| Orange belonging to genus Rutaceae of Citrus family,is one kind of the important agricultural economic crops.Oranges which are rich in bioactive substances play an important role in maintaining human health and preventing diseases.Orange fleshes are usually used for fresh food,juicing and processing into cans.However,the agricultural wastes such as peels and flesh residues produced at the same time are neglected,which greatly reduces the added values of agricultural products and causes resource wastes.In this study,the compositions and contents of phenolics,antioxidant activity,and anti-proliferation activities towards human liver cancer cells of three parts of oranges(peel,flesh and flesh residue)were investigated.The effects of orange extracts on cell cycle and apoptosis were analyzed by flow cytometry,and the mechanism of antiproliferation was explored at the protein level.Moreover,the aging model of Caenorhabditis elegans was used to determine the anti-aging effects and their mechanisms of orange flesh extracts,providing a theoretical basis for the further development of related products.There are mainly the following research results:(1)Peels,fleshes and flesh residues of oranges have antioxidant activity,and the activities of different parts of oranges varies greatly.The orange peels contained the highest amounts of vitamin C,total phenolic compounds and total flavonoid compounds,but these contents in the orange fleshes were relatively low(p < 0.05).Twelve kinds of phenolic compounds including four kinds of phenolic acids and eight kinds of flavones were identified in each part of oranges by HPLC.The contents of hesperidin were the highest among them.The antioxidant activity in vitro was significantly positively correlated with the contents of total phenolic compounds and total flavonoid compounds.Generally,orange peels have excellent antioxidant activities.(2)The free extracts of orange peels had the best anti-proliferative activity against human liver cancer cell line Hep G2.Among individual compounds,hesperidin,nobiletin,tangeretin,hesperetin and sinensetin showed strong anti-proliferative activities.(3)Cell cycle analysis and cell apoptosis experiment were determined to reveal the anticancer mechanism of orange extracts.The results of cell cycle experiment showed that orange peel extract could inhibit the growth of cancer cells by blocking Hep G2 cells in G0/G1 phase.In the cell apoptosis experiment,it was found that the apoptosis rate of Hep G2 cells in orange peel extract treatment groups were significantly higher than that in the control group after 24 hours,with a dose-dependent manner.Western blot analysis revealed that the orange peel extracts significantly down-regulated the expression levels of Cyclin D1 and CDK4 proteins in human liver cancer Hep G2 cells in a dose-dependent manner,indicating that orange peel extracts inhibited Hep G2 cells proliferation by down-regulating Cyclin D1 and CDK4 via G1/G0 cell cycle arrest.(4)In this study,C.elegans model was used to evaluate the effects of orange extracts on lifespan and healthspan of nematodes.Results showed that the orange extracts can prolong the mean lifespan of C.elegans from 20.91 ± 1.60 days of control to 26.38 ± 2.06 days in orange extracts treatment group with highest concentration,with an increase from 10.5 to 26.2%.The resistant abilities against thermal stress,uv radiation stress and oxidative stress(paraquat induced)of nematodes were improved by orange extracts treatment.Meanwhile,orange extracts promoted the healthspan by improving their locomotor,and decreasing the accumulation of lipofuscin without damaging reproductive ability and changing of body length.In addition,orange extracts could also reduce the accumulation of intracellular reactive oxygen species and malondialdehyde and improve the activities of antioxidant enzymes including SOD and CAT.(5)To reveal the molecular mechanisms of anti-aging,the RT-PCR experiment were carried out.The results showed that orange extracts increased the expressions of daf-16 and down-regulated the expression of age-1 in the IIS pathway.The expressions of skn-1 and sek-1 in MAPK pathway were both up-regulated to improve the stress resistant ability of nematodes.The expressions of antioxidant enzyme genes(sod-3,gst-4)were increased,and the oxidative stress resistances of nematodes were improved.Compared with the normal N2 nematodes,there were no significant differences in the lifespan of mutant strains GR1307 and LG333,indicating that orange extracts might prolong the lifespan of C.elegans via daf-16 and skn-1 genes. |
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