Study On Glycosylation And Application Of Flavonoid Glycosides From Citrus

Flavonoids from Citrus are a kind of natural plant components with broad application prospects.Orange peel is used as medicine in the name of orange peel.It has been recorded for a long time in the history of Chinese traditional medicine.The main active ingredient is flavonoids,including hesperidin,a flavonoid glycoside with important pharmacological activity.Flavonoids from Citrus have good activity in anti-inflammatory,anti-oxidation,anti-tumor and immune regulation.Some of them have obvious taste characteristics,which can affect the taste of fruits and processed products.For example,naringin has a strong bitter taste,which is one of the main sources of bitter taste of pomelo fruits;the product of neohesperidin extracted from limes by hydrogenation,neohesperidin dihydrochalcone,has a significant sweet taste,which can be used as a high-power sweetener.However,the water solubility of flavonoids is generally poor,which limits their development in the fields of food,medicine,health care and cosmetics.At present,the main method to improve the water solubility of flavonoids is glycosylation modification,that is to use the catalysis of glycosyltransferase to transfer the active sugar donor to the flavonoid receptor molecule and form glycosidic bond to achieve glycosylation.It has been reported in recent years that the stability,water solubility and biological activity of glycosylated products have been improved.In addition,the research shows that glycosylation can improve the taste.In this paper,cyclodextrin glucosyltransferase was used as catalyst,and dextrin,which was cheap and easy to get,was used as sugar donor to glycosylate the flavonoid glycoside from citrus.By optimizing the experimental conditions,the conversion of the reaction was improved,and the water-soluble,cytotoxic and mouthfeel properties of the glycosylated products were investigated.The main works are as follows:1.Study on the glycosylation of flavonoid glycosides from citrus.Withβ-cyclodextrin as the sugar donor and CGTase as the catalyst,seven kinds of Citrus flavone glycosides（a～g）-G_n were glycosylated,including naringin（a）,naringin dihydrochalcone（b）,hesperidin（c）,neohesperidin（d）,neohesperidin dihydrochalcone（e）,rutin（f）and neodiosmin（g）.Among them,the conversion of a～f is 61.0～77.7%,and the conversion of compound g is only 33.5%due to its low solubility.The effects of reaction time,the type of glycosylated donor,the ratio of reactants,temperature,p H and the amount of enzyme added on the reaction were investigated.The results showed that the effects ofα-cyclodextrin andβ-cyclodextrin were similar.When the mass ratio ofβ-cyclodextrin to b is 10 and the amount of enzyme added is 100μL,the conversion rate of b can reach 64.6%after reaction in water at 65℃for 24 hours.Macroporous resin adsorption and Sephadex column chromatography were used to separate and purify glycosylated products.4 compounds（a～c,e）-G₁,which were attached to a glucose group,were successfully obtained.The structures were confirmed by ¹H NMR,¹³C NMR and ESI-HRMS.The structure of the compound was confirmed by b-G₁ and e-G₁.2.Study on the performance test of Citrus glucosylated products.The water solubility of flavonoid glycosides and their glycosylated products from citrus were studied by shaking flask method.The results show that the solubility of（a,b,d,e）-G_n is about 1000 times of that of their respective raw materials,while the solubility of c-G_n and f-G_n is about 5000 times of that of their raw materials.It can be seen that the effect of glycosylation on the solubilization of flavonoids glycosides from citrus is significant.The taste of 7 citrus flavone glycosides and their glycosylated products were evaluated by electronic tongue.The results showed that compared with the raw materials,the sweetness of all glycosylated products was improved.Glycosylated products can improve the sweetness and taste.The cytotoxicity of glycosylated products was studied by MTT method.The survival rate of the cells was above 90%at the experimental concentration,and the glycosylated products showed no obvious toxicity to the cells.3.Study on the application of glycosylation modification products of citrus flavonoids glycosides.The effect of glycosylated product（a～f,h）-G_n on the photostability of curcumin was investigated.The results showed that the decomposition rate of curcumin was41.3～66.9%after 15 days of light source irradiation,which was significantly lower than75.2%of the control group.Therefore,（a～f,h）-G_n can improve the light stability of curcumin in the order of h-G_n>b-G_n>f-G_n>d-G_n>c-G_n>a-G_n>e-G_n.The inhibitory effect of（a～g）-G_n on the bitter taste of quinine hydrochloride was studied by electronic tongue test.The results showed that（a～g）-G_n had inhibitory effect on the bitter taste of quinine hydrochloride.g-G_n had the highest inhibition rate of bitterness,which was 29.1%higher than other products.The order of the effect was g-G_n>f-G_n>e-G_n>b-G_n>f-G_n>d-G_n>c-G_n>a-G_n.In conclusion,this paper can provide a scientific reference for the practical application of glycosylated modified products of citrus flavonoids glycosides,and promote the high-value utilization of citrus plant resources.