| Ionizing radiation could induce oxidative stress and cause the serious damage of hematopoietic system and gastrointestinal function in the body, or even lead to genetic mutation and carcinogenic. Therefore, looking for natural and non-toxic radioprotective substances which could effectively reduce oxidative damage, enhance immune activity become a hot topic. This study selected polyphenols from Malus baccata (Linn.) Borkh.(MBP) as the research object, obtained the best antioxidant component-actone extract MBP-3by separating of MBP extracts using different solvent, optimized the separation and purification processes of MBP-3and characterized the structure of40%(v/v) ethanol eluate MBP-3b, which possessed the best antioxidant activity; systematically studied the protective effect of MBP-3b on oxidative damage of organism induced by radiation and revealed the mechanism of radioprotective effect.MBP-1~4were obtained respectively by extracting using four kinds of solvents with different polarity and analyzed the contents of polyphenols. Established of different antioxidant system in vitro, the free radical scavenging activity, inhibition of lipid peroxidation and reducing power of MBP were systematically researched. The results showed that MBP-3possessed the most abundant of total polyphenols, flavonoids and anthocynins. All MBPs could scavenge physiological and non-physiological free radicals in different degree. MBP also had significant reducing power and could effectively inhibit lipid peroxidation of rat liver, and. The biological activity of MBP-3was better than the others. This implied that MBP-3possess the better ability of supplying electron and hydrogen, which could terminate free radical chain reaction and inhibite oxidative stress, as well as inhibited lipid peroxidation.Ultrasonic-assisted extraction process was used to separate MBP, the optimized extraction conditions obtained by response surface methodology were as follows: acetone as the extraction solvent, liquid/material was13:1, extraction temperature47℃and ultrasonic power330W, and the content of polyphenols was reached to1170.45mg GAE/100g. The best purification parameters of MBP-3on NKA-9chromatography resins were as follows: loading sample volume1/5BV, loading flow rate1.5BV/h, elution flow rate2.0BV/h and40%(v/v) ethanol eluate-MBP-3b possessed the highest content of total phenolic with the best antioxidant activity.MBP-3b constituents were analyzed and identified by UPLC-Q-TOF-MS, UV-Vis and FT-IR spectrums The results indicated that MBP-3b was composed of ten kinds of phenolic compounds, including delphinidin-3,5-diglucoside, cyanidin-3-glucoside, chlorogenic acid, proanthocyanidins C1, quercetin-3-galactoside, quercetin-3-glucoside, quercetin-3-xyloside/arabinoside, phloretin-2-xyloseglucoside, quercetin-3-rhamnoside and phlorizin, their relative quality percents were respectively3.60%,9.29%,11.96%,1.64%,18.74%,6.63%,4.75%,7.16%,4.32%and18.24%.The oxidative damage model of splenocytes and peripheral blood leukocytes induced by60Co γ radiation was established in vitro. The survival of splenocytes, redox state in splenocytes, peripheral blood leukocyte morphology, DNA damage of splenocytes and contents of ROS in splenocytes after radiation were analyzed. The results indicated that MBP-3b could effectively reduce the amount of damaged leukocytes, increase the activity of antioxidant enzymes SOD and CAT in splenocytes, and accordingly reduce the contents of lipid peroxidation products MDA. Thereby, MBP-3b inhibited the generation of ROS, then lowered the process of cellular oxidative stress and inhibited DNA damage, thus increased the survival of splenocytes after radiation.The oxidative damage model of mice induced by60Coγ radiation was established. By analyzing DNA damage, immune activity and activity of antioxidant defense system to investigate the radioprotective effect of MBP-3b. The results found that MBP-3b could promote immune function, enhance the enzymatic (SOD, CAT, GSH-Px and GST) and non-enzymatic (T-AOC and GSH) antioxidant defense system and lower lipid peroxidation after radiation, then effectively inhibit the micronucleus and chromosome aberrations. These indicated that MBP-3b possessed radioprotective effect on oxidative damage of mice.The morphology and apoptosis of small intestine and spleen of mice after radiation were studied. The results suggested that MBP-3b could alleviate the lesions of spleen and small intestinal tissues, and reduce a large number of immune cells apoptosis induced by radiation. The related proteins of apoptosis in mitochondrial pathway were determined using immunohistochemical methods. The results showed that MBP-3b could significantly inhibit the expression of pro-apoptosis protein Bax, promote the expression of anti-apoptosis protein Bcl-2, thereby inhibited the release of Cytochrome c and the expression of activity subunit of Caspase-3, and then effectively protect the splenocytes away from oxidative damage induced by radiation.The research indicated that MBP-3b possessed significant antioxidant activities and could promote the function of enzymatic and non-enzymatic antioxidant system, reduce the excessive production of ROS induced by60Co γ radiation. It could also regulate the expression of associated protein in mitochondrial apoptotic signal pathway. Accordingly, MBP-3b showed protective effect on oxidative damage of organism. The present study possesses theoretical and practical implication to revealing radioprotective mechanism of polyphenols and exploiting natural raidioprotectors. |
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