水杨酸刺激下半夏试管块茎悬浮培养及其生物碱的含量
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摘要
目的:研究水杨酸刺激下半夏试管块茎悬浮培养及其体内生物碱含量的变化。方法:以半夏悬浮试管小块茎为材料,对其处理不同浓度的外源水杨酸刺激,分析块茎的生长情况,利用高效液相色谱法(HPLC)测定块茎中生物碱的含量。检测采用Agilent C_(18)色谱柱(4. 6 mm×250 mm,5μm),流动相乙腈-水(4∶96),柱温35℃,肌苷检测波长250 nm,鸟苷检测波长260 nm,流速1. 0 m L·min~(-1),进样量10μL。结果:外源添加不同浓度的水杨酸,处理不同的时间,半夏块茎的鲜重在第25天,水杨酸150μmol·L~(-1)时达到最大7. 483 8 g。同时积累了一定量的生物碱,鸟苷在0. 03～0. 45μg(R2=0. 999 6)线性关系良好,当水杨酸浓度为50μmol·L~(-1)时,培养10 d,半夏块茎中鸟苷的质量分数达到最大值1. 353 3 mg·g~(-1);肌苷在0. 003～0. 045μg(R2=0. 999 5)线性关系良好,在水杨酸浓度为200μmol·L~(-1)时,培养30 d,半夏块茎中肌苷的质量分数达到最大值0. 149 8 mg·g~(-1)。结论:该研究结果为半夏试管块茎的组培快繁和生物碱的调控研究提供参考,对半夏产业的发展具有十分重要的意义。
Objective: To study the suspension culture of tuber and its alkaloid content based on the stimulation of salicylic acid. Method: The tubers of Pinelliae Rhizoma in suspension tube were treated with different concentrations of exogenous salicylic acid to analyze the growth status. The content of alkaloids in tuber was detected by HPLC. Test conditions: chromatographic column for Agilent Eclipse plus C_(18) column( 4. 6 mm ×250 mm,5 μm),mobile phase of acetonitrile water( 4 ∶ 96),the column temperature was maintained at 35 ℃,detection wavelength for inosine 250 nm, guanosine 260 nm, volume flow rate 1. 0 m L·min~(-1). Result: The results showed that the exogenous salicylic acid had a certain effect on the growth of suspension tuber of Pinelliae Rhizoma. When the salicylic acid concentration was 150 μmol·L~(-1),the culture lasted for 25 days and the fresh weight reached the maximum value of 7. 483 8 g. It also accumulates a certain amount of alkaloids. The linear range of guanosine was 0. 03-0. 45 μg( R2= 0. 999 6). After 10-days cultivatation in the salicylic acid concentration of 50 μmol ·L~(-1), guanosine content of Pinelliae Rhizoma tubers reached a maximum of1. 353 3 mg·g~(-1). The linear range of inosine 0. 003-0. 045 μg( R2= 0. 999 5). When the salicylic acid concentration was 200 μmol·L~(-1),cultured for 30 days,the content of inosine in Pinelliae Rhizoma tubers reached the maximum value of 0. 149 8 mg·g~(-1). Conclusion: The results of this experiment provide a reference for the study of tissue culture and rapid propagation of Pinelliae Rhizoma tubers and regulation of alkaloids,which are of great significance for the development of Pinelliae Rhizoma industry.
Objective: To study the suspension culture of tuber and its alkaloid content based on the stimulation of salicylic acid. Method: The tubers of Pinelliae Rhizoma in suspension tube were treated with different concentrations of exogenous salicylic acid to analyze the growth status. The content of alkaloids in tuber was detected by HPLC. Test conditions: chromatographic column for Agilent Eclipse plus C_(18) column( 4. 6 mm ×250 mm,5 μm),mobile phase of acetonitrile water( 4 ∶ 96),the column temperature was maintained at 35 ℃,detection wavelength for inosine 250 nm, guanosine 260 nm, volume flow rate 1. 0 m L·min~(-1). Result: The results showed that the exogenous salicylic acid had a certain effect on the growth of suspension tuber of Pinelliae Rhizoma. When the salicylic acid concentration was 150 μmol·L~(-1),the culture lasted for 25 days and the fresh weight reached the maximum value of 7. 483 8 g. It also accumulates a certain amount of alkaloids. The linear range of guanosine was 0. 03-0. 45 μg( R2= 0. 999 6). After 10-days cultivatation in the salicylic acid concentration of 50 μmol ·L~(-1), guanosine content of Pinelliae Rhizoma tubers reached a maximum of1. 353 3 mg·g~(-1). The linear range of inosine 0. 003-0. 045 μg( R2= 0. 999 5). When the salicylic acid concentration was 200 μmol·L~(-1),cultured for 30 days,the content of inosine in Pinelliae Rhizoma tubers reached the maximum value of 0. 149 8 mg·g~(-1). Conclusion: The results of this experiment provide a reference for the study of tissue culture and rapid propagation of Pinelliae Rhizoma tubers and regulation of alkaloids,which are of great significance for the development of Pinelliae Rhizoma industry.
引文
[1]刘永红,郭建宏,刘文婷,等.药用植物半夏生物碱类成分研究进展[J].西北农林科技大学学报:自然科学版,2015,43(9):171-177.
[2]龚道锋,王甫成,纪东汉,等.中药半夏化学成分及其药理、毒理活性研究进展[J].长江大学学报:自科版,2015,12(18):77-79.
[3]肖琦,阳文武,张德伟,等.半夏总生物碱含量影响因素及药理作用研究进展[J].中国药业,2016,25(3):123-126.
[4]吴皓,李伟,张科卫,等.半夏药材鉴别成分的研究[J].中国中药杂志,2003,28(9):836-839.
[5]何丹,杨林,秦少容,等.超高效液相色谱法测定半夏不同炮制品中核苷类成分含量[J].医药导报,2015,34(2):231-234.
[6]王晖,马慧芬,徐赟晟,等.炮制对半夏淀粉基础物理化学性质的影响[J].中国实验方剂学杂志,2017,23(22):32-36.
[7]张瑾,谈献和.半夏资源研究进展[J].中国中医药信息杂志,2010,17(5):104-106.
[8]黄鲛,易进海,刘玉红,等.天南星、半夏、白附子中8种核苷成分的含量测定[J].中国实验方剂学杂志,2013,19(16):59-62.
[9]龙月红,邢朝斌,劳凤云,等.高效液相色谱测定不同生境黄芩中的黄芩苷[J].中国实验方剂学杂志,2012,18(6):97-100.
[10]汪尚,徐鹭芹,张亚仙,等.水杨酸介导植物抗病的研究进展[J].植物生理学报,2016,52(5):581-590.
[11] YUAN S,LIN H H. Role of salicylic acid in plant abiotic stress[J]. Z Naturforsch C,2008,63(5/6):313-320.
[12] DONG J,WAN G W,LIANG Z S. Accumulation of salicylic acid-induced phenolic compounds and raised activities of secondary metabolic and antioxidative enzymes in Salvia miltiorrhiza cell culture[J]. J Biotechnol,2010,148(2/3):99-104.
[13] XIAO Y,DI P,CHEN J F,et al. Characterization and expression profiling of 4-hydroxyphenylpyruvate dioxygenase gene(Smhppd)from Salvia miltiorrhiza hairy root cultures[J]. Mol Biol Rep,2009,36(7):2019-2029.
[14]张悦,王义,蒋世翠,等.水杨酸诱导下人参培养物差异表达基因片段的克隆[J].基因组学与应用生物学,2009,28(2):245-250.
[15]孟书亦,马秀杰,韩梅,等.水杨酸对黄芩愈伤组织抗氧化酶活性及黄芩苷含量的影响[J].分子植物育种,2017,15(10):4179-4183.
[16] Manorma S,Archana S,Ashwani K,et al. Enhancement of secondary metabolites in cultured plant cells through stress stimulus[J]. Am J Plant Physiol,2011,6(2):50-71.
[17]祝军委,冯果,冯泳.小半夏加茯苓汤中鸟苷含量的高效液相色谱测定[J].时珍国医国药,2013,24(8):1818-1820.
[18]郝林.水杨酸参与生物学过程的交谈机制[J].沈阳师范大学学报:自然科学版,2010,28(4):449-457.
[19]薛建平,张爱民,方中明,等. 2007,水杨酸对半夏植株生长的影响[J].中国中药杂志,32(12):1134-1136.
[20] LIU Y H,LIANG Z S,ZHANG Y J. Induction and in vitro alkaloid yield of calluses and protocorm-like bodies(PLBs)from Pinellia ternata[J]. In Vitro Cell Dev Biol Plant,2010,46(3):239-245.
[21]刘永红,梁宗锁,杨东风,等.半夏小块茎悬浮培养及其生物碱类化合物的测定[J].西北农林科技大学学报:自然科学版,2009,37(11):168-174.
[2]龚道锋,王甫成,纪东汉,等.中药半夏化学成分及其药理、毒理活性研究进展[J].长江大学学报:自科版,2015,12(18):77-79.
[3]肖琦,阳文武,张德伟,等.半夏总生物碱含量影响因素及药理作用研究进展[J].中国药业,2016,25(3):123-126.
[4]吴皓,李伟,张科卫,等.半夏药材鉴别成分的研究[J].中国中药杂志,2003,28(9):836-839.
[5]何丹,杨林,秦少容,等.超高效液相色谱法测定半夏不同炮制品中核苷类成分含量[J].医药导报,2015,34(2):231-234.
[6]王晖,马慧芬,徐赟晟,等.炮制对半夏淀粉基础物理化学性质的影响[J].中国实验方剂学杂志,2017,23(22):32-36.
[7]张瑾,谈献和.半夏资源研究进展[J].中国中医药信息杂志,2010,17(5):104-106.
[8]黄鲛,易进海,刘玉红,等.天南星、半夏、白附子中8种核苷成分的含量测定[J].中国实验方剂学杂志,2013,19(16):59-62.
[9]龙月红,邢朝斌,劳凤云,等.高效液相色谱测定不同生境黄芩中的黄芩苷[J].中国实验方剂学杂志,2012,18(6):97-100.
[10]汪尚,徐鹭芹,张亚仙,等.水杨酸介导植物抗病的研究进展[J].植物生理学报,2016,52(5):581-590.
[11] YUAN S,LIN H H. Role of salicylic acid in plant abiotic stress[J]. Z Naturforsch C,2008,63(5/6):313-320.
[12] DONG J,WAN G W,LIANG Z S. Accumulation of salicylic acid-induced phenolic compounds and raised activities of secondary metabolic and antioxidative enzymes in Salvia miltiorrhiza cell culture[J]. J Biotechnol,2010,148(2/3):99-104.
[13] XIAO Y,DI P,CHEN J F,et al. Characterization and expression profiling of 4-hydroxyphenylpyruvate dioxygenase gene(Smhppd)from Salvia miltiorrhiza hairy root cultures[J]. Mol Biol Rep,2009,36(7):2019-2029.
[14]张悦,王义,蒋世翠,等.水杨酸诱导下人参培养物差异表达基因片段的克隆[J].基因组学与应用生物学,2009,28(2):245-250.
[15]孟书亦,马秀杰,韩梅,等.水杨酸对黄芩愈伤组织抗氧化酶活性及黄芩苷含量的影响[J].分子植物育种,2017,15(10):4179-4183.
[16] Manorma S,Archana S,Ashwani K,et al. Enhancement of secondary metabolites in cultured plant cells through stress stimulus[J]. Am J Plant Physiol,2011,6(2):50-71.
[17]祝军委,冯果,冯泳.小半夏加茯苓汤中鸟苷含量的高效液相色谱测定[J].时珍国医国药,2013,24(8):1818-1820.
[18]郝林.水杨酸参与生物学过程的交谈机制[J].沈阳师范大学学报:自然科学版,2010,28(4):449-457.
[19]薛建平,张爱民,方中明,等. 2007,水杨酸对半夏植株生长的影响[J].中国中药杂志,32(12):1134-1136.
[20] LIU Y H,LIANG Z S,ZHANG Y J. Induction and in vitro alkaloid yield of calluses and protocorm-like bodies(PLBs)from Pinellia ternata[J]. In Vitro Cell Dev Biol Plant,2010,46(3):239-245.
[21]刘永红,梁宗锁,杨东风,等.半夏小块茎悬浮培养及其生物碱类化合物的测定[J].西北农林科技大学学报:自然科学版,2009,37(11):168-174.