What are the methods of total sugar and reducing sugar extraction, separation and purification

1.Determination of polysaccharide content of Platycodon grandiflorum by indirect method. Using anthrone-sulfuric acid method to determine the content of total sugar (including polysaccharides and reducing monosaccharides), 3,5-dinitrosalicylic acid method (DNS), indirect iodine

measurement method, phenanthrene titration method to determine the content of reducing sugar. The difference between the total sugar content and the reducing sugar content was the polysaccharide content. The contents of polysaccharides of Platycodon grandiflorum measured by the three methods were 95.55%, 96.01%, and

95.75%, respectively. The results showed that the values of the three methods were similar, but the anthrone-sulfuric acid method combined with the DNS method was chosen for the determination of the polysaccharide content of Platycodon grandiflorus after considering the cost, equipment, operation and factors affecting the determination.

2. The effects of extraction temperature, extraction time, material-liquid ratio and number of extractions on the extraction rate of polysaccharides from Platycodon grandiflorus were investigated, and a four-factor, three-water

planar orthogonal test was conducted to optimize the extraction of polysaccharides from Platycodon grandiflorus on the basis of one-way experiments. The results of the orthogonal test showed that the optimal conditions for the extraction of polysaccharides from Platycodon grandiflorum were as follows: the extraction temperature was 80 ℃, the extraction time was 2 h, the number of extraction times was 2 times, and the ratio of liquid to liquid was

1:25. The extraction rate of polysaccharides of Platycodon grandiflorum was 42.51% in this program through three parallel validation experiments.

3. 3, using Sevag method, trichloroacetic acid method, trichloroacetic acid-n-butanol method, three methods were used for the study of de-proteinization of Erysipelas polysaccharides, protein removal rate and polysaccharide loss rate as a measure of indexes, and the results showed that the Sevag

method was more effective in removing proteins, and the process conditions were as follows: polysaccharides: chloroform: n-butanol = 25: 5: 1, and shaking for 20min. The decolorization method was H2O2 oxidation, and the decolorization conditions were as follows: polysaccharide

liquid: H2O2=4:1, adjusting the pH value to 8.8 with concentrated ammonia or 0.1% NaOH, and holding at 37℃ for 12 h. The salt and small molecule substances were removed by Sephadex G-25 column chromatography.

4. After purification, the polysaccharide of Platycodon grandiflorum was separated by DEAE-cellulose 52, which mainly obtained three fractions: PGPN, PGPA1, and PGPA3. The three fractions were further separated by chromatography on a Sephadex

G-100 column and all of them were obtained with a single symmetric elution peak. The purity of the three polysaccharides was determined by Sephadex

G-100 column chromatography, spinodensitometry and ultraviolet spectrophotometry, and all three polysaccharides were found to be homogeneous polysaccharides and free of proteins and nucleic acids. The molecular weights of the three polysaccharides were 10233, 4677, and 23442, respectively, as determined by Sephadex

G-200 column chromatography.

5. The monosaccharides of Platycodon grandiflorum polysaccharides were analyzed by thin-layer chromatography (TLC), which showed that the monosaccharides of PGPN contained fructose, and either glucose and galactose or both. both

; PGPA1 is composed of galactose and fructose; and PGPA3 is composed of galactose and xylose.IR spectra showed that the monosaccharides of PGPN are composed of fucose and pyranose***simultaneously; the monosaccharides of PGPA1

are also composed of fucose and pyranose***simultaneously; and the monosaccharides of PGPA3 are composed of pyranose, and the glycosidic bonds are of α-type. The monosaccharides of PGPA3 are composed of pyranose with α-type glycosidic bonds and may contain β-type glycosidic bonds.1H-NMR spectra showed that the glycosidic bonds of PGPN

are all of the α-type, and the glycosidic bond types of PGPA1 are all of the β-type; and the glycosidic bond types of PGPA3 are both of the α-type and of the β-type.