Table of Contents
Simon, J.E., D. Charles, E. Cebert, L. Grant, J. Janick, and A. Whipkey. 1990.
Artemisia annua L.: A promising aromatic and medicinal. p. 522-526.
In: J. Janick and J.E. Simon (eds.), Advances in new crops. Timber Press,
Artemisia annua L.: A Promising Aromatic and Medicinal*
James E. Simon, Denys Charles, Ernst Cebert, Lois Grant, Jules Janick, and Anna Whipkey
- Essential Oils
- Location of Natural Products
- Response to Plant Spacing and Nitrogen Application
- Influence of Planting Date and Harvest Time
- Tissue Culture
- Table 1
- Table 2
- Fig. 1
- Fig. 2
- Fig. 3
Artemisia annua L. (annual wormwood, sweet wormwood, sweet annie), a
highly aromatic annual herb (Fig. 1) of Asiatic and eastern European origin, is
widely dispersed throughout the temperate region (Bailey and Bailey 1976, Simon
et al. 1984). The species has naturalized in the United States and is sold on
a limited scale as a dried herb for the floral and craft trade where it is used
as an aromatic wreath. The plant has traditionally been grown in China as a
medicinal and, more recently in Europe for its aromatic leaves which are used
in flavoring beverages.
Recent research in the Peoples Republic of China with traditional herbal
medicine has brought attention to A. annua, the source of
qinghaosu (artemisinin), a compound that shows promise as an
anti-malarial agent (Klayman 1985). Artemisinin has also been reported to be a
potent plant inhibitor with potential as a natural herbicide (Duke et al. 1987,
Chen et al. 1987). Artemisinin and its derivatives, artemether and artesunate,
have been studied for their efficacy as antimalarial agents. In in vitro
trials conducted in China (WHO 1981), all three compounds have been effective
against the erythrocytic stages of two chloroquine-resistant Hainan strains of
Plasmodium falciparum, the malarial parasite, at lower minimum effective
concentrations than chloroquine, the most commonly used drug. Artemisinin and
its derivatives have effectively treated malaria and cerebral malaria in human
subjects with no apparent adverse reactions nor side effects (Klayman 1985).
With P. falciparum developing resistance to chloroquine and
pyrimethamine/sulfonamide (WHO 1981), alternative treatments based on new
compounds such as artemisinin and its derivatives are actively being sought.
While artemisinin and its derivatives may be synthesized (Zhou 1986, Xu et al.
1986), the synthetic compounds are unlikely to be economically competitive with
the naturally derived plant products (Schmid and Hofheinz 1983, Xu et al.
The relatively low content of artemisinin in cultivated European and New World
types of A. annua has been a limiting factor for the isolation and
evaluation of artemisinin on a technical scale. Artemisinin yields of 0.06%
have been extracted from samples of A. annua collected in the United
States (Klayman et al. 1984) which are low for commercial exploitation. Yields
of extracted artemisinin from the above-ground portions of the plant have
ranged from 0.01% to 0.5% (w/w) in the People's Republic of China (WHO 1981).
Although artemisinin yield varies with environmental and management conditions
(WHO 1981), specific effects are unknown. The extent of genetic variation on
artemisinin content was also poorly understood.
Since 1984, we have been evaluating the plant s production potential
determining its horticultural characteristics, and developed a rapid assay to
determine artemisinin content from crude plant materials in anticipation of
future improvement programs. Essential (volatile) oil composition was
characterized in order to evaluate A. annua as a source of aroma
chemicals for the fragrance industry
Artemisinin is a secondary or natural plant metabolite identified as a
sesquiterpene lactone endoperoxide (Klayman et al. 1984). Artemisinin has been
synthesized and its structure and absolute stereochemistry established by Zhou
(1986) and Xu et al. (1986), but biosynthetic pathways and the mechanisms and
regulation processes are unknown. Analysis of artemisinin is difficult because
the compound is unstable, concentrations in the plant low, the intact molecule
stains poorly, and other compounds in the crude plant extracts interfere in its
detection. A method to analyze artemisinin in crude plant extracts based upon
high pressure liquid chromatography (HPLC) with reductive mode electrochemical
detection was first developed by Acton et al. (1985) and later modified by
Charles et al. (1990). This latter method is highly sensitive, rapid, and
should be of value in analyzing large numbers of samples as needed in crop
Using this later method, we evaluated our germplasm collection of A.
annua for artemisinin content to determine whether there was genetic
variability that could be exploited and to identify plant lines and individual
plants with high concentrations (Charles et al. 1990). Wide variation in
artemisinin content was observed with accessions in our collection ranging from
0.003 to 0.21%, and with individual plants ranging from 0.00 to 0.39% (dry
weight basis). This data suggests that artemisinin productivity could be
enhanced by further selection and breeding.
Essential oils of A. annua can be extracted via steam distillation in
units similar to that used in the commercial distillation of peppermint and
spearmint. Essential oils of A. annua were extracted in our studies via
hydrodistillation with a modified clevenger trap (Simon and Quinn 1988) and
chemically characterized by gas chromatography (GC) analysis using a fused
silica capillary column (12 m x 0.2 mm id) with a OV 101 (Varian,
polydimethylsiloxane) bonded phase. Direct injection of 0.5 ml of essential
oil samples with He as a carrier gas (100:1 split vent ratio) and oven
temperatures held isothermal at 80°C for 2 min and then programmed to
increase at 3°C/min to 210°C gave complete elution of all peaks
(sensitivity 10-10). The injector and detector temperatures were 210°C and
300°C, respectively. Confirmation of essential oil constituents was based
upon comparison of retention time with standards and via GC/Mass Spectroscopy
Essential oils of A. annua are comprised of many constituents with the
major compounds including (in relative % of total essential oil) alpha-pinene
(0.032%), camphene (0.047%), ß-pinene (0.882%), myrcene (3.8%), 1,8-cineole
(5.5%), artemisia ketone (66.7%), linalool (3.4%), camphor (0.6%), borneol
(0.2%), and ß-caryophyllene (1.2%).
Essential oils and artemisinin were assumed to be associated with secretory
cells based on the association of mono- and sesquiterpenes with well-defined
secretory structures (Croteau 1986, Henderson et al. 1970). The relative
distribution of artemisinin is shown in Table 1. Leaves had 89% of the total
artemisinin in the plant with the uppermost foliar portion of the plant (top
1/3 of growth at maturity) containing almost double that of the lower leaves
(Charles et al. 1990). Kelsey and Shafizadeh (1980) had reported that 35% of
the mature leaf surface is covered with capitate glands which contain most of
the monoterpenes and virtually all of the sesquiterpene lactones. Essential
oils from A. annua are similarly distributed, with 36% of the total from
the upper third of the foliage, 47% from the middle third, and 17% from the
lower third, with only trace amounts in the main stem side shoots, and roots.
The response of A. annua to plant spacing and nitrogen fertilization was
evaluated in 1985 and 1986 with three populations established from transplants:
high density, 30 cm x 30 cm (111,111 plants/ha); intermediate density, 30 cm x
60 cm (55,555 plants/ha); and low density, 60 cm x 60 cm (27,778 plants/ha).
Plants in each spacing received three levels of nitrogen fertilization (0, 67,
and 134 kg N/ha) applied as a preplant broadcast application.
Plants from the densely populated treatment (111,111 plants/ha) produced an
average fresh weight of 275 g/plant, as opposed to 430 g/plant from the
intermediate and 750 g/plant from the lowest populations. However, total
biomass (fresh weight kg/ha), was greatest from the higher density population
(Table 2). Plants of the most densely populated treatments were slightly
taller, produced less side shoots, and had longer internodes with little
lateral growth than the lower densities. Number of side shoots per main stems
decreased as density increased. While yield increased with added nitrogen, the
greatest growth (herbage and essential oil content) was obtained with 67 kg
N/ha (Table 2).
Increasing density tended to increase essential oil production on an area
basis, but highest essential oil yields (85 kg oil/ha) was achieved by the
intermediate density at 55,555 plants/ha receiving 67 kg N/ha (Table 2).
Artemisinin content was not analyzed in this study because the instrumentation
and method of analysis had not yet been developed in our laboratory
Seedlings of A. annua were transplanted into a central Indiana field on
April 27, May 17, June 10, and July 13, 1987, and plant samples (for growth and
essential oil) were obtained every two weeks until the first frost (Fig. 2, 3).
The May transplanting date produced the highest fresh yields and tallest
plants, while the May and June plantings had the highest percentage of
essential oil. Regardless of planting date, all plants began to flower by
mid-August, with maximum concentration of essential oil produced in
mid-September (peak flowering stage). Results from the 1988 growing season
(data not presented) were similar to those obtained in 1987.
Plantlets of A. annua were regenerated using shoot tips of mature field
grown plants. Shoot tips were surface sterilized and cultured in Murashige and
Skoog (MS) media with factorial combinations of 2,4-D (0, 0.1, 1.0 mg/liter)
and benzyladenine (BA) (0, 0.1, 1.0 and 10.0 mg/liter). Both axillary and
adventitious shoots were produced in all the treatments. BA alone at 10
mg/liter produced the greatest number of shoots, but the most normal shoots
were obtained at 1.0 mg/liter. Shoots were easily multiplied on MS medium with
1 mg/liter BA and subculturing at 4 week intervals. Artemisinin was detected
from in-vitro shoots of A. annua, in concentrations from 0.03 to 0.05%
(dry weight basis).
All shoots rooted when dipped in commercial preparations of 0.3% indolebutyric
acid and placed in potting soil under mist conditions. Acclimatization to in
vivo conditions was easily achieved. This technique will permit rapid
increases of individual plant selections.
We have found that A. annua is relatively easy to grow and that very
high biomass yields (35t/ha) can be obtained in the Midwest. Plant spacing had
a highly significant affect on biomass yield and plant architecture. Wide
variation in artemisinin content has been found in our germplasm collection
with accessions reaching 0.21%, and individual plants as high as 0.39% (dry
weight basis). This suggests that high artemisinin yields lines could be
developed by further selection and breeding. The commercialization of A.
annua in this country is dependent upon whether artemisinin or its
derivatives will be approved for use in the treatment of malaria. Domestic
production of A. annua for the extraction and processing of artemisinin
for export should be considered as overseas markets develop and if they will
purchase imported materials.
Artemisia has potential as a source of essential oils and we have obtained oil
yields of 85 kg/ha. Uses of the essential oil from A. annua in the
fragrance industry would provide an additional market and a new use for this
- Acton, N. and D.L. Klayman. 1985. Artemisinin, a new sesquiterpene lactone
endoperoxide from Artemisia annua. Planta Medica 47:442-445
- Acton, N., D.L. Klayman and I.J. Rollman. 1985. Reductive electrochemical HPLC
assay for artemisinin (Qinghaosu). Planta Medica 47:445-446.
- Charles, D.J., J.E. Simon, K.V. Wood and P. Heinstein. 1990. Germplasm
variation in artemisinin content of Artemisia annua L. using an
alternative method of artemisinin analysis from crude plant extracts. J. Nat.
- Chen, P.K, G.R Leather and D.L. Klayman. 1987. Allelopathic effect of
artemisinin and its related compounds from Artemisia annua. Plant
Physiol. 83S. Abstr. 406.
- Croteau, R. 1986. Biochemistry of monoterpenes and sesquiterpenes of essential
oils. In: Craker, L.E. and J.E. Simon (eds.). Herbs, spices, and medicinal
plants: Recent advances in botany horticulture and pharmacology. Oryx Press,
Phoenix, AZ. Vol. 1:81-133.
- Duke, S.O., K.C. Vaughn, E.M. Croom Jr. and H.N. Elsohly 1987. Artemisinin, a
constituent of annual wormwood (Artemisia annua), is a selective
phytotoxin. Weed Sci. 35:499-505.
- Henderson, W, J.W. Hart, P. How and J. Judge. 1970. Chemical and morphological
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(Patchouli). Phytochemistry 9:1219-1228.
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lactones of Artemisia nova (Asteraceae). Biochem. Syst Ecol.
- Klayman, D.L. 1985. Qinghaosu (Artemisinin): an antimalarial drug from China.
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A.D. Theoharides. 1984. Isolation of artemisinin (qinghaosu) from Artemisia
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- Schmid, G., and W. Hofheinz. 1983. Total synthesis of qinghaosu. J. Amer. Chem.
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- Xu, Xing-Xiang, J. Zhu, Da-Zhong Huang and Wei-Shan Zhou. 1986. Total synthesis
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*Journal Paper No. 12,032, Purdue Univ. Agr. Exp. Sta., West Lafayette, IN
47907. This research was supported in part by a grant from the Purdue
University Agricultural Experiment Station (Specialty Crops Grant No.
Table 1. Relative distribution of artemisinin in Artemisia annua
L. (Data from Charles et al. 1990)
zEight weeks after transplanting prior to flowering.
|Plant part sampledz ||Artemisinin |
(% dry wt)
|% of total |
|Upper third ||0.15 ||41.7|
|Middle third ||0.09 ||25.0|
|Lower third ||0.08 ||22.2|
|Side shoots ||0.04 ||11.1|
|Main stem ||trace |||
|Roots ||absent |||
|Seedsy ||0.04 |||
ySeeds collected from 13 week old plants after transplanting.
Table 2. Fresh weight and oil yield of Artemisia annua in
response to plant spacing and nitrogen application (average yields from 1985
| ||N (kg/ha)|
|Density (no plants/ha) ||0 ||67 ||134|
| ||Biomass yield (t/ha)|
|27,778 ||21 ||23 ||24|
|55,555 ||23 ||30 ||28|
|111,111 ||30 ||35 ||33|
| ||Essential oil yield (kg oil/ha)|
|27,778 ||56 ||61 ||56|
|55,555 ||67 ||85 ||69|
|111,111 ||78 ||78 ||83|
Fig. 1. Young plant (left) and field view (right) of Artemisia
annua growing in Indiana.
Fig. 2. Influence of planting date and harvest time on fresh weight of
Artemisia annua, 1987.
Fig. 3. Influence of planting date and harvest time on oil yield of
Artemisia annua, 1987.
Last update September 5, 1997