Isolation,Purification,structural Identification And Immunostimulatory Activity Of Polysaccharides From The Leaves Of Moringa Oleifera

Moringa oleifera,belonging to the family of Moringa,is a medicinal food homology plant with high nutritional and high medicinal value.The plant is widely planted in India,Africa,Pakistan,and China,such as Yunnan,Guangdong,and Sichuan.As one of the most important nutritive components of Moringa oleifera,the leaves are often used to treat hypertension,diabetes,immunosuppression and other diseases.Modern pharmacological studies have indicated that its extract has anti-bacterial,anti-inflammatory,immunomodulation effects and etc.Polysaccharides are one of the most important active substances in Moringa oleifera leaves.However,to date,there are few studies on the structure and immunomodulatory activities of polysaccharides from Moringa oleifera leaves.Therefore,in the present study,the structure,immunomodulatory activity,in vitro digestion and fermentation characteristics of the polysaccharides from the leaves of PKM1 were measured.The obtained results could provide a theoretical basis for research and development of the polysaccharides from Moringa oleifera leaves used in functional foods.The main findings are as follows:(1)Three varieties of domestic more planted Moringa oleifera were selected as the research objects: Periyakulam 1 or PKM1 leaves,Improved Periyakulam 1 or PKM2 leaves,M.stenopetala leaves.Under the same extraction conditions,the extraction yield and polysaccharide content of the polysaccharides from the leaves of PKM1 was significantly higher than those of PKM2 and M.stenopetala leaves.High performance liquid chromatography(HPCL)analysis indicated that three polysaccharides had different molecular weight distributions.Fourier transform infrared(FTIR)spectrum analysis indicated that three polysaccharides were acidic furanose polysaccharides with ?-configuration.Congo red assay showed that three polysaccharides did not have triple-helical conformation in distilled water.In vitro antioxidant tests showed that three polysaccharides had good scavenging activities of DPPH,ABTS and OH radicals.The radical scavenging abilities of the polysaccharides from M.stenopetala and PKM1 leaves were significantly higher than PKM2.(2)Crude polysaccharides from PKM1 leaves were fractionated by DEAE-Sepharose fast flow column.Two main polysaccharides(MOP-2 and MOP-3)were obtained.HPLC analysis showed that MOP-2 and MOP-3 were homogeneous polysaccharides with molecular weights of 155.35 kDa and 4033.83 kDa,respectively.Chemical composition and structure analyses revealed that MOP-2 and MOP-3 had different physicochemical properties,monosaccharide compositions and glycosidic types.MOP-2 were composed of arabinose(35.80%),galactose(57.54%)and glucose(6.67%),while MOP-3 consisted of arabinose(44.87%),galactose(54.19%)and glucose(0.94%).Periodate oxidation-Smith degradation and NMR analysis showed that the main glycosidic linkages of MOP-2 were identified as ?5)-?-L-Araf-(1?,?-L-Araf-(1?,?3,6)-?-D-Galp-(1?,?6)-?-D-Galp-(1?,and ?-D-Glcp-(1?.The main glycosidic linkages of MOP-3 were identified as ?5)-?-L-Araf-(1?,?-L-Araf-(1?,?3,6)-?-D-Galp-(1?,and ?6)-?-D-Galp-(1?.In vitro immunomodulatory assay revealed that MOP-2 and MOP-3 could enhance the proliferation and pinocytic capacity of the RAW264.7 cells.Furthermore,MOP-2 and MOP-3 promoted the secretion of reactive oxygen species(ROS),nitric oxide(NO),interleukin-6(IL-6),and tumor necrosis factor-?(TNF-?)by activating the corresponding mRNA expression in RAW264.7 cells.These results suggest that MOP-2 and MOP-3 could be used as a potential immunoregulatory agent in functional foods.(3)In vitro simulated saliva medium,gastric medium and intestinal medium were used to investigate the digestion and fermentation profiles of MOP-2.The results showed that the molecular weight of MOP-2 did not change after digesting in saliva and gastric medium,and no reducing sugar was produced in the digestive medium.The molecular weight decreased from 155.29 ±0.41 to 145.02 ±0.47 kDa after digestion in intestinal medium for 6 h.In vitro simulated fermentation assay showed that MOP-2 could increase the diversity of intestinal microbiota and the content of short-chain fatty acids(SCFAs),especially butyric acid.At the phylum level,it could improve the relative abundance of Bacteroidetes and reduce the relative abundance of Firmicutes.At the class level,it could increase the relative abundance of Actinobacteria and Bacteroidia,decrease the relative abundance of Negativicutes.At the order level,it could increase the relative abundance of Bifidobacteriales,Bacteroidales,Coriobacteriales and Burkholderiales,decrease the relative abundance of Clostridates and Selenomonadales.At the family level,it could increase the relative abundance of Bifidobacteriaceae,Ruminococcaceae,Bacteroidaceae,Coriobacteriaceae,Streptococcaceae and Lactobacillaceae,decrease the relative abundance of Veillonellaceae,Alcaligonaceae and Lachnospiraceae.In addition,after 48 h fermentation,MOP-2 could promote the relative abundance of Phascolarctobacter,Coprococcus,Roseburia and Bacteroides.These results indicated that MOP-2 could display immune function by regulating the structure of intestinal flora and increasing the content of SCFAs.