| Excessive accumulation of melanin can lead to undesirable browning of fruits and vegetables and skin diseases such as melanoma.TYEosinase is the key enzyme and rate-limiting enzyme during the generation of melanin production.However,TYEosinase inhibitors such as kojic acid and arbutin are commonly used in the market,but they have some side effects on the human body.Asparagus is delicious,and it is a low-calorie and high nutritional value vegetable,which contains polyphenols,saponins and other biologically active ingredients that have great potential to inhibit TYEosinase.Thus,asparagus was employed as a raw material in this paper.We first studied the effect of different drying methods on the nutritional quality of asparagus and the inhibition of TYEosinase.Then,the asparagus polyphenols were purified by ultrasonic-assisted macroporous resin.The hydrothermal treatment was used to increase the inhibitory effect of asparagus polyphenols on TYEosinase,and the simulation system was also used to investigate synergistic effects among different polyphenols.The mechanisms of enhancing the inhibition ability of polyphenols and saponins by ultrasonic combined heat treatment and microwave combined heat treatment was finally clarified.The main results are as follows:The effects of VD,FIRD,HAD and FD on the microstructure,color,active ingredients and TYEosinase inhibitory ability of asparagus were studied.Scanning electron microscopy results showed that obvious destruction of vascular bundles and epithelial cells of asparagus were observed during VD.The total phenol content of VD asparagus powder is the highest(791.79?g/g·dw).The adsorption between different monomeric phenol molecules and cellulose is affected by molecular weight,hydroxyl group and number of benzene rings.The free phenol extract of VD asparagus powder has the highest content of individual phenols(gallic acid,rutin,cinnamic acid,ferulic acid and quercetin are 151.40?g/g·dw,117.90?g/g·dw,159.41?g/g·dw,244.46?g/g·dw,and 118.67?g/g·dw,respectively).However,FD has higher saponin and chlorophyll content,which are 2.69 mg/g·dw and 0.401 mg/g·dw,respectively.Among them,the inhibition rate of TYEosinase diphenolase in the free phenol extract of VD asparagus could reach 57.98%.The inhibition rate of FD free phenol and combined phenol extracts are 55.24%and 62.26%,respectively.Ultrasound-assisted macroporous resin was used to purify asparagus polyphenols.Under the ultrasonic treatment conditions of 12 W?120 W and 25??35?,the adsorption and desorption capacity of asparagus polyphenols by D101 resin was significantly improved.The analysis of macroporous resin adsorption kinetics shows that the first-order kinetic model can better predict the initial stage of asparagus polyphenol adsorption,and the second-order kinetic model and particle diffusion model can better predict the adsorption behavior of asparagus polyphenols.Thermodynamic analysis shows that the Freundlich model is more suitable for describing the adsorption of asparagus polyphenols on D101 macroporous resin at different temperatures.In addition,ultrasonic treatment can significantly shorten the equilibrium time from 60 min to 30 min?40 min.Ultrasonic treatment can make the concentration of rutin,ferulic acid and quercetin in the sample reach 10.25?g/m L,11.54?g/m L,and 9.86?g/m L,respectively,and the desorption capacity is 1.72,1.20 and 1.62 times higher than that of the control group.Hydrothermal treatment significantly improved the inhibitory effect of asparagus polyphenols on TYEosinase.After hydrothermal treatment at 140°C for 90 min,the inhibition rate of TYEosinase increased from 3.26%to 51.22%.Among them,trans 4-hydroxycinnamic acid,ferulic acid and quercetin exist before hydrothermal treatment and gradually disappear after hydrothermal treatment;rutin exists during the hydrothermal treatment;coumaric acid does not exist before hydrothermal treatment but detected and appeared gradually after hydrothermal treatment.According to the analysis of the decarboxylation and esterification products by the hydrothermal treatment model,the m/z of the main fragments of the newly generated compounds are 405 and 601.The B16F10 cells treated with ASE and ASHE showed morphological changes such as cell size reduction,cytoplasmic degeneration,and nuclear fragmentation.The cell viability of each ASE and ASEH group decreased significantly with the increase of concentration,and they all inhibited the proliferation of B16F10 cells in a time-and concentration-dependent manner.ASE promotes melanin production in B16F10 cells at low concentrations and inhibits it at high concentrations.ASEH has a higher inhibitory ability than ASE at all concentrations.The inhibition rate of CA or FA on TYEosinase was 12.15%and 22.17%,respectively,and the inhibition rate of CA-FA on TYEosinase activity could be improved to 90.44%.The fluorescence spectrum shows that the addition of CA-FA causes fluorescence quenching and it is accompanied by red shift.The first-order kinetic model and Weibull model describe the inactivation of TYEosinase after adding 2 m M?4 m M and 6 m M?10 m M CA-FA,respectively.Ultraviolet-visible spectroscopy shows that the characteristic groups in CA and FA,such as hydroxyl,can compete with the nucleophilic attack of intramolecular cyclization,which ultimately leads to the reduction of characteristic peaks.Molecular docking further indicate that CA and FA interact with the TYEosinase activity cavity through amino acid residues such as Ser282,His263 and Val283.Among 37 groups of different phenols combinations,Group11 has the best synergistic effect and is composed of Que,CA and FA.Group11 is a competitive inhibitor,with an inhibition constant K_I value of 0.239 m M.In general,the fluorescence quenching mechanism of Group11 belongs to static quenching,and its binding capacity to TYEosinase is far greater than Que,and the Group11-TYEosinase complex is also more stable.Que,CA and FA can be docked to different active sites of TYEosinase at the same time to synergistically inhibit TYEosinase activity.Among them,cation-?,anion-?,electrostatic interaction,and hydrogen bond are the main force of binding reaction.The enhanced mechanism of ultrasonic combined with heating treatment and microwave combined heat treatment on the inhibition of polyphenols and saponin TYEosinase was clarified.Heating at 120°C?150°C for 30 min?120 min can significantly increase the TYEosinase inhibitory activity of polyphenols and saponin,and after heating at 150°C for 120 min,the TYEosinase inhibition rate of Group11 can reach 97.8%.During the ultrasonic combined with heating treatment,600 W 30 min ultrasound combined with 150?30 min heating treatment can increase the inhibitory activity of polyphenols and saponin TYEosinase equivalent to 150?120 min heat treatment.After ultrasonic combined heat treatment,the binding constant of Group11 and TYEosinase is significantly increased.Hydrophobic interaction is the main force of the binding reaction.After ultrasonic combined with heating treatment,the new compound named 2-hydroxy-3-(3-Hydroxy-4-methoxyphenyl)-propionic acid 4-(2,3-dihydroxypropyl)-phenyl ester.In 700 W 20 min microwave treatment combined with 120?30 min heat treatment,the addition of protodioscin and dioscin can significantly improve its TYEosinase inhibitory ability.Fluorescence spectrum analysis showed that the Ka value of TYEosinase combined with polyphenols and saponins was in the range of 1.09×10~5 L/mol?2.11×10~5L/mol.UPLC-MS analysis showed that the deglycosylation of saponin and the production of compounds Est1 and Est2 are one of the main reasons for the improvement of TYEosinase inhibitory ability. |
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