Studies On The Biological Activity Of A Traditional Mongolian Medicine Veratrum Nigrum

Objective: To study the anti-inflammatory activity and anti-tumor activity of Mongolian medicine medicine herb Veratrum nigrum,and to investigate its antiinflammatory mechanism.By studying the anti-inflammatory activity,mechanism of action and anti-tumor activity of its monomeric compounds,we searched for new bioactive compounds in Veratrum nigrum,which provided references for the development of new drugs for Mongolian medicine.Methods and results:(1)Anti-inflammatory activity assay: effect on LPS-induced RAW 264.7 macrophage proliferation: CCK8 method was used to study whether it has cytotoxic effect on the model.The results showed that the concentration was in the range of 0-200 ?mol/L.At the time,it was not toxic to RAW264.7 cells.The Griess method was used to detect the effect of NO on the cell culture supernatant.The results showed that all the components except Methyl 4-coumarate(6)and 3,4-dimethoxybenzoic acid methyl ester(9)had significant NO inhibition.The levels of TNF-?,IL-1? and IL-6 inflammatory cytokines in cell culture supernatants were determined by ELISA.The results showed that 6-hydroxy-3',5',7-triimethoxy-2-arylbenzofuran(1),4,6-dimethoxy-3'-hydroxy-2-arylbenzofuran-5'-O-?-Dglucopyranoside(2),6-hydroxy-3',5'-dimethoxy-2-arylbenzofuran-4-O-?-Dglucopyranoside(3),Moracin M(4),Methyl 4-coumarate(6),Ferulic acid methyl ester(7),4-methyl-?-D-glucoside(8),Oxyresveratrol(11)can significantly down-regulate the level of some cytokines.(2)Anti-inflammatory mechanism study: Real-time PCR was used to detect the mRNA changes of related genes iNOS,TNF-?,IL-1? and IL-6 after stimulation of macrophages by monomeric 6-hydroxy-3',5',7-triimethoxy-2-arylbenzofuran(1),Methyl 4-coumarate(6)and Oxyresveratrol(11).After 6-hydroxy-3',5',7-triimethoxy-2-arylbenzofuran(1)treatment,the expression levels of iNOS and TNF-? mRNA were significantly decreased.After treatment with Methyl 4-coumarate(6),the expression of IL-1? mRNA was significantly decreased.There was no change in the expression level;after treatment with the Oxyresveratrol(11),the expression levels of iNOS,TNF-? and IL-6 mRNA were significantly decreased.Western blot was used to detect the changes of P65,phospho-ERK(1/2)and ERK(1/2)expression in NF-?B and MAPK signaling pathways after stimulation of macrophages by macromolecules 6-hydroxy-3',5',7-triimethoxy-2-arylbenzofuran(1),Methyl 4-coumarate(6),and Oxyresveratrol(11).The results showed that the expression of P65 and phospho-ERK(1/2)protein was significantly decreased after treatment with 6-hydroxy-3',5',7-triimethoxy-2-arylbenzofuran(1).The expression of P65 and ERK(1/2)protein after treatment with Methyl 4-coumarate(6)Significantly decreased;P65,phospho-ERK(1/2)and ERK(1/2)protein expression levels were significantly decreased after treatment with Oxyresveratrol(11).(3)Anti-tumor activity test: 9 monomeric compounds(1-9)were isolated by CCK-8 cell staining for 5 common human solid tumor cell lines(human lung cancer cell line A549,human colorectal cancer cell line)Growth inhibition of HCT-15,human neuroblastoma cell line SHSY5 Y,human breast cancer cell line MCF-7,and human hepatoma cell line HepG2.Among them,the chloroform layer,the ethyl acetate layer and the monomer component 6-hydroxy-3',5',7-trimethoxy-2-arylbenzofuran(1),Moracin M(4),Moracin A(5)has moderate to significant cytotoxic activity against some tumor cell lines,IC50 values range from 30-200?M,and the remaining fractions and monomer components have no obvious cytotoxic activity against the five cancer cell lines tested.IC Value > 200 ?M.Conclusions: The screening of anti-inflammatory and anti-tumor activities of Veratrum nigrum stilbene monomer compounds in vitro,anti-inflammatory activity detection and anti-inflammatory mechanism research results show that 6-hydroxy-3',5',7-triimethoxy-2-arylbenzofuran(1),Methyl 4-coumarate(6)and Oxyresveratrol(11)have significant resistance Inflammatory activity and inhibition by NF-?B and MAPK signaling pathways.The anti-tumor activity test results showed that the chloroform layer,the ethyl acetate layer and the monomer component 6-hydroxy-3',5',7-trimethoxy-2-arylbenzofuran(1),Moracin M(4),Moracin A(5)has moderate to significant anti-tumor activity against some tumor cell lines.