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J.F.S. Ferreira and J. Janick. 1996. Distribution of
artemisinin in Artemisia annua. p. 579-584. In: J. Janick (ed.),
Progress in new crops. ASHS Press, Arlington, VA.
Distribution of Artemisinin in Artemisia annua*
Jorge F.S. Ferreira and Jules Janick
- BOTANY
- ARTEMISININ DISTRIBUTION
- In Vivo
- In Vitro
- GLANDULAR TRICHOMES AS SITES OF ARTEMISININ ACCUMULATION
- CONCLUSION
- REFERENCES
- Table 1
- Table 2
- Table 3
- Fig. 1
- Fig. 2
Artemisia (Artemisia annua L., Asteraceae), known in the United States
as sweet Annie or annual wormwood, is an annual herb native to Asia, most
probably China, where it is known as qinghao. The plant has become
naturalized in many countries including Argentina, Bulgaria, France, Hungary,
Romania, Italy, Spain, the United States, and the former Yugoslavia (Gray 1884;
Bailey and Bailey 1976; Klayman 1989, 1993). Artemisia is mentioned in the
Chinese Handbook of Prescriptions for Emergency Treatments of 340 AD for
treatment of fevers. In 1971, extraction of aerial parts of A. annua
with low-boiling solvents, such as diethylether, produced a compound mixture
with antimalarial properties on infected mice and monkeys. The main active
principle, artemisinin (formerly referred to as arteannuin and as
qinghaosu in Chinese), was isolated and had its structure correctly
defined in 1972 in China as a sesquiterpene lactone with an endoperoxide
bridge. Artemisinin is now available commercially in China and Vietnam as an
antimalarial drug efficacious against drug-resistant strains of
Plasmodium, the malarial parasite. A semisynthetic drug based on
artemisinin (artemether) has been recently registered in Africa as Paluther.
Artemisinin also has phytotoxic activity, even on A. annua, and is a
candidate as a natural herbicide (Duke et al. 1987; Chen et al. 1991).
Artemisinin production by A. annua is usually in the range of 0.01% to
0.4% but some clones produce over 1% (Delabays et al. 1993). Artemisinin can
also be obtained from artemisinic acid which occurs at concentrations as much
as 10-fold higher than artemisinin (Acton et al. 1985). Recently, Vonwiller et
al. (1993) reported an extraction method which makes possible the extraction of
both compounds from the same plant material, thus increasing the final
production of artemisinin.
Artemisia annua, a vigorous weedy annual (Hall and Clements 1923), is a
short day plant with a critical photoperiod of 13.5 hr (Ferreira et al. 1995a).
The chromosome number is 2n = 36 (Benn et al. 1982). The plant is
usually single-stemmed reaching about 2m in height with alternate branches and
alternate, deeply dissected, aromatic leaves ranging from 2.5 to 5.0 cm in
length. Tiny yellow nodding flowers (capitula) only 2 or 3 mm across are
displayed in lose panicles containing numerous, greenish or yellowish, bisexual
central (disc) florets containing little nectar and pistillate marginal (ray)
florets (Fig. 1). The involucre is imbricated with several rows of bracts.
The central flowers are perfect and can be either fertile or sterile. Ovaries
are inferior and unilocular and each generates one achene, ca. 1 mm in length
and faintly nerved. The pistillate marginal florets in the capitulum produce
numerous achenes without pappus. The pollen is tricolpate and smooth, typical
of anemophilous species, and has vestigial or no spines (Stix 1960). It has an
internal, complex, columellae-tecta configuration in the exine, which is common
to all taxa of the tribe Anthemideae and varies from two to three layers in
A. annua (Skvarla and Larson 1965). The plant is naturally
cross-pollinated by insects and wind action, which is unusual in the Asteraceae
(McVaugh 1984).
Non-glandular T-shaped trichomes and 10-celled biseriate glandular trichomes
occur on leaves, stems, and inflorescences. The morphology and origin of the
glandular trichomes has been described for leaves (Duke and Paul 1993) and
capitula (Ferreira and Janick 1995) using light and/or scanning electron
microscopy. The essential oils of A. annua contains at least 40
volatile compounds and several nonvolatile sesquiterpenes, of which artemisinin
and related compounds are the ones of most interest due to their antimalarial
properties (Charles et al. 1991; Woerdenbag et al. 1994).
Artemisinin can be quantified by various analytical procedures including thin
layer chromatography, gas chromatography, high-performance liquid
chromatography with ultraviolet or electrochemical detection (see Ferreira et
al. 1994), radioimmunoassay, and enzyme-linked immunosorbant assay (see
Ferreira and Janick 1996a). Artemisinin has been detected from aerial parts of
the plant, mostly in leaves and inflorescences with low levels in stems and
none in pollen or roots (Table 1). The occurrence of artemisinin in the achene
(seed) is due to the presence of floral remnants.
Although some authors reported artemisinin being highest during preflowering
stages (Acton et al. 1985; Liersch et al. 1986; Woerdenbag et al. 1991;
El-Sohly 1990; Woerdenbag et al. 1994), others reported artemisinin reaching
its peak during flowering (Singh et al. 1988; Pras et al. 1991; Morales et al.
1993; Ferreira et al. 1995a; Laughlin 1995). Artemisinin reached its peak
during full flowering in a Chinese clone for both greenhouse and field
conditions (Ferreira et al. 1995b).
Reports on the distribution of artemisinin throughout the plant have been
inconsistent. Artemisinin has been reported to be higher at the top of the
plant in some clones (Charles et al. 1990; Laughlin 1995) and equally
distributed in others (Laughlin 1995). We analyzed six clones derived from
Chinese material during both vegetative and flowering stages with samples taken
from the bottom, middle, and top parts of the plant, and found a relatively
even distribution of artemisinin along the main stem (Table 2). Five of six
clones showed the same or higher levels of artemisinin at the flowering stage.
Although plants from all clones were harvested at the same date, they were in
different stages of development. Branches of one clone (clone 1) were
collected sequentially from the bottom to the top of the plant. In this clone
artemisinin showed a slight increase in artemisinin toward the top of the plant
(Table 3).
Artemisinin is produced by differentiated (shoots + roots) shoot cultures
(Martinez and Staba 1988; Fulzele et al. 1991; Whipkey et al. 1992; Ferreira
and Janick 1996b) but occur only in trace levels, if at all, in shoots without
roots (Martinez and Staba 1988; Jha et al. 1988; Fulzele et al. 1991;
Woerdenbag et al. 1993; Paniego and Giuliette 1994). Brown (1994) reported
0.0038% of artemisinin being produced by callus cultures bearing shoots but did
not specify whether or not they had roots. Most workers (Martinez and Staba
1988; Tawfiq et al. 1989; Fulzele et al. 1991; Kim et al. 1992) did not detect
artemisinin in roots, although Nair et al. (1986) and Jha et al. (1988)
reported trace amounts. Weathers et al. (1994) reported high levels (0.4%) of
artemisinin in hairy root cultures of A. annua transformed with
Agrobacterium rhyzogenes, but this was not confirmed by Jaziri et al.
(1995). With the exception of Jha et al. (1988), most authors (He et al. 1983;
Nair et al. 1986; Tawfiq et al. 1989; Fulzele et al. 1991; Kim et al. 1992;
Woerdenbag et al. 1993; Brown 1994; Paniego and Giuliette 1994; Ferreira and
Janick 1996b) either reported no artemisinin or only trace amounts produced
from callus, cell, or the spent liquid media from these cultures. Ferreira and
Janick (1996b) presented evidence that artemisinin production in shoots is
enhanced by the presence of roots. The highest levels of artemisinin (0.287%
DW) were obtained in hormone-free medium when the root production was maximized.
Although artemisinin immunolocalization has not been achieved, there is strong
circumstantial evidence that the compound is sequestered in the glandular
trichomes (Fig. 1D, E and 2A). Duke and Paul (1993) and Duke et al. (1994)
described the development of such glands in leaves of A. annua and
reported that neither artemisinin or artemisitene were detected from a
glandless biotype and that virtually all artemisinin could be extracted by a
5-sec leaf dip in chloroform, without visible damage to other leaf epidermal
cells, from the biotype with glands. Fig. 2B shows florets of the glandless
biotype photographed under the scanning electron microscope.
Artemisinin content (% DW) was shown to be 4 to 11 times higher in the
inflorescences as compared to leaves (Ferreira et al. 1995a) and the presence
and development of glandular trichomes in the inflorescences was associated
with artemisinin production based on extraction studies (Ferreira and Janick
1995). The glandular trichomes are more prominent in the corolla and
receptacles florets than in leaves, stems, or bracts. Although these glands
are present since the early stage of development on both leaves and
inflorescences, artemisinin increases at anthesis, suggesting that it
accumulates as the glands reach physiological maturity, a stage which coincides
with the end of cell expansion in floret development. As glands approach
maturity, there appears to be a cellular discharge into the subcuticular space
around the apical cells and the contents are spread over the epidermis when the
glands burst. After anthesis, artemisinin decreases and so does the number of
intact glands. The association of artemisinin with glandular trichomes
sequestration explains why artemisinin was not detected in parts of the plant
that do not bear glands, such as pollen or roots (Ferreira et al. 1995a) or in
a glandless biotype (Duke et al. 1994). Glandular trichomes are observed in
leaves and stems of differentiated shoot cultures and artemisinin content of
shoot cultures in vitro was similar to artemisinin content in vegetative clones
grown in the greenhouse (Ferreira et al. 1995b).
Artemisinin is a sesquiterpene lactone which is produced both in vivo and
differentiated in vitro cultures, by Artemisia annua and is equally
distributed throughout the plant. Artemisinin appears to be sequestered in
glandular trichomes which occur in stems, leaves, and inflorescences. The
association of peak artemisinin with flowering is related to the abundance of
glandular trichomes in the inflorescence, particularly florets and receptacle.
In vitro studies indicate that the biosynthesis of artemisinin is enhanced by
the presence of roots. Artemisia annua is unlikely to be produced
economically by chemical synthesis or by in vitro production, thus A.
annua is a potential new antimalarial crop for temperate areas.
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content of tissue-cultured, greenhouse-grown, and field-grown plants of
Artemisia annua. Planta Med. 61:351-355.
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*We acknowledge the Southern Weed Science Laboratory (USDA/ARS), for the use of
their electron microsopy facilities, and Rex N. Paul for taking the pictures.
Table 1. Artemisinin content of different organs and structures of
greenhouse- and field- grown Artemisia annua, determined by HPLC-EC
(Source: Ferreira et al. 1995a).
| Artemisinin (% DW x 1000) |
Organ/ structure | Greenhouse | Field |
Leaves | 3-30 | 6-60 |
Main stems | 0-3 | 0.4-7 |
Side stems | 0 | 0.4-14 |
Roots | 0 | 0 |
Flowers | 12-42 | 104-264 |
Pollen | 0 | NDz |
Seed husks | nd | 116 |
Seedsy | 36 | 81 |
zNot determined.
yContaining floral debris.
Table 2. Artemisin content of six clones of Artemisia annua by
location on the plant and date.
| Artemisinin (% DW) |
Clone | Datez | Base | Middle | Top | Mean |
1 | Sept 7 | 0.022 | 0.014 | 0.019 | 0.018 |
| Oct 8 | 0.044 | 0.055 | 0.054 | 0.051 |
2 | Sept 7 | 0.020 | 0.023 | 0.019 | 0.021 |
| Oct 8 | 0.079 | 0.070 | 0.094 | 0.081 |
3 | Sept 7 | 0.043 | 0.043 | 0.054 | 0.046 |
| Oct 8 | 0.034 | 0.026 | 0.023 | 0.027 |
4 | Sept 7 | 0.080 | 0.078 | 0.080 | 0.079 |
| Oct 8 | 0.081 | 0.146 | 0.143 | 0.123 |
5 | Sept 7 | 0.100 | 0.127 | 0.107 | 0.111 |
| Oct 8 | 0.086 | 0.123 | 0.127 | 0.112 |
6 | Sept 7 | 0.163 | 0.153 | 0.146 | 0.154 |
| Oct 8 | 0.245 | 0.181 | 0.234 | 0.220 |
7 | Sept 7 | 0.071 | 0.073 | 0.071 | 0.072 |
| Oct 8 | 0.095 | 0.100 | 0.112 | 0.102 |
Mean | Sept 7 | 0.071 ay | 0.073 a | 0.071 a | 0.072a |
| Oct 8 | 0.095 b | 0.100 b | 0.112 b | 0.102b |
zAll plants vegetative Sept 7; all plants flowering Oct 8.
yMean separation in rows and columns by Duncan's Multiple Range Test
(5% level).
Table 3. Artemisinin content of Artemisia annua branches (base
to apex) of clone 1 harvested Oct. 8, 1994.
Positionz | Artemisinin content (% DW) |
1 (apex) | 0.040 |
2 | 0.046 |
3 | 0.043 |
4 | 0.041 |
5 | 0.033 |
6 | 0.037 |
7 (base) | 0.023 |
zFive branches were composited at each position.

Fig. 1. Floral morphology of Artemisia annua. A Nodding
capitulum.

B. Expanded capitulum showing calyx with imbricated bracts (b),
receptacle (r), marginal pistillate floret (p), and central hermaphroditic (h)
florets. Glandular trichomes are found abundantly on the receptacle, bracts,
and florets of the capitulum.

C. Cross-section of the involucre showing
imbrication of bracts. D. Unexpanded floret showing orientation of glandular
trichomes. E. Fully developed, turgid, glandular trichome, based on SEM.

F. Details of hermaphroditic floret with lobed anthers attached to basal portion of the corolla (c), pistil with bifid stigma (s), style (st), and ovary (o). Note that in a hermaphroditic floret, the stigma reaches this state of
development only after pollen shed.

G. Tricolpate pollen grain with vestigial
spines, characteristic of wind-pollinated species, and germination pores (gp)
bulging from the furrows. H. Pollen cross section based on light microscopy
shows details of bulging germination pores. (Source: Ferreira and Janick
1995).
 |
Fig. 2. Florets of Artemisia annua pictured by JEOL JSM840 SEM, at 5KV. Top, hermaphroditic florets of normal biotype showing glandular trichomes. Bottom, hermaphroditic florets of glandless biotype. Bar size = 100 µm.
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Last update August 24, 1997
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