Purification,Structure Characterization And Antitumor Activity Study Of Polysaccharides From Gastrodiae Elata

Objective:To isolate and purify the crude polysaccharide from Gastrodia elata by hot water immersion,to identify its structure and to study its anti-tumor activity,so as to lay a theoretical foundation for the wide application of Gastrodia elata.Methods:(1)The polysaccharides in Gastrodia elata were extracted by water extraction and alcohol precipitation(extraction temperature 60℃,time 2 h,extraction times 3 times,solid-liquid ratio 1:30,Ethanol precipitation concentration is 80%).Then the polysaccharides were deproteinized by Sevage method.The hollow fiber ultrafiltration membrane was used to remove small molecular impurities and pigments,and the polysaccharides were segmented by replacing ultrafiltration membranes with different molecular weights.The polysaccharides from Gastrodia elata were purified preliminarily.(2)Separation and purification of the purified Gastrodia elata polysaccharides by DEAE-52 cellulose ion exchange chromatography and Sephadex G-100 gel column chromatography.(3)The molecular weight and molecular conformation of polysaccharides were studied by SEC-MALS-VI-RI technique.The purity of polysaccharides was analyzed by UV spectroscopy and the monosaccharide composition of polysaccharides was analyzed by HILIC-ELSD.The polysaccharides were selected for structural characterization and antitumor activity.(4)The primary and advanced structures of Gastrodia elata polysaccharides were studied by infrared spectroscopy(IR),nuclear magnetic resonance(NMR),Congo red,scanning electron microscopy(SEM)and X-ray diffraction(XRD).The characteristic absorption peaks of the refined polysaccharides were characterized by infrared spectroscopy,and the types,linking sequence and substitution sites of the sugar residues were analyzed by NMR.Congo red experiment was used to investigate the primary helical conformation of Gastrodia elata polysaccharides.The morphological distribution of the polysaccharides was observed by scanning electron microscopy and the spatial conformation of the polysaccharides was deduced.The regular arrangement structure of the polysaccharides was detected by XRD in order to obtain the higher conformation information of the polysaccharides of Gastrodia elata.(5)The anti-tumor activity of Gastrodia elata polysaccharides was studied by MTT method,and the correlation between the structure and activity of polysaccharides was analyzed by structural characterization.Results:(1)Gastrodia elata polysaccharide was extracted by water extraction and ethanol precipitation.The final yield of crude polysaccharide was 5.47%.Gastrodia elata polysaccharides were purified by deproteing,removing small molecules,depigmenting and segmenting with hollow fiber ultrafiltration membrane.(2)The segmented Gastrodia elata polysaccharide was separated and purified by DEAE-52 cellulose ion exchange column chromatography and Sephadex G-100 column chromatography.Finally,6 parts polysaccharides(WTM-1,WTM-2,WTM-3,WTM-4,WTM-5,WTM-6)were isolated from Gastrodia elata,the molecular weight of the polysaccharide was determined by SEC-MALS-VI-RI technique,Combined with UV spectroscopy to analyze the purity of 6 parts of polysaccharides.As a result,there were two peaks in WTM-1,and the molecular weights were not separated.The contents of TMWTM-2,WTM-3,WTM-5 and WTM-6 were all above 98.0%,which reached the standard of structural analysis and activity research,WTM-The content of 4 was 87.1%,and the molecular weights of WTM-2~WTM-6 were 40.41 KDa,30.43 KDa,151.72 KDa,17.02 KDa,and 12.76 KDa,respectively.(3)HILIC-ELSD technique was used to analyze the monosaccharide composition of polysaccharides.The results showed that the four refined polysaccharides were all heteropolysaccharides(most of them were glucose,but the other monosaccharides were less).The monosaccharides mainly consisted of glucose,xylose,mannose,fructose and glucuronic acid.The structure of Gastrodia elata polysaccharide was analyzed by Congo red solution,infrared spectroscopy,nuclear magnetic resonance,scanning electron microscopy and X-ray diffraction.The results showed that the four polysaccharides wereα-pyranose rings with 1,4-glucose as the main chain.The chain branch point is at the C-6position and does not have a triple helix structure.It is presumed that the basic backbone structure of the gastrodia elata polysaccharide is(α-D-Glcp-(1→4)-α-D-Glcp)_n.In addition,scanning electron microscopy(SEM)and viscosity values showed that the refined polysaccharides WTM-2 and WTM-3 were composed of flakes,which were wireless clusters.WTM-5 was composed of a small number of flakes with branched structure.WTM-6 had a large number of dense holes and dense structure,which was branched structure.X-ray diffraction showed that the crystallinity of the refined Gastrodia elata polysaccharides was very poor,and it was determined as amorphous structure.(4)The anti-tumor activities of four refined Gastrodia elata polysaccharides(WTM-2,WTM-3,WTM-5 and WTM-6)were studied.Three kinds of tumor cells(HepG2,Hela,A549)were used to study their anti-tumor activities.The results showed that the four polysaccharides had certain anti-tumor activities against HepG 2 and Hela,respectively.The tumorigenicity increased with the increase of concentration,and the higher the molecular weight of polysaccharide,the stronger the inhibition ability.But the activity of four polysaccharides on lung cancer A549 cells is different.WTM-6 has obvious activity(concentration has little effect on it).The other three polysaccharides are basically negative and have no inhibitory effect on it.Conclusions:In this experiment,we studied the in vitro antitumor activity of different molecular weight polysaccharides by a series of separation and purification operations of the gastrodia elata polysaccharide obtained by the same extraction method.It was found that the gastrodia elata polysaccharides in a certain molecular weight range on tumor cells(hepatoma cells HepG 2 and cervical cancer Cell Hela)has anti-tumor effect,the higher the molecular mass,the stronger the anti-tumor activity,and the higher the polysaccharide concentration,the stronger the inhibition.As a result of the production of polysaccharides on lung cancer A549 cells,it is known that refined Gastrodia elata polysaccharide is not a broad-spectrum antitumor drug and is selective.In this experiment,the research on the chemical structure and anti-tumor activity of Gastrodia elata polysaccharides was the next step to study the molecular mechanism of polysaccharides and its application in food,medicine and health care.