Isolation And Identification Of Toxic Ingredients From Lycoris Radiata And Their Antitumor And Anti-inflammatory Activities

Lycoris radiata belongs to lycoris herb,it's also called as Higanbana,Crow garlic,Black garlic and Lycoris radiate.Most of them grow in damp mountain or jungles of China east,south and southwest.According to the "Compendium of Materia Medica" and other records Lycoris radiata has functions of detoxification,diuresis and vomiting,but also have a slight toxicity.It has many pharmacological activities which is a rich traditional Chinese medicine resource.The main chemical components of Lycoris radiata are alkaloids,flavonoids,carbohydrates,lectins and trace elements.At present,domestic and foreign scholars mainly studied the pharmacological activities of alkaloids from Lycoris radiata,such as anticancer,antitumor,antibacterial,antiviral,antimalarial effects etc.However,some studies have shown that some alkaloids of Lycoris radiata have toxic to head and neck squamous cells.Therefore,it is necessary to find toxic components and study the good effect of toxic components in their safe range.The total extract was obtained by the extraction of heating reflux with 75% ethanol and then portioned with petroleum ether,ethyl acetate,n-butanol and water,which yielded petroleum ether fraction,ethyl acetate fraction,n-butanol fraction,and water fraction,respectively.Using MTT method to explore whether the 4 polar sites(12.5~400?g/m L)were toxic to human normal liver cells,and then studied inhibitory effect on tumor cells(HepG2 cells and HT-29 cells)in a safe concentration range(1~8?g/m L)and bacteriostatic effects on Pathogenic Bacterias(Staphylococcus aureus and Escherichia coli.)in a safe concentration range(1.25~401~8?g/mL).The results showed that four polar parts of Lycoris radiate had strong cytotoxicity to L02 cells,the median lethal concentration(IC50)of ethyl acetate,n-butanol and petroleum ether to L02 cells were 24.19,31.31 and 51.87? g/m L,respectively.The results also showed that there was a certain inhibitory effect on tumor cells and pathogenic bacteria in safe concentration range.In summary,the toxic components of Lycoris radiata are mainly concentrated in the ethyl acetate and n-butanol parts,so the ethyl acetate and n-butanol fractions were selected for further separation and purification.Two fractions were separated and purified by various column chromatographies(including silica gel column,Sephadex LH-20 gel column and ODS column)and the prepared HPLC.By using the method of toxicity screening three compounds were obtained and identified by mass spectrometry and nuclear magnetic resonance as?(+)-Narciclasine(NCS),?: 7-deoxynarciclasine,?: Dibutylphthalate,respectively.Discussing(+)-Narciclasine(NCS)and 7-deoxynarciclasine cytotoxicity to L02 cells and HFF-1 cells.And then researching the inhibitory effect of compounds on tumor cells(HepG2 cells and HT-29 cells)in a safe concentration.The experimental results,24 hours later,showed that the median lethal concentration(IC50)of(+)-Narciclasine(NCS)and 7-deoxynarciclasine to L02 cells was about 0.384?g/m L and 11.672?g/m L,respectively.These two compounds can also cause the apoptosis of L02 cells in the process of inhibiting the proliferation of tumor cells.Establishing macrophage inflammatory model in RAW264.7 mice induced by LPS.Then using RT-PCR and Weston-Blot to detect the expression of inflammatory cytokines,mRNA and related protein release effected of NCS in the model.To study the anti-inflammatory effect of NCS in the safe concentration range(0.00125~0.005?g/m L)and the possible anti-inflammatory pathway and mechanism.The results showed that NCS has a significant anti-inflammatory effect.The anti-inflammatory mechanism is relate to reduce the release of inflammatory cytokines NO,IL-6 and the TNF-?,depress the iNOS,IL-6 and TNF-? mRNA transcription and translation,reduce expression of protein phosphorylation of P38,COX-2,P65,Cytosol p-I?B?/?.