Preparation Of Homogeneous Codonopsis Pilosula Polysaccharides And It’s Sulfate Derivative And Preliminary Study Of Antitumor

Objective: To prepare and analysis the structure of purified homogeneous codonopsis polysaccharide and sulfated derivative. At the same time, homogeneous polysaccharide and it’s sulfate derivative on antitumor activity were evaluated. Methods: The sevege method was used to remove protein in Codonopsis pilosula polysaccharide(COP). Two fractions of COP, namely COP-1 and COP-2, were isolated by Sephacryl S-200 HR and Sephadex G-25 column chromatography. The third fraction, COP-3, was purified on Sephacryl S-200 HR and Sephadex G-75 column chromatography. High Performance Gel Permeation Chromatography(HPGPC) was used to determinate the purity and molecular weight of COP-1, COP-2 and COP-3. In addition, their purity was verified by melting point analysis. High Performance Liquid Chromatography(HPLC) was applied to analyze the monosaccharide composition of COP-1 and COP-2, while COP-3 was detected by Gas Chromatography coupled with Mass Spectrometry(GC-MS). Their structures were elucidated by IR and NMR(1H-NMR, 13C-NMR, HMBC, HSQC and 1H-1H COSY). Their sulfated derivates were prepared by amino sulfonic acid, and the degree of substitution of polysaccharide sulfate was determined by barium chloride- gelatin turbidimetric method. The effects of(COP-1,COP-1)and it’s sulfation on HT29 colon cancer cells by CCK8 method. Results: The average molecular weight of COP-1, COP-2 and COP-3 was 2.1×103 Da, 9.9×103 Da and 2.34×104 Da. The main construction of COP-1 and COP-2 was β-D-(2�'1) fructose. The monosaccharide constitution of COP-3 was glucose, galactose, mannose and its molar ratio was 4.3:1:3.7. Range of melting point of COP-1, COP-2 and COP-3 was: 250.2~252.8 ℃, 264.7~266.9 ℃ and 313.6~315.8 ℃, respectively. The substitution degree of COP-1, COP-2 and COP-3 was DSSCOP- 1 = 0.28, DSSCOP- 2 = 0.25, DSSCOP- 3 = 1.63, respectively. There were difference in instatistics between COP-2 carnitine concentration group and control group with 50 50μg/m L dose(P<0.01), but no statistical significance under other concentration. However, The differences of SCOP-2、COP-1 and SCOP-1carnitine concentration group and control group with different concentrations had no significance(P>0.05). Conclusion: The purified of COP-1 and COP-2 were homogeneous and neutral polysaccharide and mainly composed of β-D-(2�'1) fructose, while COP-3 was a complex heteroglycan. The melting process of three homogeneous polysaccharides was around 2 ℃, so they had a higher purity. COP-1, COP-2 and COP-3 were successfully sulfated, and three different substitution degree of polysaccharide sulfates were obtained. In our preliminary study: COP-1、COP-2、SCOP-1 and SCOP-2 can’t inhibit HT29 colon cancer cells growth.