Study On Biological Activities Of KGM And Its Degradation Products

Being KGM as raw materials,irradiation and enzymatic hydrolysis treatments were conducted to obtain KGM samples with different molecular weights and their oligosaccharide products. These samples were characterized via infrared spectrometry and gel permeation chromatography with laser scattering instrument analysis. Moreover, their biological activity was studied in detail so as to provide a more effective method for obtaining KGM degradation products and a theory basis for broadening the application of KGM and its degradation products. The main studies are as follows:1 KGM was irradiated by 60 Co with different irradiation dosages to get KGM degradation products with different molecular weights(10KGy-KGM, 20KGy-KGM, and 100KGy-KGM). Gel permeation chromatography with laser scattering instrument analysis showed tha the molecular weight was degraded from 923.8 k Da to 307.8 k Da,169 k Da and 53 k Da, respectively. The root mean square radius decreased from 108.5 nm to 53.4 nm, 40.2 nm and 20.2 nm. And the molecular conformation changed from a random coil to a branched structure(10KGy-KGM, 20KGy-KGM) and a spherical conformation with branches(100KGy-KGM), respectively. The infrared spectrum proved irradiation had no effect on acetyl groups.2 KOS samples were obtained by endo-β-1,4-mannanase hydrolysis of 100KGy-KGM. The infrared spectrum and mass spectrum analysis shows the degradation products should be oligosaccharides with acetyl groups and a degree of polymerization from 2 to 10.3 Based on the H2O2 oxidation damage model, the cell survival rate,LDH in cell culture supernatant, the content of antioxidant activity enzymes(CAT, GSH-Px, MDA, SOD) and oxide active enzymes(ROS) in cells, as well as the amount of free Ca2+ were measured to analyze the antioxidant activity of KGM and its degradation products. The research results showed that KGM, 10KGy-KGM, 20KGy-KGM and 100KGy-KGM had no obvious effect on the growth of normal L02 cells; KGM, 10KGy-KGM, and 20KGy-KGM did not effect on L02 cells damaged by H2O2 oxidation. But KOS and 100KGy-KGM could reduce H2O2 oxidation damage to cells. KOS and 100KGy-KGM could decrease the content of LDH in cell culture supernatant and the amount of ROS, free Ca2+ and MDA in cells, while increased the intracellular content of GSH-Px, CAT and SOD. KOS showed the most significant effect at a concentration of 7 mg/m L(P <.05), whereas 100KGy-KGM works most effectively at a concentration of 400 ng/m L(P < 0.05). Therefore, KOS and 100KGy-KGM could exhibit antioxidant activities by clearing ROS,by reducing the production of MDA and improving the activity of antioxidant enzymes. Their functional mechanism may be related to ROS clearance and the inhibition of intracellular calcium ions elevation.4 The effect of KGM and its degradation products on RAW 264.7 cell growth, neutral red phagocytosis, NO and TNF-α release was determined to study their immuno-functionality. The results indicated that KGM, 10KGy-KGM and 20KGy-KGM had not affect no RAW264.7 immuno-competence, but KOS and 100KGy-KGM could significantly enhance the capability of macrophages uptaking neutral red, NO and TNF-α release, as well as cell proliferation.KOS works best at concentrations of 2.5 and 5 mg/m L(P < 0.05), while 100KGy-KGM shows excellent effect at a concentration of 1.6 mg/m L(P < 0.05). Hence, it is speculated that KOS and 100KGy-KGM affect cell immunocompetence mainly by enhancing macrophage phagocytosis and releasing cell factors.5 The inhibitory effect of KGM and its degradation products on cancer cells was analyzed by MTT, which was then associated with DAPI staining to analyze their anti-tumor activity in vitro. The results show 10 mg/m L KOS could significantly inhibit Hep G2, Hela and Bel-7402 cell growth, with inhibition rates of 19.4%, 29.9% and 17.1% respectively, and induce Hep G2 cell apoptosis. 1.6 mg/m L 10KGy-KGM, 20KGy-KGM and 100KGy-KGM could significantly inhibit Hep G2 cell growth, with inhibition rates of 14.3%, 9.5% and 13.7% separately, and induce Hep G2 cell apoptosis; 1.6 mg/m L 10KGy-KGM and 100KGy-KGM could significantly inhibit Hela cell growth, with inhibition rates of 8.8% and 11.2% respectively, whereas 20KGy-KGM, with an inhibition rate of 9.2%, did not show significant difference. The inhibition rates of 10KGy-KGM, 20KGy-KGM and 100KGy-KGM on Bel-7402 cells were 8.1%, 4.6% and 3.8% separately, exhibiting no significant differences. In conclusion, KGM and its degradation products have antitumor activity in vitro, which is various with different molecular weights and cell types.