人参、西洋参和甘草组织培养研究
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摘要
人参（Panax ginseng C. A. Meyer）、西洋参（Panax quinquefolium L.）和甘草（Glycyrrhiza uralensis Fisch.）属于大宗类药材，应用十分广泛。本文建立了人参不定根、西洋参细胞和甘草细胞培养体系，并进行了反应器培养研究。
本文以5年生人参根为外植体诱导出愈伤组织，在附加IBA5mg·L_(-1)的MS培养基中，由愈伤组织诱导得到人参不定根。采用5L近球型鼓泡式反应器培养人参不定根40天后，生长速率达到50倍。总皂苷含量在培养第30天达到最大值。利用LC-MS鉴定了人参不定根中8种人参皂苷单体（Rg1、Re、Ro、Malonyl-Rb1、Rb1、Rc、Rb2和Rd）；人参不定根培养体系生长速度较快，皂苷合成能力也有明显优势。利用诱导子10mg/L MJ培养人参不定根24h后，人参皂苷含量显著提高，其中以二醇型皂苷尤为显著，这一结果与SE, DS, P450基因表达密切相关。抗氧化活性研究表明：人参不定根中皂苷和多糖的DPPH抑制率均高于栽培人参。不定根中皂苷含量为60mg·L_(-1)时，DPPH抑制率为96.03%。
以5年生栽培西洋参为外植体诱导出愈伤组织，将愈伤组织加入到附加2mg·L_(-1)2,4-D1，0.25mg·L-KT的MS培养基中诱导得到悬浮细胞。5L反应器培养中，西洋参细胞的生长和活性成分含量基本在第21天达到最大值，这一过程与营养成分消耗相关。添加混合诱导子后（100mg·L_(-1)LH和2mg·L_(-1)MJ），细胞的生物量和多糖含量没有显著变化，而皂苷含量要高于添加单一诱导子的处理组。第16天补加30g·L_(-1)蔗糖后，细胞干重生长率以及多糖含量均高于对照组，多糖产率在第21天达到最大值（1.608g/L），为对照组的1.96倍。西洋参两步培养法获得了较高的皂苷产率（31.52mg·L_(-1)）和多糖产率（1.72g·L_(-1)），分别是对照组的4.34倍和2.1倍。西洋参细胞中鉴定出了4种成分，分别为Rg1、Re、Rf和Rb1。较高的多糖含量是西洋参悬浮细胞的显著优势，但皂苷含量较低。抗氧化活性研究表明：西洋参细胞中皂苷的DPPH抑制率（55.72%）高于栽培西洋参，栽培西洋参和细胞中多糖的DPPH抑制率均较低。
以甘草下胚轴为外植体诱导出愈伤组织，将愈伤组织加入到附加1.0mg·L_(-1)2,4-D,1.0mg·L_(-1)NAA,0.2mg·L_(-1)6-BA的MS培养基中诱导得到悬浮细胞。采用5L近球型鼓泡式反应器培养甘草细胞20天后，干重生长率达到最大值。三萜皂苷和黄酮分别在培养第10天和15天达到最大值。采用10L近球型鼓泡反应器培养18天后，甘草悬浮细胞生长率为10.86倍。甘草细胞中鉴定出了5种成分，分别为甘草苷、甘草皂苷B、甘草皂苷J_2、甘草酸和甘草皂苷B_2；甘草细胞中三萜类成分较少且甘草酸含量较低，甘草黄酮的成分和含量与栽培甘草接近。甘草细胞中多糖含量为11.7%，高于栽培甘草。抗氧化活性研究表明：当黄酮浓度为500mg·L_(-1)时，栽培甘草和甘草细胞的DPPH抑制率分别为93.98%和100%。两种材料中甘草多糖的DPPH抑制率则较低。
以上研究为工业化生产人参、西洋参和甘草的活性成分奠定了基础。
Panax ginseng C. A. Meyer, Panax quinquefolium L. and Glycyrrhiza uralensisFisch. are widely used herb. In this study, adventitious root of P. ginseng, cells of P.quinquefolium and G. uralensis have been established. We also conducted studies onbioreactor culture.
Callus of ginseng was induced by roots of P. ginseng (5-year-old). Adventitiousroots were initiated from callus which were inoculated onto MS solid mediacontaining5.0mg·L_(-1)IBA. Growth rate of50-fold in5L balloon-type bubblebioreactor (BTBB) was obtained after40days of inoculation. The maximum totalsaponin was achieved on day30. Rg1, Re, Ro, Malonyl-Rb1, Rb1, Rc, Rb2and Rdwere identified from ginseng adventitious root. Ginseng adventitious root culturegrew faster and had a greater capability of ginsenoside production. With24h of10mg·L_(-1)MJ elicitation, level of total saponins in ginseng adventitious root increasedmuch higher than that observed in the control, especially Rb group, which wererelated to the expression of SE, DS and P450. DPPH inhibition of ginsenoside andpolysaccharide were higher in adventitious roots than in native roots, with60mg L_(-1)ginsenoside of ginseng adventitious roots, the DPPH inhibition was96.03%.
Callus of P. quinquefolium was induced by roots of P. quinquefolium (5-year-old).Cells were initiated from callus which were inoculated onto MS solid mediacontaining2mg·L_(-1)2,4-D,0.25mg·L_(-1)KT. In a bioreactor, the dry cell weight, thecontents of ginsenosides and polysaccharide reached the maximum peaksimultaneously on about21days and the results showed that cell growth andmetabolites synthesis related to nutrients consumption. LH at100mg L_(-1)with methyljasmonate (MJ) at2mg L_(-1)synergistically stimulated ginsenoside accumulation in P.quinquefolium cells compared with100mg L_(-1)LH. Using a fed-batch cultivationstrategy, polysaccharide production was enhanced to1.608g L1, which was1.96-foldgreater than with batch cultivation. Two-stage cultivation caused a significant increasein total saponins yield (31.52mg·L_(-1)) and polysaccharide yield (1.72g·L_(-1)) cellcultures. This value was increased by4.34-fold and2.1-fold compared to the batchcultivation, respectively. Rg1, Re, Rf and Rb1were identified from P. quinquefoliumcells. The higher polysaccharide content is an obvious advantage of P. quinquefolium cells. However, the ginsenoside contents in the cell cultures are still much lower thanthat in field cultivated. DPPH inhibition of ginsenoside in P. quinquefolium cells washigher than that in native root. DPPH inhibition of polysaccharide both in native P.quinquefolium and cells are lower.
Callus of G. uralensis was induced by hypocotyls of G. uralensis. Cells wereinitiated from callus which were inoculated onto MS solid media containing1.0mg·L_(-1)2,4-D,1.0mg·L_(-1)NAA,0.2mg·L_(-1)6-BA. The maximum dry weight,triterpenoids and flavonoids were achieved on day20,10and15, respectively in10LBTBB. Maximum growth rate of10.86-fold in10L BTBB were obtained after18days of inoculation. Liquiritin, licorice glycoside B, licorice saponin J2, glycyrrhizicacid and licorice saponin B2were identified from G. uralensis cells. Triterpenoids in G.uralensis cells are much less than that in native licorice. However, flavonoids in cellsare similar to the native licorice. Content of polysaccharide in cells (11.7%) is higherthan that in native. About93.98%and100%DPPH inhibition occurred with500mgL_(-1)flavonoids of native licorice and cells. DPPH inhibition of polysaccharide in nativelicorice and cells are lower.
These results provide a theoretical reference for an efficient production of activemetabolites of above three kinds of plants.
本文以5年生人参根为外植体诱导出愈伤组织，在附加IBA5mg·L_(-1)的MS培养基中，由愈伤组织诱导得到人参不定根。采用5L近球型鼓泡式反应器培养人参不定根40天后，生长速率达到50倍。总皂苷含量在培养第30天达到最大值。利用LC-MS鉴定了人参不定根中8种人参皂苷单体（Rg1、Re、Ro、Malonyl-Rb1、Rb1、Rc、Rb2和Rd）；人参不定根培养体系生长速度较快，皂苷合成能力也有明显优势。利用诱导子10mg/L MJ培养人参不定根24h后，人参皂苷含量显著提高，其中以二醇型皂苷尤为显著，这一结果与SE, DS, P450基因表达密切相关。抗氧化活性研究表明：人参不定根中皂苷和多糖的DPPH抑制率均高于栽培人参。不定根中皂苷含量为60mg·L_(-1)时，DPPH抑制率为96.03%。
以5年生栽培西洋参为外植体诱导出愈伤组织，将愈伤组织加入到附加2mg·L_(-1)2,4-D1，0.25mg·L-KT的MS培养基中诱导得到悬浮细胞。5L反应器培养中，西洋参细胞的生长和活性成分含量基本在第21天达到最大值，这一过程与营养成分消耗相关。添加混合诱导子后（100mg·L_(-1)LH和2mg·L_(-1)MJ），细胞的生物量和多糖含量没有显著变化，而皂苷含量要高于添加单一诱导子的处理组。第16天补加30g·L_(-1)蔗糖后，细胞干重生长率以及多糖含量均高于对照组，多糖产率在第21天达到最大值（1.608g/L），为对照组的1.96倍。西洋参两步培养法获得了较高的皂苷产率（31.52mg·L_(-1)）和多糖产率（1.72g·L_(-1)），分别是对照组的4.34倍和2.1倍。西洋参细胞中鉴定出了4种成分，分别为Rg1、Re、Rf和Rb1。较高的多糖含量是西洋参悬浮细胞的显著优势，但皂苷含量较低。抗氧化活性研究表明：西洋参细胞中皂苷的DPPH抑制率（55.72%）高于栽培西洋参，栽培西洋参和细胞中多糖的DPPH抑制率均较低。
以甘草下胚轴为外植体诱导出愈伤组织，将愈伤组织加入到附加1.0mg·L_(-1)2,4-D,1.0mg·L_(-1)NAA,0.2mg·L_(-1)6-BA的MS培养基中诱导得到悬浮细胞。采用5L近球型鼓泡式反应器培养甘草细胞20天后，干重生长率达到最大值。三萜皂苷和黄酮分别在培养第10天和15天达到最大值。采用10L近球型鼓泡反应器培养18天后，甘草悬浮细胞生长率为10.86倍。甘草细胞中鉴定出了5种成分，分别为甘草苷、甘草皂苷B、甘草皂苷J_2、甘草酸和甘草皂苷B_2；甘草细胞中三萜类成分较少且甘草酸含量较低，甘草黄酮的成分和含量与栽培甘草接近。甘草细胞中多糖含量为11.7%，高于栽培甘草。抗氧化活性研究表明：当黄酮浓度为500mg·L_(-1)时，栽培甘草和甘草细胞的DPPH抑制率分别为93.98%和100%。两种材料中甘草多糖的DPPH抑制率则较低。
以上研究为工业化生产人参、西洋参和甘草的活性成分奠定了基础。
Panax ginseng C. A. Meyer, Panax quinquefolium L. and Glycyrrhiza uralensisFisch. are widely used herb. In this study, adventitious root of P. ginseng, cells of P.quinquefolium and G. uralensis have been established. We also conducted studies onbioreactor culture.
Callus of ginseng was induced by roots of P. ginseng (5-year-old). Adventitiousroots were initiated from callus which were inoculated onto MS solid mediacontaining5.0mg·L_(-1)IBA. Growth rate of50-fold in5L balloon-type bubblebioreactor (BTBB) was obtained after40days of inoculation. The maximum totalsaponin was achieved on day30. Rg1, Re, Ro, Malonyl-Rb1, Rb1, Rc, Rb2and Rdwere identified from ginseng adventitious root. Ginseng adventitious root culturegrew faster and had a greater capability of ginsenoside production. With24h of10mg·L_(-1)MJ elicitation, level of total saponins in ginseng adventitious root increasedmuch higher than that observed in the control, especially Rb group, which wererelated to the expression of SE, DS and P450. DPPH inhibition of ginsenoside andpolysaccharide were higher in adventitious roots than in native roots, with60mg L_(-1)ginsenoside of ginseng adventitious roots, the DPPH inhibition was96.03%.
Callus of P. quinquefolium was induced by roots of P. quinquefolium (5-year-old).Cells were initiated from callus which were inoculated onto MS solid mediacontaining2mg·L_(-1)2,4-D,0.25mg·L_(-1)KT. In a bioreactor, the dry cell weight, thecontents of ginsenosides and polysaccharide reached the maximum peaksimultaneously on about21days and the results showed that cell growth andmetabolites synthesis related to nutrients consumption. LH at100mg L_(-1)with methyljasmonate (MJ) at2mg L_(-1)synergistically stimulated ginsenoside accumulation in P.quinquefolium cells compared with100mg L_(-1)LH. Using a fed-batch cultivationstrategy, polysaccharide production was enhanced to1.608g L1, which was1.96-foldgreater than with batch cultivation. Two-stage cultivation caused a significant increasein total saponins yield (31.52mg·L_(-1)) and polysaccharide yield (1.72g·L_(-1)) cellcultures. This value was increased by4.34-fold and2.1-fold compared to the batchcultivation, respectively. Rg1, Re, Rf and Rb1were identified from P. quinquefoliumcells. The higher polysaccharide content is an obvious advantage of P. quinquefolium cells. However, the ginsenoside contents in the cell cultures are still much lower thanthat in field cultivated. DPPH inhibition of ginsenoside in P. quinquefolium cells washigher than that in native root. DPPH inhibition of polysaccharide both in native P.quinquefolium and cells are lower.
Callus of G. uralensis was induced by hypocotyls of G. uralensis. Cells wereinitiated from callus which were inoculated onto MS solid media containing1.0mg·L_(-1)2,4-D,1.0mg·L_(-1)NAA,0.2mg·L_(-1)6-BA. The maximum dry weight,triterpenoids and flavonoids were achieved on day20,10and15, respectively in10LBTBB. Maximum growth rate of10.86-fold in10L BTBB were obtained after18days of inoculation. Liquiritin, licorice glycoside B, licorice saponin J2, glycyrrhizicacid and licorice saponin B2were identified from G. uralensis cells. Triterpenoids in G.uralensis cells are much less than that in native licorice. However, flavonoids in cellsare similar to the native licorice. Content of polysaccharide in cells (11.7%) is higherthan that in native. About93.98%and100%DPPH inhibition occurred with500mgL_(-1)flavonoids of native licorice and cells. DPPH inhibition of polysaccharide in nativelicorice and cells are lower.
These results provide a theoretical reference for an efficient production of activemetabolites of above three kinds of plants.
引文
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