Medicinal plants chemistry, biology and omics woodhead publishing series in biomedicine no 732015UnitedVRG

Medicinal Plants
Chemistry, Biology and Omics


Related title
Medicinal Plant Biotechnology
(ISBN 978-1-84593-678-5)


Woodhead Publishing Series in Biomedicine:
Number 73

Medicinal Plants
Chemistry, Biology and Omics

Authored by

Da Cheng Hao
Xiao-Jie Gu
Pei Gen Xiao

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Preface

Medicinal plants provide myriad pharmaceutically active components, which has

been commonly used in traditional Chinese medicine (TCM) and worldwide ethnomedicine for thousands of years. Increasing interest in plant-based medicinal
resources has led to additional discoveries of many novel compounds, such as steroidal alkaloids, saponins, terpenoids, glycosides, in various angiosperm and gymnosperm species, and to investigations on their chemotaxonomy, molecular
phylogeny, and pharmacology. In continuation with our studies on pharmacophylogeny, in this book we review the phytochemistry, chemotaxonomy, molecular biology, and phylogeny of selected medicinal plant tribes and genera and their
relevance to drug efficacy. Literature search is used to characterize the global scientific effort in the flexible technologies being applied. The interrelationship within traditional Chinese medicinal plant groups and between Chinese species and species
outside of China is clarified by the molecular phylogenetic inferences based on
nuclear and chloroplast DNA sequences. The incongruence between chemotaxonomy
and molecular phylogeny is revealed and discussed. It is indispensable to study more
species, according to the principles of pharmacophylogeny, for both the sustainable
utilization of medicinal resources and finding novel compounds with potential clinical
utility. Systems biology and omics technologies (genomics, transcriptomics, proteomics, metabolomics, etc.) will play an increasingly important role in future pharmaceutical research involving bioactive compounds of land plants.
Biodiversity represents an endless source for the discovery of pharmacological
active compounds in the development of new medicinal drugs. Bioactive compounds
occur in a broad diversity of organisms ranging from bacteria to flowering plants. Discoveries are made through systematic taxonomical investigations and the analysis of
herbal medicines used for thousands of years in TCM, Ayurvedic medicine, and
worldwide ethnomedicine. The medicinal drugs represent a broad spectrum of chemical structures such as steroidal alkaloids, saponins, terpenoids, polyphenol, and glycosides. Promising compounds are further investigated and developed for improved
analog drugs through approaches such as chemotaxonomy, molecular phylogeny,
and poly-pharmacology. Evolutionary biology and chemical ecology, especially when
approached by high-throughput genomic technology, are closely related to the above
fields and would significantly facilitate both, a more systematic approach toward the
conservation of medicinal plant diversity as well as drug discovery and development.
Some researchers would argue that the discovery of many compounds is passe´—the
chemical side has been looked at for many years. Evolutionary approaches could thus
provide new twist and add new dimension in medicinal plant studies. For example,
approved and clinical-trial natural product drugs are obtained from clustered and


x

Preface

disjunct taxonomic clades and similarly are herbal indigenous pharmacopeias biased
toward the selection of certain phylogenetic clusters.
Evolutionary analyses have been performed at different levels, including genes
involved in the biosynthesis of secondary metabolites, its pathways and networks,
population dynamics and the molecular interaction of species with the ecosystem
or parts thereof (chemical ecology). Also, have studies focusing on the evolution
of biosynthesis pathways provided novel insights and directed approaches in synthetic
biology and metabolic engineering. Evolutionary approaches offer a rich sciencebased method to prospect plant diversity having revealed predictive power for bioprospecting traditional medicines. The trend of integrating evolution into studies of
medicinal plants is perceivable and therefore it is time to summarize the current progress in the relevant fields in order to make full use of evolutionary biology and revolutionize the roadmap of medicinal plant research.
This book wish to reflect the current progress in phylogeny, chemotaxonomy,
molecular biology, and phytochemistry of selected medicinal plant tribes and genera
in the light of evolutionary biology and genomics. In the context of evolution, each
chapter of this book is a kind of fusion of commentary, perspective, and review, which
aims to characterize the global scientific effort as well as the flexible technologies and
methods applied, and also illustrate how evolutionary biology could further our understanding of a number of aspects of medicinal plant research.

Five features of this book
1. reviews and summarizes best practice and essential developments in medicinal plant chemistry and biology;
2. discusses the principles and applications of various chemical, biology, and omics techniques
used to discover medicinal compounds, bioactivities, and underlying evolutionary relationship;
3. explores the analysis and classification of novel plant-based medicinal compounds;
4. includes case studies on pharmacophylogeny;
5. compares and integrates traditional knowledge and current perception of worldwide medicinal plants.

The book is designed for use by senior undergraduate and graduate students,
researchers, and professionals in medicinal plant, phytochemistry, pharmacognosy,
molecular biology, biotechnology, agriculture, and pharmacy working in the academic and industrial sectors. Students and researchers in pharmacology, medicinal
chemistry, plant systematics, food and nutrition, clinical medicine, evolution and
ecology, as well as professionals in pharmaceutical industries might also be interested
in plants included in this book.

Chapter authorship
Chapters 1, 3, 6, 10, 11, and 15, Da Cheng Hao (DH) and Xiao Jie Gu (XG); Chapter 2,
DH; Chapters 4, 5, 7–9, 12–14, DH, Pei Gen Xiao and XG.


Preface

xi

This book is supported by Academic Publication Fund of Dalian Jiaotong University. Friends and colleagues in many parts of the world lent support to this book. We
would like to thank all those who have published their findings that we cite in the chapters. Special thanks go to the project editor Dr. Glyn Jones and the project manager
Mr. Harriet Clayton from Elsevier UK (Woodhead) and other team members for their
interest, support and encouragement.

About the authors
1. Dr. Da Cheng Hao, associate professor/principle investigator, School of Environment and
Chemical Engineering/Biotechnology Institute, Dalian Jiaotong University, Dalian, China
Dr. Hao got his Bachelor’s degree in Medicine, Master’s degree in Science, and PhD
degree in Biotechnology from Xi’an Jiaotong University, National University of Singapore
and Chinese Academy of Sciences, respectively. He had the post-doc training in Institute of
Medicinal Plant Development (IMPLAD), Chinese Academy of Medical Sciences (CAMS),
under the supervision of Prof. Pei Gen Xiao and Prof. Shi Lin Chen. He was a visiting scholar
of John Innes Centre, UK for 1 year (2012–2013), supported by Ministry of Education,
China.
2. Dr. Xiao Jie Gu, lecturer, School of Environment and Chemical Engineering/Biotechnology
Institute, Dalian Jiaotong University, Dalian, China
Dr. Gu got her Bachelor’s degree and PhD degree in pharmaceutical science from
Liaoning University of Traditional Chinese Medicine and China Pharmaceutical University,
respectively. Her major research interest is medicinal plant and pharmacognosy.

3. Prof. Pei Gen Xiao, the founder of pharmacophylogenetics and the leading scientist of
Chinese medicinal plant and Chinese Materia Medica studies, and also a well-known
ethnopharmacologist.
Prof. Xiao graduated in 1953 from Xiamen University majoring in Biology. After
graduation, he served at the Institute of Materia Medica, CAMS. Since 1983 he has been
a professor and was designated as the director of IMPLAD, CAMS. Starting from 1996
he has been designated as honorary director of IMPLAD, CAMS, and the head of Key
Laboratory on Resource Utilization and Conservation of Chinese Materia Medica, State
Administration of Traditional Chinese Medicine. Due to his outstanding scientific achievements, Prof. Xiao has been elected as member (academician) of Chinese Academy of
Engineering, Division of Medicine and Health Engineering since 1994 and was also elected
as president of the International Society on Ethnopharmacology in 1994.



Chemotaxonomy: a phylogenybased approach

1.1

1

Introduction

Chemotaxonomy, also called chemosystematics, is used to classify and identify
organisms (mainly plants), according to perceptible differences and similarities in
their biochemical compositions. The compounds studied in all cases are either primary
metabolites or secondary metabolites (SMs). Examples of chemotaxonomic markers
used in recent years are summarized below. Chemotaxonomy contributes to the classification of plants when uncertainty exists using classical botanical methods. Chemosystematics can be regarded as a fusion science that complements available
morphological and molecular data to improve plant systematics and to facilitate pharmaceutical resource discovery.

1.2

1.2.1

Chemotaxonomic marker
Primary metabolite

1.2.1.1 Fatty acid
Among the various biochemical markers, fatty acids (FAs) or lipid profiles represent a
chemically relatively inert class of compounds that is easy to isolate from biological
material. FA (Figure 1.1) profiles are chemotaxonomic markers that define groups of
various taxonomic ranks in flowering plants, trees, and other embryophytes. The FA
profiles of 2076 microalgal strains from the Culture Collection of Algae at G€ottingen
University (SAG) were determined in the stationary phase (Lang et al., 2011).
Seventy-six different FAs and 10 other lipophilic substances were identified and quantified. The FA profiles were added into a database. FA distribution patterns were found
to reflect phylogenetic relationships at the level of phyla and classes. At lower taxonomic levels, for example, between closely related species and among multiple isolates of the same species, FA contents may be rather variable. FA distribution patterns
are suitable chemotaxonomic markers to define taxa of higher rank in algae. Due to
their extensive variation at the species level, it is difficult to make predictions about
the FA profile in a novel isolate.
The distribution of FAs in 13 species of macroalgae (Chlorophyta, Ochrophyta, and
Rhodophyta) and one sea grass (Spartina sp.), collected on the Rio de Janeiro state
coast, was determined (Fleury et al., 2011). Statistical analyses showed the
Medicinal plants: chemistry, biology and omics. />© 2015 Elsevier Ltd. All rights reserved.


2

Medicinal plants: chemistry, biology and omics
OH

(E)-phytol
O

OH

Hexadecanoic acid (palmitic acid)
O
OH

Linoleic acid

O

OH

Oleic acid
O
OH

Stearic acid
O
C

OH

C

6-Octadecynoic acid

Figure 1.1 Fatty acids.

effectiveness as taxonomic and phylogenetic markers of the distribution of the methyl
FA esters in these macrophytes.

In Geranium (Geraniaceae) and highly related Erodium taxa from Serbia and
Macedonia, the investigated essential oils consisted mainly of FAs and FA-derived
compounds (45.4–81.3%), with hexadecanoic acid and (E)-phytol as the major components (Radulovic´ and Dekic´, 2013). Geranium and Erodium taxa are phylogenetically closely related, and there is no great intergeneric oil-composition variability.
The FA composition of 12 Brassica species (Brassicaceae) was analyzed by GCFID and confirmed by gas chromatography–mass spectrometry (GC–MS) (Barthet,
2008). According to the C18:1 (n À 7)/(n À 9) ratios for chemotaxonomy, the surveyed
species could be arranged into three groups. The first group includes Brassica napus,
B. rapa, and B. tournefortii with Eruca sativa branching only related to B. napus. The
second group includes B. tournefortii, Raphanus sativus, and Sinapis alba. The last
group includes B. juncea, B. carinata, and B. nigra with no similarity/relationship
between them and between the other species.


Chemotaxonomy: a phylogeny-based approach

3

C.charrieriana FA1
C.congensis
FA2 Lower Guinea/Congolian
C.pseudozanguebariae
FA3 East Africa
C.sessiliflora
C.racemosa
FA4 East Africa
C.salvatrix
C.liberica C
C.canephora N
C.kapakata
C.liberica C
FA5 Lower Guinea/Congolian

C.liberica W
C.heterocalyx
C.canephora C
C.canephora W
C.humilis
C.stenophylla FA6 Upper Guinea
C.eugenioides
FA7 East-Central Africa
C.humblotiana

Figure 1.2 A simplified scheme showing the hierarchical clustering analysis of the seed
fatty acid composition of 59 Coffea genotypes (according to Dussert et al., 2008).
Correspondence between groups of species obtained through HCA of seed FA data and
clades inferred from DNA sequences (Maurin et al., 2007) is shown.

The FA composition of the seed oil of 23 Stachys (Labiatae) taxa was analyzed by
GC–MS (G€
oren et al., 2012). The main compounds were linoleic (27.1–64.3%), oleic
(20.25–48.1%), palmitic (4.3–9.1%), stearic (trace to 5.2%), and 6-octadecynoic (2.2–
34.1%) acids. The latter compound could be a chemotaxonomic marker of the genus
Stachys.
FAs and sterols were determined in 59 genotypes of 17 distinct Coffea species
(Rubiaceae) (Dussert et al., 2008). Interestingly, while groupings based on seed FA
composition showed remarkable ecological and geographic coherence (Figure 1.2),
no phylogeographic explanation was found for the clusters retrieved from sterol data.
When compared with previous phylogenetic studies, the groups deduced from seed
FA composition were remarkably congruent with the clades inferred from nuclear
and plastid DNA sequences (Table 1.1). Leaf FA composition is useful in chemotaxonomy of Rubiaceae (Mongrand et al., 2005). Principal component analysis (PCA)
allowed a clear-cut separation of Coffeae, Psychotrieae, and Rubieae.

1.2.1.2 Protein, amino acid, and carbohydrate
The complete amino acid sequence of [2Fe–2S] ferredoxin from Panax ginseng (Araliaceae) was determined (Mino, 2006). Phylogenetic analysis based on the amino acid
sequence of ferredoxin suggests that P. ginseng is related taxonomically to umbelliferous plants.
Eighteen species of the genus Euphorbia (Euphorbiaceae) have proteolytic
enzymes in their lattices, nine of them are characterized by the type of endopeptidases
(cysteine endopeptidase, serine endopeptidase, metallo-endopeptidase, and aspartic
endopeptidase), which are responsible for the activity (Domsalla et al., 2010), and


4

Medicinal plants: chemistry, biology and omics

Table 1.1 Distribution of phenylethanoid glycoside
in Gesneriaceae species
Species
Beccarinda
tonkinensis
Hemiboea
subcapitata
Hemiboea flaccida
Chirita macrodonta
Chirita pumila
Chiritopsis repanda
Paraboea peltifolia
Paraboea nutans
Paraboea rufescens
Rhabdothamnopsis
sinensis
Lysionotus

pauciflorus

Paraboside
Acteoside B

Isonuomioside
A

Paraboside
II

Paraboside
III

*

_

_

_

_

*

_

_

_

_

*
*
*
*
*
*
*
*

_
_
_
_
_
_
*
*

*
_
_
_
_
*
_
_

_
_
_
_
_
_
_
_

_
_
_
_
_
_
_
_

*

_

_

_

*

*, present; _, not detected.

all nine are serine endopeptidases. The lattices of 64 different species were examined
concerning proteolytic activity and serine protease activity, five of which are mentioned in the literature to be proteolytic active and four are known to contain at least
one serine endopeptidase. All tested samples were able to degrade labeled casein; the
activity of six lattices was completely inhibited by specific serine protease inhibitors;
15 samples were not influenced; and in 43 lattices, a remaining activity was measured,
indicating that other types of endopeptidases seem to be involved.
Differences in cell-wall composition and structure, corresponding to the carbohydrate fingerprint region (1200–800 cmÀ1) of the FT-IR spectrum, can provide the
basis for chemotaxonomy of flowering plants (Kim et al., 2004).

1.2.1.3

Alkanes

The PCA of the contents of nine n-alkanes showed a clear separation of the Serbian
spruce populations from those of the two investigated pine species, which partially
overlapped (Nikolic´ et al., 2013). The separation of the species was due to high contents of the n-alkanes C29 and C31 (Picea omorika); C19, C20, C21, C22, C23, and
C24 (Pinus heldreichii); and C28 (Pinus peuce).
Samples of 195 Pinus nigra trees from seven populations belonging to several
infraspecific taxa (Pinus nigra ssp. nigra, Pinus nigra var. gocensis, Pinus nigra
ssp. pallasiana, and Pinus nigra var. banatica) were analyzed (Bojovic´ et al.,
2012). The size of the n-alkanes ranged from C16 to C33, with the exception of Pinus


Chemotaxonomy: a phylogeny-based approach

5

nigra ssp. nigra, for which it ranged from C18 to C33. The most abundant were C23,
C25, C27, and C29 alkanes. The needle waxes of populations I–III and V were characterized by a higher content of C23, C25, and C27 alkanes and a lower content of

C24, C26, C28, and C30 alkanes, compared to the other populations, and the trees
of these populations could be assigned to Pinus nigra ssp. nigra. The samples of population VI were characterized by higher amounts of C22, C24, C30, and C32 alkanes
and lower amounts of C25 and C27 alkanes, and the trees could be Pinus nigra ssp.
pallasiana. The samples of population VII, consisting of trees belonging to Pinus
nigra var. banatica, were richer in C29, C31, and C33 alkanes. The wax compositions
of populations IV and V, both composed of trees previously determined as Pinus nigra
var. gocensis, showed a tendency of splitting. The alkane composition of population
IV was closer to that of Pinus nigra ssp. pallasiana pines, while that of population V
was more similar to that of Pinus nigra ssp. nigra pines. In the central part of the
Balkan Peninsula, significant diversification and differentiation of the populations
of black pine exist, and these populations could be defined as different intraspecific
taxa. n-Alkanes are valid as chemotaxonomic characters within this aggregate.
Analyses by GC and GC–MS of an essential oil sample obtained from dry fruits of
Scandix balansae (Apiaceae) allowed the identification of 81 components (Radulovic´
and Denic´, 2013), comprising 91.4% of the total oil composition. The major identified
volatile compounds were medium-chain length n-alkanes, that is, tridecane (6.7%;
Figure 1.3), pentadecane (13.4%), and heptadecane (19.3%), and a long-chain homologue nonacosane (7.6%). A number of minor oil constituents, among them tetradecyl
3-methylbutanoate, and octadecyl 2-methylpropanoate, 3-methylbutanoate, and pentanoate, have a restricted natural occurrence not only in umbellifers but also in the
plant kingdom, whereas the last ester is a new natural compound in general. The identity of these rare plant constituents that present excellent chemotaxonomic marker
candidates for Scandix was unambiguously confirmed by coinjection of the oil sample
with appropriate standards. These samples and additional 58 oils obtained from Scandiceae were compared using multivariate statistical analyses (MVAs), which demonstrated that the evolution of the volatiles’ metabolism of Scandiceae taxa neither was
genus-specific nor follows their morphological evolution.
The leaf cuticular n-alkane chain length distribution pattern was used as an alternative taxonomic marker for eggplant (Solanaceae) and related species (Halinski
et al., 2011). The results are in good agreement with current knowledge of the systematics of these plants.

1.2.1.4 Alkynes
Among 106 and 81 constituents, S-containing polyacetylene (Figure 1.4) compounds
and triquinane sesquiterpenoids made up $80% of Echinops bannaticus (Compositae)
and E. sphaerocephalus oils, respectively (Radulovic´ and Denic´, 2013). A multivariate statistical comparison of the essential oil composition data for these two and additional six taxa of this genus available from the literature permitted an examination of
the mutual relationships of the taxa within this morphologically highly uniform genus.

PCA and agglomerative hierarchical clustering revealed a grouping of E. bannaticus


6

Medicinal plants: chemistry, biology and omics

Tridecane

Pentadecane

Heptadecane

Nonacosane
O

O

Tetradecyl 3-methylbutanoate
O

O

Octadecyl 2-methylpropanoate
O

O
OH

OH

3-Methylbutanoate

Pentanoate

Figure 1.3 Some alkanes as chemotaxonomic markers mentioned in the text.

n

n

Polyacetylene

Figure 1.4 Alkynes.

and E. sphaerocephalus (section Echinops), and their close relationship with E. grijsii,
suggesting a circumscription of this Chinese taxon to the section Echinops. PCA correlation matrix offered valuable insight into the biosynthetic links between essential
oil constituents, and these agreed excellently with the currently proposed ones for the
polyacetylene S-containing compounds, triquinanes, and monoterpenes.

1.2.1.5

Carotenoid

Berries and leaves from six varieties of Carpathians’ sea buckthorn (Hippophae rhamnoides L., ssp. carpatica) were analyzed for their carotenoid composition (free and
esterified) using HPLC-PAD, GC–MS, and UHPLC–PAD-ESI-MS techniques


Chemotaxonomy: a phylogeny-based approach

7
OH

H

HO

Lutein

β-Carotene
OH
O

O
HO

Violaxanthin

C
O
HO

Neoxanthin

HO

OH

Figure 1.5 Carotenoids.

(Pop et al., 2014). GC–MS revealed the FA profile specific for each berry variety,
while targeted UHPLC–MS identified the FAs involved in carotenoid esterification:
palmitic (C16:0), myristic (C14:0), and stearic (C18:0). Total carotenoid content varied between 53 and 97 mg/100 g dry weight in berries and between 3.5 and 4.2 mg/
100 g DW in leaves. The carotenoid diesters were the main fraction among berry varieties having zeaxanthin dipalmitate as major compound, while leaves contained only
free carotenoids like lutein (Figure 1.5), b-carotene, violaxanthin, and neoxanthin.
PCA identified the suitable carotenoid biomarkers characteristic for the Carpathians’
sea buckthorn from Romania with contribution to their taxonomic classification and
authenticity recognition.

1.2.2

Secondary metabolite

The bryophytes contain the Marchantiophyta (liverworts), Bryophyta (mosses), and
Anthocerotophyta (hornworts). The Marchantiophyta have a cellular oil body that produces various mono-, sesqui-, and diterpenoids; aromatic compounds like bibenzyl;
bisbibenzyls; and acetogenins (Asakawa et al., 2013). Most sesqui- and diterpenoids


8

Medicinal plants: chemistry, biology and omics

obtained from liverworts are enantiomers of those found in higher plants. Many of
these compounds display a characteristic odor and have interesting biological activities, including antimicrobial, antifungal, antiviral, cytotoxic, insecticidal, insect
antifeedant, NO production-inhibitory, antioxidant, piscicidal, neurotrophic, and
muscle-relaxing activities, and are involved in allergenic contact dermatitis and in
the release of superoxide anion radicals, 5-lipoxygenase, calmodulin, hyaluronidase,
cyclooxygenase, DNA polymerase b, and a-glucosidase. Each liverwort synthesizes
unique components, which are valuable for their chemotaxonomic classification.
Initial collection of Cameroon herbs was composed of 3742 phytochemicals previously isolated from 67 families, along with 319 hemisynthetic products, giving a

total of 4061 chemical structures (Ntie-Kang et al., 2013). Removal of duplicates gave
2770 pure compounds. Emphasis was laid on those plant families from which at least
2.5% of the SMs have been isolated, which include Leguminosae (13.9%), Moraceae
(10.6%), Guttiferae (10.1%), Rutaceae (6.5%), Meliaceae (4.5%), Euphorbiaceae
(4.4%), Compositae (3.9%), Zingiberaceae (3.4%), Ochnaceae (3.2%), Bignoniaceae
(3.1%), Sapotaceae (3.1%), and Apocynaceae (2.8%). Terpenoids were most abundant in Cameroon medicinal plants (26.0% of the isolated compounds). This was followed by flavonoids (19.6%), alkaloids (11.8%), xanthones (5.4%), quinones (5.0%),
and glycosides (4.9%), showing a similar trend with a previous analysis of 1859
metabolites.

1.2.2.1

Essential oil and volatile terpene

The structures of some monoterpenoids, sesquiterpenoids, aromatic compounds, aliphatic hydrocarbons, and other compounds included in essential oil are shown in
Figure 1.6a–e.
It might be possible to use chemical analysis of SMs emitted from the trees to differentiate clones growing in various diverse environments, as their terpenoid emissions are directly influenced by the environmental conditions in which they grow
(Niogret et al., 2013). The endogenous factors are related to anatomical and physiological characteristics of the plants and to the biosynthetic pathways of the volatiles,
which not only might change either in the different tissues of the plants or in different
seasons but also could be influenced by DNA adaptation (Barra, 2009). Those factors
lead to ecotypes or chemotypes in the same plant species. In recent years, chemotaxonomy has been widely used to classify plants with essential oils characterized by
intraspecific chemical polymorphism.
Considerable intra- and interspecific essential oil component variation was detected
in six subspecies of Phebalium squamulosum (Rutaceae: Boronieae), suggesting the
existence of distinct chemotypes and supporting previously observed segregate species
based on morphological evidence (Sadgrove et al., 2014). GC–MS identified 145
(including 64 tentatively identified) volatile compounds from peel oils of Citrus, Poncirus, and Fortunella (Rutaceae) (Liu et al., 2013a). The chemotaxonomic results based
on peel oils are congruent with the Swingle taxonomy system, and Citrus, Poncirus, and
Fortunella were almost completely separated. Citrophorum, Cephalocitrus, and Sinocitrus, which belong to the subgenus Citrus, can be differentiated by chemotaxonomy.

Chemotaxonomy: a phylogeny-based approach

9

Mangshanyegan (Citrus nobilis Lauriro), a wild germplasm in the citrus family, contains volatile compounds similar to those from pomelo.
The major monoterpenes among the volatiles, that is, b-phellandrene (4), limonene
(6), and g-terpinene (5), and phenylpropanoids, that is, estragole (3), (E)-anethole (7),
and myristicin (1), showed to be useful chemotaxonomic markers of six Gingidia
(Umbelliferae) species from New Zealand and Australia (Sansom et al., 2013).

OH

β-Phellandrene

Limonene

OAc

γ-Terpinene

Terpinen-4-ol

α-Terpinyl acetate

O
O

α-Pinene

1,8-Cineole

β-Pinene

Verbenone

Camphor

O
O
O

β-Dihydroionone

(–)-(1S,2R,4S)-Borneol acetate

O
HO
O

O

O

HO

OH

H

OH

H
O

O

O

O
HO

O

OGlc

Asperuloside
O
HO

O

H
OGlc

H
OGlc

Deacetylasperulosidic acid

O

Asperulosidic acid

OCH3

H

O
HO

H
OGlc

(a)

6α-Hydroxygeniposide

Figure 1.6 Essential oil: (a) monoterpenoids;
(Continued)


10

Medicinal plants: chemistry, biology and omics

H
HO

δ-Cadinene

OH

α-Eudesmol

Epi-α-cadinol

OH

β-Eudesmol

HO
O
H

Epi-β-bisabolol

Caryophyllene oxide

HO

HO

(b)

Spathulenol

Germacrene D

Guaiol

O

O
H3CO

H3CO

Estragole

(E)-anethole

(E)-methyl cinnamate

OH
O
OH

O
OCH3

(c)

Myristicin

Thymol

Carvacrol

Figure 1.6 Continued. (b) sesquiterpenoids; (c) aromatic compounds;
(Continued)

Essential oils, as chemotaxonomic markers, could be useful to classify Artemisia
(Compositae) species and to characterize biodiversity in the different populations
(Maggio et al., 2012).
The hydrodistilled essential oils obtained from aerial flowering parts of Teucrium
stocksianum ssp. stocksianum (TSS) and T. stocksianum ssp. gabrielae (TSG) from
Iran were analyzed by capillary GC and GC–MS (Sonboli et al., 2013). The oil analysis of two subspecies led to the identification of 65 compounds that accounted for


Chemotaxonomy: a phylogeny-based approach

11

O

(2E, 4E)-decadienal

(Z )-β-ocimene

HO

Linalool

Myrcene
O
OH

(d)

Hexadecanoic acid

O

S

(e)

Menthofuran

Sabinene

2,3,4-Trimethylthiophene

Figure 1.6 Continued. (d) aliphatic hydrocarbons; (e) others.

93.3% and 95.1% of the total oil compositions, respectively. Sesquiterpenoids
(52.9%) constituted the main compounds in the essential oil of TSS represented
mainly by cis-sesquisabinene hydrate (12.0%), epi-b-bisabolol (6.6%), guaiol
(5.4%), and b-eudesmol (4.4%), while monoterpenoids (61.2%) were found to be
the major components of the oil of TSG, represented by a-pinene (23.0%), b-pinene
(13.0%), myrcene (6.3%), and sabinene (6.3%). The principal component in both subspecies was a-pinene (22.0 and 23.0%, respectively) and b-pinene (6.5 and 13.0%,
respectively). epi-a-Cadinol, myrcene, and sabinene, detected as principal compounds of TSG, were characterized in lower amounts (<1.5%) in the oil of TSS. Seven
components were identified in the oil of TSS corresponding to 25.9% of total oil,
which were absent in TSG, in which cis-sesquisabinene hydrate (12.0%), guaiol
(5.4%), and b-eudesmol (4.4%) were abundant.
The oils of Teucrium polium (Lamiaceae) and T. montanum consisted mainly of
sesquiterpenes (64.3 and 72.7%, respectively), with germacrene D (4; 31.0%) and
d-cadinene (10; 8.1%) as the main constituents, respectively (Radulovic´ et al.,
2012). In contrast, the monoterpene menthofuran (1; 11.9%) predominated in the
oil of T. scordium ssp. scordioides, which clearly distinguished this species from

the other Teucrium taxa.
Hydrodistilled essential oils of 21 accessions of Ocimum basilicum belonging to
two different varieties (Ocimum basilicum var. purpurascens and Ocimum basilicum
var. dianatnejadii) from Iran were characterized by GC-FID and GC–MS analyses
(Pirmoradi et al., 2013). The oil yield was found to be between 0.6 and 1.1%
(v/w). Forty-nine compounds, accounting for 96.6–99.7% of the oil compositions,


12

Medicinal plants: chemistry, biology and omics

Monoterpene hydrocarbons
Oxygenated monoterpenes

60

Sesquiterpene hydrocarbons
Oxygenated sesquiterpenes
Aromatic compounds

Essential oil content (%)

50

40

30

20

10

0
.3

no

.5 o.1 .12 .11 o.2 o.9 o.4 o.6 o.7 o.8 .10 .17 .19 .13 .16 .14 .20 .18 .21 .15
n no no
n
n
n
n
n no no no no no no no no no no
n

no

O. basilicum var. purpurascens

O. basilicum var. dianatnejadii

Figure 1.7 Essential oil content (percentage) and composition of 21 O. basilicum accessions.

were identified. Aromatic compounds, represented mainly by methyl chavicol (33.6–
49.1%), and oxygenated monoterpenes, represented by linalool (14.4–39.3%), were
the main components in all essential oils (Figure 1.7). Monoterpene hydrocarbons
were present in the essential oils of all accessions of the Ocimum basilicum var. purpurascens, whereas they were completely absent in those of the Ocimum basilicum
var. dianatnejadii, indicating that monoterpene hydrocarbons might be considered

as marker constituents of the Ocimum basilicum var. purpurascens. The cluster analysis (CA) showed a clear separation of the Ocimum basilicum var. purpurascens
accessions and the Ocimum basilicum var. dianatnejadii accessions, although the data
showed no major chemotype variation between the studied varieties. The CA revealed
only one principal chemotype (methyl chavicol/linalool) for both varieties.
The essential oil variability in seven native populations belonging to different
infraspecific taxa of Pinus nigra (Pinus nigra ssp. nigra, Pinus nigra var. gocensis,
Pinus nigra ssp. pallasiana, and Pinus nigra var. banatica) growing wild in Serbia
was analyzed (Sarac et al., 2013). In the needles of 195 trees from seven populations,
58 essential oil components were identified. The major components were a-pinene
(43.6%) and germacrene D (29.8%), composing 73.4% of the total oil composition.
Based on the average chemical profile of the main terpene components (with contents
>5%), the studied populations were found to be most similar to populations from central Italy and Greece (Pinus nigra ssp. nigra). CA showed the division of the populations into three principal groups: the first group consisted of populations I, II, III, IV,


Chemotaxonomy: a phylogeny-based approach

13

and V (considered as Pinus nigra ssp. nigra group); the second of population VI
(Pinus nigra ssp. pallasiana group); and the third of population VII, which had the
most distinct oil composition (Pinus nigra ssp. banatica group).
Three relict conifers are clearly separated according to terpene profile with 22
common compounds (Nikolic´ et al., 2011). In addition, Picea omorika has the most
abundant O-containing monoterpenes and sesquiterpenes; Pinus heldreichii and Pinus
peuce have the largest abundance of sesquiterpene and monoterpene hydrocarbons.
The chemosystematic value of the total ketone content, especially of thujone isomers
and fenchone, is confirmed (Tsiri et al., 2009), as oil analysis for Thuja genus (Cupressaceae) has been proved as a reliable chemosystematic tool in previous studies on different species and subspecies.
The essential oil compositions of leaves, flowers, and rhizomes of Alpinia galanga
(Zingiberaceae), A. calcarata, A. speciosa, and A. allughas were examined and compared by capillary GC and GC–MS (Padalia et al., 2010). Monoterpenoids were the
major oil constituents. 1,8-Cineole, alpha-terpineol, (E)-methyl cinnamate, camphor,

terpinen-4-ol, and a- and b-pinenes were the major constituents commonly distributed
in leaf and flower essential oils. The presence of endo-fenchyl acetate, exo-fenchyl
acetate, and endo-fenchol was the unique feature of rhizome essential oils of
A. galanga, A. calcarata, and A. speciosa. The rhizome oil of A. allughas was dominated by b-pinene. Significant qualitative and quantitative variations were observed
in essential oil compositions of the different parts of Alpinia species growing in
subtemperate and subtropical regions of northern India. CA was performed to find
similarities and differences in essential oil compositions based on representative
molecular skeletons. 1,8-Cineole, terpinen-4-ol, camphor, pinenes, (E)-methyl cinnamate, and fenchyl derivatives were used as chemotaxonomic markers.
To evaluate the chemotaxonomic significance of the essential oils of 23 populations of 18 Iranian Ferula (Umbelliferae) species, the chemical composition of the
oils was investigated by GC-FID and GC–MS (Kanani et al., 2011). Eighty-four
constituents, representing 81.3–99.7% of the total composition of the oils, have been
identified. The main constituents were a-terpinyl acetate (73.3%), 2,3,4trimethylthiophene (2; 49.0%), sabinene (75.3%), verbenone (5; 69.4%), b-pinene
(59.0–66.3%), and (Z)-b-ocimene (41.7%). CA of the percentage content of the essential oil components of the Ferula species resulted in the characterization of four
groups, that is, taxa containing either (i) monoterpene hydrocarbons, (ii) oxygenated
monoterpenes, (iii) organosulfur compounds, or (iv) monoterpene, sesquiterpene, and
aliphatic hydrocarbons (Figure 1.6d) as the principal classes of compounds. The
chemical independence of F. hirtella from F. szowitsiana and of F. galbaniflua from
F. gummosa at the specific level was concluded and their positions as distinct species
were confirmed.
The essential oil analysis is also useful in chemotaxonomy of Hypericum (Guttiferae; Yuce and Bagci, 2012).
Iridoid, derivative of monoterpene, is useful in chemotaxonomy of Linaria (Scrophulariaceae; Guiso et al., 2007) and Veronica L. (Plantaginaceae; Saracoglu et al.,
2011). From a qualitative point of view, the iridoidic pattern of the two accessions
of Crucianella maritima was similar (Venditti et al., 2014), since the same compounds


14

Medicinal plants: chemistry, biology and omics

(asperuloside, asperulosidic acid, and deacetyl asperulosidic acid) were isolated.

Asperuloside was the main compound in both accessions. Asperulosidic acid was
the second most abundant compound in the accession from Sardinia, while the accession from Latium exhibited a similar amount of asperulosidic acid and deacetyl asperulosidic acid. These iridoids can be chemotaxonomic markers for Rubiaceae family,
especially for the Rubioideae subfamily to which C. maritima belongs.
Several roots or rhizomes of rubiaceous species are used as the emetic and antiamoebic drug ipecac. True ipecac (Carapichea ipecacuanha) is chemically well characterized, in contrast to striated or false ipecac derived from the rhizomes of Ronabea
emetica (syn. Psychotria emetica; Rubiaceae). Besides its previous use as substitute of
ipecac, the latter species is applied in traditional medicine of Panama, and fruits of its
relative Ronabea latifolia are reported as curare additives from Colombia. Compounds of R. emetica were isolated using standard chromatographic techniques
(Berger et al., 2011) and structurally characterized by NMR spectroscopy and MS.
Organ-specific distribution in R. emetica as well as in R. latifolia was assessed by
comparative HPLC analysis. Four iridoid glucosides, asperuloside, 6a-hydroxygeniposide, deacetylasperulosidic acid, and asperulosidic acid, were extracted from
leaves of R. emetica. Rhizomes, used in traditional medicine, were dominated by deacetylasperulosidic acid. HPLC profiles of R. latifolia were largely corresponding.
These results contrast to the general tendency of producing emetine-type and indole
alkaloids in species of Psychotria and closely related genera and merit chemotaxonomic significance, characterizing the newly delimited genus Ronabea. Chemotaxonomy resolves the historic problem of adulteration of ipecac by establishing the
chemical profile of R. emetica, the false ipecac, as one of its less known sources.
Licorice (Glycyrrhiza glabra, Leguminosae) is a plant of considerable commercial
importance in traditional medicine and for the flavor and sweets industry. Glycyrrhiza
species are very competitive targets for phytochemical studies, and knowledge about
the volatile composition is important for understanding the olfactory and taste properties. Volatile constituents from G. glabra, G. inflata, and G. echinata roots were
profiled using steam distillation and solid-phase microextraction (Farag and
Wessjohann, 2012). Two phenols, thymol and carvacrol, were found exclusively in
essential oil and headspace samples of G. glabra and with highest amounts for
samples that originated from Egypt. In G. echinata oil, (2E, 4E)-decadienal (21%)
and b-caryophyllene oxide (24%) were main constituents, whereas 1a, 10a-epoxyamorpha-4-ene (13%) and b-dihydroionone (8%) predominated G. inflata. Principal
component and hierarchical cluster analyses clearly separated G. echinata and
G. inflata from G. glabra, with phenolics and aliphatic aldehydes contributing mostly
for species segregation. Thymol and carvacrol, exclusively in G. glabra, could serve
as chemotaxonomic markers and might be considered as potentially relevant for taste.
Vibrational spectroscopy can be used to discriminate between different essential
oil profiles from individual oil plants of the same species (chemotypes) (Baranska
et al., 2005). The spectroscopic data correlate very well with those found by GC analysis. Electronic-nose (e-nose) instruments, derived from numerous types of aromasensor technologies, have been used in wood chemotaxonomy (Wilson, 2013). The

volatile compound BinBase mass spectral database is well suited for between-study


Chemotaxonomy: a phylogeny-based approach

15

comparisons of chemotaxonomy investigations (Skogerson et al., 2011). Together, oil
analysis can be of considerable help, providing basic information needed for the chemosystematic approach of a genus.

1.2.2.2 Sesquiterpene
The sesquiterpene dialdehyde contents can be used to differentiate Pseudowintera
(Winteraceae) species (Wayman et al., 2010). P. insperata individuals had high levels
(3.0–6.9% of leaf dry wt.) of the coumarate, P. axillaris had high levels (2.2–6.9%) of
paxidal, and P. colorata from different areas of New Zealand contained varying levels
of polygodial (1.4–2.9%) and 9-deoxymuzigadial (0–2.9%).
The New Caledonian endemic Treubia isignensis var. isignensis, which is a morphologically primitive liverwort, was extracted with diethyl ether, and the crude
extract analyzed by TLC and GC–MS (Coulerie et al., 2014). The species is chemically very primitive since it produces only maaliane, eudesmane, aristolane, and gorgonane sesquiterpene hydrocarbons, which are significant chemical markers of the
species; neither oxygenated terpenoids nor aromatic compounds were detected.
a-Bisabolol (Figure 1.8) is a commercially important aroma chemical currently
obtained from the candeia tree (Vanillosmopsis erythropappa; Asteraceae). Continuous overharvesting of the candeia tree has prompted the urgent need to identify alternative crops as a source of this sesquiterpene alcohol. A chemotaxonomic assessment
of two Salvia species indigenous to South Africa recommended them as a potential
source of a-bisabolol (Sandasi et al., 2012). The essential oil obtained by hydrodistillation of the aerial parts was analyzed by GC–MS and mid-infrared spectroscopy
(MIRS). Orthogonal projections to latent structures discriminant analysis (OPLSDA) were used for multivariate classification of the oils based on GC–MS and MIRS
data. Partial least squares (PLS) calibration models were developed on the MIRS data
for the quantification of a-bisabolol using GC–MS as the reference method. A clear
distinction between Salvia stenophylla and S. runcinata oils was observed using
CHO

H

CHO

HO

H

α-Bisabolol

Polygodial
HO
O

O

HO
O

O

Cnicin

OH

Figure 1.8 Sesquiterpene.