Article on bioactives in plants in China.
Science Bulletin 61(1) · November 2015 .


Yang, Lei & Yang, Changqing & Chenyi, Li & Zhao, Qing & Liu, Ling & Xin, Fang & Chen, Xiao-Ya. (2015). Recent advances in biosynthesis of bioactive compounds in traditional Chinese medicinal plants. Science Bulletin. 61. 10.1007/s11434-015-0929-2.
DOI: 10.1007/s11434-015-0929-2;

Recent advances in biosynthesis of bioactive compounds
in traditional Chinese medicinal plants.
Lei Yang •Changqing Yang •Chenyi Li •
Qing Zhao •Ling Liu •Xin Fang •Xiao-Ya Chen
Received: 24 August 2015/Accepted: 15 October 2015
ÓScience China Press and Springer-Verlag Berlin Heidelberg 2015. This article is published with open access at Springerlink.com
Abstract Plants synthesize and accumulate large amount
of specialized (or secondary) metabolites also known as
natural products, which provide a rich source for modern
pharmacy. In China, plants have been used in traditional
medicine for thousands of years. Recent developmentof
molecular biology, genomics and functional genomics as
well as high-throughput analytical chemical technologies
has greatly promoted the research on medicinal plants. In
this article, we review recent advances in theelucidation of
biosynthesis of specialized metabolites in medicinal plants,
including phenylpropanoids, terpenoids and alkaloids. Th-
ese natural products may share a common upstream path-
way to form a limited numbers of common precursors, but
are characteristic in distinct modifications leading tohighly
variable structures. Although this review is focused on
traditional Chinese medicine, other plants with a great
medicinal interest or potential are also discussed.Under-
standing of their biosynthesis processes is critical for pro-
ducing these highly value molecules at large scale and low
cost in microbes and will benefit to not only human health
but also plant resource conservation.
Keywords Medicinal plant Biosynthesis 
Phenylpropanoid Terpenoid Alkaloid
1 Introduction
China is rich in plant resources. Of the *300,000 species
of higher plants on the earth, around 10% can be found in
China. As in many other countries, people in China have
used plants for treatment of diseases for thousands of years.
Compendium of Materia Medica has been held in high
esteem since it was first published in 1593, and this ancient
encyclopedia of traditional Chinese medicine (TCM)
described more than 1,000 species of plants. Plants produce
a wealth of specialized (or secondary) metabolites also
known as natural products, which are small molecular
weight compounds with enormous structural diversity and
show various biological activities. It is estimated that there
are approximately 200,000 secondary metabolites in plant
kingdom [1], which, based on biosynthetic origins, can be
classified into three major categories: phenylpropanoids,
terpenoids and alkaloids, plus a few other less abundant
groups. The usage records of China’s ancient medical
books, such as Sheng Nong’s Herbal Classic,Huang Di’s
Canon of Medicine and Compendium of Materia Medica,
already recognized that plant extracts contain active prin-
ciples in treating illness and classified them into assump-
tive, intuitive or largely philosophic categories, such as
cold, neutral or hot, toxic or nourishing. Over the past
century, hunting the active ingredients has led to important
findings, such as artemisinin for malaria, huperzine A for
Alzheimer’s disease, ephedrine for cold and camptothecin
SPECIAL TOPIC: Advances in Artemisinin Study
L. Yang L. Liu X.-Y. Chen (&)
Plant Science Research Center, Shanghai Chenshan Botanical
Garden, Shanghai Key Laboratory of Plant Functional Genomics
and Resources, Shanghai 201602, China
e-mail: xychen@sibs.ac.cn
C. Yang C. Li Q. Zhao X. Fang X.-Y. Chen
National Key Laboratory of Plant Molecular Genetics and
National Center for Plant Gene Research, Institute of Plant
Physiology and Ecology, Shanghai Institutes for Biological
Sciences, Chinese Academy of Sciences, Shanghai 200032,
China
C. Li
University of Chinese Academy of Sciences, Beijing 100049,
China
123
Sci. Bull.www.scibull.com
DOI 10.1007/s11434-015-0929-2www.springer.com/scp



for cancer, which were isolated from Artemisia annua,
Huperzia serrata,Ephedra sinica,Camptotheca acumi-
nate, respectively [2]. Very recently, tetrandrine, an alka-
loid isolated from the TCM plant Stephania tetrandra
previously used for reducing blood pressure, were reported
to have the therapeutic efficacy against Ebola [3], and
celastrol, a triterpene extracted from Tripterygium Wil-
fordi, has the potential as an anti-obesity agent [4]. These
findings strongly support that TCMs are the reliable source
for new therapies in treatment of lethally epidemic disease
and long unsolved disease.
However, multi-classes of natural products are generated
by each plant species. In addition, geographic distributions,
growth conditions and harvesting seasons could significantly
affect chemical compositions of the plant. Whereas one
component may act as the active ingredient, the effects of a
mixture of many ingredients are often uncertain and this has
caused increasing concerns [5]; thus, the traditional practice
of herbology has to face the challenges from modern medi-
cine and the manufactures’ requirement.
While plant natural products continue to be a prime source
for drug discovery and development, supply of these com-
pounds is often curtailed due to limitation of natural
resources and/or low contents in plant. The biotechnological
platforms, such as metabolic engineering of effective plant
and microbial production, are urgently needed to ensure that
the supply of bioactive natural products is sustainable and
environmentally friendly, rather than at the expense of
resource exhaustion [6–9]. A prerequisite to these solutions
is the understanding of the biosynthetic pathways of these
specialized metabolites, in particular the cloning and iden-
tification of enzymes and the regulatory factors.
In the past two decades, the rapid development in
genomics and high-throughput technologies of chemical
analysis, in combination with molecular biology tools, has
accelerated the research of medicinal plants. In this review,
we summarize the recent advances in the elucidation of
biosynthetic pathways of secondary metabolites in, not
exclusively, TCM plants. Although alkaloids are probably
the most important resource for drug discovery and
biosynthesis of these amino acid-derived compounds has
been investigated intensively, there are, surprisingly to
some extent, relatively few studies of alkaloids fromTCM
plant; thus, this review is emphasized on phenylpropanoids
and terpenoids. In addition to enzymes, transcription fac-
tors characterized from medicinal plants are also discussed.
2 Phenylpropanoids
Phenylpropanoids, commonly found in plants, are derived
from the six-carbon aromatic phenyl group and the three-
carbon propene tail [10], and form a large group of special-
ized metabolites including monolignols, lignans, flavonoids,
phenolic acids and stilbenes [11]. They serve as basic com-
ponents of a number of structural polymers, as well as floral
pigments, scent compounds or signaling molecules to
mediate bio-interactions, phytoalexins against herbivores
and pathogens, and protective components against ultravi-
olet light radiation and other abiotic stresses [12]. In many
TCM plants, such as the plants of Lamiaceae, Fabaceae
(Leguminasae) and Asteraceae, phenylpropanoids are also
the bioactive principles (Table1), which have been shown to
act as anti-oxidants, free radical scavengers, anti-inflam-
matories and anticancer compounds [13].
The majority of phenylpropanoids are derived from
phenylalanine. The first three steps are catalyzedby
phenylalanine ammonia lyase (PAL), cinnamate
Table 1List of examples of TCM plants rich in phenylpropanoids
Plant speciesChinese name in Pin-yinFamilyRepresentative compounds
Salvia miltiorrhizaDanshenLamiaceaeSalvianolic acid A, B and C
Scutellaria baicalensisHuangqinLamiaceaeBaicalin, wogonin, scutellarin
Glycyrrhiza uralensisGancaoLeguminosaeLiquiritin, isoliquiritin, 7,40-dihydroxyflavone
Astragalus membranaceusHuangqiLeguminosaeCalycosin-7-glucoside, ononin
Sophora flavescensKushenLeguminosaeSophoraflavecromane A, B, C
Sophora tonkinensisShandougenLeguminosaeSophoranone, sophoradin
Pueraria lobataGeLeguminosaePuerarin, daidzin, genistein
Lonicera japonicaJinyinhuaCaprifoliaceaeChlorogenic acid, luteolin
Dendranthema morifoliumJuhuaAsteraceaeChlorogenic acid, acacetin-7-O-b-D-glucoside,
apigenin-7-O-b-D-glucoside, and
luteolin-7-O-b-D-glucoside
Ginkgo bilobaYinxingGinkgoaceaeGinkgetin, isoginkgetin
Epimedium brevicornuYinyanghuoBerberidaceaeIcariine, icarisid
Isatis indigoticaSonglanBrassicaceae Lariciresinol
Sci. Bull.
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4-hydroxylase (C4H) and p-coumaroyl coenzyme A ligase
(4CL), which are commonly referred as ‘‘general phenyl-
propanoid pathway’’ [14,15]. The product of 4CL is used
as precursor for the biosynthesis of various phenyl-
propanoids in plants (Fig.1). Parts of phenylpropanoids are
synthesized from L-tyrosine, and the transformation is more
restricted, being mainly limited to members of several
families. For instance, 3,4-dihydroxyphenyllactic acid, one
precursor of rosmarinic acid, is synthesized from tyrosine-
derived pathway in some species of Lamiaceae, such as
Salvia miltiorrhiza [16,17].
2.1 Flavonoids
Flavonoids constitute a highly diverse class of secondary
metabolites composed of more than 9,000 structures [18].
They are commonly found in land plants, including all
vascular plants and some mosses [19]. Based on the agly-
cone core, they are generally further grouped into fla-
vanones, flavones, flavonols, isoflavonoids, anthocyanins
and proanthocyanidins. All flavonoids are basically
derivatives of 1,3-diphenylpropan-1-one (C6–C3–C6),
which is derived from the condensation of three malonyl-
CoA molecules with one p-coumaroyl-CoA to form a
chalcone intermediate [20]. Chalcone isomerase converts
chalcone into flavanones, and respective enzymes trans-
form flavanones to various flavones, isoflavones, dihydor-
flavonols, flavonols and anthoanidins. Every class of
flavanones possesses the compounds with pharmaceutical
activity and is widely used in folk medicines [21].
2.1.1Flavanones and flavones
Two completely different flavone synthase (FNS) proteins
have been found to catalyze the biosynthesis of flavones in
plants. The first member of the FNS I type was identified
from parsley (Petroselinum crispum) cell suspension cul-
tures and classified as 2-oxoglutarate-dependent dioxyge-
nase [22]. The FNS I cDNA was then cloned and
functionally expressed in yeast [23], and it shares a high
sequence identity to the flavanone 3-b-hydroxylase (FHT).
Interestingly, characterized FNS I enzymes appear to be
mainly in the family of Apiaceae [18,24]. Molecular and
phylogenetic analysis revealed a gene duplication of FHT,
and a subsequent neofunctionalization occurred early in the
development of the Apiaceae subfamilies [25]. Formation
of most flavones in a wide range of plant species is cat-
alyzed by FNS II, cytochrome P450 proteins of CYP93B
subfamily. The FNS II activity was first demonstrated in
extract of Antirrhinum majus flowers [26], and the cDNAs
were then isolated from other plants, including Perilla
frutescens (CYP93B6) [27] and Gentiana triflora [28].
Glycyrrhiza uralensis is one of the most popular TCM
plants and also widely used in food flavoring. Although the
sweeting agent of this plant is glycyrrhizin, a triterpenoid
saponin [29], flavanones and flavones are also important
components in its root, which include liquiritigenin,
isoliquiritigenin and 7,40-dihydroxyflavone [30]. A P450
enzyme from Glycyrrhiza echinata, CYP93B1, was
Fig. 1(Color online) Biosynthesis of phenylpropanoids in TCM
plants. aBiosynthesis of flavonoids and isopentenyl flavonoids;
bformation of phenolic acids from the L-phenylalanine- and the L-
tyrosine-derived pathways in Salvia miltiorrhiza, a medicinal plant of
Lamiaceae. Phenylpropanoids are mainly synthesized from pheny-
lalanine via the ‘‘general phenylpropanoid pathway’’, catalyzed by
phenylalanine ammonialyase (PAL), cinnamate 4-hydroxylase (C4H)
and p-coumaroyl coenzyme A ligase (4CL). The product of
p-coumaroyl-CoA is used for the biosynthesis of flavonoids, isopen-
tenyl flavonoids and phenolic acids. CHS, chalcone synthase; CHI,
chalcone isomerase; FNS, flavone synthase; IFS, isoflavone synthase;
FPT, flavonoid prenyltransferase; TAT, tyrosine aminotransferase;
HPPR, 4-hydroxyphenylpyruvate reductase; RAS, rosmarinic acid
synthase; P450, cytochrome P450 monooxygenase. Dotted lines
represent multiple enzymatic catalyzed steps
Sci. Bull.
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identified as flavanone 2-hydroxylase (F2H), a member of
FNS II [31]. The products, 2-hydroxyflavanones, were
transformed into flavones in vitro in acid treatment, sug-
gesting that an additional enzyme, probably a dehydratase,
was involved in catalyzing the formation of flavones. A
full-length cDNA of cytochrome P450 CYP93C2 was iso-
lated from the elicited G. echinata cells, which was shown
to encode 2-hydroxyisoflavanone synthase [32].
The flavones baicalin and wogonoside, as well as their
aglycones baicalein and wogonin, represent the dominant
flavonoids in Scutellaria baicalensis, a perennial species of
Lamiaceae and an important herb in Chinese traditional
and clinical-orientated medicine. The flavones, such as
baicalin and wogonin, are distinct for lacking a 40-OH
group but having a 6-OH group on their A-ring [33]. Genes
encoding the upstream enzymes of the pathway, including
PAL, C4H, 4CL, chalcone synthase (CHS) and chalcone
isomerase (CHI), have been isolated [34,35]. However, the
enzymes committed to the formation of the S. baicalensis-
type flavones remain unknown. It is also possible that
specific enzyme isoforms are involved in the formation of
cinnamoyl-CoA [36]. It has been reported that accumula-
tion of these flavones was enhanced by jasmonate (JA)
treatment, and a R2R3-MYB transcription factor,
SbMYB8, was found involved in the regulation [37,38].
2.1.2 Isoflavones
The isoflavones are well studied for their substantial health
promoting benefits. They are found mainly in leguminous
plants and are the major bioactive ingredients in soybean,
Astragalus,Pueraria lobata [39]. Isoflavones are converted
from flavanones by the isoflavone synthase (IFS). By using
EST-based approach combined with enzymatic assays,
P450s of CYP93C subfamily from soybean were shown to
have such activities [40,41]. Members of this subfamily with
IFS activity were also reported in other leguminous plants,
such as Lotus japonicus [42] and Trifolium pratense [43].
Astragalus membranaceus, a species of Fabaceae, has
been used in TCM for thousands of years. Astragaus is
considered an adaptogen because it is believed to help
protect the body against stresses, including those of phys-
ical, mental or emotional [44,45]. In China, Astragalus has
been used to help patients with severe forms of heart dis-
ease in relieving symptoms, lowering cholesterol levels and
improving heart function. Constituents of the Astragalus
roots (radix astragali) include polysaccharides, triter-
penoids (astragalosides) and isoflavones [46,47]. Iso-
flavones such as calycosin-7-glucoside and ononin are
considered the important active components in this medi-
cine. Hairy root system of Astragalus was developed a long
time ago to produce these ingredients [48,49]. Research at
molecular level in this plant is limited, but will help reveal
the biosynthetic pathway in this leguminous medicinal
plant [50].
Pueraria lobata, also a species of Fabaceae, is com-
monly known as ‘‘kudzu’’. Puerariae radix, the dried root
of the kudzu, has been used in China as herbal medicine for
the prevention of cardiovascular disease and rehabilitation
of stroke patients [51]. The major secondary metabolites
accumulated in kudzu roots are isoflavones, including
daidzein, genistein, formononetin and their glucosides
Puerarin [52], among which the 8-C-glucoside of daidzein
is considered the major active compound [53]. The co-
occurrence of both O — and C-linked glycosides in root is of
particular interests and worthy of further investigation.
Using a functional genomics approach, He et al. identified
enzymes associated with the isoflavone biosynthesis in
kudzu roots, including 15 UDP-dependent glycosyltrans-
ferases (UGTs), among which one, GT04F14, exhibitedthe
in vitro activity of glycosylation of a wide range of sub-
strates, including coumarins, flavones, flavonols, and iso-
flavones. The isoflavones are converted region-specifically
to their 7-O-glucosides, whereas C-glycosylation might
take place at the 2,7,40-trihydroxyisoflavanone precursor of
daidzein, rather than directly on daidzein. Conceivablythe
intermediate 8-C-b-glucopyranosyl-2,7,40-trihydroxy-
isoflavanone is converted to puerarin under in vivo con-
ditions by the action of 2-hydroxyisoflavanone dehydratase
(HID). A candidate gene encoding HID was identified from
the EST library of kudzu root [54]. In addition, a partially
purified preparation from kudzu root was shown to have
the C-glucosyltransferase activity that converted isoliquir-
itigenin (20,40,4-trihydroxychalcone) and UDP-Glc to
puerarin [55].
2.1.3Isopentenyl flavonoids
Prenylation, the addition of prenyl groups, contributes to
the diversification of flavonoids, and the occurrence of
more than 1,000 prenylated flavonoids in plants has been
recorded [56]. This prenylation represents the coupling
process of the aromatic moiety from shikimate pathway
and the prenyl (isoprenoid) chain from the isoprenoid
pathways. Many prenylated flavonoids were identified as
active components in medicinal plants and thus are of
particular interests as lead compounds for drugs and
functional food ingredients [57].
Species Sophora, family Fabaceae, are widely dis-
tributed in Asia. Sophora flavescens has a long history of
use in China, and the root, known as Ku Shen, is a typical
TCM. It is used to dispel heat, dry dampness and eliminate
intestinal parasites. It is thus administered in formulas for
the treatment of dysentery and jaundice (damp-heat syn-
dromes), edema and dysuria (dampness syndromes), and
eczema and pruritis (damp-heat-wind syndromes). The S.
Sci. Bull.
123



flavescens prenyltransferase SfN8DT-1 is the first enzyme
identified to be responsible for the prenylation of narin-
genin at the 8-position, with dimethylallyl diphosphate
(DMAPP) as substrate [58]. Later, two new flavonoid
prenyltransferases (FPTs) were isolated from S. flavescens
at the molecular level: one is the isoflavone-specific
prenyltransferase (SfG6DT) for the prenylation of the
genistein at the 6-position and the other a chalcone-specific
prenyltransferase designated as isoliquiritigenin dimethy-
lallyltransferase (SfiLDT) [29].
Herba epimedii is prepared from the aerial parts of
Epimedium brevicornum or Epimedium sagittatum, species
of Berberidaceae. Herba epimedii contains various bioac-
tive components and has been utilized extensively in China
as the tonic and anti-rheumatic herb for thousands of years,
and in the treatments of diseases such as impotence, fre-
quency/urgency of urination, coronary heart disease,
chronic bronchitis and neurasthenia [59,60]. The isopen-
tenyl flavonoids icariine and icarisid are the major active
compounds [61]; however, their biosynthesis remains
poorly understood [62]. Recently, Huang et al. isolated 12
structural genes and two putative transcription factors
(TFs) in the flavonoid pathway. Transcriptional analysis
revealed that two R2R3-MYB TFs (EsMYBA1 and
EsMYBF1), together with a bHLH TF (EsGL3) and WD40
protein (EsTTG1), are probably involved in coordinated
regulation of biosynthesis of the anthocyanins and the
flavonol-derived bioactive components [63].
2.2Phenolic acids
Salvia miltiorrhiza is a perennial herb in the mint family
(Lamiaceae). Its dried root or rhizome is called Danshen in
TCM and was recorded in first pharmaceutical monograph
Shennong’s Classic of Materia Medica (A.D. 102-200). S.
miltiorrhiza has been cultivated throughout Eastern Asia
and used to prevent and cure cardiovascular, cerebrovas-
cular, hyperlipidemia and acute ischemic stroke diseases
[64]. Both the hydrophilic and lipophilic components in S.
miltiorrhiza are considered active ingredients. The hydro-
philic compounds are mainly phenolic acids including
rosmarinic acid, salvianolic acid B, lithospermic acid and
dihydroxyphenyllactic acid or Danshensu, and they may
also function as antioxidative, anti-bacterial and anti-viral
reagents [65,66].
The biosynthetic pathway for phenolic acids in S. mil-
tiorrhiza is distinct and has attracted many interests.
Labeling experiments using [ring-(13)C]-phenylalanine
suggested two intermediates derived from the phenylala-
nine-derived general phenylpropanoid pathway and the
tyrosine-derivedpathway,respectively(Fig. 1): 4-cou-
maroyl-CoA and 3,4-dihydroxyphenyllactic acid (DHPL),
which are coupled by a acyl-CoA-dependent
acyltransferase BAHD family enzyme rosmarinic acid
synthase (SmRAS) to form 4-coumaroyl-30,40-dihydrox-
yphenyllactic acid (4C-DHPL). The 3-hydroxyl groupis
introduced later in the pathway by a P450 monooxygenase
(SmCYP98A14) to form rosmarinic acid (RA) [16]. This
type of P450 was first reported in Coleus blumei (Lami-
aceae), and it catalyzes the 3-hydroxylation of 4-coumar-
oyl-30,40-dihydroxyphenyllactate and the 30-hydroxylation
of caffeoyl-40 — hydroxyphenyllactate, in both cases forming
rosmarinic acid [67]. Recent genome assembly to search
the putative enzymes involved in biosynthesis of phenolics
in S. miltiorrhiza revealed twenty-nine candidates, among
which 15 were predicted in the phenylpropanoid pathway,
seven in the tyrosine-derived pathway and six encoding
putative hydroxycinnamoyltransferases [17].
3 Terpenoids
Terpenoids are formed from sequential assembly of five-
carbon building blocks (C
5
H
8
) called isoprene units.
Accordingly, single or assemblies of two, three and four
units constitute hemiterpenes, monoterpenes, sesquiterpe-
nes and diterpenes, respectively. After the formation of the
basic carbon skeletons, subsequent modifications, such as
oxidation, reduction, isomerization and conjugation, lead to
enormous numbers of structures, which represent the most
abundant class of plant specialized metabolites, with more
than 36,000 individual compounds [68].
In plant cells, the common precursors of terpenoids,
isopentenyl diphosphate (IPP) and dimethylallyl diphos-
phate (DMAPP) are synthesized via two independent
pathways: the cytosolic mevalonic acid (MVA) pathway
that starts with the condensation of acetyl-CoA, and the
plastid-localized methylerythritol phosphate (MEP) path-
way that uses pyruvate and glyceraldehydes 3-phosphate as
substrates(Fig. 2). The IPP and DMAPP are condensed
into geranyl diphosphate (GPP, C
10
), farnesyl diphosphate
(FPP, C
15
) and geranylgeranyl diphosphate (GGPP, C
20
)by
the respective prenyltransferases and then converted to
terpenes by terpene synthases (TPSs), which catalyze the
critical step that determines the structures of terpen
skeletons [69].
Generally, the cytosolic MVA pathway provides the
precursor of FPP for the biosynthesis of sesquiterpenes and
triterpenes, whereas the plastid MEP pathway is responsi-
ble for the biosynthesis of GPP and GGPP for mono-, di-,
and tetra-terpenes [70]. Although cross-talk between these
two spatially separated IPP pathways is prevalent, partic-
ularly in a direction from plastid to cytosol, our under-
standing of the molecular mechanism behind remains
primitive.
Sci. Bull.
123



3.1 Sesquiterpenoids
Monoterpenoids (C
10
) and sesquiterpenoids (C
15
) are
widely distributed in plants, and they are the common
constituents of volatile compounds in flowers, fruits, stems
and leaves, playing important roles in plant–environment
interactions, many of them also possess great commercial
value and some are used in pharmaceuticals.
3.1.1 Artemisinin
One of the most famous plant-sourced medicines is arte-
misinin, an endoperoxide sesquiterpene lactone isolated
from Artemisia annua L., an annual herb of Asteraceae.
Due to its effectiveness against drug-resistant cerebral
malaria, it is the essential component of the combinational
therapies recommended by the World Health Organization
[8]. It has saved millions of lives globally, especially in
developing countries. The 2011 Lasker DeBakey Clinical
Research Award and the 2015 Nobel Prize in Physiology or
Medicine honor the Chinese scientist Youyou Tu who
made the important contribution to the discovery of arte-
misinin [71–73].
As a sesquiterpenoid, artemisinin is believed to be
synthesized from the cytosolic MVA pathway. However, a
recent report suggested that the MEP pathway may also
contribute to its biosynthesis. GPP, which is synthesized in
plastids, can be transported to cytoplasm, forming FPP with
the addition of another IPP unit [74]. The FPP is converted
to the artemisinin skeleton by amorpha-4,11-diene synthase
(ADS), a sesquiterpene synthase [75], and then oxidated by
the cytochrome P450 CYP71AV1. When expressed in
Saccharomyces cerevisiae, CYP71AV1 catalyzed the
continuous oxidation of amorpha-4,11-diene into artemi-
sinic alcohol and artemisinic aldehyde [76], with signifi-
cantly increased production of artemisinic acid and
artemisinic aldehyde when co-expressed with a cyto-
chrome b5 (CYB5) in yeast [8]. The artemisinic aldehyde
D11(13) reductase (Dbr2), a double-bond reductase, cat-
alyzes the formation of dihydroartemisinic aldehyde [77],
which is further converted into dihydroartemisinic acid by
aldehyde dehydrogenase 1 (ALDH1) [78]. Moreover, an
additional alcohol dehydrogenase (ADH1) was also found
to be involved in the oxidation of amorpha-4,11-diene to
artemisinic acid, with specificity toward artemisinic alco-
hol in A. annua plants [8].
Several transcription factors have been shown to par-
ticipate in the regulation of artemisinin biosynthesis [79].
Two jasmonate responsive AP2/ERF proteins, AaERF1
and AaERF2, were found to up-regulate the transcription of
ADS and CYP71AV1 genes, by binding to the
CRTDREHVCBF2 (CBF2) and RAV1AAT (RAA) motifs
present in their promoters [80]. A WRKY family tran-
scription factor, AaWRKY1, was demonstrated to be cap-
able of binding to the W-box in the ADS promoter and
involved in the regulation of artemisinin biosynthesis [81].
A deep sequencing on the transcriptome of A. annua to
identify genes and markers for fast-track breeding was per-
formed, and a detailed genetic map with nine linkage groups
was built. Replicated field trials resulted in a quantitative
trait loci (QTL) map that accounts for a significant amount of
the variation in key traits controlling artemisinin yield, and
positive QTLs in parents of new high-yielding hybrids were
enriched, which made it available to convert A. annua into a
robust crop [82]. Ma et al. [83] recently reported an inte-
grated approach combining metabolomics, transcriptomics
and gene function analyses to characterize gene-to-terpene
and terpene pathway scenarios in a self-pollinating variety of
A. annua. Forty-seven genes that mapped to the terpenes
biosynthesis pathway were identified by sequence mining,
and such metabolites-transcriptome network associated with
different tissues is fundamental to metabolic engineering to
artemisinin.
Fig. 2(Color online) Biosynthesis of terpenoids in TCM plants.
Terpenoids are synthesized via the cytosol MVA pathway and plastid
MEP pathway. Generally, isopentenyl diphosphate (IPP) and
dimethylallyl diphosphate (DMAPP) synthesized from the MVA
pathway are converted to farnesyl diphosphate (FPP) for the
biosynthesis of sesquiterpenoids and triterpenoids, whereas those
derived from the MEP pathway contribute to the formation of geranyl
diphosphate (GPP) and geranylgeranyl diphosphate (GGPP) for
biosynthesis of monoterpenoids, diterpenoids and tetraterpenoids.
HMG-CoA, 3-hydroxy-3-methylglutaryl-CoA; MEP, 2-C-methyl-D-
erythritol 4-phosphate; GGPP, geranylgeranyl diphosphate; HMGR,
3-hydroxy-3-methylglutaryl-CoA reductase; DXS, 1-deoxy-D-xylu-
lose-5-phosphate synthase; DXR, 1-deoxy-D-xylulose-5-phosphate
reductoisomerase; GPPS, geranyl diphosphate synthase; GPP, geranyl
diphosphate; FPPS, farnesyl diphosphate synthase; GGPPS, geranyl-
geranyl diphosphate synthase; TPS, terpene synthase; SS, squalene
synthase; SE, squalone epoxidase; OSC, oxidosqualene cyclase.
Dotted lines represent multiple enzymatic catalyzed steps
Sci. Bull.
123



3.1.2 Patchoulol
Patchouli (Pogostemon cablin), a perennial herbaceous
species of Lamiaceae, is not only a fragrant plant produc-
ing patchouli oil for cosmetics industry, but also a
medicinal plant for the treatment of medical ailments, such
as removing dampness, relieving summer heat and exterior
syndrome, and serving as an anti-emetic and appetite
stimulant [84]. The patchouli oil is composed of
sesquiterpenoids dominated by (-)-patchoulol. The
sesquiterpene synthase, patchoulol synthase, was firstly
purified from patchouli leaves by chromatofocusing, anion
exchange, gel permeation and hydroxylapatite chromatog-
raphy [85]. Then, its cDNA was cloned and the recombi-
nant patchoulol synthase was shown to produce patchoulol
as the major product, plus at least 13 additional
sesquiterpenes [86].
Patchouli oil in leaves accumulates with plant age: The
content is low at juvenile stage and increases during plant
growth and reaches a high level in mature plant. The
microRNA156 (miR156)-targeted SQUAMOSA promoter
binding protein-like (SPL) factors, which function as the
major plant age cue in regulating developmental phase
transition and flowering, play a key role in the age-de-
pendent progressive up-regulation of the patchoulol syn-
thase gene expression, and the patchouli oil biosynthesis.
Interestingly, expression of a miR156-resistant form of
SPL not only accelerated plant maturation but also pro-
moted patchouli oil production [87].
3.2 Diterpenoids
Certain groups of diterpenoids (C
20
), such as gibberellins,
are regulators (phytohormones) of plant growth and
development. Many other specialized diterpenoids, like
tanshinone from Salvia miltiorrhiza and taxol from Taxus,
are highly valuable in medicine. A few more examples
include: stevioside, extracted from Stevia rebaudiana of
Asteraceae, is a natural sweetest [88–90]; adenanthin, from
the leaves of Rabdosia adenantha, induces differentiation
of acute promyelocytic leukemia (APL) cells [91]; ori-
donin, from Lamiaceae plants Isodon rubescens and Isodon
amethystoides, is a potential compound for molecular tar-
get-based therapy of leukemia [92]; and triptolide, a highly
oxygenated diterpene isolated from Tripterygium wilfordii,
was shown to have anti-leukemic activity [93].
3.2.1 Tanshinone
Besides the phenolic acids discussed above, tanshinones
are another class of active diterpenoid compounds of S.
miltiorrhiza, which include tanshinone I, tanshinone IIA,
cryptotanshinone and dihydrotanshinone I. They are all
abietane-type derivatives, among which tanshinone IIAis
considered to be an important bioactive component in
protecting cardiovascular system [94,95], and tanshinone I
was reported to be an apoptosis inducer and display anti-
cancer activities [95].
As diterpenoid compounds, tanshinones are expected to
be traced to the plastid MEP pathway, and their biosyn-
thesis starts from the conversion of geranylgeranyl
diphosphate (GGPP) to ent-copalyl diphosphate (CPP) and
then to miltiradiene. The subsequent extensively structural
tailing converts miltiradiene to cryptotanshinone, tanshi-
none I, tanshinone IIA or tanshinone IIB [96].
Based on sequence homology, enzymes shared by other
diterpenoid biosynthesis have been characterized [96,97].
To date, two enzymes specifically committed to the tan-
shinone biosynthetic pathway have been identified: the
kaurene synthase-like (SmKSL), a diterpene synthasethat
utilizes CPP as substrate to produce miltiradiene [96], and
a P450 monooxygenase CYP76AH1 which transforms
miltiradiene to ferruginol [98], both representing the mile-
stone achievement in the research of TCM plant. Recently,
functional divergence of SmCPSs and SmKSLs was
reported, which specified the roles of individual CPSs in
tanshinone production in different tissues, including
SmCPS1 in roots and SmCPS2 in aerial part, and SmCPS4
and SmKSL2 were found to oxidize ent-13-epi-manoyl in
floral sepals, and the conserved SmCPS5 involved in the
plant growth hormone gibberellin biosynthesis. This study
is a typical example of how the evolutionary diversification
of diterpenoids in plants in molecular level [99].
With the rapid development of sequencing technologies,
several transcriptome datasets and the draft genome of S.
miltiorrhiza have been reported. For examples, the cDNA
library of whole plant contained 10,228 ESTs [100], the
transcriptome of nearly entire growing cycle generated by
Illumina revealed 56,774 unigenes [101], and the searching
of the draft genome resulted in 40 putative genesencoding
enzymes involved in the biosynthesis of universal isoprene
precursors of IPP and DMAPP [102]. Genes encoding
cytochrome P450 monooxygenases, dehydrogenases and
reductases, as well as several groups of transcription fac-
tors were predicted to be involved in tanshinone biosyn-
thesis by comparative analysis of transcriptomes generated
from different tissues [103]. Recently, next-generation
sequencing (NGS) and single-molecule real-time (SMRT)
sequencing were combined to generate a more com-
plete/full-length set of S. miltiorrhiza transcriptome, which
provides a valuable resource for further investigation of
tanshinone biosynthesis [104].
Organ- or tissue-specific patterns are common feature
observed in biosynthesis and accumulation of specialized
metabolites, as well as the expression patterns of corre-
sponding genes [105–107]. Tanshinones are actively
Sci. Bull.
123



synthesized and stored in roots, whereas only alow or trace
amount was detected in aerial organs like leaves [108].
Moreover, both the accumulation and the expression of the
related genes of tanshinones in hairy root cultures can be
induced by biotic elicitors, such as the carbohydrate frac-
tion of yeast extract, and phytohormones of salicylic acid
and jasmonate [97,109–114]. Further investigation can be
directed to the characterization of the signaling compo-
nents and transcription factors that regulate the diterpenoid
biosynthesis in S. miltiorrhiza.
3.2.2Taxol (paclitaxel)
Taxol (paclitaxel) is a diterpenoid isolated from the bark of
Taxus trees. The anti-mitotic and cytotoxic properties of
taxol are derived from its activity in disrupting normal
tubulin dynamics, leading to dysfunction of microtubules
[115]. Fourteen enzymes involved in taxol biosynthesis have
been identified, they are geranylgeranyl diphosphate syn-
thase [116], taxadiene synthase [117], taxadien-5a-ol-O-
acetyl transferase [118], taxane 2a-O-benzoyltransferase
[119], baccatin III: 3-animo-3phenylpropanoyltransferase
[120], 10-deacetylbacctin III-10-O-acetyltransferase [121],
30-N-debenzoyl-20-deoxytaxol N-benzoyltransferase [122],
taxane 5-alpha hydroxylase [123], taxane 10-alpha hydrox-
ylase [124], taxane 13-alpha hydroxylase [125], taxane
2-alpha hydroxylase [126], taxane 7-alpha hydroxylase
[127], taxane 14-alpha hydroxylase [128] and phenylalanine
aminomutase [129].
In addition to elucidation of the biosynthetic enzymes,
progresses have been made in identification of transcription
factors involved in taxol biosynthesis, which include mem-
bers of the AP2 and WRKY families [130]. A recent report
showed that the bHLH transcription factors of TcJAMYC1,
TcJAMYC2 and TcJAMYC4 act as negative regulators of
taxol biosynthesis in T. cuspidata cultured cells [131].
Due to the extremely low content of taxol (at ppm level)
in plant, it requires massive harvesting to obtain sufficient
amounts of the drug; thus, productions by total synthesis,
semi-synthesis, tissue or cell cultures, endophytic fungal
fermentation and more recently metabolic engineering and
synthetic biology have attracted great interests [132]. Pre-
cursors of taxol biosynthesis have been produced in
Escherichia coli [7] and Saccharomyces cerevisiae [123,
133], and the integration of parts (modules) of the whole
pathway in separate organisms cultured together led to the
combination of production of taxadiene in E. coli and
oxygenation of taxadiene by S. cerevisiae [9].
3.3 Triterpenoids
Triterpenoids are cyclization product of squalene which is
condensed by two molecules of FPP. In general,
triterpenoids are formed from MVA pathway in cytoplasm,
as sesquiterpenoids.
3.3.1 Ginsenosides
Ginseng, the root of Panax ginseng, is one of the oldest tra-
ditional medicines and is widely regarded as a tonic in East
Asia [88–90]. Theprinciple bioactive constituents of Ginseng
are ginsenosides, a group of tetra- or pentacyclic triterpene
glycosides belonging to saponins [134]. The clinical and
pharmacological activities of ginsenosides include anti-dia-
betic, anticancer, anti-amestic hypoglycemic, radioprotec-
tive, immunomodulatory, neuroprotective and anti-stress
[135–139]. More than 40 ginsenosides have been isolated
from the white and the red ginseng, and they show different
biological activities based on their structural differences
[140]. Generally, the major pharmacologically active gin-
senosides belong to tetracyclic dammarane- and pentacyclic
oleanane-type triterpene saponins [141].
The common precursor of ginsenosides is squalene,
which is formed by condensation of two FPPs with squa-
lene synthase (SS) [135,142,143]. In Ginseng, squalene is
converted into dammarenediol-II by squalone epoxidase
(SE). The cyclization of 2,3-oxidosqualene can result in
two different type of triterpenoids: dammarane and olea-
nane type. Ginsenosides belonging to dammarane-type
triterpenoids are biosynthesized from 2,3-oxidosqualene by
dammarenediol synthase (DS) to form dammarenediol-II
[144], whereas the biosynthesis of oleanane-type ginseno-
sides is started by b-amyrin synthase (PNY1) that trans-
forms 2,3-oxidosqualene into b-amyrin [145,146]. SS is
considered a rate-limiting enzyme in the pathway and
catalyzes the initial biosynthetic step for both steroids and
triterpenoids [147]. PgPDR, a member of ABC trans-
porters, was found to be involved in the ginsenosides
accumulation upon MeJA induction [148].
3.3.2 Cucurbitacins
Cucurbitacins, conferring a bitter taste in cucurbits such as
cucumber, melon, watermelon, squash, and pumpkin,
belong to a class of highly oxidized tetracyclic triter-
penoids mainly found in the plant of Cucurbitaceaefamily,
in which Gynostemma pentaphyllum,Hemsleya chinesis,
Siraitia grosvenorii and Bolbostemma paniculatum are
well-known TCM plants. Recent studies suggest that
cucurbitacins repress cancer cell progression [149] and
inhibit neuroblastoma cell proliferation through up-regu-
lation of PETN (phosphatase and tensin homolog) [150].
By genome-wide association study based on the genome
variation map of 115 diverse cucumber lines, the gene of
Csa6G088690 (Bi) encoding oxidosqualene cyclase is
found to be correlated to the cucurbitacin C (CuC)
Sci. Bull.
123



biosynthesis. Co-expression and co-regulation studies
revealed a 9-gene module responsible for CucC biosyn-
thesis, of which, four enzymes, including Bi, two P450s
and one ACT, were characterized. Moreover, two bHLH
transcription factors, Bl (bitter leaf) and Bt (bitter fruit),
were found to directly regulate the expression of 9-gene
module in cucumber leaf and fruit, respectively. During the
cucumber domestication, mutations occurred within Bt
promoter region which decreased its expression in the fruit
tissue which may have been selected and fixed and resulted
in nonbitter fruit we eat nowadays [151].
3.3.3 Glycyrrhizin
The roots and stolons of Glycyrrhiza plants (G.uralensis and
G. glabra) contain a large amount ofoleanane-type triter-
penoid glycyrrhizin. It is not only used worldwide as a
natural sweetener and flavoring additive due to its sweet
taste, but alsoexhibit a wide range of pharmacological
activities, including anti-inflammatory [152], immunomod-
ulatory [153], anti-ulcer [154], anti-allergy [155], and anti-
viral activity [156–158].
From G. glabra, genes that encode enzymes responsible
for triterpene skeleton formation, including the squalene
synthase (SS) and b-amyrin synthase (bAS), were isolated
[159,160]. Later biosynthesis steps of glycyrrhizin involve
a series of oxidative reactions at positions C-11 and C-30
and glucuronylation of the C-3 hydroxyl group. Enzymes
that catalyze the oxidation steps have been found to be
cytochrome P450 monooxygenases. One of them,
CYP88D6, was characterized to catalyze the sequential
two-step oxidation of b-amyrin at C-11 to produce 11-oxo-
b-amyrin by both in vitro assay with recombinantprotein
and co-expression with b-amyrin synthase in yeast [161].
Another P450, CYP72A154, was identified to be respon-
sible for three sequential oxidations at C-30 to transform
11-oxo-b-amyrin to glycyrrhetinic acid, a glycyrrhizin
aglycone [162]. Both CYP88D6 and CYP72A154 tran-
scripts were detected in the roots and stolons, but not in the
leaves or stems, which is consistent with the accumulation
pattern of glycyrrhizin in planta [161,162].
4 Alkaloids
Alkaloids are a group of nitrogen-containing compounds
with basic properties, most of which are derivatives of
amino acids [163–166]. Biosynthesis of alkaloids usually
starts from modification of amino acids, mostly decar-
boxylation or deamination, and undergoes further steps like
methylation, hydroxylation and oxidation, and/or coupled
with other compounds. There are over 12,000 alkaloids that
have been identified from plants. Although widely dis-
tributed in plants, they are particularly enriched in certain
families, such as Solanaceae, Manispermaceae, Papaver-
aceae, Berberidaceae and Fabaceae (Table 2).
It is noteworthy that the most of alkaloids display
bioactivities to certain degrees, often derived from their
nitrogen-containing properties. Not surprisingly, alkaloids
constitute the major portion of drugs both in history and
nowadays. The discovery and isolation of morphine from
the opium poppy (Papaver somniferum) by Friedrich Ser-
tu
¨rner in 1806 is a milestone in the history of pharmacy.
Investigations of biosynthesis of natural alkaloids such as
morphinan, vindoline and noscapine have been intensive
and led to the complete elucidation of the pathway [167–
170], and increasing alkaloid biosynthesis in plant through
co-expression of enzymes genes was also reported [114].
Unfortunately, although alkaloids with TCM background
like camptothecin, higenamine, huperzine A and tetran-
drine have been used in pharmacy, reports of their
biosynthesis are relatively rare. We list in Table 3several
Table 2List of examples of TCM plants rich in terpenoids
Plant speciesChinese name in Pin-yinFamilyRepresentative compounds
Pogostemon cablinGuanghuoxiangLamiaceae Patchoulol
Artemisia annuaHuanghuahao or QinghaoAsteraceaeArtemisinine
Salvia miltiorrhizaDanshenLamiaceae Tanshinone
Taxus chinensisHongdoushanTaxaceae Paclitaxel
Andrographis paniculataChuanxinlianAcanthaceae Andrographolide
Isodon rubescensDonglingcaoLamiaceaeOridonin, ponicidin
Isodon amethystoidesXiangchacaiLamiaceaeOridonin, ponicidin
Tripterygium wilfordiiLeigongtengCelastraceae Triptolide
Panax ginsengGinsen or RenshenAraliaceaeGinsenosides
Panax notoginsengSanqiAraliaceae Notoginsenosides
Radix liquiritiaeGancaoFabaceae Glycyrrhizin
Dioscorea polystachyaShuyuDioscoreaceae Dioscin
Sci. Bull.
123



typical alkaloids in TCM plants, and the relevant refer-
ences. Various aspects on the alkaloid biosynthesis, regu-
lation and metabolites trafficking can be found in review
articles [178–183]. Without doubt more efforts are needed
to study alkaloids in TCM plants to further explore their
biological activities and facilitate their usage.
5 Perspective
Unlike model plant or staple crops, medicinal plants often
lack a well-studied genetic background and a high-quality
genome sequence. Due to the recently developed high-
throughput sequencing technologies, it is possible to gen-
erate transcriptomic data of medicinal plants in a shorttime
at an affordable cost. Comparative analysis of chemical
constituents, transcriptomes and correlation of spatial and
temporal patterns of gene expressions with those of
metabolite accumulation have led to the identification of
candidate genes of the biosynthesis pathway [184]. GWS
combined with metabolomics analysis (mGWAS) provides
a powerful platform which screens a large number of
accessions simultaneously to understand genetic contribu-
tions to the metabolic diversity [185,186].
Throughout the history, herbal plants are an integral part
of our lives. In addition to curing illness, they are grown in
elegant gardens, provide natural fragrance, delicate acces-
sories and stimulate appetite. The biosynthesis of
metabolites in medicinal plants is complex and specialized
and involves many sequence-similar but functionally
diverged enzymes. With the fast development of new
technologies of analytical chemistry, bioinformatics and
synthetic biology, more and more achievements will be
made in this genomic or post-genomic era and bring us
better life.
Acknowledgments This work was supported by the National Nat-
ural Science Foundation of China (31200222), and Special Fund for
Shanghai Landscaping Administration Bureau Program (F132424,
F112418 and G152421).
Conflict of interestThe authors declare that they have no conflict
of interest.
Open AccessThis article is distributed under the terms of the
Creative Commons Attribution 4.0 International License (http://
creativecommons.org/licenses/by/4.0/), which permits unrestricted
use, distribution, and reproduction in any medium, provided you give
appropriate credit to the original author(s) and the source, provide a
link to the Creative Commons license, and indicate if changes were
made.
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