绞股蓝提取液对蛋白核小球藻的化感效应及影响机理研究
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摘要
以中草药植物绞股蓝[Gynostemma pentaphyllum(Thunb.) Makino]为化感供体材料,研究其不同浓度的提取液(0、5、10、25、50 g/L)对蛋白核小球藻(Chlorella pyrenoidesa)生长及生理生化特征的化感效应。结果表明:(1)绞股蓝提取液对蛋白核小球藻生长均具有抑制作用,其抑制作用随提取液质量浓度增大和培养时间延长均呈增强趋势,且25 g/L绞股蓝提取液培养15 d时的抑制率达到最大(79.41%)。(2)各浓度绞股蓝处理组蛋白核小球藻细胞内的叶绿素a含量均低于对照组,且随着提取液浓度升高以及处理时间延长叶绿素a含量较对照的降低量越多,表明蛋白核小球藻光合作用受到的影响也越大。(3)绞股蓝处理组蛋白核小球藻细胞的膜透性(吸光度OD_(264))显著高于对照,且膜透性随着提取液浓度增大而增强;高浓度提取液处理下,藻细胞内部的可溶性蛋白质(OD_(280))及核酸(OD_(260))含量均显著高于对照,且随着处理时间延长,细胞膜透性增大,细胞内部的可溶性蛋白质及核酸向胞外渗透增多。研究发现,绞股蓝提取液能够抑制蛋白核小球藻生长,并随着提取液质量浓度增大而增强;绞股蓝提取液能促进藻细胞叶绿素分解、增加细胞膜透性,引起可溶性蛋白质和核酸向胞外渗透量升高,导致藻细胞结构受损,代谢功能紊乱,从而达到化感抑制作用。
The growth and physiology of Chlorella pyrenoidesa were studied by using different concentrations of G. pentaphyllum extract(0, 5, 10, 25, 50 g/L). The results showed that:(1) G. pentaphyllum extract could inhibit the growth of C. pyrenoidesa, with the increasing concentration and the prolonging time of cultivate, the inhibition increased. And the inhibition rate reached the maximum that was 79.41% when C. pyrenoidesa was cultured for 15 days with 25 g/L of G. pentaphyllum extract.(2) Chlorophyll a content in the cells of each concentration treatment group was lower than that of control group. With the increase of extraction concentration and the extension of treatment time, chlorophyll a content decreased more than that of control group. This result indicated that the photosynthetic effect of C. pyrenoidesa was affected.(3) The cell membrane permeability(absorbance OD_(264)) of the treatment groups was significantly higher than that of control group, which meant the cell membrane permeability increased with the increase of the concentration of the extract. At high concentration, both OD_(260) and OD_(280) were significantly higher than that of control group. And the cell membrane permeability was increased with the extension time, and more soluble proteins and nucleic acid were infiltrate out of the cells. The experimental results indicated that inhibition was enhanced with the increase of the concentration, and its mechanism of action was to promote chlorophyll decomposition and increase cell membrane permeability as well as promote soluble protein and nucleic acid exosmosis. So the structure of algal cells damaged and metabolic disorders happened, thus allelopathic inhibition was achieved.
The growth and physiology of Chlorella pyrenoidesa were studied by using different concentrations of G. pentaphyllum extract(0, 5, 10, 25, 50 g/L). The results showed that:(1) G. pentaphyllum extract could inhibit the growth of C. pyrenoidesa, with the increasing concentration and the prolonging time of cultivate, the inhibition increased. And the inhibition rate reached the maximum that was 79.41% when C. pyrenoidesa was cultured for 15 days with 25 g/L of G. pentaphyllum extract.(2) Chlorophyll a content in the cells of each concentration treatment group was lower than that of control group. With the increase of extraction concentration and the extension of treatment time, chlorophyll a content decreased more than that of control group. This result indicated that the photosynthetic effect of C. pyrenoidesa was affected.(3) The cell membrane permeability(absorbance OD_(264)) of the treatment groups was significantly higher than that of control group, which meant the cell membrane permeability increased with the increase of the concentration of the extract. At high concentration, both OD_(260) and OD_(280) were significantly higher than that of control group. And the cell membrane permeability was increased with the extension time, and more soluble proteins and nucleic acid were infiltrate out of the cells. The experimental results indicated that inhibition was enhanced with the increase of the concentration, and its mechanism of action was to promote chlorophyll decomposition and increase cell membrane permeability as well as promote soluble protein and nucleic acid exosmosis. So the structure of algal cells damaged and metabolic disorders happened, thus allelopathic inhibition was achieved.
引文
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[19] 高苗,刘娟娟,王栋.绞股蓝总皂苷对宫颈癌HeLa细胞的生长抑制作用及可能的分子机制[J].肿瘤,2013,33(10):868-872.GAO M,LIU J J,WANG D.The anti-proliferation effect of gypenosides on cervical cancer HeLa cells and its molecular mechanism[J].Tumor,2013,33(10):868-872.
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[22] 李锋民,胡洪营.大型水生植物浸出液对藻类的化感抑制作用[J].中国给水排水,2004,(11):18-21.LI F M,HU H Y.Allelopathy and inhibitory effect of extracts from macrophytes on algae growth[J].China Water & Wastewater,2004,(11):18-21.
[23] 冯彬.香蒲耐营养盐的生理机制及其对两种水华藻类的化感作用研究[D].杭州:浙江农林大学,2017.
[24] 王赛君,吴湘,王奕棉,等.水生入侵植物对常见水华的抑藻效应及其影响机理[J].海洋与湖沼,2017,48(4):798-805.WANG S J,WU X,WANG Y M,et al.Inhibitory effect of invasive aquatic plants on common algae bloom species and the underlying physiological mechanisms[J].Oceanologia et Limnologia Sinica,2017,48(4):798-805.
[25] 周绪申,孟宪智,王迎,等.两种计数板在藻类定量中的比较研究[J].水利技术监督,2016,24(4):59-61.ZHOU X S,MENG X Z,WANG Y,et al.Comparative study of two kinds of counting plates in algae quantification[J].Technical Supervision in Water Resources,2016,24(4):59-61.
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[27] 王学奎.植物生理生化实验原理和技术(第二版)[M].北京:高等教育出版社,2006,134-136.
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[29] 谢田,徐中际.测定细胞膜透性的紫外吸收法[J].植物生理学通讯,1986,(1):45-46.XIE T,XU Z J.Ultraviolet absorption method for determination of cell membrane permeability[J].Plant Physiology Communications,1986,(1),45-46.
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[2] 封志明,杨艳昭,游珍.中国人口分布的水资源限制性与限制度研究[J].自然资源学报,2014,29(10):1 637-1 648.FENG Z M,YANG Y Z,YOU Z.Research on the water resources restriction on population distribution in China[J].Journal of Natural Resources,2014,29(10):1 637-1 648.
[3] WANG J,LIU Q,FENG J,et al.Photosynthesis inhibition of pyrogallol against the bloom-forming cyanobacterium Microcystis aeruginosa TY001[J].Polish Journal of Environmental Studies,2016,25(6):2 601-2 608.
[4] 周立红.基于化感作用的环境友好型抑藻剂的研究[D].福建厦门:厦门大学,2008.
[5] 胡洪营,门玉洁,李锋民.植物化感作用抑制藻类生长的研究进展[J].生态环境,2006,15(1):153-157.HU H Y,MEN Y J,LI F M.Research progress on phyto-allelopathic algae control[J].Ecology and Environment,2006,15(1):153-157.
[6] 金相灿.湖泊富营养化调查规范[M].北京:中国环境科学出版社,1990.
[7] 李凌燕,杜桂森.水生高等植物化感抑藻作用研究[J].世界科技研究与发展,2010,32(4):505-508.LI L Y,DU G S.Researches on the allelopathic effects of macrophytes on the algae[J].World Sci-Tech R & D,2010,32(4):505-508.
[8] 李锋民,胡洪营,种云霄,等.2-甲基乙酰乙酸乙酯对藻细胞膜和亚显微结构的影响[J].环境科学,2007,(7):1 535-1 538.LI F M,HU H Y,ZHONG Y X,et al.Effects of allelochemical EMA isolated from Phragmites communis on algal cell membrane lipid and ultrastructure[J].Environmental Science,2007,(7):1 535-1 538.
[9] QIAN H F,XU X Y.Allelochemical stress causes oxidative damage and inhibition of photosynthesis in Chlorella vulgaris[J].Chemosphere,2009,75(3):368-375.
[10] SHAO J H,WU Z X.Allelopathic mechanism of pyrogallol to Microcystis aeruginosa PCC7806 (Cyanobacteria):From views of gene expression and antioxidant system[J].Chemosphere,2009,75(7):924-928.
[11] 张爱华,郜玉钢,许永华,等.我国药用植物化感作用研究进展[J].中草药,2011,42(10):1 885-1 890.ZHANG A H,GAO Y G,XU Y H,et al.Advances in studies on allelopathy of medicinal plants in China[J].Chinese Traditional and Herbal Drugs,2011,42(10):1 885-1 890.
[12] LI F M,HU H Y.Isolation and effects on green alga Chlorella pyrenoidosa of algal-inhibiting allelochemicals in the Macrophyte,Phragmites communis Tris[J].Environment Science,2004,25(5):89-92.
[13] BAO F X,TAO L X,ZHANG H Y.Research progress on pharmacological effects of Gynostemma pentaphyllum active ingredients[J].Chinese Journal of New Drugs & Clinical Remedies,2018,37(1):11-17.
[14] 杨靓,王攀,成晓霞,等.绞股蓝总皂苷对小鼠白血病L1210细胞抑制作用初探[J].中药材,2010,33(10):1 588-1 592.YANG L,WANG P,CHENG X X,et al.Suppressive effect of gypenosides on murine leukemia L1210 cell lines[J].Journal of Chinese Medicinal Materials,2010,33(10):1 588-1 592.
[15] LU K W,TSAI M L,CHEN J C,et al.Gypenosides inhibited invasion and migration of human tongue cancer SCC4 cells through down-regulation of NF-kappaB and matrix metalloproteinase-9[J].Anticancer Research,2008,28(2):1 093-1 099.
[16] WANG P,GAO L,LI W X,et al.Neuroprotective effect of gypenosides against oxidative injury in the substantia nigra of a mouse model of Parkinson's disease[J].Journal of International Medical Research,2010,38(3):1 084-1 092.
[17] 范冬冬,匡艳辉,向世勰,等.绞股蓝化学成分及其药理活性研究进展[J].中国药学杂志,2017,52(5):342-352.FAN D D,KUANG Y H,XIANG S X,et al.Research progress in chemical constituents and pharmacological activities of Gynostemma pentaphyllum[J].Chinese Pharmaceutical Journal,2017,52(5):342-352.
[18] 郎志芳,王洪伟,李姝,等.绞股蓝总皂苷对糖尿病肾病大鼠肾皮质基因表达谱的影响研究[J].糖尿病新世界,2018,21(21):169-170.LANG Z F,WANG H W,LI S,et al.Effect of gypenosides on gene expression profile of renal cortex in diabetic nephropathy rats[J].Diabetes New World,2018,21(21):169-170.
[19] 高苗,刘娟娟,王栋.绞股蓝总皂苷对宫颈癌HeLa细胞的生长抑制作用及可能的分子机制[J].肿瘤,2013,33(10):868-872.GAO M,LIU J J,WANG D.The anti-proliferation effect of gypenosides on cervical cancer HeLa cells and its molecular mechanism[J].Tumor,2013,33(10):868-872.
[20] 赵艳,汪成.低氮胁迫对蛋白核小球藻生化组分和絮凝性能的影响[J/OL].植物营养与肥料学报:1-10[2019-04-12].http://kns.cnki.net/kcms/detail/11.3996.S.20190309.1613.002.html.ZHAO Y,WANG C.Effects of low nitrogen stress on the biochemical components and flocculation property of Chlorella pyrenoidosa[J/OL].Journal of Plant Nutrition and Fertilizers:1-10[2019-04-12].http://kns.cnki.net/kcms/detail/ 11.3996.S.20190309.1613.002.html.
[21] RAMOS E U,VAES W H J,MAYER P,et al.Algal growth inhibition of Chlorella pyenoidosa by polar narcotic pollutants:Toxic cell concentrations and QSAR modeling[J].Aquatic Toxicology,1999,46:1-10.
[22] 李锋民,胡洪营.大型水生植物浸出液对藻类的化感抑制作用[J].中国给水排水,2004,(11):18-21.LI F M,HU H Y.Allelopathy and inhibitory effect of extracts from macrophytes on algae growth[J].China Water & Wastewater,2004,(11):18-21.
[23] 冯彬.香蒲耐营养盐的生理机制及其对两种水华藻类的化感作用研究[D].杭州:浙江农林大学,2017.
[24] 王赛君,吴湘,王奕棉,等.水生入侵植物对常见水华的抑藻效应及其影响机理[J].海洋与湖沼,2017,48(4):798-805.WANG S J,WU X,WANG Y M,et al.Inhibitory effect of invasive aquatic plants on common algae bloom species and the underlying physiological mechanisms[J].Oceanologia et Limnologia Sinica,2017,48(4):798-805.
[25] 周绪申,孟宪智,王迎,等.两种计数板在藻类定量中的比较研究[J].水利技术监督,2016,24(4):59-61.ZHOU X S,MENG X Z,WANG Y,et al.Comparative study of two kinds of counting plates in algae quantification[J].Technical Supervision in Water Resources,2016,24(4):59-61.
[26] 陈坤,张前前,史海燕,等.浮游植物计数方法比较研究[J].海洋环境科学,2007,26(4):383-385.CHEN K,ZHANG Q Q,SHI H Y,et al.Calibration of phytoplankton numeration according to Uterm?hl method[J].Marine Environment Science,2007,26(4):383-385.
[27] 王学奎.植物生理生化实验原理和技术(第二版)[M].北京:高等教育出版社,2006,134-136.
[28] 中华人民共和国水利部.SL 88-2012水质叶绿素的测定-分光光度法[S].北京:中国水利水电出版社,2012.
[29] 谢田,徐中际.测定细胞膜透性的紫外吸收法[J].植物生理学通讯,1986,(1):45-46.XIE T,XU Z J.Ultraviolet absorption method for determination of cell membrane permeability[J].Plant Physiology Communications,1986,(1),45-46.
[30] 王立新,张玲,张余霞,等.黑藻(Hydrilla verticillata)养殖水对铜绿微囊藻(Microcystic aeruginosa)的抑制效应及其机制[J].植物生理与分子生物学学报,2006,32(6):672-678.WANG L X,ZHANG L,ZHANG Y X,et al.The inhibitory effect of Hydrilla verticillata culture water on Microcystic aeruginosa and its mechanism[J].Journal of Plant Physiology and Molecular Biology,2006,32(6),672-678.
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