Malaria, one of the oldest known diseases, was referred to in Egyptian writings of the 16th century B.C. In the 17th century, Italians believed that breathing bad air (mal aria) arising from swamps was responsible for the disease, and the term malaria first entered the English medical literature in the first half of the 19th century. Each year, this disease afflicts over 300 million people worldwide, killing up to 2.7 million, mostly children. Most of these cases occur in Africa, but large areas of Asia, Central, and South America have high incidences of the disease. Out of 37 countries and territories, which are members of the Pan American Health Organization (PAHO), World Health Organization (WHO), 21 still have active malaria transmission (PAHO/WHO 1998).
Malaria has been treated for over 40 years with quinine-derived drugs. However, Plasmodium falciparum has developed resistance against these drugs in several areas of the world. Artemisinin (qinghaosu) (), a sesquiterpene lactone belonging to the cadinane series, is an antimalarial compound first isolated from Artemisia annua L. by Chinese scientists in 1972. In addition to a lactone group, artemisinin contains an endoperoxide bridge, which is rarely found in secondary metabolites. One of the few compounds, which contain a peroxide group, is 10, 12-peroxycalamanene. It is extracted from tubers of Cyperus rotundus, and is also effective against Plasmodium falciparum (). Schmid and Hofheinz, Xu, Ravindranathan, and Avery achieved complete chemical (de novo) synthesis of artemisinin. The procedures require several steps, and can start from different raw materials. A comprehensive review of the chemistry of artemisinin has been recently published. However, low yield, complexity, and high cost make extraction from plants the most economically feasible method for artemisinin production at present. Artemisia annua plants normally produce from 0.01 to 0.5% (w/w) artemisinin. However, researchers in Switzerland have developed artemisinin hybrids which produce over 0.8% (w/w) artemisinin, in the field, in about 8 months from sowing to harvest. Pure artemisinin (98%) presently has a value of approximately $300.00/g, while crude artemisinin sells for ca. $300.00/kg and costs $1,200.00/ton for extraction, plus the costs for purification and crystallization.
Artemisinin has been detected in leaves, small green stems, buds, flowers, and seeds of artemisia. Artemisinin has not been reported in roots of field-grown plants or pollen, and the detection of artemisinin from seeds appears to be due to the presence of floral debris. The highest concentration of artemisinin is found in the inflorescence, which at anthesis may contain more than ten times as much artemisinin as leaves. Artemisinin accumulates in glandular trichomes, which are present in leaves, stems, and flowers of the plant.
Production of artemisinin under in vitro conditions has attracted the attention of several investigators due to the progress achieved in the production of other medicinal natural compounds. Examples include shikonins and related naphtoquinones, produced from cell cultures of Lithospermum erythrorhizon by Mitsui Petrochemicals in China and Japan (); sanguinarine, produced by elicited cell cultures of Papaver somniferum (); and taxol, the commercial production of which is being pursued by Phyton Catalytic, Inc. and ESCA Genetics.
Attempts to produce artemisinin and related compounds by tissue culture systems have been reviewed by Woerdenbag and Ferreira. This review will focus on recent results on the search of artemisinin production by in vitro cultures of untransformed and transformed tissues of A. annua.
Distribution and Importance of Artemisia annua
Artemisia annua L. (Asteraceae or Compositae), also known as qinghao (Chinese), annual or sweet wormwood, or sweet Annie, is an annual herb native to Asia, most probably China. Artemisia annua occurs naturally as part of the steppe vegetation in the northern parts of Chahar and Suiyuan provinces (40°N, 109°E) in China, at 1000-1500 m above sea level. The plant now grows wild in many countries, such as Argentina, Bulgaria, France, Hungary, Romania (cultivated for its essential oil), Italy, Spain, the United States, and the former Yuguslavia. Artemisinin, along with taxol, is considered one of the novel discoveries in recent medicinal plant research, and its isolation and characterization has increased interest in A. annua worldwide. Artemisinin is the base compound for the synthesis of more potent and stable antimalarial drugs with reduced toxicity for humans. Artemisinin is effective against Plasmodium species, including P. vivax and P. falciparum, two of the four species that cause human malaria; with P. falciparum being responsible for the often fatal cerebral malaria, an advanced stage of the disease.
Conventional Practices for the Propagation and Production of Artemisinin, and the Demand on the World Market
Propagation of artemisia is normally done by seeds. Seeds keep their vigor for at least 3 years if stored under dry and cool conditions. Several researchers transplant artemisia to the field at the five- to six-leaf stage, which requires 4-6 weeks of greenhouse growth. Vegetative propagation of artemisia is achieved easily from cuttings. The shoots can be taken from juvenile or adult plants and have a rooting rate of 95-100%. Cuttings will root in about 2 weeks in a mist chamber.
Traditionally, artemisinin has been extracted from wild stands in China, with artemisinin concentrations varying from 0.01 to 0.5% (w/w). Material from Sichuan Province is reported to have the highest artemisinin levels. Macro- and micronutrients have minor influence on artemisinin production by field plants. Applications of 50 mgl-1 gibberellic acid (GA3) to field grown plants increased artemisinin content from 0.77 to 1.10 mgg-1; kinetin (10 and 20 mgl-1) increased leaf yield and oil content, but decreased artemisinin content; and triacontanol had no effect on artemisinin content. The levels of artemisinin increased from 0.77 to 1.3% when 80 mgl-1 GA3 was applied to field crops, but artemisinin levels were not correlated to the levels of GA3 applied to the crop. Salinity stress did not influence artemisinin production.
China and Vietnam are the main producers of artemisinin and its derivatives either for oral or parenteral use. Malaria control program officials have distributed, between 1991-1998, 31.6 million tablets of artemisinin, 10.5 million of artesunate, and 793,500 vials of injectable artesunate in Vietnam. Although recent data from China are not available, sales of artesunate tablets rose from 185,000 to 2,545,000 between 1991 and 1995. In Thailand, consumption of artesunate rose from 2880 tablets in 1993 to 653,199 tablets in 1997.
Artemisinin, artemether, arteether, artesunate, and dihydroartemisinin (the last four being artemisinin semi-synthetic derivatives) can all be purchased as drug substances from producers in China, while artemisinin, artemether, and artesunate can be purchased from Vietnam (WHO/MAL 1998). According to the World Health Organization, artemisinin and its derivatives are widely registered as antimalarial drugs in countries where malaria is endemic. Paluther, a commercial brand of artemether (Aventis Pharma), is available in over 100 countries, including 20 countries from Asia and Africa. Combined sales of Paluther in Peru and Brazil reached $200,000.00 in 1999. Currently, no formulation has been registered in Europe or North America, but intramuscular artemether can be made available in France and Denmark upon request. Because countries such as Bangladesh and the Philippines have no problem with multi-drug resistant malaria, artemisinin-derived drugs are unavailable. However, countries such as Myanmar and Vietnam require the use of artemisinin drugs due to the existence of multi-drug resistant Plasmodium strains. The development and spread of multi-drug resistant Plasmodium falciparum currently dictate the demand of artemisinin-derived drugs in the world.
Artemisia annua L.: Conclusions and Prospects
Secondary plant metabolites result from the activity of several enzymes in a multi-step pathway. These enzymes are encoded by genes, which are activated at a certain time and in a specific tissue during the plant life cycle. Currently, the de novo synthesis of most compounds has not yet been mastered with profitable results. While results have been promising for some classes of compounds, such as alkaloids, only a few cDNAs have been cloned, which regulate the production of enzymes needed early in the sesquiterpene pathway. Artemisinin, a sesquiterpene lactone, is an efficient herbicide and esquizontocide, which requires over ten steps to be produced from raw materials. An alternative approach for artemisinin synthesis would be to clone genes regulating late enzymes in the pathway, and then to provide bacterial cultures with immediate precursors, such as artemisinic acid and/or arteannuin B. However, it is not clear if these cultures could perform all the steps required for artemisinin biosynthesis, even if they contained all the genes coding for the needed enzymes. The positive association of artemisinin with light, both in in vivo and in vitro cultures, indicates that its biosynthesis is associated with chloroplasts, lacking in bacteria. Also, if successfully produced in large amounts, artemisinin could very well quench its own biosynthesis, or kill the cultures, due to its anti-microbial effects.
Previous attempts to produce artemisinin by undifferentiated in vitro cultures have yielded inconsistent results. Recent studies have concentrated on the transformation of organs of the artemisia plant with Agrobaterium rhizogenes to produce hairy roots, or with A. tumefasciens to produce shooty teratomas. However, we suggest that some key factors have been overlooked:
- 1. Past successes in producing secondary metabolites by hairy root cultures in a number of species have been limited to compounds which are originally produced by roots of differentiated plants. Roots of Artemisia annua do not normally produce artemisinin.
- 2. Hairy roots exposed to light might behave as “pseudoshoots.” The detection of artemisinin in green hairy roots suggests an association of artemisinin production with chloroplasts.
- 3. Artemisinin is synthesized and stored in leaves, stems, flower receptacles, and the corolla, all bearing glandular trichomes. This suggests that artemisinin production is best approached through differentiated cultures or shooty teratomas, which have the potential to bear glandular trichomes or, at least, to exert photosynthetic activity.
- 4. Elicitation has been a successful approach to trigger the production of certain metabolites, particularly antifungal or antibacterial compounds. However, elicitors have not succeeded in triggering artemisinin production either in vivo or in vitro. This suggests that artemisinin function to the plant might not be as an antifungal or bactericidal compound.
- 5. The only phytohormone that appears to influence artemisinin production is gibberellic acid (GA3) which is also known to promote flowering. Artemisinin production is reported to reach its peak close to or during full flowering, and GA3 is reported to promote flowering in shoot cultures of A. annua (). Thus, gibberellins might be one of the biochemical signals produced by the plant, which leads to an increased production of artemisinin. The influence of gibberellins could be verified by further investigating the effect of GA3 antagonizers, such as abscisic acid (ABA), on artemisinin production.
- 6. Artemisia annua is a highly cross-pollinating species, and plants derived from seeds will have highly variable levels of artemisinin. Preferably, high artemisinin-producing clones should be used for in vitro studies.
Although field production of artemisia is presently the most commercially feasible approach to produce artemisinin and related compounds, molecular studies with green hairy roots and shooty teratomas could contribute to our understanding of artemisinin biosynthesis. In addition to research on the genetic potential of artemisinin biosynthesis by Artemisia annua, factors that affect temporal (when artemisinin reaches its maximum) or spatial (where it is stored) accumulation must not be ignored.
Selections from the book: “Medicinal and Aromatic Plants XII” (2002).