中草药对铜绿微囊藻的抑制作用及机理研究
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摘要
铜绿微囊藻(Microcystis aeruginosa)水华的发生,导致养殖水环境恶化、对养殖对象造成危害,破坏水体生态环境平衡,为此本文研究了中草药对铜绿微囊藻的抑制作用及机理,为研制高效环保的铜绿微囊藻抑制剂提供参考。本文通过一系列的抑制铜绿微囊藻毒性实验,筛选出抑藻能力较强的中草药黄连,并就其主要成分生物碱对室内外铜绿微囊藻、模拟微生态系统及主要环境因子对抑藻效果的影响进行实验研究,并对生物碱抑制铜绿微囊藻的机理从藻类光合作用、抗氧化系统及超微结构等方面进行了深入系统的研究。获得以下有价值的研究结果:
(1)首次筛选出高效的铜绿微囊藻抑制剂中草药黄连。室内毒性实验结果表明,黄连对铜绿微囊藻的有效抑制浓度为0.100%(W/V),96 h抑制率达到95.15%,线性方程为Y = 1 .6510?4.5192×C?0.008×T(Y:藻细胞密度,C:黄连浓度,T:作用时间)。
(2)揭示了黄连对铜绿微囊藻的抑制是通过其主要成分生物碱的化感作用完成的。实验结果表明,无论是从黄连中提取的还是工业合成的小檗碱对室内培养及天然生长的铜绿微囊藻均有很好的抑制效果,其中0.020%的小檗碱96 h时对室内培养及天然生长的铜绿微囊藻的抑制率均达90.00%以上。小檗碱对模拟微生态系统水体主要水化指标和其它浮游生物虽然也有不同程度的影响,但总体对养殖对象草金鱼(Carassius aurutus)的成活并没有产生明显的不良影响(p > 0.05)。
(3)比较系统地揭示了生物碱抑制铜绿微囊藻的机理。生物碱处理铜绿微囊藻细胞后,生物碱通过与铜绿微囊藻细胞DNA结合,破坏DNA结构,使铜绿微囊藻细胞超微结构受到不同程度损伤,藻细胞的类囊体及藻胆体数目减少,藻青素颗粒、质体小球及多磷酸盐体增多,铜绿微囊藻细胞膜及细胞壁出现破裂,使细胞膜选择通透性发生改变,藻胆蛋白各组分相对百分含量发生变化,Ca~(2+)Mg~(2+)-ATPase酶活性受到抑制,光合作用受到影响,同时藻细胞MDA含量和O_2~-活性随生物碱浓度升高和处理时间延长而增加。低浓度的生物碱使SOD活性和GSH含量升高,高浓度的生物碱SOD活性降低,进而引起藻细胞氧化损伤。由此可见,生物碱可以破坏铜绿微囊藻细胞DNA和超显微结构,改变藻细胞膜离子通透性,抑制Ca~(2+)Mg~(2+)-ATPase活性,使光合作用无法正常进行,引起O_2~-活性升高,降低SOD活性,破坏抗氧化酶防御系统,进而导致膜脂质过氧化,使新陈代谢过程受抑制,最终导致藻细胞死亡。
Microcystis blooms could deteriorate aquatic environments, cause harm to aquatic animals, and destroy the equilibrium of the aqua-ecosystem. To provide the basic knowledge for developing environment-friendly algicides with high efficiency,the inhibitory effects of Chinese herb on Microcystic aeruginosa and its mechanism involved are studied in present paper.
Based on the antialgal results of sampled Chinese herb, golden thread (Coptidis rhizome, Huang lian in Chinese) showing best algal inhibition was screened. Further studies are conducted to reveal the effects of several alkaloids in golden thread on M. aeruginosa in laboratory and field, as well as on the micro-ecosystem. Meanwhile, the effects of several main environment factors on the algal inhibition are studied. Moreover, the antialgal mechanisms of the alkaloids have been studied from the view of photosynthesis, oxidation defense system, and the ultrastructure of M. aeruginosa. The main results of the present study are presented as follow:
1. Golden thread, exhibited the best inhibitory effects, can act as effective algicide. Effective concentration of golden thread for suppressing M. aeruginosa is 0.100%(W/V). 96 h inhibitory rate (IR) is 95.15%, and the linear equation is Y= 1.6510-4.5192×C-0.008×T (Y: algal density, C: concentration of golden thread, T: exposure time).
2. The results show that alkaloids might be the allelopathic substance of golden thread implementing inhibitory effects on M. aeruginosa. The berberine, either extracted from the golden thread or synthesized by industry, exhibits strong inhibitory effects on M. aeruginosa in laboratory and field, 96 h IR of 0.020% berberine is more than 90.00%. Although the main physical and chemical index of micro-ecological water has been affected in varying degrees by berberine, Carassius aurutus are influenced insignificantly (p > 0.05) under the stress of berberine.
3. Ultrastructure destruction, oxidative damage and photosynthetic pigments change might be involved in alkaloids -induced antialgal mechanisms. alkaloids binds with DNA and destroys DNA structure, leading to ultrastructure destruction of algal cell. It is observed that the number of algal thylakoids and phycobilisomes decrease, accompanying with the increases in the number of cyanophycin granules, plastoglobuli and polyhedral bodies. Moreover, the smooth cell membrane and the multiple-layered cell wall are ruptured. The selective permeability of cell membrane has changed, while Ca~(2+)Mg~(2+)-ATPase activity is inhibited. Photosynthesis is influenced by alkaloids too. Meanwhile, MDA content and O2- activity of M. aeruginosa under alkaloids stress increase with the enhancement of exposure concentration and the prolongation of exposure time. Berberine upregulates SOD activity and GSH content at low concentrations while downregulating SOD activity at high concentrations. In general, alkaloids could destroy DNA and ultrastructure of M. aeruginosa, change the selective permeability of algal cell membrane, inhibit Ca~(2+)Mg~(2+)-ATPase activity and photosynthesis, induce increase in O2- activity and decrease in SOD activity, destroy the antioxidant defense system, lead to lipid peroxidation and oxidative damage, and inhibit the metabolism processes of M. aeruginosa, eventually result in the death of the algal cell.
(1)首次筛选出高效的铜绿微囊藻抑制剂中草药黄连。室内毒性实验结果表明,黄连对铜绿微囊藻的有效抑制浓度为0.100%(W/V),96 h抑制率达到95.15%,线性方程为Y = 1 .6510?4.5192×C?0.008×T(Y:藻细胞密度,C:黄连浓度,T:作用时间)。
(2)揭示了黄连对铜绿微囊藻的抑制是通过其主要成分生物碱的化感作用完成的。实验结果表明,无论是从黄连中提取的还是工业合成的小檗碱对室内培养及天然生长的铜绿微囊藻均有很好的抑制效果,其中0.020%的小檗碱96 h时对室内培养及天然生长的铜绿微囊藻的抑制率均达90.00%以上。小檗碱对模拟微生态系统水体主要水化指标和其它浮游生物虽然也有不同程度的影响,但总体对养殖对象草金鱼(Carassius aurutus)的成活并没有产生明显的不良影响(p > 0.05)。
(3)比较系统地揭示了生物碱抑制铜绿微囊藻的机理。生物碱处理铜绿微囊藻细胞后,生物碱通过与铜绿微囊藻细胞DNA结合,破坏DNA结构,使铜绿微囊藻细胞超微结构受到不同程度损伤,藻细胞的类囊体及藻胆体数目减少,藻青素颗粒、质体小球及多磷酸盐体增多,铜绿微囊藻细胞膜及细胞壁出现破裂,使细胞膜选择通透性发生改变,藻胆蛋白各组分相对百分含量发生变化,Ca~(2+)Mg~(2+)-ATPase酶活性受到抑制,光合作用受到影响,同时藻细胞MDA含量和O_2~-活性随生物碱浓度升高和处理时间延长而增加。低浓度的生物碱使SOD活性和GSH含量升高,高浓度的生物碱SOD活性降低,进而引起藻细胞氧化损伤。由此可见,生物碱可以破坏铜绿微囊藻细胞DNA和超显微结构,改变藻细胞膜离子通透性,抑制Ca~(2+)Mg~(2+)-ATPase活性,使光合作用无法正常进行,引起O_2~-活性升高,降低SOD活性,破坏抗氧化酶防御系统,进而导致膜脂质过氧化,使新陈代谢过程受抑制,最终导致藻细胞死亡。
Microcystis blooms could deteriorate aquatic environments, cause harm to aquatic animals, and destroy the equilibrium of the aqua-ecosystem. To provide the basic knowledge for developing environment-friendly algicides with high efficiency,the inhibitory effects of Chinese herb on Microcystic aeruginosa and its mechanism involved are studied in present paper.
Based on the antialgal results of sampled Chinese herb, golden thread (Coptidis rhizome, Huang lian in Chinese) showing best algal inhibition was screened. Further studies are conducted to reveal the effects of several alkaloids in golden thread on M. aeruginosa in laboratory and field, as well as on the micro-ecosystem. Meanwhile, the effects of several main environment factors on the algal inhibition are studied. Moreover, the antialgal mechanisms of the alkaloids have been studied from the view of photosynthesis, oxidation defense system, and the ultrastructure of M. aeruginosa. The main results of the present study are presented as follow:
1. Golden thread, exhibited the best inhibitory effects, can act as effective algicide. Effective concentration of golden thread for suppressing M. aeruginosa is 0.100%(W/V). 96 h inhibitory rate (IR) is 95.15%, and the linear equation is Y= 1.6510-4.5192×C-0.008×T (Y: algal density, C: concentration of golden thread, T: exposure time).
2. The results show that alkaloids might be the allelopathic substance of golden thread implementing inhibitory effects on M. aeruginosa. The berberine, either extracted from the golden thread or synthesized by industry, exhibits strong inhibitory effects on M. aeruginosa in laboratory and field, 96 h IR of 0.020% berberine is more than 90.00%. Although the main physical and chemical index of micro-ecological water has been affected in varying degrees by berberine, Carassius aurutus are influenced insignificantly (p > 0.05) under the stress of berberine.
3. Ultrastructure destruction, oxidative damage and photosynthetic pigments change might be involved in alkaloids -induced antialgal mechanisms. alkaloids binds with DNA and destroys DNA structure, leading to ultrastructure destruction of algal cell. It is observed that the number of algal thylakoids and phycobilisomes decrease, accompanying with the increases in the number of cyanophycin granules, plastoglobuli and polyhedral bodies. Moreover, the smooth cell membrane and the multiple-layered cell wall are ruptured. The selective permeability of cell membrane has changed, while Ca~(2+)Mg~(2+)-ATPase activity is inhibited. Photosynthesis is influenced by alkaloids too. Meanwhile, MDA content and O2- activity of M. aeruginosa under alkaloids stress increase with the enhancement of exposure concentration and the prolongation of exposure time. Berberine upregulates SOD activity and GSH content at low concentrations while downregulating SOD activity at high concentrations. In general, alkaloids could destroy DNA and ultrastructure of M. aeruginosa, change the selective permeability of algal cell membrane, inhibit Ca~(2+)Mg~(2+)-ATPase activity and photosynthesis, induce increase in O2- activity and decrease in SOD activity, destroy the antioxidant defense system, lead to lipid peroxidation and oxidative damage, and inhibit the metabolism processes of M. aeruginosa, eventually result in the death of the algal cell.
引文
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