State of the art of the production of the antimalarial compound artemisinin in plants

For more than three centuries we have relied on the extracts of the bark of Cinchona species to treat malaria. Now, it seems we may be changing to the leaves of a Chinese weed, Artemisia annua, and its active compound artemisinin. Artemisinin-derived drugs have been proved particularly effective treatments for severe malaria, even for multi-drug-resistant malaria. However, this promising antimalarial compound remains expensive and is hardly available on a global scale. Therefore, many research groups have directed their investigations toward the enhancement of artemisinin production in A. annua cell cultures or whole plants in order to overproduce artemisinin or one of its precursors. This article provides a brief review of the state of art of the different aspects in A. annua research.     

不同产地与类型及采收方法对黄花蒿中青蒿素含量的影响

以来源于重庆、广西、湖南、广东、江苏、陕西等省区的72分种质资源为试验材料,研究不同产地与类型及采收方法对黄花蒿中青蒿素含量的影响,为良种的选育和药材的采收提供科学的依据。结果表明:(1)早熟型与中、晚熟型青蒿素含量的差异显著,其顺序为:中熟型>晚熟型>早熟型;(2)在同一管理条件下不同茎秆颜色的青蒿素含量之间差异显著,其顺序为:白杆型>黄绿秆型>紫秆型>绿秆型;(3)不同产地的黄花蒿青蒿素含量动态变化规律一致,青蒿素含量最高时期为孕蕾期,是最佳采收期;(4)不同部位之间青蒿素含量差异显著,以叶片的青蒿素含量最高。     

Quantification of artemisinin in Artemisia annua extracts by 1H-NMR

Artemisinin is a polycyclic sesquiterpene lactone that is highly effective against multidrug-resistant strains of Plasmodium falciparum, the etiological agent of the most severe form of malaria. Determination of artemisinin in the source plant, Artemisia annua, is a challenging problem since the compound is present in very low concentrations, is thermolabile and unstable, and lacks chromophoric or fluorophoric groups. The ain of this study was to develop a simple protocol for the quantification of artemisinin in a plant extract using an (1)H-NMR method. Samples were prepared by extraction of leaf material with acetone, treatment with activated charcoal to remove chlorophylls and removal of solvent. (1)H-NMR spectra were measured on samples dissolved in deuterochloroform with tert-butanol as internal standard. Quantification was carried out using the using the delta 5.864 signal of artemisinin and the delta 1.276 signal of tert-butanol. The method was optimised and fully validated against a reference standard of artemisinin. The results were compared with those obtained from the same samples quantified using an HPLC-refractive index (RI) method. The (1)H-NMR method gave a linear response for artemisinin within the range 9.85-97.99 mm (r(2) = 0.9968). Using the described method, yields of artemisinin in the range 0.77-1.06% were obtained from leaves of the A. annua hybrid CPQBA x POP, and these values were in agreement with those obtained using an HPLC-RI.     

Comparative quantitative analysis of artemisinin by chromatography and qNMR

Introduction – Since the discovery of artemisinin in the 1970s, many techniques based on diverse chromatography techniques have been developed to detect and quantify this important antiplasmodial compound. The accurate quantification of this compound in the Artemisia annua plant material is mainly needed for breeding purposes in order to cultivate higher yielding varieties. It is also important for the quality control of herbal preparations containing A. annua plant material. Objective – To evaluate the most common validated quantification techniques (LC‐MS, HPLC‐ELSD and TLC) and compare the results to quantitative nuclear magnetic resonance spectroscopy (qNMR) in eight different A. annua samples collected from around the world. Methodology – The leaf material were extracted according to standard procedures and analysed with the validated quantification techniques. For the qNMR analysis we did not employ a standard curve but instead used an internal standard (maleid acid) which is not chemically related to artemisinin. Results – We found a significant difference between the results in this study. Compared with the qNMR results the HPLC‐ELSD corresponded closely, followed by LC‐MS. Quantitation with TLC led to an estimation range of ?0.5 to +3.2 mg artemisinin/g of A. annua. Conclusion – These results imply that qNMR, with the addition of an internal standard, can be used to quantify artemisinin in A. annua samples in a rapid and reproducible manner. Copyright © 2010 John Wiley & Sons, Ltd.     

青蒿毛状根生长、青蒿素合成以及 营养物消耗的动力学

诱导产生的青蒿毛状根培养物置于MS培养基(含30 g/L蔗糖)进行悬浮培养,并对悬浮培养过程中毛状根生长、青蒿素合成、蔗糖、磷酸盐和不同氮源的消耗、pH和电导率的动力学过程进行分析。经30 d培养,生物量干重和青蒿素产量分别达到13.7 g/L和0.23 g/L,碳源和氮源在培养过程中被逐渐利用,而磷酸盐的利用速率最快,培养至15 d所有的磷酸盐均被吸收,pH在培养初期降低,后又逐渐上升,电导率由于毛状根生长对无机离子的吸收而逐渐减低。     

温度对青蒿毛状根生长和青蒿素生物合成的影响

本实验研究了不同温度(15℃～35℃)对青蒿毛状根生长和青蒿素生物合成的影响,发现25℃有利于毛状根生长,30℃促进了青蒿素生物合成。通过温度改变的二步培养技术(培养前20d温度控制在25℃,后10d温度提高到30℃),青蒿素的产量得到明显提高,高于在恒温培养时(25℃或30℃)的结果。     

Enhanced artemisinin accumulation and metabolic profiling of transgenic Artemisia annua L. plants over-expressing by rate-limiting enzymes from isoprenoid pathway

Artemisinin, a natural product isolated from aerial parts of Artemisia annua L. plant, is a potent antimalarial drug against drug-resistant malaria. In recent times, the demand (101–119 MT) for artemisinin is exponentially increasing with the increased incidence of drug-resistant malaria throughout the world, especially African and Asian continents. However, the commercial production of artemisinin-based combination therapies has limitation because of the presence of low concentration of artemisinin in plants. Therefore, transgenic lines of A. annua L. plants over-expressing both HMG-Co A reductase (hmgr) and amorpha-4, 11-diene synthase (ads) genes were developed to enhance the content of artemisinin. The selected transgenic lines (TR4, TR5, and TR7) were found to accumulate higher artemisinin (0.97–1.2%) as compared to the non-transgenic plants (0.63%). The secondary metabolite profiles of these lines were also investigated employing gas chromatography mass spectrometry, which revealed a clear difference in these metabolites in transgenic and non-transgenic lines of A. annua L. at different growth and developmental stages. The major metabolites reported in these lines at pre-flowering stage were related to essential oil and chlorophyll biosynthesis (71.33% in TR5 transgenic lines vs. 61.70% in non-transgenic line). Based on these results, we concluded that over-expression of both hmgr and ads genes in A. annua L. plants results not only increase in artemisinin content, but also enhances synthesis of other isoprenoid including essential oil. It is also evident from this study that the novel artemisinin-rich varieties of A. annua L. could be developed by suppressing essential oil biosynthesis, so that more carbon could preferentially be diverted from mevalonate pathway to artemisinin biosynthesis.     

无载体固定化黄花蒿细胞生产青蒿素

疟疾是一种严重危害人类健康的流行病,主要由疟原虫经蚊虫叮咬引起。目前,在临床上疟原虫对治疗疟疾的药物(如氯奎等)有较强的耐药性,并表现出明显的交叉耐药性。来自黄花蒿的青蒿素具有极其明显的抗疟活性,成为临床首选的药物,因此青蒿素的获取成为关键。本研究采用无载体固定化法培养黄花蒿生产青蒿素,初步研究了无载体固定化细胞的生长特性。检测发现,利用该方法生产的青蒿素是常规细胞培养法的9倍,因此该方法有望成为青蒿素生产的首选方法。     

Nitric oxide potentiates oligosaccharide-induced artemisinin production in Artemisia annua hairy roots

The purpose of the present study was to characterize the generation of nitric oxide （NO） in Artemisia annua roots induced by an oligosaccharide elicitor （OE） from Fusarium oxysporum mycelium and the potentiation role of NO in the elicitation of artemisinin accumulation. The OE （0.3 mg total sugar/mL） induced a rapid production of NO in cultures, which exhibited a biphasic time course, reaching the first plateau within 1.5 h and the second within 8 h of OE treatment. Artemisinin content in 20-day-old hairy roots was increased from 0.7mg/g dry wt to 1.3 mg/g dry wt by using the OE treatment for 4d. In the absence of OE, the NO donor sodium nitroprusside （SNP） at 10, 50 ~1 and 100 ~1 enhanced the growth of hairy roots, but had no effect on artemisinin synthesis, The combination of SNP with OE increased artemisinin content from 1.2 mg/g drywt to 2.2 mg/g dry wt, whereas the maximum production of artemisinin in cultures was 28.5 mg/L, a twofold increase over the OE treatment alone. The effects of SNP on the OE-induced artemisinin were suppressed strongly by the NO scavenger 2-（4- carboxyphenyl）-4,4,5,5-tetramethylimidazoline-l-oxyl-3-oxide （cPTIO）. The results suggest that NO can strongly potentiate elicitor-induced artemisinin synthesis in A. annua hairy roots.     

Modulation of artemisinin biosynthesis by elicitors,inhibitor, and precursor in hairy root cultures of Artemisia annua L.

Artemisinin is frequently used in the artemisinin-based combination therapy to cure drug-resistant malaria in Asian subcontinent and large swath of Africa. The hairy root system, using the Agrobacterium rhizogenes LBA 9402 strain to enhance the production of artemisinin in Artemisia annua L., is developed in our laboratory. The transgenic nature of hairy root lines and the copy number of transgene (rol B) were confirmed using polymerase chain reaction and Southern Blot analyses, respectively. The effect of different concentrations of methyl jasmonate (MeJA), fungal elicitors (Alternaria alternate, Curvularia limata, Fusarium solani, and Piriformospora indica), farnesyl pyrophosphate, and miconazole on artemisinin production in hairy root cultures were evaluated. Among all the factors used individually for their effect on artemisinin production in hairy root culture system, the maximum enhancement was achieved with P. indica (1.97 times). Increment of 2.44 times in artemisinin concentration by this system was, however, obtained by combined addition of MeJA and cell homogenate of P. indica in the culture medium. The effects of these factors on artemisinin production were positively correlated with regulatory genes of MVA, MEP, and artemisinin biosynthetic pathways, viz. hmgr, ads, cyp71av1, aldh1, dxs, dxr, and dbr2 in hairy root cultures of A. annua L.     

Abscisic acid (ABA) treatment increases artemisinin content in <Emphasis Type="Italic">Artemisia annua</Emphasis> by enhancing the expression of genes in artemisinin biosynthetic pathway

Artemisinin, a sesquiterpene lactone endoperoxide derived from Artemisia annua L., is the most effective antimalarial drug. In an effort to increase the artemisinin production, abscisic acid (ABA) with different concentrations (1, 10 and 100 μM) was tested by treating A. annua plants. As a result, the artemisinin content in ABA-treated plants was significantly increased. Especially, artemisinin content in plants treated by 10 μM ABA was 65% higher than that in the control plants, up to an average of 1.84% dry weight. Gene expression analysis showed that in both the ABA-treated plants and cell suspension cultures, HMGR, FPS, CYP71AV1 and CPR, the important genes in the artemisinin biosynthetic pathway, were significantly induced. While only a slight increase of ADS expression was observed in ABA-treated plants, no expression of ADS was detected in cell suspension cultures. This study suggests that there is probably a crosstalk between the ABA signaling pathway and artemisinin biosynthetic pathway and that CYP71AV1, which was induced most significantly, may play a key regulatory role in the artemisinin biosynthetic pathway.     

TLC-densitometric analysis of artemisinin for the rapid screening of high-producing plantlets of Artemisia annua L

A simple TLC-densitometric technique has been developed for the rapid and accurate analysis of artemisinin in a large number of Artemisia annua plantlets cultured in vitro. This new analytical method is based on the structural conversion of artemisinin on a silica gel layer by ammonia vapour to form 10-azadesoxyartemisinin, a chromophore-containing compound (lambdamax 320 nm) that can be detected by UV-based TLC densitometry. The TLC system was evaluated quantitatively in terms of product stability, precision, accuracy and calibration. Good linearity was obtained in the range of 0.01-0.12 microg artemisinin. The technique appeared to be accurate and sensitive as compared with the complicated pre-column reaction-HPLC technique. Among 90 samples of A. annua plantlets, the artemisinin content in the leaves appeared to be highly variable, ranging from 0.02 to 0.67% w/w dry weight. These results demonstrate that densitometric TLC can be a cheap and simple technique for the accurate screening of high-artemisinin-producing plants.     

衍生化条件的优化及不同产地青蒿中青蒿素含量的柱前衍生化-HPLC法测定

Derivation conditions of pre-column derivation-HPLC method used for determinating artemisinin content were compared and selected,and artemisinin content in above-ground part of Artemisia annua L.from seventeen locations was compared by optimal pre-column derivation-HPLC method.The optimal derivation condition is selected via comparing of 0.2% NaOH solution addition(3,4,5,6 and 7 mL),derivation temperature(30 ℃,35 ℃,40 ℃,45 ℃ and 50 ℃) and derivation time(0,2,5,10,20,40 and 60 min) during derivation process.The optimal derivation condition is adding 5 mL 0.2% NaOH solution,then insulating in 40 ℃ water bath for 10 min.Differences of artemisinin content in A.annua from seventeen locations are obvious,the general trend is that artemisinin content decreases gradually with location from south to north,in which artemisinin content in A.annua from Youyang of Chongqing is the highest with a value of 7.08 mg·g-1.The method is simple and accurate with good reproducibility,and can be used to determinate artemisinin content in medicinal material of A.annua.     

氮对黄花蒿生长、光合特性和青蒿素含量的影响

对不同氮处理黄花蒿生长、生物量分配、青蒿素含量和光合特性进行测定,结果表明:(1)供氮量在0～0.4g.kg-1之间,黄花蒿叶片单位重量的氮含量、最大净光合速率、光饱和点和表观量子效率均随供氮量的增大而增加,之后开始下降。在较大范围内,环境氮含量越高,黄花蒿的光合能力越强,能够利用的光强也更高;(2)黄花蒿根生物量分数和根冠比均随供氮量的减少而显著增大,低氮时分配更多的生物量到养分吸收器官,有利于减少氮素对生长的限制,供氮量在0.1～0.6g.kg-1之间,黄花蒿叶生物量分数随供氮量的增加而增大,高氮时更多的生物量投入到碳同化器官,提高了植株的竞争能力;(3)无论以最大净光合速率、地径、叶片生物量还是以总生物量来衡量,均以0.4g.kg-1氮处理的植株生长得最好,0.2g.kg-1氮处理的青蒿素含量最高,生产中推荐使用0.2g.kg-1剂量的氮更经济。     

Metabolic engineering of ketocarotenoid formation in higher plants

Although higher plants synthesize carotenoids, they do not possess the ability to form ketocarotenoids. In order to generate higher plants capable of synthesizing combinations of ketolated and hydroxylated carotenoids the genes responsible for the carotene 4,4' oxygenase and 3,3' hydroxylase have been transformed into tomato and tobacco. The gene products were produced as a polyprotein. Subsequent cleavage of the polyprotein, targeting of the two enzymes to the plastid and enzyme activities have been shown for both gene products. Metabolite profiling has shown the formation of ketolated carotenoids from beta-carotene and its hydroxylated intermediates in tobacco and tomato leaf. In the nectary tissues of tobacco flowers a quantitative increase (10-fold) as well as compositional changes were evident, including the presence of astaxanthin, canthaxanthin and 4-ketozeaxanthin. Interestingly, in this tissue the newly formed carotenoids resided predominantly as esters. These data are discussed in terms of metabolic engineering of carotenoids and their sequestration in higher plant tissues.     

代谢工程的发展及其应用

本首先论述了代谢工程的代谢网络理论、代谢分析、节点分析和中心代谢物作用机理等代谢工程理论基础。然后,分析了代谢工程的各种具体设计思路,并以实际例子作了详细说明。另外还对代谢工程的新兴研究方向－逆代谢工程进行了简单说明。     

Improved growth of Artemisia annua L hairy roots and artemisinin production under red light conditions

Hairy root cultures of Artemisia annua L were cultivated for 30 days under either white, red, blue, yellow or green light. Red light at 660 nm gave the highest biomass of hairy roots (5.73 g dry wt cells l^–1 medium) and artemisinin content (31 mg arteminsinin g^–1 dry cells) which were, respectively, 17% and 67% higher than those obtained under white light.