| Citrus flavonoids are widely distributed in citrus plants in the form of glycosides or glycosides,many of which possess pharmacological activities such as antibacterial,anti-oxidant,anti-tumor and anti-inflammatory.In recent years,it has been found that some citrus flavonoid glycosides can be added to some foods as flavor agents.For example,neodisosimin,which is isolated from lime,has the effect of suppressing bitterness.neohesperidin dihydrochalcone and naringin dihydrochalcone are citrus flavonoid glycoside derivatives,the sweetness is 10001500 times and 500700 times of sucrose respectively,and have been applied in the food field as healthy sweeteners.These dihydrochalcone glycoside compounds also have important pharmacological activities such as anti-diabetes,anti-oxidation and anti-tumor.However,the development of citrus flavonoids has been limited in the field of food and health care products,cosmetics and medicine due to the poor solubility.Therefore,it is of practical significance to modify and transform citrus flavonoids to improve bioavailability by increasing water solubility.Previous work showed that monoglucoside has the best water solubility,which is higher than diglycosides?including rutinoside and neohesperidin?and free aglycones.Japanese scholars reported that the water solubility of hesperidin glycosylated was significantly improved,so it can be speculated that the introduction of glucose group into citrus flavonoid compounds or derivatives has a solubilizing effect.In this paper,the biological enzymatic modification of citrus flavonoids and their water-solubility and taste properties were studied.1.Study on glycosylation modification of citrus flavonoids.?1?Six citrus flavonoids monoglucoside compounds?2af?were obtained by hydrolysis of terminal rhamnose with?-L-rhamnosidase from citrus flavonoids,which are cheap and readily available in nature.Then,the process conditions of glycosylation catalyzed by transferase were explored and optimized.In pure water or phosphate buffer,transferase catalyzed glycosylation modified trilobatin?2b?reaction was used as a model to investigate the effects of the types of glycosylated donors,pH,temperature,time and enzyme dosage.The results showed that the conversion of trilobatin could reach 80%under optimal conditions with dextrin as glycosyl donor.?2?Dextrin was used as the glycosylation donor,and transferase was used to catalyze the glycosylation reaction of 6 substrates,including 2af,respectively.Glycosylation products?2af?-G1n were obtained,which contained the substrates of 2af that were not used up.After multi-step separation and purification,a single compounds?2af?-G1 were obtained.The enzyme modification products 2af,?2af?-G1 were analyzed and characterized by NMR and MS,and confirmed.2b-G1 has not been reported in literature.?3?The 2b-G1n was hydrolyzed by glycolytic enzyme and analyzed by HPLC.The chromatogram showed that the substrate 2b was almost completely run out;2d-G1n was hydrolyzed by amylase and analyzed by HPLC.The chromatogram showed that the content of 2d-G2n is reduced greatly,which is beneficial to the separation of 2d-G1,and is advantageous for obtaining a single compound 2d-G1 of higher purity.2.Study on water solubility and water partition coefficient.The water solubility and water partition coefficient of citrus flavonoids before and after enzyme modification were tested by using shake flask method and ultraviolet spectrophotometer.The results at 25°C showed that 1ag<2af<?2af?-G1<?2af?-G1n,the type and the number of glucosides is important factors affecting water solubility.The results showed that the water solubility of citrus flavonoids was significantly increased after glycosylation.The oil-water partition coefficients of the above compounds were determined by shaking flask method.The results showed that log Po/w values were in the range of 0-1,which met the requirements of drug for oil-water partition coefficients?-l<log P<2?.3.Cytotoxicity study.The cytotoxicity of glycosylated products?2af?-G1 on human immortalized keratinocyte?Hacat?and prostate cancer cells?PC3?was investigated by MTT assay.No significant toxicity was detected for these two kinds of cells.4.Taste evaluation study.The Intelligent Taste Evaluation system was used to evaluate the taste of citrus flavonoids.The results also showed that the introduction of glucose group increased sweetness.The above work provides a scientific reference for the development and application of these compounds in the fields of food,health products and pharmaceuticals. |
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