柔毛淫羊藿愈伤组织诱导及其总黄酮和淫羊藿苷含量测定
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摘要
本研究旨在探讨柔毛淫羊藿愈伤组织的最佳诱导条件和愈伤组织中总黄酮、淫羊藿苷含量。采用正交试验设计方法研究不同外植体、培养基、培养温度和光照条件对柔毛淫羊藿愈伤组织诱导的影响,分光光度法测定愈伤组织中总黄酮含量,高效液相色谱法测定其中淫羊藿苷含量。结果显示,以幼叶为外植体,培养基为N6+2, 4-D 3 mg/L+6-BA 1 mg/L+NAA 0.5 mg/L,在黑暗或弱光(1 000 Lx)和(21±1)℃条件下愈伤组织诱导效果最好;愈伤组织中总黄酮和淫羊藿苷含量分别达5.24%和0.564%,与叶中含量无显著差异。初步建立了柔毛淫羊藿愈伤组织诱导的适宜培养条件,为高产淫羊藿总黄酮和淫羊藿苷的组培苗生产和细胞悬浮培养体系的建立提供了依据。
The aim of our study was to investigate the best conditions of callus induction and the contents of total flavonoids and Icariin in callus from Epimedium pubescens Maxim. The effects of different explants, media, culture temperature and light on callus induction of Epimedium pubescens were studied by orthogonal experimental design method. The contents of total flavonoids and Icariin in callus were determined by spectrophotometry and HPLC,respectively. The results showed that callus induction was the best under dark or low light(1 000 Lx) and(21±1)℃conditions when young leaves were used as explants and the medium was N6+2, 4-D 3 mg/L+6-BA 1 mg/L+ NAA0.5 mg/L. The contents of total flavonoids and Icariin in callus were 5.24% and 0.564% respectively, which was not significantly different from those in leaves. The suitable culture conditions of callus induction of Epimedium pubescens Maxim. were preliminarily established, which would provide a basis for the production of tissue culture seedlings and the establishment of cell suspension culture system of high-producing total flavonoids and Icariin of Epimedium pubescens.
The aim of our study was to investigate the best conditions of callus induction and the contents of total flavonoids and Icariin in callus from Epimedium pubescens Maxim. The effects of different explants, media, culture temperature and light on callus induction of Epimedium pubescens were studied by orthogonal experimental design method. The contents of total flavonoids and Icariin in callus were determined by spectrophotometry and HPLC,respectively. The results showed that callus induction was the best under dark or low light(1 000 Lx) and(21±1)℃conditions when young leaves were used as explants and the medium was N6+2, 4-D 3 mg/L+6-BA 1 mg/L+ NAA0.5 mg/L. The contents of total flavonoids and Icariin in callus were 5.24% and 0.564% respectively, which was not significantly different from those in leaves. The suitable culture conditions of callus induction of Epimedium pubescens Maxim. were preliminarily established, which would provide a basis for the production of tissue culture seedlings and the establishment of cell suspension culture system of high-producing total flavonoids and Icariin of Epimedium pubescens.
引文
Chen K.K.,and Chiu J.H.,2006,Effect of Epimedium brevicomum Maxim extract on elicitation of penileerection in the rat,Urology,67(3):631-635
Chen W.Y.,and Lv Y.T.,2009,Medicinal plant cell suspension culture and the progress of new drug research and development,Yaredai Zhiwu Kexue(Subtropical Plant Science),38(4):85-88(陈文源,吕一婷,2009,药用植物细胞悬浮培养与新药研发进展,亚热带植物科学,38(4):85-88)
Fu L.,Yuan J.Y.,Ding C.B.,and Zhang Z.H.,2015,The content determination analysis of general flavones and icariin for Epimedium in different habitats of Sichuan province,Xiandai Zhongyao Yanjiu Yu Shijian(Research and Practice on Chinese Medicines),29(5):27-30(付亮,袁璟亚,丁春邦,张中华,2015,四川不同产地淫羊藿总黄酮及淫羊藿苷含量的分析测定,现代中药研究与实践,29(5):27-30)
Fu L.,Yuan J.Y.,Zhou Y.H.,and Ding C.B.,2012,Research progress and development prospects of Epimedium polysaccharides,Shipin Kexue(Food Science),33(3):261-266(付亮,袁璟亚,周永红,丁春邦,2012,淫羊藿多糖的研究进展及开发前景,食品科学,33(3):261-266)
Gong N.,Yang Z.,Li X.L.,Yang T.,Wang N.,and Xiao J.,2015,Key technology of tissue culture for tiller buds of Epimedium koreanum,Guangdong Nongye Kexue(Guangdong Agricultural Sciences),42(5):15-18(龚娜,杨镇,李学龙,杨涛,王娜,肖军,2015,朝鲜淫羊藿组织培养关键技术研究,广东农业科学,42(5):15-18)
H L.L.,H S.Q.,and Yang X.,2006,Advances in studies on bioengineering in medicinal plantsⅠ,tissue and cell culture,Zhongcaoyao(Chinese Traditional and Herbal Drugs),37(4):486-491(黄璐琳,胡尚钦,杨晓,2006,药用植物生物工程研究进展Ⅰ,组织和细胞培养研究,中草药,37(4):486-491)
Meng F.H.,Li Y.B.,Xiong Z.L.,Jia Z.M.,and Li F.M.,2005,Osteoblastic proliferative activity of Epimedium brevicornum Maxim,Phytomedicine,12(3):189-193
Meng N.,Kong K.,and Li S.W.,2010,Advances in studies on chemical constituents and pharmaceutical activitiesin species of Epimedium,Xibei Zhiwu Xuebao(Acta Botanica Boreali-Occidentalia Sinica),30(5):1063-1073(孟宁,孔凯,李师翁,2010,淫羊藿属植物化学成分及药理活性研究进展,西北植物学报,30(5):1063-1073)
Nian H.,Xu L.L.,Ma M.H.,Qin L.P.,Zheng H.C.,and Zhang Q.Y.,2006,Prevention of bone loss by aqueous extract of epimedium sagittatum in an ovariectomized rat model of osteoporosis,Zhongxiyi Jiehe Xuebao(Journal of Integrative Medicine),4(6):628-633(年华,徐玲玲,马明华,秦路平,郑汉臣,张巧艳,2006,箭叶淫羊霍对去卵巢大鼠骨量丢失的保护作用,中西医结合学报,4(6):628-633)
Ouyang J.,Wu Y.W.,Wang X.D.,Zhao B.,and Wang Y.C.,2008,Establishment of Cistanche deserticola cell lines in suspension culture,Zhongcaoyao(Chinese Traditional and Herbal Drugs),39(3):426-429(欧阳杰,武彦文,王晓东,赵兵,王玉春,2008,肉苁蓉细胞悬浮培养体系的建立,中草药,39(3):426-429)
Wang D.M.,Huang X.L.,and Huang S.Z.,1996,The action mechanism of cytokins in plant tissiue culture,Zhiwu Shenglixue Tongxun(Plant Physiology Communications),32(5):373-377(王东梅,黄学林,黄上志,1996,细胞分裂素物质在植物组织培养中的作用机制,植物生理学通讯,32(5):373-377)
Yin J.Y.,and Chen H.F.,2009,Research advances in the mechanisms of antitumor activity of Epimedium brevicornum Maxim.and its effective components,Zhongxiyi Jiehe Xuebao(Journal of Integrative Medicine),12(7):1184-1187(尹剑云,陈红风,2009,淫羊藿及其有效成分抗肿瘤机制的研究进展,中西医结合学报,12(7):1184-1187)
Zhou H.Q.,Zhu G.S.,Guo Q.S.,Liu Y.Z.,and Zhou N.,2012,Tissue cultivation of tiller buds of Epimedium wushanense,Zhongguo Zhognyao Zazhi(China Journal of Chinese Materia Medica),37(14):3723-3727(周海琴,朱国胜,郭巧生,刘易作,周宁,2012,巫山淫羊藿分蘖芽组织培养研究,中国中药杂志,37(14):3723-3727)
Chen W.Y.,and Lv Y.T.,2009,Medicinal plant cell suspension culture and the progress of new drug research and development,Yaredai Zhiwu Kexue(Subtropical Plant Science),38(4):85-88(陈文源,吕一婷,2009,药用植物细胞悬浮培养与新药研发进展,亚热带植物科学,38(4):85-88)
Fu L.,Yuan J.Y.,Ding C.B.,and Zhang Z.H.,2015,The content determination analysis of general flavones and icariin for Epimedium in different habitats of Sichuan province,Xiandai Zhongyao Yanjiu Yu Shijian(Research and Practice on Chinese Medicines),29(5):27-30(付亮,袁璟亚,丁春邦,张中华,2015,四川不同产地淫羊藿总黄酮及淫羊藿苷含量的分析测定,现代中药研究与实践,29(5):27-30)
Fu L.,Yuan J.Y.,Zhou Y.H.,and Ding C.B.,2012,Research progress and development prospects of Epimedium polysaccharides,Shipin Kexue(Food Science),33(3):261-266(付亮,袁璟亚,周永红,丁春邦,2012,淫羊藿多糖的研究进展及开发前景,食品科学,33(3):261-266)
Gong N.,Yang Z.,Li X.L.,Yang T.,Wang N.,and Xiao J.,2015,Key technology of tissue culture for tiller buds of Epimedium koreanum,Guangdong Nongye Kexue(Guangdong Agricultural Sciences),42(5):15-18(龚娜,杨镇,李学龙,杨涛,王娜,肖军,2015,朝鲜淫羊藿组织培养关键技术研究,广东农业科学,42(5):15-18)
H L.L.,H S.Q.,and Yang X.,2006,Advances in studies on bioengineering in medicinal plantsⅠ,tissue and cell culture,Zhongcaoyao(Chinese Traditional and Herbal Drugs),37(4):486-491(黄璐琳,胡尚钦,杨晓,2006,药用植物生物工程研究进展Ⅰ,组织和细胞培养研究,中草药,37(4):486-491)
Meng F.H.,Li Y.B.,Xiong Z.L.,Jia Z.M.,and Li F.M.,2005,Osteoblastic proliferative activity of Epimedium brevicornum Maxim,Phytomedicine,12(3):189-193
Meng N.,Kong K.,and Li S.W.,2010,Advances in studies on chemical constituents and pharmaceutical activitiesin species of Epimedium,Xibei Zhiwu Xuebao(Acta Botanica Boreali-Occidentalia Sinica),30(5):1063-1073(孟宁,孔凯,李师翁,2010,淫羊藿属植物化学成分及药理活性研究进展,西北植物学报,30(5):1063-1073)
Nian H.,Xu L.L.,Ma M.H.,Qin L.P.,Zheng H.C.,and Zhang Q.Y.,2006,Prevention of bone loss by aqueous extract of epimedium sagittatum in an ovariectomized rat model of osteoporosis,Zhongxiyi Jiehe Xuebao(Journal of Integrative Medicine),4(6):628-633(年华,徐玲玲,马明华,秦路平,郑汉臣,张巧艳,2006,箭叶淫羊霍对去卵巢大鼠骨量丢失的保护作用,中西医结合学报,4(6):628-633)
Ouyang J.,Wu Y.W.,Wang X.D.,Zhao B.,and Wang Y.C.,2008,Establishment of Cistanche deserticola cell lines in suspension culture,Zhongcaoyao(Chinese Traditional and Herbal Drugs),39(3):426-429(欧阳杰,武彦文,王晓东,赵兵,王玉春,2008,肉苁蓉细胞悬浮培养体系的建立,中草药,39(3):426-429)
Wang D.M.,Huang X.L.,and Huang S.Z.,1996,The action mechanism of cytokins in plant tissiue culture,Zhiwu Shenglixue Tongxun(Plant Physiology Communications),32(5):373-377(王东梅,黄学林,黄上志,1996,细胞分裂素物质在植物组织培养中的作用机制,植物生理学通讯,32(5):373-377)
Yin J.Y.,and Chen H.F.,2009,Research advances in the mechanisms of antitumor activity of Epimedium brevicornum Maxim.and its effective components,Zhongxiyi Jiehe Xuebao(Journal of Integrative Medicine),12(7):1184-1187(尹剑云,陈红风,2009,淫羊藿及其有效成分抗肿瘤机制的研究进展,中西医结合学报,12(7):1184-1187)
Zhou H.Q.,Zhu G.S.,Guo Q.S.,Liu Y.Z.,and Zhou N.,2012,Tissue cultivation of tiller buds of Epimedium wushanense,Zhongguo Zhognyao Zazhi(China Journal of Chinese Materia Medica),37(14):3723-3727(周海琴,朱国胜,郭巧生,刘易作,周宁,2012,巫山淫羊藿分蘖芽组织培养研究,中国中药杂志,37(14):3723-3727)