Study On Inhibition Of Neurogenic Inflammatory Substance Basis And Mechanism Of N-hexane Extract From Amillariella Mellea

Armillaria mellea belongs to the Agaricales,Armillaria,has been used to treatment of nervous system diseases,such as headache,epilepsy,convulsion.The inflammatory reaction and the pathogenesis of these diseases may be related to the nervous system suggest that Armillaria may have certain anti-inflammatory activity.The previous study from our labortory found that n-hexane and ethyl acetate extracts from Armillaria mellea fermentation product had anti-inflammatory activity.In this study,firstly,Parkinson syndrome(PD)mice model induced by MPTP was used to evaluate the ant-inflammatory effect of n-hexane extract from A.mellea fermentation products(Hex-AM).Then lipopolysaccharide(LPS)induced BV-2 microglia was used todo bioacrivity guided fractionation of the Hex-AM.We also explored the anti-inflammatory mechanism and chemical composition of the active components.The main results are shown as follows:We extracted A.mellea fermentation product with n-hexane,concentrated to dryness to get Hex-AM.We used MPTP induced C57BL/6 mice to establish PD in vivo model to evaluate the anti-inflammatory effect of Hex-AM.The results showed that PD mices fed with Hex-AM(10 and 20mg/kg/d)pole climbing time can significantly shortened pole climbing time compared with that of the model mice.They also increased the number of the substantia nigra pars compacta(increased by 22% and 32% compared to the model group),reduced the number of activated microglia and reduced the activity of i NOS.These data suggest that Hex-AM can inhibit the occurrence of neurogenic inflammation of microglia in PD.The further separation of Hex-AM was based on the anti-inflammatory activity.First of all,we determined the petroleum ether ethyl acetate as the mobile phase to separate Hex-AM by thin layer chromatography.Hex-AM was separated by silica gel column chromatography(petroleum-ether ethyl acetate as eluent: 100:0~0:100)to obtain six components Fr.1~Fr.6.We screened the anti-inflammatory activity of Fr.1~Fr.6 and the residual phase using LPS induced BV-2 microglia inflammatory model.The results showed that Fr.3,Fr.5 and Fr.6 could inhibit the secretion of NO in the cell mediums dose-dependently.Then,Fr.3,Fr.5 and Fr.6 were further separated to obtain Fr.3-1,Fr.5-1 and Fr.6-1 by using 300-400 silica gel column,and the characteristic fingerprint was determined by HPLC.Finally,we found Fr.5-1 showed the highest anti-inflammatory activity among the three fractions,which inhibited LPS-induced NO production in BV-2 cells.We performed Fr.5-1 chemical composition analysis and explored the anti-inflammatory mechanisms.We found that the main compounds in Fr.5-1 were lauric acid,palmitic acid,linoleic acid and stearic acid by GC-MS analysis.The main component of Fr.5-1 was daidzein revealed by LC-MS analysis.Four fractions were isolated from Fr.5-1 by preparative liquid chromatography.However,their anti-inflammatory activities were not further increased.We concluded that Fr.5-1 was the smallest unit of anti-inflammatory activity in Hex-AM.We further explored the anti-inflammatory mechanism of Fr.5-1,we found that Fr.5-1 can significantly inhibit the transcription and secretion of proinflammatory cytokines TNF-?,IL-6,IL-10 in BV-2 microglial induced by LPS.The inhibition rates of TNF-?and IL-6 were up to 36.0±0.3% and 55.7±0.6%.In addition,the phosphorylation of NF-?B/p65 was significantly decreased after Fr.5-1 treated in LPS activated BV-2 microglia.These results suggested that Fr.5-1 has anti-inflammation activity by blocking the NF-?B signaling pathway.