Detection And Purification Of Polymethoxyflavones From Citrus(Citrus L.) And Its Potential Anti-fatigue Activity And Mechinsms Involved

China is one of the most important origin of Citrus fruits with abundant resources of Citrus plants.Studies of Chinese Citrus resources has been carried out for over half a century,but little investigation of Polymethoxylated Flavones（PMFs）in Chinese Citrus landraces has been reported.PMFs are a kind of flavonoids that specifically exist in Citrus fruits with great bio-activities of anti-inflammation,antioxidant,cardiovascular disease prevention and cancer-combating.And PMFs have been a focus of many scholars because of its good oral administration and utilization efficiency compared with flavanones.However,as isomery exist widely in PMFs,the preparation of polymethoxylated flavone（PMF）was sometimes limited by numerous problems,for instance,the complexity of separation,deficiency of purification and impurity,and environmental pollution.Therefore,it is the key to future studies on PMFs in Citrus plants and to utilize resources of landraces is to clarify the component and content of PMFs in Citrus landraces and to develop efficient and green preparation technologies.Fatigue makes the organisms in sub health state and has been proven to be a health hazard by decreasing the immunity,disordering the endocrine and nervous system and causing cardiovascular diseases.Mitochondria are the main organelle that produces cellular energy and reactive oxygen species.Improvement in optimal mitochondrial activity is essential to not only the stability of energy supply but the endurance of the oxidant,which makes mitochondria an important target for anti-fatigue studies.Recent researches have found that by activating the expression of some proteins in muscle tissues,natural phenolic substances can improve the mitochondrial activity and,therefore,relief the physical fatigue,whereas such functions in PMFs has not been reported to date.It has been proven that the mixture of PMFs in Citrus peel regulates energy metabolism indicating a potential of improvement on mitochondrial activity,according to the latest results in our lab.We hypothesized that the PMF relief the physical fatigue by regulating mitochondrial activity and improving the function of skeletal muscle based on literature and our former studies.Aims:1)By analyzing the component and the content of PMFs in different Citrus species and landraces peel,this work aimed to provide information of Citrus resources and evidences for selecting materials of PMFs preparation;2)By developing efficient and green separation and purification methods of PMFs in Citrus peel,this work aimed to set up new field of Citrus peel utilization and to provide guidelines to large-scale production of Citrus PMFs;3)By screening PMFs in vitro and investigating its pharmacodynamics in vivo,this work aimed to find new molecular targets for functional studies and to provide experimental evidences to anti-fatigue function of PMFs.Methods and results:1.Detection and analysis of the component and the content of PMFs in 13 landracesMethods:Using the UPLC-PDA method,we detected the component and the content of PMFs in the peel of 13 Citrus materials,including mandarin,oranges,pomelo,lemons and citron.Results:In terms of the component of PMFs,Nianju（NJ）,Shantoujiaogan（JG）,Yanxiwanlu（WL）and Dahongpao（DHP）had the richest PMFs containing all the components used in the standard curve.Followed by Jincheng S26（JC）,Pingxiangtuningmeng（TNM）and Limeng（LM）which had 8 components of PMFs.When it comes to the content of PMFs,DHP has the highest PMFs content of283.40±13.20（mg/100g FW）,followed by WL（233.40±9.74 mg/100g FW）,JG（222.35±20.65 mg/100g FW）,NJ（191.64±29.60 mg/100g FW）and LM（69.30±8.02 mg/100g FW）.From the point of flavanones,Limeng（LM,778.00±22.54mg/100g FW）,Dahongpao（DHP,617.93±82.04 mg/100g FW）,Gailiangcheng（GLC,585.10±115.67 mg/100g FW）,Yanxiwanlu（WL,517.42±28.15 mg/100g FW）and Fengjieqicheng（QC,464.01±53.93 mg/100g FW）contained the richest component and content of flavanones.2.Separation,purification and identification of the PMFs in DHP peelMethods:Macroporous adsorptive resins combined with MS Directed prep-HPLC was used to purificate and separate PMFs at one time.The PMFs were analyzed and identified by UPLC-UV-NMR-MS detection.Results:Among the 7 resin materials which were commonly used to absorb flavonoids,HPD 100 showed the best performance on absorption and separation that could efficiently absorb or separate the PMFs in Dahongpao peel.In the present study,the absorption process of PMFs in HPD100 fit the pseudo-second order model and first particle diffusion kinetics model.The mechanism of the absorption was that PMFs transferred from the edge of the liquid membrane to the surfaces of HPD 100 followed by an exchange of electrons between PMFs and HPD 100 leading to absorption.Absorption was exothermic reaction and was proven to fit the Langmuir model indicating that lower temperature benefit the absorption of PMFs.The key conditions（loading concentration,loading flow rate,elution liquid concentration and elution flow rate）of HPD 100 dynamic absorption were optimized.The best performance on absorption was observed when samples loaded at a concentration of 8.5 mg/mL and loading flow rate at a speed of 4 BV,while eluting at4.0 BV/h with 90%ethanol sufficiently improved the efficiency of separation.The concentration of PMFs improved by 11.43 fold after HPD 100 adsorption.Five PMFs were separated and purified by MS Directed prep-HPLC.The PMFs were identified as Sinensetin(C₂₀H₂₀O₇,molecular weight 372.12,purity 97.4%),5,6,7,4’-Tetramethoxy flavone(C₁₉H₁₈O₆,molecular weidght 342.11,purity 95.6%),Nobiletin(C₂₁H₂₂O₈,molecular weight 402.13,purity 99.1%),Tangeretin(C₂₀H₂₀O₇,molecular weight372.12,purity 98.6%)and 5-hydroxyl-6,7,8,3’,4’-Pentamethoxy flavone(C₂₀H₂₀O₈,molecular weight 388.11,purity 97.8%).3.The DHP tangerine PMFs screening of anti-fatigue efficiency in vitro and pharmacodynamic test in vivoMethods:The C2C12 myotube model was established to screen PMFs depending on their effects of cell viability and mitochondrial regulatory activity,and then mechanism was investigated in PMF screened.Followed by experiments in vivo,the anti-fatigue function of the PMF was evaluated and investigated by behavioral experiments,biochemical marks,and transmission electron microscope detection.Results:C2C12 cells were cultured in 3%horse plasma medium and formed basic morphological structures of mature myotubular cells at Day 6.The symbolic metabolites,myosin heavy chain and myogenin,were highly expressed,indicating that the model was successfully built.The results of CCK-8 showed that a 6-hour treatment of Nobiletin,Sinensetin,Tageretin and 5-hydroxyl-6,7,8,3’,4’-Pentamethoxy flavone did not significantly inhibited the cellular activities with a concentation ranged 0-200μmol/L.5,6,7,4’-Tetramethoxy flavone did not significantly inhibited the cellular activities at a concentation of 100μmol/L.Nobiletin and Tageretin were proved to upregulate the expression of TFAM,an important regulator of mitochondrial biogenesis,and to active the symbolic molecular of mitochondial biogenesis,mtDNA.Moreover,Tangeretin showed a better performance on the activity regulation of mitochondrial biogenesis camepared with nobiletin.By investigating the mechanisms,Tangeretin probably changed the levels of cellular energy by adjusting the rate of AMP/ATP,leading to the enhancement of the phosphorylation of Ser428 site in kinase LKB1 and the activation of AMPK-PGC1-α-NRF1-TFAM signaling pathway.Animal experiments witnessed that the 4-week tangeretin preventable treatment could markedly increase time of hanging wire and run to fatigue,enhance the storage of muscle glucogen,and decrease the level of plasma lactic acid.Transmission electron microscope detection showed a robust improvement on the mitochondria number and volume in muscle tissue,While WB and IHC further validated that the mitochondrial biogenesis signaling pathways in mice muscles had been activated.Conclusions:1)Among all 13 Citrus materials selected in the experiments,Dahongpao had the richest component and the highest content of PMFs（283.40±13.20mg/100g FW）,making the cultivar an ideal material for PMFs preparation;2)Macroporous resin HPD 100 assisted with MS Directed prep-HPLC could green and efficiently separated and purified the PMFs in Dahongpao peel.The 5 PMFs were identified as Sinensetin,Nobiletin,Tageretin,5-hydroxyl-6,7,8,3’,4’-Pentamethoxy flavone and 5,6,7,4’-Tetramethoxy flavone.Each of the PMF had a purity of over 95%;3)PMF tangeretin could enhance mitochondrial biogenesis in skeletal muscle and relif the physical fatigue by activating AMPK-PGC1-α-NRF1-TFAM signaling pathway.