Study On Degradation Method Of Flavonoids In Aurantii Fructus Immaturus And The Chemical Constituents And Biological Activity Of Its Degradation Products

Fructus Aurantii Immaturus（FAI）is a commonly used traditional Chinese medicine.It has the functions of breaking Qi,eliminating food accumulation,expectorating phlegm and removing sputum.It is often used to treat gastroptosis,dyspepsia,constipation,abdominal pain and other adverse symptoms.The main chemical components in Aurantii Fructus Immaturus are flavanones with two glycosides,among which the contents of narirutin,naringin,hesperidin and neohesperidin are in higher lever.After degradation of flavanones with two glycosides in Aurantii Fructus Immaturus,the corresponding flavanones with one glycoside and its aglycones can be obtained.Studies have shown that the antibacterial effect of flavanones with one glycoside obtained after degradation of flavanones with two glycosides in Aurantii Fructus Immaturus is better than the corresponding flavanones with two glycosides,which prompts that after degradation of flavanones with two glycosides,it may obtain stronger activity even discover new biological activity,which can expand the application of Aurantii Fructus Immaturus in the field of disease treatment and daily health care.Therefore,this article is based on the degradation of Flavanones with two glycosides in Aurantii Fructus Immaturus to obtain its flavanones with one glycoside and its aglycones as the research idea.So degradation methods of total flavonoids（The main ingredients are narirutin,naringin,hesperidin and neohesperidin）in Aurantii Fructus Immaturus,the chemical composition of degradation products,bioactivities of its degradation products were studied in this paper.First,through single factor experiment,the optimal extraction conditions for total flavonoids from Aurantii Fructus Immaturus were established as follows: using water decocting method,the ratio of liquid to material was 25:1,decocting 3 times in total,and the decocting time was 90 min,60min and 30 min respectively.The decoction liquid was combined,then DM-301 macroporous adsorption resin was used to adsorb the target copositions,80% ethanol was used to desorb,after recovered ethanol and dried,the total flavonoids have been obtained.As determined by HPLC,narirutin,naringin,hesperidin and neohesperidin are the main components,and the sum contents was greater than 50%.In order to obtain two types of flavanones with one glycoside and its aglycones,this article conducts degradation conditions for the total flavonoids of the Aurantii Fructus Immaturus.Through single-factor experiments and orthogonal tests,the effects of hydrochloric acid concentration,degradation temperature and degradation time on the degradation efficiency of flavanones with two glycosides in Aurantii Fructus Immaturus.Add 40 times of 6% hydrochloric acid aqueous solution and heat to dissolve,heat and hydrolyze in a water bath at 80°C for 2.5 h,cooled down to room temperature,adjust the pH of the reaction solution to neutrality with Na OH solution（5%）,then extract three times with ethyl acetate,and combined the ethyl acetate layers,ethyl acetate was recovered under reduced pressure and dried at 105°C.Under these conditions,the average conversion rate of flavanones with two glycosides in the total flavonoids of Aurantii Fructus Immaturus can reached to 78%.In order to clarify the main chemical constituents in the degradation products of total flavonoids from Aurantii Fructus Immaturus,the degradation products of total flavonoids were separated and identified by means of column chromatography and preparative liquid chromatography.A total of 6 compound monomers were isolated and identified as 5,7-Dihydroxy chromone,hesperetin-7-O-glucoside,hesperetin,pruning,naringenin and rutnaringin.Among them,hesperetin-7-O-glucoside and hesperetin are the degradation products of hesperidin and neohesperidin,respectively,while pruning and naringenin are the degradation products of narirutin and naringin,respectively.These four compounds are the main components in the degradation products.Next,this paper established an HPLC method for the determination of the contents of pruning,hesperetin-7-O-glucoside,naringenin and hesperetin in the degradation products of total flavonoids of Aurantii Fructus Immaturus,in order to use these four components as the content measurement indicators for Quality control of degradation products.The chromatographic conditions and methodology were investigated successively.The results showed that under the selected chromatographic conditions,the injection amounts of pruning,hesperetin-7-O-glucoside,naringenin,and hesperetin were all within the range of 0.05～9.80μg.Good linear relationship,the RSD values of the precision,repeatability,stability and sample recovery rate of these four components are all less than 3.0%,indicating that the content determination method established in this experiment has high sensitivity,good precision and nice accuracy.Finally,in this paper,DPPH free radical scavenging assay was used to study the antioxidant activities of 10 samples including total flavonoids,degradation products of total flavonoids,narirutin,naringin,hesperidin,neochesperidin,pruning,hesperetin-7-O-glucoside,naringenin,and hesperetin.The experimental results show ed that 10 samples have the effect of clearing DPPH free radicals.Among them,the total flavonoid degradation products is the highest,followed by the total flavonoids of Aurantii Fructus Immaturus,both of which are higher than the monomer compounds.The results of this study prove that the feasibility of degradation products with stronger biological activity through degradation of total flavonoids,providing new research ideas for the development and application of Aurantii Fructus Immaturus flavonoids in the future.As for the reasons for the removal of the DPPH free radical activity of the unilateral compound,the reasons of the corresponding flavonoids and the total flavonoid degradation of the Aurantii Fructus Immaturus may be related to the existence of multiple components in the mixture or other ingredients in the mixture.In each monomers,the removal of the DPPH free radical activity of the aglycone is stronger than that with one glycoside,and the one glycoside is stronger than two glycoside,indicating that the sugar substrate will affect its antioxidant activity,and the longer the sugar chain,the weaker this antioxidant effect.The mechanism of its role needs to be studied in depth.In conclusion,The results of this study provided a new idea for the study of Chinese medicine glycoside compounds,especially for the development and utilization of flavonoids in Citrus Aurantii.