Study On Degradation Methods Of Flavanones In Aurantii Fructus Immaturus And Bioactivity Of Its Degradation Products

Flavanoneshavemanypharmacologicalactivities,suchas anti-bacterial,anti-inflammatory,anti-tumor,anti-atherosclerosis,anti-allergic and so on.Their chemical structures are composed of aglycones and glycosyls.According to the number of glycosyls,they are usually divided into single glycoside type and two glycoside type.The contents of flavanones with two glycosides are higher than others in Citrus plants of Rutaceae,such as narirutin,naringin,hesperidin and neohesperidin.Hesperidin and neohesperidin are isomer,whose position of rhamnose and glucose are different.After degradation,hesperetin-7-O-glucoside and hesperetin are obtained.Similarly,prunin and naringenin are obtained after degradation of narirutin and naringin.These four natural compounds have strong bioactivities,but the contents are low in plants.So flavanones with single glycosides or aglycones can be prepared by degradation of flavanones with two glycosides.According to the multi-component and multi-target theory of traditional Chinese medicine,we can speculate that degradation products,which mainly consist of prunin and hesperetin-7-O-glucoside and naringenin and hesperetin,may have stronger bioactivities than any single compound,and also exhibit new bioactivities.So far,there are no methods for preparing the four compounds mentioned above at the same time.So degradation methods of flavanones with two glycosides and bioactivities of its degradation products were studied in this paper.First,Aurantii Fructus Immaturus was chosen as materials,and hydrochloric acid was chosen as degradation solvent.After column chromatography and crystallization,four compounds were identified prunin,hesperetin-7-O-glucoside,naringenin and hesperetin by NMR,respectively.On the basis of above research,we found optimum degradation conditions by single factor investigation and orthogonal experiment.The optimum degradation condition of prunin and hesperetin-7-O-glucoside was as follows:The dosage of HCl was 1:10?V/V,mL/m L?;The ratio of sample to solution was 1:40;Degradation temperature was 70?;Degradation time was 2.5 h.The transformation rates of prunin and hesperetin-7-O-glucoside were 70.7%and 48.1%,respectively.The optimum degradation condition of naringenin and hesperetin was as follows:The dosage of HCl was 3:10?V/V,m L/mL?;The ratio of sample to solution was 1:30;Degradation temperature was 80?;Degradation time was 2.0 h.The transformation rates of naringenin and hesperetin were 82.9%and 62.7%,respectively.The optimum degradation condition for the mixture of four compounds was as follows:The dosage of HCl was 1.5:10?V/V,m L/mL?;The ratio of sample to solution was 1:20;Degradation temperature was 75?;Degradation time was 2.0 h.The transformation rates of prunin and hesperetin-7-O-glucoside and naringenin and hesperetin were47.9%,35.5%,44.4%,27.8%,respectively.The methods provided a new and effective idea for the preparation with high transformation rate and easy operation.In addition,the methods for purifying degradation products of Aurantii Fructus Immaturus by macroporous adsorption resin was studied.It was found that the mixture of prunin,hesperetin-7-O-glucoside,naringenin and hesperetin can be purified effectively by DM301 resin,which could make total content of four compounds over 50%in degradation products.Besides,we established a method for detemining contents of prunin,hesperetin-7-O-glucoside,naringenin and hesperetin by HPLC.The results showed that there was good linear relationship between concentration and peak area,LODs were between 2.6×10^-3 and 9.6×10^-3?g,average recoveries were 98.10%¹04.32%?n=6?,RSDs were less than 3%,which indicated that the method had good precision,high accuracy and sensitivity.Finally,the effect of four compounds and their mixture on Antigen induced?-HEX releasing in RBL-2H3 cells was studied.The inhibitory activity of mixture was higher than monomers,and the combination of prunin and hesperetin had the highest inhibitory activity.The results indicated that there were synergistic effects between monomers.Thereinto,prunin and hesperetin have stronger synergistic effects.In conclusion,Aurantii Fructus Immaturus was chosen as materials,hydrochloric acid was chosen as degradation solvent.The preparation and purification methods of a mixture composed of prunin,hesperetin-7-O-glucoside,naringenin and hesperetin were firstly studied,and a method was established for detemining contents of prunin,hesperetin-7-O-glucoside,naringenin and hesperetin by HPLC.And the anti-allergic activity of compounds and their mixture was studied.This paper provides a new scientific basis for their application.