| The oxidation of the organism is one of the inducement causes of the diseases related to the cancer, arteriosclerosis, heart and cerebral vessels, etc. Therefore, the research and development about the antioxidant has been aroused great concern all along. As the pathways of the antioxidation in the organism are complex and variable, the single antioxidant has difficulty in reaching the ideal antioxidation. To improve the high efficiency of the current antioxidant, this research selects the black fungus with much polysaccharides and Pinus koraiensis with rich polyphenols as the main raw material, and divides the Auricularia Auricular polysaccharide into several segments to conduct active tracking, and deals with sulfating modification on the classified polysaccharide with relatively high oxidative activity, and jointly uses the classified polysaccharide of the black fungus, sulfating polysaccharide segment, and the Pinus koraiensis polyphenol, and carries out antioxidation analysis on the drug combination through the in vitro and in vivo experiment. The specific content and research result are as follows:1. Select three kinds of solution with different concentration (pure water, 0.1mol/L hydrochloric acid, and O.lmol/L sodium hydroxide) to extract the Auricularian, and then apply ethanol with different concentration to the extracted Auricularian to precipitate different classified polysaccharide, and screen out ABTS radical scavenging activity towards each classified polysaccharide. Obtain three kinds of superior classified polysaccharides and analyze their radical scavenging activity, lipid peroxidation inhibitory activity and the antioxidant activity of the gross reducing power in vitro. It reaches the conclusion that: the antioxidant activity of NTAAP90% in vitro is the strongest.2. Adopt concentrated sulfuric acid method to carry out molecular modification towards the selected optimal classified polysaccharide, and optimize the optimal technological condition of preparing the sulfating Auricularian and select preparation conditions according to the process optimization result: reaction time 1h, reaction temperature -2℃, and reaction agent proportion 1:3, and then the obtained substitute value of the sulfating polysaccharide is 0.5170. Through the infrared spectroscopy, it displays that NTAAP90% successfully connects to the sulfuric acid group through the sulfating.3. Measure IC50 value of the antioxidant indexes in vitro (ABTS radical scavenging activity, OH radical scavenging activity, superoxide radical scavenging activity and inhibitory activity of the lipid peroxidation) under the single action, and take the specific value of IC50 of the active substance composing the compound group as the reference, select the proportions of 50:1,100:1 and 150:1 as the transactional analysis of ComboNP and ComboSP. Use the drug combination analysis software to conduct "CompuSyn" analysis and achieve the following result: the measurement of ComboNP in ABTS radical scavenging activity is antagonism; the superoxide radical scavenging activity and inhibitory activity of the lipid peroxidation present slight synergy. However, ComboSP shows relatively good synergy of eliminating ABTS free radical and superoxide radical, and it is also reflected on inhibiting the lipid peroxidation activity. Besides, its interaction has obvious correlation with the proportion of SNTAAP90% and PP. When SNTAAP90% and PP compound at the proportion of 150:1, ComboSP presents strong synergistic effect.4. Adopt 60Co-γ ray radiation on the mouse, establish an oxidative damage model, and study the effect of ComboSP on the antioxidation in the body of the mouse. The result shows that, ComboSP can effectively prevent DNA from dispersing, reduce the level of lipid peroxidation product MDA in the mouse, and strengthen the synthesis and regeneration of the antioxidant substance (GSH, VE, VC and COQ10). Moreover, it indicates that ComboSP can effectively prevent the ionizing radiation damage.This research carries out sulphating modification on the classified polysaccharide of the black fungus with certain oxidation resistance, and conducts compound study on the classified polysaccharide and Pinus koraiensis polyphenol after the sulfating, and obtains compound with strong synergistic effect, which is also proved through the in vivo experiment. This finding has certain theoretical value on revealing the antioxidation pathway, and also has practical significance on developing the antioxidant with high natural efficiency. |
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