The Anti-colon Cancer Active Components And Molecular Mechanism Of Bound Polyphenols From Seabuckthorn Berries

Seabuckthorn,called vinegar willow as well,which produce small orange berries,also be used to treat diseases in addition to food.Studies showed that Hippophae rhamnoides is rich in many kinds and high content of polyphenols,has the functions of anti-oxidation,anti-inflammatory,enhancement of immunity,protecting cardio-cerebrovascular and preventing cancer.As a characteristic resource of Shanxi Province,wild seabuckthorn resources account for more than 70 % in China,but such rich resources are still not fully utilized.According to the data,the morbidity and mortality rate of cancer are increasing year by year,and these in China ranks the first in the world.Studies show that tumor cells often alter energy metabolism to cope with hypoxia and low nutrition microenvironment,and provide a large number of intermediate metabolites to meet the needs of rapid proliferation.Glucose taking in tumor cells is metabolized mainly through aerobic glycolysis,in which the key kinase PKM2 is abnormally high expression.Therefore,the intervention and regulation about PKM2 is an effective strategy for cancer treatment.PKM2 is generated by alternative splicing of PKM m RNA,and in which the splicing factor hn RNP proteins(hn RNPA1?hn RNPA2?PTBP1)are involved.The high expression of these proteins promotes the expression of PKM2 and enhances metabolism and proliferation of cancer cells.Studies have shown that polyphenols from seabuckthorn have significant anti-tumor activity,but the active components and molecular mechanism are still unknown.In this study,Bound polyphenols were extracted,isolated and identified from fresh seabuckthorn fruit,and the specific molecular mechanism of its anti-colon cancer effect through reversing PKM alternative splicing mediated tumor glycolysis was explored.The research contents and results include the following aspects:(1)To explore the polyphenols with significant anti-tumor activity in different parts of seabuckthorn.Different parts of seabuckthorn(fruits,leaves and seeds)were used to obstain polyphenols.Free fraction were extracted by organic reagent and bound fraction were extracted by alkaline hydrolysis in seabuckthorn berries,leaves and seeds,respectively.The results showed that the bound polyphenols from seabuckthorn fruit exhibited significant anti-tumor activity.Then the extraction solvent and hydrolysis method were selected in the process of extracting the polyphenols from seabuckthorn berries.Finally,80% ethanol and alkaline hydrolysis method were determined to obtain free and combined polyphenols of seabuckthorn fruit respectively.(2)To determine the anti-colon cancer effects and the active ingredient in bound polyphenols from seabuckthorn berries.The polyphenols including Erucic acid,Ferulic acid,4-coumaric acid,7-hydroxycoumarin,and flavonoids including Kaempferol,Kaempferol-3-o-?-glucopyranosyl-7-o-?-rhamnoside,Afzelin and 5,7-Dihydroxy-2-(4-hydroxy-3-methoxyphenyl)-4-oxo-4H-chromen-3-yl-6-O-(6-deoxy-?-L-mannopyranosyl)hexopyranoside were separated and identified by LC-MS technique.The consequences of MTT,WB analysis and flow cytometry expriment implied that bound polyphenols from seabuckthorn berries induce apoptosis and G1 arrest,thereby the inhibition of the growth of colon cancer cells.(3)To elucidate the molecular mechanism of bound polyphenols from seabuckthorn berries reversing PKM2-mediated aerobic glycolysis to exert anti-colon cancer effect.The results showed that the bound polyphenols reduce the consumption of glucose,the production of lactic acid and ATP,then affect the glycolysis process.Western blot was used to detect the expression of PKM1,PKM2 and splicing factors hn RNPA1,hn RNPA2,and PTBP1,the expression of PKM1 was increased,and the expression of PKM2 and splicing factor PTBP1 were significantly decreased,indicating that the alternative splicing of PKM was regulated by bound polyphenol from seabuckthorn berries.mi RNAs are involved in the regulation of more than one-third of protein-coding genes in vivo,including PTBP1.Subsequently,q PCR was used to compare the expression of mi RNAs upstream of PTBP1 in colon cancer cells treated with or without bound polyphenols from berries,and increased levels of mi R-339-5p were found in treated colon cancer cells.Furthermore,its mimic and inhibitor were transfected into colon cancer cells,it was indicated that the expression level of mi R-339-5p was negatively correlated with the expression of PKM2 and PTBP1.Finally,the dual-luciferase reporter assay disclosed that mi R-339-5p was directly bound to the 3'UTR region of PTBP1,thereby inhibiting the expression of PKM2.In conclusion,in this study,the antitumor active ingredients in bound polyphenols from seabuckthorn fruit were separated and identified,and the molecular mechanism of its anti-tumor activity was elucidated by remodeling tumor glucose metabolism through mi R-339-5p/PTBP1/PKM2 axis,which lays a theoretical foundation for making full use of seabuckthorn resources and provides new ideas for the intervention of PKM2 involved in the metabolic regulation and treatment of cancer.