Structural Characterization And Functional Evaluation Of Anti-inflammatory Polysaccharide From Cultivated Dendrobium Huoshanense

Dendrobium huoshanense is a traditional Chinese medicinal plant,which is used as a precious tonic for thousands in China.The cultivated D.huoshanense stems were firstly subjected to extraction with different reagents under the guidance of anti-inflammatory activity evaluation.Subsequently,the structural features of polysaccharide with high anti-inflammatory activity were elucidated and its anti-inflammatory mechanism was investigated.(1)With ethanol extraction,water extraction,ethanol precipitation and anion exchange chromatography,five different extracts from D.huoshanense stems were obtained.The evaluation of anti-inflammatory activity based on the inhibition of inflammatory mediator NO release from macrophages showed that distinct activities occurred in five different extracts and the highest activity was from polysaccharide c DHP-W1.(2)The analysis of physicochemical properties showed that c DHP-W1,which consisted of Man and Glc in a molar ratio of 1.00 : 8.20,was a homogeneous polysaccharide fraction with a relative molecular weight of 24 k Da,carbohydrate content of 97.79%,intrinsic viscosity of 0.7192,uronic acid content of 1.4% and acetyl content of 4.3%.The combined analysis of periodate oxidation,FT-IR,methylation,and 1D and 2D NMR indicated that c DHP-W1 was composed of 1,4-linked-Glcp,1,4-linked-Manp and 1,4,6-linked-Glcp as the backbone,which was attached with T-linkedGlcp as branches.(3)Using LPS-induced macrophage inflammation model,the anti-inflammatory activity and mechanism of c DHP-W1 were investigated.Concentration experiments showed that c DHP-W1 could inhibit the secretion of LPS-induced NOs,PGE2,TNF-α and IL-1 in a dose dependent manner.When the concentration reached 200 μg/m L,the inhibition effect was the most significant.LPS and c DHP-W1 treatment time experiments displayed that when c DHP-W1 pretreatment time was 1 h and LPS treatment time was 24 h,c DHP-W1 had the best inhibitory effect on the secretion of NO,PGE2,TNF-α and IL-1β.RT-PCR analysis showed that the different expression levels of i NOS,COX-2,TNF-α and IL-1β were found with different LPS and c DHP-W1 treatment time.When the macrophages were pretreated with c DHP-W1 for 1h and LPS for 12 h,c DHP-W1 could markedly down-regulated ts the expression of i NOS,COX-2,TNF-α and IL-1β m RNA.Western blot analysis showed that c DHP-W1 could effectively inhibit the LPStriggered phosphorylation of p38,ERK and JNK as well as the LPS-triggered nuclear shift of NF-κB and p65,suggesting that c DHP-W1 might inhibit the LPS-triggered expression and secretion of inflammatory mediators in macrophages through inhibiting NF-κB and MAPK signaling pathway.