Recovery, Purification And Biological Activities Of Polysaccharide From Cordyceps Fermentation Broth

Cordyceps is one of the most valued traditional Chinese medicines. Some bioactive components extracted from Cordyceps were found to have antitumor, immunomodulating, antioxidant, anti-inflammatory and hypoglycemic activities. Submerged fermentation is an important way to enhance the yield of Cordyceps and avoid destroying the scarce resource, but the fermentation broth used to be discharged as liquid waste which polluted environment seriously.In this paper, the fundamental ingredients in Cordyceps （Paecilonyces hepialid chen & Dai Cs-4） fermentation broth were analysed. The results showed that 421.9 mg/g total sugar, 122.8 mg/g reducing sugars, 32.7 mg/g D-mannitol, 0.18 mg/mL protein and 10.34 U/ml SOD were in the fermentation broth.The optimization for polysaccharide recovery from Cordyceps fermentation broth was investigated by using response surface methodology （RSM） . Aiming at the rate of recovery, the levels of pH value, amount of ethanol and deposited time were tested with one-factor experimental design, and the Box-Benhnken center-united experimental design was subsequently employed for further optimization. Eventually, the optimum extraction condition was confirmed by using Design Expert V7.1 as follows: pH value 4.88, 4.0 times amount of ethanol for precipitation and 20 minutes, the rate of recovery was 7.8%.To compare the bioactivities of the intracellular polysaccharide and the polysaccharide in fermentation broth in vitro, the reducing power and the scavenging activity of superoxide radical, hydroxyl radical, ABTS⁺ free radical, DPPH·free radical were measured. Moreover, the activity of anti-tumor including the mouse gastric carcinoma MFC cell, hepatoma HepG2 cell, cervical cancer Hela cell and mouse sarcoma S180 cell were analyzed. The obtained results were as follows: the intracellular polysaccharide had higher antioxidant activity than the polysaccharide in fermentation broth, but the polysaccharide in fermentation broth had higher activity of anti-tumor （MFC cell, HepG2 cell, Hela cell and S180 cell） and hydroxyl free radical scavenging. Especially, with regard to the Hela cell, the anti-tumor activity of the polysaccharide in fermentation broth is significant, IC₅₀ is 4.98 mg/mL, but the IC₅₀ of the intracellular polysaccharide is 13.8 mg/mL.CPS- I and CPS- II were obtained from crude polysaccharide by removing proteins using Sevag method, decolourization using H₂O₂ method, dialysis and column chromatography, CPS-I was collected subsequently. Homogeneity of CPS-I was verified by UV scan and polyacrylamide gel electrophoresis. Its average molecular weight was 93,325 Da, detected by Sepharose 4B column chromatography. The component of CPS-I was determined to be composed of carubinose and glucose in molar ratio of 1.94:1 by thin-layer chromatography and gas chromatography.