PMFs Component Analysis And Biological Activity Study Of Citrus Species Native To China

China is one of the most important origins of the genus Citrus L.,with abundant citrus wild and cultivated germplasm.Although the study related to the Citrus resources has been last for more than half a century,the classification,evolution,origin and scientific utilization of Citrus are still the key and hot points.Citrus fruits are rich in flavonoids,which are mainly divided into flavanones,flavonoid glycosides and polymethoxyflavones（PMFs）.Among them,PMFs have attracted the attention of domestic and foreign scholars because of their special chemical structure and good biological activity.Among the different biological activities of citrus PMFs,antioxidant,anti-inflammatory,and anti-aging have been proved to be directly related to various major chronic diseases of humans,such as cancer,obesity,and cardiovascular and cerebrovascular diseases.Recently,citrus PMFs-enriched extract can regulate gut microbial to prevent obesity induced by high-fat diet.However,research on the key component of citrus PMFs（tangeretin）by regulating the gut microbial to prevent obesity is still has not been reported yet.In this study,UPLC-PDA was used to make a preliminary comparison of PMFs of27 Chinese local citrus cultivar fruits（peel,pulp and juice）.On this basis,macroporous resin（HPD300）was used to enrich the PMFs of the peel extracts,and the UPLC-Q-TOF-MS/MS technology was used to analyze the PMFs component of the extracts from 27 different citrus cultivars.Based on the PMFs metabolic map of citrus-enriched extracts,a citrus chemical classification model based on PMFs metabolites was established,and the classification model was used to analyze the four basic species of Citrus（citron,pummelo,papeda,and mandarin）,Kumquat and three important cultivated or wild（hybrid）species.Combined with the in vitro cell model,the antioxidant,anti-inflammatory,anti-aging and anti-cancer of citrus PMFs-enriched extract were systematically studied.Finally,the main component of PMFs（tangeretin）in the metabolism and gut microbial disorders of obese mice induced by high-fat diet was discussed.The main results obtained in our study were listed as follows:1.Analysis of flavonoids and antioxidant capacity in different tissues of citrusTwenty-seven accessions native to China were investigated on the total phenolic content（TPC）,total flavonoid content（TFC）,and flavonoids were measured using a Folin–Ciocalteau reagent,Al（NO₃）-Na NO₂ complexometry,and UPLC-PDA,respectively.The antioxidant capacities of the extracts were evaluated by DPPH,ABTS and FRAP method,respectively.The results showed that citrus peel contained more TPC and TFC,compared with pulp and juice.Among citrus studied,the peel of Guihuadinanfeng（C.reticulata）had the highest TPC of 23.46 mg equivalent gallic acid/g DW（dry weight）and TFC of 21.37 mg equivalent rutin/g DW.Similar to TPC and TFC,citrus peel is rich in flavonoids,especially PMFs.Different citrus species were characterized by their individual predominant flavonoids,contributing largely to the antioxidant activity,such as mandarin was characterized by hesperidin,nobiletin and tangeretin,while pummelo and papeda were characterized by naringin.Shiyueju（C.reticulata）peel showed the highest antioxidant capacity based on the antioxidant potency composite（APC）analysis.Overall,mandarin（C.reticulata）fruits peel contained more TPC and TFC,exhibiting higher antioxidant capacities than other species,and were good natural sources of flavonoids and antioxidants.2.PMFs component analysis of in citrus peel extract and their biological activityThe UPLC-Q-TOF-MS/MS system was used to detect the components of 27 citrus peel enriched extracts in the positive ion mode.52 PMFs were preliminarily identified based on the retention time,accurate molecular weight,fragment ions with reference standards.Kumquat,citron,pummelo and papeda were gathered together and mandarin was clearly discriminated based upon their PMFs metabolite profiles using cluster analysis and principal component analysis（PCA）.In addition,important cultivated or wild hybrid genotypes including sweet orange,sour orange and lemon were located between their parents.Eleven PMFs,such as nobiletin,tangeretin,and3,5,6,7,8,3’,4’-heptamethoxyflavone,are characteristic components in the metabolic map of citrus PMFs enriched extracts.The main biomarkers or characteristic compounds of the citrus chemical classification model based on PMFs metabolites.The quantitative results showed that the variation range of total PMFs content in different extracts was0.40-595.08 mg/g DW,and Wangcangzhoupigan had the highest content.The antioxidant capacity of 27 citrus PMFs enriched extracts was evaluated by DPPH,ABTS and FRAP methods.In addition,using different cell models,the anti-oxidation,anti-inflammatory,anti-aging,and anti-cancer activities were discussed.From the results of chemical antioxidant analysis,the antioxidant capacity is not related to the content of PMFs.In the cell model,the results of the transcriptional activity of the dual luciferase Nrf2 reporter gene showed that the extracts of Guanximiyou,Juyuan,Huaihuayichangcheng,Guihuadinanfeng,Yanxiwanlu,Gailiangcheng,Lixingtiancheng and Bingtangcheng（5μg/L）,as well as sinensetin（25μM）,can significantly activate Nrf2 activity.The PMFs enriched extracts（2μg/L）and individual compounds（5μM）can significantly inhibit the production of NO.The comparison of nobiletin and5-OH-6,7,8,3′,4′-pentamethoxyflavone,the hydroxyl group weakened the ability of PMFs to inhibit NO production.Citrus extract can effectively inhibit the production of IL-1β,IL-6,and TNF-α,and inhibit the expression of COX-2 and i NOS.The extracts of PMFs from citrus peel have the ability to inhibit the active oxygen content in yeast cells,except for Gailiangcheng,which all exhibit a concentration-dependent enhancement.In addition,PMFs extracts showed significant positive regulation on the chronological lifespan of Saccharomyces cerevisiae.The IC₅₀ value of citrus extract against DU145and PC3 prostate cancer cells is between 7.35 and 114.06μg/m L,and its antiproliferative activity against PC3 is stronger than that of DU145.Among the five PMFs,tangeretin has stronger antiproliferative activity.3.Evaluation of the efficacy of tangeretin in preventing obesity in mice induced by high fat diet（HFD）The effect of tangeretin on obesity was studied in C57BL/6 mice model fed with HFD.The results showed that tangeretin can significantly reduce the weight of liver,kidney,and fat in mice.Tangeretin can significantly inhibit the hypertrophy of white fat and keep the size of fat cells at a normal level.Tangeretin inhibits the expression of adipogenic genes（FAS and ACC-1）and FAS protein.In addition,tangeretin significantly reduces the levels of inflammatory cytokines（TNF-αand IL-2）and serum endotoxin（LPS）,and increases the expression of antioxidant activity-related genes（NRF2,NQO-1 and HO-1）,And inhibited the expression of inflammation-related genes（TNF-αand IL-1β）.Tangeretin protects the liver by significantly reducing the concentration of alanine aminotransferase and aspartate aminotransferase in serum.Tangeretin significantly reduces fasting blood glucose and insulin levels,and improves serum triglyceride and total cholesterol levels.Tangeretin stimulates the browning of brown fat（BAT）and inhibits the whitening of inguinal white fat（Ing-WAT）,and enhances the ability of mice to adapt to heat.16S r DNA-amplicon sequencing results showed that tangeretin significantly changed the composition（richness and diversity）of the gut microbial of high-fat fed mice,and reversed 14 OTUs back to NCD structure.It is worth noting that tangeretin reduces the ratio of Firmicutes/Bacteroides,significantly increases the abundance of Bacteroides and Lactobacillus,thereby improving the obesity phenotype.