Extraction Of Total Flavonoids From Mulberry Leaves And Anti-cancerous Potential Of Morin

National Ministry of health has identified the Mulberry leaves as important plant material for their use in medicine and functional foods.In our study,taking mulberry leaves as raw materials,the optimum extraction technology and antioxidant activity of flavonoids from mulberry leaves were investigated.Meanwhile,we used the polyamide column chromatography to separate and purify of Morin and also study its antitumor activity and molecular mechanism on cervical cancer He la cells in vitro.The present research work provides theoretical basis and technical support for the comprehensive utilization of mulberry leaf resources.The single factor and response surface optimization method was used to determine the optimum extraction conditions of Flavonoids from mulberry leaves through ultrasonic assisted extraction.The optimum extraction conditions used were as follow:Ethanol concentration of 75%,liquid-solid ratio of 25:1,extraction temperature at 70? and extraction time of 1.5 h.The extraction rate of Flavonoids under this condition was 2.96%.The antioxidant activity of flavonoids from mulberry leaves were tested through reducing power,DPPH radical scavenging ability,ABTS+ radical scavenging activity and Fe2+ chelating ability.These results showed a concentration dependent mode of the antioxidant activity.At the concentration of lmg/mL,the DPPH radical scavenging,ABTS +radical scavenging and Fe2e chelating activities were 93.21%,96.79%and 75.24%,respectively.In our study,98.81%of the purity of Morin was obtained through polyamide column chromatography and the structure of the product was identified by UPLC-TOF-MS/MS and NMR technology.In addition,we further studied the antitumor activity of Morin in Human Cervical Carcinoma Hela cells.The present study indicated that Morin have good anticancer abilities in a concentration-dependent manner in vitro,which inhibited the proliferation of Hela cells at IC50 of 214.28?M;Morin could induce cell cycle arrest in G2/M phase;in addition,Morin could also induce cell apoptosis,after the treatment with 50?M,150?M and 220?M of Morin on Hela cells for 48 h,the apoptotic cells reached up to 22.3%,29.1%and 41.2%,respectively.Morin-induced G2/M arrest was observed in Hela cells through decreased mRNA expression of CDK1,Cdc25c,Survivin,cyclin B1 and CHK2 genes and increased mRNA expression of p53,p21 and Wee 1 genes.Moreover,Morin derived apoptosis was regulated via multiple pathways,including intrinsic and extrinsic pathway.The underlying mechanisms include increased mRNA expression of Bax,Bad,cytochrome c,Apaf-1,caspases-9,DR3,DR5,FasL,FADD,caspases-10,PARP,PI3K,AKT,mTOR,P70S6K and Smac genes;while,decreased expression of Bcl-2,Bcl-xL,AMPK,cIAP-1,cIAP-2,PKC? and NF-k?.In addition,Morin treatment resulted in the generation of intracellular ROS,which play an essential role in the induction of apoptosis.After the treatment with 50?M,150?M and 220?M of Morin,the percentage of ROS reached to 10.27%11.25%and 32.31%,respectively.