苦瓜素Ⅰ和苦瓜苷B对亚洲玉米螟Ofh细胞的增殖抑制和致坏死作用
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摘要
【目的】研究从苦瓜Momordica charantia叶乙醇提取物中分离出的苦瓜素Ⅰ和新化合物苦瓜苷B对亚洲玉米螟Ostrinina furnacalis幼虫血细胞(Ofh)的毒力作用,并探讨其作用机理。【方法】用CCK-8法比较了苦瓜素Ⅰ、苦瓜素Ⅱ、苦瓜苷B和印楝素A对Ofh细胞的增殖抑制作用;用倒置相差显微镜和荧光倒置显微镜观察了苦瓜素Ⅰ及苦瓜苷B对Ofh细胞形态结构的影响,并用台盼蓝染色法和流式细胞术研究了其对Ofh细胞的致坏死作用。【结果】苦瓜素Ⅰ、苦瓜素Ⅱ和苦瓜苷B对Ofh细胞有明显的增殖抑制作用,36 h后的IC50值分别为7.566,24.340及8.514μg/m L,苦瓜素Ⅰ及苦瓜苷B对Ofh细胞的增殖抑制作用明显强于苦瓜素Ⅱ及对照药物印楝素A。苦瓜素Ⅰ和苦瓜苷B处理后的Ofh细胞表现为体积膨大、表面变得粗糙并有明显的空泡现象,严重破坏了Ofh细胞的形态结构。进一步的研究结果表明,8μg/m L的苦瓜素Ⅰ及苦瓜苷B处理Ofh细胞12~48 h,细胞死亡率均高于对照组,呈一定的时间依赖关系,说明苦瓜素Ⅰ对Ofh细胞的急性毒力高于苦瓜苷B。Ofh细胞经AO/EB染色后,用荧光显微观察发现,苦瓜素Ⅰ和苦瓜苷B处理组细胞膜破裂,细胞核受损严重,细胞呈不均匀桔黄色-橙红色荧光,呈典型的坏死特征。流式细胞仪检测结果表明,苦瓜素Ⅰ和苦瓜苷B对Ofh细胞具有明显的致坏死作用。8μg/m L苦瓜素Ⅰ和苦瓜苷B处理Ofh细胞36 h后,总坏死率分别为74.92%±2.02%和49.77%±1.69%。【结论】苦瓜素Ⅰ和新化合物苦瓜苷B对Ofh细胞均具有有效的增殖抑制和致坏死作用,这可能是苦瓜素类化合物对亚洲玉米螟的拒食作用及抑制其生长发育和生殖力的主要机制之一。
【Aim】To ascertain the toxic effect of momordicin I and a new compound( charantin B) from ethanol extracts of Momordica charantia leaves on larval Ostrinina furnacalis hemocytes( Ofh),and to illuminate its action mechanism as well. 【Methods】The inhibition effects of momordicin I,momordicin II,charantin B and azadirachtin A on Ofh cells were compared by using CCK-8 assay. The effects of momordicin I and charantin B on Ofh cell morphology structure were observed by using inverted phase contrast microscopy and fluorescence microscopy,and the induced necrosis effects on Ofh cells were studied by using trypan blue staining and flow cytometry( FCM) technologies. 【Results】Momordicin I,momordicin II and charantin B had apparent inhibition effects on Ofh cell proliferation,with the IC50 values of 7. 566,24. 340 and 8. 514 μg / m L,respectively,at 36 h after treatment. The cytotoxicity of momordicin I and charantin B were significantly higher than that of momordicin II and azadirachtin A.Using inverted phase contrast microscopy,we found that after exposure to momordicin I and charantin B,Ofh cell shapes changed to circular and swelling increased, and the cellular membrane bubbled,suggesting that both momordicin I and charantin B could destruct cell morphology structure. Further research indicated that the mortality rates of cells in the groups treated with 8 μg / m L momordicin I and charantin B were significantly higher than that of the control group at 12- 48 h after treatment,and in a time-dependent manner,indicating that the cytotoxicity of momordicin I is significantly higher than that of charantin B. Fluorescence microscopy observation revealed that nuclear morphology was irregular,the cell membrane dissolved and the nucleus appeared to be severely damaged after AO / EB staining,and cells exhibited a non-uniform orange-yellow fluorescence after treatment with 8 μg / m L momordicin I and charantin B,showing a typical characteristic of necrosis. Flow cytometry analysis showed that Ofh cells treated with momordicin I and charantin B were dramatically induced to undergo necrosis,and the total cell necrosis rates were 74. 92% ± 2. 02% and 49. 77% ± 1. 69%,respectively,after 36 h treatment with 8 μg / m L momordicin I and charantin B. 【Conclusion 】 Momordicin I and charantin B have significant inhibition effects on cell proliferation and can induce necrosis,which may account for the antifeedant effect of momordicins on O. furnacalis and the inhibition of growth and fecundity of O.furnacalis treated by momordicins.
【Aim】To ascertain the toxic effect of momordicin I and a new compound( charantin B) from ethanol extracts of Momordica charantia leaves on larval Ostrinina furnacalis hemocytes( Ofh),and to illuminate its action mechanism as well. 【Methods】The inhibition effects of momordicin I,momordicin II,charantin B and azadirachtin A on Ofh cells were compared by using CCK-8 assay. The effects of momordicin I and charantin B on Ofh cell morphology structure were observed by using inverted phase contrast microscopy and fluorescence microscopy,and the induced necrosis effects on Ofh cells were studied by using trypan blue staining and flow cytometry( FCM) technologies. 【Results】Momordicin I,momordicin II and charantin B had apparent inhibition effects on Ofh cell proliferation,with the IC50 values of 7. 566,24. 340 and 8. 514 μg / m L,respectively,at 36 h after treatment. The cytotoxicity of momordicin I and charantin B were significantly higher than that of momordicin II and azadirachtin A.Using inverted phase contrast microscopy,we found that after exposure to momordicin I and charantin B,Ofh cell shapes changed to circular and swelling increased, and the cellular membrane bubbled,suggesting that both momordicin I and charantin B could destruct cell morphology structure. Further research indicated that the mortality rates of cells in the groups treated with 8 μg / m L momordicin I and charantin B were significantly higher than that of the control group at 12- 48 h after treatment,and in a time-dependent manner,indicating that the cytotoxicity of momordicin I is significantly higher than that of charantin B. Fluorescence microscopy observation revealed that nuclear morphology was irregular,the cell membrane dissolved and the nucleus appeared to be severely damaged after AO / EB staining,and cells exhibited a non-uniform orange-yellow fluorescence after treatment with 8 μg / m L momordicin I and charantin B,showing a typical characteristic of necrosis. Flow cytometry analysis showed that Ofh cells treated with momordicin I and charantin B were dramatically induced to undergo necrosis,and the total cell necrosis rates were 74. 92% ± 2. 02% and 49. 77% ± 1. 69%,respectively,after 36 h treatment with 8 μg / m L momordicin I and charantin B. 【Conclusion 】 Momordicin I and charantin B have significant inhibition effects on cell proliferation and can induce necrosis,which may account for the antifeedant effect of momordicins on O. furnacalis and the inhibition of growth and fecundity of O.furnacalis treated by momordicins.
引文
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Cao X,Zhu CY,Zhang MX,Ling B,2015.Bioactivity of momordicin against Ostrinia furnacalis(Lepidoptera:Pyralidae)and its effect on the metabolizing enzyme activities in larval bodies of the moth.Acta Entomologica Sinica,58(6):625-633.[曹溪,朱春亚,张茂新,凌冰,2015.苦瓜素Ⅰ对亚洲玉米螟的生物活性及对其幼虫体内代谢酶活性的影响.昆虫学报,58(6):625-633]
Chandravadana MV,1987.Identification of triterpenoid feeding deterrent of red pumpkin beetles(Aulacophora foveicollis)from Momordica charantia.J.Chem.Ecol.,13(7):1689-1694.
Chen C,Jiao N,Jing JY,Xu RR,2013.Significance of the research of trypan blue dye exclusion,MTT and CCK-8 methods on the cytotoxicity of As2O3.China Medical Herald,10(12):24-26.[陈冲,焦宁,靖景艳,徐瑞荣,2013.台盼蓝拒染法、MTT法、CCK-8法在研究As2O3细胞毒性作用中的意义.中国医药导报,10(12):24-26]
Feng CJ,Dai HG,Fu WJ,2004.Effect of juvenile hormone analogue and ecdysteroids on the activity of phenoloxidase in larvae of the Asian corn borer,Ostrinia furnacalis(Guenée)(Lepidoptera:Pyralidae).Acta Entomologica Sinica,47(5):562-566.[冯从经,戴华国,符文俊,2004.保幼激素类似物及蜕皮甾类对亚洲玉米螟幼虫酚氧化酶活性的影响.昆虫学报,47(5):562-566]
Feng HL,Sun HY,Li KB,Xi JH,Yin J,Cao YZ,2011.Progress of molecular biology researches in insect flight muscle cell apoptosis.Chinese Journal of Applied Entomology,48(3):701-709.[冯红林,孙红岩,李克斌,席景会,尹姣,曹雅忠,2011.昆虫飞行肌细胞凋亡的分子生物学研究进展.应用昆虫学报,48(3):701-709]
Fukuda T,Takeda S,Xu R,Ochi H,Sunamura S,Sato T,Shibata S,Yoshida Y,Gu Z,Kimura A,Ma C,Xu C,Bando W,Fujita K,Shinomiya K,Hirai T,Asou Y,Enomoto M,Okano H,Okawa A,Itoh H,2013.Sema3A regulates bone-mass accrual through sensory innervations.Nature,497(7450):490-493.
Hu XN,Liang GW,Pang XF,2004.Ecological control of Asian corn borer(ACB),Ostrina furnacalis in the sweet corn field.Acta Ecologica Sinica,24(11):2573-2578.[胡学难,梁广文,庞雄飞,2004.甜玉米地亚洲玉米螟为害的生态控制系统研究.生态学报,24(11):2573-2578]
Li DS,Huang SH,Zhang BX,Wang YJ,Li HL,2007.The seasonal occurrences of Ostrinia furnacalis and its egg parasitic wasps on sweet corn in Pearl River Delta Region.Acta Phytophylacica Sinica,34(2):173-176.[李敦松,黄少华,张宝鑫,王燕君,李惠陵,2007.珠江三角洲地区甜玉米地亚洲玉米螟及其卵寄生蜂的发生规律.植物保护学报,34(2):173-176]
Li SQ,Deng XW,Zhang QD,2001.The killing action and growth influence of Momordica charantia extracts on Liriomyza sativae.Journal of Huazhong Agricultural University,20(6):539-543.[李绍勤,邓喜望,张求东,2001.苦瓜叶片提取液对美洲斑潜蝇杀虫作用与生长发育的影响.华中农业大学学报,20(6):539-543]
Ling B,Wang GC,Ya J,Zhang MX,Liang GW,2008.Studies on antifeeding activity and active ingredients against Plutella xylostella from Momordica charantia leaves.Scientia Agricultura Sinica,41(10):3116-3122.[凌冰,王国才,轧霁,张茂新,梁广文,2008.苦瓜叶提取物对小菜蛾的拒食活性及有效成分研究.中国农业科学,41(10):3116-3122]
Ling B,Xiang YL,Wang GC,Chen SH,Zhang MX,2009.Antifeedant and antioviposition activities of Momordica charantia leaf ethanol extract against Liriomyza sativae.Chinese Journal of Applied Ecology,20(4):836-842.[凌冰,向亚林,王国才,陈少华,张茂新,2009.苦瓜叶提取物对美洲斑潜蝇取食和产卵行为的抑制作用.应用生态学报,20(4):836-842]
Liu H,Wang GC,Zhang MX,Ling B,2015.The cytotoxicology of momordicins I and II on Spodoptera litura cultured cell line SL-1.Pestic.Biochem.Physiol.,122:110-118.
Liu H,Zhu CY,Zhang MX,Ling B,2014.Bioactivity of extracts of Momordica charantia leaves against Ostrinina furnacalis and their cytotoxicity to Spodoptera litura ovary cells.Chinese Journal of Applied Entomology,51(1):212-220.[刘欢,朱春亚,张茂新,凌冰,2014.苦瓜叶提取物对亚洲玉米螟的生物活性及对斜纹夜蛾卵巢细胞的毒力.应用昆虫学报,51(1):212-220]
Luo Y,Ling B,Xie JF,Zhang MX,2012.Inhibition effects of ethyl acetate extracts of Momordica charantia leaves on the experimental population of Spodoptera litura.Acta Ecologica Sinica,32(13):4173-4180.[骆颖,凌冰,谢杰峰,张茂新,2012.苦瓜叶乙酸乙酯提取物对斜纹夜蛾实验种群的抑制作用.生态学报,32(13):4173-4180]
Mekuria DB,Kashiwagi T,Tebayashi S,Kim CS,2005.Cucurbitane triterpenoid oviposition deterrent from Momordica charantia to the leafminer,Liriomyza trifolii.Biosci.Biotechnol.Biochem.,69(9):1706-1710.
Pan YQ,Gao F,Cheng J,2000.Research progress between apoptosis and cellular nectosis.Progress in Veterinary Medicine,21(4):5-8.[潘耀谦,高丰,成军,2000.细胞凋亡与细胞坏死比较的研究进展.动物医学进展,21(4):5-8]
Tao SX,Li Y,Shi SS,Zhu SR,Liu JF,Deng T,Fang XJ,2010.Effect of Beauveria bassiana on hemolymph defensive response from Ostrinia furnacalis larvae.Journal of Jilin Agricultural University,32(5):492-497.[陶淑霞,李玉,史树森,朱殊蕊,刘家富,邓涛,房晓君,2010.球孢白僵菌对亚洲玉米螟幼虫血淋巴防御反应的影响.吉林农业大学学报,32(5):492-497]
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