Chemical Modification And Bioactivities Of Polysaccharides From Stems And Leaves Of Abelmoschus Manihot(L.) Medicus

Abelmoschus manihot(L.)Medicus,firstly recorded in "Jiayou Materia Medica",is a traditional herbal medicine from the family of Malvaceae and each part of this herb could be used as traditional medicine.A.manihot was widely distributed in the whole country,however,only the flower was developed as traditional Chinese medicine while the stems and leaves were mainly discarded and burned,which directly caused enormous waste of these natural resources.Researches about the active ingridients in the stems and leaves of A.manihot was very little,especially the polysaccharides.Our previous studies found that polysaccharides in the stems and leaves was not very little,thus in this study,the extraction condition of polysaccharides from disused parts of A.manihot was opitimized,and chemical modification,characterization,immunomodulatory and antitumor activities of polysaccharides was investigated which could provide theoretical reference for the development and utilization of A.manihot stems and leaves resources.1.Extraction,separation and composition of polysaccharides from stems and leaves of A.manihot.Effects of solid-liquid ratio,extraction time,extraction temperature and extraction times on the extraction rate was investigated,and the orthogonal design was applied to optimize the extraction process,and the optimum extraction parameters were obtained as follows:solid-liquid ratio 30:1,extraction time 1 h,extraction temperature 80 ?,extracting 3 times.The protein removal rate and polysaccharide retention rate were considered to evaluate the number of protein removal,and the optimal number of protein removal by Sevag was 4 times.The total sugar of the crude polysaccharide from stems and leaves of A.manihot(SLAMP)is 39.94%with 15.94%uronic acid as well as about 5%sulfates.Monosaccharide composition analysis showed that SLAMP was composed of mannose,glucuronic acid,rhamnose,glucose,galactose and arabinose.DEAE-52 was applied for the classification of SLAMP,and two homogeneous components SLAMP-c and SLAMP-d and two heterogeneous components SLAMP-a and SLAMP-b were obtained.SLAMP-a was almost insoluble in water and its molecular weight distribution was broad,and the total sugar was 99.76%.SLAMP-a was mainly composed of glucose with a few mannose,galactose and arabinose.SLAMP-b,with bad water-solubility,contained 66.17%total sugar and about 6%protein,and the molecular weight was mainly distributed around 1474.5 kDa,SLAMP-b was composed of six monosaccharides(with SLAMP),in which glucose is the highest proportion and rhamnose was the lowest.SLAMP-c and SLAMP-d,with better water solubility and triple-helical structure,were much close in chemical and monosaccharide composition.Both of them contained about 50%total sugar,about 40%uronic acid as well as a few sulfates and protein,and the content of uronic acid in SLAMP-c was higher.The molecular weight of SLAMP-c and SLAMP-d were 477.8 kDa and 264.2 kDa respectively.Both SLAMP-c and SLAMP-d were composed of six monosaccharides(with SLAMP)and the molar ratio was close in which glucuronic acid is the highest while the proportion of mannose and arabinose was very low.2.Sulfation,acetylation and characterization of neutral polysaccharides from stems and leaves of A.manihot.Three sulfated derivatives and three acetylated derivatives of SLAMP-a with poor water-solubility were prepared by amino sulfonic acid and acetic anhydride respectively.The molecular weight distribution showed that only S-SLAMP-a3 wase homogeneous among the six derivatives.S-SLAMP-a3 exhibited much better water solubility,and the molecular weight,DS and yield was 1044.2 kDa,0.57 and 126.57%,respectively.S-SLAMP-a3 mainly contained glucose with a few mannose,galactose and arabinose.Both S-SLAMP-al and S-SLAMP-a2 were slightly soluble in water,and the molecular weight mainly distributed in 1000?13 00 kDa with a few low molecular polysaccharides.The DS of them is about 0.1 and the characteristic peaks of sulfate in IR spectra couldn't be observed.The monosaccharide composition of S-SLAMP-al and S-SLAMP-a2 was close to that of S-SLAMP-a3.Additionally,the triple-helical structure was only identified in S-SLAMP-al.All of the three acetylated derivatives showed poor water-solubility and the molecular weight distribution is similar,which was composed of relatively homogeneous macromolecule and low molecule region.The DS and yield of the three products distributed in 0.62?0.85 and 40?50%,respectively.Monosaccharide composition analysis showed that mannose and galactose and Arabia sugar decreased sharply and the proportion of glucose increased a lot in these acetylated derivatives.Congo-Red assay demonstrated that Ac-SLAMP-al and Ac-SLAMP-a2 possessed the triple-helical structure.3.Immunomodulatory and anti-tumor activity of polysaccharides from stems and leaves of A.manihot and derivatives.In vitro immunomodulatory activity study showed that SLAMP,SLAMP-c and SLAMP-d could significantly stimulate the proliferation of spleen lymphocytes and activate RAW264.7 release NO,TNF-? and IL-6,while SLAMP-a showed little immunomodulatory activity and SLAMP-b possessed some immunosuppression.Among the six derivatives,only S-SLAMP-a3 and Ac-SLAMP-al could significantly stimulate lymphocyte proliferation and activate RAW264.7 produce NO,TNF-a and IL-6.SLAMP-a obtained immunomodulatory activity after sulfation and acetylation.Based on the sensitivity of polysaccharides on different tumor cells or cultured with different administration time on the inhibition,the tumor cell was determined to be A549 and the administration time was 72 h.SLAMP-c and SLAMP-d showed better anti-tumor activity,while anti-tumor activity of SLAMP was relatively weak.The anti-tumor activity of SLAMP-a is very poor,while the derivatives S-SLAMP-a3 and Ac-SLAMP-al could significantly enhance its anti-tumor activity.Compared with S-SLAMP-a3,Ac-SLAMP-al exhibited better antitumor activity.