Characterization And Detection Of Bioactive Compounds And Studies On Their Biosynthesis Regulation In Hairy Root Cultures Of Artemisia Annua

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.