proved by applied this method to 6 blueberry products. |
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