Cloning and functional characterization of a beta-pinene synthase from Artemisia annua that shows a circadian pattern of expression. (1/50)

Artemisia annua plants produce a broad range of volatile compounds, including monoterpenes, which contribute to the characteristic fragrance of this medicinal species. A cDNA clone, QH6, contained an open reading frame encoding a 582-amino acid protein that showed high sequence identity to plant monoterpene synthases. The prokaryotically expressed QH6 fusion protein converted geranyl diphosphate to (-)-beta-pinene and (-)-alpha-pinene in a 94:6 ratio. QH6 was predominantly expressed in juvenile leaves 2 weeks postsprouting. QH6 transcript levels were transiently reduced following mechanical wounding or fungal elicitor treatment, suggesting that this gene is not directly involved in defense reaction induced by either of these treatments. Under a photoperiod of 12 h/12 h (light/dark), the abundance of QH6 transcripts fluctuated in a diurnal pattern that ebbed around 3 h before daybreak (9th h in the dark phase) and peaked after 9 h in light (9th h in the light phase). The contents of (-)-beta-pinene in juvenile leaves and in emitted volatiles also varied in a diurnal rhythm, correlating strongly with mRNA accumulation. When A. annua was entrained by constant light or constant dark conditions, QH6 transcript accumulation continued to fluctuate with circadian rhythms. Under constant light, advanced cycles of fluctuation of QH6 transcript levels were observed, and under constant dark, the cycle was delayed. However, the original diurnal pattern could be regained when the plants were returned to the normal light/dark (12 h/12 h) photoperiod. This is the first report that monoterpene biosynthesis is transcriptionally regulated in a circadian pattern.  (+info)

Pharmacokinetic study of artemisinin after oral intake of a traditional preparation of Artemisia annua L. (annual wormwood). (2/50)

Artemisia annua L. (annual wormwood) contains the antimalarial artemisinin. Aqueous preparations of the dried herb are included in the pharmacopoeia of the People's Republic of China for treatment of fever and malaria. Fourteen healthy male volunteers received one liter of tea prepared from nine grams of Artemisia annua leaves. Blood samples were taken and artemisinin was detected by reversed phase high-performance liquid chromatography. The mean +/- SD maximum plasma concentration of artemisinin was 240 +/- 75 ng/mL and the mean +/- SD area under the plasma concentration-time curve was 336 +/- 71 ng/mL x hr. Artemisinin was absorbed faster from herbal tea preparations than from oral solid dosage forms, but bioavailability was similar. One liter of an aqueous preparation of nine grams of Artemisia annua contained 94.5 milligrams of artemisinin (approximately 19% of the usually recommended daily dose). Artemisinin plasma concentrations after intake of this herbal tea are sufficient for clinical effects, but insufficient to recommend such preparations as equivalent substitutes for modern artemisinin drugs in malaria therapy.  (+info)

Artemisia annua L. (Asteraceae) trichome-specific cDNAs reveal CYP71AV1, a cytochrome P450 with a key role in the biosynthesis of the antimalarial sesquiterpene lactone artemisinin. (3/50)

Artemisinin, a sesquiterpene lactone endoperoxide derived from the plant Artemisia annua, forms the basis of the most important treatments of malaria in use today. In an effort to elucidate the biosynthesis of artemisinin, an expressed sequence tag approach to identifying the relevant biosynthetic genes was undertaken using isolated glandular trichomes as a source of mRNA. A cDNA clone encoding a cytochrome P450 designated CYP71AV1 was characterized by expression in Saccharomyces cerevisiae and shown to catalyze the oxidation of the proposed biosynthetic intermediates amorpha-4,11-diene, artemisinic alcohol and artemisinic aldehyde. The identification of the CYP71AV1 gene should allow for the engineering of semi-synthetic production of artemisinin in appropriate plant or microbial hosts.  (+info)

Ensuring sustained ACT production and reliable artemisinin supply. (4/50)

INTRODUCTION: This paper reviews recent trends in the production, supply and price of the active ingredients as well as finished ACT products. Production and cost data provided in this paper are based on an ongoing project (Artepal). Stability data are derived from a development project on rectal artesunate. DISCUSSION: The artemisinin raw material and its derivatives appear to be very stable compared to the finished products. Supply of artemisinin changed in May 2004 when the Global Fund shifted financial support to qualified countries from chloroquine or sulphadoxine-pyrimethamine to an ACT for treatment of malaria. First, there was a sudden shortage of the starting material, and short term scarcity led to a steep rise in API price: it increased dramatically in 2004, from $350 per kg to more than $1000. Second, there was a parallel increase in the number of companies extracting artemisinin from 10 to 80 between 2003 and 2005 in China, and from 3 to 20 in Vietnam. Commercial cultivation began also in East Africa and Madagascar.A steady and predictable demand for the crop can eliminate such wide fluctuations and indirectly contribute to price stability of the herb, the API and ACT. With appropriate mechanisms to reduce those fluctuations, the cost of artemisinin might decrease sustainably to US$ 250-300 per kg. CONCLUSION: Today the global health community is facing the risk of another cyclical swing with lower demand feeding into reduced planting of A. annua and, thereafter, a new shortage of the raw material and higher API prices. International donors, the largest purchasers for ACTs could better coordinate their activities, in order to guarantee purchase of ACTs and consequently of API with manufacturers. In parallel, the base of quality producers of APIs and finished ACT products needs to be broadened.While the ACT programme is still in its early stages, the consequences of another wave of artemisinin and ACT shortages would permanently discredit it and impede any progress in rolling malaria back.  (+info)

The rapid determination of artemisinin by post-column derivatization high-performance liquid chromatography using matrix solid-phase dispersion method. (5/50)

Artemisinin (an antimalaric compound) is isolated as the active compound of the medicinal plant Artemisia annua L. A simple, rapid, and high-efficient method of extraction is developed, in which it is extracted by matrix solid-phase dispersion (MSPD) and directly analyzed by post-column derivatization high-performance liquid chromatography (HPLC). The quantitation results from the MSPD method are compared with two conventional liquid solvent extraction processes, Soxhlet and ultrasonic wave by F-test, and the result indicates no significant difference. The production rate of Artemisinin during wild plant growth (tested over two years, acquired from Yunnan, provincial Yuan Yan country) is determined. The recovery range of determination is 88.1-91.2%. The relative standard deviation (RSD) is 4.55-6.43%. The limits of detection are 0.1 microg/mL, and the limits of quantitation are 0.5 microg/mL.  (+info)

The molecular cloning of artemisinic aldehyde Delta11(13) reductase and its role in glandular trichome-dependent biosynthesis of artemisinin in Artemisia annua. (6/50)

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Induction of multiple pleiotropic drug resistance genes in yeast engineered to produce an increased level of anti-malarial drug precursor, artemisinic acid. (7/50)

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Recent advances in artemisinin production through heterologous expression. (8/50)

Artemisinin the sesquiterpene endoperoxide lactone extracted from the herb Artemisia annua, remains the basis for the current preferred treatment against the malaria parasite Plasmodium falciparum. In addition, artemisinin and its derivatives show additional anti-parasite, anti-cancer, and anti-viral properties. Widespread use of this valuable secondary metabolite has been hampered by low production in vivo and high cost of chemical synthesis in vitro. Novel production methods are required to accommodate the ever-growing need for this important drug. Past work has focused on increasing production through traditional breeding approaches, with limited success, and on engineering cultured plants for high production in bioreactors. New research is focusing on heterologous expression systems for this unique biochemical pathway. Recently discovered genes, including a cytochrome P450 and its associated reductase, have been shown to catalyze multiple steps in the biochemical pathway leading to artemisinin. This has the potential to make a semi-synthetic approach to production both possible and cost effective. Artemisinin precursor production in engineered Saccharomyces cerevisiae is about two orders of magnitude higher than from field-grown A. annua. Efforts to increase flux through engineered pathways are on-going in both E. coli and S. cerevisiae through combinations of engineering precursor pathways and downstream optimization of gene expression. This review will compare older approaches to overproduction of this important drug, and then focus on the results from the newer approaches using heterologous expression systems and how they might meet the demands for treating malaria and other diseases.  (+info)

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