青蒿毛状根中活性化合物的分离分析及其生物合成调控探索
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摘要
青蒿(Artemisia annua)作为一种传统中药,被用来抗疟、抗血吸虫、镇咳和祛痰平喘等。青蒿毛状根培养物被认为具有与天然青蒿植物相似的遗传稳定性和生化性质,可用来有效生产活性次级代谢产物,如倍半萜类、黄酮类以及香豆素类等。
本学位论文从青蒿毛状根培养物中分离纯化并鉴定了三种化合物,多炔类的(E)-反式-本都山蒿环氧化物((E)-trans-Ponticaepoxide, PO),倍半萜香豆素醚类的锥满醇A(Drimartol A, DA)和一种新的倍半萜类的(Z)-7-乙酰-甲基-11-甲基-3-亚甲基-十二碳-1,6,10-三烯((Z)-7-Acetoxy-methyl-11-methyl-3-methylene-dodeca-1,6,10-triene, AMDT)。多炔PO据报道是一种天然的除虫剂,而DA和AMDT的活性未见报道。在本研究中,我们研究了DA和AMDT对多种肿瘤细胞株的生长抑制作用,它们的IC50范围为17.9-73.3μM。着眼于致死率最高的肺癌,我们使用人高转移肺癌细胞株95-D作为研究对象,研究了AMDT和DA诱导95-D细胞凋亡的能力。DA能抑制95-D细胞生长并剂量依赖性地阻滞95-D细胞于G2期,AMDT能抑制95-D细胞生长并剂量依赖性地阻滞95-D细胞于G1期；它们均能引起95-D细胞凋亡,并且诱导的细胞凋亡伴随着线粒体膜电位的下降。半胱氨酸天冬氨酸蛋白酶-3(Caspase-3)和半胱氨酸天冬氨酸蛋白酶-9(Caspase-9)均参与了DA和AMDT诱导的95-D细胞凋亡过程。这些结果表明AMDT和DA可以通过线粒体依赖的途径有效诱导了95-D的细胞凋亡,它们有望成为化疗药剂的候选物。
由于以上三种活性化合物的定量分析方法尚未见报道,在本研究中,我们建立了一种可以同时检测它们的反相高效液相光二极管阵列检测法(RP-HPLC-PAD)。该方法使用C18反相色谱柱,以乙腈和水为流动相进行梯度洗脱。DA, PO和AMDT的检测波长分别为220 nm、254 nm和220 nm。该方法线性好,相关系数R>0.99。研究了该方法的精密度、准确度和回收率,并证明它可以作为分析青蒿毛状根细胞培养物中三种活性化合物积累的方法。
为了提高毛状根细胞中这些生物活性成分的含量,我们研究了改变毛状根培养细胞营养环境并添加诱导子的方法。实验结果表明,添加甲基茉莉酸酯(MJ, 100μM)可使毛状根细胞中PO、DA和AMDT的含量从0.7 mg/gDW,1.7 mg/gDW和0.2 mg/gDW分别提高到2.07mg/gDW,4.77 mg/gDW和0.48 mg/gDWo当细胞在缺氮培养又用MJ处理后,PO, DA和AMDT的含量可进一步提高至3.43 mg/gDW,28.9 mg/gDW和0.9mg/gDW。这表明缺氮培养和MJ添加的组合策略可有效提高青蒿毛状根细胞培养中三种活性化合物的积累。
为了探讨上述策略的调控机制,以缺氮和MJ添加作为典型条件,以两种抗肿瘤倍半萜化合物DA和AMDT为目标产物,我们检测了胞外H202、胞内信号分子茉莉酸(JA)和苯丙氨酸氨裂解酶酶活的变化,以及倍半萜合成途径相关基因的转录。尽管胞外过氧化氢未见迸发,但苯丙氨酸氨裂解酶酶活及内源茉莉酸的合成被诱导增强；当使用JA生物合成途径的抑制剂或苯丙氨酸氨裂解酶的抑制剂后,青蒿毛状根细胞的DA和AMDT的合成也受到抑制。在MJ处理以及在缺氮培养和MJ处理组合的时候,法呢基焦磷酸合酶(fps),1-脱氧-D-木酮糖-5-磷酸合酶(dxr),和紫穗槐-4,11-双烯C-12加氧酶(cyp71av)的转录均得到上调,而倍半萜合成的支路酶鲨烯合酶(sqs)的基因转录下调,且这种趋势被苯丙氨酸氨裂解酶抑制剂氨氧基乙酸(AOA)所抑制。以上结果说明苯丙氨酸氨裂解酶和茉莉酸介导了外源MJ添加和缺氮条件下诱导的DA和AMDT的生物合成。
总之,本文从青蒿毛状根中分离纯化了三种化合物,建立了它们的分析方法。并对它们的抗肿瘤活性进行了鉴定,结果表明,AMDT和DA可通过线粒体依赖的途径有效诱导了95-D的细胞凋亡。为了提高毛状根细胞中这些生物活性成分的含量,我们采用了改变毛状根细胞的营养微环境并添加诱导子的策略,结果表明青蒿毛状根在缺氮并添加MJ诱导的条件下,可使细胞的DA和AMDT的含量大幅提高,同时从信号分子和基因转录的水平探讨了这些细胞培养策略的调控机制。这些工作为今后从青蒿毛状根中开发新的活性药物奠定了良好基础,也为这些培养策略应用到植物组织细胞培养的生产实践提供了一定的理论基础。
Artemisia annua is traditionally used as folk medicine for antimalaria, anti-schistosomiasis, antitussis, expectoration and antiasthma in China. Hairy root cultures of A. annua, which present genetic and biochemical stabilities similar to those of the mother plant, are established for more stable and efficient production of their active constituents such as sesquiterpenes, flavornoids and courmains.
In this thesis, (E)-trans-ponticaepoxide (polyacetylene, PO), drimartol A (sesquiterpene coumarin ether, DA) and a new sesquiterpene, (Z)-7-acetoxy-methyl-11-methyl-3-methylene-dodeca-1,6,10-triene (AMDT), were isolated, purified and identified from the cultured hairy roots of A. annua. PO had been reported as a natural insecticide, while the bioactivity of DA and AMDT were unknown and was investigated in this work. Both AMDT and DA showed moderate cytotoxic activities against several human tumor cell lines by MTT assay, whose IC50 values were ranged within 17.9-73.3μM. As treatment of lung cancer, the No.1 killer of global cancer patients, is our interest, the ability of AMDT and DA to induce apoptosis of the human lung tumor cell line 95-D was focused. It was found that the 95-D cell growth was inhibited and its cell cycle was arrested in the G2 phase by DA and in the G1 phase by AMDT. The apoptotic rate of the cells increased in a dose-dependent manner. AMDT and DA lowered the mitochondrial membrane potential and increased the expression of caspase-9 and-3. The results revealed that AMDT and DA could efficiently induce 95-D cell apoptosis through mitochondrial dependent pathway, and they may be potential chemotherapeutic agents.
Quantitative determination methods for any of those bioactive metabolites are not yet available, not to mention their simultaneous analysis. In this work, simultaneous determination of those useful compounds was achieved by using reversed-phase high-performance liquid chromatography-photo diode array detector (RP-HPLC-PAD). The HPLC operating conditions were optimized and the chromatographic separation was performed on a C18 column with a gradient acetonitrile-water as mobile phase. DA, PO and AMDT was detected at 220 nm,254 nm and 220 nm, respectively, and a good linear relationship within the range of investigated concentrations was observed with their correlation coefficient R higher than 0.99. The method was validated for precision, accuracy and recovery, and it was further successfully applied to the time-course detection of the accumulation of those three bioactive compounds in the genetically transformed root cultures of A. annua.
To improve the accumulation of these bioactive compounds, different nutrition state and elicitor treatment were applied in the hairy root culture of A. annua. Production of PO, DA and AMDT was increased from 0.7 mg/gDW,1.7 mg/gDW and 0.2 mg/gDW to 2.07mg/g DW, 4.77 mg/gDW and 0.48 mg/gDW, respectively, by adding 100μM methyl jasmonic acid (MJ). When the cultures were treated with MJ in nitrogen deficient medium, the content of PO, DA and AMDT was improved in a higher level to 3.43 mg/gDW,28.9 mg/gDW and 0.9 mg/gDW.
To provide an insight into the molecular mechanism of the inducing effect of MJ treatment and nitrogen deficiency, in this work sesquiterpene biosynthetic pathway and signal molecules jasmonic acid (JA), H2O2 and phenylalanine ammonia lyase (PAL) were examined in the hairy root culture of A. annua. Although the oxidative burst (induced H2O2 production) was not detected, the induction of PAL activity and intracellular JA synthesis were observed. When the cultures were treated with the inhibitor of JA biosynthsis or the inhibitor of PAL, the contents of DA and AMDT were decreased. The gene transcription of fps (farnesyl diphosphate synthase gene), dxr (1-deoxy-D-xylulose-5-phosphate synthase gene), and cyp71av (amorpha-4,11-diene C-12 oxidase gene) were up-regulated and the sqs (squalene synthase gene) was down-regulated by the combination treatment of MJ elicitation and nitrogen deficiency. Meanwhile, these regulation trends of gene transcription could be inhibited by the PAL inhibitor (BOC-aminooxy) acetic acid (AOA). The results implied that the sesquiterpene biosynthesis induction by MJ treatment and nitrogen deficiency may be mediated by the JA synthesis, PAL activation and the sesquiterpene biosynthetic gene regulation.
Collectively, in this work three bioactive compounds were separated and identified from A. annua hairy root culture. AMDT was a new sesquiterpene. Their simultaneous quantitative analysis method was developed. Their antitumor activities were found. The results revealed that DA and AMDT induced the 95-D apoptosis through mitochondrial dependent pathway. To improve the content of these bioactive compounds, the strategies of using nutrition deficiency medium, adding elicitor and their combination were applied. Results showed the content of DA and AMDT could be increased respectively to 21.1 folds and 9 folds when the hairy root cultures were treated with the combination of nitrogen starvation and MJ addition. Meanwhile, the mechanism involved in the stratege was studied and revealed from the levels of signal transduction and gene transcription. This work provided a good basis for further investigation on the discovery of new drugs from the A. annua hairy root culture. This study is also helpful for application of the strategy to efficient production of other useful secondary metabolites in plant cells.
本学位论文从青蒿毛状根培养物中分离纯化并鉴定了三种化合物,多炔类的(E)-反式-本都山蒿环氧化物((E)-trans-Ponticaepoxide, PO),倍半萜香豆素醚类的锥满醇A(Drimartol A, DA)和一种新的倍半萜类的(Z)-7-乙酰-甲基-11-甲基-3-亚甲基-十二碳-1,6,10-三烯((Z)-7-Acetoxy-methyl-11-methyl-3-methylene-dodeca-1,6,10-triene, AMDT)。多炔PO据报道是一种天然的除虫剂,而DA和AMDT的活性未见报道。在本研究中,我们研究了DA和AMDT对多种肿瘤细胞株的生长抑制作用,它们的IC50范围为17.9-73.3μM。着眼于致死率最高的肺癌,我们使用人高转移肺癌细胞株95-D作为研究对象,研究了AMDT和DA诱导95-D细胞凋亡的能力。DA能抑制95-D细胞生长并剂量依赖性地阻滞95-D细胞于G2期,AMDT能抑制95-D细胞生长并剂量依赖性地阻滞95-D细胞于G1期；它们均能引起95-D细胞凋亡,并且诱导的细胞凋亡伴随着线粒体膜电位的下降。半胱氨酸天冬氨酸蛋白酶-3(Caspase-3)和半胱氨酸天冬氨酸蛋白酶-9(Caspase-9)均参与了DA和AMDT诱导的95-D细胞凋亡过程。这些结果表明AMDT和DA可以通过线粒体依赖的途径有效诱导了95-D的细胞凋亡,它们有望成为化疗药剂的候选物。
由于以上三种活性化合物的定量分析方法尚未见报道,在本研究中,我们建立了一种可以同时检测它们的反相高效液相光二极管阵列检测法(RP-HPLC-PAD)。该方法使用C18反相色谱柱,以乙腈和水为流动相进行梯度洗脱。DA, PO和AMDT的检测波长分别为220 nm、254 nm和220 nm。该方法线性好,相关系数R>0.99。研究了该方法的精密度、准确度和回收率,并证明它可以作为分析青蒿毛状根细胞培养物中三种活性化合物积累的方法。
为了提高毛状根细胞中这些生物活性成分的含量,我们研究了改变毛状根培养细胞营养环境并添加诱导子的方法。实验结果表明,添加甲基茉莉酸酯(MJ, 100μM)可使毛状根细胞中PO、DA和AMDT的含量从0.7 mg/gDW,1.7 mg/gDW和0.2 mg/gDW分别提高到2.07mg/gDW,4.77 mg/gDW和0.48 mg/gDWo当细胞在缺氮培养又用MJ处理后,PO, DA和AMDT的含量可进一步提高至3.43 mg/gDW,28.9 mg/gDW和0.9mg/gDW。这表明缺氮培养和MJ添加的组合策略可有效提高青蒿毛状根细胞培养中三种活性化合物的积累。
为了探讨上述策略的调控机制,以缺氮和MJ添加作为典型条件,以两种抗肿瘤倍半萜化合物DA和AMDT为目标产物,我们检测了胞外H202、胞内信号分子茉莉酸(JA)和苯丙氨酸氨裂解酶酶活的变化,以及倍半萜合成途径相关基因的转录。尽管胞外过氧化氢未见迸发,但苯丙氨酸氨裂解酶酶活及内源茉莉酸的合成被诱导增强；当使用JA生物合成途径的抑制剂或苯丙氨酸氨裂解酶的抑制剂后,青蒿毛状根细胞的DA和AMDT的合成也受到抑制。在MJ处理以及在缺氮培养和MJ处理组合的时候,法呢基焦磷酸合酶(fps),1-脱氧-D-木酮糖-5-磷酸合酶(dxr),和紫穗槐-4,11-双烯C-12加氧酶(cyp71av)的转录均得到上调,而倍半萜合成的支路酶鲨烯合酶(sqs)的基因转录下调,且这种趋势被苯丙氨酸氨裂解酶抑制剂氨氧基乙酸(AOA)所抑制。以上结果说明苯丙氨酸氨裂解酶和茉莉酸介导了外源MJ添加和缺氮条件下诱导的DA和AMDT的生物合成。
总之,本文从青蒿毛状根中分离纯化了三种化合物,建立了它们的分析方法。并对它们的抗肿瘤活性进行了鉴定,结果表明,AMDT和DA可通过线粒体依赖的途径有效诱导了95-D的细胞凋亡。为了提高毛状根细胞中这些生物活性成分的含量,我们采用了改变毛状根细胞的营养微环境并添加诱导子的策略,结果表明青蒿毛状根在缺氮并添加MJ诱导的条件下,可使细胞的DA和AMDT的含量大幅提高,同时从信号分子和基因转录的水平探讨了这些细胞培养策略的调控机制。这些工作为今后从青蒿毛状根中开发新的活性药物奠定了良好基础,也为这些培养策略应用到植物组织细胞培养的生产实践提供了一定的理论基础。
Artemisia annua is traditionally used as folk medicine for antimalaria, anti-schistosomiasis, antitussis, expectoration and antiasthma in China. Hairy root cultures of A. annua, which present genetic and biochemical stabilities similar to those of the mother plant, are established for more stable and efficient production of their active constituents such as sesquiterpenes, flavornoids and courmains.
In this thesis, (E)-trans-ponticaepoxide (polyacetylene, PO), drimartol A (sesquiterpene coumarin ether, DA) and a new sesquiterpene, (Z)-7-acetoxy-methyl-11-methyl-3-methylene-dodeca-1,6,10-triene (AMDT), were isolated, purified and identified from the cultured hairy roots of A. annua. PO had been reported as a natural insecticide, while the bioactivity of DA and AMDT were unknown and was investigated in this work. Both AMDT and DA showed moderate cytotoxic activities against several human tumor cell lines by MTT assay, whose IC50 values were ranged within 17.9-73.3μM. As treatment of lung cancer, the No.1 killer of global cancer patients, is our interest, the ability of AMDT and DA to induce apoptosis of the human lung tumor cell line 95-D was focused. It was found that the 95-D cell growth was inhibited and its cell cycle was arrested in the G2 phase by DA and in the G1 phase by AMDT. The apoptotic rate of the cells increased in a dose-dependent manner. AMDT and DA lowered the mitochondrial membrane potential and increased the expression of caspase-9 and-3. The results revealed that AMDT and DA could efficiently induce 95-D cell apoptosis through mitochondrial dependent pathway, and they may be potential chemotherapeutic agents.
Quantitative determination methods for any of those bioactive metabolites are not yet available, not to mention their simultaneous analysis. In this work, simultaneous determination of those useful compounds was achieved by using reversed-phase high-performance liquid chromatography-photo diode array detector (RP-HPLC-PAD). The HPLC operating conditions were optimized and the chromatographic separation was performed on a C18 column with a gradient acetonitrile-water as mobile phase. DA, PO and AMDT was detected at 220 nm,254 nm and 220 nm, respectively, and a good linear relationship within the range of investigated concentrations was observed with their correlation coefficient R higher than 0.99. The method was validated for precision, accuracy and recovery, and it was further successfully applied to the time-course detection of the accumulation of those three bioactive compounds in the genetically transformed root cultures of A. annua.
To improve the accumulation of these bioactive compounds, different nutrition state and elicitor treatment were applied in the hairy root culture of A. annua. Production of PO, DA and AMDT was increased from 0.7 mg/gDW,1.7 mg/gDW and 0.2 mg/gDW to 2.07mg/g DW, 4.77 mg/gDW and 0.48 mg/gDW, respectively, by adding 100μM methyl jasmonic acid (MJ). When the cultures were treated with MJ in nitrogen deficient medium, the content of PO, DA and AMDT was improved in a higher level to 3.43 mg/gDW,28.9 mg/gDW and 0.9 mg/gDW.
To provide an insight into the molecular mechanism of the inducing effect of MJ treatment and nitrogen deficiency, in this work sesquiterpene biosynthetic pathway and signal molecules jasmonic acid (JA), H2O2 and phenylalanine ammonia lyase (PAL) were examined in the hairy root culture of A. annua. Although the oxidative burst (induced H2O2 production) was not detected, the induction of PAL activity and intracellular JA synthesis were observed. When the cultures were treated with the inhibitor of JA biosynthsis or the inhibitor of PAL, the contents of DA and AMDT were decreased. The gene transcription of fps (farnesyl diphosphate synthase gene), dxr (1-deoxy-D-xylulose-5-phosphate synthase gene), and cyp71av (amorpha-4,11-diene C-12 oxidase gene) were up-regulated and the sqs (squalene synthase gene) was down-regulated by the combination treatment of MJ elicitation and nitrogen deficiency. Meanwhile, these regulation trends of gene transcription could be inhibited by the PAL inhibitor (BOC-aminooxy) acetic acid (AOA). The results implied that the sesquiterpene biosynthesis induction by MJ treatment and nitrogen deficiency may be mediated by the JA synthesis, PAL activation and the sesquiterpene biosynthetic gene regulation.
Collectively, in this work three bioactive compounds were separated and identified from A. annua hairy root culture. AMDT was a new sesquiterpene. Their simultaneous quantitative analysis method was developed. Their antitumor activities were found. The results revealed that DA and AMDT induced the 95-D apoptosis through mitochondrial dependent pathway. To improve the content of these bioactive compounds, the strategies of using nutrition deficiency medium, adding elicitor and their combination were applied. Results showed the content of DA and AMDT could be increased respectively to 21.1 folds and 9 folds when the hairy root cultures were treated with the combination of nitrogen starvation and MJ addition. Meanwhile, the mechanism involved in the stratege was studied and revealed from the levels of signal transduction and gene transcription. This work provided a good basis for further investigation on the discovery of new drugs from the A. annua hairy root culture. This study is also helpful for application of the strategy to efficient production of other useful secondary metabolites in plant cells.
引文
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