Extraction,Purification And Bioactive Properties Of Polysaccharides From Sphallerocarpus Gracilis

Sphallerocarpus gracilis is a perennial plant belongs to the Apiaceae family and the only species of the genus Sphallerocarpus.This research focuses on the extraction,purification,characterization and bioactive properties of polysaccharides from Sphallerocarpus gracilis.At the same time,this research also discusses the analysis of the nutritional components of Sphallerocarpus gracilis.It is hoped that the research will provide a theoretical basis for the in-depth utilization and further development of resources.The crude polysaccharide primary product S-W was acquired by water extraction and alcohol precipitation and the crude polysaccharide primary product S-A was obtained by alkali extraction and alcohol precipitation.The protein content was determined by Coomassie Brilliant Blue G-250 method and the results showed that the protein content of S-W was8.49%,and the protein content of S-A was 13.73%.The phenol sulfuric acid method was used to determine the sugar content and showed that the sugar content of SGP-W was 29.39%,and the sugar content of SGP-A was 58.04%.SGP-W and SGP-A were further separated and purified by DEAE-Cellulose 52 column chromatography and Sephadex G-100 column chromatography,respectively,and four purified polysaccharides SGP-1,SGP-2,SGP-3 and SGP-4 were obtained(SGP-1 and SGP-2 were obtained from SGP-W,SGP-3 and SGP-4 were obtained from SGP-A).No absorption was detected by the UV spectrum at 260 and 280 nm,indicating there is no free proteins in the samples.Their average molecular weights of SGP-1～4 were 2.44×10~4,3.16×10~4,5.41×10~4and 2.28×10~4Da,respectively.The physical and chemical properties of SGP-1～4 were studied,and showed that SGP-1,SGP-2,SGP-3,and SGP-4 were all anionic polysaccharides with potentials of-18.36 m V,-18.96 m V,-20.07 m V and 23.4 m V.The morphology of SGP-1～4 were characterized by scanning electron microscopy(SEM).It showed that the four polysaccharides of SGP-1～4 had no regularity at low magnifications,and exhibited different morphological characteristics as the magnification increasing.The structure of SGP-1～4 was studied by gas chromatography-mass spectrometer(GC-MS),Congo red experiment,Fourier transform infrared(FT-IR)spectroscopy and nuclear magnetic resonance(NMR).The GC-MS showed that SGP-1～4were mainly composed of five monosaccharides with different ratios.Congo red test showed that no triple helical structure was detected in the four polysaccharides.In this paper,the antioxidant activity,tumor cytotoxicity activity,and hypolipidemic activity of the polysaccharide of Sphallerocarpus gracilis were explored.Three free radical scavenging experiments including DPPH free radicals,superoxide anion free radicals and ABTS free radicals were carried out to evaluate the antioxidant activity of the polysaccharides.All samples have shown no cytotoxic activity on L02(Human normal liver cells)cells.Therefore,the safety of the samples are all considerable.The inhibitory effects of SGP-1～4 on MGC-803(Human gastric cancer cells),A2780(Human ovarian cancer cells),T24(Human bladder cancer cells),CNE-2(Human nasopharyngeal cancer cells),and 4T1(Mouse breast cancer cells)cells in vitro were investigated.The inhibition ratio of MGC-803,A2780,T24,CNE-2,and 4T1 cells induced by the SGP-1～4 was not more than 20%at the concentration of0.2 mg/m L,suggesting that the purified polysaccharides had no significant effects on these cancer cells in vitro and SGP-2 had the highest inhibition rate on 4T1 cells.The MTT was used to study the inhibitory effects of SGP-2 and DOX on 4T1 cells.The results showed that the inhibition rate of 4T1 tumor cells was 71.42%,and the tumor inhibition rate was increased by more than 25%(compared with 20μM DOX alone)after the combined use of 20μM DOX and 0.5 mg/m L SGP-2.Calcein-AM/PI staining confocal imaging method and JC-1 staining mitochondrial membrane experimental studies showed that SGP-2 and DOX have a synergistic anti-tumor effect.It was forwards to a certain extent reduce the toxic and side effects of the chemotherapy drug DOX on normal cells.The binding capacities of SGP-1～4,SPG-W,and SGP-A against sodium taurocholate and sodium glycocholate were examined.The experimental results showed that the bile salt binding rate of SGP-A was significantly higher than that of SGP-W.At the same time,the bile salt binding rate of the alkali-extrated polysaccharides(SGP-3,SGP-4)were significantly higher than that of the water-extrated polysaccharides(SGP-1,SGP-2).The binding rate of SGP-3 with sodium taurocholate is36.39%,and the binding rate with sodium glycocholate was 53.36%,which exhibited the strongest binding ability.Therefore,SGP-3 had the best hypolipidemic activity...