Screening Of Anti-inflammatory Activity And Mechanism Of Action Of Fermented Forsythia Leaf Extract

Forsythia leaves are the leaves of Forsythia suspensa(Thunb.)Vahl,plant of the family Myrtaceae,which is recorded in the Chinese Materia Medica as "Forsythia stems and leaves,cool in nature and bitter in taste,clearing heat and detoxifying the toxins,belonging to the heart and lung meridians,and treating heat accumulation in the heart and lung".Most of the non-medicinal parts of forsythia leaves are used as forsythia tea,but most of them are not effectively utilized,resulting in a great waste of resources,so we need to exploit forsythia leaves efficiently and reasonably.Forsythia leaves have various pharmacological activities,such as anti-inflammatory,hepatoprotective and antioxidant.The group found that the chemical composition of forsythia leaves changed significantly after fermentation,and the types and contents of lignin sapogenins increased;the lignin sapogenins-rich extracts were prepared by macroporous adsorption and purification technology,and the main lignin sapogenins,forsythia lipocalin,were obtained by further preparation;animal experiments demonstrated that The animal experiments demonstrated that lignan has regulatory and ameliorative effects on ulcerative colitis and acute lung injury mice inflammation models.After literature research,no studies related to the anti-inflammatory activity and mechanism of action of fermented forsythia leaf extracts rich in lignan sapogenins and other major lignan sapogenins in the extracts have been reported so far.In this paper,based on the work of our group,we screened fermented forsythia leaf extracts and evaluated the anti-inflammatory activity of the main lignan sapogenins in the extracts using the LPS-induced RAW264.7 cell inflammation model,and conducted a preliminary study of the anti-inflammatory mechanism.Details are as follows.1.Optimization of inflammation model conditions,screening of fermented forsythia leaf extract activity and evaluation of anti-inflammatory activity of major lignan sapogeninsFirstly,the cell seed plate density and LPS modeling concentration were optimized,and the cell viability was detected by CCK-8 method to determine the safe concentrations of F40,F50,F80 and F95 extracts of fermented forsythia leaves and the main lignan sapogenins in the extracts acting on the cells;Using the Griess method to detect the effect of the above samples on the release of NO,and this was used as an indicator for the preliminary screening of the best anti-inflammatory active sites and lignan monomer components.The effect of the extract and lignan sapogenins on the secretion of inflammatory factors in RAW264.7 cells was examined by ELISA to further confirm the anti-inflammatory activity.Increased NO release with increasing cell seed plate density;cell viability was not affected when LPS concentration was ≤10μg/ml,Extracts in the concentration range of 1.5625 to 100μg/m L and four lignan components concentration was≤100μg/m L;RAW264.7 cells stimulated by LPS promoted a significant increase of NO content,but by extracts(1.5625-100μg/m L)and four lignan components(1.5625-100μg/m L)intervention significantly reduced NO secretion.ELISA experiments further demonstrated that the four lignan components and F80 extract significantly reduced the secretion of TNF-α、IL-6 and IL-1β.The above results indicate that the fermented forsythia leaf extract and the main lignan sapogenins components can inhibit NO、TNF-α、IL-6 and IL-1β expression,and have a protective effect on inflammation-damaged cells.2.Effects of F80 extract and pinealin on TLR4-NF-κB/MAPKs signaling pathway in RAW264.7 cells.The effects of F80 extract and pinoresinol(25,50and100μg/ml)on TLR4-NF-κB/MAPKs inflammatory signaling pathway were investigated by Western blot,RT-q PCR and immunofluorescence techniques to determine the pathways through which F80 extract and pinealin exert their anti-inflammatory effects;in addition,NF-κB/MAPKs pathway inhibitors BAY11-7082,SP600125,PD98059 were added to detect the signaling pathway The expression of IκBα,p65,JNK,and ERK,which are related proteins in the signaling pathway,was examined.Combined with RT-q PCR and immunofluorescence experiments,the mechanisms of protective effects of extracts and pinoresinol on inflammatory damage of RAW264.7 cells were revealed.This suggests that LPS can increase the expression of the above protein content,and that F80 extract and pinoresinol intervention can inhibit the upregulation of the above phosphorylated proteins with similar effects as inhibitors;meanwhile,the intervention of F80 extract and pinoresinol could inhibit the expression of p65 m RNA and hinder the nucleation of p65,thus exerting anti-inflammatory effects.