| Fructus Aurantii Immaturus(FAI)is a traditional Chinese medicine and contains extremely rich flavonoids(5%-28%),including narirutin,naringin,hesperidin.Modern pharmacological studies demonstrated that flavonoids in FAI have antiviral,sedative,analgesic,antioxidant and other pharmacological effects,but its extraction method is also limited to traditional organic solvent extraction,which do not meet the green pharmaceutica concepts and limits its development and utilization to a certain extent.In addition,there is no literature report on the therapeutic effect of FAI on chemotherapy induced diarrhea.Therefore,in this paper,the ultrasound-assisted eutectic extraction of flavonoids from Fructus AurantiiImmaturus and its therapeutic effect on chemotherapy-induced diarrhea were studied.The main research contents and results are as follows.Method: 1.Ultrasonic-assisted deep eutectic solvent extraction of flavonoids from FAIAs many as 16 kinds of deep eutectic solvents(DESs)including choline chloride-sucrose,choline chloride-xylitol,choline chloride-proline,choline chloride-citric acid,choline chloride-glucose,Choline chloride-malic acid,choline chloride-acetic acid,choline chloride-lactic acid,sodium acetate-lactic acid,ammonium acetate-lactic acid,choline chloride-ethylene glycol,choline chloride-1,2-Propylene glycol,choline chloride-,6-hexanediol,choline chloride-1,4-butanediol,choline chloride-1,3-butanediol,choline chloride-glycerin were prepared and screened to extract flavonoid from AFI to determine the most suitable DES.After the composition ratio selection for the most suitable DES,single factor test,including temperature,ultrasonic power and time was conducted.Finally,taking the total flavonoid content as quality indicator,Box-Behnken response surface method was applied to design and optimize the extraction process.2.The therapeutic effect of flavonoids in FructusAurantii Immaturus on 5-FU induce diarrhea in miceChemotherapy mouse model was induce by by intraperitoneal injection of 5-FU at 50 mg/kg for 5 days.Brefly,total of 60 KM mice in female genders,weighting 20±2g,were randomly divided into six groups: normal control group(C),model group(M),LHC(0.3 mg/kg)group,AFIF.L(50 mg/kg),AFIF.M(100mg/kg)and AFIF.H(200 mg/kg).In the first 5 days,mice in M,LHC and AFIF groups were given 5-FU at 50 mg/kg by intraperitoneal injection while mice in C group were received same volume of saline in the same manner.Thereafter,mice in LHC and AFIF groups were orally given corresponding drugs at designed doses onece a day for 7 days and mice in C group reveived saline similary.Manifestations of mice during the experiment were recorded.Morphology and pathology of colon,inflammatory cytokines,oxidative indicators,as well as gut microbiota were detected.Using female Kunming mice and 5-fluorouracil to build an intestinal mucosal injury model,explore the therapeutic effect of different doses of flavonoids inFructusAurantii Immaturus on intestinal mucosal injury model.Judging the initial efficacy of flavonoids inFructusAurantiiImmaturus by weight,food intake,water intake,spleen index,thymus index,degree of diarrhea,and stool status of mice.The length of the colon of mice was measured and the material was stained with H&E(Hematoxylin and Eosin)to further judge the efficacy of flavonoids inFructusAurantii Immaturus by pathological observation and diagnosis.Using the world and the detection of oxidative stress parameters and inflammatory cytokine levels in the colon tissue of mice,the next-generation sequencing method was used to detect the intestinal flora of mice,and the effects of flavonoids inFructusAurantii Immaturus on intestinal mucosa injury mice were studied from the above aspects Influence,put forward a reasonable mechanism of drug effect.Results: 1.Optimal extraction process of flavonoid from AFICholine-acetic acid(1:2)was screened as the most suitable DES system to extract flavonoid from AFI.The results of single factor test were: temperature 60℃,ultrasonic power 80 W and extraction time 80 min.The optimal extraction conditions obtained by BBD method were: temperature 42℃,ultrasonic power 90 W and extraction time 82 min.Under the optimized conditions,the percentages of narirutin,naringin,hesperidin were 3.36%,0.629% and 15.96% respectively,which were significantly higher than that of ethanol.D101 macroporous resin exerted the highest rate of absorption and desorption of flavonoid in DES of choline-acetic acid(1:2)among other resins tested.After treatment with D101 macroporous resin,the recoveries of rutinnaringin,naringin and hesperidin were 83.59%,73.41%,83.43%,respectively.The recovered DES of choline-acetic acid was 81.2%.the recoveried DES was further used to extract flavonods from AFI for two times without obivious decrease in extraction capacity.In this experiment,16 kinds of deep eutectic solvents were prepared,and the extraction effect of 16 kinds of deep eutectic solvents was tested by single factor experiment with the content of three flavonoids of narirutin,naringin,hesperidin in Fructus Aurantii Immaturus.After screening,the type,composition ratio and water content of DES were investigated,and the choline chloride/acetic acid composition system was selected as the best solvent for extracting flavonoids from Fructus Aurantii Immaturus,composition ratio is 1:2,water content is 0%,ultrasonic assist the conditions of eutectic solvent extraction of Narirutin,Naringin,Hesperidin in Fructus Aurantii Immaturus were experimentally designed and process optimized,and the process parameters of ultrasonic-assisted eutectic solvent extraction were determined: the eutectic solvent was chlorinated Choline/acetic acid=1: 2(molar ratio,water content 0%),extraction temperature 42°C,extraction time 82 min,extraction power 90 W.Under the optimized conditions,the extraction of citrus aurantium was carried out,and the percentages of narirutin,naringin,hesperidinwere 3.36%,0.629% and 15.96% respectively,and the extraction effect was better than the traditional organic solvent ethanol.Seven types of macroporous adsorption resins of HPD-722,HPD-826,AB-8,D-101,NKA-9,X-5 are used to directly adsorb and separate the three flavonoids in the eutectic solvent extract.The D-101 resin has the highest overall recovery rate.After treatment with the D-101 macroporous adsorption resin,the recovery rates of the three flavonoid components of rutin naringin,naringin and hesperidin were 83.59%,73.41%,83.43%.The recovered deep eutectic solvent extracts flavonoids in Fructus Aurantii Immaturus a second time,and the recovery rate of flavonoids can reach 81.21%.2.The effect of flavonoids from FAI on 5-FU-induced diarrhea in miceIntraperitoneal injection of 5-FU at 50 mg/kg for 5 days,mice developed mild to severe diarrhea judged from DS,body weight,fecal morphologies and spontaneous activities.Treatment with flavonods from FAI(50,100,200 mg/kg)for 7 consecutive days effectively relieves symptoms such as diarrhea and weight loss caused by 5-FU.The results of morphological and pathological analysis of the colon further confirmed the efficacy.At the same time,flavonoids from FAI also improved the changes of oxidative stress and inflammatory cytokines.5-FU interfered with the overall structure of intestinal flora by reducing the richness and diversity of species The 100 mg/kg flavonoids from FAI significantly restored the species richness and diversity.Further investigation revealed that flavonoids from FAI promoted the growth of probiotics and inhibited the overgrowth of Pathogenic bacteria.Conclusion:DES of choline chloride-acetic acid(1:2)is a suitable DES system for the extraction of flavonoids from AFI and under the ultrasonic assisted condition,choline-acetic acid(1:2)can effectively extract the flavonoids from AFI.As our result is based on laboratory scale,there are still some questions such as the reuse the DES need to be further studied.Flavonods from AFI effectively ameliorated 5-FU induced diarrhea in mice and antioxidant,anti-inflammatory and gut microbiota modulation are involved in this effect. |
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