Study On Extraction,Purification,Structure And Antioxidant Activity Of Polysaccharides From The Stems Of Dendranthema Indicum Var.Aromaticum

Dendranthema indicum var.aromaticum is a new variety of Dendranthema Compositae,its flowers and leaves have the effect of heat-cleaning and detoxifying.D.var.aromaticum has been widely used to treat influenza,sore throat,swelling pain in the eyes and so on.Domestic and foreign scholars have isolated volatile oil,flavonoids,terpenoids and other components from stems and leaves of D.var.aromaticum,with anti-bacterial,anti-oxidant,anti-inflammatory and other activities.However,few systematic studies have been reported on the polysaccharides from the stems of D.var.aromaticum.In this paper,the extraction,purification,structure and antioxidant activity of polysaccharides from the stem of D.var.aromaticum in Shennongjia,Enshi were systematically studied,in order to provide a new direction and reference basis for the comprehensive development and utilization of D.var.aromaticum stem resources in China.The main research contents were as follows:?1?The extraction rate of polysaccharides was compared from the stems of D.var.aromaticum by hot water extraction and ultrasound-assisted extraction.The optimum technological conditions of hot water extraction were as follows:the ratio of material to liquid was 1?40,the extraction temperature was 90?,the extraction time was 2h,the extraction rate of polysaccharide was 5.735%on the optimum conditions;and the optimum conditions of ultrasonic-assisted extraction were as follows:the ratio of material to liquid was 1?40,the ultrasonic time was60min,the concentration of ethanol precipitation was 70%,and the extraction rate of polysaccharide was 11.385%on the optimum conditions,which was 1.99 times higher than that of hot water extraction.The advantages of ultrasonic-extraction method are to increase the yield,shorten the extraction time and simplify the equipment conditions.?2?The decolorization efficiency of activated carbon,macroporous resin and DEAE-52 cellulose on ultrasonic preparation of crude polysaccharide from stems of D.var.aromaticum were compared by taking the decolorization rate and recovery rate of polysaccharide as evaluation indexes.The results were as follows:when the dosage of activated carbon was 4%,the decolorization temperature was 60?and the decolorization time was 45min,the maximum decolorization rate of polysaccharide was 81.586%,but the recovery rate of polysaccharide was only 20.445%.Seven microporous resins were screened by static experiment.The decolorization rate of D301 resin was 91.736%while the recovery rate of polysaccharide was only34.759%.Speaking of DEAE-52,different eluents were used to elute the polysaccharide solution of D.var.aromaticum stem.A₄₅₀nm?distilled water?<0.002,no pigment absorption,the decolorization rate of this section was high;A₄₉₀nm?distilled water?>5.5,the content of polysaccharide was high which meant the recovery rate of polysaccharide was high.In a word,the best method for decolorization of polysaccharides from stem of D.var.aromaticum was DEAE-52cellulose,which compared with activated carbon and macroporous resin.The crude polysaccharides from the stems of D.var.aromaticum was grayish brown,and the polysaccharide decolorized by DEAE-52 was white spongy solid.?3?The polysaccharides from the stem of D.var.aromaticum were further isolated and purified by ion exchange chromatography.The isolation of polysaccharides in different ionic systems?OH^-,Cl^-,HCO₃^-?were investigated.The purification effect of HCO₃^-type DEAE-52 cellulose was the best,the resolution was high.Besides,the number of elution peaks was the largest.The four fractions were obtained by HCO₃^-DEAE-52chromatography:DILP-1,DILP-2,DILP-3,DILP-4.?4?The homogeneity of the four components was determined by HPLC,and DILP-1 was determined as the detection object.The structure of DILP-1 from the stem of D.var.aromaticum was analyzed by UV scanning analysis,high performance liquid chromatography,¹H-NMR,¹³C-NMR and et al.The results of UV scanning analysis showed that there was no protein or nucleic acid in DILP-1.The mass average molar mass of DILP-1 was determined to be 2.182*10⁴Da by multiple angle laser light scattering.The monosaccharide composition of DILP-1was composed of glucose,galactose,rhamnose and fructose with a molar ratio of1?1.992?1.577?2.379.DILP-1 was further identified as a polysaccharide component by ¹H-NMR and ¹³C-NMR spectra.?5?The scavenging capacity of polysaccharides?DILWP,DILUP?from D.var.aromaticum stems obtained by hot water extraction and ultrasonic extraction on DPPH and ABTS free radicals and total reduction capacity were studied.The results showed that both DILWP and DILUP had antioxidant activity in vitro,the efficiency of scavenging was increased with the concentrations in a certain concentration range.The antioxidant capacity of DILUP was stronger than that of DILWP.