Study On Structure-activity Relationship Of Polysaccharide From Seeds Of Plantago Asiatica L. And Its Immunoregulatory Mechanism

There are abundant polysaccharide resources around the world.It is until 1940 s that polysaccharide was proved to be the potent medicinal resource,since then a great deal of effort has been devoted in development and utilization of polysaccharide.Recently,hypotheis of “pattern recognition receptor” has been proved by t he discovery of Toll-like receptors(TLRs)family,which provides a n ew direction to explore the molecule mechanism for the biological activities of polysaccharide.Besides,structure modifications have been applied to modulated physicochemical or biological properties of polysaccharides.And such structure modifications also provide an effective approach to explore the structure-activity relationship of polysaccharides.The seeds from Plantago asiatica L.have a long history to be used as a traditional medicine in China.The polysaccharide from the seeds from Plantago asiatica L.have been certainly demonstrated to possess several biological activites,such as laxative effect,hypoglycemic effect,hypolipidemic effect,andti-tumor effect and so on.Particularly,Plantago asiatica L.seeds polysaccharide exhibited a notable immune-regulatory effect by inducing bone-marrow derived dendritic cells in vitro.In this study,the mechanism for Plantago asiatica L.seeds polysaccharide to induce dendritic cells maturation has been study.And a lots of structure modifications have been appled to Plantago asiatica L.seeds polysaccharide to investigate influences of these modifications on the immune-regulatory effect of the natural polysaccharide.The conclusions of this study were list as follows.1.Crude polysaccharide from the seeds of Plantago asiatica L.(PLCP)was carboxymethylated(CM-PLCP).CM-PLCPs with gradient degree of substitution(DS)from 0.40 to 0.62 exhibited significant effects on inducing DCs maturation.And CM-PLCP with DS of 0.62 possessed highest immunoregulatory activity.Acetylated PLCP(Ac-PLCP)with DS from 0.06 t o 0.1 possessed higher maturation stimulating activity on DCs than the natural polysaccharide PLCP.Particularly,Acetylated PLCP with DS of 0.82 exhibited highest immunoregulatory activity.However,sulfation modification significantly decreased the immunoregulatory activity of PLCP.2.Fractions of PLCP were prepared by hydrolysis using trifluoroacetic acid(TF-PLCP I and Peri-PLCP II).Both of TF-PLCP I and TF-PLCP II could effectively induce the maturation of DCs.But only TF-PLCP II showed a significantly higher effect than PLCP,indicating that TF-PLCP II may contain a active space.Besides,fractions of PLCP prepared through periodate oxidation showed no effect on inducing maturation of DCs.3.PLCP was treated by glucamylase or pectinase to obtained Glu-PLCPs and Pec-PLCPs,respectively.All the Glu-PLCPs and Pec-PLCPs could induce maturation of DCs,however,there was no significant difference compared with PLCP,indicating that enzymolysis by glucamylase or pectinase has no influence on the immunoregulatory activity of PLCP.4.Uronic acid content in PLCP was reduced using carbodiimide-sodium borohydirde method to prepared CN-PLCPs.CN-PLCPs exhibited immunoregulatory activities since they could induce maturation of DCs.But there was no significant difference between CN-PLCP and PLCP.It indicates that the immunoregulatory activity of PLCP is not denpended on its uronic acid content.5.The comparison among the immunoregulatory activieites of all the structure modified polysaccharides was investigated.Carboxymethylation,acetylation,and trifluoroacetic acid hydrolysis could significantly improve the immunoregulatory activity of PLCP.Particularly,acetylation PLCP showed highest effect on inducing DC maturation.Treatment of glucamylase enzymolysis,pectinase enzymolysis and uronic acid reduction showed no i nfluence on t he immuoregulatory activity of PLCP.While sulfation and periodate oxidation significantly decreased the immuoregulatory activity of PLCP.6.Purified polysaccharide from seeds of Plantago asiatica L.(PLP-2)can induce maturation of bone-marrow-derived dendritic cells in vitro.Blocking toll-like receptor 4 and its downstream cascades MAPK and NF-κB signal transduction pathways could significantly decreased the PLP-2 induced expression of MHC II,CD86 surface molecules on bone-marrow-derived dendritc cells.It also significantly inhibited the cytokine secretion of TNF-α and IL-12p70 of DCs as well.Meanwhile,DCs in the signal-blocking groups exhibited higher endocytosis activities.Especially,DCs in the signal-blocking groups showed dramatically attenuated effects on s timulating proliferation of T lymphocytes in the mixed lymphocyte reaction.These data suggests that toll-like receptor 4 mediates the PLP-2 induced maturation of DCs,and both of its downstream MAPK and NF-κB pathway play key roles in the signal transduction.This study not only provides a new perspective for exploration and utilization of polysaccharide resource,but also provides theory support for polysaccharide drug design and development.