| Polygonum Multiflorum which is the dryed roots of Polygonum Multiflorum Thunb,is one of the best genuine medicinal materials in Chongqing,China,and has been widely used in treatment of hyperlipemia,anti-aging,antibiosis,strengthening immunity,improving cardiovascular function,et al.In present study,stibene glucoside,regarded as main active component,has been studied thoroughly.However,the polysaccharides of Polygonum Multiflorum gained few attention.Therefore,study on the bioactivity of polysaccharides and structural features is good for exploring the phamaceutical value of Polygonum Multiflorum,promoting the research of Polygonum Multiflorum polysaccharides based drug and health food,and boosting the development of bioengineering and pharmaceutical industry of Chongqing.In this study,the immunomodulatory activity and structural features of Polygonum Multiflorum polysaccharides were focused on,and immunomodulatory activity-structure relationship was revealed.Furthermore,a polymer-drug conjugate based on immunomodulatory active Polygonum Multiflorum polysaccharide was designed and constructed for potential synergistic anti-tumor.The main results and conclusions are as follows:(1)Two crude polysaccharides named WPMP and APMP were obtained from medicinal materials and water-extracted residues of Polygonum Multiflorum by reflux extraction with water and dilute NaOH solution,respectviely.And,three homogeneous polysaccharides WPMP-1,WPMP-2 and APMP-2 were obtained from WPMP and APMP after purfication by DEAE-52 and Sephadex G-100 column chromatography.The molecular weight of three homogeneous polysaccharides were 2.04 kDa,92.13 kDa,7.72 kDa,respectively,detected by GPC method.The content of uronic acid of WPMP-2 and APMP-2 were 16.5% and 3.2%,respectively,which indicated that they were acid polysaccharides.During the decoloration process,the SBC MCI GEL,which was of double peculiarities of reversion phase and molecular sieve chromatography,was selected as the separating medium,to achieve a well separation between polysaccharides and other coloring matter in the aqueous solution on the basis of differences in polarity and molecular weigh between polysaccharides and other coloring matter.As a result,the content of polysaccharide was over 80%,and the retention rate of polysaccharides was exceed 90%.(2)The structure of polysaccharides of Polygonum Multiflorum were analysed by infrared spectroscopy,HPLC,methylatio-GC-MS,and NMR.It was showed that WPMP-1 was only composed by glucose,it’s backbone constituted by 1,4-linked α-D-Glcp,and there was a side branch at O-6 of 1,4,6-linked α-D-Glcp.WPMP-2 was consist of 1,2-linked α-D-Rhap,1,2,4-linked α-D-Rhap,and 1,4-linked α-D-GalpA as the backbone with side chain of either arabinan(1,3,5-linked α-L-Ara,1,5-linked α-L-Araf,terminal α-L-Araf)or galactooligosaccharides(1,4-linked β-D-Galp,4-linked α-D-GalpA)on O-2/4 of Rhap.APMP-2 has complexed monosaccharide composition,which contained mannose,ribose,rhamnose,glucose,galacturonic acid,galactose,arabinose,and xylose with a molar ratio of 0.14∶0.44∶0.15∶4.31∶0.24∶1.06∶0.86.There was no report on the chain structure of WPMP-1,chain structure and monosaccharide composition of WPMP-2,and monosaccharide composition of APMP-2 before.(3)The immunomodulatory activity of Polygonum Multiflorum polysaccharides were studied by measuring the cell viability and immunologic function of splenocytes and peritoneal macrophage in vitro.It was showed that the Polygonum Multiflorum polysaccharides could promote the proliferation of splenocytes and phagocytosis of peritoneal macrophages,and even significantly better than those in LPS group while the polysaccharides at high concentration(≥0.3 mg·m L-1).When used in combination with 5-FU,ploysaccharides could reduce the cellular damage and immunosupression induced by 5-FU.With the increasing of concentration of polysaccharides,the cell viability and immunologic function of splenocytes and peritoneal macrophage would recover to the mormal level,and the secretion of IL-2 and TNF-α aslo significantly rised.WPMP-2 showed more significantly immunomodulatory activity than the others,which could improve the secretion of IL-2(≥1257.9±64.2 ng·L-1)significantly higher than that of normal control group(##p <0.01)while at a concentration of 0.3 mg·mL-1,and possess more obviously improvement effect on secretion of TNF-α while at a concentration of 0.1 mg·mL-1.As a result,the immunomodulatory activity order could be expressed as: WPMP-2 > WPMP > APMP > APMP-2 > WPMP-1.Further study on the structure-activity relationship proceed on the basis of differences in immunomodulatory activity,molecular weight,monosaccharide composition,content of uronic acid,and branching degree of polysaccharides.It revealed that there was a positive correlation between immunomodulatory activity with the content of uronic acid,percentage of rhamnose,arabinose and galactose,and branching degree.(4)WPMP-2 was utilized as macromolecular carrier to construct WPMP-2@5-FU conjugates.The 5-Fu derivatives 5-fluorouracil-1-acetic acid(5-FUAc)was successful conjugated to WPMP-2 through acetyl spacer group,and the content of 5-FUAc in conjugate system was 7.79%.The release behavior of 5-FUAc from conjugate was related to pH value of medium.It showed significantly better control release behavior in phosphate buffer(pH7.4)than others.In vitro culture of tumor cells,the inhibition effect of WPMP-2@5-FU on HepG-2 and HT-29 was increased with the action time of the drug administration extending from 24 h to 96 h,the inhibition rate of conjugates on HepG-2 increased from 20.20% to 47.69%,and that of HT-29 increased from 5.43% to 51.09%.After the normal mice were fed with intraperitoneal injection of WPMP-2@5-FU conjugtes for one month,the change of body weight,level of IL-2/TNF-α in serum,proliferation of splenocytes and phagocytosis of macropahges of tested mice were all close to normal level.These findings revealed that WPMP-2@5-FU was a potential colon-specific drug delivery system.The inhibition effect of conjugates on the proliferation of HepG-2 and HT-29 was significantly increased,due to the 5-FUAc was released slowly from conjugate.And it also illustrated that the conjugate could significantly mitigate inhibition and damage effects of 5-FU on immune system. |
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