Determination And Analysis Of The Polysaccharides From Codonopsis Pilosula And Its Protection Activity Against DNA Damage

Radix Codonopsis is the dry root of Codonopisis pilosula (Franch.)Nannf., Codonopsis pilosula Nannf. var. modesta (Nannf.) L. T. Shen or Codonopisis tangshen Oliv. belonging to codonopsis genus, campanulaceae family. It is a well-known traditional Chinese herbal medicine, and beneficial to the immune, digestive and hematopoieric systems. It can be used to treat fatigue, thirst and loss of appetite. Fengdang, trade name Xidang, one of C. pilosula, which is produced in Fengxian, Shaanxi province, is famous for its good quality and commonly used by herbalist doctors. The polysaccharide from Radix Codonopsis is one of the main effective composition, playing a very important role in organisms, such as immunity regulating effect, radical scavenging activity, hematopoiesis, as well as resisting senility, so people are taking more of an interest in it. Here, we studied Fengdang from three aspects.Firstly, we studied the dynamic features of yield, the polysaccharide content and the extraction by 45% ethanol in the roots of Fengdang harvested in different years and different times. The results suggested that the best harvest time for Fengdang was in late October and early November of the third year. The results provided scientific data for the GAP research.Secondly, we analyzed the monosaccharides of polysaccharides from Fengdang by a PMP procolumn derivatization HPLC method. The fats of air-dried roots of Fengdang were removed with alcohol and polysaccharides extracted with hot distilled water. After dialysis and deproteinization, the extracts were hydrolyzed by TFA, derived by PMP, and then determined by HPLC. The effect of kalium phosphate and ammonium acetate on the HPLC were compared in order to optimize the mobile phase. We extended this HPLC method to polysaccharides from Gentiana macrophylla and gynostemma pentaphyllum. The results were as follows: (1) For ammonium acetate phase, the optimal mobile condition consisted of 100 mmol/L ammonium acetate (pH 5.5) with (A) 10% and (B) 25% acetonitrile, using a gradient elution of 45%→75%→75%→85% →85%→100% buffer B by a linear increase from 0 min→10 min→18 min→40 min→50 min 4→80 min. (2) In the optimal ammonium acetate phase, there were 8 peaks in the mixture of 9 monosaccharides. Xylose and arabonose did not separate. (3) For kalium phosphate phase, the optimal mobile condition consisted of 50 mmol/L kalium phosphate (pH 6.9) with (A) 15% and (B) 40% acetonitrile, using a gradient elution of 10%→11.6%→14%→20% buffer B by a linear increase from 0 min→5 min→10 min→60 min. (4) In the optimal kalium phosphate phase, there were 9 peaks in the mixture of 9 monosaccharides. Though galactose, xylose and arabonose did not sepatate completely, its effect was better than ammonium acetate phase. (5) Polysaccharide sample of Fengdang was analyzed using optimal kalium phosphate phase. Six monosaccharides were detected, which were mannose, rhamnose, galacturonic acid, glucose, galactose and arabonose with molar ratio of 0.07:0.22:1.00:4.38:0.81:1.40. (6) Polysaccharides from Gentiana macrophylla and gynostemma pentaphyllum were performed by this method. The former comprised mannose, rhamnose, glucuronic acid, galacturonic acid, glucose, galactose and arabonose, whose molar ratio was 0.06:0.25:0.03:1.00:1.23:0.87:3.52; the latter consisted of mannose, rhamnose, glucuronic acid, galacturonic acid, glucose, galactose, xylose and arabonose, whose molar ratio was 1.01:4.01:0.35:1.00:31.6:7.45:4.65:8.56. (7) Differences in the kinds and contents of the monosaccharides from the polysaccharides of different Chinese herbs were found by comparing polysaccharide samples from Fengdang, Gentiana macrophylla and gynostemma pentaphyllum.Thirdly, the protecting activity of polysaccharides against DNA damage caused by UV-photolysis of H₂O₂ was investigated. The results were as follows: (1) The effect of polysaccharide from Fengdang on protecting DNA against damage increased at first, then decreased with the increase of concentration. 0.3 mg/mL of polysaccharide was optimal, and had the same effect as rutin. (2) Polysaccharide from Gentiana macrophylla had no effect. (3) The effect of polysaccharide from gynostemma pentaphyllum on protecting DNA against damage showed a concentration dependent activity. 3.0 mg/mL was optimal.