Preparation of iristectorin A and iristectorin B and their anti-inflammatory mechanism in vitro
Objective:To isolate and prepare high-purity iristectorin A and iristectorin B from Belamcanda chinensis,and explore their anti-inflammatory activity and mechanism in vitro.Methods:The iristectorin A and iristectorin B monomers were isolated and purified from Belamcanda chinensis by polyamide column chromatography-recrystallization method.The effects of iristectorin A and iristectorin B on acetylcholinesterase(AChE)activity were determined by modified Ellman colorimetric method.The effects of different concentrations of iristectorin A and iristectorin B on cell viability were determined by CCK-8 method,and the range of administration was determined.Lipopolysaccharide(LPS)-induced mouse RAW264.7 macrophages were used as an inflammatory model.The content of nitric oxide(NO)in the cell supernatant was detected by Griess method,and the contents of tumor necrosis factor-α(TNF-α),interleukin-1β(IL-1β)and IL-6 were detected by ELISA.The protein expressions of cyclooxygenase-2(COX-2),inducible nitric oxide synthase(iNOS)and α7 nicotinic acetylcholine receptor(α7nAChR)were detected by Western blot.Results:Polyamide showed good separation and purification effect for iristectorin A and iristectorin B,and iristectorin A and iristectorin B monomers were obtained by recrystallization,and their purity was greater than 98％as calculated by HPLC peak area normalization method.Iristectorin A and iristectorin B had certain inhibitory effect on acetylcholinesterase;iristectorin A and iristectorin B had no inhibitory effect on RAW 264.7 cells in the range of 12.5～200 μg·mL-1.Compared with LPS model group,iristectorin A and iristectorin B could inhibit the release of NO,TNF-α,IL-1β and IL-6 in cells in a dose-dependent manner(P＜0.05),up-regulate the expression of α7nAChR protein in cholinergic anti-inflammatory pathway(P＜0.05),and down-regulate the expression of COX-2 protein in arachidonic acid pathway and iNOS protein in NO pathway(P＜0.05).Conclusion:Iristectorin A and iristectorin B have certain anti-inflammatory activity in vitro.The anti-inflammatory mechanism is to reduce the release of inflammatory mediators such as NO,TNF-α,IL-1β and IL-6 in macrophages by regulating cholinergic anti-inflammatory pathway,arachidonic acid metabolic pathway and NO pathway,so as to achieve multi-channel anti-inflammatory effect.
iristectorin Airistectorin Bpolyamide column chromatography-recrystallizationacetylcho-linesteraseanti-inflammatory in vitroRAW264.7 cells
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