Optimization Of The Synthesis Of Quinalizarin And Its Mechanism Of Inducing Apoptosis Of Melanoma A375 Cells

Malignant melanoma is a highly dangerous skin tumor,which has the characteristics of poor prognosis,easy metastasis and invasion,also seriously threatens human life and health.Relevant studies have shown that advanced melanoma is resistant to most chemotherapeutic drugs.Therefore,it is particularly important to find effective drugs to treat melanoma.Quinaliarzin is an anthraquinone compound extracted from the plants of the family Rubiaceae,and It has anti-inflammatory and anti-tumor effects.It is considered to be an effective and highly selective drug for cell membrane permeability.However,the amount of quinaliarzin extracted from plants is very low.In this study,we hope to improve the synthetic yield of quinaliarzin by optimizing the reaction time,reaction temperature and substrate molar ratio in the synthetic quinaliarzin reaction.By verifying its biological activity,to explore the mechanism of action of quinaliarzin on human melanoma A375 cells,and provide a theoretical basis for melanoma treatment and new drug development.In this experiment,1,4-dihydroxyanthraquinone was used as substrate to prepare quinaliarzin in the presence of metaboric acid and 98% concentrated phosphoric acid by redox reaction.Through optimized reaction conditions,when the reaction time was 2.1 h,the reaction temperature was 221? and the molar ratio of substrates was 1.2:1.4:1.6,the yield of quinaliarzin reached 96.24%.At the same time,the relative molecular weight of the product was examined by mass spectrometry,and the relative molecular mass of the product was the same as that of quinaliarzin.The cost of synthesis was reduced by using the optimized synthesis process.MTT assay was used to detect the inhibitory effect of quinaliarzin on proliferation of human melanoma A375 cells and the results showed that quinaliarzin inhibited the proliferation of human melanoma A375 cells in a concentration-dependent manner,which was significantly different from the positive control drug 5-FU.Annexin V-FITL/PI double staining was used to observe the morphology of apoptotic cells by fluorescence microscopy,as the quinaliarzin treatment time prolongs the fluorescence intensity of apoptosis and necrosis increases.Meanwhile,flow cytometry and western blotting are used to detect apoptosis.The apoptotic rate is increasing in a time-dependent manner by inhibiting the expression of anti-apoptotic protein Bcl-2 and activating the expression levels of apoptotic proteins Bad,caspase-3 and PARP.The cell cycledistribution of quinaliarzin-induced melanoma A375 was detected by flow cytometry,the results show that quinaliarzin triggered cell cycle arrest in G2/M phase in melanoma A375 cells by inhibiting cyclin A and cyclin B expression and activating CDK1/2,p21 and p27 protein expression levels;quinaliarzin could up-regulate the expression levels of p-JNK,p-p38 and I-?B,down-regulate the expression levels of p-AKT,p-ERK,p-STAT3 and NF-?B;The effects of quinaliarzin on reactive oxygen species(ROS)in melanoma A375 cells were detected by flow cytometry and Western blotting.The results showed that quinaliarzin accelerated the production of intracellular ROS,and ROS scavenger(NAC)inhibited the production of ROS and cell apoptosis by promoting the expression levels of p-AKT,p-ERK,p-STAT3,NF-?B and Bcl-2,and inhibited the expression levels of p-JNK,p-p38,I-?B,Bad,caspase-3 and PARP.The relationship between MAPK and STAT3 in quinaliarzin-induced apoptosis was detected by Western blot and the results showed that STAT3 was regulated by MAPK in quinaliarzin-induced apoptosis of melanoma A375 cells.After optimizing the reaction time,temperature and molar ratio of substrates,the yield of quinaliarzin was increased and the cost of synthesis was reduced.Quinaliarzin can inhibit proliferation and induce apoptosis of melanoma A375 cells by inhibiting cell cycle arrest at G2/M phase and promoting ROS-mediated AKT,MAPK,STAT3 and NF-?B signaling pathways.