| Crabapples, belonging to the Malus genus(Rosaceae), are widely grown in China as ornamental trees for their colourful leaves, beautiful flowers or fruit. They are not only have the high ornamental value, but also have a rich source of bioactive substances, and these natural bioactive compounds can be used in pharmaceutical, chemical, food and other fields, these bioactive products manufacturing have attracted more and more attention in the development of China’s agriculture. In which dihydrochalcone as ‘a handful of flavonoids’ compounds and have a potential value; which the dihydrochalcone phloridzin is rich in apples, the high-potency, low-caloric and whitening property, so it could be used as a natural sweeteners and whitening agent added to food and cosmetics industry. Crabapples are a wild resources and the flavonoids content is larger than the cultivated apples, it has a great value of research and exploitation, but the study of flavonoids have little reported in Malus crabapples, especially the dihydrochalcones. Therefore, a comprehensive study of the flavonoids distribution, explore the bioactivator, screening the high content of dihydrochalcone in crabapples is necessary; and find the key genes and transcription factors in dihydrochalcones synthese pathway is necessary for their opening up and utilization in future.In this study, the typical varietie with new colored leaf ‘Radient’ as the main materials, the typical varieties with ever-red color leaves ‘Royal’ and ever-green leaves‘Flame’ as contrast materials. Firstly, we detect the flavonoids in roots, leaves, petals, peel and pulp of crabapples by HPLC-MS and UPLC-MS; according to the HPLC analysis, we analyze the flavonoids content of the leaves, petals and fruits in their development stage; besides, we focus on analysised the dihydrochalcones distribution of crabapples. Secondly, we analyze the diversity indices, principal component analysis and cluster analysis to select the best development period of spatiotemporal pattern and the dominant plant material. Thirdly, according the on-line and off-line methods, animal model experiments, the study show that each flavonoids compounds cooperate with each other and produced a synergistic antioxidant function in crabapples. Fourth, we extract and purify 20 kinds of flavonoid monomers, including 7 kinds of dihydrochalcone monomers. Fifth, our study verify the dihydrochalcones anticancer activity by MTT method. Finally, we verify the key MYB transcription factor and main structural genes in regulation of dihydrochalcone compound metabolic pathway by molecular biology technology.The main important results obtained are as follows:1. According to HPLC-MSn and UPLC-Qtof-MSn analysis methods, we find that the crabapples leaves contain the most kinds of flavonoids, ‘Royalty’ is 17, ‘Flame’ is 21, ‘Flame’ is 15; followed was the flowers, ‘Royalty’, ‘Radiant’ and ‘Flame’ all are 17; the next are peel and fresh, ‘Royalty’ peel is 15 and fresh is 9, ‘Radiant’ peel is 15 and fresh is 10, ‘Flame’ peel is 11 and fresh is 9; the roots with the least flavonoids and only 3 types in these three cultivates. These flavonoids compounds included anthocyanin, flavonol, dihydrochalcone, flavanols and dihydroflavonol, in which there are 13 kinds of dihydrochalcones in crabapples and the cultivar ‘Radiant’ contains the most types of dihydrochalcones; by HPLC analysis, the most flavonoids content is leaves, next is petals, peel and fresh, and the most dihydrochalcone phloridzin content is root; in addition, according to the diversity analysis, principal component analysis and cluster analysis, we established an analysis method and this method can be applied to the analysis of the complex flavonoid system in other plants.2. To compare the antioxidant capacity of RL and GL, we measured the SOD and CAT activities, MDA content and T-AOC in vitro and vivo, our results showed that SOD and CAT activities were significantly higher and the MDA content was significantly lower in RL than in GL. T-AOC was significantly higher in RL than in GL. According the HPLC-ABTS analysis, our result showed that the high antioxidant capacity of RL might result from not only the high concentrations of most compounds but also the high antioxidant activities of several individual compounds and their synergistic effects with the flavonoid compounds. The linear regression equation show that the GLU, TC and TG levels are negatively correlated with T-AOC, and indicate that consumption of the RL extract may improve antioxidant capacity, decreasing pathological indices in rat blood. The results show that intaked of the antioxidant could enhance the resistance against disease, increase the body protect function.3. We extract 20 flavonoids monomers from crabapples by separation and purification system and NMR techniques. They are included 8 kinds of flavonols, HTY-1(juglanin,418.35), HTY-2(avicularin),HTY-3(quercetin-3-O-glucoside),HTY-5(quercetin-3-O-rhamnoside),HTY-8(quercetin-3-O-xyloside), HTY-9(quercetin-4’-O-glucoside), HTY-10(rutin),HTY-30-1(quercetin); two kinds of dihyflavonols, HTY-14(astilbin), HTYZB-4(dihyquercetin-3-O-glucoside); 7 kinds of dihydrochalcones, HTY-4(trilobatin),HTY-12(phloridzin), HTY-30-2(phloretin), HTY-30-5(3-hydroxyl-phloretin), HTYZB-1-3(6’’-O-coumaroyl-41-O-glucopyranosylphlo retin, crabapples dihychalcones A),HTYZB-1-4(3’’’-methoxy-6’’-O-feruloy-4’- O-glucopyranosyl-phloretin, crabapples dihychalcones B),HTGL-4(phloretin rutinoside); 1 kind of flavanol, HTY-11(epicatechin); 1 kind of phenolic acid, HTY-15(4-O- caffeoylquinic acids); 1 kind of dihyflavone, HTY-30-3(eriodictyol); in which the two new rare dihydrochalcones are HTYZB-1-3(6’’-O-coumaroyl-4’-O-glucopyranosylphloretin, crabapples dihychalcones A) and HTYZB-1-4(3’’’-methoxy-6’’-O-feruloy-4’-O-glucopy ranosyl- phloretin, crabapples dihychalcones B).4. MTT cancer cell growth inhibition assay demonstrate that seven dihydrochalcone compounds separate and purify from Malus crabapple var. ‘Radiant’ leaves display remarkable biological activities. They show significant positive anticancer activities against several human cancer cell lines, including the A549 human lung cancer cell line, Bel 7402 liver cancer cell line, Hep G2 human ileocecal cancer cell line, and HT-29 human colon cancer cell line. A7 have significant effects on all cancer cell lines, suggesting potential applications for phloretin and its derivatives. Adding a methoxyl group to phloretin dramatically increases phloretin’s anticancer activity. This article shows that Malus crabapples may be a valuable resource of anticancer effects, and the two new rare compounds A6 6’’-O-coumaroyl-4’-Oglucopyranosyl-phloretin and A7 3’’’-methoxy-6’’-O-feruloy-4’-O-glucopyranosyl-phloretin could be developed into promising anticancer agents. Therefore, we propose that the dihydrochalcones may have beneficial effects on human health and can be considered as possible therapeutic agents against cancer; at the same time, our results show that the ornamental crabapple may be a natural significant anticancer effect can also use of natural resources.5. We conducte MYB10 silence experiment by VIGS and real time PCR techniques, and the results show that the content of dihydrochalcones compounds and relative genes expression decreased. It illustrate that MYB10 transcription factor can regulate dihydrochalcones biosynthesis in leaves and peels of crabapples. Transcription factor MYB16 silence up-regulated dihydrochalcones biosynthesis. Acetylation regulate NADPH synthesis by down-regulating ENRL gene and inhibited dihydrochalcones phloridzin biosynthesis. Deacetylation promote dihydrochalcones phlorizin biosynthesis by up regulating NADPH biosynthesis. Our results show that the key enzymes in the regulation of dihydrochalcones phloridzin metabolism pathways in ornamental crabapples, NADPH and Mc CHS can promote the biosynthesis of phloridzin in a certain extent, but not plays a decisive role in the regulation of phloridzin biosynthesis. The up-regulation of Mc ENRL has a direct effect on phlorizin biosynthesis.Based on the above results, the results of this study are demonstrated that the crabapples are rich in antioxidant flavonoids, especially, the dihydrochalcone compounds are natural bioactive substances and displayed remarkable anticancer activities. The two new rare compounds A6 6’’-O-coumaroyl-4’-O-glucopyranosyl-phloretin and A7 3’’’-methoxy-6’’-O-feruloy-4’-O-glucopyranosyl-phloretin could be developed into promising anticancer agents. At the same time, we reveale the key genes and transcription factors in regulation of dihydrochalcone compounds, our study support the basal important theory and technical support of flavonoids in Malus crabapples. |
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