悬浮培养瑞香狼毒不定芽过程中黄酮和香豆素类物质的累积
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摘要
本文以瑞香狼毒不定芽为材料,研究了在250 mL培养瓶悬浮培养过程中,影响其生长及活性物质(伞形花内酯、东莨菪内酯、总黄酮)积累的四类因子(蔗糖浓度、培养基强度、接种量、接种长度),初步建立了不定芽的悬浮培养体系。在此基础上,研究了3 L生物反应器内,影响不定芽生长及活性物质积累的因素(蔗糖浓度、6-BA浓度、初始接种量)。结果显示,不定芽在250 mL培养瓶中悬浮培养周期是15 d,最佳蔗糖浓度、培养基强度、接种量、接种长度分别是30 g·L~(-1)、MS培养基、1.0 g、3 cm。在此培养条件下,不定芽中伞形花内酯、东莨菪内酯和黄酮累积量都在15 d达到最大值,分别为0.351、0.035和2.459 mg。3 L生物反应器内不定芽悬浮培养周期为24 d,最佳培养条件为蔗糖30 g·L~(-1)、6-BA 0.5 mg·L~(-1)和初始接种量15 g。此条件下,伞形花内酯、东莨菪内酯和黄酮累积量分别为6.041、0.763和51.334 mg。本研究建立了瑞香狼毒不定芽的小型生物反应器培养体系,为进一步大规模培养,高效生产上述三种活性物质奠定了基础。
In this study, during the suspension culture of the shoots of Stellera chamaejasme in 250 mL culture flask, four kinds of factors(sucrose concentration, medium strength, quantity and length of inoculum) affecting the growth of shoots and the active substances(umbelliferone, scopoletin, total flavonoids) in them were studied, and the culture-flask suspension culture system of adventitious shoots was established. On this basis, the 3 L bioreactor culture system was established, by analyzing the effects of sucrose concentration, 6-BA concentration, initial inoculum quantity on shoots growth and accumulation of active substances. The results showed that the suspension culture period of adventitious shoots in a 250 mL culture flask was 15 d. The optimal sucrose concentration, medium strength, inoculum quantity and length were 30 g·L~(-1), MS medium, 1.0 g and 3 cm, respectively. Under the optimal culture conditions, the accumulation of coumarins and flavonoids in adventitious shoots reached the maximum on the 15 th day, i.e., umbelliferone 0.351 mg, scopoletin 0.035 mg and flavonoid 2.459 mg, respectively. The suspension culture period of adventitious shoots in the 3 L bioreactor was 24 d. The optimal culture conditions were sucrose 30 g·L~(-1), 6-BA 0.5 mg·L~(-1) and initial quantity of inoculum 15 g. Under these conditions, the cumulative amounts of umbelliferone, scopoletin and flavonoids were 6.041, 0.763 and 51.334 mg, respectively. In this study, a small bioreactor culture system for adventitious shoots of Stellera chamaejasme was established, which laid a foundation for further large-scale cultivation and production of the above active substances.
In this study, during the suspension culture of the shoots of Stellera chamaejasme in 250 mL culture flask, four kinds of factors(sucrose concentration, medium strength, quantity and length of inoculum) affecting the growth of shoots and the active substances(umbelliferone, scopoletin, total flavonoids) in them were studied, and the culture-flask suspension culture system of adventitious shoots was established. On this basis, the 3 L bioreactor culture system was established, by analyzing the effects of sucrose concentration, 6-BA concentration, initial inoculum quantity on shoots growth and accumulation of active substances. The results showed that the suspension culture period of adventitious shoots in a 250 mL culture flask was 15 d. The optimal sucrose concentration, medium strength, inoculum quantity and length were 30 g·L~(-1), MS medium, 1.0 g and 3 cm, respectively. Under the optimal culture conditions, the accumulation of coumarins and flavonoids in adventitious shoots reached the maximum on the 15 th day, i.e., umbelliferone 0.351 mg, scopoletin 0.035 mg and flavonoid 2.459 mg, respectively. The suspension culture period of adventitious shoots in the 3 L bioreactor was 24 d. The optimal culture conditions were sucrose 30 g·L~(-1), 6-BA 0.5 mg·L~(-1) and initial quantity of inoculum 15 g. Under these conditions, the cumulative amounts of umbelliferone, scopoletin and flavonoids were 6.041, 0.763 and 51.334 mg, respectively. In this study, a small bioreactor culture system for adventitious shoots of Stellera chamaejasme was established, which laid a foundation for further large-scale cultivation and production of the above active substances.
引文
Cui XH,Chakrabary D,Lee EJ,et al(2010).Production of adventitious roots and secondary metabolites by Hypericum perforatum L.in a bioreactor.Bioresour Technol,101:4708-4716
Feng WJ,Ikekawa T,Yoshida M(1995).The antitumor activities of gnidimacrin isolated from Stellera chamaejasme L.Chin J Oncol,17:24-26(in Chinese with English abstract)[冯威健,池川哲郎,吉田光二(1995).瑞香狼毒提取物尼地吗啉的抗癌活性.中华肿瘤杂志,17:24-26]
Fulzele DP,Heble MR,Rao PS(1995).Production of terpenoid from Artemisia annua L.plantlet cultures in bioreactor.J Biotechnol,40:139-143
Guo WJ,Zhang SQ,Wu H,et al(2007).Study on extraction process for total flavonoids in leaves of Acanthopanax senticosus harms at ultra high pressure.Chem Eng,35:66-69(in Chinese with English abstract)[郭文晶,张守勤,吴华等(2007).超高压提取刺五加叶中总黄酮的研究.化学工程,35:66-69]
Huang JQ,Zhao Y,Sun FY,et al(2013).Accumulation of flavonoids and coumarins during adventitious bud growth in Stellera chamaejasme.Chin Trad Herbal Drugs,44:1328-1333(in Chinese with English abstract)[黄嘉琦,赵越,孙凤阳等(2013).瑞香狼毒不定芽生长过程黄酮类和香豆素类成分累积研究.中草药,44:1328-1333]
Jiang YJ,Piao XC,Li H,et al(2016).Effect of several factors on saponin-accumulation in adventitious roots of Oplopanax elatus cultured with bioreactor culture.J Forest Eng,1:68-72(in Chinese with English abstract)[姜银姬,朴炫春,李贺等(2016).几种因素对生物反应器培养东北刺人参不定根中皂苷积累的影响.林业工程学报,1:68-72]
Kan XX,Wang ZX,Yang QX,et al(2013).Antitumor effect of alcohol extracts from Stellera chamaejasme.Chin J Chin Materia Med,38:1219-1225(in Chinese with English abstract)[阚晓溪,王志鑫,杨乾栩等(2013).瑞香狼毒醇提物的抗肿瘤活性研究.中国中药杂志,38:1219-1225]
Li HJ,Zhang Y,Guo Y(1997).Effects of inoculated mass on cell growth of Artemisia annua L.in suspension culture.JJinan Univ(Nat Sci Med Ed),18:89-92(in Chinese with English abstract)[李弘剑,张毅,郭勇(1997).生物量对黄花蒿悬浮培养细胞生长的影响.暨南大学学报(自然科学与医学版),18:89-92]
Park JM,Hu WS,Staba EJ(1989).Cultivation of Artemisia annua L.plantlets in a bioreactor containing a single carbon and a single nitrogen source.Biotechnol Bioeng,34:1209-1213
Piao XC,Lian ML,Wang SM(2007).Mass producing of adventitious roots of Ginseng by using bioreactor.For Sci Tech,32:54-56(in Chinese with English abstract)[朴炫春,廉美兰,王守明(2007).利用生物反应器大量生产人参不定根.林业科技,32:54-56]
Qiao LR,Yang L,Zhang DW,et al(2011).Studies on chemical constitutes from callus cultures of Stellera chamaejasme.Chin J Chin Materia Med,36:3457-3462(in Chinese with English abstract)[乔立瑞,杨林,张德武等(2011).瑞香狼毒细胞培养物的化学成分研究.中国中药杂志,36:3457-3462]
Shi CQ,Wang C(2011).Study on the insecticidal activity of Stellera chamaejasme.Nat Sci J Harbin Nor Univ,27:84-86(in Chinese with English abstract)[史传奇,王臣(2011).瑞香狼毒粗提取物杀虫活性研究.哈尔滨师范大学自然科学学报,27:84-86]
Shu JF(2014).Determination of scopoletin,baicalin,baicalein and farrerol in Qinbaohong Zhike Capsule by HPLC.Chin J Mod Appl Pharm,31:1114-1117(in Chinese with English abstract)[舒金富(2014).HPLC测定芩暴红止咳胶囊中东莨菪内酯、黄芩苷、黄芩素和杜鹃素的含量.中国现代应用药学,31:1114-1117]
Wang L(2016).The seed germination conditions and metabolism analysis of Stellera chamaejasme(dissertation).Xi’an:Northwest University(in Chinese with English abstract)[王琳(2016).瑞香狼毒种子萌发条件及代谢分析(学位论文).西安:西北大学]
Wang Q(2015).Adventitious roots culture in bioreactor of medicinal plants of Panax quinquefolium L.and Pseudostellaria heterophylla(dissertation).Tianjin:Tianjin University of Science Technology(in Chinese with English abstract)[王琴(2015).药用植物西洋参和太子参不定根反应器培养研究(学位论文).天津:天津科技大学]
Wang WX,An Q,Wang Y,et al(2010).A study on cell suspension culture and flavonoids accumulation of Stellera chamaejasme.Acta Prataculturae Sin,19:132-139(in Chinese with English abstract)[王文星,安琪,汪莹等(2010).瑞香狼毒细胞悬浮培养及黄酮积累的研究.草业学报,19:132-139]
Wu X,Zhang P,Zhang MT,et al(2017).Simultaneous determination of daphnetin,umbelliferone and scopoletin in Stellera chamejasme L.by HPLC.Chin J Mod Appl Pharm,34:1171-1174(in Chinese with English abstract)[武雪,张平,张明童等(2017).HPLC同时测定瑞香狼毒中瑞香素、伞形花内酯和东莨菪内酯的含量.中国现代应用药学,34:1171-1174]
Xing CB,Long YH,Lao FY,et al(2012).Effect of endophytic fungi on expression amount of key enzyme genes in saponins biosynthesis and Eleutherococcus senticosus saponins content.Chin J Chin Materia Med,37:2041-2045(in Chinese with English abstract)[邢朝斌,龙月红,劳凤云等(2012).内生真菌对刺五加皂苷合成关键酶基因表达及皂苷含量的影响.中国中药杂志,37:2041-2045]
Yao R(2014).Optimizing bioreactor culture system of protocorm-like bodies in Dendrobium candidum(dissertation).Yanbian,Jilin:Yanbian University(in Chinese with English abstract)[姚睿(2014).优化铁皮石斛原球茎生物反应器的培养体系(学位论文).吉林延边:延边大学]
You XL,Tan X,Jia HB,et al(2011).Ginsenosides accumulation in adventitious roots of Panax ginseng cultured in small-type bioreactors.Acta Bot Boreal-Occident Sin,31:1700-1705(in Chinese with English abstract)[由香玲,谭啸,贾洪柏等(2011).小型生物反应器内人参不定根的人参皂苷累积.西北植物学报,31:1700-1705]
Yu FK,Ma YQ,Li XW(2005).Tissue culture and rapid propagation of Stellera chamaejasme.Plant Physiol Commun,41:59(in Chinese with English abstract)[于福科,马永清,李秀维(2005).瑞香狼毒的组织培养及快速繁殖.植物生理学通讯,41:59]
Zheng BJ,Hu HQ(2006).Variety trends of coumarin in Stellera chamejasme L.in Songnen grassland of Heilongjiang,China.J Nat Sci Heilongjiang Univ,23:223-226(in Chinese with English abstract)[郑宝江,胡海清(2006).松嫩草地瑞香狼毒香豆素类化合物含量动态变化.黑龙江大学自然科学学报,23:223-226]
Feng WJ,Ikekawa T,Yoshida M(1995).The antitumor activities of gnidimacrin isolated from Stellera chamaejasme L.Chin J Oncol,17:24-26(in Chinese with English abstract)[冯威健,池川哲郎,吉田光二(1995).瑞香狼毒提取物尼地吗啉的抗癌活性.中华肿瘤杂志,17:24-26]
Fulzele DP,Heble MR,Rao PS(1995).Production of terpenoid from Artemisia annua L.plantlet cultures in bioreactor.J Biotechnol,40:139-143
Guo WJ,Zhang SQ,Wu H,et al(2007).Study on extraction process for total flavonoids in leaves of Acanthopanax senticosus harms at ultra high pressure.Chem Eng,35:66-69(in Chinese with English abstract)[郭文晶,张守勤,吴华等(2007).超高压提取刺五加叶中总黄酮的研究.化学工程,35:66-69]
Huang JQ,Zhao Y,Sun FY,et al(2013).Accumulation of flavonoids and coumarins during adventitious bud growth in Stellera chamaejasme.Chin Trad Herbal Drugs,44:1328-1333(in Chinese with English abstract)[黄嘉琦,赵越,孙凤阳等(2013).瑞香狼毒不定芽生长过程黄酮类和香豆素类成分累积研究.中草药,44:1328-1333]
Jiang YJ,Piao XC,Li H,et al(2016).Effect of several factors on saponin-accumulation in adventitious roots of Oplopanax elatus cultured with bioreactor culture.J Forest Eng,1:68-72(in Chinese with English abstract)[姜银姬,朴炫春,李贺等(2016).几种因素对生物反应器培养东北刺人参不定根中皂苷积累的影响.林业工程学报,1:68-72]
Kan XX,Wang ZX,Yang QX,et al(2013).Antitumor effect of alcohol extracts from Stellera chamaejasme.Chin J Chin Materia Med,38:1219-1225(in Chinese with English abstract)[阚晓溪,王志鑫,杨乾栩等(2013).瑞香狼毒醇提物的抗肿瘤活性研究.中国中药杂志,38:1219-1225]
Li HJ,Zhang Y,Guo Y(1997).Effects of inoculated mass on cell growth of Artemisia annua L.in suspension culture.JJinan Univ(Nat Sci Med Ed),18:89-92(in Chinese with English abstract)[李弘剑,张毅,郭勇(1997).生物量对黄花蒿悬浮培养细胞生长的影响.暨南大学学报(自然科学与医学版),18:89-92]
Park JM,Hu WS,Staba EJ(1989).Cultivation of Artemisia annua L.plantlets in a bioreactor containing a single carbon and a single nitrogen source.Biotechnol Bioeng,34:1209-1213
Piao XC,Lian ML,Wang SM(2007).Mass producing of adventitious roots of Ginseng by using bioreactor.For Sci Tech,32:54-56(in Chinese with English abstract)[朴炫春,廉美兰,王守明(2007).利用生物反应器大量生产人参不定根.林业科技,32:54-56]
Qiao LR,Yang L,Zhang DW,et al(2011).Studies on chemical constitutes from callus cultures of Stellera chamaejasme.Chin J Chin Materia Med,36:3457-3462(in Chinese with English abstract)[乔立瑞,杨林,张德武等(2011).瑞香狼毒细胞培养物的化学成分研究.中国中药杂志,36:3457-3462]
Shi CQ,Wang C(2011).Study on the insecticidal activity of Stellera chamaejasme.Nat Sci J Harbin Nor Univ,27:84-86(in Chinese with English abstract)[史传奇,王臣(2011).瑞香狼毒粗提取物杀虫活性研究.哈尔滨师范大学自然科学学报,27:84-86]
Shu JF(2014).Determination of scopoletin,baicalin,baicalein and farrerol in Qinbaohong Zhike Capsule by HPLC.Chin J Mod Appl Pharm,31:1114-1117(in Chinese with English abstract)[舒金富(2014).HPLC测定芩暴红止咳胶囊中东莨菪内酯、黄芩苷、黄芩素和杜鹃素的含量.中国现代应用药学,31:1114-1117]
Wang L(2016).The seed germination conditions and metabolism analysis of Stellera chamaejasme(dissertation).Xi’an:Northwest University(in Chinese with English abstract)[王琳(2016).瑞香狼毒种子萌发条件及代谢分析(学位论文).西安:西北大学]
Wang Q(2015).Adventitious roots culture in bioreactor of medicinal plants of Panax quinquefolium L.and Pseudostellaria heterophylla(dissertation).Tianjin:Tianjin University of Science Technology(in Chinese with English abstract)[王琴(2015).药用植物西洋参和太子参不定根反应器培养研究(学位论文).天津:天津科技大学]
Wang WX,An Q,Wang Y,et al(2010).A study on cell suspension culture and flavonoids accumulation of Stellera chamaejasme.Acta Prataculturae Sin,19:132-139(in Chinese with English abstract)[王文星,安琪,汪莹等(2010).瑞香狼毒细胞悬浮培养及黄酮积累的研究.草业学报,19:132-139]
Wu X,Zhang P,Zhang MT,et al(2017).Simultaneous determination of daphnetin,umbelliferone and scopoletin in Stellera chamejasme L.by HPLC.Chin J Mod Appl Pharm,34:1171-1174(in Chinese with English abstract)[武雪,张平,张明童等(2017).HPLC同时测定瑞香狼毒中瑞香素、伞形花内酯和东莨菪内酯的含量.中国现代应用药学,34:1171-1174]
Xing CB,Long YH,Lao FY,et al(2012).Effect of endophytic fungi on expression amount of key enzyme genes in saponins biosynthesis and Eleutherococcus senticosus saponins content.Chin J Chin Materia Med,37:2041-2045(in Chinese with English abstract)[邢朝斌,龙月红,劳凤云等(2012).内生真菌对刺五加皂苷合成关键酶基因表达及皂苷含量的影响.中国中药杂志,37:2041-2045]
Yao R(2014).Optimizing bioreactor culture system of protocorm-like bodies in Dendrobium candidum(dissertation).Yanbian,Jilin:Yanbian University(in Chinese with English abstract)[姚睿(2014).优化铁皮石斛原球茎生物反应器的培养体系(学位论文).吉林延边:延边大学]
You XL,Tan X,Jia HB,et al(2011).Ginsenosides accumulation in adventitious roots of Panax ginseng cultured in small-type bioreactors.Acta Bot Boreal-Occident Sin,31:1700-1705(in Chinese with English abstract)[由香玲,谭啸,贾洪柏等(2011).小型生物反应器内人参不定根的人参皂苷累积.西北植物学报,31:1700-1705]
Yu FK,Ma YQ,Li XW(2005).Tissue culture and rapid propagation of Stellera chamaejasme.Plant Physiol Commun,41:59(in Chinese with English abstract)[于福科,马永清,李秀维(2005).瑞香狼毒的组织培养及快速繁殖.植物生理学通讯,41:59]
Zheng BJ,Hu HQ(2006).Variety trends of coumarin in Stellera chamejasme L.in Songnen grassland of Heilongjiang,China.J Nat Sci Heilongjiang Univ,23:223-226(in Chinese with English abstract)[郑宝江,胡海清(2006).松嫩草地瑞香狼毒香豆素类化合物含量动态变化.黑龙江大学自然科学学报,23:223-226]