Study On Extraction, Isolation, Purification And Structure Of Polysaccharides From Platycodon Grandiflorum(Jacq.)A.DC.

Platycodon grandiflorum(Jacq.)A.DC. as a traditional Chinese medicine was planted widely in China. In many countries, it was used for both medicinal materials and edibles. Pharmacological studies indicated that polysaccharides of Platycodon grandiflorum (PGP) had important activities of antitumor and imm-unoenhancement. So in this study, the auther conducted a systematic study on PGP, which were content determination, extraction, isolation, purification and structure analysis. Research methods and results were sumarized as follows:1. The content of polysaccharides was determined indirectly. The total sugar content (including polysaccharides and reducing sugars) of crude polysaccharides was determined by anthrone-sulfuric acid method. The monosaccharide content was determined by 3,5-dinitrosalicylic acid (DNS) method, indirect iodometric method, and fehling titration method, respectively. The difference between the total sugar content and the monosaccharide content was the content of polysaccharides. The content of PGP that measured by three methods was 95.55%,96.01%,95.75%, respectively. The data showed that the results of three methods were similar. While considered to experiment costs, equipments, operation and the factors that affected the measurement, anthrone-sulfuric acid method combined with DNS method was used to determine the content of polysaccharides.2. The extraction of PGP was used the traditional hot water extraction and alcohol precipitation. During the extraction process, temperature, time, solid-liquid ratio and times of extraction were examined. On the basis of the single factor, orthogonal experiment was used to optimize the extraction process of PGP. The optimum conditions by orthogonal experiment with hot water were solid-liquid ratio 1:25 with temperature of 80℃for 2 hours and extracting 2 times. The extraction percent of PGP was 42.51%by three sub-parallel test with the optimum conditions.3. Three methods were used to remove the protein which existed in the crude polysaccharides, such as Sevag method, trichloroacetic acid method, and trichloroac-etic acid-n-butanol method. Using of protein removal rate and loss rate of polysacch-arides as the measurement standard, the result showed that the effect of Sevag method removing the protein was better than the two other methods. Its process conditions were as follows:polysaccharides:chloroform:n-butanol= 25:5:1, stirring for 20 minu-tes. H2O2 oxidation was used to remove the colour of the crude polysaccharides, which process conditions were as follows:polysaccharides:H2O2=4:1, using concent-rated ammonia or 0.1%NaOH adjusted pH value 8.8 and putting at temperature of 37℃for 12 hours. Sephadex G-25 was used to desalt and remove small molecules.4. Purified polysaccharides were isolated by DEAE-cellulose 52 column, and three fractions were mainly got which were named as PGPN, PGPA1 and PGPA3. The three fractions were further isolated by Sephadex G-100 column, respectively, which were all single symmetrical elution peak. The purity of three fractions was tested by Sephadex G-100 column chromatography, optical rotation and UV spectro-photometry assay, and the results showed that they were all homogeneous polysacch-ride and didn't have protein and nucleic acid. The molecule weight of three fractions were determined by Sephadex G-200 column chromatography, which were 10233, 4677,23442, respectively.5. The monosaccharide compositions of PGP were analyzed by TLC. The result of TLC showed that PGPN was composed of fructose and contained either glucose or galactose and maybe had the two both, and PGPA1 was composed of galactose and fructose, while PGPA3 was consisted of xylose and galactose. IR spectrum confirmed that the type of monosaccharides existed in PGPN was furan sugar and pyranose, which existed in PGPA1 was as same as in PGPN, and PGPA3 only contained pyran-ose while IR spectrum showed that it had a-type glycosidic bond and maybe hadβ-type glycosidic bond.'H-NMR spectrum indicated that PGPN was all a-type glycol-sidic bond, PGPA1 was allβ-type glycosidic bond, and PGPA3 not only had a-type glycosidic bond, but also had P-type glycosidic bond, which confirmed the results of IR spectrum.