| With the rapid development of economy and society, large quantities of pollutants have been discharged into water bodies, resulting in serious eutrophication. Therefore, the frequency of harmful algal blooms, especially cyanobacteria, has increased dramaticlly in recent years. It is crucial to develop an effective technique for algal blooms control. Recent findings suggest that allelopathy presents an effective and environmentally friendly method to control cyanobacterial growth. Flavonoids are one kind of natural allelochemicals with multiple unique biological properties, and they exist broadly in the plant kingdom.In this dissertation, Microcystis aeruginosa was chosen as experimental microorganisms to explore the law of algal inhibition by a serious of natural plant flavonoids. The "chemical structures-antialgal ability" relationships of congeneric flavonoids were systematically studied using different theoretical tools including 2D-QSAR and 3D-QSAR, to construct predictive model for antialgal activity and explain the antialgal active sites and mechanism. Based on the study on physiological changes of cyanobacterial cells by FCM and PAM, mechanisms of algal control by natural flavonoids were also clarified. Last but not least, by utilizing sustained release microsphere technology, simulated-allelopathic beads of natural flavonoid were developed as antialgal agent, which offers great potential to control harmful cyanobacterial blooms. The main original conclusions of this dissertation are listed as following:(1) The inhibitory effects of 22 kinds of natural flavonoids on the growth of M. aeruginosa were investigated. The results showed that 5,4’-dihydroxyflavone, kaempferol, and luteolin exhibited the best anti-cyanobacterial activities among all tested flavonoids. Based on the "structure-activity" analysis, the effects of the number of hydroxyl groups on antialgal ability were reveals:The antialgal ability increases with the increased number of hydroxyl groups, but when there are 2 hydroxyl groups existing in the flavonoid molecule, the influence of the positions are more significant for the antialgal activity.(2) The quantitative structure-activity relationship (QSAR) method was innovatively introduced to predict the antialgal activity of allelochemicals. Using an antialgal activity dataset of thiazolidinediones for 2D-QSAR modelling, the modeling strategy for antialgal allelochemicals was summarized. Through the above strategy, the action of 22 kinds of flavonoids on M. aeruginosa was modeled using multiple linear regression (MLR) based on 2D-QSAR. The model was shown to have highly predictive ability and stability through strict internal and external validations, and provided insight that the inhibitory ability becomes better with the higher polarity, greater permeability, and better hydrophobicity of the molecule.(3) 3D-QSAR methods including CoMFA and CoMSIA were employed to investigate the antialgal activities of flavonoids. Both the constructed CoMFA and CoMSIA models exhibited satisfied robustness and predictive ability. Based on the 3D-QSAR analysis, the 3 position with hydrophilic groups, the 5 position of A ring with negatively charged and hydrogen bond donor groups, the 4’ position of B ring with hydrophilic and hydrogen bond donor groups, and 3’,4’ position with negatively charged groups are beneficial for the antialgal activity of flavonoids. However, the 6,7 position with negatively charged and hydrogen bond donor group, and the 5 position of A ring with negatively charged and hydrogen bond donor groups disfavor the antialgal activity of flavonoids. According to the 3D-QSAR model, one of the main inhibitory processes might be that flavonoid compound interacts with phospholipid molecules on the algal cell membrane through hydrogen bonding, resulting in the breakdown of the membrane function. And 5 and 4’ position with hydroxyl group were probably the active sites of the hydrogen bonding in this inhibitory effect. Furthermore, the interaction between flavonoid and phospholipid molecules was analyzed by TEM and FTIR. and the results proved the above hypothesis.(4) The biological responses of cell systems, especially the photosynthesis and membrane system, in an environment where flavonoids (5,4’-dihydroxyflavone, apigenin, luteolin) and cyanobacteria coexisted, were investigated. The results showed that luteolin interrupted the electron transport in the PS Ⅱ reaction center and then affected the photosynthesis efficiency, while 5,4’-DHF and apigenin induced membrane depolarization, and compromised the membrane integrity. The above physiological phenomena of cyanobacterial cells under flavonoid stress were coincidence to the 3D-QSAR analysis:5,4’-dihydroxyflavone and apigenin, which have 5 and 4’ hydroxyl groups, have negative effects on the cell membrane, while the ortho hydroxyl groups in B ring enhance the impacts of the flavonoid (luteolin) on the photosynthesis system.(5) Based on the sustained release microsphere technology, a novel antialgal agent, the simulated-allelopathic beads of natural flavonoid, were prepared, which is a more controllable application of allelochemicals than direct-added mode. The flavonoid release time of the beads can be consistently prolonged to ca.120 d through a diffusion-controlled release process, which is nearly equivalent to the time of allelochemicals released by natural macrophytes during the cyanobacterial bloom season. Based on the cyanobacterial inhibition bioassay and mesocosm experiment, the simulated-allelopathic beads showed better sustainable inhibitory activity and stronger oxidative stress against M. aeruginosa than direct flavonoid treatment in a long-term experiment. Applying the beads in the season before the recruitment of cyanobacteria can be even more effective. Therefore, the simulated-allelopathic beads of natural flavonoid have been shown to be a promising environmentally friendly anti-cyanobacteria product, which could be used to control the excessive growth of harmful cyanobacteria in eutrophic waters. |
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