Separation,Purification And Functional Study Of Catechins Polymers MAF

Mitochondrial activation factors(MAF)are a series of catechins polymers that isolated from black tea,which is named for its ability to enhance the potential gradient across the inner membrane of mitochondria in Tetrahymena.Subsequent in-vivo studies have shown that MAF administration can cause skeletal muscle hypertrophy through increasing the protein synthesis in skeletal muscle fibers.Besides,MAF administration could improve the endurance of skeletal muscle in mice under endurance training,performing a certain auxiliary effect during exercise training.These studies suggest that MAF could be used as a supplement to improve skeletal muscle status.However,only few studies have focus on MAF for now,resulting in limited knowledge on the preparation process,structure analysis and biological function of MAF.Therefore,this study based on the improvement of the existing laboratory preparation process of MAF and applied a series of chromatographic and mass spectrometry analysis techniques to analyze the structure of MAF.Meanwhile,through a label-free quantitative proteomic analysis,the effects of MAF administration on mice skeletal muscle with and without exercise training were firstly studied under protein level.The biological functions of MAF were validated and possible mechanisms were illustrated,providing theoretical basis for the future study and application of MAF.The main findings and conclusions are as follows:1.The laboratory preparation method of MAF was simplified.By changing the gradient elution during the column chromatography process to a two-step isometric elution,elution components could be determined more easily according to the volume of eluent.This modification reduced the technical requirements in the preparation process and increased the repeatability of MAF preparation experiment.2.Through the application of size exclusion chromatography,high performance liquid chromatography,matrix-assisted laser desorption ionization time of flight mass spectroscopy,ultra-performance liquid chromatography-electrospray ionization-triple quadrupole mass spectroscopy,and ultra-performance liquid chromatography-electrospray ionizationquadrupole-time of flight mass spectroscopy,the main components of MAF-1 and MAF-2fraction were firstly studied and illustrated.The main component of MAF-1 fraction should be isoquercitrin or its epimers.The molecular weight of the main component in MAF-2fraction should be 888,and quercetin or its derivatives might participate in the formation of this class of polymers.3.The effects of MAF administration on the skeletal muscle of mice under different exercise conditions were studied through an 8-week swimming training schedule.Under normal conditions,100 mg/kg/d MAF administration increased the body weight,gastrocnemius muscle mass,and the content of structural proteins in the muscle fiber,resulting in the hypertrophy of skeletal muscle in ICR mice.By contrast,MAF administration combined with exercise training resulted in increased energy consumption,improved serum glucose and lipid status,and thus the least body weight.These results are consistent with published studies and confirm the biological function of MAF in-vivo.4.Label-free quantitative proteomics analysis showed that MAF administration can cause skeletal muscle hypertrophy through reducing glucose and fatty acids catabolism and improve actin and myosin anabolism in muscle fibers under normal conditions.Combined with exercise training,MAF administration significantly increased the expression of mitochondrial proteins,especially the expression of pyruvate dehydrogenase and succinate dehydrogenase in muscle fibers,resulting in elevated efficiency in the citrate cycle and electron transport chain and thus more ATP production,contributing to the adaptation to exercise training.5.The different biological functions of MAF in the skeletal muscle of mice under different exercise states are probably realized by the increased expression of the 5-AMPactivated protein kinase(AMPK)catalytic subunit alpha.Without extra exercise,the expression of AMPK subunit increased by MAF administration,but the energy state in muscle tissue was normal and thus the AMPK signaling pathway was not activated,resulting in normal catabolism efficiency.By contrast,exercise training resulted in extra consumption of ATP and thus the activation of AMPK,resulting in the activation of downstream glucose and fatty acid catabolism and further increase the efficiency of the citrate cycle and electron transport chain to produce more ATP for the compensation of energy consumption.This process could finally lead to the lower body weight and better serum glucose and lipid status in the exercised mice.