Updates on artemisinin: an insight to mode of actions and strategies for enhanced global production

Abstract

Application of traditional Chinese drug, artemisinin, originally derived from Artemisia annua L., in malaria therapy has now been globally accepted. Artemisinin and its derivatives, with their established safety records, form the first line of malaria treatment via artemisinin combination therapies (ACTs). In addition to its antimalarial effects, artemisinin has recently been evaluated in terms of its antitumour, antibacterial, antiviral, antileishmanial, antischistosomiatic, herbicidal and other properties. However, low levels of artemisinin in plants have emerged various conventional, transgenic and nontransgenic approaches for enhanced production of the drug. According to WHO (2014), approximately 3.2 billion people are at risk of this disease. However, unfortunately, artemisinin availability is still facing its short supply. To fulfil artemisinin's global demand, no single method alone is reliable, and there is a need to collectively use conventional and advanced approaches for its higher production. Further, it is the unique structure of artemisinin that makes it a potential drug not only against malaria but to other diseases as well. Execution of its action through multiple mechanisms is probably the reason behind its wide spectrum of action. Unfortunately, due to clues for developing artemisinin resistance in malaria parasites, it has become desirable to explore all possible modes of action of artemisinin so that new generation antimalarial drugs can be developed in future. The present review provides a comprehensive updates on artemisinin modes of action and strategies for enhanced artemisinin production at global level.

Full text

REVIEW ARTICLE
Updates on artemisinin: an insight to mode of actions
and strategies for enhanced global production
Neha Pandey
1
&Shashi Pandey-Rai
1
Received: 19 January 2015 /Accepted: 16 March 2015
#Springer-Verlag Wien 2015
Abstract Application of traditional Chinese drug,
artemisinin, originally derived from Artemisia annua L., in
malaria therapy has now been globally accepted.
Artemisinin and its derivatives, with their established safety
records, form the first line of malaria treatment via artemisinin
combination therapies (ACTs). In addition to its antimalarial
effects, artemisinin has recently been evaluated in terms of its
antitumour, antibacterial, antiviral, antileishmanial,
antischistosomiatic, herbicidal and other properties.
However, low levels of artemisinin in plants have emerged
various conventional, transgenic and nontransgenic ap-
proaches for enhanced production of the drug. According to
WHO (2014), approximately 3.2 billion people are at risk of
this disease. However, unfortunately, artemisinin availability
is still facing its short supply. To fulfil artemisinin’s global
demand, no single method alone is reliable, and there is a need
to collectively use conventional and advanced approaches for
its higher production. Further, it is the unique structure of
artemisinin that makes it a potential drug not only against
malaria but to other diseases as well. Execution of its action
through multiple mechanisms is probably the reason behind
its wide spectrum of action. Unfortunately, due to clues for
developing artemisinin resistance in malaria parasites, it has
become desirable to explore all possible modes of action of
artemisinin so that new generation antimalarial drugs can be
developed in future. The present review provides a compre-
hensive updates on artemisinin modes of action and strategies
for enhanced artemisinin production at global level.
Keywords Artemisia annua L.Artemisinin .Malaria .
Cancer .Mode of action .Yield enhancement
Introduction
Artemisinins (artemisinin and its chemical derivatives), a fam-
ily of unique sesquiterpene trioxane with endoperoxide
bridge, is surely a natures’gift to the mankind. Artemisinins
are derived from an Asteraceae member Artemisia annua and
has long been used in traditional medication in the form of
herbal preparations to cure intermittent fevers. With the redis-
covery of artemisinin in 1970s, its tremendous potential to
cure malaria infections have been established (Hsu 2006). In
the present scenario, WHO recommended that artemisinin
combination therapies (ACT) form the backbone of the global
struggle to cure malaria. According to WHO (2014), 97 coun-
tries worldwide had ongoing malaria transmission, and ap-
proximately 3.2 billion people are at risk of this disease.
Pharmacological action of artemisinin is now no more restrict-
ed to treat malaria but to other medical conditions such as
various cancers, inflammatory diseases, viral (e.g. Human
cytomegalovirus), protozoal (e.g. Toxoplasma gondii), hel-
minths (Schistosom sp., Fasciola hepatica)andfungal
(Cryptococcus neoformans) infections (Ho et al. 2013). Very
recently, an artemisinin derivative, artesunate has been shown
to inhibit autoimmune arthritis (Hou et al. 2014) and possess
antihistaminic activity (Favero Fde et al. 2014). The unique-
ness of artemisinin as potential drug for various diseases lies
in its complex structure. Various studies have revealed that
Handling Editor: Peter Nick
*Shashi Pandey-Rai
shashi.bhubotany@gmail.com
1
Laboratory of Morphogenesis, Department of Botany, Banaras
Hindu University, Varanasi, India
Protoplasma
DOI 10.1007/s00709-015-0805-6

there is no single standard mode of action that exists for
artemisinin. It executes its action through multiple mecha-
nisms, and this could be the probable reason for its wide spec-
trum of action against many diseases. Being a frontline treat-
ment for malaria and a promising drug for cancer treatment,
there are continuous efforts to elucidate antimalarial and anti-
cancer mechanisms of action for artemisinin. Therefore, there
is a need to review different modes of action in order to carry
forward the research of development and application of
artemisinin in a better way.
Artemisinin has a low ratio of its production to demand.
The various factors contributing to short falls in artemisinin
availability are its low concentration in plants (less than 0.01
to 1.4 % of the plant dry weight) (Liu et al. 2006) and depen-
dence of its yield on environmental variables such as temper-
ature, humidity and soil types (White 2008). Owing to its
complex structure, chemical synthesis is very uneconomical
and low yielding (Geldre et al. 1997). All these factors cumu-
latively make artemisinin relatively expensive causing diffi-
culty to fulfil demand of over 392 million courses of ACT
(equivalent to 180 metric tonnes of artemisinin) each year
(WHO 2014). Various efforts have been made, and research
is still on to find out the most suitable way to enhance
artemisinin production. However, any individual method is
not enough to produce higher artemisinin at global level,
and orchestrating various yield enhancing strategies is desir-
able to speed up the artemisinin supply to needy. The present
review summarizes recent progress on artemisinin’smecha-
nism of action and strategies for enhanced artemisinin produc-
tion globally.
Mode of action of artemisinin
No single mechanism exists for artemisinins’action. It func-
tions through multiple modes. Despite of having plenty of
biological properties, artemisinins’modes of action have been
widely investigated in terms of its antimalarial and anticancer
effects. There exist a number of reviews dealing with mecha-
nism of antimalarial/anticancer individually. This review pro-
vides a compilation of latest advances in both action mecha-
nisms together.
Antimalarial modes of action
The exact mechanism of action of artemisinin is still a debated
topic. Multiple antimalarial modes of action have been postu-
lated for artemisinin (Fig. 1). Prior to its action, artemisinin
needs to be activated that generate free radical species. The
nature of ‘artemisinin activator’is a bit controversial. Various
groups have demonstrated a covalent reaction between
artemisinin and haemoglobin-bound iron (Selmeczi et al.
2004;Kannanetal.2005; Messori et al. 2006); however, its
probability was denied due to steric hindrance caused by
bulky artemisinin when approaching heme moiety of
haemoglobin (Creek et al. 2009). Afterwards, it has been sug-
gested that ferrous heme (heme-Fe
2+
) is probably the most
efficient activator of artemisinin (Zhang and Gerhard
2008).
Two most acceptable models for artemisinin activation are
(i) reductive scission model and (ii) open peroxide model
(O’Neill et al. 2010). In reductive scission, ferrous
heme/non-heme exogenous Fe
2+
bind to artemisinin and via
reductive scission of the peroxide bridge produce oxygen-
centred radicals which self-arranges to give carbon-centred
radicals. Further, iron-peroxide interaction occurs in different
ways to form either primary carbon-centred radicals (via C
3
-
C
4
bond scission) or secondary carbon-centred radicals (via 1,
5 H-shifts). In contrast, open peroxide model suggests in-
volvement of a Fanton reaction of Fe
2+
and the endoperoxide
to produce secondary carbon-centred radicals (Haynes et al.
2007) that are short-lived and are damaging to parasite’svital
biomolecules (Edikpo et al. 2013). This ‘carbon-centred rad-
ical theory’may be strengthened by the fact that artemisinin
activity greatly diminished in the presence of free radical scav-
engers and antioxidants (Hamacher-Brady et al. 2011)where-
as pro-oxidants enhanced its activity (Krungkrai and
Yuthawang 1987). A recent report by Gopalakrishnan and
Kumar (2014) revealed that artesunate (an artemisinin deriv-
ative) induces DNA double-strand breaks in P. falciparum
with simultaneous increase in intercellular ROS level in the
parasite ultimately causing parasite death. In addition to oxi-
dative stress-mediated parasite damage, artemisinins have
been suggested to covalently interact with essential
plasmodial biomolecules (Asawamahasakda et al. 1994).
One such identified molecule is PfTCTP, a histamine releasing
factor that is involved in regulation of cell cycle, histamine
release, malignant transformation, immunological functions
(Bommer and Thiele 2004) as well as a target molecule in
tumour reversion and malaria treatment (Telerman and
Amson 2009; Bhisutthibhan et al. 1998). Though artemisinin
interacts covalently by alkylation of TCTP, it was unclear
whether it might function as target for artemisinin; however,
recently, Eichhorn et al. (2013) demonstrated a role of TCTP
in mediating artemisinin effect owing to its very high Kd
values (77–120 μM) that are several orders of magnitude
higher than the artemisinin IC
50
s for parasite growth
inhibition.
A decade ago, another potential target for artemisinin ac-
tion was identified as PfATP6, the P. falciparum sarco-
endoplasmic reticulum calcium ATPase (SERCA) (Eckstein-
Ludwig et al. 2003). It reduces cytosolic free Ca
2+
by
pumping two Ca
2+
into the endoplasmic reticulum, in ex-
change for <4H
+
, a mechanism critical for parasite survival
(Brini and Carafoli 2009). Of various Ca
2+
-ATPases in
Plasmodium, only one enzyme orthologous to SERCA;
N. Pandey, S. Pandey-Rai

PfATP6 is found in P. falciparum (Eckstein-Ludwig et al.
2003) that consists of 10 transmembrane α-helix and three
cytosolic domains—the effector (A) and the phosphorylation
(P) that are connected to the M domain and third, the
nucleotide-binding domain (N) that is connected to P domain
(Brini and Carafoli 2009). Eckstein-Ludwig et al. (2003)have
also suggested that mechanism of artemisinin-mediated
PfATP6 inhibition is highly specific, not affecting any other
malarial transporter including the non-SERCA Ca
2+
ATPase
(PfATP4) and possibly function through allostericmechanism
(Shandilya et al. 2013). Further experiments dealing with mu-
tation of a single residue (L263) that directly modulates
PfATP6 sensitivity to artemisinin suggest a potential resis-
tance mechanisms (Uhlemann et al. 2005). Artemisinin resis-
tance is generally characterized by a delayed clearance of ma-
larial parasite. Efforts are being implicated for using PfATP6
as a molecular marker for artemisinin resistance. However,
mutations in the Kelch 13 (K13) propeller domain have re-
cently been demonstrated as important determinants for
artemisinin resistance both in vivo and in vitro (Ariey et al.
2014;Straimeretal.2015). Mok et al. (2015) conducted pop-
ulation transcriptomics of 1043 human malaria parasites and
revealed that elevated expression of unfolded protein response
(UPR) pathways including the major PROSE and TRiC chap-
eron complexes is the possible underlying mechanism behind
artemisinin resistance. Further, they provided the mechanistic
evidence that artemisinin-resistant parasites upregulate UPR
pathways in order to overcome the protein damage caused by
artemisinin.
Recently, Hartwig et al. (2009) have demonstrated that
artemisinin may cause parasite membrane damage by accu-
mulating within its neutral lipids and suggested the role of
endoperoxide moiety of artemisinin behind such effects.
Experiments were conducted on Sacchramyces cereviciae
where targeted deletion of genes encoding mitochondrial
NADH dehydrogenases (NDE 1 or NDI 1) resulted in resis-
tance to inhibitory effects of artemisinin whereas their over-
expression increased sensitivity to artemisinin, suggesting a
Fig. 1 Different antimalarial
modesofactionofartemisinin
Artemisinin: modes of action and its enhanced global production

mode of action associated with mitochondrial functions (Li
et al. 2005). This idea was further attested by Wang et al.
(2010), demonstrating artemisinin-induced mitochondrial
membrane depolarization via locally generated reactive oxy-
gen species (ROS) that lead to mitochondrial malfunctioning
and ultimately parasite death. Additional mechanistic evi-
dence was provided by the study conducted on mammalian
cells with non-functional electron transport chain where ac-
tively respiring mitochondria have been shown to generate
reactive oxygen species that plays central role in
endoperoxide-induced cytotoxicity of artesunate (Mercer
et al. 2011).
Within the host erythrocytes, parasite degrades host
haemoglobin into ‘heme’and ‘globin’moiety through a series
of proteases in to its food vacuole. Further, catabolism releases
short peptides and amino acids which are needed for parasite
nutrition. This releases a toxic by-product ‘hematin’which
undergoes bio-mineralization to form non-toxic ‘hemozoin’
(malaria pigment). It has been proposed that Fe
2+
-heme-acti-
vated artemisinin interfere this catabolic pathway through
heme alkylation at α,βand δ-carbon atoms that later create
toxicity to parasite (Meunier et al. 2010; Cazelles et al. 2001).
Two proteins related to this catabolic pathway—heme detox-
ification protein (HDP) and histidine-rich protein II (HRP
II)—have been posited as targets of artemisinin action
(Chugh et al. 2013).
An interesting demonstration regarding interaction of heme
and mitochondria with artemisinin has recently been revealed
(Sun et al. 2015). It has been suggested that heme and mito-
chondria playdistinct roles for mediating artemisinin damage.
In tumour cells, heme pathway has been shown to mediate
artemisinin’s killing whereas antimalarial action of artemisinin
is driven by specific mitochondrial pathways.
To further uncover the artemisinins’mode of action, gene
expression analysis was undertaken that revealed alterations in
two genes (encoding heat shock protein, HSP70, and a hypo-
xanthine phosphoribosyltransferase, PF10_0121, related to
purine biosynthesis) associated with decreased sensitivity to
artemisinin. However, whether or not these proteins are direct-
ly a part of artemisinin mode of action is unclear.
Anticancer modes of action
Similar to its antimalarial actions, artemisinins exert their an-
ticancer effects through endoperoxide bridge-dependent ROS
generation and subsequent oxidative damage to the target
cell(s) (O’Neill et al. 2010). Many workers have established
ROS-dependent artemisinin effects. Further boost to this the-
ory has recently been given by Lu et al. (2014)where
dihydroartemisinic acid (DHA) significantly reduced viability
of human colorectal cancer HCT-116 cells in vitro through G1
cell arrest, apoptotic cell death and ROS accumulation. A
consensus opinion is that artemisinin and its derivatives exert
their therapeutic effects through multiple modes (Fig. 2).
Though oxidative damage remains in the prime focus, italone
cannot describe everything about anticancer actions of
artemisinins. This hypothesis was strengthened when DHA
was demonstrated to activate p38 MAPK pathway in an
ROS-independent way (Lu et al. 2008) through inhibiting
vascular endothelial growth factor (VEGF)-induced endothe-
lial cell migration (Guo et al. 2014). Further refinements were
brought by workers who revealed an anticancer specific action
of artemisinin through modulation of transferrin receptor-1
(TfR1), a type II transmembrane protein (Ba et al. 2012).
TfR1 is required for iron import in to the cells by binding to
diferric transferrin and subsequent internalization of the Tf-
TfR1 complex through receptor-mediated endocytosis (Ponka
and Lok 1999). For rapid proliferation of cancer cells,
high iron concentration is required; therefore, TfR1
overexpresses in cancer. DHA deplete cellular iron
levels via palmitoylation and subsequent lipid raft-
mediated non-classical endocytosis of TfR1, leading to
iron deficiency in cancer cells (Ba et al. 2012).
To uncover the probable molecular network governing an-
ticancer mechanisms of artemisinins, recent efforts by Huang
et al. (2013) have revealed key signalling pathways of
artemisinin actions including Pi3k-akt, T cell receptor, toll-
like receptor and TGF-beta signalling pathways. Further, dif-
ferent targets of artemisinins from these pathways were also
predicted. Pi3k-akt (phosphatidylinositol-3 kinases–Akt), an
intracellular signalling pathway, is activated in cancers and
plays central role in cancer cell progression and survival
(Courtney et al. 2010). However, a current report by Chen
et al. (2014) suggests that dihydroartemisinin inhibits glioma
cell proliferation and invasion possibly via suppressing
disintegrin and metalloproteinase 17 (ADAM17) levels and
downregulating epidermal growth factor receptor (EGFR)-
Pi3k-akt signalling. Consequently, Pi3k-akt pathway is being
developed as a potential drug target to cure cancers.
Artemisinins, recently emerged potent anticancer drugs, target
this pathway as a major mechanism of action and thereby
efficiently regulate various cellular processes like cell growth,
proliferation, survival and immunity probably through
targeting several membrane receptors such as EGFR, EPOR,
IGF1R, IL2RA, IL4R, ITGAV, KDR, KIT, MET, NGFR,
PDGFRB and TLR4 (Huang et al. 2013). Additionally,
artemisinins’derivatives such as DHA and artesunate were
also postulated to induce apoptosis in prostate cancer cells
and regulate immunological responses via modulating Pi3k-
akt pathway (He et al. 2010;Xuetal.2007).
In addition to Pi3k-akt, more targeted approaches for can-
cer treatment involve inhibition of molecular/signalling path-
ways that are crucial for tumour cell progression and mainte-
nance, and hence, artemisinins are making their place for ad-
vance cancer therapies owing to its capability to target toll-like
receptors (TLRs), TFG-beta and T cell signalling pathways.
N. Pandey, S. Pandey-Rai

TLRs are integral membrane protein receptors which are be-
lieved to promote tumour growth and progression (Muccioli
et al. 2012). Similarly, due to their growth promoting abilities,
TFG-beta (transforming growth factor-beta) and T cell recep-
tor signalling pathways are believed as excellent targets for
treatment of cancers and other diseases (Akhurst and Hata
2012; Watanabe et al. 2014). Artemisinin-mediated targets
for T cell receptor signalling pathways have been predicted
as CD28, CD4, CD8A and CTLA4 whereas for toll-like re-
ceptor and TGF-beta signalling pathways, TLR4 and
TGFBR1, respectively, are predicted targets (Huang et al.
2013). A recent report by Lee et al. (2014) suggests that
artesunate, an artemisinin derivative, has a negative mitogenic
effect on CD+ T cells as it inhibits CD4+ T cell proliferation
and IL-2 (T cell growth factor) production and reduction in
expression of cell surface protein CD25 (IL-2 receptor alpha
chain) and CD69 on CD4+ T cells.
Emerging resistance to artemisinin: an unfortunate
condition
Although tremendous efforts have been implicated to eluci-
date possible mechanisms of action, many points are still con-
troversial and unclear which are needed to be looked for.
While attempts have been made and are still in process to
eliminate malaria, unfortunately, spread of artemisinin resis-
tance in malarial parasites has imperilled efforts for malaria
control and elimination. As per the latest WHO report
(February 2015), artemisinin resistance has been confirmed
in Combodia, the Lao People’s Democratic Republic,
Myanmar, Thailand and Viet Nam, and many other regions
are at high risk of emergence of multidrug resistance. Of var-
ious factors that have contributed towards evolution of
artemisinin resistance, widely available oral artemisinin
monotherapies and other substandard antimalarial drugs have
played major roles. WHO along with other organizations are
working with the strategy to remove oral artemisinin-based
monotherapies and substandard antimalarial drugs from the
market and emphasizing the use of artemisinin combination
therapies to check further spread of drug resistance. However,
this requires strong financial capital, long-term political com-
mitments and cross-border cooperation. Besides various ma-
laria elimination and control policies that is being developed,
it is now highly desirable to uncover depth of every possible
mechanism underlying artemisinin action so as to open new
doors for development of accurate and potential new genera-
tion antimalarial drugs and modes of malaria treatment in case
of further spreading of artemisinin resistance in future.
Strategies for enhanced artemisinin production
Extensive efforts have been made to improve artemisinin syn-
thesis through many approaches. The present review provides
compilation of recent advancement in both conventional and
modern approaches of yield enhancement such as (i) metabol-
ic engineering of the plant using various genetic engineering
tools; (ii) regulation of artemisinin biosynthesis through stress
signals to plants, hairy root cultures and cell cultures and (iii)
conventional, mutational and molecular breeding of A. annua.
Fig. 2 Proposed model for
various anticancer modes of
action of artemisinin
Artemisinin: modes of action and its enhanced global production

Covello (2008) had suggested the implementation of mix
mode production system involving both biological and chem-
ical synthesis, as a good option for artemisinin production.
Besides various approaches for improved artemisinin synthe-
sis, recent success has been achieved in its semi-synthetic
production with the completion of The Bill and Melinda
Gates Foundation funded ‘The semi-synthetic Artemisinin
Project’in a partnership between University of California
(Berkeley, USA), Amyris Inc. and the Institute for One
World Health (a non-profit pharmaceutical company and
now known as PATH Drug Solutions). The project involved
combined use of metabolic engineering of microorganisms
and synthetic biology to produce semi-synthetic artemisinin.
They achieved 25 gm per litre artemisinic acid production
from engineered S. cerevisiae (Baker’s yeast) as well as 40–
45 % conversion of artemisinic acid into artemisinin (Paddon
et al. 2013). Details of the complete scheme and approaches
adopted for semi-synthetic artemisinin production have been
very recently reviewed by the project leaders themselves
(Paddon and Keasling 2014). Even though successful produc-
tion of semi-synthetic artemisinin to supplement the plant-
derived production, research is still on to produce WHO rec-
ommended potent artemisinin derivatives at large scale to be
used in ACTs. Therefore, it is still highly desirable not to be
dependent solely on semi-synthetic artemisinin, and efforts
should be implemented to enhance drug production at global
level by other means as well.
It is interesting to note that dried leaves of A. annua con-
taining artemisinin and artemisinin synergistic flavonoids are
more effective to cure malaria than a comparable dose of pure
artemisinin as ACT oral therapy (Weathers et al. 2014). As
discussed earlier in this review, use ofcombination therapy (as
ACT) was promoted to overcome the increasing resistance to
existing antimalarial drugs. However, very recently, consump-
tion of dried A. annua leaves has been demonstrated to over-
come existing resistance to pure artemisinin in Plasmodium
yoelii (Elfawal et al. 2015). These recent reports promote the
idea of increasing artemisinin synthesis through in-planta
yield enhancement over the semi-synthetic production of pure
artemisinin for better and more effective cure of human
malaria.
Artemisinin biosynthetic pathway and overexpression
of concerned pathway gene(s)
Like other terpenes, artemisinin biosynthesis involves two
pathways, the cytosolic ‘mevalonate (MVA) pathway’stem-
ming from acetyl co-A and the plastidic ‘non-mevalonte/MEP
pathway’originating from glyceraldehyde-3-phosphate and
pyruvate. These two pathways result in the formation of two
isoprenoid precursors, isopentenyl diphosphate (IPP) and di-
methyl allyl diphosphte (DMAPP). These two precursors de-
rived from two pathways do not lead rest of the pathway
separately and independently, rather it has been demonstrated
that cytosolic DMAPP (mevalonate origin) is transferred to
the plastid and it is the plastidic IPP unit that form the central
isoprenoid unit of majority of fernesyl diphosphate (FPP) en-
gaged in artemisinin biosynthesis (Schramek et al. 2009). Two
stages of artemisinin biosynthesis have now been completely
elucidated: first stage involves the formation of FPP while
second is the cyclization of FPP to form amorpha-4,11-diene,
which ultimately lead to the synthesis of artemisinin and its
various derivatives, all steps catalyzed by a series of enzymes.
Key enzymes involved are as follows: HMGS, HMGR, DXS,
DXR, FPS, ADS, CYP71AV1, DBR2 and ALDH1 (Fig. 3).
All the biosynthetic pathway genes have been shown to be
differentially expressed in various plant parts. Glandular tri-
chomes of A. annua have been well characterized as the site of
artemisinin biosynthesis and tissues with high density of glan-
dular trichomes such as flower buds and young leaves show
relatively higher expression of biosynthetic pathway genes
(Olofsson et al. 2011). In contrast to the all enzyme-
catalyzed steps of artemisinin biosynthesis, the conversion
of dihydroartemisinic acid to artemisinin is, however, a non-
enzymatic photooxidative step. It has been hypothesized that
in A. annua, there are many oxygenated sesquiterpenoids in-
cluding artemisinin, which are formed by spontaneous autox-
idation of terpene precursors via highly reactive allyllic hydro-
peroxide intermediate (Brown 2010).
Manipulations of biosynthetic pathways to channelize
the carbon flux for enhanced synthesis of a particular
metabolite involves either upregulation of desired path-
way or downregulation of competing pathways through
overexpression and suppression of their respective path-
way genes, respectively.
Overexpression of key biosynthetic genes for hyperproduc-
tion of precursors of different stages and hence greater syn-
thesis of product (artemisinin) is an excellent idea. Much suc-
cess has been achieved in engineering almost all the key genes
from artemisinin biosynthetic pathway. Basic chemical skele-
ton of artemisinin involves fusion of three C-5 isoprenoid unit
(IPP or DMAPP). Two important key enzymes leading to C-5
isoprenoid unit are HMGR and DXR. HMGR shunts HMG
Co-A into the mevalonate pathway, leading to the synthesis of
isoprenoid unit in cytosol, whereas in plastidic non-
mevalonate pathway DXR catalyzes the first committed step
of isoprenoid (C5) synthesis by converting 1-deoxy-D-
xylulose-5-phosphate in to 2-C-methyl-D-erythritol-4-
phosphate.
These two key genes have been overexpressed in A. annua
by many workers. Agrobacterium-mediated HMGR gene
transfer from Catheranthus roseus to A. annua was first per-
formed by Aquil et al. (2009), and this resulted in 38.9 %
higher artemisinin level in one of the transgenic line.
However, when HMGR was co-expressed along with ADS
gene, artemisinin enhanced up to 7.65-folds in one of the
N. Pandey, S. Pandey-Rai

transgenic line as compared to non-transgenic plants (Alam
and Abdin 2011).
Isotopologue (
13
CO
2
) profiling (Schramek et al. 2009)and
fosmidomycin (MEP pathway blocker) treatment (Towler and
Weathe rs 2007) confirmed DXR to be an important regulator
of artemisinin biosynthesis. Further, genetic map (Graham
et al. 2010) itself did not bring direct evidence for DXR to
be regulator of artemisinin biosynthesis; however, it
pointed the fact that DXR locus co-localizes with QTL
for artemisinin yield and concentration. Recently, CaMV
35S promoter driven overexpression of DXR gene in
A. annua depicted 1.21–2.35-folds higher artemisinin
in transgenic plants (Xiang et al. 2012).
C5 isoprenoid unit thus produced (via actions of key en-
zymes HMGR and DXR) undergo two sequential 1-4 conden-
sations catalyzed by fernesyl diphosphate synthase (FPS) to
give C15 fernesyl diphosphate (FPP), which is further utilized
to produce variety of sesquiterpenoids and triterpenoids in-
cluding artemisinin. Thus, overexpression of FPS gene may
probably contribute to greater metabolic flux towards
artemisinin synthesis and both heterologous (from
Gossypium arboreum) and homologous (from A. annua)
FPS gene overexpression resulted in 2–3-folds higher
artemisinin content in transgenic lines in comparison to
wild-type plants (Chen et al. 2000;Banyaietal.2010).
Further, when FPS gene was coexpressed with HMGR gene
(both were homologous), they boosted artemisinin content
1.8-fold in transgenic plants.
The first committed step of artemisinin biosynthesis is the
cyclization of FPP in to amorpha-4,11-diene catalyzed by
amorpha-4,11-diene synthase (ADS). Thus, the overexpres-
sion of ADS is a promising approach for upregulated
artemisinin biosynthesis in A. annua. Tang et al. (2008)cloned
and expressed ADS gene in A. annua and observed 2.3-folds
more artemisinin as compared to control. However, spraying
of artemisinic acid to A. annua plants reduced ADS transcript
level suggesting a possibility of some feedback inhibition on
artemisinin biosynthesis (Arsenault et al. 2010a)Further,
Fig. 3 Transgenic approaches for enhanced artemisinin biosynthesis in
A. annua. (3-hydroxy-3-methylglutaryl-CoA synthase: HMGS, 3-
hydroxy-3-methylglutaryl-CoA reductase: HMGR, 1-deoxyxylulose 5-
phosphate synthase: DXS, 1-deoxyxylulouse 5-phosphate
reductoisomerase: DXR, amorpha-4,11-diene synthaase: ADS,
cytochrome P 450 CYP71AV1: CYP, double bond reductase 2: DBR2,
dihydroartemisinic aldehyde reductase 1: RED1, aldehyde
dehydrogenase 1: ALDH1, farnesyl diphosphate synthase: FPS,
squalene synthase: SQS, caryophyllene synthase: CPS)
Artemisinin: modes of action and its enhanced global production

diversion of amorpha-4,11-diene towards artemisinin synthe-
sis is carried out by cytochrome P450 monooxygenase
(CYP71AV1) that catalyzes three-step sequential conversion
of amorpha-4,11-diene to artemisinin alcohol, then artemisinic
aldehyde, and ultimately to artemisinic acid. The activity of
CYP71AV1 also requires a reducing companion cytochrome
P450 oxidoreductase (CPR), which is considered to be co-
expressed with CYP71AV1 (Ro et al. 2006). In addition,
Chen et al. (2012) tried to co-overexpress CYP71AV1 and
CRP with FPS gene. This led to 3.6-fold greater accumulation
of artemisinin in FPS+CYP71AV1+CPR co-overexpressing
transgenic lines in comparison to wild-type plants. In a similar
approach, CYP71AV1 and CPR were co-overexpressed with
ADS and resulted in 2.4-fold higher artemisinin in one of the
transgenic line as compared to control plants. Further in the
artemisinin biosynthetic pathway, reduction of artemisinic al-
dehyde in to dihydroartemisinic aldehyde has been proposed
to be an important step which is catalyzed by artemisinic al-
dehyde Δ11(13) reductase (DBR2). Very recently, Yuan et al.
(2014)madeanefforttooverexpressDBR2 gene driven by the
CaMV 35S promoter and observed remarkable increase in
artemisinin and its direct precursor DHA, as confirmed by
HPLC analysis. In addition, DBR2-overexpressing lines also
produced more arteannuin B and its direct precursor
artemisinic acid.
All the important genes encoding key enzymes of upstream
and downstream artemisinin biosynthetic pathway have been
cloned and transgenic lines overexpressing single or multiple
genes have so far resulted in 1.8–to 7.6-folds elevated
artemisinin production, suggesting overexpression of pathway
genes as a promising approach to effectively improve
artemisinin synthesis.
Indirect upregulation of artemisinin biosynthetic pathway
genes
The expression of genes is largely regulated by specific tran-
scription factors (TFs). Therefore, overexpression of TFs of-
fers an alternative and complementary strategy to upregulate
biosynthetic pathway genes (Fig. 3). Promoters of three key
genes of artemisinin biosynthesis, i.e. ADS,CYP71AV1 and
DBR2, have been characterized till date. The first ever tran-
scription factor of A. annua that has been characterized is
AaWRKY1 (a WRKY type transcription factor) and is pro-
posed to bind to the W-boxes in ADS and CYP71AV1 pro-
moters. Transient expression of AaWRKY cDNA in leaves
of A. annua was shown to increase the transcript level of the
majority of artemisinin biosynthetic genes (Ma et al. 2009)
and AaWRKY-overexpressing transgenic lines produced
4.4-folds higher artemisinin as compared to control lines
(Tang et al. 2012). Recently, in a more targeted approach,
trichome-specific overexpression of AaWRKY1 resulted in
more effective activation of CYP71AV1 transcription as
compared to the constitutive overexpression of AaWRKY
(Han et al. 2014). However, this did not lead to any significant
improvement in transcript level of other key genes such as
FDS,ADS and DBR2 and promoted artemisinin content 1.8
times as compared to wild-type plants. Further, CRTD
REHVCBF2 (CBF2) and RAV1AAT (RAA) motifs in pro-
moters of both ADS and CYP71AV1 have been proposed to
be the binding sites of two TFs, AaERF1 and AaERF2, which
belong to JA-responsive AP2 family of TF. Both the TFs were
cloned from A. annua and overexpression of either of the two
promoted ADS and CYP71AV1 transcripts level and
artemisinin as well. The fourth TF that has been successfully
cloned and overexpressed in A. annua is AaORA which is a
trichome-specific APETALA2/ethylene response factor (AP2/
ERF) family TF. Its overexpression in A. annua significantly
boosted transcription of ADS,CYP71AV1,DBR2 and
AaERF1. AaORA-overexpressing and AaORA-RNAi trans-
genic lines showed significant increase (53 %) and decrease in
artemisinin content, respectively, as compared to control (Lu
et al. 2013). Recently, a fifth TF, AabHLH1, a bHLH TF has
been successfully isolated from a cDNA library of glandular
secretary trichomes (GSTs) of A. annua and has been
demonstrated to be able to bind to the E-box-cis ele-
ment in the promoter of ADS and CYP71AV1.Transient
expression of AabHLH1 in the leaves of A. annua sig-
nificantly enhanced transcript levels of ADS,CYP71AV1
and HMGR (Jietal.2014).
In addition to TF engineering, artemisinin biosynthesis
may also be improved by blocking/silencing artemisinin com-
petitive pathway/genes and hence ensuring diversion of com-
mon isoprenoid precursors more towards artemisinin biosyn-
thetic pathway (Fig. 3). So far, two competitive pathway en-
zymes have been engineered in A. annua: squalene synthase
(SQS) and β-caryophyllene synthase (CPS). RNA interfer-
ence (RNAi) offers a potential ‘reverse genetics’approach to
knock down the expression of particular gene(s) and in
A. annua Zhang et al. (2009) applied RNAi technology for
the first time to knock down the expression of SQS. SQS
catalyzes the first committed step in sterol biosynthetic path-
way by converting FPP into squalene and thus competes with
ADS for utilizing FPP in the biosynthesis of sterols.
Suppression of SQS expression in A. annua through hairpin
RNA-mediated RNAi technique reported a 3.14-fold higher
artemisinin content along with significantly lower sterol level
in transgenic plants as compared to control. Another gene that
has been artificially modulated for its expression in A. annua
is CPS. CPS, being a sesquiterpene synthase, competes with
ADS for utilizing FPP and converts FPP into β-caryophyllene.
Using antisense RNA technology, Chen et al. (2011)triedto
suppress the expression of CPS gene by Agrobacterium-me-
diated insertion of 750-bp antisense fragment of CPS cDNA
into A. annua via pBI121 plant expression vector. This
lowered the endogenous CPS expression and elevated
N. Pandey, S. Pandey-Rai

artemisinin content by 54.9 % in one of the transgenic line as
compared to wild-type plants.
Regulation of artemisinin biosynthesis through stress
signals
Various environmental stresses in a moderate dose and dura-
tion can act as a potential elicitor for enhanced secondary
metabolite production. When plants recognize stress signals
at cellular level, a stress response is induced. A number of
researchers have exposed A. annua to a number of stress sig-
nals and had reported alterations in artemisinin content along
with transcript level of its biosynthetic genes which is sum-
marized in Table 1. Environmental stress signals such as tem-
perature (low or high), salinity, water (drought or flooding),
radiations, chemical, mechanical and various kinds of biotic
stresses often result in induced accumulation of
phenylpropanoids (Dixon and Paiva 1995). In recent years,
many of them have been explored in terms of their potential
to boost artemisinin level in A. annua. Chilling has been dem-
onstrated to affect artemisinin content in A. annua.Yangetal.
(2010) had revealed chilling-mediated induction in
artemisinin level along with its biosynthetic genes such as
ADS,CYP and DXS. Similarly, night frost also increased
artemisinin content (Wallaart et al. 2000). Influence of salinity
on artemisinin accumulation has been investigated by differ-
ent groups. Qureshi et al. (2005) showed that salt treatment
(0–160 mM) increased artemisinin content during the early
phase of plant growth, possibly due to sudden non-
enzymatic conversion of artemisinic acid/DHA (artemisinin
precursors) in to artemisinin under increasing oxidative stress.
However, at later stages, it reduced artemisinin. In contrast,
when A. annua seedlings were subjected to salinity stress (4–
6 g/l NaCl) significantly boosted artemisinin (2–3%dry
weight) compared to non-treated plants (1 % dry weight)
(Qian et al. 2007). Role of nutrient deficiency in artemisinin
production was first reported by Ferreira (2007). Higher level
of artemisinin was found in plants grown in potassium defi-
ciency as compared to plants grown with full supplementation
of macronutrients and had suggested that artemisinin produc-
tion per hectare may be enhanced by growing A. annua under
a mild potassium deficiency. In contrast, supplementation of
few elements reportedly enhances artemisinin content. Mild
boron stress (1.0 mM) enhanced artemisinin as compared to
control (Aftab et al. 2010). Similarly, arsenic (As) (3000 μg/l)
for 7 days and cadmium (Cd) treatment (20 and 100 μmol/l) to
Tabl e 1 Different stress signals for enhanced artemisinin biosynthesis in-planta
Stress signals for artemisinin enhancement Experiments conducted on Reference
Chilling Soil-grown plants/In vitro propagated plants Yang et al. 2010;Luluetal.2008
Night frost Soil-grown plants Wallaart et al. 2000
Salinity Soil-grown plants Qian et al. 2007; Qureshi et al. 2005
Potassium deficiency Soil-grown plants Ferreira 2007
Boron Soil-grown plants Aftab et al. 2010
Arsenic Soil grown plants/hydroponic culture Rai et al. 2011a; Paul and Shakya 2013
Phosphorus Soil grown plants Kapoor et al. 2007
Cadmium Hydroponic culture Li et al. 2012
Drought Soil-grown plants Marchese et al. 2010
Post harvest drying Soil grown plants Laughlin 2002
Miconazole Hydroponic culture Towler and Weathers 2007
UV-B In vitro propagated plants Pandey and Pandey-Rai 2014a
UV-C Soil-grown plants Rai et al. 2011b
Jasmonic acid Exogenous application to soil-grown plants Wang et al. 2009;Maesetal.2011
Salicylic acid Exogenous application to soil-grown plants Pu et al. 2009; Guo et al. 2010
Phytohormones (GA3+BAP+ ABA) Soil-grown plants Maes et al. 2011
Abscisic acid (alone) Pot culture of in vitro germinated seedlings Jing et al. 2009
Sugars In-vitro propagated plants Wang and Weathers; Arsenault et al. 2010b
Methyl jasmonate (MeJA)+Miconzole Cell suspension culture Caretto et al. 2011
MeJA (alone) Hairy root culture Patra et al. 2013
Oligosaccharide elicitor (OE) of fungal origin Hairy root culture Zheng et al. 2008;Wangetal.2006; Wang et al. 2009
Yeast extract+MeJA+Chitosan Hairy root culture Putalun et al. 2007
MeJA+ cell homogenate of Piriformospora indica Hairy root culture Ahlawat et al. 2014
Artemisinin: modes of action and its enhanced global production

hydroponically grown A. annua promoted synthesis and ac-
cumulation of artemisinin (Rai et al. 2011a;Lietal.2012).
RT-PCR analysis revealed upregulated transcript levels of
HMGR,FDS,ADS and CYP71AV1 genes under arsenic stress
(Rai et al. 2011a). In tune with boron and As, phosphorus also
enhanced artemisinin level (Kapoor et al. 2007). Not all the
metal stress signals affect artemisinin positively. A re-
cent study by Paul and Shakya (2013) has demonstrated
that As(III) (at 5 and 7.5 μg/ml) and NaCl favour
growth of A. annua and artemisinin biosynthesis while
higher doses of As(III) (10 μg/ml) and Cr(VI) even at
mild doses have adverse effects on growth and
artemisinin level (Paul and Shakya 2013).
There are reports in support of drought stress affecting
artemisinin accumulation (Marchese et al. 2010). Water deficit
of 38 h (W=−1.39 MPa) significantly increased both leaf and
plant artemisinin up to 29 % without causing any serious harm
to biomass production. This suggests that pre-harvest water
deficit may reduce time and costs in crop drying along with
gain in artemisinin content. Further, different ways of post-
harvest drying of crops also play role in overall artemisinin
availability. Field crop drying is beneficial over artificial dry-
ing (such as oven-drying) and gives higher artemisinin; how-
ever, mode of post-harvest drying does not seem to affect
artemisinic acid level (Laughlin 2002). The exact mechanism
behind such abiotic factor-mediated elevation in artemisinin is
still not clear. However, a common factor behind these stresses
is the ROS-mediated oxidative stress which may probably be
one of the reasons for greater artemisinin accumulation.
Though there are many reports of deciphering roles of abi-
otic stress signals in influencing artemisinin content in
A. annua, relatively fewer work have been conducted to depict
artemisinin regulation through biotic stress signals. A study by
Kapoor et al. (2007)showedthatinoculationbytwo
arbuscular mycorrhizal fungi, Glomus macrocarpum and
Glomus fassiculatum, significantly enhanced artemisinin con-
centration density of leaf glandular trichomes (GT) which are
the accumulation sites of artemisinin. They also observed a
strong positive linear correlation between leaf GT density and
artemisinin, suggesting that certain biotic stress signals may
be potential tools to improve in-planta improvement of
artemisinin synthesis and accumulation. Few more biotic elic-
itors have been demonstrated to improve artemisinin but in
hairy root culture which will be discussed later in this section.
Exogenous application of signalling molecules such as
jasmonic acid (Wang et al. 2009;Maesetal.2011)and
salicylic acid (Pu et al. 2009; Guo et al. 2010) also has role
in boosting biosynthesis of artemisinin probably due to in-
duced burst of ROS and subsequent conversion of DHA in
to artemisinin as well as upregulation of key artemisinin bio-
synthetic genes. In addition, few phytohormones such as
gibberellic acid cytokinins (BAP) and abscisic acid also boost
artemisinin level (Maes et al. 2011;Jingetal.2009).
Elicitation and plant cell/tissue culture
Plants respond quickly to even a small fluctuation in its phys-
ical or chemical environment. These fluctuations are very well
known to elicit the plant’s secondary metabolism and hence
are exploited to get the desired secondary metabolite. Plant
tissue culture offers an opportunity to grow plants in vitro in
a controlled environment and manipulation of plant physical
and chemical environment to get the desired product. Rapidly
increasing world population is putting more and more pres-
sure on availability of cultivation land. Therefore, in the pres-
ent scenario, there is a need to explore plant cell/tissue culture
technique in integration with elicitation to get higher second-
ary metabolite production with minimum land usage. This will
reduce the land use by medicinal plants which are largely
grown for production of pharmaceuticals and other phyto-
chemicals and better utilization of lands for various other
motives.
There are established standard protocols for in vitro prop-
agation of A. annua. It can easily be propagated through
micro-cuttings in MS media. Since accumulation of secondary
metabolites in plants in response to abiotic factors is a com-
mon phenomenon, manipulation of abiotic environment of
in vitro propagated A. annua plantlets may lead to differential
accumulation of artemisinin. We applied this hypothesis in our
recent work and had demonstrated that UV-B radiation expo-
sure (3 h) to in vitro propagated A. annua plantlets can double
the artemisinin content (Pandey and Pandey-Rai 2014a)with-
out causing any serious damage to physiology including pho-
tosynthesis and hence survival (Pandey and Pandey-Rai
2014b). Chilling (4 °C) treatment for 30 min also affected
artemisinin content as well as transcript level of ADS (11-
fold) and CYP (7-fold) genes in in vitro cultured plants as
revealed by HPLC and qPCR analysis, respectively (Lulu
et al. 2008). Different sugar supplementation during in vitro
plantlet development also affects artemisinin content in plants.
Seedlings were inoculated in Gamborg’sB5mediumcontain-
ing3%(w/v) sucrose, glucose or fructose. Fructose inhibited
artemisinin synthesis while seedlings growing in 3 % glucose
produced more artemisinin (Wang and Weathers 2007). These
results were further confirmed by Arsenault et al. (2010)
through expression analysis of biosynthetic genes as well as
quantification of artemisinin and its various derivatives.
Various efforts have been made to get higher artemisinin
through integrated approach of elicitation with hairy root or
cell suspension culture in A. annua. Among these, hairy root
culture seems advantageous over cell suspension culture in
terms of high growth rate, better genetic stability and indepen-
dency of hormone supplemented media requirement for
growth (Guillon et al. 2006). Plant signalling compounds,
precursors, inhibitors and various other kinds of molecules
have been explored in terms of their potential to elicit
artemisinin biosynthesis. Caretto et al. (2011) investigated
N. Pandey, S. Pandey-Rai

efficacy of methyl jasmonate (MeJA) and miconazole on
artemisinin biosynthesis in cell suspension cultures of
A. annua. Three-fold increment in just 30 min of MeJA
(22 μmol) treatment and 2.5-fold after 24 h of miconazole
treatment were observed. However, miconazole treatment se-
verely affected cell viability. Earlier, this antifungal compound
miconazole significantly induced artemisinin level in
A. annua seedlings (Towler and Weathers 2007).
Miconazole-mediated induction in artemisinin is due to its
ability to inhibit sterol biosynthesis and redirection of carbon
flux towards terpenoid pathway (Zarn et al. 2003). Using
MeJA alone (40 μg/l, 15 days) as elicitor, 3.45 mg/g
artemisinin was produced (Patra et al. 2013). Nitric oxide
(NO) also stimulates artemisinin synthesis in hairy root cul-
ture. Zheng et al. (2008) studied that NO generation induced
by an oligosaccharide elicitor (OE) from Fusarium oxysporum
mycelium elevated artemisinin from 0.7 to 1.3 mg/g DW by
treating 20-day-old hairy roots with OE for 4 days. Further,
combined treatment of OE with NO donor sodium nitroprus-
side increased artemisinin from 1.2 to 2.2 mg/g DW. Various
other elicitors have been reported to stimulate artemisinin bio-
synthesis in hairy root culture such as OE from Colletotrichum
gloeosporioides (Wang et al. 2006), cerebroside (Wang et al.
2009), yeast extract, methyl jasmonate and chitosan (Putalun
et al. 2007). Very recently, in an interesting approach of com-
bined application of abiotic and biotic elicitor (MeJA and cell
homogenate of Piriformospora indica) to hairy root culture,
2.44 times greater artemisinin was obtained (Ahlawat et al.
2014). This increase was probably due to upregulation of bio-
synthetic genes. They also reported for the first time a positive
correlation between elicitor application and expression of bio-
synthetic pathway genes viz. HMGR,ADS,CYP71V1,ALDH1,
DXR,DXS and DBR2 in hairy root cultures of A. annua.
Combined approaches utilizing in vitro techniques and
elicitation is a promising idea for large-scale production of
artemisinin through bioreactor cultivation technology.
Breeding approaches for enhanced artemisinin
production
According to the factsheets on the World Malaria Report 2014,
issued by WHO (December 2014), in the year 2013, there
were an estimated 198 million cases of malaria (uncertainty
range 124–283 million) and an estimated 584,000 deaths (un-
certainty range 367,000–755,000) in the world, of which 90 %
deaths occur in Africa. Further, the second most affected part
of the world is South-East Asia where India has the highest
malaria burden, followed by Indonesia and Myanmar. These
facts suggest that major risk and burden of this disease is inthe
developing countries which are generally with insufficient ad-
vanced infrastructure/techniques. Therefore, despite of various
efforts to produce high artemisinin by diverse means, there is
still a need to think on ground level as well because
agricultural production is not a problem or limiting factor in
developing countries. Integrating cultivation of A. annua with
different breeding techniques may be a viable alternative to
produce more artemisinin thereby aiding in overall enhanced
production of artemisinin globally. By bringing Artemisia
annua into cultivation, conventional and new biotechnological
plant-breeding techniques can be applied at the genetic level to
improve yield and uniformity with stable high performing
phenotypes across adverse/variable environments and to mod-
ify desired valuable compounds. Conventional plant breeding
practices include collection/creation of population or germ-
plasm with useful desired genetic variation, identification of
superior individuals and development of improved variety
from selected individuals. Success of conventional breeding
is dependent on the selection process. Numerous selection
methods can be adopted for A. annua. Mass selection is de-
pendent mainly on selection of plants according to their phe-
notypes and performance and can be used to improve the
overall population of A. annua by positive or negative mass
selection. One drawback of mass selection is the influence of
the environment on the development, phenotype and perfor-
mance of single plants. Another method is the recurrent selec-
tion, which is more suitable for the A. annua as it is cross-
pollinating species. In this method, the seed from selected
plants is not added together but is kept apart and used to
perform offspring tests. Through different selection strategies,
certain high yielding varieties of A. annua such as ‘CIM-
Arogya’Jeevan Raksha’and Asha were released by Central
Institute for Medicinal and Aromatic Plants (CIMAP), India,
as superior lines rich in artemisinin (Patra and Kumar 2005).
Recently, Townsend et al. (2013) have reported that selection
of material for breeding using combining ability analysis of a
diallele cross can be used for the identification of elite parents
to produce improved A. annua hybrids. This selection method
was reportedly consistent with advanced QTL-based molecu-
lar breeding approaches.
Modernization of plant breeding using different biotechno-
logical tools is opening new avenues for crop improvement.
Two such tools are mutation and molecular breeding (Fig. 4).
Mutation breeding involves induced mutation through
chemicals or radiation with the advantage of improving one
or two characters without modifying the rest of genotypes.
A successful breeding effort was made by Mediplant (a
swiss not-for-profit organization) by developing high-
yielding F1 hybrid populations of A. annua known as
‘Artemis’with mean annual artemisinin production of about
32 kg/ha. Further, they also created new high yielding hybrids
with 40.5–52.0 kg/ha artemisinin (Simonnet et al. 2008).
There has been significant progress in the development of
biotechnological plant-breeding techniques using various mo-
lecular tools. Different DNA markers (for example, RFLP,
RAPD, AFLP, SSR, SNP, STMS etc) and functional markers
(such as EST, microarray and qRT-PCR) can be variously
Artemisinin: modes of action and its enhanced global production

used to speed up the selection for recognition of desired ge-
notypes at an early stage. Although these marker-assisted se-
lections are being applied in various crops, there are relatively
few studies of molecular marker-based approaches in medic-
inal plants. One such effort was made by CIMAP (India) for
developing high-artemisinin variety CIM-arogya, through
marker-assisted selection breeding.
For the production of high-yield varieties of A. annua,afast
track molecular breeding project known as ‘The CNAP
Artemisia Research Project’has been initiated at the Centre
for Novel Agricultural Products (CNAP) which is a part of
university of York. This project led by CNAP’s Director
Dianna Bowles and Deputy Director Ian Graham was funded
by Bill & Melinda Gates Foundation. With the aim to produce
high yielding non-GM variety of A. annua, over 23,000 pa-
rental lines were screened for desired traits and 768 different
hybrid crosses were made, of which 268 most promising hy-
brids were forwarded to field trials. After rigorous selection
procedure, two best performing hybrids viz. Hyb1209r
(Shennong) and Hyb8001r (Zenith) with 36.3 and 54.5 kg/ha
artemisinin have been commercially released. Using various
molecular markers, they also developed the genetic map of
A. annua with nine linkage groups (Graham et al. 2010). For
this, they used the Artemis (high yielding variety) pedigree and
established genetic linkage and QTL maps. Positive QTL re-
lated to artemisinin yield were also independently validated.
Conclusion and future directions
Various approaches have been adopted for improvement in the
yield of artemisinin. However, it is still a challenge for any
individual approach alone to meet the global demand of
artemisinin. Though recent achievement of semi-synthetic
production of artemisinin by Keasling’s group is a major
breakthrough in bringing artemisinin production at industrial
level, there still exist many hurdles for sustainable production
of this drug to be used in ACTs. Its insecure supply leads to
price hike and to reduce the drug cost there is a need to speed
up the artemisinin production worldwide.
Therefore, in-planta yield enhancement through various
ways as discussed in this review remains the prime focus for
sustainable drug supply. It is worth mentioning that the all
Fig. 4 Different proposed
breeding approaches in A. annua
for enhanced production of
artemisinin
Fig. 5 Intercropping model with special reference to Indian cropping for
growing A. annua
N. Pandey, S. Pandey-Rai

transgenic or non-transgenic tools for enhancement of
artemisinin biosynthesis can be more fruitful if implemented
on high yielding chemotypes/hybrids of A. annua. Integration
of conventional methods with advanced techniques will serve
to extend and enhance the continued production of drug. As
discussed earlier, more than 90 % burden of malaria is in
African and South-East Asian developing countries. As ma-
jority of these countries have agricultural economies, agri-
based enhancement of artemisinin production in-planta can
be a promising approach for overall improved production.
Various agricultural strategies can be posited depending on
the local scenario of agricultural practices in different coun-
tries. Deficiency in the supply chain of artemisinin can be
remediated by integrating agri-based technologies with mo-
lecular tools for affordable drug production. As for example
in India, we hope that intercropping of high yielding varieties
of A. annua (developed through molecular/mutation breeding)
with staple food crops can be good option for its cultivation
(Fig. 5) without engaging additional cultivable land and mean-
time execution of multiharvesting and advanced drug extrac-
tion practices may result in greater artemisinin production
with lower production cost. Similar approaches may also be
applied in other countries as well which can give a further
boost to artemisinin production at the same time bettering life
standards of their farmers.
Keeping in mind, benefits and limitations associated with
every yield enhancement strategies, we emphasize that to
bring artemisinin supply up to the level of its demand requires
its production collectively through semi-synthesis, in-planta
yield enhancement by both transgenic and non-transgenic
methods as well as modern breeding.
Conflict of interest The authors (NP and SPR) of the review article
entitled BUpdates on Artemisinin: an insight to mode of actions and
strategies for enhanced global production^have no conflict of interest.
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