The Research On Some Extraction,Purification And Anti-canceer Activity Of Polyphenols From Lactarius Hatsudake

Lactarius hatsudake Tanaka is a world-famous delicious wild edible mushroom.Lactarius hatsudake Tanaka is distributed in Anhui,Jilin,Liaoning,Jiangsu,Fujian,Guangdong,Guangxi,Sichuan,Yunnan,Guizhou and Tibet.It was born on the ground of pine forest in early summer and autumn.It can be processed into mushroom oil or preserved for a long time after quick freezing,which have a market prospect for large-scale development.In this paper,the ultrasonic-assisted extraction of polyphenols from Lactarius hatsudake Tanaka was studied and optimized by using Lactarius hatsudake Tanaka as raw material.The anticancer activity of polyphenols from Lactarius hatsudake Tanaka was studied in vitro.In the process optimization of ultrasonic-assisted extraction of Lactarius hatsudake Tanaka,the response surface was optimized with the total phenol content as the response value,and the factors such as solvent type,solvent concentration,material-liquid ratio,ultrasonic time and extraction times were selected.The fitting equation Y = 4.83+0.13*A+0.30*B-0.037*C-0.089*A*B+0.037*A*C-0.11*B*C-0.54*A2-0.16*B2(A,B,C are time,material-liquid ratio,solvent concentration respectively).The effects of three factors on the extraction of polyphenols were material-liquid ratio>extraction time>extraction concentration.By solving the equation and predicting the equation,and combining with the experimental parameter setting requirements,the optimum technological conditions for ultrasonic-assisted extraction of Lactarius hatsudake Tanaka were obtained:ethanol concentration 56%,solid-liquid ratio 1:25(g/mL),extraction time 80 min,extraction twice.The error between the experimental results and the optimized results is 1.05%.Composition of polyphenols in Lactarius hatsudake Tanaka:Purchaing the polyphenols which may be contained in Lactarius hatsudake Tanaka were found by reading references.The purified polyphenols of Lactarius hatsudake Tanaka were analyzed by high-performance liquid at flow rate of 1 ml/min.Solvent A:0.1%formic acid,B:acetonitrile,the elution conditions:0-2min,8-10%acetonitrile;2-10min,10-20%acetonitrile;10-30min,20-80%acetonitrile;30-31min,80-100%acetonitrile;31-40min,100%acetonitrile;40-43min,100-8%acetonitrile.Chlorogenic acid,ferulic acid,gallic acid,pyrogallol,cinnamic acid,myricetin,p-hydroxybenzoic acid,homogentisate and seven other unknown substances were found in Lactarius hatsudake Tanaka.Anti-cancer experiments in vitro showed that the purified polyphenols from Lactarius hatsudake Tanaka could inhibit HCT116 colorectal cancer cells at a lower concentration.Polyphenols can inhibit the proliferation of colorectal cancer cells,and the inhibition mechanism is:Lactarius hatsudake Tanaka polyphenols purified ?MAPK signaling pathway inhibited?AP-1 and NF-?B activity decreased ?cyclin D1 expression,increased CDKI expression?inhibited cell cycle inhibited proliferation of HCT116 cells;polyphenols can also promote apoptosis of colorectal cancer cells.It was speculated that the inhibition mechanism was as follows:the polyphenol purified from Lactarius hatsudake Tanaka?the expression of Bcl-2 decreased,the expression of Bax increased,the ratio of Bcl-2/Bax decreased ?the activation of Caspase-3?the apoptosis of HCT116 cells.