| Dendrobium officinale is a valuable traditional Chinese medicinal material with a variety of medicinal effects.Polysaccharides are high in Dendrobium officinale and are its main biologically active substances.At present,most of the Dendrobium polysaccharides used in the market are crude polysaccharide extracts.Due to the presence of impurities such as protein,the activity and practical application of Dendrobium polysaccharides are affected to a certain extent.This paper takes Dendrobium officinale polysaccharide as the main research object,studies and establishes the extraction and purification process of polysaccharide,and studies the immunomodulatory activity of polysaccharide,which is intended to provide theoretical basis and method reference for the industrial production and application of Dendrobium polysaccharide.The main research contents and results are as follows:1.Research on the extraction process of Dendrobium officinale polysaccharidesThe hot water extraction method was used to extract polysaccharides from Dendrobium officinale.Single factor method and orthogonal method were used to study the influence of factors such as the ratio of material to liquid,extraction time,extraction times,and extraction temperature on the extraction rate of polysaccharides from Dendrobium officinale.Results The optimal hot water extraction process for Dendrobium officinale polysaccharides was as follows: the material-to-liquid ratio was 1:20,the extraction time was 15 min,the number of extractions was 2 times,and the extraction temperature was 60 ℃.Under these conditions,the average extraction rate of polysaccharides in Dendrobium candidum was 12.45%.2.Study on the purification process of Dendrobium officinale polysaccharide in two aqueous phasesThe ethanol-ammonium sulfate aqueous two-phase system was used to extract and purify Dendrobium officinale polysaccharides.The cloud point method was used to determine the ethanol-ammonium sulfate aqueous two-phase diagram.The single factor method and response surface method were used to study the effects of ammonium sulfate concentration,ethanol concentration and extraction temperature on the iron sheet.The effect of Dendrobium polysaccharide extraction rate and protein removal rate.Results The optimal extraction and purification process conditions of Dendrobium officinale polysaccharide by ethanol-ammonium sulfate two-phase system were as follows: in 30.0 g two-phase system,the mass concentration of ammonium sulfate was 18.8%,the mass concentration of ethanol was 25.00%,and the extraction temperature was 25 ℃.Under these conditions,the polysaccharide extraction rate and protein removal rate were 94.39% and 91.36%,respectively.The purity of polysaccharide was increased from 85.20% before purification to95.96% after purification,and the purity of polysaccharide was significantly improved.3.Characterization of Dendrobium officinale polysaccharideThe crude polysaccharides and purified polysaccharides of Dendrobium officinale obtained by extraction,purification and separation were analyzed by ultraviolet spectroscopy,infrared spectroscopy and gel permeation chromatography to study the effect of aqueous two-phase extraction and purification methods on the structure of Dendrobium officinale polysaccharides.Ultraviolet spectroscopy showed that the polysaccharide purified by the aqueous two-phase method did not contain impurities such as protein.The results of infrared spectroscopy showed that the structure of the polysaccharide of Dendrobium officinale did not change significantly before and after purification.Gel permeation chromatography showed that the purified polysaccharide had no small molecules.The results show that the aqueous two-phase extraction method can remove proteins and other polymer impurities without changing the structure and other properties of polysaccharides.The monosaccharide composition of the purified polysaccharide was analyzed by ion chromatography.The polysaccharide of Dendrobium officinale was mainly composed of glucose,mannose and a small amount of galactose.4.Evaluation of immune activity of Dendrobium officinale polysaccharideUse mouse macrophage RAW264.7 to establish an immune evaluation model to explore the stimulating effects of four polysaccharides(Dendrobium officinale crude polysaccharide,purified polysaccharide,neutral polysaccharide,and acid polysaccharide)on macrophages through cell proliferation activity and phagocytic activity,NO release and ROS production to evaluate the stimulating effect of polysaccharides on macrophages.The results show that Dendrobium polysaccharide can effectively improve the immune activity of macrophages.The purified polysaccharide has stronger immune activity than the crude polysaccharide,and the acidic polysaccharide in the polysaccharide has stronger immune activity than the neutral polysaccharide. |
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