| Cordyceps militaris is one of the edible and pharmaceutical mushrooms, which contains many nutrients and active substances. Crude cordycepin and cordyceps polysaccharide were extracted with hot water and deposited with ethanol. Comparing the effects of cordycepin separation with four chromatographic columns, Sephadex G-10 was choosen to obtain purified cordycepin; purified polysaccharides were further obtained by DEAE Cellouse-52 chromatography. The basic structure of polysaccharide fractions, the in vitro immune activity of the cordyceps polysaccharide and cordycepin were also investigated in the research.1. The crude extracts percent of Cordyceps militaris fruiting body were extracted with hot water, deposited with ethanol and separated. The yields of the extracts I and II were 16.99% and 3.24% respectively. By comparing 717 anion exchange resin (A), JK006 cationic exchange resin (B), D101 macroporous resin (C) and Sephadex G-10 (D), four different chromatography columns for separating cordycepin from extract I, the effect of separation was:D> C> A> B respectively. Finally,14.0 mg cordycepin was obtained from the Sephadex G-10 chromatographic column, with 98.78% purity.2. Crude extract II was applied onto DEAE Cellouse-52 chromatography, and three purified fractions, CP-1, CP-2 and CP-3 were obtained. The polysaccharide yields of the three fractions were 46.02%,10.86% and 7.54% respectively. As to crude polysaccharide, CP-1, CP-2 and CP-3, their polysaccharides content was 54.71% ,96.86%,68.28% and 92.86% respectively; uronic acid content was 7.86%,16.81%,5.36% and 7.86% respectively; protein content was 4.29%, not detected,6.92% and 0.07% respectively; sulfuric acid content was 10.53%,0.85%,3.43% and 1.54% respectively.Ultraviolet spectra indicated, CP-1 and CP-3 have no or less than detection of trace proteins, but CP-2 may contain a small amount of protein. Characteristic absorption peaks of polysaccharides, carboxyl and sulphuric acid base were revealed in FT-IR of the three fractions, which indicated the presence of pyranose in CP-1 and CP-3, and furanose in CP-2. With the HPAEC-PAD, CP-1 and CP-3 were mainly composed of glucose, galactose and mannose, and the molar ratios were 6:1:1 and 38:8:5 respectively; CP-2 was composed of glucose and fucose, and the content fucose was more than 20%.3. With macrophage proliferation and devouring of neutral red experiment in vitro, CP-1 and CP-3 revealed a remarkable ability in promoting macrophage proliferation and enhancing macrophage ability to devour neutral red in a dose-depedent manner. However, CP-2 and cordycepin showed certain inhibitory effect on proliferation and devouring neutral red of macrophage. The results of digital eyepiece pictures revealed, the number of macrophages cultured in the medium with CP-1 and CP-3, increased obviously, and the cells grew well; the number of macrophages treated with CP-2 decreased, but some cells still presented divisive status; macrophages treated with cordycepin displayed the irregular growth as small spots and the shrinkage nucleus which was obviously late form of cells.4. Through the cultivation of the liver cancer cells HepG-2, proved that CP-1, CP-2 and CP-3 could restrain HepG-2 proliferation in a certain degree. The highest growth inhibition rate of HepG-2 proliferation was 63.1%, which was caused by 20 μg/mL cordycepin. Digital eyepiece pictures showed, the liver cancer cells cultured with the three polysaccharides remained dividing, but the number of cells had certain reduction. The cells treated with cordycepin showed the irregular forms, protuberant shapes and little adhesive, and the cell number was obviously less than the blank control group.5. By CPs and macrophages co-culture medium stimulating liver cancer experiment, proved that the co-culture medium had significant growth inhibition on HepG-2 cells. The effect of inhibition was:CP-2> CP-3> CP-1 respectively.6. The results of IL-6 detection by ELISA displayed, the three fractions of cordyceps polysaccharides could stimulate macrophages to secrete IL-6. The macrophages cultured with 75 μg/mL CP-2 secreted the most IL-6 (99.5 μg/mL), which was 40.7% higher than the blank control group. |
PDF Full Text Request