| More and more attention has been paid to the functional quality of fruit.And the functional quality has also become one of the important indicators for evaluating fruit quality.However,research on functional qualities of fruits was limited to the field of horticulture in the previous study.Zhique(Citrus wilsonii Tanaka)is a hybrid of trifoliate orange and pummelo.It is produced in the northernmost margin of citrus in China(Hanzhong,Shaanxi).Its fruit is bitter and can be used for medicine,but its medicinal value of functional ingredients has not yet been developed and applied.There are few reports on the medicinal value of the functional components in Zhique frutis,and the research mainly focused on the separation and determination of part functional components.Research methods in horticulture and medical are combined,Taking Zhique fruits as the research material,and its main components were determined and analyzed.Inflammation model in vitro with the mouse macrophage cell line RAW 264.7 and primarydendritic cell,and myocardial ischemia/reperfusion(I/R)injury model in rats were established to explore the medicinal value of the functional components of Zhique,evaluate the anti-inflammatory activity and screen its main components,and investigate myocardial protection effects.The main contents and results are listed in detail as follows:(1)Analysis of metabolites of different citrus and ZhiqueZhique metabolites were determinated by extensive-targeted methods,and it was found that the flavonoids were the most aboundant.Naringin,hesperidin,neohesperidin,naringin and poncirin of the flavonoids in the Zhique were further quantitative analyzed,and the results suggested that naringin content was the highest.Comparing the naringin content in different citrus and Zhique,it was found that there naringin contents in sour orange,Zhique and pomelo were higher than others and the lowest in wide-peel citrus,and and the naringin content in Zhique is equivalent to that of Huazhoujuhong and sour orange used to medicine.Determination of Zhique at different developmental stages showed that the naringin content gradually decreased as the Zhique fruit developed,and finally stabilized.In addition,environmental factors(light and soil)have also been found to affect the accumulation of naringin.(2)The anti-inflammatory and myocardial protection evaluation of Zhique crude extractThe anti-inflammatory activity of Zhique crude extract was evaluated using an inflammation model in vitro.Treating lipopolysaccharide(LPS)-stimulated mouse macrophages RAW 264.7 and bone marrow-derived dendritic cells with crude extract of Zhique,it was found that crude extract of Zhique significantly inhibited the expression level of the pro-inflammatory factor prostaglandin E2,cyclooxygenase-2,interleukin(IL)-1β,IL-6 and tumor necrosis factor-α(TNF-α)induced by LPS,and further inhibited the inflammatory response in cells.It indicated that the Zhique crude extract has a good antiinflammatory activity.To evaluate the anti-inflammatory activity and myocardial protective effect of Zhique in vivo,myocardial I/R injury model in rats were established.SD male rats were treated by Zhique crude extracts,and the results showed that Zhique crude extract can significantly inhibited I/R induced the degree of myocardial injury,the myocardial infarction area,the indicators of myocardial damage(creatine kinase-MB,cardiac troponin I),cardiomyocyte apoptosis,superoxide dismutase(SOD)activity and malondialdehyde(MDA)content,and cardiomyocyte secretion of inflammatory factor IL-6 and TNF-α,and reduced the phosphorylation level of p38 MAPK and the expression of pro-inflammatory mediator high mobility group box B1(HMGB1).(3)The screening and purification of the main anti-inflammatory effect components of Zhique crude extractSemi-preparative liquid chromatography was used to divide the crude extract of Zhique into three fractions according to the peak time of naringin.Three fragments and/or LPS were used to treat mouse macrophages RAW 264.7,and the anti-inflammatory activities were analyzed.The results showed that second fraction(naringin)has the strongest anti-inflammatory activity in the three fractions,indicating that naringin is the main anti-inflammatory effect compound.Subsequently,the naringin with a purity greater than 98% was purified from the Zhique by recrystallization.(4)The potential mechanism investigation of myocardial protection of naringin isolated from ZhiqueSD male rats were injected with naringin in the tail vein,and it was found that naringin can significantly inhibit myocardial injury and reduced the marker levels(creatine kinase,lactate dehydrogenase).It reversed SOD activity,MDA content,and the secretion of inflammatory factors IL-6 and TNF-α caused by myocardial I/R in rats.Immunoblotting results showed that naringin can increase the expression of anti-apoptotic protein Bcl-2 anddecrease the expression of pro-apoptotic protein Bax in cardiomyocytes,increase the Bcl-2/Bax ratio,and significantly reduces the expression of cleaved-caspase3.In addition,naringin suppressed the decrease of the expression of deacetylase-1 Sirtuin-1(SIRT1)and SIRT3 and the increase of HMGB1 expression caused by I/R in rat myocardium. |
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