| Nowadays,Noni fresh juice,fermented juice and wine are significant players in the growing Noni product market.In the production process of these products,a large amount of pomace is generated after juice squeezing.Generally,these by-products are discarded as waste,which increases the risk of environmental pollution.Analyses of Noni pomace have shown that the pomace is rich in flavonoids.There has been a great interest in reusing Noni pomace due to its potential as a source of bioactive substances with highly added value but vastly underutilized currently.However,there are still some problems to be solved in the utilization of flavonoids in Noni pomace.Conventionally,hot-organic solvent extraction is the most common method to extract flavonoids from plant tissues.But traditional extraction techniques are usually associated with high organic solvent consumption,long extraction time and high cost.Meanwhile,the preparation of flavonoid aglycones in Noni pomace by hydrolysis reaction also requires the use of acidic organic solvents,which produce non-degradable acidic organic wastes.In addition,the bioactivities of the flavonoid extract from Noni pomace are generally affected by the type and ratio of the original flavonoids in the raw material,which might limit the application of the flavonoid extract.At present,there is still a lack of a method that can directionally regulate the composition of flavonoids in flavonoid extracts to improve their biological activity.In recent years,pulsed electric fields（PEF）and deep eutectic solvents（DES）have shown some potential for the extraction of bioactive substances and the enhancement of chemical reactions.In this study,the effect and mechanism of PEF combined with DES on the extraction of flavonoids and preparation of flavonoid aglycones from Noni pomace were investigated.The feasibility of improving the bioactivity of Noni pomace flavonoids by PEF combined with DES targeted regulation of flavonoid composition was also investigated.The main research contents and conclusions are as follows:（1）The impacts of pulsed electric field-assist extraction（PEF-AE）using deep eutectic solvents（DESs）on the recovery of flavonoids from Noni pomace were investigated.Compared to the hot-water extraction（HWE）,PEF-AE can significantly increase the extraction amounts of the two main flavonoids（rutin and quercetin）in Noni pomace by at least 21.32%and 37.72%,respectively.Surprisingly,rutin could be converted to quercetin in choline chloride/oxalic acid（Ch/Oxa）under PEF and HWE treatments,but PEF had a higher conversion efficiency.The rutin conversion rate of PEF was 25.4-fold to 206.4-fold higher than that of HWE at the same treatment time and temperature.Outstanding rutin conversion efficiency（8.72%/min）for PEF was achieved by adjusting the key reaction conditions.Moreover,degraded rutin has been almost 100%converted to quercetin.Based on the screening for 18 DESs and the phenomenon of rutin conversion,choline chloride/ethylene glycol（Ch/Eth）and Ch/Oxa were found to be the most suitable solvents for the selective extraction of rutin and quercetin,respectively.After response surface methodology optimization,the highest extraction amounts of rutin and quercetin were 16.21 mg/g and 19.85 mg/g,respectively.Overall,PEF-AE combined with DESs is an efficient,selective and sustainable method for extracting flavonoids from Noni pomace waste.This work also demonstrated the feasibility to degrade flavone glycosides to flavone aglycones using PEF and DESs without the use of enzymes,acidic organic solvents,high temperature and pressure.（2）Based on the rutin conversion phenomenon found in the previous chapter,the existing PEF equipment was upgraded.The influence of the new equipment processing conditions on the conversion of rutin was discussed.In addition,the optimal concentration of Ch/Oxa and the electrode material of the treatment chamber were also screened.The results showed that 100%Ch/Oxa and Ti4O7 was the most suitable reaction solvent and electrode material,respectively.The single-factor experiments revealed that the electric field strength,pulse width,pulses number and frequency of PEF were positively correlated with the conversion of rutin.Response surface experiments（RSM）showed that 2 k V/cm,10 Hz,30μs and 3000 pulses were the optimal PEF treatment parameters.Under these conditions,PEF can completely convert rutin（30 mg/m L）into quercetin within 5 min.The results of HPLC and FTIR revealed that Ch/Oxa could be recycled at least two times after PEF treatment.（3）The effect of reaction temperature on the conversion reaction of rutin was investigated.The mechanism of the promotion of rutin conversion by PEF combined with Ch/Oxa was also investigated based on the molecular dynamics analysis.The results showed that the temperature was positively correlated with the conversion of rutin.The rutin can be converted to quercetin when the solvent temperature was beyond 50°C.The effect of an electric field alone did not cause the conversion of rutin.The mechanism of PEF to promote the conversion of rutin is mainly divided into two parts.First,the ohmic effect during the PEF treatment can rapidly warm up the Ch/Oxa solvent to above the conversion temperature threshold of rutin.Molecular dynamics simulations revealed that the pulsed electric field can increase the diffusion coefficient of molecules and ions in the reaction system when the solvent temperature exceeds50°C,which increases the chance of proton and chloride ions contacting the rutin glycosidic bond.The bond-breaking reaction of rutin might be related to the increase in the concentration of protons and chloride ions around the rutin glycosidic bond.In addition,we found that PEF combined with Ch/Oxa can be applied in the conversion of other glycosidic substances.Under the optimal PEF treatment conditions（2 k V/cm,10 Hz,30μs）,the flavonoid compositions in buckwheat and Sophora japonica extracts can be directionally adjusted by changing the number of pulses,resulting in the enhancement of their antioxidant activities.（4）The effects of PEF combined with Ch/Oxa on the conversion and the protein glycation inhibition ability of Noni pomace flavonoids were investigated.The component 1（NF 1）of Noni pomace flavonoids as the raw material in this part.The increase of quercetin content in NF1 was positively correlated with the pulses number under the optimal PEF treatment conditions（2 k V/cm,10 Hz,30μs）.After 1500 and 3000 pulses,NF1 was converted into NF2and NF3 samples with molar ratio of rutin to quercetin of 1.87:1 and 0.5:1,respectively.All NF samples showed the inhibition effect on the production of protein glycation products,especially for the advanced glycation end products（AGEs）.Among the tested concentrations（100-300μg/m L）,NF2 and NF3 were significantly stronger than NF1 in the inhibition of fructosamine,α-dicarbonyl and AGEs.At the highest treatment concentration（300μg/m L）,the ability of NF3to inhibit fructosamine,α-dicarbonyl and AGEs was 2.53 times,2.98 times,1.33 times（AGEs in the BSA-fructose system）and 1.23 times（BSA-AGEs in the MGO system）of the NF1.The stronger inhibition activity of NF3 to protein glycation is mainly related to its better protein binding ability,protein structure protection ability and MGO capture ability.（5）The effect of PEF combined with Ch/Oxa on the inhibition of xanthine oxidase activity by NF1 was investigated.The XOD inhibition activity of NF1 increased at least 10-fold after PEF treatment.Among the treated samples,the NF sample treated 3000 pulses（NF3）showed the best XOD inhibition activity,and the IC50 value was 12.13μg/m L.The kinetic analysis showed that the type of inhibition by NF2 was reversible and competitive.The type of inhibition by NF3 was reversible and mix.NF3 possesses the best XOD inhibitory ability due to its stronger binding ability with XOD and antioxidant activities.Molecular docking analysis showed that quercetin the main component of NF3,is bound to XOD through hydrophilic interaction and hydrogen bonding. |
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