Effects Of Acetyl Group Contents On The Immunomodulatory Activity Of Glucomannans From Aloe Barbadensis Miller And Amorphophallus Konjac

The plant genus Amorphophallus belongs to the family Araceae（aroidae）.Tubers of Amorphophallus have been used as food for a long time in areas such as China,Japan and southeast of Asia.Konjac glucomannan is the main chemical component of A.konjac,which is composed ofβ-1,4-linked D-mannosyl and D-glucosyl residues at a molar ratio of 1.6:1.0 with some acetyl groups linked in the main chain.Aloe barbadensis Miller（or Aloe vera）,belonging to the Lily family,is a kind of evergreen fleshy plant.Polysaccharides extracted from the pulp of Aloe vera were proved to be acetylated glucomannans which mainly consisted of 1,4-linkedβ-D-mannopyranosyl units with minor amounts of 1,4-linkedβ-D-glucopyranosyl units.When comparing the structures of glucomannans from Aloe vera and Amorphophallus konjac,great differences in the monosaccharide compositions,side chains,the content and distribution of acetyl groups were found.Do these differences contributed to differences in their corresponding immunomodulatory activity and to what extent?There are few reports which can answering these questions.Interestingly,relevant investigations in the structure-function relationship of polysaccharides become more and more popular in recent years.The elucidation of structure-function relationship of polysaccharides could help the directional development of functional factors in the food or functional foods in the food industry.Therefore,the acetyl groups in glucomnannan were focused in the present study.The acetyl groups in the polysaccharides from Aloe vera and Amorphophallus konjac were modified using chemical methods and then the effects of the modified acetylated polysaccharides on the immunomodulatory activities were investigated.The main contents and results of the current study were as follows.1.The isolation,purification,physicochemical properties and primary structure of the polysaccharide from Aloe vera were conducted.Crude polysaccharide（AGP）was extracted from fresh Aloe vera after peeling.A fraction with high purity(AGP₄₀)was obtained by ammonium sulfate precipitation method at a saturation of 40%（w/w）.The purity and molecular weight（Mw）were determined by high performance gel permeation chromatography（HPGPC）.The chemical components,monosaccharide compositions,spectroscopic and structural properties of AGP₄₀ were analyzed.Results showed that AGP₄₀ was neutral polysaccharides with high purity.Its relative molecular mass was 3.38×10⁵ Da.No uronic acid and protein were detected.AGP₄₀ was mainly composed of mannose,containing a small amount of glucose and a high content of acetyl group（29.3%）.Methylation analysis showed that the polysaccharide is linked by 1→4 glycosidic bonds.In conclusion,AGP₄₀ is probably a highly acetylated glucomannan linked by 1→4 glycosidic bonds.In contrast,glucomannan from Amorphallus konjac（KGM）was prepared in our lab.It was neutral polysaccharide with relative molecular mass 9.19×10⁵ Da.KGM is composed ofβ-1,4-linked D-mannosyl and D-glucosyl residues at a molar ratio of 1.15:1.00 with some acetyl groups（10.4%）linked in the main chain.2.To obtain a series of glucomannans with different content of acetyl groups,AGP₄₀was deacetylated using sodium carbonate and KGM was acetylated by acetic anhydride.For the Aloe polysaccharides,derivatives(designated as Dac₁-AGP₄₀,Dac₂-AGP₄₀,Dac₃-AGP₄₀)with different degree of deacetylation were obtained in Na₂CO₃aqueous solution at different concentration.The content of acetyl groups of Dac₁-AGP₄₀,Dac₂-AGP₄₀,Dac₃-AGP₄₀were determined to be 24.6%,16.6%,10.7%,successively.Compared with the orginated Aloe polysaccharide（29.9%）,the degree of deactylation were 17.7%,44.5%,64.2%.For the acetylated konjac glucomannan containg lower content of acetyl groups,acetylation were carried out.The contents of acetyl groups ranged from 17.8%to 23.0%with the increase of acetic anhydride volume.The modifications of acetyl groups were confirmed by the infrared spectroscopy and scanning electron microscopy images.3.To assess the effects of the acetyl groups on the immunomodulatory activities of glucomannans from Aloe vera and Amorphophallus konjac,mouse macrophage cell line RAW 264.7 was used as the in vitro model.AGP₄₀ had potent immunomodulatory activity on macrophage,the effects of deacetylated AGP₄₀ on the phagocytosis and cytokines secretion（TNF-αand IL-1β）decreased with the decrease of acetyl groups in polysaccharides.On the other hand,modified konjac polysaccharides after acetylation showed no obvious difference in the secretion of NO and IL-1β.However,under relatively high concentration of 20μg/m L and 80μg/m L,konjac acetylated polysaccharides Ac₂-KGM,Ac₃-KGM showed a rising trend to phagocytosis and reduction of Ac₁-KGM.Moreover,the TNF-αsecretion of all konjac acetylated polysaccharides was declined in 20μg/m L concentration.The results showed that different degrees of acetylation of konjac polysaccharide had different effects on the immunoregulation of macrophages at different concentrations,and the deacetylation of aloe polysaccharide could reduce its immunomodulatory activity.