INAUGURAL DISSERTATION
submitted to the
Combined Faculties for the Natural Sciences and for Mathematics
of the Ruperto-Carola University of Heidelberg, Germany
for the degree of
Doctor of Natural Sciences
Presented by
Florian Herrmann
Apotheker (Staatsexamen)
born in Karlsruhe, Germany
Oral examination: 12.10.2011
TCM Plants: DNA Barcoding, Cytotoxic and
Trypanocidal Properties of Drugs and Their Active
Constituents
This work was carried out in the
Department of Biology,
Institute for Pharmacy and Molecular Biotechnology (IPMB),
Im Neuenheimer Feld 364, 69120 Heidelberg
at the Ruprecht-Karls-University of Heidelberg
between February 2006 and August 2011.
Head of division: Prof. Dr. Michael Wink
Supervisor:
Prof. Dr. Michael Wink
Department of Biology, IPMB
Ruprecht-Karls-University Heidelberg
1st Referee:
Prof. Dr. Michael Wink
Department of Biology, IPMB
Ruprecht-Karls-Universiität Heidelberg
2nd Referee:
Prof. Dr. Thomas Efferth
Institute for Pharmacy and Biochemistry
Johannes Gutenberg-Universität Mainz
2
Publications
F. Herrmann, R. Hamoud, F. Sporer, A. Tahrani, M. Wink: Carlina oxide – a natural
polyacetylene from Carlina acaulis (Asteraceae) with potent antitrypanosomal and
antimicrobial properties!
In press, Planta Medica; published online June 15, 2011
DOI http://dx.doi.org/10.1055/s-0031-1279984
F. Herrmann, M. Wink: Synergistic interactions of saponins and monoterpenes in HeLa cells,
Cos7 cells and in erythrocytes
In press, Phytomedicine
F. Herrmann, F. Sporer, A. Tahrani, M. Wink: Antitrypanosomal properties of Panax ginseng
CA Meyer – new possibilities for a remarkable traditional drug!
Submitted, Phytotherapy Research
F. Herrmann, M. Wink: Cytotoxicity of structurally diverse ginsenosides from Panax ginseng
in HeLa cells is linked to membrane disturbances
Submitted, Journal of Pharmacy and Pharmacology
F Herrmann, M. R. Romero, A.G. Blazquez, D. Kaufmann, M. L. Ashour, S. Kahl, J.J.G.
Marin, T. Efferth, M. Wink: Cytotoxicity, antiviral and antitrypanosomal screening of 82
plants from Chinese and European phytomedicine
Submitted, Diversity
F. Herrmann, M. Wink: Use of rbcL sequences for DNA barcoding and authentication of
plant drugs used in Traditional Chinese Medicine
Submitted, Zeitschrift für Naturforschung
N.Z. Mamadalieva§, F. Herrmann§, M.Z. El-Readi, A. Tahrani, R. Hamoud, D. Egamberdieva,
Sh.S. Azimova, M. Wink: Flavonoids in Scutellaria immaculata and S. ramosissima and their
biological activities
In press, Journal of Pharmacy and Pharmacology
§
Equal distributed authors
3
T. Efferth, F. Herrmann, A. Tahrani, M. Wink: Cytotoxic activity towards cancer cells of
arteanuine B, artemisitene, scopoletin and 1.8-cineole derived from Artemisia annua L. in
comparison to artemisinin
In press, Phytomedicine 2011
DOI 10.1016/j.phymed.2011.06.008
D. Hamdan, M. Z. El-Readi, A. Tahrani, F. Herrmann, D. Kaufmann, N. Farrag, A. El-Shazly,
M. Wink: Chemical composition and biological activity of Citrus jambhiri Lush.
Food Chemistry 2011, 127: 394-403
D. Hamdan, M. Z. El-Readi, A. Tahrani, F. Herrmann, D. Kaufmann, N. Farrag, A. El-Shazly,
M. Wink: Secondary metabolites of ponderosa lemon (Citrus pyriformis Hassk) and their
antioxidant, anti-inflammatory and cytotoxic activities
In press, Zeitschrift für Naturforschung 2011
S. Mulyaningsih, M. Youns, M. Z. El-Readi, M. L. Ashour, E. Nibret, F. Sporer, F. Herrmann,
J. Reichling, M. Wink: Biological activity of the essential oil of Kadsura longipedunculata
(Schisandraceae) and its major components
Journal of Pharmacy and Pharmacology 2010, 62: 1037–1044
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Table of Contents
Summary………………...………………………………………………………………..….…....7
Summary (German)……………………………………………………………………...………..8
1. General Introduction…………………………………………...………...…………….………9
1.1 Traditional Chinese Medicine……………………………………………….………..9
1.1.1 Traditional Chinese Medicine…………………………………….……………...9
1.1.2 Chinese herbal medicine………………………………………………………..11
1.2 Secondary metabolites………………………………………………………………12
1.2.1 Ecological function…………………………………………………………...12
1.2.2 Biosynthesis…………………………………………………………………..12
1.2.3 Storage………………………………………………………………………..12
1.2.4 Classes of secondary metabolites……………………………………………….13
1.2.5 Mode of action………………………………………………………………..14
1.3 African Trypanosomiasis……………………………………………………………17
1.3.1 Epidemiology…………………………………………………………………17
1.3.2 Classification……………………………………………………………………17
1.3.3 Biology………………………………………………………………………..17
1.3.4 Control………………………………………………………………………..19
1.3.5 Targets……...………...……………………………………………………….21
1.4 References…………………………………………………………………………...24
2. Use of rbcL sequences for DNA barcoding and authentication of plant drugs used in
Traditional Chinese Medicine………………..………………………...……………………….31
2.1 Abstract……………………………………………………………………………...31
2.2 Introduction………………………………………………………………………….31
2.3 Material and Methods……………………………………………………………….34
2.4 Results……………………………………………………………………………….34
2.5 Discussion…………………………………………………………………………...36
2.6 References…………………………………………………………………………...42
3. Cytotoxicity, antitrypanosomal and antiviral screening of 82 plants from Chinese and
European phytomedicine………………………..………………………………………………45
3.1 Abstract……………………………………………………………………………...45
3.2 Introduction………………………………………………………………………….45
3.3 Material and Methods……………………………………………………………….48
3.4 Results and Discussion……………………………………………………………...56
3.5 References…………………………………………………………………………...74
5
4. Cytotoxicity of 12 structurally diverse ginsenosides from Panax ginseng in HeLa cells is
linked to membrane disturbance…………………………...………...…………………………81
4.1 Abstract……………………………………………………………………………...81
4.2 Introduction………………………………………………………………………….81
4.3 Material and Methods……………………………………………………………….84
4.4 Results……………………………………………………………………………….85
4.5 Discussion…………………………………………………………………………...85
4.6 References…………………………………………………………………………...91
5. Antitrypanosomal properties of Panax ginseng CA Meyer – new possibilities for a
remarkable traditional drug!.......................................................................................................95
5.1 Abstract……………………………………………………………………………...95
5.2 Introduction………………………………………………………………………….95
5.3 Material and Methods……………………………………………………………...101
5.4 Results…………………………………………………………………………..….105
5.5 Discussion……………………………………………………………………….....107
5.6 References………………………………………………………………………….116
6. Carlina oxide – a natural polyacetylene from Carlina acaulis (Asteraceae) with potent
antitrypanosomal and antimicrobial properties!......................................................................126
6.1 Abstract…………………………………………………………………………….136
6.2 Introduction………………………………………………………………………...126
6.3 Material and Methods……………………………………………………………...127
6.4 Results……………………………………………………………………………...132
6.5 Discussion………………………………………………………………………….133
6.6 References………………………………………………………………………….138
7. Synergistic Interactions of Saponins and Monoterpenes in HeLa cells, Cos7 Cells and in
Erythrocytes.…………………...…………………………………...…………………………..141
7.1 Abstract…………………………………………………………………………….141
7.2 Introduction………………………………………………………………………...141
7.3 Material and Methods……………………………………………………………...144
7.4 Results……………………………………………………………………………...146
7.5 Discussion………………………………………………………………………….148
7.6 References………………………………………………………………………….157
8. General conclusion and outlook………………………………………..…………………...160
9. Acknowledgements…..………………………………………………..……………………..163
10. Appendix………………………………………………………………………..…………...165
6
Summary
Our extensive screening of 82 herbal drugs used in traditional Chinese and European
Phytomedicine revealed the great potential still hidden in this field of research. The correct
authentication of plants used in research on traditional medicinal systems such as TCM is
crucial before any serious research can be started. DNA barcoding offers a reliable, fast
possibility to overcome this obstacle. Only one adulteration could be detected where Arctium
lappa was wrongly labelled Fraxinus rhynchophylla.
After screening 246 extracts of herbal drugs against a variety of targets such as trypanosomes,
viruses and cancer cells we were able to select several highly promising plants for further
research. We decided to focus our research on Panax ginseng, Araliaceae, a well known
herbal medicine of international fame and a long history of traditional use.
Our experiments revealed that ginsenosides, a highly variable group of saponins, disturb the
biomembrane of cells, while the exact position of the sugar moieties and the resulting ability
to be incorporated into the lipid bilayer of the cell membrane explain the great differences in
their cytotoxicity. The membrane activity of the ginsenosides however was not responsible for
the trypanocidal effect of ginseng revealed in our screening. Panaxynol, the major
polyacetylene of ginseng was with an IC50 concentration of 0.01 µg/ml and a selectivity index
of 858 three times as selective against Trypanosoma brucei brucei as established
antitrypanosomal drugs like suramin. We were able to discover with Carlina oxide isolated
from Carlina acaulis, Asteraceae, a second polyacetylene highly selective in its cytotoxicity
against T. b. brucei with an IC50 of 1.0 µg/ ml and a selectivity index of 446. We suggest that
the inhibition of the trypanothione reductase, essential for the resistance of the parasite against
oxidative stress and unique to trypanosomes, is the primary principle of this selectivity.
Our previous results obtained from the activity of ginsenosides had shown us the efficacy, but
also the structure dependency of the membrane effects of saponins. With monoterpenes we
have a second group of structurally completely different natural products that also target the
biomembrane. The question arose if their omnipresence in phytomedicine and their
application in combination would be more than just a coincidence. We combined several
saponins with three monoterpenes and explored their effect on biomembranes modelled by
erythrocytes and living cells. Astonishingly, the combination lowered the dosis necessary for
haemolysis up to the factor 100, which was also confirmed in living cells. This revealed that
monoterpenes and saponins are working together synergistically by enhancing their activity
far above a simple additive effect.
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Summary (German)
Wir untersuchten in einem umfangreichen Screening 82 Pflanzendrogen der traditionellen
Chinesischen und Europäischen Phytomedizin. Dabei bildet die korrekte Identifizierung der
zu testenden Arten die entscheidende Voraussetzung für jegliche weiterführende
Untersuchung. Wir benutzten erfolgreich DNA Barcoding zur Identifizierung und fanden nur
eine einzige Verfälschung: Arctium lappa war fälschlicherweise als Fraxinus rhynchophylla
ausgegeben worden. Durch unser umfangreiches Screening von 246 Extrakten gegen
Trypanosomen, Viren und Krebszellen gelang es uns, mehrere vielversprechende Pflanzen für
weiterführende Untersuchungen auszuwählen. Besonders interessant erschien uns dabei
Panax ginseng, Araliaceae, eine bemerkenswerte Heilpflanze mit langer Geschichte.
Die Wirkung von Ginseng wird üblicherweise den Ginsenosiden, einer speziellen Gruppe von
Saponinen, zugeschrieben. Dementsprechend untersuchten wir die Wirkung von 12
Ginsenosiden und konnten feststellen, dass die Position der Zuckergruppe am Molekül
hauptverantwortlich für die großen Unterschiede in ihrer zytotoxischen Wirkung auf
Zellmembranen ist. Eine Erklärung für die im Screening beobachtete Wirkung der
Ginsengextrakte gegenüber Trypanosomen lieferten diese Ergebnisse jedoch nicht. Bei
näherer Untersuchung der Hexanextrakte konnten wir feststellen, dass Panaxynol, das
Hauptpolyacetylen von Ginseng Trypanosoma brucei brucei gegenüber hoch selektiv ist (IC50
1.0 µg/ ml). Der Selektivitätsindex war mit 858 dreimal höher als der gängiger Arzneistoffe
wie Suramin. Mit Carlinaoxid aus Carlina acaulis, Asteraceae, konnten wir ein weiteres
gegenüber Trypanosomen hochselektives Polyacetylen entdecken (IC50 1.0 µg/ ml bei einem
Selektivitätsindex von 446). Als Wirkungsmechanismus für beide Polyacetylene schlagen wir
die Hemmung der Trypanothionreduktase vor; dieses Enzym kommt nur in Trypanosomen
vor und ist unverzichtbar für deren Schutz vor oxidativem Stress.
Die Membranaktivität der Ginsenoside veranlasste uns, die Wirkung von Saponinen in der
Phytomedizin näher zu untersuchen. Besonders auffällig ist dabei, dass Saponindrogen oft mit
Monoterpendrogen in Erkältungstees kombiniert werden. Da sich beide Stoffgruppen
strukturell stark unterscheiden, aber trotzdem denselben Angriffspunkt, die Biomembran,
besitzen, wollten wir wissen, ob diese Kombination die Wirkung verstärken würde. Wir
kombinierten mehrere Saponine mit Monoterpenen und untersuchten ihre Wirkung auf
kernlose Erythrozyten und lebende Zellen. Erstaunlicherweise senkte diese Kombination die
für Hämolyse nötige Dosis an Saponin um den Faktor 100, was sich auch in lebenden Zellen
bestätigte. Dies zeigt, dass Monoterpene und Saponine ihre Aktivität weit über einen
additiven Effekt hinaus synergistisch verstärken.
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1. General Introduction
1.1 Traditional Chinese Medicine
1.1.1 Traditional Chinese Medicine
Traditional Chinese Medicine is as old as China itself. The beginnings of acupuncture and
herbal medicine seem to date back to
even before the Shang Dynasty (c. 1600
to 1045 BC) [1-4]. Our main knowledge
besides historical descriptions from later
dynasties such as the Shiji by Sima Qian
is based on oracle bones with an archaic
form of Chinese characters and bronze
vessels. Basic Chinese imperial ideas
such as the legitimization of the ruler by
the Mandate of Heaven or ancestor
worship date to this period [2-4].
The philosophical concept of the TCM
theory however developed during the
Han Dynasty (202 BC to 220 AD) [1,5].
The Han Dynasty is generally regarded
as the period when classical Chinese
culture
Fig. 1: Shi si jing fa hui (Expression of the Fourteen
Meridians)
Tokyo : Suharaya Heisuke kanko, Kyoho gan 1716
became
consolidated.
Confucianism was adopted as state
philosophy, and the country made great
advances in many areas of the arts and sciences. Military expansion enabled the Han Dynasty
to form trade links across the Silk Road to the west [2-4]. In this climate of progress medicine
could also flourish and changed from local traditions to a theoretical concept that was
accepted all over China. Confucianism was essential for this development by creating the
ideological framework the different local adaptations could be fitted into [1]. The famous
Huangdi Neijing, commonly regarded as the beginning of TCM, dates to the second century
BC [1]. From then, the medicinal theory developed continuously until the system we know
today was created. This development is always linked to individual, outstanding physicians,
such as Hua Tuo, who lived during the late Han Dynasty and introduced surgery, or Sun
Simiao who wrote the first Chinese encyclopedia of medicine during the Tang Dynasty (618
to 907 AD) [1]. Li Shizhen wrote the essential book on the Chinese herbal medicine, the
9
Compendium of Materia Medica (Bencao Gangmu) during the Ming Dynasty (1368 to 1644
AD) [1]. TCM experienced a revival in simplified form under Mao who wanted the system
standardized to overcome the catastrophic healthcare situation in the countryside. Based on
this, the modern TCM developed and became increasingly popular in the west.
Theoretical concept of TCM
Contrary to western medicine, TCM has a holistic approach to illness and medical treatment.
The main concept is to balance the different functions of the body [5]. According to TCM, all
aspects of the human body are linked, which can be exemplified by the pulse or tongue
diagnosis used to draw conclusions about the general state of health of the patient [6]. An
illness is commonly regarded as a disturbance of the harmony, which can be diagnosed in
various ways [5]. Due to the holistic approach, the treatment also has to be a holistic one.
Rather than to treat the symptoms or a specific illness, the concept seeks to restore the
harmony and thus to treat the roots of the illness.
To understand TCM, it is necessary to know some of the basic principles. The concept of
dualism, commonly refered to as yin and yang is crucial for TCM [5,6]. Yin and yang, the two
aspects of everything, are the basis of all treatment. Stemming from this are the qi, the five
elements, and the meridians [5,6]. Qi is often explained as life energy, but has a far broader
application in Chinese philosophy. The five elements are closely linked to the organs of the
body and are essential for the general diagnosis. The meridian system, on the other hand, is
used in the treatment and detailed analysis of the health disorder (Fig. 1). Disturbances of the
flow of qi, the life energy, can be diagnosed in this way. The theoretical basis of acupuncture
can be found here as well [5].
From the western point of view the complex theoretical system of TCM is a way to discribe
symptoms. The resulting treatment is based on long evidence based experimental knowledge
integrated into the philosophical world view of the Chinese cultural tradition.
Major methods of treatment
Acupuncture is the best known aspect of TCM and is increasingly used in the west as well to
treat pain related health problems [6]. Patients are treated by insertion of needles into the body
on the meridian points. It is often combined with moxibustion, the burning of Artemisia
closely above the meridian points. Two variations exist – either the moxa (Artemisia spec.) is
10
held above the meridian points and burned or it is added on top of an acupuncture needle and
then ignited. Another common method to treat health problems is the cupping (Ba Guan) [6].
Several glass cups are placed on top of specific body parts, commonly the back. Due to an
applied vacuum, the cups form pressure on the skin. The physician can diagnose the health
problem from the effect on the skin. It is also used to treat muscle pain and back ache, and is
partly related to massage techniques. The Tui Na massage is another method to restore the
undisturbed flow of qi in the body and thus forms an important part in the holistic treatment
traditionally applied in TCM [6].
1.1.2 Chinese herbal medicine
Chinese herbal medicine is integrated
into the theoretical concept of TCM by
sorting the herbs according to attributed
properties such as cooling, heating, etc
[7-11]. According to the theoretical
concept, they are used to counterbalance
deficiencies caused by diseases. Diseases
causing a lack of heat thus are treated
with herbs that possess heating properties
to restore the balance in the human body
[5,6,9]. However, we should not regard it
as so simple since the long historical use
and the huge number of herbs used in
TCM allows a very individualized and
specific
treatment
(Fig.
2).
The
combination of 10 or more herbs per
formulation gives the physician the
Fig. 2: Chinese herbal medicine store in
Beijing, 2006
possibility to individualize the treatment in a way not possible in western medicine [9].
Behind the theoretical concept of TCM thus is a solid phytotherapy based on thousands of
years of experience. The large area of China combines various climatic zones from tropics
over deserts to high altitudes, providing the basis for an especially rich flora [2].
Consequently TCM uses a huge number of medicinal plants that by far exceeds those used in
European phytotherapy [7-11]. Currently, 4773 herbs are officially recognised for treatment in
TCM, not to mention locally used plants that are not part of the Chinese pharmacopoeia [8].
11
1.2 Secondary metabolites
1.2.1 Ecological function
Lacking the ability to avoid predators by flight or fight and lacking an immune system to fight
microbes, plants developed two other methods to survive during 500 million years of
evolution. One is the physical protection by bark or thorns, the other, secondary metabolites
[12-18]. They are present in one form or another in all plants and form the most effective yet
often unspecific defence system imaginable. So far more than 100 000 different substances
have been identified that can be grouped in nitrogen-containing (alkaloids, amines, nonprotein amino acids, peptides, etc) and nitrogen-free (terpenes, phenolics, saponins,
polacetylenes, etc) molecules [12]. They are synthesized in specific pathways and stored in
specifically for this task developed compartments and cells. Due to their complexity, they
form a highly effective defence system against herbivores, fungi, bacteria and even viruses.
Additionally, they are also used in the struggle between plants for ecological advantages by
attracting animals for pollination or by hindering the growth of competing plants for light or
water [12]. Especially rich in secondary metabolites are usually those parts vital for
reproduction such as seeds, flowers, etc [12].
1.2.2 Biosynthesis
Contrary to the huge variability of natural products, the pathways used for their biosynthesis
are few. Precursors are usually products of the primary metabolic pathways such as glucose or
amino acids. These primary metabolites are the basis for the secondary metabolism. Here, the
primary metabolites are transformed into secondary metabolites such as terpenes, saponins,
alkaloids or tannins. Especially three pathways are essential for all plants: The glycolysis, the
Krebs cycle and the Shikimate pathway. They are not only essential for the common
processes in the cells such as respiration but also transform the primary metabolites into
secondary metabolites. The different classes of secondary metabolites can generally be
attributed to different pathways. While terpenes, saponins or fatty acids are among the
products of the glycolysis, alkaloids are either products of the Krebs cycle or the Shikimate
pathway. The Shikimate pathway is also involved in the production of flavonoids and tannins
[19-25].
1.2.3 Storage
Secondary metabolites usually need to be transported from their place of origin to the storage
compartments. This transportation can occur within the cell or via the xylem and phloem to
specific storage cells in remote parts of the plant.
12
Hydrophilic secondary metabolites are mainly stored in the vacuole but also in laticifers and
cell walls [19,26-32]. Most alkaloids, saponins and flavonoids are commonly found in the
vacuole, while several specific alkaloids are concentrated in the laticifers. Tannins are usually
found in the vacuoles and cell walls. One peculiar feature is that many hydrophilic molecules
such as saponins are stored as prodrugs and are only activated once the cell is hurt. The
disruption of the cell compartments leads to the release of enzymes that activate the molecules
to their final form.
Lipophilic secondary metabolites can be stored in specific oil cells, the cuticle, resin ducts or
simply in the plastid membranes [19,26,33]. Oil cells are a specific defence mechanism rich in
terpenoids. Closely related is the storage of waxes and terpenoids in the cuticle. Resin ducts
and plastid membranes also contain terpenoids and even lipophilic flavonoids.
1.2.4 Classes of secondary metabolites
Alkaloids are the most variable group of secondary metabolites [14-17,34-38]. They usually
interact with specific target proteins such as ion channels, receptors or enzymes. Similarities
to neurotransmitters like acetylcholine or noradrenalin enable them to modify neuronal signal
transduction [15,34,39-45].
Non-protein amino acids are analogues of the protein amino acids. They can be incorporated
into proteins by enzymes instead of common amino acids, resulting in the malfunction of vital
processes [34,46].
Cyanogenic glucosides are in the vacuole stored prodrugs that are activated by the rupture of
the cell. β-glucosidases cleave the sugar off the molecule which is hydrolyzed into
hydrocyanic acid (HCN) and an aldehyde. HCN is extremely reactive and blocks the
mitochondrial respiration [34,40,47-50].
Glucosynolates are also stored as prodrugs and only activated by cell damage. They are
cleaved into mustard oil by myrosinase and interact with biomembranes, enzymes or DNA
[34,48-50].
Terpenes are a group of highly variable lipophilic molecules that interact with biomembranes
and the lipophilic core of proteins. Their membrane activity is rather unspecific, resulting in
an increase of membrane fluidity [34,50].
Saponins also target the biomembrane [34,50,51]. They are glycosides of triterpenes or
steroids and appear as mono- and bidesmosidic molecules. Bidesmosidic saponins are
prodrugs that need enzymatic activation prior to be able to interact with biomembranes.
Monodesmosidic saponins are amphiphilic molecules that interact with the lipid bilayer and
13
proteins of the biomembrane. One subgroup of the saponins is the cardiac glycosides that
target the Na+-K+-ATPase.
Flavonoids and polyphenols are phydrophilic molecules that interact with proteins via
phenolic hydroxyl groups. While flavonoids are sometimes target specific and interact with
enzymes, the polyphenols with more phenolic hydroxygroups are usually much less specific
and interact with all proteins present [14,34,50,52,53].
Fig. 3: Targets for secondary metabolites in animal cells after Wink 2008 [14]
1.2.5 Mode of action
The animal cell offers a wide range of targets for secondary
metabolites (Fig. 3). The principal ones are proteins, the
biomembrane and nucleic acids.
Proteins are responsible for almost all processes in the cell. They
act as receptors, ion channels, regulatory molecules such as
transcription factors or signal molecules, transporters, catalytic
enzymes and structural molecules. Their functionality depends on
their three dimensional structure – any conformational change
usually modifies the activity of the protein [19,34]. Most
secondary metabolites interact with proteins unselectively (Fig. 4).
14
Fig. 4: Unspecific interaction of polyphenols with
proteins after Wink 2008 [14]
They are not specific for certain proteins but interact with all proteins by forming covalent or
ionic bonds with free amino-, SH- or OH-groups. Molecules with epoxide or aldehyde groups
bind to amino groups, while molecules with double or triple bonds interact with free SHgroups and epoxides. The Hydroxylgroups of polyphenols dissociate under physiological
conditions into O- ions that form hydrogen and ionic bonds with electronegative atoms and
positively charged side chains of the amino acids.
The presence of several polyphenols leads to
interactions that are strong enough to inactivate
proteins
[19,34].
Besides
the
unselective
interaction of polyphenols a lot of secondary
metabolites interact quite specifically with
enzymes and receptors. They bind to the active
binding site of the molecule due to structural
similarities to endogenous ligands. There, they
either activate or inactivate proteins and disturb
for example the signal transduction of vital
Fig. 5: Interaction of saponins [1], mono- [2,3]
and sesquiterpenes [4] with the biomembrane of
animal cells after Wink and Schimmer 2009 [34]
processes. This effect can be either reversible or,
in worst case, irreversible. Many neurotoxic
alkaloids work this way [19,34,35,40].
The biomembrane (Fig. 5) is the second major target of secondary metabolites [19,34].
Contrary to many interactions with proteins is the interference with the biomembrane purely
unspecific. Monodesmosidic saponins are amphiphilic molecules whose lipophilic part
integrates into the biomembrane while the hydrophilic part interacts with proteins or sugars on
the membrane surface. Thus the fluidity of the lipid bilayer is enhanced while the
conformation of proteins is disturbed.
Additionally
the
interactions
with
cholesterol in the lipid bilayer influence the
microstructure of the biomembrane which
also leads to conformational changes of
receptors on the surface. Monoterpenes
interact with the same target by integrating
into the lipid bilayer and the lipophilic core
Fig. 6: Intercalation of planar natural products into the
DNA after Wink 2008 [14]
15
of membrane proteins. Their interaction is
usually even less specific than that of saponins. They simply increase the membrane fluidity
which results in leakage and eventually the rupture of the biomembrane.
The third major target of secondary metabolites is nucleic acids such as DNA (Fig. 6) and
RNA [15,19,34,41,44,45]. Several natural products interfere with proteins necessary for the
replication of DNA and mRNA. However, there exist also natural products that interact with
the DNA directly. Epoxides and aldehydes like pyrrolizidine alkaloids or furanocoumarins
bind covalently to functional groups of the DNA bases. Planar molecules with aromatic rings
like the alkaloids berberine, emetine, quinidine or furanocumarins intercalate into the DNA by
fitting between adenine-thymine or guanine-cytosine base pairs. There, they can cross-link
DNA strands; a disturbed replication leads to apoptosis, mutations and even cancer.
16
1.3 African Trypanosomiasis
1.3.1 Epidemiology
African trypanosomiasis or sleeping sickness is a parasitic disease found in most sub-Saharan
countries. Human trypanosomiasis is caused by the protozoon Trypanosoma brucei
rhodesiense and T. b. gambiense, while T. b. brucei, T. congolense, T. simiae and T. vivax are
responsible for the cattle disease nagana [54-56]. T. b. gambiense is responsible for the
chronic form of sleeping sickness with 90% of the cases. It occurs in west and central Africa,
while T. b. rhodesiense, found in eastern and southern Africa, represents less than 10% of the
cases and causes the acute form of the disease [57].
African trypanosomiasis is responsible for severe health and economic problems. After being
nearly extinct in the middle of the 20th century, it has been increasing since the independence
of the African countries due to several reasons. The development of resistances against the
commonly used drugs together with a decentralisation of the measures taken to fight African
trypanosomiasis is among the reasons to explain this unfortunate trend. Nowadays, African
trypanosomiasis is endemic again to 36 countries with 60 million people threatened by its
consequences. Estimations of the WHO speak of 300 000 to 500 000 cases of human
trypanosomiasis per year, while at least 46 million cattle are exposed to nagana, the cattle
version of the disease [58-60].
1.3.2 Classification
The different trypanosome species with Trypanosoma
brucei and Trypanosoma cruzi as famous representatives
form part of the order of the Trypanosomatida, a group of
exclusively
parasitic,
single-cell
uniflagellates.
The
Trypanosomatida are an order of the class of the
Domain
Kingdom
Phylum
Class
Order
Genera
Eukaryota
Excavata
Euglenozoa
Kinetoplastida
Trypanosomatida
Trypanosoma
Kinetoplastida, which consists mainly of parasitic, single-cell flagellates. Kinetoplastida can
be separated into uniflagellates (Trypanosomastida) and biflagellates (Bodonida). The
Kinetoplastida are part of the phylum Euglenozoa. Euglenozoa, which are separated into
Kinetoplastida and Euglenida are unicellular organisms. While Euglenida often contain
chloroplasts Kinetoplastida mostly are parasitic organisms. Euglenozoa are part of the
kingdom of Excavata, one of the six kingdoms of the Eukaryota [61].
17
1.3.3 Biology
Human African trypanosomiasis and nagana are transmitted to their mammalian hosts by the
tsetse fly (Glossina species) [54]. Accordingly, the occurrence of sleeping sickness is limited
to the distribution of the tsetse fly. They inhabit most of mid-continental Africa between the
Sahara and the Kalahari deserts. Tsetse flies are usually separated into savannah flies, forest
flies and riverine flies based on their usual habitat and behaviour [62]. Only high altitudes and
deserts are free of them. The name tsetse derives from Tswana, a language spoken in southern
Africa and simply means fly [63].
Fig. 7: Life cycle of Trypanosoma brucei after CDC [64]
Life Cycle
The life cycle of African trypanosomiasis includes a Tsetse fly stage and a human stage (Fig.
7). By taking a blood meal, the infected tsetse fly injects the metacyclic trypomastigotes into
the host. Now the human stage starts with the transformation of the metacyclic
trypomastigotes into bloodstream trypomastigotes. These are distributed in the human body
and multiply by binary fission. At first they occur mostly in the blood and the lymphatic
system. After crossing the blood-brain barrier, they invade the central nervous system and
18
cause irreparable damage to the brain. If left untreated, the disease is in most cases fatal. The
human stage ends for the parasite with the uptake of the bloodstream trypomastigotes by a
new tsetse fly bite. Once the parasite is in the tsetse fly midgut, the bloodstream
trypomastigotes transform into the procyclic trypomastigotes which multiply again by binary
fission. In the next step the procyclic trypomastigotes leave the midgut and transform into
epimastigotes. These wander into the salivary glands of the fly where they multiply once more
by binary fission. Finally, they transform into metacyclic trypomastigotes and the cycle is
closed [64,65].
Kinetoplast
The Kinetoplastida are named after their kinetoplast, a unique feature existing only in this
class of single cell eucaryots. It is found at the base of the flagella and connected to the
flagellum basal body via the cytoskeleton. The kinetoplast is a dense granule containing the
mitochondrial genome with the associated structural proteins and DNA and RNA polymerases
in the single mitochondrion of the cell [66]. The genome of T. brucei is made up of 11 pairs of
large chromosomes, 3 to 5 intermediate chromosomes containing mostly genes responsible
for the antigenic variation such as the VSG genes and up to 100 mini chromosomes [66]. The
genes are organised in polycistronic units, so that they are transcribed at an equivalent rate.
This requires post-transcriptional regulations to control the gene expression. The advantage of
this system is the avoidance of transcription factors which allows a fast reaction to adapt to a
new environment that occurs during the change of the host from tsetse fly to human [67].
Variable Surface Glycoprotein
The Variable Surface Glycoprotein (VSG) hides T. brucei from the host’s immune system,
enabling a chronic infection. The surface of the trypanosome is densely covered with
approximately 1x107 molecules of highly variable VSG, which shields the plasma membrane
from the immune reaction of the host. Each trypanosome expresses only one VSG gene at a
time, while the other genes are inactive [68,69]. VSGs occur in dimers and have a highly
variable N terminal domain of between 300 and 350 amino acids, a conserved C terminal
domain of approximately 100 amino acids and are anchored in the cell membrane by a
glycophosphatidylinositol (GPI) anchor. Their tertiary structure is conservative enough to
form a physical barrier to protect the cell membrane [70]. The immune system reacts strongly
to the VSG protein and would destroy the trypanosome. However, each cell division of the
parasite results in a stochastic genetic modification of the VSG, which makes an immune
19
reaction against T. brucei almost impossible due to the short period between each division
cycle. Accordingly, the trypanosomes occur in waves in the blood and the disease persists
chronically in the host [71].
1.3.4 Control
The control of African trypanosomiasis includes the control of its vector, the tsetse fly and the
treatment of the disease in the patient. Frequently applied measures of vector control include
pesticides, trapping and the release of sterile males. The use of sterile male tsetse flies has
been especially successful due to the specific breeding habit of the flies since tsetse fly
females mate only once in their life. Sterile males are released in an infected area in great
numbers, where they will compete with the local males and mate with the local females.
Accordingly, all unions with sterile males will have no offspring, thus dropping the numbers
of tsetse flies in the area significantly. Land clearing can be locally of use by removing the
habitat of the tsetse fly but are not practicable in the larger scale [54].
Fig. 8: Approved drugs against African trypanosomiasis
20
The medical treatment of African trypanosomiasis is still problematic. Only four drugs are
licensed for the treatment of African trypanosomiasis (Fig. 8): Suramin, pentamidine,
melarsoprol and eflornithine [56,72-78].
Pentamine is the drug of choice against T. b. gambiense while suramin can be used as
alternative. T. b. rhodesiense is preferably treated with suramin. Once the disease invades the
central nervous system, either eflornithine or melarsoprol are applied [79]. Except for
eflornithine, which was introduced in 1990, all drugs for the treatment of African
trypanosomiasis date to the first half of the 20th century. The mode of action of suramin and
melarprosol are unknown, while pentamidine is enriched in the parasite and attacks a whole
range of unspecific targets. Eflornithine is a suicide inhibitor of the ornithine decarboxylase
[80,81]. Unfortunately, all of these drugs have strong side effects and several resistances were
reported recently [82-85], so that the discovery of new drugs is an urgent necessity [86-88].
1.3.5 Targets
Trypanothione reductase
Fig. 9: Trypanothione reductase with binding site after Bond et al., 1999 [92]
The trypanothione reductase (Fig. 9) is an enzyme specific to trypanosomastids and essential
for the protection of the cell against oxidative stress [81,89-92] and an exciting new target for
21
drugs against African trypanosomiasis. Mammalian cells use the closely related gluthatione
reductase instead. Both enzymes are members of the FAD-dependent NADPH oxidoreductase
family. These dimeric molecules protect the cell against reactive oxygen species formed
during the aerobic metabolism, since trypanosomes are extremely sensitive to oxidative stress.
Fig. 10: The equilibrium of trypanothione disulfide and trypanothione is kept on the side of trypanothione by
NADPH and the trypanothione reductase after Krauth-Siegel et al., 2005 [81]
Trypanothione is kept in its reduced form in the cell by NADPH and the trypanothione
reductase (Fig. 10). In the presence of reactive oxygen species like hydroperoxides
trypanothione reacts to trypanothione disulfide while the reactive oxygen species are reduced
[92]. The equilibrium of TS2 and T(SH)2 can only be kept on the side of T(SH)2 if the
trypanothione reductase is working properly. Once
it is inactive, the equilibrium changes rapidly to
TS2 which can not inactive the reactive oxygen
species resulting in the death of the cell.
The two enzymes are able to distinguish their
substrates trypanothione disulfide and glutathione
disulfide by the factor 1000 because of differences
in size and charge [92]. Glutathione disulfide is
smaller and -2 charged at physiological pH, while
trypanothione disulfide is +1 charged with a
hydrophobic body of 7 methylene groups (Fig. 11).
The trypanothione disulfide binding site in the
trypanothione reductase is larger with a negatively
22
Fig. 11: Glutathione disulfide (a) and
trypanothione disulfide (b) after Bond et
al., 1999 [92]
charged glutamate side chain and a hydrophobic cleft for the polyamine moiety. Contrary to
this, the glutathione reductase has a positively charged, smaller active centre. Recently, the
importance of the trypanothione reductase as a target for the selective inhibition of
trypanosomes has gained wide recognition. Research is focusing on the discovery of selective
inhibitors with several promising findings such as polyamine-based inhibitors or tricyclic
compounds [81]. However, none have reached the clinical phase yet. Other trypanothione
disulfide dependent enzymes can also be regarded as potential targets for a selective inhibition
of T. brucei. Especially interesting are those enzymes important for the biosynthesis of
trypanothione disulfide such as S-adenosylmethionine decarboxylase [93,94] or trypanothione
synthetase [93,95].
Trypanosome alternative oxidase
The bloodstream forms of trypanosomes entirely depend on the conversion of glucose into
pyruvate for their ATP production. Under aerobic conditions, only pyruvate is produced,
while under anaerobic conditions glucose is converted into similar amounts of pyruvate and
glycerol. Characteristically for trypanosomes is that glycolysis occurs to the biggest part in
compartments and not in the cytoplasma [96]. The production of ATP depends on a
reoxidation system with the trypanosome alternative oxidase (TAO) as one of the essential
enzymes [93]. TAO is unique for trypanosomes and located in the inner membrane of the
mitochondrion. In the process of ATP production glycerol 3-phosphate is converted into
glycerol, while ADP is converted into ATP. As an essential part of the ATP production TAO
is accordingly an interesting target for the development of new antitrypanosomal drugs.
Without it, the production of ATP depends on high glycerol 3-phosphate concentrations and a
high ADP/ATP ratio. The inhibition of TAO decreases the ATP concentration significantly; in
combination with additional glycerol the ATP production is completely blocked [97].
Accordingly, the complete energy production of trypanosome is inhibited with fatal
consequences for the parasite.
Topoisomerase and kinetoplast DNA
A second important target is the kinetoplast with its unique mitochondrial DNA and
topoisomerases [66]. Several natural products are known to inhibit cell growth by
intercalation into the DNA. Examples are berberine and sanguinarine [93,98]. Camptothecin
is a DNA topoisomerase inhibitor known for its selective cytotoxicity against tumor cells. It
exhibits also a strong cytotoxitic effect against T. brucei [93,99].
23
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Merschjohann K, Sporer F, Steverding D, Wink M (2001). In vitro effect of alkaloids on
bloodstream forms of Trypanosoma brucei and Trypanosoma congolense. Planta Med. 67,
623-627.
99
Bodley AL, Shapiro TA (1995). Molecular and cytotoxic effects of camptothecin, a
topoisomerase I inhibitor, on trypanosomes and Leishmania. Proc Natl Acad Sci USA. 92,
3726-3730.
30
2. Use of rbcL sequences for DNA barcoding and authentication of
plant drugs used in Traditional Chinese Medicine
2.1 Abstract
Traditional Chinese medicine has become increasingly popular in Europe and North America.
There is evidence that quality control in terms of species authentication is sometimes
inappropriate. Repeated incidents of adulterations and wrong identification, some even with
serious consequences have occurred recently. The necessity of a quality control for TCM
plants to avoid these incidents is given since many years. DNA barcoding was used in this
study to authenticate drugs which are often used in Chinese herbal medicine. 37 plants from
28 families were identified using the rbcL gene. Only one adulteration could be detected
where Fraxinus rhynchophylla was substituted with Arctium lappa. Both the advantages and
limitations of rbcL as a marker gene for identification were analysed and discussed. We could
show that DNA barcoding is a valid and fast method to identify medicinal herbs, showing
some advantages over chemical profiling because of its universal application even for
unknown plant species.
2.2 Introduction
The complex nomenclature in Traditional Chinese Medicine is an acknowledged yet unsolved
problem, responsible for potentially fatal confusions [1]. Currently, 4773 botanicals are listed
as used in TCM [2]. The scientific term “species” often does not correlate with the
nomenclature used in TCM. We would like to elaborate this in a few examples. Four main
classifications are common in TCM nomenclature.
1. Drug and plant name are identical, e.g. Panax ginseng is called ren shen ( 参) both
as a drug and as a plant species.
2.
Drug and plant name differ, even though the drug is derived from a single species.
Ginkgo biloba is called bai guo ( 果) as a drug while the species is referred to as yin
xing (银
).
3. The different parts of a particular species have different names as drugs. Trichosanthes
kirilowii is a good example where the plant is called gua lou (瓜蒌); the fruit bears the
same name, while the seed is called gua lou zi (瓜蒌
(瓜蒌
) and the root tian hua fen (
), the pericarpum gua lou pi
).
4. Several plant species can be combined under one drug name, making it impossible to
know which exact species has been used. One example is lao guan cao ( 鹳
31
)
which can be either Erodium stephanianum, Geranium carolinianum or G. wilfordii
(Tab. 1).
Table 1: Examples for the complex nomenclature in Traditional Chinese Medicine
Case
Scientific name of the
plant
Chinese name of
the plant
Part used
Chinese name of
the drug
Case 1
Panax ginseng
ren shen
参
root
ren shen
参
Case 2
Ginkgo biloba
yin xing
银
seed
bai guo
果
Case 3
Trichosanthes kirilowii
gua lou
瓜蒌
fruit
gua lou
瓜蒌
gua lou zi
瓜蒌
chao gua lou zi
瓜蒌
gua lou pi
瓜蒌
tian hua fen
seed
roasted seed
pericarpum
root
Case 4
Erodium stephanianum
Geranium wilfordii
Geranium
carolinianum
mang niu er miao
牻 儿
lao guan cao
鹳
ye lao guan cao
鹳
herb
lao guan cao
鹳
lao guan cao
鹳
lao guan cao
鹳
herb
herb
To make things worse, several substitutions are allowed in TCM, so that han fang ji (
Stephania tetrandra can be substituted by mu fang ji (
orbiculatus or by guang fang ji (廣
己),
己), Cocculus trilobus or C.
己), Aristolochia fangchi [1]. Intoxication with renal
failure due to carcinogenic aristolochic acids, as reported in 1993, might be due to such
nomenclature difficulties [3]. These difficulties are intrinsic problems based on the complex
nature of TCM. However, frequent adulterations of expensive drugs with cheap, similar
looking species cause additional problems [4-6]. Therefore, TCM needs rigorous quality
control which allows a reliable authentication of the plant material.
32
Plant drugs can be authenticated by several methods:
1. Microscopic and macroscopic analysis
2. Identification via the phytochemical profiling
3. Identification via DNA sequences of marker genes
Place of origin, age, season and treatment all affect the chemical profile [7], while they have
no influence on the DNA. Chemical markers are further sensible to severe errors, since they
need to be specific for the species, stable during storage and modification processes and
should represent the therapeutically relevant compound. Especially the latter is often
extremely difficult to achieve, since the active principle is either not known or ignores the
other compounds responsible for modifying the pharmaceutical effect [8]. A more holistic
approach is chemical profiling by HPLC and mixture NMR (metabolomics).
Here, many of the problems of individual chemical markers are avoided since the profile
represents the whole spectrum of compounds [9-11]. One major drawback is that the profile is
extremely sensitive to origin, age, season and modification processes the drug went through
before being sold on the market and thus no profile is identical. To cope with these problems,
authentication of the herbs can be accessed from a less variable character, the DNA. The
genetic information is not affected by the factors mentioned before but remains constant
allowing the reliable identification of a plant [12,13]. The comparison of nucleotide sequences
of marker genes is often referred to as DNA barcoding. Using this method, conclusions
regarding the relationship between plant families, species and even individuals can be drawn.
The choice of the marker gene determines the grade of separation that can be detected.
Different DNA methods to identify Chinese medical materials have recently been reviewed
[14,15].
As discussed above, several aspects require to be considered addressing the complex problem
of quality control of TCM. To get reproducible results, a combination of chemical profiling
together with the identification of the plant via DNA is crucial to avoid toxic substitutions.
We will show in this study the practicability of DNA barcoding to authenticate TCM plants
using sequences of the chloroplast gene rbcL, which is widely used in plant systematics.
33
2.3 Material and Methods
Plant material
We analysed 37 herbal drugs purchased in the herbal market of Shanghai, China, belonging to
29 families and 23 orders. Plant samples were deposited at the IPMB, Heidelberg. Authentic
species were obtained from the Botanical Garden, Heidelberg, and 886 DNA sequences were
retrieved from the online database GenBank.
DNA extraction, amplification and sequencing
Chloroplast DNA was extracted from the herbal material using the chloroform extraction
method [16]. Chloroplast DNA was amplified using a primer pair for ribulose-bisphosphate
carboxylase large chain (rbcL) obtained from MWG Biotech AG. As forward primer rbcL-N
(5’ ATGTCACCACAAACAGAAACTAAAGC 3’) was used, as reverse primer rbcL-leg7
(5’ TTCRCATGTACCYGCAGTAGCA 3’), obtaining a PCR product of approximately 700
bp length (TRIO Thermoblock Biometra). The PCR-mix contained 5 µl buffer, 1.5 µl
nucleotide mix (100µM), 0.5 µl BSA (10mg/ ml), 0.2 µl Taq polymerase (5 units/ µl), 0.5 µl
primer rbcL-N and 0.5 µl primer rbcL-leg7 (concentration: 10 pM/ µl) and 2 µl DNA solution.
The temperature program was 94 °C 5 min, 94 °C 43 sec, 50 °C 1 min, 72 °C 2 min (38
times), 72 °C 20 min. The purified PCR products were sequenced on a MegaBace 1000
instrument (GE Healthcare). Dye-terminator sequencing provided reliable >1000 nucleotide
long fragments [17,18], sufficient for the 700 bp fragments obtained in the PCR.
Sequence alignment and data analysis
Clustal W was used to align the sequences [19]; the genetic distances were calculated using
MEGA 4.0 following the Kimura 2-Parameter (K2P) model [20]. BLAST database search
was performed as described previously [21]; Neighbour-joining (NJ) and Maximum
Likelihood (ML) performed with MEGA 4.0 were used to reconstruct the phylogenetic tree
[22].
2.4 Results
37 herbal drugs traditionally used in TCM were authenticated according to partial nucleotide
sequences of rbcL. The 37 plants belong to 28 families and 23 orders; they were chosen to
represent the high diversity of plants used in TCM and to test the utility of rbcL for barcoding
of herbal medicine. In 75% of the plants, species identity could be confirmed by comparison
34
with authentic DNA sequences (plants or GenBank accessions); in 25% this was possible only
at the genus level, which is similar to the findings of Arif et al. [23] (Tab. 2).
Table 2: Plants studied and identified by rbcL
A. Identification: Species level
No
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
IPMB
Accession
number
P6839 / 05
P6843 / 09
P6883 / 49
P6846 / 12
P6859 / 25
P6860 / 26
P6863 / 29
P6864 / 30
P6865 / 31
P6866 / 32
P6894 / 60
P6872 / 38
P6875 / 41
P6879 / 45
P6882 / 48
P6885 / 51
P8088 / 81
P6888 / 54
P6891 / 57
P6893 / 59
P6896 / 62
P6897 / 63
P6898 / 64
P6901 / 67
P6903 / 69
P6904 / 70
P6908 / 74
P6910 / 76
Genbank
Accession
number
JF949994
JF949995
JF949996
JF949997
JF949998
JF949999
JF950000
JF950001
JF950002
JF950003
JF950004
JF950005
JF950006
JF950007
JF950008
JF950009
JF950028
JF950010
JF950011
JF950012
JF950013
JF950014
JF950015
JF950016
JF950017
JF950018
JF950019
JF950020
Species, Family, Order
Arctium lappa, Asteraceae, Asterales
Belamcanda chinensis, Iridaceae, Asparagales
Berberis bealei, Berberidaceae, Ranunculales
Capsella bursa-pastoris, Brassicaceae, Brassicales
Cyrtomium fortunei, Dryopteridaceae, Polypodiales
Dendrobium loddigesii, Orchidaceae, Asparagales
Eclipta prostrata, Asteraceae, Asterales
Ephedra sinica, Ephedraceae, Gnetales
Epimedium koreanum, Berberidaceae, Ranunculales
Equisetum hiemale, Equisetaceae, Equisetales
Fallopia japonica, Polygonaceae, Caryophyllales
Ginkgo biloba, Ginkgoaceae, Ginkgoales
Houttuynia cordata, Saururaceae, Piperales
Kadsura longipedunculata, Schisandraceae, Austrobaileyales
Magnolia officinalis, Magnoliaceae, Magnoliales
Ophioglossum vulgatum, Ophioglossaceae, Ophioglossales
Panax ginseng, Araliaceae, Apiales
Paris polyphylla, Melanthiaceae, Liliales
Platycladus orientalis, Cupressaceae, Pinales
Polygonum aviculare, Polygonaceae, Caryophyllales
Prunella vulgaris, Lamiaceae, Lamiales
Punica granatum, Lythraceae, Myrtales
Rheum officinale, Polygonaceae, Caryophyllales
Sanguisorba officinalis, Rosaceae, Rosales
Scutellaria baicalensis, Lamiaceae, Lamiales
Selaginella tamariscina, Selaginellaceae, Selaginellales
Taraxacum officinale, Asteraceae, Asterales
Verbena officinalis, Verbenaceae, Lamiales
B. Identification: Genus level
29
30
31
32
33
34
35
36
P6844 / 10
P6849 / 15
P6853 / 19
P6855 / 21
P6873 / 39
P6886 / 52
P6887 / 53
P6892 / 58
JF950021
JF950022
JF950023
JF950024
JF950025
JF950026
JF950030
JF950027
Bupleurum chinense, Apiaceae, Apiales
Centella asiatica, Apiaceae, Apiales
Cinnamomum cassia, Lauraceae, Laurales
Coptis chinensis, Ranunculaceae, Ranunculales
Glycyrrhiza inflata, Fabaceae, Fabales
Paeonia lactiflora, Paeoniaceae, Saxifragales
Panax notoginseng, Araliaceae, Apiales
Polygonatum kingianum, Ruscaceae, Asparagales
C. Identification: Substitution of the TCM drug Fraxinus rhynchophylla with Arctium lappa
37
P6871 / 37
Arctium lappa, Oleaceae, Lamiales
35
In 8 cases, the interspecific variations of rbcL within a genus were too small to allow the
distinction of species while in 28 cases, this was possible. One drug (Fraxinus rhynchophylla)
was substituted with Arctium lappa. BLAST search resulted in 100% identity (627/ 627 bp)
with 0 gaps. DNA isolation and amplification of the rbcL gene sequence was repeated three
times with similar results.
Two examples are given to exemplify this (Tab. 3, 4). Equisetum hiemale, (Equisetaceae)
could be identified with p-distances within the genus ranging between 0.003 and 0.03. The
next family, Lycopodiaceae, has already p-distances of 0.15. The second example is the genus
Coptis. TCM does not differentiate between the three used species; rbcL did not allow the
exact identification since the sequence of C. chinensis and C. deltoidea was identical. C. teeta
could be excluded because of a difference at position 498. The p-distances within the genus
range between 0.000 and 0.009, within the family Ranunculaceae between 0.03 and 0.04 and
within the order between 0.05 and 0.09. The phylogenetic trees of these two examples further
visualize the relationships (Fig. 1, 2).
2.5 Discussion
The genetic authentication via DNA barcoding is an important aspect of quality control to
increase the safety of TCM drugs. Unfortunately, substitutions and adulterations with cheaper
plants are a well-known phenomenon in TCM [15]. DNA barcoding is increasingly used to
identify these substitutes [14,24]. In one study by Mihalov et al. [25], soybean was detected as
an adulteration in P. ginseng preparations. We could also detect an alteration in our sample of
Fraxinus rhynchophylla. Instead, A. lappa was present, as BLAST and comparison of the
gene sequence revealed.
However, adulterations are not the only problem quality control of TCM has to face. The
more common problem lies in the system of TCM itself. As explained above, the complex
nomenclature of TCM plants can be responsible for unintentional substitutions with fatal
consequences [1].
A major problem every DNA barcoding approach of TCM drugs has to face is the often
problematic condition of the DNA [14]. Due to the various processes such as drying, steaming,
bleaching etc TCM drugs have to undergo before being sold, the DNA is often badly damaged.
36
Table 3: Phylogeny of Equisetum hiemale, Equisetaceae, with Lycopodium and Polypodium
as outgroups
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
Species
Family
Order
Genbank
Accession
number
Equisetum hiemale (TCM)
Equisetum hiemale
Equisetum hiemale Bot. Garden Heidelberg
Equisetum arvense
Equisetum bogotense
Equisetum diffusum
Equisetum fluviatile
Equisetum palustre
Equisetum pratense
Equisetum sylvaticum
Equisetum telmateia
Equisetum variegatum
Equisetum x ferrissii
Lycopodium annotinum
Polypodium scouleri
Equisetaceae
Equisetaceae
Equisetaceae
Equisetaceae
Equisetaceae
Equisetaceae
Equisetaceae
Equisetaceae
Equisetaceae
Equisetaceae
Equisetaceae
Equisetaceae
Equisetaceae
Lycopodiaceae
Polypodiaceae
Equisetales
Equisetales
Equisetales
Equisetales
Equisetales
Equisetales
Equisetales
Equisetales
Equisetales
Equisetales
Equisetales
Equisetales
Equisetales
Lycopodiales
Filicales
JF950003
EU677110
L11053
AY226139
AY226141
DQ463101
GQ248601
AY226137
AY226136
AF313580
AY226134
AF313579
EU352290
FJ825693
87 Equisetum arvense
95 Equisetum fluviatile
32
Equisetum diffusum
Equisetum telmateia
97
Equisetum sylvaticum
Equisetum palustre
51
69
42
Equisetum pratense
78
Equisetum variegatum
Equisetum x ferrissii
100
100
Equisetum hiemale TCM
Equisetum hiemale
63
Equisetum hiemale Bot. Garden Heidelberg
Equisetum bogotense
Lycopodium annotinum
Polypodium scouleri
0.02
Fig. 1: Phylogenetic NJ tree of E. hiemale based on nucleotide sequences of the rbcL gene with bootstrap values
37
Table 4: Phylogeny of Coptis chinensis, Ranunculaceae, and other families of the
Ranunculales
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
Species
Family
Genbank
Accession
number
Coptis chinensis (TCM)
Coptis chinensis
Coptis deltoidea
Coptis teeta
Coptis aspleniifolia
Coptis japonica_var._anemonifolia
Coptis japonica_var._major
Coptis laciniata
Coptis lutescens
Coptis occidentalis
Coptis omeiensis
Coptis quinquefolia
Coptis quinquesecta
Coptis ramosa
Coptis trifolia
Coptis trifoliolata
Aconitum napellus
Anemone hupehensis
Aquilegia vulgaris
Clematis montana
Delphinium bonvalotii
Glaucidium palmatum
Hydrastis canadensis
Ranunculus japonicus
Thalictrum simplex
Berberis bealei
Circaeaster agrestis
Euptelea pleiosperma
Lardizabala biternata
Menispermum dauricum
Papaver rhoeas
Ranunculaceae
Ranunculaceae
Ranunculaceae
Ranunculaceae
Ranunculaceae
Ranunculaceae
Ranunculaceae
Ranunculaceae
Ranunculaceae
Ranunculaceae
Ranunculaceae
Ranunculaceae
Ranunculaceae
Ranunculaceae
Ranunculaceae
Ranunculaceae
Ranunculaceae
Ranunculaceae
Ranunculaceae
Ranunculaceae
Ranunculaceae
Ranunculaceae
Ranunculaceae
Ranunculaceae
Ranunculaceae
Berberidaceae
Circaeasteraceae
Eupteleaceae
Lardizabalaceae
Menispermaceae
Papaveraceae
JF950024
AB163775
AB163774
AB163773
AB163777
AB163764
AB163765
AB163778
AB163766
AB163779
AB163776
AB163770
AB163772
AB163769
AF093730
AB163768
EU053898
FJ626577
FJ449851
FJ449855
FJ626583
L75848
L75849
FJ449862
FJ449863
FJ449858
FJ626607
AY048174
D85693
FJ026493
FJ626614
38
Coptis chinensis
Coptis omeiensis
63
Coptis deltoidea
Coptis sp. TCM
38
Coptis teeta
22
Coptis quinquesecta
Coptis aspleniifolia
Coptis laciniata
3852
94 Coptis occidentalis
Coptis japonica var. anemonifolia
87
Coptis japonica var. major
77
Coptis lutescens
Coptis quinquefolia
99
57 Coptis ramosa
46 Coptis trifoliolata
30
Coptis trifolia
Glaucidium palmatum
Aquilegia vulgaris
89
Thalictrum simplex
13
61
Aconitum napellus
97
36
Delphinium bonvalotii
Ranunculus japonicus
43
24
Anemone hupehensis
45
Clematis montana
96
35
Hydrastis canadensis
Euptelea pleiosperma
75
Menispermum dauricum
73
Berberis bealei
Lardizabala biternata
Papaver rhoeas
Circaeaster agrestis
0.01
Fig. 2: Phylogenetic NJ tree of Coptis species TCM (C. chinensis or C. deltoidea) based on nucleotide sequences
of the rbcL gene with bootstrap values. The rbcL gene does not allow the distinction between these two Coptis
species.
39
Additionally, the secondary metabolites such as flavonoids, tannins or alkaloids inhibit the
PCR amplification of the selected marker gene. Intercalating substances can disturb the PCR
resulting in mismatched base pairs and erroneous DNA sequences.
Purification of the DNA is essential to reduce the secondary metabolites before a successful
amplification can be tried [26,27].
The challenge is to discover a DNA marker that is general enough not to raise false alarm but
specific enough to discover all adulterations. Furthermore, the marker must be universal to
cover the large variety of plant species applied in TCM. And, last but not least, the DNA must
ideally exist in many copies to increase the chance of detection in TCM drugs which are
usually dried and grounded and thus often contain degraded DNA. Several studies showed
that a marker fulfilling all these requirements hardly exists and we need to live with certain
restrictions [15,28]. However, the level of identification can be directed by carefully choosing
the adequate target region of the genome. Promising for the identification of herbal medicine
are especially chloroplast genes since chloroplasts contain many copies of the same gene and
thus increase the chances of successful detection [29]. The interspecific variations change
from plant species to plant species which means the decision for the right marker gene can not
be absolute but needs to be adapted to the particular situation [15,30].
Nevertheless, two marker genes, rbcL and ITS (a commonly used nuclear marker), are widely
used in DNA barcoding. Several examples of successful identification of TCM drugs both of
rbcL [25,30] and ITS [24,31-33] were published recently.
To choose the right gene, we have to remember the nature of TCM. Quite often, several
closely related members of the same genus are used as one drug; a marker gene distinguishing
between these species or even subspecies might raise false alarm. ITS is useful to detect a
plant at the species level, while rbcL is in 75% of the cases precise enough to determine the
species but can not distinguish drugs on the species level in the remaining 25% of the cases
[15,23,29]. This disadvantage of rbcL for phylogenetic research might be an advantage in
quality control of herbal medicine. It is important to detect adulterations beyond doubt, but
the exact species within a genus is only of secondary importance, since TCM itself does not
differentiate to that level and also phytochemical profiles are similar between closely related
species. rbcL can fulfil these requirements successfully and can guarantee the safety of the
drug, as we could demonstrate in our study. The level of detection of rbcL is high enough to
40
discover possibly toxic substitutions within the traditional system of TCM, such as
Aristolochia species as substitutes for Stephania. Adulterations can be identified using
database search since an extensive library of most families and even most genera exists
already for these two marker genes. Furthermore, the rbcL marker is present in many copies
in each plant cell, making a successful amplification more probable than for nuclear genes.
In our study we have demonstrated the utility of rbcL as marker for DNA barcoding, but have
to point out its limitations as well. Since genetic information does not cover the morphology,
chemical profile, quality control should always try to consider different techniques. The
Medicinal Materials DNA Barcode Database [34] offers with 1297 species the possibility to
authenticate TCM herbal medicine. For roughly 400 species the rbcL gene sequence is
provided, while currently 4773 botanicals are officially used in TCM [2]. It is advisable to
extend this and similar TCM libraries with the rbcL and ITS sequences of all botanicals
officially used in TCM for international use to allow rapid detection since authentic species
examples are rather difficult to obtain outside of Asia. This would improve the acceptance of
TCM internationally beyond the image of traditional medicine.
41
2.6 References
1
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traditional Chinese medicine (TCM): Lessons learned from the misuse of Aristolochia-related species
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2
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CFSAN/US FDA, Office of Nutritional Products, Labelling and Dietary Supplements (2001). Letter
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http://www.fda.gov/Food/DietarySupplements/Alerts/ucm096374.htm
Accessed June 30, 2011
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But PP (1993). Need for correct identification of herbs in herbal poisoning. Lancet. 341, 637.
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11
Zhang Q, Ye M (2009). Chemical analysis of the Chinese herbal medicine Gan-Cao (licorice). J
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Chang WT, Thissen U, Ehlert KA, Koek MM, Jellema RH, Hankemeier T, van der Greef J, Wang
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Ma XQ, Shi Q, Duan JA, Dong TT, Tsim KW (2002). Chemical analysis of Radix Astragali
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14
Heubl G (2010). New aspects of DNA-based authentication of Chinese medicinal plants by
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Yip PY, Chau CF, Mak CY, Kwan HS (2007). DNA methods for identification of Chinese
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Doyle JJ, Doyle JL (1987). A rapid DNA isolation procedure for small quantities of fresh leaf tissue.
Phytochem Bull, BSA. 19, 11–15.
42
17
Olsvik O, Wahlberg J, Petterson B, Uhlén M, Popovic T, Wachsmuth IK, Fields PI (1993). Use of
automated sequencing of polymerase chain reaction-generated amplicons to identify three types of
cholera toxin subunit B in Vibrio cholerae O1 strains. J Clin Microbiol. 31, 22-25.
18
Kress WJ, Wurdack KJ, Zimmer EA, Weigt LA, Janzen DH (2005). Use of DNA barcodes to
identify flowering plants. Proc Natl Acad Sci. 102, 8369-8374.
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44
3. Cytotoxicity, antiviral and antitrypanosomal screening of 82
plants from Chinese and European phytomedicine
3.1 Abstract
In an extensive screening the antiviral, antitrypanosomal and anticancer properties of extracts
from 82 plants used in traditional Chinese medicine and European phytomedicine were
determined. Several promising plants that were highly effective against hepatitis B virus
(HBV), bovine viral diarrhoea virus (BVDV) - a flavivirus used here as a surrogate in vitro
model of hepatitis C virus -, trypanosomes (Trypanosoma brucei brucei) and several cancer
cell lines were identified. Six aqueous extracts from Celosia cristata, Ophioglossum vulgatum,
Houttuynia cordata, Selaginella tamariscina, Alpinia galanga and Alpinia oxyphylla showed
significant antiviral effects against BVDV without toxic effects on host EBTr cells, while
Evodia lepta, Hedyotis diffusa and Glycyrrhiza spp. demonstrated promising activities against
the HBV without toxic effects on host HepG2 2.2.15 cells. Seven organic extracts from
Alpinia oxyphylla, Coptis chinensis, Kadsura longipedunculata, Arctium lappa, Panax
ginseng, Panax notoginseng and Saposhnikovia divaricata inhibited T. b. brucei. Moreover,
among fifteen water extracts that combined high antiproliferative activity (IC50 0.5-20 µg/ml)
and low acute in vitro toxicity (0-10% reduction in cell viability at IC50), Coptis chinensis
presented the best beneficial characteristics. In conclusion, traditional herbal medicine from
Europe and China still has a potential for new therapeutic targets and therapeutic applications.
3.2 Introduction
Traditional Chinese medicine (TCM) has a long history of almost 5000 years and officially
uses approximately 4773 herbs, while the number of locally used plants is probably much
higher [1]. Clinical efficacy was shown in various examples, one of the best known is that of
artemisinin from Artemisia annua, commonly used against malaria, but also effective against
T. b. brucei, viral infections and cancer [2-8].
European medicine also has a long tradition of at least 2500 years with the two important
early scholars Hippocrates and Dioscorides who described more than 400 medicinal plants
2000 years ago of which many are still in use today [9]. Many pure therapeutic agents used in
modern medicine were originally based on herbal medicine; in fact, the process of developing
new drugs from European herbal medicine is still alive and important discoveries are
regularly made [10,11]. Even though the theoretical concept of traditional medicine differs
between Europe and China, often the same plants were and are still used in both cultures to
45
treat the same or similar health disorders. Modern European phytotherapy also includes
important herbal medicines from Africa and America.
Even though the diversity of plants and possible natural products is vast, the number of targets
is usually limited (Tab. 1). Most natural products target proteins, biomembranes or DNA
unselectively. Selective interaction is often the case when especially alkaloids mimic signal
molecules and interact with receptors or enzymes. It is often possible to conclude from the
type of the natural products to their most likely mode of action. Saponins and monoterpenes
are active on the biomembrane, while polyphenols usually interact with proteins. Alkaloids
also interact with proteins or the DNA.
The formations of covalent and of non-covalent bonds are the two modes of action that form
the basis of all interactions between proteins and natural products.
The two main targets for the formation of covalent bonds are free amino and free SH groups.
Aldehydes, isothiocyanates and epoxids can form covalent bonds with free amino groups
while sesquiterpene lactones, disulfides (e.g. allicin), polyacetylenes and epoxides can form
covalent bonds with free SH groups.
The second mechanism of maybe even greater importance due to its universality is the
formation of non-covalent bonds between phenolic OH-groups and amino groups. The proton
of the phenolic OH-group can partly dissociate under physiological conditions so that
unspecific interactions by forming strong, ionic bonds occur with proteins. Tannins are
especially effective due to their large number of hydroxygroups.
All of these interactions will change the three dimensional structure of the protein and thus
inactivate it. The omnipresence of these unspecific natural products in plants explains the
efficacy of many plant extracts. They are responsible for the great number of “hits” usually
occurring in extended screenings of medicinal plant extracts (Tab. 2).
Hepatitis B (HBV) and hepatitis C (HCV) are responsible for 75% of all cases of liver
diseases worldwide, often causing cirrhosis and hepatocellular carcinoma [12,13]. HBV and
HCV account for the most problematic viral infections, since the standard treatment with
pegylated IFN-α and the purine nucleoside analogues lamivudine and ribavirin have severe
side effects while being at the same time ineffective for 50% of the patients [12,14]. Thus,
new drugs are urgently needed [15]. Together with the bovine viral diarrhoea virus (BVDV),
46
and the Japanese Encephalitis virus, HCV belongs to the Flaviviridae family. As BVDV,
whose cytopathic strains induce a lytic infection in some cell lines, such as embryonic bovine
trachea (EBTr) cells, is easier to manipulate and lacks human infectivity, this is commonly
used as in vitro model for infections of this viral family [16].
Our knowledge of the natural products of many plants used in European and Chinese
phytomedicine is broad (Tab. 2), however, many new discoveries are still possible. Previously,
several studies demonstrated the promising potential of traditional phytomedicine for the
discovery of new antiviral drugs. Artemisinin and related compounds proved effective in
screening assays against viral hepatitis [6,7,17]. In water extracts of Terminalia chebula,
Sanguisorba officinalis, Rubus coreanus and Rheum palmatum Kim et al. [18] discovered
prominent anti-HBV activities. The ethanolic extract of Hypericum perforatum, a wellestablished drug for treatment of depression [9] was also shown to be active against the
hepatitis B virus [19]. Laxative anthraquinones isolated from Rheum palmatum demonstrated
significant effects against hepatitis B virus [20] and saikosaponins from Bupleurum species
were previously shown to lower significantly the hepatitis B virus level in the HepG2 2.2.15
assay [21]. HepG2 2.2.15 is a stable cell line infected with the hepatitis B virus. The assay
measures the production of secreted HBV from the cell by using real time quantitative PCR.
Parasites such as protozoa and helminths cause a major health threat in many tropical
countries [22], while suitable drugs are still rare [23]. Blood parasites of the genus
Trypanosoma (Trypanosoma brucei rhodesiense and T. b. gambiense) are responsible for
African trypanosomiasis (sleeping sickness) with serious consequences for human health and
economy. Due to the high infectivity of African human trypanosomes, T. b. brucei is
commonly used as model organism with similar morphology and biochemical processes,
while being only infective for cattle [22,24,25]. This subspecies causes the cattle epidemic
nagana, it is responsible for severe financial loss of 1340 billion USD per year [26].
Currently, only four drugs are approved internationally for the treatment of humans against
sleeping sickness: suramin, pentamidine, melarsoprol and eflornithine. Diminazene, another
effective antitrypanosomal drug, is only approved for the use on animals because of severe
side effects [24]. Even the drugs approved for human use are responsible for serious side
effects, and furthermore, the parasites develop increasing resistance to them [27-30]. This
47
situation makes the discovery of new, effective drugs an urgent task of the 21st century [3133].
When considered together, enterohepatic tumours, i.e., those affecting the liver, the biliary
duct, gallbladder and the intestine, constitute the first cause of death due to cancer. Although
in many cases surgery and radiotherapy are efficacious, these therapeutic strategies cannot
always be applied. Moreover, even when the removal of tumours is possible, pre- and postoperative pharmacological adjuvant regimens are often needed. However, one important
limitation to the use of cytostatic drugs to treat enterohepatic tumours is that they generally
exhibit marked resistance to currently available pharmacological approaches and the
development of resistance during treatment [34].
Many natural products and derivatives thereof belong to the standard repertoire of cancer
chemotherapy. Examples are Vinca alkaloids, such as vincristine, vinblastine and vinorelbine,
obtained from Madagascar periwinkle (Catharanthus rosea). Also taxanes such as paclitaxel
and docetaxel, which are produced from the bark of Pacific yew (Taxus), podophyllotoxins,
such as etoposide and teniposide, derivatives of the genus Podophyllum, and camptothecin,
derived from the Asian "Tree of Happiness" (Camptotheca acuminata) and its derivatives,
irinotecan and topotecan, are natural products from TCM plants [4].
In this study extracts from 82 traditional medicinal plants were screened against hepatitis B
virus and flaviviruses, T. b. brucei and several cancer cell lines. Our aim was to detect new
sources of active compounds for the possible treatment of these important causes of diseases.
3.3 Material and Methods
Chemicals
Dimethylsulfoxide (DMSO), trypsin-EDTA, DMEM and MEM with GLUTAMAX media,
fetal bovine serum (FBS) and supplementary chemicals were bought from Gibco
Invitrogen; Germany. Antibiotic/antimycotic solution, gentamicin, Neutral Red (NR, 3amino-7-dimethylamino-2-methylphenazine), NaHCO3, L-glutamine and MEM media were
purchased from Sigma-Aldrich (Madrid, Spain). Geneticin® (G418) was from Roche
(Barcelona, Spain). Dried TCM plants were obtained in Shanghai; South African plants were
provided by Prof. van Wyk, University of Johannesburg, South Africa.
48
Table 1: Targets in animal cells, bacteria cells and viruses [35]
Target
Activity
Biomembrane
Membrane disruption
Disturbance of membrane
fluidity
Disturbance of membrane
proteins
Proteins
(unspecific
interaction)
Secondary metabolites
Saponins
Saponins, monoterpenes
Monoterpenes
Phenolic molecules (flavonoids,
catechins, tannins, anthraquinones,
quinones, lignans, phenylpropanoids)
Allicin, furanocoumarins,
Covalent bonding (change of 3D
isothiocyanates, sesquiterpene lactones,
protein conformation)
aldehydes, epoxids, triple bonds
Non-covalent bonding (change
of 3D protein conformation)
Proteins
(specific
interaction)
Structural mimetics of signal molecules
(many alkaloids, e.g. nicotin), hydrogen
cyanide from cyanogens
Cardiac glycosides
Colchicine, podophyllotoxin, taxol,
vinblastine
Inhibition of enzymes
Inhibition of Na+Ka+ pumps
Inhibition of microtubule
formation
Inhibition of protein
biosynthesis
Inhibition of transporters
Modulation of hormone
receptors
Modulation of ion channels
Emetine, lectins
Non-protein amino acids
Isoflavonoids
Modulation of neuroreceptors
Modulation of regulatory
proteins
Modulation of transcription
factors
Many alkaloids, aconitine
Many alkaloids, some non-protein amino
acids
Caffeine, phorbol esters
Structural mimetics of hormones (e.g.
isoflavones genistein, daidzein)
DNA/ RNA
Aristolochic acids, furanocoumarins,
pyrrolizidine alkaloids, molecules with
epoxy groups
Covalent modification
(alkylation)
Inhibition of DNA
topoisomerase I
Inhibition of transcription
Berberine, camptothecin
Amanitine
Planar, aromatic and lipophilic molecules
(anthraquinones, berberine, emetine,
quinine, sanguinarine, furanocoumarins)
Intercalation
49
Table 2: Main Compounds of Plants used in this study [9,36]
Family
Acanthaceae
Species
Andrographis paniculata
Amaranthaceae Celosia cristata
Apiaceae
Bupleurum chinense
Bupleurum marginatum
Centella asiatica
Cnidium monnieri
Saposhnikovia divaricata
Araliaceae
Eleutherococcus senticosus
Panax ginseng China
Panax ginseng Korea
Panax notoginseng
Arecaceae
Areca catechu
Asclepiadaceae
Cynanchum paniculatum
Asteraceae
Artemisia annua
Artemisia capillaris
Arctium lappa
Centipeda minima
Chrysanthemum indicum
50
Main Compounds
Diterpenelactones
Lectins (amarathin, isoamaranthin,
celosianin), ferulic acid
Flavonoids (quercetin, rutin,
isoquercetin, isorhamnetin), β-sitosterol,
β-sitosterol-3-O-glucosid, α-spinasterol,
α-spinasterol-3-O-glucoside
Flavonoids (quercetin, rutin,
isoquercetin, isorhamnetin), β-sitosterol,
β-sitosterol-3-O-glucosid, α-spinasterol,
α-spinasterol-3-O-glucoside
Triterpenes (asiaticoside, asiatic acid,
madecassic acid), flavonoids
(kaempferol), monoterpenes (camphor),
fatty acids (palmitic acid)
Monoterpenes (pinene), cnidium lactone
Polyacetylenes, furanocoumarins,
chromones
Saponins (ginsenosides), polyacetylenes,
fatty acids, amino acids, polysaccharides
Saponins (ginsenosides Rb1, Rb2, Rc, Rd,
Re and Rg1), polyacetylenes (panaxynol,
panaxydol, panaxytriol, falcarindiol),
fatty acids, amino acids, polysaccharides
Saponins (ginsenosides Rb1, Rb2, Rc, Rd,
Re and Rg1), polyacetylenes (panaxynol,
panaxydol, panaxytriol, falcarindiol),
fatty acids, amino acids, polysaccharides
Saponins (ginsenosides Rb1, Rb2, Rc, Rd,
Re and Rg1), polyacetylenes (panaxynol,
panaxydol, panaxytriol, falcarindiol),
fatty acids, amino acids, polysaccharides
Alkaloids (arecoline, arecaidin,
arecolidin, guvacolin, guvacin)
Glucosides (cynanchocerin, cynanchin)
Sesquiterpene lactones (artemisinin,
arteannuin, artemisitene), monoterpenes
(1,8 cineol, borneol, camphor, menthol),
coumarins (coumarin, scopoletin)
Sesquiterpene lactones, monoterpenes
(1,8 cineol, borneol, camphor, menthol),
coumarins (coumarin, scopoletin)
Monoterpenes, polyacetylenes
(falcarinol), fatty acids, sterols
Monoterpenes (thymol), terpene
glycosids, sesquiterpene lactones
Monoterpenes (1,8 cineole, pinene,
Chrysanthemum morifolium
Eclipta prostrata
Senecio scandens
Siegesbeckia orientalis
Taraxacum officinale
Berberidaceae
Berberis bealei
Dysosma versipellis
Epimedium koreanum
Brassicaceae
Capsella bursa-pastoris
Isatis indigotica (root)
Isatis indigotica (leaf)
Caprifoliaceae
Lonicera confusa
Convallariaceae
Crassulaceae
Cupressaceae
Dryopteridaceae
Ephedraceae
Equisetaceae
Polygonatum kingianum
Rhodiola rosea
Platycladus orientalis
Cyrtomium fortunei
Ephedra sinica
Equisetum hiemale
Euphorbiaceae
Croton tiglium
Fabaceae
Abrus cantoniensis
Acacia catechu
Cassia tora
51
borneol, camphor), tannins
Monoterpenes (1,8 cineole, pinene,
borneol, camphor), tannins
Monoterpenes, volatile compounds
(Heptadecane, 6,10,14-trimethyl-2pentadecanone, n-hexadecanoic acid,
pentadecane, eudesma-4(14),11-diene,
phytol, octadec-9-enoic acid, 1,2benzenedicarboxylic acid diisooctyl
ester, (Z,Z)-9,12-octadecadienoic acid)
Pyrrolizidine alkaloids, terpenoids
Phytosterols (β-sitosterol)
Sesquiterpene lactones, phenolic acids,
triterpene saponins, inulin, phytosterols
(β-sitosterol)
Alkaloids (berberine, columbamine,
jatrorrhizine, palmatine)
Flavonoids, podophyllotoxin lignans
Flavonoids (quercetin, maohuoside B,
epimedin A, epimedin B, icariin, icriside
II, icariside I, epimedoside A,
hexandraside E)
Flavonoids, terpenes, glucosinolates,
saponins, tannins
Flavonoids, glucosinolates, alkaloids
(isatisine A, indican, isatin, indirubin and
indigotin)
Flavonoids, glucosinolates, alkaloids
(isatisine A, indican, isatin, indirubin,
indigotin)
Flavonoids (rutin, quercetin, luteilin-7O-beta-D-galactoside, lonicerin),
chlorogenic acid, beta-sitosterol,
tetratriacontane)
Flavonoids, steroidal saponins
Glucosides (salidroside, tyrosol)
Monoterpenes
Flavonoids
Phenylethylamine alkaloids (ephedrine)
Flavonoids, silicic acids
Glyceryl crotonate, crotonic acid,
crotonic resin, phorbol esters (phorbol
formate, phorbol butyrate, phorbol
crotonate)
Lectins, indolalkaloids
Flavonoids (quercetin, rutin), catechin,
epicatechin
Flavonoids, dianthrone glycosides
(sennoside A, B), anthraquinones
(anthrones, emodin, rhein)
Desmodium styracifolium
Monoterpenes, alkaloids
Flavonoids, isoflavonoids, chalcone
(liquiritin, isoliquiritin), saponins
(glycyrrhizic acid, 4-hydroxyGlycyrrhiza inflata
glycyrrhtinic acid), monoterpenes (1-(2Furyl)propan-2-one, pyrazine (2-acetyl1-furfuryl pyrrole), benzene (1-methoxy4-isopropylbenzene)
Spatholobus suberectus
Flavonoids, catechin, pyranoside
Flavonoids, triterpene saponins, LSutherlandia frutescens
canavanin, pinitol
Geranium
wilfordii
Flavonoids, tannins, monoterpenes
Geraniaceae
Flavonoids, tannins, coumarines,
Pelargonium sidoides
monoterpenes
Flavonoids (glycosides of kaempferol,
quercetin, isorhamnetin), bisflavonoids,
Ginkgo
biloba
proanthocyanidins, ginkgolic acid, the
Ginkgoaceae
sesqiterpene alcohol bilobalide, terpene
lactones, diterpene lactones (ginkgolides)
Hypericin, hyperforin, monoterpenes,
Hypericum japonicum
Hypericaceae
flavonoids, tannins, saponins
Belamcanda chinensis
Flavonoids (belamcandin, iridin)
Iridaceae
Mentha haplocalyx
Monoterpenes (menthol)
Lamiaceae
Triterpene saponins, flavonoids (rutin)
Prunella vulgaris
tannins, rosmarinic acid, monoterpenes
(camphor)
Scutellaria baicalensis
Flavonoids, iridoid glycosides
Monoterpenes (1,8-cineol, pinene,
Cinnamomum cassia
Lauraceae
cinnamaldehyde), coumarins, tannins
Taxillus chinensis
Flavonoids (avicularin, quercetin)
Loranthaceae
Tannins (punicalin, punicalagin),
Punica granatum
Lythraceae
piperidine alkaloids
Tannins, flavonoids (rutin),
Magnolia
officinalis
sesquiterpenes, monoterpenes (1,8Magnoliaceae
cineol)
Steroidal saponins (dioscin, polyphyllin
Melanthiaceae Paris polyphylla
D)
Lysimachia christinae
Flavonoids, tannins, triterpene saponins
Myrsinaceae
Monoterpenes (1,8-cineol)
Eucalyptus robusta
Myrtaceae
sesquiterpenes
Quercetin 3-O-methyl ether, ophioglonin
Ophioglossaceae Ophioglossum vulgatum
Dendrobium loddigesii
Alkaloids (dendrobine, nobiline)
Orchidaceae
Flavonoids, (kaempferol), β-sitosterol,
Paeonia
lactiflora
resveratrol derivatives, phytoestrogens,
Paeoniaceae
monoterpene glycosid (paeoniflorin)
Iridoid glycosides (harpagide,
Harpagophytum procumbens
Pedaliaceae
harpagoside)
52
Poaceae
Cymbopogon distans
Polygonaceae
Fallopia japonica (syn.
Polygonum cuspidatum)
Fallopia multiflora (syn.
Polygonum multiflorum)
Polygonum aviculare
Rheum officinale
Ranunculaceae
Coptis chinensis
Rosaceae
Rosa chinensis
Rosa laevigata
Sanguisorba officinalis
Rubiaceae
Hedyotis diffusa
Rutaceae
Evodia lepta
Evodia rutaecarpa
Phellodendron chinense
Saururaceae
Houttuynia cordata
Schisandraceae Kadsura longipedunculata
Selaginellaceae
Selaginella tamariscina
Valerianaceae
Patrinia scabiosaefolia
Verbenaceae
Violaceae
Verbena officinalis
Viola yezoensis
Zingiberaceae
Alpinia galanga
Alpinia oxyphylla
53
Monoterpenes (1,8-cineol, pinene,
cymbopogone, cymbopogonol)
Anthraquinones (emodin, rhein,
chrysophanol), tetrahydroxystilbene
glucosides, steroidal saponins, tannins
Flavonoids, tannins
Flavonoids, tannins
Flavonoids, tannins, anthraquinone
glycosides (emodin, rhein)
Alkaloids (berberine, palmatine,
coptisine, columbamine, epiberberine)
Flavonoids, tannins, carotinoids, vitamin
C
Flavonoids, tannins, carotinoids, vitamin
C
Tannins, flavonoids, saponins,
proanthocyanidins
Iridoid glycosides
Indole alkaloids, (evodiamin, rutecarpin),
chromenes
Indole alkaloids, (evodiamin, rutecarpin)
Isoquinoline alkaloids (berberine,
palmatine, jatrorrhizine), sesquiterpene
lactones
Flavonoids (quercetin, quercetin 3rhamnoside), norcepharadione B
Lignans (kadsurilignans), triterpenoid
acids, triterpene dilactones, camphene,
borneol
Flavonoids (amentoflavone,
isocryptomerin, biflavonoids), sterols
Triterpene saponins, iridoid glycosides
(patrinoside)
Iridoid glycosides, flavonoids
Falavonoids, saponins
Monoterpenes (camphor, cineole, dpinene, eugenol, cadinene), flavonoids
(galangin, riboflavin), niacin, 1'acetoxychavicol acetate, ascorbic acid
Monoterpenes (camphor, cineole, dpinene, eugenol, cadinene), flavonoids
(galangin, riboflavin), niacin, 1'acetoxychavicol acetate, ascorbic acid
Genetic authentication of plant material
The TCM plants were genetically identified by DNA barcoding. DNA was isolated from plant
drugs; their chloroplast rbcL gene was amplified and sequenced. The obtained sequences
were authenticated with sequences obtained from sample species of the Botanical Garden of
Heidelberg and databases. Voucher specimens of the plant material were deposited at the
Department of Biology, Institute of Pharmacy and Molecular Biotechnology, Heidelberg
University, Germany.
Extract preparation
500 g of dry plant material was powdered and extracted with dichloromethane, methanol and
water under moderate heat using a reflux condenser for 4 hours. The extracts obtained were
concentrated using the rotation evaporator, stored at -40 °C under exclusion of light and dried
under vacuum prior to the experiments. Dried extracts were dissolved in DMSO for the
experiments.
Test organisms
T. b. brucei TC 221 were originally obtained from Prof. Peter Overath (Max-Plank Institut für
Bioloie, Tübingen) by Dr. D. Steverding before being cultured at the IPMB, Heidelberg since
1999. HeLa cancer cells and Cos7 fibroblast cells (African green monkey kidney cells
immortalized with the monkey virus SV40) were cultured at the IPMB, Heidelberg for several
years; Hep G2, Sk Hep1 and LS 174 T, HepG2 2.2.15 and EBTr cells were cultured at the
Laboratory of Experimental Hepatology and Drug Targeting (HEVEFARM), University of
Salamanca, CIBERehd, Spain.
Methods
Cancer Cells (Hep G2, Sk Hep1 and LS 174 T) were basically grown as previously described1
[35], HeLa and Cos72 cells were grown at 37 °C with 5% CO2 in DMEM complete media
(10% heat-inactivated FBS; 5% penicillin/ streptomycin; 5% non-essential amino acids). Hep
G2, Sk Hep1, and LS 174 T cells were grown at 37 ºC with 5% CO2 in MEM complete media
(10% heat-inactivated FBS; 1% antibiotic-antimycotic solution).
1
Experiments performed by Laboratory of Experimental Hepatology and Drug Targeting (HEVEFARM),
University of Salamanca, CIBERehd, Spain: Marta R. Romero, Alba G. Blazquez, Jose J.G. Marin
2
Experiments performed by Institute of Pharmacy and Molecular Biotechnology, Heidelberg University,
Heidelberg, Germany: Dorothea Kaufmann
54
HepG 2.2.15 cells were cultured as previously described3 [7] in DMEM complete medium
with 10% FBS and 20 mg/ml gentamicin. EBTr cells were cultured as described elsewhere3
[6], they were maintained in MEM-GLUTAMAX medium with 10% heat-inactivated FBS;
1% penicillin/ streptomycin, and 0.1 % gentamicin.
T. b. brucei TC221 cells were cultured in BALTZ medium [36] supplemented with 20%
inactivated FBS and 0.001% β-mercaptoethanol.
The MTT cell viability assay was used to determine cytotoxicity in Cos74 and HeLa cells
[37,38]. Cells during the logarithmic growth period were seeded in 96 well plates (Greiner
Labortechnik) at concentrations of 2 x 104 cells/ well and grown for 24 h. Dried and powdered
extracts were dissolved in DMSO before being serially diluted to 10 concentrations in 96 well
plates. Cells were incubated with the extract for 24 h before the medium was removed and
replaces with fresh medium containing 0.5 mg/ml 3-(4,5-dimethylthiazol-2-yl)-2,5diphenyltetrazolium bromide (MTT). The formazan crystals were dissolved in DMSO 4 h
later; the absorbance was measured at 570 nm with a Tecan Safire II Reader.
T. b. brucei TC221 cell viability was additionally to the MTT assay confirmed and evaluated
using microscopic techniques.
Toxicity of the extracts for T. b. brucei was compared to HeLa and Cos7 cells and the
Selectivity index (SI) was calculated. SI: ratio of the IC50 value of mammalian cells divided
by the IC50 value of trypanosomes.
To test the antiproliferative effect, 5x103 or 15x103 cells per well (depending on the cell line)
were seeded in 96 well plates and incubated with 5, 10, 25, 50, 100, and 200 µg/ml water
extract for 72 h. The cell viability was also determined using the MTT assay with minor
modifications. Acute toxicity was similarly measured using MTT assay but after short-term (6
h) incubation with the extracts at the concentrations of IC50 calculated for each cell line.
To determine the antiviral effect of the extracts, BVDV was used here as a substitute in vitro
model for hepatitis C virus infection3. Bovine epithelial cells obtained from embryonic
3
Experiments performed by Laboratory of Experimental Hepatology and Drug Targeting (HEVEFARM),
University of Salamanca, CIBERehd, Spain: Marta R. Romero, Alba G. Blazquez, Jose J.G. Marin
4
Experiments performed by Institute of Pharmacy and Molecular Biotechnology, Heidelberg University,
Heidelberg, Germany: Dorothea Kaufmann
55
trachea (EBTr) were cultured in MEM medium as described previously [6]. They were seeded
in 96 well plates (15 x 103 cells/well; 50 µl/well) and left to attach for 2 h. Afterwards, the
cells were infected with 50 µl/well of the desired dilution in culture medium of an initial
suspension of BVDV (cytopathic strain Oregon C24V, genotype I, subgenotype b) to reach
40% cytopathic effect. After 48 h of incubation the medium was replaced with dilutions in
culture medium of the extracts (1, 5, 10, 50, 100 µg/ml). The viability of the EBTr cells was
measured using the MTT assay after 72 h incubation.
An HBV antiviral assay based on the HepG2 2.2.15 model was used to determine the antiviral
activity of the extracts5 [39]. HepG2 2.2.15 cells were seeded in six-wells plates (35x104 per
well) before being incubated for 21 days with 50 µg/ml, 25 µg/ml and 12.5 µg/ml extract. The
culture medium was replaced every 3 days with fresh medium, containing the extract dilutions.
Quantitative real-time PCR (QPCR) was used to measure the HBV-DNA levels in the culture
medium (representing HBV virion production) as described previously [7]. Cytotoxicity was
determined using the uptake of NR dye at the end of treatment [40].
At least three cultures for each experimental condition were carried out. Data points were
obtained in triplicate form (T. b. brucei, cancer cell lines, Cos7, HepG2 2.2.15 cells) and in 8
different wells (EBTr). The IC50 value was calculated using SigmaPlot® 11.0 (4 parameter
logistic curve). Statistical significance determined via paired t-test or the Bonferroni method
of multiple-range testing.
3.4 Results and Discussion
The great diversity of natural products occurring in plants is of the utmost importance for the
discovery of new pharmaceutical lead compounds. Through millions of years of evolution the
defence mechanisms of plants were perfected. The great variety of natural products clearly
demonstrates the efficacy of this defence strategy against herbivores, but also fungi, bacteria
and viruses (Tab. 2). In many cases the plants do not rely on specific interactions but also rely
on unspecific molecules that interact with a great number of targets (Tab. 1). Of highest
importance are the interactions with free amino and free SH groups. While aldehydes,
isothiocyanates and epoxids are able to form covalent bonds with free amino groups,
sesquiterpene lactones, disulfides, polyacetylenes and epoxides interact with free SH groups.
5
Experiments performed by Laboratory of Experimental Hepatology and Drug Targeting (HEVEFARM),
University of Salamanca, CIBERehd, Spain: Marta R. Romero, Alba G. Blazquez, Jose J.G. Marin
56
Phenolic OH-groups interact on a non-covalent basis with free amino groups by forming
strong hydrogen and ionic bonds.
The cytotoxicity of water and organic solvent extracts from 82 medicinal plants was
determined in the fibroblast cells Cos7 and in four cancer cell lines: HeLa, HepG2, SK Hep1
and LS 174T (Tab. 3, 4). The aqueous extracts were also screened against BVDV and HBV
(Tab. 3), whereas organic solvent extracts were assayed on T. b. brucei (Tab. 4). Our results
revealed promising results in order to use several of these plants as sources for therapeutic
agents.
The viral cell offers three main targets to the natural products (Tab. 1). They can interact with
the surface proteins, the biomembrane and the DNA or RNA. While most plants interact
unselectively with the virus, selective interactions do also occur.
Ten plants demonstrated antiviral protection against BVDV in combination with low
cytotoxicity. Four plants (Panax ginseng, Cassia tora, Ginkgo biloba and Viola yezoensis)
exerted protective antiviral effect only at high doses, whereas other six plant extracts (Celosia
cristata, Ophioglossum vulgatum, Houttuynia cordata, Selaginella tamariscina, Alpinia
galanga and Alpinia oxyphylla) were effective at lower concentrations (Tab. 3).
Regarding the six plants with higher potential interest as a source of anti-HCV drugs, antiviral
glycoproteins, CCP-25 and CCP-27, purified from the leaves of Celosia cristata [43] have
been previously studied [44-48]. Their ability to inhibit viral RNA translation activities
against several plant viruses have been described [49].
Quercetin 3-O-methyl ether and ophioglonin obtained from plants belonging to
Ophioglossaceae genus have shown slight activity against HBV [50]. Since 1995 when
antiviral activities against enveloped viruses were discovered in extracts of Houttuynia
cordata [51], such as influenza, HIV, herpes, SARS and also in enteroviruses [51-54], 40
compounds have been isolated from the whole plant [55].
Among all of them, norcepharadione B has been identified as anti-herpes virus type 1
compound [55], quercetin may reduce virions production of HCV [56], but not against HBV
[7] and quercetin 3-rhamnoside may be effective against influenza A virus [57].
57
Selaginella tamariscina has been a source of several drugs with anti-bacterial and antifungal
activities such as amentoflavone [58], isocryptomerin [59-61], or with antitumor effects such
as sterols [62] and biflavonoids [63]. Alpinia galanga crude extracts have been shown to have
antibacterial activities [64] which seem to be enhanced in combination with other plants such
as rosemary and lemon iron bark [65]. Compounds obtained from this plant, have also
demonstrated other antimicrobial activities, such as anti-leishmanial phenylpropanoids [66] or
1'-acetoxychavicol acetate, and its halogenated derivatives (inhibitors of HIV-regulator
protein Rev-export) [67-70].
The insecticidal properties of diarylheptanoid [71] as well as protective effects on
anaphylactic reactions of the aqueous extracts from the fruit of Alpinia oxyphylla [72] have
been described in the past. Recently anti-angiogenic properties of the fruit have been also
discovered [73].
The water extracts were also screened against HBV in HepG2 2.2.15 cells (Tab. 3). Evodia
lepta, Hedyotis diffusa and several Glycyrrhiza species lowered the HBV DNA significantly
while being not toxic to the HepG2 2.2.15 cell line (Fig. 1).
Hardly anything is known about the other natural products of Evodia lepta, while the highly
bioactive chromenes seem to be among the major constituents [74]. Glycyrrhiza species, on
the other hand, are well known for their anti-inflammatory effects due to glycyrrhizic acid [9].
This genus is also known for its antiviral, especially antihepatitis properties [15,75]. Its ability
to reduce the HBV-DNA in the culture medium of HepG2 2.2.15 at high doses has been
previously reported [7]. Heydiotis diffusa again is a plant rich in iridoid glycosides with antiinflammatory and hepatoprotective activities [76-78]. These compounds are most likely to be
responsible for the effects against HBV.
Three enterohepatic cancer cell lines, HepG2 and SK Hep1 (from human hepatoblastoma and
hepatoma) and LS 174T (from human colon adenocarcinoma), were used to determine the
antitumor ability of water extracts (Tab. 3). Twenty extracts were found to induce a
significant antiproliferative effect with IC50 values between 0.5 and 20 µg/ml on these cell
lines. These were further investigated to elucidate whether this was due to cytotoxicity.
58
Fig. 1: Effect of water extracts on hepatitis B virus (HBV) release as determined by HBV-DNA content in the
culture medium and cell viability as determined by Neutral Red uptake by human hepatoblastoma cells HepG2
2.2.15 infected with HBV. Values are means ± SD of three experiments carried out in triplicate by incubation
with the extracts for 21 days.
59
Fig. 2: Acute cell toxicity as determined by MTT assay in human hepatoblastoma HepG2 cells. Values are
means ± SD of four experiments carried out in triplicate.
Fig. 3: Acute cell toxicity as determined by MTT assay in human hepatoma SK-Hep1 cells. Values are means ±
SD of four experiments carried out in triplicate.
60
Fig. 4: Acute cell toxicity as determined by MTT assay in human colon adenocarcinoma LS 174T cells. Values
are means±SD of four experiments carried out in triplicate.
In HepG2, none of the four extracts with ability to inhibit cell growth (Coptis chinensis,
Epimedium brevicornum, Equisetum hiemale and Senecio scandens) were found to induce
acute cell toxicity when they were incubated with the IC50 of the extracts for 6 h (Fig. 2).
In SK Hep1, among the ten extracts with antitumour effect seven did not induce acute toxicity
(Arctium lappa, Cassia tora, Centipeda minima, Chrysanthemum indicum, Coptis chinensis,
Phellodendron chinense and Rheum palmatum), whereas Dysosma versipellis was especially
active by lowering the cell viability in comparison to the control to 40% (Fig. 3). This is
consistent with the inhibitory effects known for the lignans of D. versipellis against prostate
cancer cell lines [79].
Evodia lepta and Kadsura longipedunculata lowered also the cell viability of Sk Hep1 in
comparison to the control to 50-60%. Recently, it has been reported that the essential oil of
Kadsura longipedunculata and its major components (delta-cadinene, camphene, borneol,
cubenol, and delta-cadinol) have some degree of cytotoxic activity against some human cell
lines [80]. In LS 174T cells, water extracts from Coptis chinensis, Dysosma versipellis,
Epimedium brevicornum, Hedyotis diffusa and Houttunia cordata have antiproliferative
effects without affecting cell viability (Fig. 4), whereas Paeonia lactiflora, Platycladus
orientalis, and Polygonum aviculare, in addition to inhibition of cell growth were able to
acutely lower cell viability in comparison to the control to 60-70%.
61
Paeonia lactiflora, which belongs to the Paeoniacea family, is known as one of the richest
sources of various resveratrol derivatives [81]. These phytoestrogens are known to exert
strong antioxidant activity [81] and to inhibit growth of several cancer cell lines [82,83],
including a colon human cell line [84]. Recently, the antiproliferative effects of essential oils
obtained from Platycladus orientalis on human renal adenocarcinoma and amelanotic
melanoma cells have been reported [85].
Coptis chinensis, which has been found active against the three enterohepatic cell lines,
belongs to TCM formulations commonly used to treat liver diseases associated to infections
by gastrointestinal parasite such as Blastocystis hominis [86]. Coptisine, which is used as
gastric mucosa protector, and berberine, which has very interesting properties as
antiinflamatory, antidiabetes, antidiarrhea, and hypocholesterolemic drug, have been obtained
from this plant. Berberine has also shown antitumoral activities in other in vitro models [8791].
Animal cells offer several targets to natural products (Tab. 1). Of great importance are the
biomembrane, the proteins and the DNA. Since human cells and trypanosomes share many
similarities in the structure of the cells, it is extremely important to select those plant extracts
where a great selectivity index occurred. The different cytotoxicity strongly hints to selective
interactions between natural products and trypanosomes that do not occur in human cells. A
great selectivity index also hints to the relative absence or relative insignificance of general
cytotoxic mechanisms like the unspecific interaction of phenolic OH-groups compared to
more specific interactions with certain structures in trypanosomes.
The CH2Cl2 and MeOH extracts of 82 medicinal plants were screened against the cell lines
HeLa, Cos7 and trypanosomes (T. b. brucei) (Tab. 4). The Selectivity index (SI) of the IC50 of
mammalian cell/ trypanosomes was regarded as significant if it was over 80. According to this
criterium, seven extracts were highly selective towards trypanosomes.
The CH2Cl2 extract of Alpinia oxyphylla showed IC50 values of 119.6 µg/ ml, 30.4 µg/ ml and
0.7 µg/ ml against HeLa, Cos7 and T. b. brucei respectively with SI of 170 and 43 between
HeLa and T. b. brucei and Cos7 and T. b. brucei respectively. The MeOH extract of A.
oxyphylla also was effective with IC50 values of 213.8 µg/ ml, 110.2 µg/ ml and 2.0 µg/ ml
62
against HeLa, Cos7 and T. b. brucei respectively. The SI was 107 and 55 between HeLa and T.
b. brucei and Cos7 and T. b. brucei respectively. A. oxyphylla is basically an essential oil plant,
so that we suspect the active principle to be based on the sesquiterpenes already known for
their cytotoxic properties [92].
For Kadsura longipedunculata only the CH2Cl2 extract exhibited a significant selectivity.
Here, the IC50 values of HeLa, Cos7 and T. b. brucei were 9.9 µg/ ml, 1.8 µg/ ml and 0.1 µg/
ml respectively, resulting in SI of 99 and 18 between HeLa and T. b. brucei and Cos7 and T. b.
brucei respectively. Essential oil and lignans form the major natural compounds of K.
longipedunculata [80,93]. The specific trypanocidal effect rather seems to be based on the
lignans than on the more unspecific essential oil. Further studies would be necessary to
confirm this assumption.
For Arctium lappa only the CH2Cl2 extract showed a significant selectivity with IC50 values
of 345.0 µg/ ml, 344.2 µg/ ml and 3.6 µg/ ml against HeLa, Cos7 and T. b. brucei respectively
and SI of 96 between HeLa and T. b. brucei and Cos7 and T. b. brucei.
In Panax ginseng and P. notoginseng the selectivity was again limited to the CH2Cl2 extract.
P. ginseng gave IC50 values of 152.4 µg/ ml, 47.7 µg/ ml and 0.9 µg/ ml against HeLa, Cos7
and T. b. brucei respectively with SI of 169 and 53 between HeLa and T. b. brucei and Cos7
and T. b. brucei respectively. P. notoginseng demonstrated IC50 values of 263.0 µg/ ml, 6.4
µg/ ml and 0.9 µg/ ml with SI of 292 and 7 between HeLa and T. b. brucei and Cos7 and T. b.
brucei respectively.
Also an extremely active plant was Saposhnikovia divaricata. Here as well, lipophilic CH2Cl2
extract was selective with IC50 values of 410.1 µg/ ml, 45.9 µg/ ml and 5.1 µg/ ml against
HeLa, Cos7 and T. b. brucei respectively and SI of 80 and 9 between HeLa and T. b. brucei
and Cos7 and T. b. brucei respectively.
The trypanocidal effects of A. lappa, P. ginseng, P. notoginseng and S. divaricata are based
on the presence of highly reactive polyacetylenes, especially panaxynol.
Only the methanolic extract of Coptis chinensis showed a significant selectivity, but not the
dichloromethane extract. The IC50 values of 81.8 µg/ ml, 3.7 µg/ ml and 0.4 µg/ ml against
HeLa, Cos7 and T. b. brucei respectively gave SI of 205 and 9 between HeLa and T. b. brucei
and Cos7 and T. b. brucei respectively. Our analytical data confirmed berberine as the main
63
alkaloid of C. chinensis. The toxicity of C. chinensis is probably an effect of the DNA
intercalation of its alkaloids into the DNA double helix of T. b. brucei [94,95].
The trypanocidal effect of berberine against different trypanosome species has been
demonstrated previously. Merschjohann et al. [96] showed that T. congolense are sensitive to
berberine at concentrations of 83 µM, while Rosenkranz and Wink [97] demonstrated a
sensitivity of T. brucei to berberine at concentrations of only 0.5 µM. Recently, the effect of
berberine against T. rhodesiense was also established by Freiburghaus et al. [98].T.
rhodesiense was sensitive to 4.2 µg/ml.
The significant differences in sensitivity of different trypanosome species to berberine could
be of high interest regarding resistance mechanisms against mutagenic compounds. Berberine
might due to its mutagenic activity never become a lead structure for the development of
trypanocidal drugs, but the differences in sensitivity of these three trypanosome species might
help to understand defence mechanisms against DNA intercalating substances.
Traditional Chinese and European Medicine comprise promising plants that might be used for
antiviral, antitrypanosomal and anticancer therapy. The promising discoveries of highly
effective plants against viral hepatitis, trypanosomiasis and liver and intestinal cancer cells
however require further research to establish new lead structures or their combinations for the
treatment of these important diseases.
64
Table 3: Cytotoxicity against cancer cells, Cos7 fibroblasts, and antiviral activity against HBV and flaviviruses of water extracts obtained from 82
medicinal plants
Antiumour Effect
O
Family
Species
IPMB / N
GenBank
Acanthaceae
Amaranthaceae
Andrographis paniculata
Celosia cristata
Bupleurum chinense
Bupleurum marginatum
Centella asiatica
Cnidium monnieri
Saposhnikovia divaricata
Eleutherococcus senticosus
Panax ginseng
Panax notoginseng
Areca catechu
Cynanchum paniculatum
Artemisia annua
Artemisia capillaris
Arctium lappa
Centipeda minima
Chrysanthemum indicum
Chrysanthemum morifolium
Eclipta prostata
Senecio scandens
Siegesbeckia orientalis
Taraxacum officinale
Berberis bealei
Dysosma versipellis
Epimedium koreanum
Isatis indigotica (root)
Isatis indigotica (leaf)
Lonicera confusa
Polygonatum kingianum
Rhodiola rosea
Platycladus orientalis
Cyrtomium fortunei
Ephedra sinica
Equisetum hiemale
Croton tiglium
Abrus cantoniensis
Acacia catechu
Cassia tora
P6838 / 04
P6848 / 14
P6844 / 10
P6845 / 11
P6849 / 15
P6854 / 20
P6902 / 68
P6919 / 79
P8088 / 81
P6887 / 53
P6840 / 06
P6858 / 24
P6841 / 07
P6842 / 08
P6839 / 05
P6850 / 16
P6851 / 17
P6852 / 18
P6863 / 29
P6905 / 71
P6906 / 72
P6908 / 74
P6883 / 49
P6862 / 28
P6865 / 31
P6877 / 43
P6878 / 44
P6880 / 46
P6892 / 58
P6920 / 84
P6891 / 57
P6859 / 25
P6864 / 30
P6866 / 32
P6856 / 22
P6835 / 01
P6836 / 02
P6847 / 13
JF949965
JF949970
JF950021
JF949968
JF950022
JF949973
JF949988
JF950028
JF950030
JF949975
JF949966
JF949967
JF949994
JF949971
JF949972
JF950000
JF949989
JF949990
JF950019
JF949996
JF950002
JF949981
JF949981
JF949982
JF950027
JF950011
JF949998
JF950001
JF950003
JF949964
JF949969
Apiaceae
Araliaceae
Arecaceae
Asclepiadaceae
Asteraceae
Berberidaceae
Brassicaceae
Caprifoliaceae
Convallariaceae
Crassulaceae
Cupressaceae
Dryopteridaceae
Ephedraceae
Equisetaceae
Euphorbiaceae
Fabaceae
Cos 7
IC50
(µg / ml)
255.6
263.9
15.6
350.6
325.8
339.7
153.0
130.5
151.7
182.3
16.6
220.7
288.6
201.9
355.5
55.6
320.9
760.4
291.7
114.2
159.2
156.9
270.0
1276.9
140.7
557.2
93.5
446.8
298.4
61.9
97.7
30.4
69.1
265.9
166.2
575.2
35.7
481.3
HeLa
IC50
(µg / ml)
576.0
2773.5
339.3
838.1
1436.8
775.5
1024.7
430.0
2594.6
1574.9
378.1
693.9
775.5
561.7
516.3
207.2
583.4
1045.8
667.0
607.9
542.8
708.5
659.4
1274.8
280.2
2427.4
1223.5
812.2
2321.7
144.4
428.2
567.4
193.1
1058.2
1052.7
587.1
157.5
1519.3
65
Antiviral Effect
a
HepG2
IC50
Sk Hep1
IC50
LS 174T
IC50
170
200
120
ND
> 200
> 200
> 200
> 200
> 200
> 200
40
> 200
177
142
200
72
130
> 200
> 200
3.5
40
130
63
> 200
5
> 200
170
> 200
> 200
160
> 200
> 200
200
5
140
> 200
> 200
> 200
80
180
100
ND
40
> 200
200
125
140
> 200
21
130
50
30
5
0,5
8
180
30
50
100
147
60
3.5
32
> 200
98
> 200
130
110
155
> 200
150
> 200
50
100
25
0.5
> 200
> 200
100
ND
70
> 200
155
160
> 200
200
90
> 200
> 200
> 200
> 200
130
200
> 200
120
25
50
65
70
3.5
10
> 200
80
> 200
42
40
10
> 200
> 200
> 200
> 200
> 200
> 200
> 200
anti-BVDV
Toxicity
on EBTr cells
0
0
++
ND
++++
++++
++
++
0
0
++++
++
++++
++
++
++
++++
++
++
++++
++
++
ND
++++
++
0
++
++
++
++
++
++
++++
++
++++
++
++
0
b
anti-BVDV
protection in
EBTr cells
0
++
0
ND
0
0
0
++
+
0
0
++
0
0
0
++
0
0
++
++
++
++
ND
0
0
0
++
0
0
++
0
++
++
0
0
++
++
+
c
anti-HBV
effect in Hep
G2 2.2.15
++
0
ND
ND
0
0
++
0
0
ND
ND
0
++
++
0
ND
0
0
0
ND
ND
0
ND
0
ND
0
0
0
0
0
0
0
ND
ND
++
0
++
++
d
Toxicity
at effective
doses
++
ND
ND
ND
ND
ND
++
ND
0
ND
ND
ND
++
++
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
++
ND
++
++
Geraniaceae
Ginkgoaceae
Hypericaceae
Iridaceae
Lamiaceae
Lauraceae
Loranthaceae
Lythraceae
Magnoliaceae
Melanthiaceae
Myrsinaceae
Myrtaceae
Ophioglossaceae
Orchidaceae
Paeoniaceae
Pedaliaceae
Poaceae
Polygonaceae
Ranunculaceae
Rosaceae
Rubiaceae
Rutaceae
Saururaceae
Schisandraceae
Selaginellaceae
Valerianaceae
Verbenaceae
Violaceae
Zingiberaceae
Desmodium styracifolium
Glycyrrhiza inflata
Spatholobus suberectus
Sutherlandia frutescens
Geranium wilfordii
Pelargonium sidoides
Ginkgo biloba
Hypericum japonicum
Belamcanda chinensis
Mentha haplocalyx
Prunella vulgaris
Scutellaria baicalensis
Cinnamomum cassia
Taxillus chinensis
Punica granatum
Magnolia officinalis
Paris polyphylla
Lysimachia christinae
Eucalyptus robusta
Ophioglossum vulgatum
Dendrobium loddigesii
Paeonia lactiflora
Harpagophytum procumbens
Cymbopogon distans
Fallopia japonica
Polygonum aviculare
Polygonum multiflorum
Rheum officinale
Coptis chinensis
Rosa chinensis
Rosa laevigata
Sanguisorba officinalis
Hedyotis diffusa
Evodia lepta
Evodia rutaecarpa
Phellodendron chinense
Houttuynia cordata
Kadsura longipedunculata
Selaginella tamariscina
Patrinia scabiosaefolia
Verbena officinalis
Viola yezoensis
Alpinia galanga
Alpinia oxyphylla
P6861 / 27
P6873 / 39
P6907 / 73
tba / 83
P6867 / 33
tba / 82
P6872 / 38
P6876 / 42
P6843 / 09
P6884 / 50
P6896 / 62
P6903 / 69
P6853 / 19
P6909 / 75
P6897 / 63
P6882 / 48
P6888 / 54
P6881 / 47
P6868 / 34
P6885 / 51
P6860 / 26
P6886 / 52
tba / 80
P6857 / 23
P6894 / 60
P6893 / 59
P6895 / 61
P6898 / 64
P6855 / 21
P6899 / 65
P6900 / 66
P6901 / 67
P6874 / 40
P6869 / 35
P6870 / 36
P6890 / 56
P6875 / 41
P6879 / 45
P6904 / 70
P6889 / 55
P6910 / 76
P6911 / 77
P6837 / 03
P6917 / 78
JF949976
JF950025
JF949991
JF949977
JF950005
JF949980
JF949995
JF949984
JF950013
JF950017
JF950023
JF949992
JF950014
JF950008
JF950010
JF949983
JF950009
JF949999
JF950026
JF949974
JF950004
JF950012
JF949987
JF950015
JF950024
JF950016
JF949979
JF949978
JF949986
JF950006
JF950007
JF950018
JF949985
JF950020
JF949993
-
333.5
583.9
16.6
857.6
225.8
15.2
450.8
151.8
222.1
285.7
21.5
46.4
453.9
181.7
8.6
73.0
38.4
152.1
94.1
344.0
104.0
148.2
242.9
257.7
39.8
82.6
61.3
51.5
118.3
24.3
93.6
20.5
158.7
419.2
1176.9
282.9
633.2
6.8
103.9
147.3
93.9
135.0
952.8
105.8
651.4
2288.0
174.1
1670.7
62.1
62.2
1717.0
445.5
1378.8
519.1
341.3
150.0
713.6
1023.2
152.4
451.5
42.6
431.4
15.8
1780.1
294.4
287.3
733.4
486.1
596.4
488.6
928.0
670.9
101.0
135.8
781.7
87.0
1542.7
971.0
185.6
750.3
2835.9
167.6
703.4
525.5
416.9
1459.2
2357.3
1802.2
66
> 200
> 200
100
> 200
80
200
> 200
165
> 200
70
100
80
180
> 200
100
ND
54
> 200
ND
> 200
> 200
> 200
160
> 200
> 200
> 200
ND
200
10
ND
190
ND
> 200
> 200
> 200
50
> 200
> 200
> 200
168
100
170
> 200
> 200
> 200
> 200
135
70
45
> 200
9
80
> 200
82
145
50
> 200
> 200
100
ND
168
> 200
ND
> 200
70
> 200
190
> 200
> 200
> 200
ND
25
2
ND
135
ND
> 200
20
25
10
135
20
> 200
87
168
200
> 200
> 200
150
185
70
> 200
200
45
> 200
100
> 200
> 200
80
120
> 200
155
60
ND
8
> 200
ND
> 200
160
10
100
> 200
80
10
ND
200
18
ND
60
ND
5
> 200
100
85
5
20
200
35
117
140
> 200
155
0
0
++
0
++
++
0
++
++
++++
++
++
++++
0
++++
ND
++++
++
ND
0
++
0
++
++++
++++
++
ND
++
++++
ND
++
ND
++
++++
++++
++
0
++
0
++
++
0
0
0
0
0
0
0
++
0
+
++
0
0
0
0
0
0
0
ND
++
0
ND
++
++
0
++
0
++
0
ND
0
0
ND
++
ND
++
0
++
++
++
++
++
++
0
+
++
++
0
++++
ND
0
ND
0
0
0
++
ND
ND
ND
++
0
ND
ND
ND
0
ND
0
0
ND
0
++
0
0
ND
0
ND
ND
0
ND
++++
++++
0
ND
0
0
0
0
ND
ND
++
0
ND
0
ND
ND
ND
ND
ND
ND
++
ND
ND
ND
++
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
++
ND
ND
ND
ND
ND
ND
ND
ND
0
0
ND
ND
ND
ND
0
ND
ND
ND
++
ND
a
Toxicity on EBTr cells: 0, not toxic; ++, toxic at high concentrations; ++++, toxic in all concentrations.
b
Anti-BVDV protection in EBTr cells: 0, without effect; +, protection at high concentrations; ++, protection at low concentrations
c
Anti-HBV effect in Hep G2 2.2.15 cells: 0, without effect; ++, effect comparable to toxicity; ++++, high ability to reduce HBV DNA.
d
Toxicity at effective dose on Hep G2 2.2.15 cells: 0, not toxic; ++, effect comparable to reduction in HBV DNA.
ND: Not determined
67
Table 4: Cytotoxicity against HeLa cancer cells, Cos7 fibroblasts and Trypanosoma brucei brucei of organic extracts obtained from 82 medicinal
plants
CH2Cl2
CH2Cl2
CH2Cl2
MeOH
MeOH
MeOH
HeLa
Cos 7
T. b. brucei
Family
Species
IPMB / N
GenBank
HeLa
Cos 7
T. b. brucei
Acanthaceae
Amaranthaceae
Andrographis paniculata
Celosia cristata
Bupleurum chinense
Bupleurum marginatum
Centella asiatica
Cnidium monnieri
Saposhnikovia divaricata
Eleutherococcus senticosus
Panax ginseng
Panax notoginseng
Areca catechu
Cynanchum paniculatum
Artemisia annua
Artemisia capillaris
Arctium lappa
Centipeda minima
Chrysanthemum indicum
Chrysanthemum
morifolium
Eclipta prostata
Senecio scandens
Siegesbeckia orientalis
Taraxacum officinale
Berberis bealei
Dysosma versipellis
Epimedium koreanum
Isatis indigotica (root)
Isatis indigotica (leaf)
Lonicera confusa
Polygonatum kingianum
Rhodiola rosea
Platycladus orientalis
Cyrtomium fortunei
Ephedra sinica
Equisetum hiemale
Croton tiglium
Abrus cantoniensis
Acacia catechu
P6838 / 04
P6848 / 14
P6844 / 10
P6845 / 11
P6849 / 15
P6854 / 20
P6902 / 68
P6919 / 79
P8088 / 81
P6887 / 53
P6840 / 06
P6858 / 24
P6841 / 07
P6842 / 08
P6839 / 05
P6850 / 16
P6851 / 17
JF949965
JF949970
JF950021
JF949968
JF950022
JF949973
JF949988
JF950028
JF950030
JF949975
JF949966
JF949967
JF949994
JF949971
188.4
472.0
235.2
176.0
175.0
127.1
410.1
300.0
152.4
263.0
1023.3
395.6
107.9
93.5
345.0
63.3
152.1
104.7
136.0
87.1
67.4
64.9
37.0
45.9
61.4
47.7
6.4
117.0
114.2
34.5
29.4
344.2
10.4
63.5
16.8
55.2
17.0
16.2
14.0
14.9
5.1
13.5
0.9
0.9
22.5
53.1
8.1
10.6
3.6
2.2
16.0
Ratio
HeLa/ T.
b. brucei
11
9
14
11
13
9
80
22
169
292
45
7
13
9
96
29
10
P6852 / 18
JF949972
129.4
42.8
19.3
7
2
349.2
P6863 / 29
P6905 / 71
P6906 / 72
P6908 / 74
P6883 / 49
P6862 / 28
P6865 / 31
P6877 / 43
P6878 / 44
P6880 / 46
P6892 / 58
P6920 / 84
P6891 / 57
P6859 / 25
P6864 / 30
P6866 / 32
P6856 / 22
P6835 / 01
P6836 / 02
JF950000
JF949989
JF949990
JF950019
JF949996
JF950002
JF949981
JF949981
JF949982
JF950027
JF950011
JF949998
JF950001
JF950003
JF949964
-
266.4
268.6
101.5
232.8
93.8
213.9
48.7
196.4
321.5
226.5
279.6
164.1
121.7
572.4
95.3
125.6
422.9
494.4
164.1
112.0
143.5
17.7
177.1
13.3
49.9
3.5
42.3
0.6
58.9
53.9
74.6
21.8
132.1
41.8
35.7
225.9
129.4
31.5
38.1
13.1
7.9
17.5
5.9
39.5
4.2
2.9
45.3
16.2
52.6
43.9
17.7
37.1
20.9
30.9
86.5
14.5
13.1
7
21
13
13
16
5
12
68
7
14
5
4
7
15
5
4
5
34
12
3
11
2
10
2
1
0.8
14
0.01
3
1
1
1
3
2
1
2
9
2
329.7
299.3
237.5
636.7
149.7
385.2
257.5
674.3
274.2
923.5
1517.9
705.5
722.0
163.5
241.2
297.0
612.4
318.0
Apiaceae
Araliaceae
Arecaceae
Asclepiadaceae
Asteraceae
Berberidaceae
Brassicaceae
Caprifoliaceae
Convallariaceae
Crassulaceae
Cupressaceae
Dryopteridaceae
Ephedraceae
Equisetaceae
Euphorbiaceae
Fabaceae
O
68
Ratio
Cos7/ T. b.
brucei
6
2
5
4
4
2
9
4
53
7
5
2
4
3
96
5
4
28.8
77.2
120.8
111.9
44.7
17.9
999.5
17.3
319.0
469.6
118.1
39.3
51.2
51.9
2229.0
13.3
15.3
Ratio
HeLa/ T.
b. brucei
11
6
5
10
17
14
2
40
4
2
4
13
6
6
0.7
16
23
Ratio
Cos7/ T. b.
brucei
12
0.3
3
5
8
6
1
11
1
0.4
0.2
5
4
4
0.8
4.0
18
323.3
499.8
646.4
1147.0
773.0
251.1
1515.6
692.0
1427.9
1241.6
414.2
500.5
287.2
314.9
1467.7
219.1
355.7
344.7
28.4
358.7
576.0
392.8
120.0
1575.4
190.1
510.8
229.5
31.0
227.7
201.1
215.4
1813.0
54.2
287.2
166.7
24.9
14
6
186.1
126.2
84.4
485.3
35.3
54.9
30.7
324.4
90.6
118.9
1535.3
87.4
158.2
348.7
36.7
243.5
222.1
733.1
34.8
39.6
18.6
12.3
64.9
7.8
53.2
12.6
94.6
14.6
38.0
119.5
84.2
61.0
23.4
51.6
150.4
73.5
50.8
8
16
19
10
19
7
20
7
19
24
13
8
12
7
4
2
8
6
4.6
6
6
7
4
1.0
2
3
6
3
12
1
5
1
4
1
10
0.6
Geraniaceae
Ginkgoaceae
Hypericaceae
Iridaceae
Lamiaceae
Lauraceae
Loranthaceae
Lythraceae
Magnoliaceae
Melanthiaceae
Myrsinaceae
Myrtaceae
Ophioglossaceae
Orchidaceae
Paeoniaceae
Pedaliaceae
Poaceae
Polygonaceae
Ranunculaceae
Rosaceae
Rubiaceae
Rutaceae
Saururaceae
Schisandraceae
Selaginellaceae
Valerianaceae
Verbenaceae
Violaceae
Zingiberaceae
Cassia tora
Desmodium styracifolium
Glycyrrhiza inflata
Spatholobus suberectus
Sutherlandia frutescens
Geranium wilfordii
Pelargonium sidoides
Ginkgo biloba
Hypericum japonicum
Belamcanda chinensis
Mentha haplocalyx
Prunella vulgaris
Scutellaria baicalensis
Cinnamomum cassia
Taxillus chinensis
Punica granatum
Magnolia officinalis
Paris polyphylla
Lysimachia christinae
Eucalyptus robusta
Ophioglossum vulgatum
Dendrobium loddigesii
Paeonia lactiflora
Harpagophytum
procumbens
Cymbopogon distans
Fallopia japonica
Polygonum aviculare
Polygonum multiflorum
Rheum officinale
Coptis chinensis
Rosa chinensis
Rosa laevigata
Sanguisorba officinalis
Hedyotis diffusa
Evodia lepta
Evodia rutaecarpa
Phellodendron chinense
Houttuynia cordata
Kadsura longipedunculata
Selaginella tamariscina
Patrinia scabiosaefolia
Verbena officinalis
Viola yezoensis
Alpinia galanga
Alpinia oxyphylla
P6847 / 13
P6861 / 27
P6873 / 39
P6907 / 73
tba / 83
P6867 / 33
tba / 82
P6872 / 38
P6876 / 42
P6843 / 09
P6884 / 50
P6896 / 62
P6903 / 69
P6853 / 19
P6909 / 75
P6897 / 63
P6882 / 48
P6888 / 54
P6881 / 47
P6868 / 34
P6885 / 51
P6860 / 26
P6886 / 52
JF949969
JF949976
JF950025
JF949991
JF949977
JF950005
JF949980
JF949995
JF949984
JF950013
JF950017
JF950023
JF949992
JF950014
JF950008
JF950010
JF949983
JF950009
JF949999
JF950026
1335.4
156.0
26.4
299.1
367.7
99.1
488.2
768.3
163.3
324.4
108.5
282.1
90.9
138.9
417.8
583.3
23.6
952.6
53.4
188.9
83.0
166.9
189.1
139.8
6.9
154.6
259.3
17.0
218.2
15.3
10.8
89.2
34.1
90.4
287.9
23.2
68.6
126.6
5.4
24.0
137.3
62.8
25.7
34.0
185.9
16.3
6.4
25.4
41.8
23.0
52.1
71.9
21.3
22.3
14.7
13.2
7.4
11.0
27.2
14.6
0.9
73.6
20.6
19.8
13.5
9.1
7
10
4
12
9
4
9
11
8
15
7
21
12
13
15
40
26
13
3
1
8
1
6
6
0.7
4
0.2
0.5
4
2
7
39
2
2
8
6
0.3
7
10
6
18
tba / 80
-
36.2
15.8
0.9
P6857 / 23
P6894 / 60
P6893 / 59
P6895 / 61
P6898 / 64
P6855 / 21
P6899 / 65
P6900 / 66
P6901 / 67
P6874 / 40
P6869 / 35
P6870 / 36
P6890 / 56
P6875 / 41
P6879 / 45
P6904 / 70
P6889 / 55
P6910 / 76
P6911 / 77
P6837 / 03
P6917 / 78
JF949974
JF950004
JF950012
JF949987
JF950015
JF950024
JF950016
JF949979
JF949978
JF949986
JF950006
JF950007
JF950018
JF949985
JF950020
JF949993
-
425.9
88.0
118.5
469.4
22.5
100.0
559.4
712.3
66.5
147.8
232.0
50.4
370.1
279.9
9.9
339.2
140.5
298.1
59.6
55.7
119.6
114.5
2.8
53.3
107.7
39.5
141.5
151.8
26.7
45.3
42.0
8.7
71.3
48.2
1.8
98.8
38.7
145.9
60.8
5.7
30.4
31.1
13.1
18.2
98.6
34.0
12.9
20.1
20.6
12.3
13.3
13.9
16.8
15.6
68.3
0.1
13.6
13.7
16.5
3.3
1.4
0.7
69
3
2
3
670.9
324.3
528.3
237.5
586.6
236.0
112.3
302.9
177.5
522.6
375.0
475.4
367.6
272.4
1213.4
211.2
49.1
35.0
1752.6
181.4
469.0
232.8
294.6
75.9
104.1
126.8
54.8
352.0
169.8
95.7
260.1
100.9
319.5
147.8
494.5
28.8
108.4
378.2
218.6
13.1
5.5
436.3
15.2
68.8
61.6
309.8
276.9
40.1
39.0
67.8
87.4
13.3
18.3
39.3
23.6
80.2
16.2
25.1
86.2
13.4
59.2
8.1
4.3
11.8
52.1
16.3
33.2
27.6
11.7
2
8
14
4
7
18
6
8
8
7
23
19
4
20
20
26
11
3
34
11
14
8
25
0.2
2
3
0.8
4
12
5
6
4
4
9
19
0.3
8
6
27
3
0.4
8
1
2
2
26
40
17
692.6
217.2
21.4
32
10
14
7
7
5
0.6
8
28
35
5
11
17
3
24
4
99
25
10
18
18
39
170
3
0.2
3
1
3
7
7
2
3
3
0.5
4
0.7
18
7
3
9
18
4
43
98.8
317.3
342.3
437.4
270.9
81.8
266.6
1855.4
158.5
796.1
350.7
297.4
487.6
575.2
86.1
393.9
159.4
334.7
297.5
111.7
213.8
17.6
19.5
226.5
48.8
35.3
3.7
36.7
1100.1
41.6
418.1
427.7
178.5
85.5
63.9
43.9
150.9
15.9
37.7
19.1
53.4
110.2
18.9
19.0
49.1
62.1
24.5
0.4
12.5
102.9
4.0
24.9
44.4
29.5
14.1
97.6
11.8
33.4
19.0
20.5
24.7
15.4
2.0
5
17
7
7
11
205
21
18
40
32
8
10
35
6
7
12
8
16
12
7
107
1
1
4
0.7
1
9
3
10
10
16
9
6
6
0.6
3
4
0.8
1
0.7
3
55
Origin and current area of use of the medicinal plants included in our study
Family
Acanthaceae
Species
Andrographis paniculata
Amaranthaceae
Celosia cristata
Apiaceae
Bupleurum chinense
Bupleurum marginatum
Centella asiatica
Cnidium monnieri
Saposhnikovia divaricata
Araliaceae
Eleutherococcus senticosus
Panax ginseng China
Panax ginseng Korea
Panax notoginseng
Arecaceae
Areca catechu
Asclepiadaceae
Asteraceae
Cynanchum paniculatum
Artemisia annua
Artemisia capillaris
Arctium lappa
Centipeda minima
Chrysanthemum indicum
Chrysanthemum morifolium
Eclipta prostrata
Senecio scandens
Siegesbeckia orientalis
70
Origin
Area of Use
India, Sri Lanka,
India
SE Asia, East Asia
India, SE Asia,
Tropical Asia
China, Africa,
South America
China
East Asia, China
China
East Asia, China
East Asia, India,
East Asia, India,
Sri Lanka,
Sri Lanka,
Australia,
northern Australia,
Melanesia, Papua
Iran, Melanesia,
New Guinea,
Papua New
Middle East,
Guinea
Africa
China
East Asia, China
Central Asia
Central Asia, East
(steppe region)
Asia, China
Siberia, China,
Siberia
Korea, Japan
Siberia, China,
China
Korea, Japan
Siberia, China,
Korea
Korea, Japan
Siberia, China,
China
Korea, Japan
SE Asia, East
Malaysia,
Asia, India, Sri
Philipines
Lanka, Papua New
Guinea
SE Asia
East Asia, SE Asia
Asia, introduced
worldwide
worldwide
Asia
Asia
Northern
Hemisphere
Europe, Asia
(Europe, Asia,
North America)
Asia, Himalaya
Asia
India
Asia
Asia
Asia
Tropical Asia,
Tropical Asia,
East Asia, South
South America
America
Asia
Asia
Tropical Asia
Tropical Asia,
East Asia, Africa
Taraxacum officinale
Berberidaceae
Berberis bealei
Brassicaceae
Dysosma versipellis
Epimedium koreanum
Capsella bursa-pastoris
Isatis indigotica (root)
Isatis indigotica (leaf)
Caprifoliaceae
Convallariaceae
Crassulaceae
Cupressaceae
Lonicera confusa
Polygonatum kingianum
Rhodiola rosea
Platycladus orientalis
Dryopteridaceae Cyrtomium fortunei
Ephedraceae
Equisetaceae
Euphorbiaceae
Fabaceae
Ephedra sinica
Equisetum hiemale
Croton tiglium
Abrus cantoniensis
Acacia catechu
Cassia tora
Desmodium styracifolium
Glycyrrhiza inflata
Spatholobus suberectus
Sutherlandia frutescens
Geraniaceae
Geranium wilfordii
Northern
Hemisphere
(Europe, Asia,
North America)
Asia, introduced in
America, Europe
East Asia
East Asia (Korea)
Northern
Hemisphere
(Europe, Asia,
North America)
Central Asia
(steppe region)
Central Asia
(steppe region)
East Asia
Asia
Northern
Hemisphere
(Europe, Asia,
North America)
China, introduced
in most of Asia
Asia, introduced in
America, Europe
China
Northern
Hemisphere
(Europe, Asia,
North America)
SE Asia
Southern China
East Asia, SE Asia
East Asia, SE
Asia, introduced to
Middle and South
America, Africa,
Middle East
SE Asia
Central Asia
(Mongolia, China)
Tropical Asia
South Africa
East Asia
71
Europe, Asia,
North America
Asia, America,
Europe
East Asia, China
East Asia, China
Europe, Asia
Central Asia, East
Asia, China
Central Asia, East
Asia, China
East Asia, China
Asia
Europe, Asia,
North America
Asia
Asia
East Asia
Europe, Asia,
North America
East Asia, SE Asia
East Asia, SE Asia
East Asia, SE Asia
Europe, Asia,
America, Africa
East Asia, SE Asia
East Asia, Central
Asia
India, East Asia,
SE Asia
South Africa,
Europe
East Asia, China
Pelargonium sidoides
Ginkgoaceae
Ginkgo biloba
Hypericaceae
Iridaceae
Lamiaceae
Hypericum japonicum
Belamcanda chinensis
Mentha haplocalyx
Prunella vulgaris
South Africa
China
Scutellaria baicalensis
Lauraceae
Cinnamomum cassia
Loranthaceae
Lythraceae
Taxillus chinensis
Punica granatum
Magnoliaceae
Melanthiaceae
Myrsinaceae
Myrtaceae
Magnolia officinalis
Paris polyphylla
Lysimachia christinae
Eucalyptus robusta
Japan
China
China
Northern
Hemisphere
(Europe, Asia,
North America)
Central Asia
(Russia, Mongolia,
China)
Tropical Asia
(India, East Asia,
SE Asia)
China
Middle East,
Himalaya
Himalaya, China
Himalaya, China
China
East Australia
Ophioglossaceae Ophioglossum vulgatum
Orchidaceae
Paeoniaceae
Pedaliaceae
Dendrobium loddigesii
Paeonia lactiflora
Harpagophytum procumbens
Poaceae
Polygonaceae
Cymbopogon distans
Fallopia japonica (syn.
Polygonum cuspidatum)
Fallopia multiflora (syn.
Polygonum multiflorum)
Polygonum aviculare
Rheum officinale
Ranunculaceae
Rosaceae
Northern
Hemisphere
(Europe, Asia,
North America)
SE Asia
China
South Africa
Himalaya, China
East Asia
East Asia
Northern
Hemisphere
(Europe, Asia,
North America)
Asia
Coptis chinensis
Rosa chinensis
Rosa laevigata
China
China
SE Asia, China
72
South Africa,
Europe
Asia, Europe,
North America
East Asia, China
East Asia, China
East Asia, China
Europe, Asia,
North America
East Asia, Central
Asia
India, East Asia,
SE Asia
East Asia, China
Europe, Asia,
America, Africa
East Asia, China
East Asia, China
East Asia, China
Europe, Asia,
America, Africa,
Australia
Europe, Asia,
North America
East Asia, SE Asia
East Asia
South Africa,
Europe
East Asia, China
East Asia, China
East Asia, China
Europe, Asia,
North America
Europe, Asia,
North America
East Asia, China
East Asia, China
Europe, Asia,
North America
Sanguisorba officinalis
Rubiaceae
Rutaceae
Saururaceae
Schisandraceae
Selaginellaceae
Valerianaceae
Verbenaceae
Violaceae
Zingiberaceae
Hedyotis diffusa
Evodia lepta
Evodia rutaecarpa
Phellodendron chinense
Houttuynia cordata
Kadsura longipedunculata
Selaginella tamariscina
Patrinia scabiosaefolia
Verbena officinalis
Viola yezoensis
Alpinia galanga
Alpinia oxyphylla
Northern
Hemisphere
(Europe, Asia,
North America)
East Asia
East Asia
East Asia
Himalaya, China
East Asia, SE Asia
East Asia
East Asia
East Asia
Europe
East Asia
SE Asia
SE Asia
73
Europe, Asia,
North America
East Asia, China
East Asia, China
East Asia, China
East Asia, China
East Asia, SE Asia
East Asia, China
East Asia, China
East Asia, China
Europe, Asia,
North America
East Asia, China
East Asia, SE Asia
East Asia, SE Asia
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4. Cytotoxicity of 12 structurally diverse ginsenosides from Panax
ginseng in HeLa cells is linked to membrane disturbance
4.1 Abstract
Panax ginseng is widely regarded as an important herbal medicine with a broad range of
pharmacological properties. We investigated the cytotoxicity of 12 structurally diverse
ginsenosides to gain further insight in their mode of action. The monodesmosidic
ginsenosides Rg3 and Rh2 with the sugar moiety attached to C3 exhibit a stronger cytotoxicity
with IC50 values of 245 and 29 µg/ml, respectively than monodesmosidic ginsenosides with
the sugar moiety attached to C6 or bidesmosidic ginsenosides. Only Rg3 and Rh2 behave as
typical monodesmosidic saponins which can intercalate in cell membranes and interact with
membrane proteins and cholesterol. The disturbance of membrane fluidity and permeability
can lead to apoptosis and cell death.
4.2 Introduction
Panax ginseng (Araliaceae) has been used in China, Korea, Japan and eastern Russia as a
medicinal plant for several thousand years. Its first record was on Chinese oracle bones 1600
BC while the first surviving detailed description of its medicinal properties dates to the Han
dynasty around 200 AD [1]. Since then ginseng root has always been regarded as one of the
most precious herbal medicines known. Recently, it got worldwide attention due to its various
medicinal properties.
The main secondary metabolites of ginseng are ginsenosides, but polyacetylenes, fatty acids,
polysaccharides and peptides are also present [2,3]. Today, 38 ginsenosides have been
isolated from P. ginseng. Taking P. quinquefolius and P. notoginseng into account, 66
ginsenosides are presently known [4]. The ginsenosides Rb1, Rb2, Rc, Rd, Re and Rg1
contribute to over 90% of the total ginsenosides in P. ginseng while the other 32 ginsenosides
are present as minor components [5].
Ginsenosides can be regarded as steroidal saponins with four trans-ring rigid steroid skeletons.
Several structural types of ginsenosides are known, such as bidesmosidic and
monodesmosidic ginsenosides and their aglycones. The bidesmosidic and monodesmosidic
ginsenosides again differ according to the sugar moieties and the position of their attachment
to the steroidal moiety. Sugars are attached to C3, C6 or C20 [6]. The structural diversity of
ginsenosides is due to the variation of the sugar moieties, their chain length and the position
81
of glycoside attachment. Common sugars are glucose, maltose, fructose and sucrose. The
position and number of hydroxyl groups on the steroidal moiety is variable as well. The
structural elements influence the biological activity.
Traditionally, P. ginseng has been used as a general tonic, while modern research revealed
many more applications such as the positive influence on reaction time, concentration and
mental fitness [7-10]. The ginsenosides Rg1 and Rb1 modulate the neurotransmission in the
brain [11,12] while the ginsenosides Rb1, Rg1, Rg2 and Re prevent scopolamine-induced
memory deficits by increasing the cholinergic activity [4,13-15]. Neuroprotective effects of
the ginsenosides Rb1, Rg1, Rg3, and Rh2 were shown in several studies [16-20].
Effects on the cardiovascular system are also well documented. The ginsenosides Rg1 and Rg3
were shown to relax vascular smooth muscles [21,22] and to inhibit endothelin production
[23], causing an antihypertensive effect. The ginsenosides Rb1, Re and Rg1 were shown to
enhance the recovery of the brain and organs after damage [20,24]. Anti-atheriosclerotic and
wound healing effects were demonstrated [25], which together with the inhibition of platelet
aggregation [26] might be one reason for the traditionally assumed increase of life expectancy
attributed to P. ginseng.
Another aspect of P. ginseng is the stimulation of the immune system [27,28]. The
ginsenoside Rb1 inhibits leukotriene release while Rg1 increases the activity of T-helper cells
[24]. The ginsenosides Rb1, Rg1 and Rg3 further inhibit cytokine production, COX-2 gene
expression and block histamine release [29-31]. P. ginseng was shown to lower the blood
glucose level, thus having a positive effect on diabetes [32-34].
One of the most important aspects of P. ginseng, however, is the induction of apoptosis in
tumour cells attributed to the ginsenosides Rg3 and Rh2 [35-38]. Several studies demonstrated
that P. ginseng has cancer preventive effects [39-41].
In this study, we investigated the cytotoxicity of 12 selected ginsenosides (Fig. 1) in cancer
cells (HeLa) representing all major groups with a special focus on mono- and bidesmosidic
molecules and their possible interference with membrane fluidity and permeability.
82
Fig. 1: Ginsenosides
Group I: Bidesmosidic saponins
Ginsenoside Rd
Ginsenoside Rc
Ginsenoside Rb1
Ginsenoside Re
Ginsenoside Rg1
Group II: Monodesmosidic saponins (C3-gly)
Ginsenoside Rg3
Ginsenoside Rh2
Group III: Monodesmosidic saponins (C6-gly)
Ginsenoside Rg2
Ginsenoside Rh1
Pseudoginsenoside F11
83
Group IV: Aglycones
Protopanaxadiol
Protopanaxatriol
4.3 Material and Methods
Chemicals
DMEM media, supplements, fetal bovine serum (FBS), trypsin-EDTA and dimethyl sulfoxide
(DMSO) was purchased from Gibco Invitrogen; Germany. Ginsenosides of known purity
were purchased in China by Wan Chuangxing, Tarim University, PR China.
Methods
Cytotoxicity
HeLa (human cervical cancer) cells were cultured in DMEM complete medium supplemented
with 10% FBS, 5% antibiotics (penicillin/ streptomycin) and 5% non-essential amino acids.
Cultures were maintained in a humidified atmosphere of 5 % CO2 at 37 °C.
Sample preparation
Ginsenosides were dissolved in DMSO prior to the experiment. The test samples were serially
diluted in medium, so that the final concentrations of DMSO did not exceed 0.05%.
Doxorubicin was used as positive control.
MTT assay
The MTT viability assay [42] was used to determine the cytotoxicity of the ginsenosides. 2 x
104 cells/well were seeded in 96-well plates (Greiner Labortechnik, Frickenhausen), cultured
for 24 h to 80% confluence before the test samples were added. Cells were incubated for 24 h
before the medium was replaced by fresh medium containing 0.5 mg/ml MTT. After 4 h, the
medium was discarded to be replaced with 100 µl DMSO. The absorbance was recorded at
570 nm with a Tecan Safire II Reader.
84
Statistical analysis
The IC50 values were calculated from 9 data points per concentration using a four parameter
logistic curve (SigmaPlot® 11.0).
4.4 Results
The cytotoxicity of 12 ginsenosides
with differing structures was analysed
in the cervical cancer cell line HeLa.
The commonly occurring ginsenosides
Rb1, Rc, Re and Rg1, had IC50 values
around 1.2 mg/ml. However, the
ginsenoside
Rd,
another
major
ginsenosides had an IC50 value of 161
µg/ml, ten times lower than of the other
main compounds. Rh2, protopanaxadiol
and protopanaxatriol had IC50 values of
HeLa IC50 [µg/ml]
Bidesmosidic saponins
Ginsenoside Rb1
Ginsenoside Rc
Ginsenoside Rd
Ginsenoside Re
Ginsenoside Rg1
1373.3
1431.5
161.3
1291.8
1408.8
Monodesmosidic saponins:
C3-gly
Ginsenoside Rg3
Ginsenoside Rh2
245.7
29.1
Monodesmosidic saponins:
C6-gly
Ginsenoside Rg2
Ginsenoside Rh1
Pseudoginsenoside F11
1172.7
452.9
2332.9
Aglycones
Protopanaxadiol
Protopanaxatriol
13.2
31.6
below 30 µg/ml, indicating a high
cytotoxicity (Tab. 1).
4.5 Discussion
Ginsenosides are generally regarded as
the main active secondary metabolites
Reference substance
Doxorubicin (positive
control)
0.6
of ginseng. They are saponins with
amphiphilic
properties
which
can
Table 1: Cytotoxic effects of ginsenosides
exhibit three main modes of action. Monodesmosidic ginsenosides can intercalate into the
biomembrane of a cell, complex membrane cholesterol and thus can change membrane
fluidity and permeability. By modifying the lipid environment of ion channels, transporter
proteins and receptors in the lipid bilayer of the cell membrane it is possible to modify the
function of membrane proteins [43,44].
Membrane proteins are either in cholesterol rich or cholesterol poor parts of the
biomembrane; the exact environment is crucial for their correct function [45]. Ginsenosides
are able to interact with the polar head of the phospholipids and the C3-OH group of
cholesterol through polar function groups such as hydroxyl groups of the sugar moieties. They
are also able to intercalate into the hydrophobic interior of the bilayer with their lipophilic
85
steroidal part. In the biomembrane, they are able to displace cholesterol from the immediate
environment of membrane proteins, thus increasing membrane fluidity and permeability. Any
change in the close environment of the membrane proteins can facilitate conformational
changes that can modulate the activity of the protein. In accordance with this observation,
ginsenosides were shown to modify the activity of voltage-dependent and ligand-gated ion
channels [16]. The steroidal structure of lipophilic ginsenoside aglycones facilitates diffusion
across the cell membrane. Another mode of action is based on their structural similarities to
steroid hormones. Thus they are likely to modify gene expression [43,46,47]. Their presence
in the nucleus was shown by Wakabayashi et al. [48]; their ability to bind to the
glucocorticoid receptor has also been demonstrated [49,50], leading to a modification of the
transcription of mRNA and the protein synthesis.
We selected 12 representative ginsenosides of the three general saponin types, the
bidesmosides, the monodesmosides and the aglycones (Fig. 1). The monodesmosides were
again subdivided into two groups with the sugar moiety attached to C3 or C6. The first group,
the bidesmosidic saponins, contains the ginsenosides Rb1, Rc, Rd, Re and Rg1. The second
group, the monodesmosidic saponins with the sugar moiety attached to C3, contains the
ginsenosides Rg3 and Rh2. The third, also monodesmosidic group with the sugar moiety
attached to C6, includes the ginsenosides Rg2, Rh1 and PF11, while the fourth group, the
aglycones, comprises the protopanaxadiol and protopanaxatriol.
The monodesmosidic ginsenosides Rg3 and Rh2 have glucopyranose sugar chains attached
only to C3 similar to the situation in many other steroidal and triterpenoid saponins. The IC50
values of Rg3 and Rh2 with 245 and 29 µg/ml demonstrated strong cytotoxic activity.
Although ginsenoside Rd is a bidesmosidic saponin, it acts more like the monodesmosidic
ginsenosides Rg3 and Rh2. Its IC50 of 161 µg/ml is in the range of the IC50 values of Rg3 and
Rh2. The strong cytotoxicity of ginsenoside Rd had already been reported by Yang et al. [37]
with a similar IC50 of 150 µg/ml also in HeLa cells. Like the ginsenosides Rg3 and Rh2,
ginsenoside Rd has a glucopyranose sugar chains on C3, but has also a one-molecule
glucopyranose sugar chain on C20. We suppose a hydrolysis of the glucopyranose at C20,
transforming the bidesmosidic ginsenoside Rd into a monodesmosidic molecule. Despite its
originally bidesmosidic structure, we group the ginsenoside Rd for the discussion together
with the monodesmosidic saponins Rg3 and Rh2 because of structural and cytotoxic
86
similarities. The same cleavage probably happens with the ginsenosides Re and Rg1, changing
them into monodesmosidic ginsenosides of the group III. Due to the low cytotoxic activity of
monodesmosidic ginsenosides with the sugar moiety on C6, this transformation is not visible
in the cytotoxic results.
Rd was found to induce apoptosis in HeLa cells through down-regulation of Bcl-2 expression
[37], which explains the observed cytotoxicity (Tab. 1). Furthermore, it decreases MDR in
MCF-7/ADR cells [51], which makes it a highly promising cancer drug.
The monodesmosidic ginsenosides Rg3 and Rh2 have been regarded as potent anticancer
drugs [38]. Mochizuki [52] reported a decrease of lung metastasis of B16-BL6 melanoma
cells due to ginsenoside Rg3. Ginsenoside Rg3 has been shown to be angiosuppressive [53],
thus adding another mode of action to suppress cancer growth Rh2 induces apoptosis through
ligand-independent Fas activation [54] and arrests the cell cycle in the G1 phase [55]. This is
in agreement with the substantial cytotoxicity of Rg3 and Rh2 against the cancer cell line
HeLa (Tab. 1).
The activity of the three ginsenosides Rd, Rg3 and Rh2 is connected to their sugar moiety on
C3, leaving C6 free. The sugar moiety on C3 enables the molecule to be incorporated into the
bilayer of the biomembrane next to cholesterol. This is not possible for either the
bidesmosidic or monodesmosidic ginsenosides with the sugar moiety attached to C6 due to
sterical reasons.
We conclude that a monodesmosidic structure with the sugar moiety attached to C3 seems to
be crucial for the cytotoxic effect of ginsenosides.
The monodesmosidic ginsenosides Rg2, Rh1 and PF11, tested in group III, all showed a very
low cytotoxicity. They were up to 80 times less cytotoxic than the ginsenosides with the sugar
moiety attached to C3. The sugar moiety attached to C6 prevents the integration of the
steroidal part into the bilayer.
Ginsenosides with the sugar moiety attached to C6 such as Rg2 or Re and bidesmosidic
ginsenosides such as Rb1 were shown to be active in the CNS [4,14]. Most likely they are
specific ligands for receptor proteins involved in psychologic effects. A direct integration into
the biomembrane does not seem to be necessary for this effect.
87
The bidesmosidic ginsenosides of group I were, as expected, inactive. Their bidesmosidic
structures prevent their intercalation into the biomembrane.
The aglycones protopanaxadiol and protopanaxatriol (group IV) showed strong cytotoxicity
with IC50 values of 13 and 31 µg/ml, respectively. As aglycones, they are highly lipophilic, so
that they can easily cross the cell membrane and enter the nucleus or other compartments in
the cell. Their high cytotoxicity is probably based on their effects either on membrane
proteins or on cell compartments such as the nucleus. Due to their structural similarity to
steroidal hormones, they are likely to have an impact on gene transcription and might even
provoke apoptosis.
88
Fig. 2: Cytotoxic effects of ginsenosides against HeLa cells
Survival [%]
150
Ginsenoside Rb1
Ginsenoside Rc
Ginsenoside Rd
Ginsenoside Re
Ginsenoside Rg1
100
50
2500
1250
625
312
156
78
39
20
10
5
2.5
1.25
0.63
0
Concentration [µg/ml]
A: Cytotoxic effect of the ginsenoside group I
Survival [%]
150
Ginsenoside Rg3
Ginsenoside Rh2
100
50
Concentration [µg/ml]
B: Cytotoxic effect of the ginsenoside group II
89
312
156
78
39
20
10
5
2.5
1.25
0.63
0
Survival [%]
150
Ginsenoside Rg2
Ginsenoside Rh1
Pseudoginsenoside F11
100
50
5000
2500
1250
625
312
156
78
39
20
10
0
Concentration [µg/ml]
C: Cytotoxic effect of the ginsenoside group III
Survival [%]
150
Protopanaxatriol
Protopanaxadiol
100
50
Concentration [µg/ml]
D: Cytotoxic effect of the ginsenoside group IV
90
312
156
78
39
20
10
5
2.5
1.25
0.63
0
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94
5. Antitrypanosomal properties of Panax ginseng CA Meyer – new
possibilities for a remarkable traditional drug!
5.1 Abstract
African trypanosomiasis is still a major health problem in many sub-Saharan countries in
Africa. We investigated the effects of three preparations of Panax ginseng, P. notoginseng,
isolated ginsenosides and the polyacetylene panaxynol on Trypanosoma brucei brucei and the
human cancer cell line HeLa. Hexane extracts and the pure panaxynol were toxic and at the
same time highly selective against T. b. brucei, while methanol extracts and 12 isolated
ginsenosides were significantly less toxic and showed only weak selectivity. Panaxynol was
cytotoxic against T. b. brucei at the concentration of 0.01 µg/ml with a selectivity index of
858, superior even to established anti-trypanosomal drugs. We suggest that the inhibition of
trypanothione reductase which is only found in trypanosomes, might explain the observed
selectivity. The high selectivity together with a cytotoxic concentration in the range of the
bioavailability make panaxynol and other polyacetylenes in general very promising lead
compounds for the treatment of African trypanosomiasis.
5.2 Introduction
Protozoa and helminths still cause severe damage to the health of people and their properties
in many tropical countries [1]. Nevertheless, suitable drugs are scarce since these diseases are
often neglected by commercially oriented research and development [2], so that new and
reliable drugs are hardly available.
One of the most important parasitic diseases in sub-Saharan Africa is African trypanosomiasis,
causing major health and economic problems. The parasites are protozoa of the genus
Trypanosoma: Trypanosoma brucei rhodesiense and T. b. gambiense are causing human
sleeping sickness while T. b. brucei is causing the cattle disease nagana. T. b. brucei, lacking
human infectivity, is commonly used as a model for human sleeping sickness due to its
morphological and biochemical similarities [1,3,4].
After becoming nearly extinct in the 1960s, sleeping sickness is again continually increasing
due to various reasons. Endemic to 36 countries, 60 million people are threatened by its
consequences. The WHO estimates 300 000 to 500 000 cases of human trypanosomiasis to
95
occur per year [5,6]. 46 million cattle are exposed to nagana, which causes annual costs of
1340 billion USD [7].
The medical situation is far from ideal with only four approved drugs: Suramin, pentamidine,
melarsoprol and eflornithine. Diminazene is approved for the treatment of animals but not
humans [3]. All of these drugs have strong side effects and several resistances were reported
recently [8-10]. Thus, the discovery of new drugs is an urgent necessity [11-13].
The root of ginseng (Panax ginseng C. A. Meyer) has been used as a highly praised medicinal
plant in China, Korea, Japan and eastern Russia since several thousand years. The first
surviving records of its use are Chinese oracle bones from around 1600 BC. Later descriptions
dating to the Han dynasty around 200 AD elaborate on its medicinal properties [14]. It is still
regarded as the most valuable herbal medicine in the traditional medicinal concept of China,
Korea and Japan. Research is ongoing worldwide to explain its various medical properties.
Traditionally, P. ginseng from Korea is regarded to be of higher quality compared to the
Chinese product. Korean red ginseng is a special preparation of P. ginseng where the root is
steamed and boiled over several hours before drying it [15]. This method of preparation has a
long history in Korea and is said to improve the pharmaceutical qualities of the ginseng root.
Korean red ginseng was first mentioned in the Sonhoa Bongsa Goryeo Dogyoung in the year
1123 AD. Successively, Korean red ginseng is valued much higher and accordingly is much
more expensive compared to the untreated, sundried root.
The main compounds of ginseng are ginsenosides and polyacetylenes, while fatty acids,
polysaccharides and peptides seem to be of minor importance [16,17]. Till now, 38
ginsenosides have been isolated from P. ginseng; by further adding P. quinquefolius and P.
notoginseng, a total of 66 ginsenosides have been isolated [18]. The ginsenosides Rb1, Rb2,
Rc, Rd, Re and Rg1 form over 90% of the total amount of ginsenosides in P. ginseng while
the other 32 ginsenosides are present in only minor quantities [19].
Ginsenosides (Fig. 1, Tab. 1), generally regarded as the main pharmaceutical compounds of P.
ginseng, are steroidal saponins with a four trans-ring rigid steroid skeleton and modified side
chains at C3, C6 and C20 [20]. The chain length, site of attachment and the different sugar
moieties result in a high diversity of structures. Common sugars are glucose, maltose, fructose
and sucrose. The structural modifications influence on the biological activity and explain the
96
various effects described for P. ginseng. We focused especially on their individual effects on
HeLa and T. b. brucei. The tested 12 ginsenosides can be sorted into four groups according to
their structure as mono- or bidesmosidic saponins and depending on the C atom to which the
sugar chain is linked (Fig. 1). These variations explain the strong differences in toxicity, since
aglycones and monodesmosidic saponins are able to enter and modify the biomembrane while
bidesmosidic saponins remain outside of the lipid bilayer [21].
Fig. 1: Ginsenosides
Group I: Bidesmosidic saponins
Ginsenoside Rd
Ginsenoside Rc
Ginsenoside Rb1
Ginsenoside Re
Ginsenoside Rg1
Group II: Monodesmosidic saponins (C3-gly)
Ginsenoside Rg3
Ginsenoside Rh2
97
Group III: Monodesmosidic saponins (C6-gly)
Ginsenoside Rg2
Ginsenoside Rh1
Pseudoginsenoside F11
Group IV: Aglycones
Protopanaxadiol
Protopanaxatriol
Table 1: Ginsenosides
Compound: Ginsenoside
Molecular Formula
Molar Mass
Bidesmosidic saponins
Ginsenoside Rb1
Ginsenoside Rc
Ginsenoside Rd
Ginsenoside Re
Ginsenoside Rg1
C54H92O23
C53H90O22
C48H82O18
C48H82O18
C42H72O14
1109.29
1079.27
947.15
947.15
801.01
Monodesmosidic saponins: C3-gly
Ginsenoside Rg3
Ginsenoside Rh2
C42H72O13
C36H62O8
785.01
622.87
Monodesmosidic saponins: C6-gly
Ginsenoside Rg2
Ginsenoside Rh1
Pseudoginsenoside F11
C42H72O13
C36H62O9
C41H70O14
785.01
638.87
786.99
Aglycones
Protopanaxadiol
Protopanaxatriol
C30H52O3
C30H52O4
460.73
476.73
As a traditional tonic, P. ginseng has surprised scientists with a broad series of pharmaceutical
effects [22]. One of the main applications could be confirmed by the positive effect on
reaction time, concentration and mental fitness [23-25]. Many studies were conducted to
98
demonstrate the effects on the cardiovascular system [26,27]. Additionally, P. ginseng has
positive effects on the immune system [28,29], lowered the blood glucose level and thus
having a positive effect on diabetes [30-33].
One of the most important aspects of P. ginseng, however, is inhibition of tumour cell growth
and induction of apoptosis [34,35]. Cancer preventive effects could be demonstrated by
several studies both using extracts and pure ginsenosides [36,37].
Polyacetylenes (Fig. 2, Tab. 2) are the second major group of compounds found in P. ginseng.
They are lipophilic molecules and constitute the main active group of compounds in the
hexane extracts. In P. ginseng, panaxynol, panaxydol, panaxytriol and falcarindiol are the
main polyacetylenes (Fig. 2). Polyacetylenes are not limited to the genus Panax, but are
widely distributed in Apiaceae, Araliaceae and Asteraceae. Nearly all polyacetylenes found in
food and medicinal plants (carrot, celeriac, parsnip, parsley and ginseng) are of the panaxynol
type [32,38-48]. Panaxynol was discovered twice; the first isolation was performed by
Takashi and Yoshikura [49], 1964, who named the molecule panaxynol. Bohlman et al. [50]
isolated the same compound shortly afterwards, not knowing that it was already described and
called it falcarinol. Unfortunately, both names became established in scientific literature and
are used as synonyms, adding to the confusion. We give credit to the first isolation and will
use the name panaxynol. Panaxynol is sensitive to heat and not stable in pure form or DMSO
at room temperature while being relatively stable in organic solvents such as hexane and
dichloromethane [51-53].
Polyacetylenes possess a surprisingly wide range of biological activities: They are direct
antioxidants due to their CC triple bond [40,41], while at the same time up regulating the
antioxidant response element (ARE) [54]. The triple bonds are reactive and can covalently
bond to SH-groups of proteins [21].
Polyacetylenes are potent anti-inflammatory compounds [55-57].
The inhibition of
lipoxygenases and the modulation of prostaglandin catabolism by inhibiting the
prostaglandin-catabolizing enzyme 15-hydroxy-prostaglandin dehydrogenase seem to be the
major mechanism [58-60]. However, the inhibition of the inducible nitric oxide synthase
(iNOS) [61] and the inhibition of COX-1 [62] seem to be important as well.
99
Fig. 2: Polyacetylenes
Panaxynol
Falcarindiol
Panaxytriol
Panaxydol
Table 2: Polyacetylene
Compound: Polyacetylene
Panaxynol
Falcarindiol
Panaxydol
Panaxytriol
Molecular Formula
C17H24O
C17H24O2
C17H24O2
C17H26O3
Molar Mass
244.37
260.37
260.37
278.39
Polyacetylenes were shown to be cytotoxic to various cancer cell lines [63-79]. Both in vitro
and in the rat model an anticancer activity could be shown [80-82]. The effect of cancer drugs
was shown to be enhanced in combination with polyacetylenes due to membrane effects and
multiple attacks on proteins and other targets [83]. The antioxidant, the anti-inflammatory and
the cytotoxic effects seem to be important and connected for cancer prevention by
polyacetylenes.
An anti-platelet-aggregation effect by regulating the levels of cGMP and TXA2 to inhibit
platelet aggregation induced by thrombin was shown, too [84-86].
Panaxynol causes allergic contact dermatitis and irritant skin reactions while related
polyacetylenes do not seem to be allergenic [51,87-89].
100
Polyacetylenes are toxic to a wide range of organisms:
Crustaceans were sensitive in the brine shrimp assay [76]. Antiviral activity was also
demonstrated [90]. Antifungal and spore germination inhibiting properties are well known
[46,91-96], as are the effects against bacteria and mycoplasma [97-102].
Interestingly, polyacetylenes possess high antimalarial activities in vitro and in vivo [103,104],
whereas medium antiprotozoal activities were demonstrated for some rare polyacetylenes of
Tanzanian medicinal plants in vitro [105]. The mode of action, however, was not established
by these authors.
We studies the effects of different ginseng preparations as well as of 12 pure ginsenosides and
panaxynol on African trypanosomiasis, revealing a new, very promising application for this
important medicinal plant.
5.3 Material and Methods
Chemicals and plant material
DMEM media, supplements, fetal bovine serum (FBS), trypsin-EDTA and dimethyl sulfoxide
(DMSO) were purchased from Gibco Invitrogen (Germany). The positive controls
diminazene (D7770, ≥90% purity), DL-α-difluoromethylornithine (D193, ≥97% purity),
metronidazole (M3761, analytical purity), ornidazole (O5879, analytical purity), suramin
(S2671, ≥95% purity) were purchased from Sigma-Aldrich, Germany. Chinese Panax
ginseng C. A. Meyer was bought in a store specialized on P. ginseng in China (P8088).
Korean Panax ginseng C. A. Meyer (P8086) and red Panax ginseng C. A. Meyer (P8087) (6
years old; land of origin Korea) were purchased from Cheong Kwan Jang ginseng store in
Korea. Panax notoginseng (Burkill) F. H. Chen (P6887) was provided by Prof. Dr. T. Efferth,
Johannes Gutenberg University, Mainz. Ginsenosides and panaxynol were purchased in China
by Wan Chuangxing, Tarim University, PR China.
Genetic identification of plant material
P. ginseng and P. notoginseng were authenticated by visual and microscopic identification
and by DNA barcoding. DNA was isolated from dried plant material; their chloroplast rbcL
gene was amplified and sequenced. The obtained sequences were authenticated with
sequences obtained from sample species of the Botanical Garden of Heidelberg and databases.
Voucher specimens of the plant material were deposited at the Department of Biology,
101
Institute of Pharmacy and Molecular Biotechnology, Heidelberg University (P. ginseng
China: IPMB number P8088, GenBank accession number JF950028; P. ginseng Korea: IPMB
number P8086, GenBank accession number JF950029; P. ginseng Red IPMB number P8087;
P. notoginseng IPMB number P6887, GenBank accession number JF950030).
Extract preparation
100 g of dried fine milled root powder was exhaustively extracted in a Soxhlet Extractor first
with hexane, then dichloromethane and finally methanol under moderate heat for 20 h each.
The solvents were removed with a rotavapour. The resulting residues were dried in oil
vacuum and stored under exclusion of light at -40 °C. The yield of the hexane extracts were
0.46 g, 0.48 g, 0.47 g and 0.35 g dry extract. The yield of the dichloromethane extracts were
0.31 g, 0.39 g, 0.33 g and 0.28 g dry extract. The yield of the methanol extracts were 9.87 g,
9.75 g, 9.99 g and 8.76 g dry extract for P. ginseng (Korea), P. ginseng (China), P.
notoginseng and P. ginseng (red) respectively. Prior to the experiments the remaining solvent
was evaporated and the extract solved in DMSO before being applied to the cells. This was
performed prior to each experiment to minimize the error due to the instability of
polyacetylenes in DMSO.
Mass spectrometry
GLC-MS:
GLC-MS analysis6 was done on a 5890 Series II Gas Chromatograph (Hewlett-Packard, Palo
Alto, California, USA) coupled to a Finnigan SSQ 7000 Mass Spectrometer (Thermo
Finnigan, Bremen, Germany). The head pressure of the 30 meter long capillary column with
an inner diameter of 0.25 mm and an OV-1 bonded phase layer of 0.25 micrometer (Ohio
Valley Speciality Company Marietta, Ohio, USA) was 100 kPa Helium. The temperature of
the split injector (split ratio 1:30) was 250 deg C. Oven temperature was kept at 120 deg C
for 1 min isotherm at the start. Then the temperature was raised at a rate of 6 deg C/min up to
300 deg/C and kept isotherm for 10 min. The Quadrupol Mass Spectrometer was operated in
MID mode (m/z =40-500), ion source temperature was 175 deg C, electron energy 70 eV.
The datasystem (Xcalibur version 1.3, Thermo Finnigan) was started after a delay time of 5
min. 2 mikro l samples of solutions in hexane were analysed.
6
Experiments performed by Institute of Pharmacy and Molecular Biotechnology, Heidelberg University,
Heidelberg, Germany: Frank Sporer
102
HPLC-MS:
Mass spectrometry7 was performed on a VG Quattro II system form Micromass provided with
ESI source at a negative scan mode under the parameters Capillary: 3.00 kV, HV Lens: 0.50
kV, Cone: 40 V. Drying and nebulising gas N2. Source temp. 120 oC. Scan range: 400-1200.
Ginsenosides were identified using pure authentic compounds. HPLC was performed on a
Merck-Hitachi L-6200A system with 1 mL/min step gradient flow rate. The used stationary
phase was an RP18e column (LiChrospher, 250-4mm, 5 µm, Darmstadt, Germany). Data
were processed using MassLynx 4.0 software. HPLC and LC-MS were used to analyse
ginseng preparations, extracts and the purity of ginsenosides and polyacetylenes.
Cell culture
Trypanosomes:
T. b. brucei TC 221 derived from stock 427 and were originally obtained from Prof. Peter
Overath (Max-Plank Institut für Biologie, Tübingen) by Dr. D. Steverding; they were cultured
in the IPMB since 1999.
HeLa and T. b. brucei culture:
HeLa (cervical cancer) cell lines were maintained in DMEM complete media (L-glutamine
supplemented with 10% heat-inactivated fetal bovine serum, 5% penicillin/ streptomycin and
5% non-essential amino acids). Cells were grown at 37 °C in a humidified atmosphere of 5%
CO2. Bloodstream forms of T. b. brucei cells were grown in BALTZ medium [106],
supplemented with 20% inactivated fetal bovine serum (FBS) and 0.001% β-mercaptoethanol.
DMEM media, supplements, fetal bovine serum (FBS), trypsin-EDTA and dimethyl sulfoxide
(DMSO) were purchased from Gibco Invitrogen (Germany). All experiments were
performed with cells in the logarithmic growth phase.
MTT assay:
Cytotoxicity was determined in triplicate using the 3-(4.5-dimethylthiazol-2-yl)-2.5diphenyltetrazolium bromide (MTT) cell viability assay [107]. Extracts, ginsenosides and
panaxynol were dissolved in dimethylsulfoxide (DMSO) and serially diluted in medium into
ten different concentrations. A 100 µl sample containing medium was dispensed into each
well. The concentration of the solvent, DMSO, did not exceed 0.05% in the medium that
contained the highest concentration tested. Controls included wells with untreated cells and
without cells. Cells (2 x 104 cells/well) were seeded in 96-well plates (Greiner Labortechnik).
7
Experiments performed by Institute of Pharmacy and Molecular Biotechnology, Heidelberg University,
Heidelberg, Germany: Ahmad Tahrani
103
Cells were cultured for 24 h to 80% confluence before treating them with the test samples.
After incubation for 24 h the medium was replaced with fresh medium containing 0.5 mg/ml
MTT. 4 h later the formed formazan crystals were dissolved in 100 µl DMSO; the absorbance
was detected at 570 nm with a Tecan Safire II Reader. Doxorubicin was used as positive
control for HeLa cells, artemisinin, diminazene, DL-α-difluoromethylornithine, metronidazole,
ornidazole and suramin were used as positive control for T. b. brucei. Cell viability of T. b.
brucei was additionally confirmed and evaluated using microscopical techniques.
Screening:
T. b. brucei was compared to HeLa cells and the Selectivity index (SI) was calculated as:
SI: ratio of the IC50 value of mammalian cells divided by the IC50 value of trypanosomes.
Statistical analysis:
The results were obtained as means and standard errors of triplicates repeated at least three
times for each measurement point. The IC50 values were calculated using a four parameter
logistic curve (SigmaPlot® 11.0) representing 50% reduction of viability compared to the
positive control.
Table 3: Relative abundance of polyacetylenes detected in the Panax extracts via GLC-MS
P. ginseng Korea
P. ginseng China
P. ginseng Red
P. notoginseng
C6H14
CH2Cl2
CH4O
C6H14
CH2Cl2
CH4O
C6H14
CH2Cl2
CH4O
C6H14
CH2Cl2
CH4O
Panaxynol
9.4 %
trace
-
10.9 %
trace
-
30.7 %
trace
-
7.8 %
trace
-
Peak 2
60.1 %
trace
-
67.7 %
trace
-
5.9 %
trace
-
20.4 %
trace
-
Peak 3
2.6 %
-
-
1.9 %
-
-
20.4 %
trace
-
21.7 %
trace
-
Table 4: Relative abundance of ginsenosides detected in the Panax extracts via HPLC-MS
P. ginseng Korea
P. ginseng China
C6H14
CH2Cl2
CH4O
C6H14
CH2Cl2
CH4O
C6H14
CH2Cl2
CH4O
C6H14
CH2Cl2
CH4O
Rd
-
-
ND
-
-
3.9 %
-
ND
3.4 %
-
-
Rg1
-
2.2 %
ND
-
1.4 %
1.3 %
-
ND
10 %
-
0.4 %
PF11
Rg2
-
0.3 %
0.5 %
-
ND
ND
ND
-
trace
trace
0.8 %
1.7 %
trace
1.5 %
-
ND
ND
ND
2%
1.8 %
0.3 %
-
0.3 %
-
Rb1
-
-
ND
-
-
6.4 %
-
ND
6%
-
-
Rc
-
-
ND
ND
ND
ND
-
trace
2.1 %
5.2 %
4.5 %
trace
trace
-
ND
ND
ND
ND
0.5 %
3%
0.9 %
-
-
-
3.2 %
28.8
%
1.1 %
trace
4.3 %
16.1
%
1.4 %
6.9 %
-
-
trace
-
-
-
-
-
trace
-
-
trace
-
-
-
-
-
-
-
-
-
-
-
-
-
Rh1
Re
Rg3
Rh2
Protopanaxa
diol
Protopanaxat
riol
P. ginseng Red
104
P. notoginseng
5.4 Results
The GLC-MS data of the four hexane extracts revealed that panaxynol is one of the main
polyacetylenes and present in all extracts (Fig. 3,4, Tab. 3). The RI (OV-1) was similar to the
one published previously [108]. The second major polyacetylene is an unidentified
polyacetylene similar in structure to panaxynol. The MS spectrum shows the close structural
relationship to panaxynol (Fig. 4). The third major polyacetylene also of the panaxynol type is
present in significant amounts only in the hexane extract of P. notoginseng and the red P.
ginseng.
The HPLC-MS data of the extracts showed that ginsenosides are the major compounds of the
methanol extracts. They appear only in traces in the dichloromethane extracts and are
completely absent in the hexane extracts (Tab. 4). The major ginsenosides of all four ginseng
variations are the ginsenosides Rd, Rg1, Rb1 and Re while the exact amount of the
ginsenosides varies significantly.
Three variations of P. ginseng (origin China, origin Korea and red ginseng from Korea), P.
notoginseng, panaxynol and 12 ginsenosides were tested against the human cancer cell line
HeLa and T. b. brucei. Suramin, DL-α-difluoromethylornithine and diminazene plus three
further drugs were used as control substances in comparison to P. ginseng extracts, 12
isolated ginsenosides and panaxynol (Tab. 5,6). Suramin and DL-α-difluoromethylornithine
are established drugs for the treatment of human trypanosomiasis, while diminazene is
licensed for veterinary treatment only [109,110].
The lipophilic hexane and dichloromethane extracts of the P. ginseng varieties and P.
notoginseng showed a remarkably selective cytotoxicity against T. b. brucei, while the polar
methanol extracts were significantly less toxic and less selective (Tab. 5; Fig. 5-8). The
cytotoxicity of the hexane extracts against T. b. brucei and HeLa cells was 0.1 to 2.6 µg/ml
and 52.9 to 1085.9 µg/ml, respectively. The resulting selectivity index was between 101 and
529, while the selectivity index of the polar methanol extracts was only between 1 and 4 (Tab.
5).
The effects of the pure compounds of P. ginseng were even more remarkable.
The polyacetylene panaxynol showed a toxicity of 0.01 µg/ml against T. b. brucei and 8.58
µg/ml against HeLa, giving a substantially high selectivity index of 858 (Tab. 6; Fig. 9).
105
Table 5: Cytotoxic effects of the hexane, dichloromethane and methanol extracts of four
Panax drugs
Panax
ginseng
China
Panax
ginseng
Korea
Panax
ginseng
Red
Panax
notoginseng
T. b.
brucei
IC50
[µg/ml]
HeLa
IC50
[µg/ml]
T. b.
brucei
IC50
[µg/ml]
HeLa
IC50
[µg/ml]
C6H14
C6H14
CH2Cl2
CH2Cl2
2.1
506.1
241.0
0.9
152.4
0.6
138.6
231.0
3.3
0.1
52.9
529.0
0.4
150.3
Ratio
(HeLa/
T. b.
brucei)
375.7
Ratio
(HeLa/
T. b.
brucei)
T. b.
brucei
IC50
[µg/ml]
HeLa
IC50
[µg/ml]
CH4O
CH4O
169.3
319.0
1427.9
4.4
173.1
52.4
1075.0
1317.9
1.2
3.1
279.3
90.0
782.7
1310.6
1.6
0.9
263.0
469.6
1241.6
Ratio
(HeLa/
T. b.
brucei)
292.2
Table 6: Cytotoxic effect of panaxynol and ginsenosides
Cytotoxic effect of
polyacetylenes
HeLa
IC50 [µg/ml]
8.58
Ratio
Panaxynol
T. b. brucei
IC50 [µg/ml]
0.01
Cytotoxic effects of
ginsenosides
T. b. brucei
IC50 [µg/ml]
HeLa
IC50 [µg/ml]
Ratio
Bidesmosidic saponins
Ginsenoside Rb1
Ginsenoside Rc
Ginsenoside Rd
Ginsenoside Re
Ginsenoside Rg1
411.1
157.3
71.9
974.5
1003.4
1373.3
1431.5
161.3
1291.8
1408.8
3.3
9.1
2.2
1.3
1.4
Monodesmosidic saponins: C3-gly
Ginsenoside Rg3
Ginsenoside Rh2
37.0
21.3
245.7
29.1
6.6
1.3
Monodesmosidic saponins: C6-gly
Ginsenoside Rg2
Ginsenoside Rh1
Pseudoginsenoside F11
240.3
295.8
2181.0
1172.7
452.9
2332.9
4.8
1.5
1.0
Aglycones
Protopanaxadiol
Protopanaxatriol
12.4
11.5
13.2
31.6
1.0
2.7
T. b. brucei
IC50 [µg/ml]
98.0
0.1
11.7
18.8
18.8
4.7
HeLa
IC50 [µg/ml]
451.8
170.6
1502.6
1180.5
849.8
1317.1
Ratio
Cytotoxicity of control
compounds
Artemisinin
Diminazene
DL-α-difluoromethylornithine
Metronidazole
Ornidazole
Suramin
106
(HeLa/ T. b. brucei)
858.00
(HeLa/ T. b. brucei)
(HeLa/ T. b. brucei)
4.6
1706.0
128.4
62.7
45.2
280.2
2.6
Compared to this, the established drugs against human trypanosomiasis, DL-αdifluoromethylornithine and suramin have only a selectivity index of 128 and 277,
respectively. The selectivity index of panaxynol is with 858 three times higher than the one of
the standard drug suramin with 277. Only diminazene, which is not licensed for the treatment
of humans, showed a higher selectivity index of approximately 1706 as compared to
panaxynol (Tab. 6).
The 12 ginsenosides had a toxicity range from 11.5 to 2181.0 µg/ml for T. b. brucei and 13.2
to 2332.9 µg/ml for HeLa with selectivity indices between 1 and 9 (Tab. 6; Fig. 10-13).
5.5 Discussion
Due to the high importance of P. ginseng in Asian medicine, we chose to investigate three
types of P. ginseng: P. ginseng with the origin of China, P. ginseng with the origin of Korea
and Red ginseng from Korea. Additionally, we decided to compare these three varieties of P.
ginseng with P. notoginseng. Being closely related, it also contains ginsenosides as well as
polyacetylenes. Just as in P. ginseng, its main polyacetylene is panaxynol.
Our HPLC-MS results confirm that ginsenosides are mainly present in the methanol extracts;
tiny amounts could also be found in the dichloromethane extracts, while none were present in
the hexane extracts (Tab. 4). Our GLC-MS data demonstrated that polyacetylenes with
panaxynol as main representative are major compounds of the hexane extracts of all ginseng
variations (Fig. 3).
Red ginseng is traditionally believed to be of higher efficacy than the untreated root. Certain
ginsenosides are said to be degraded, while others are concentrated [36,111]. Yun [36]
describes the monodesmosidic ginsenosides Rh1, Rh2, Rg3 and Rg5 as the major saponin
components of red ginseng, while our results showed that they occur only in low amounts.
Contrary to this, the bidesmosidic ginsenosides Rb1, Rd, Re and Rg1 were most abundant in
our red ginseng (Tab. 4). These findings show that unlike to the common opinion,
bidesmosidic saponins are not necessarily degraded but can survive the preparation process of
red ginseng unaltered, which explains the similar cytotoxicity of the methanol extracts of the
four ginseng variations. The preparation method of steaming and boiling seems to be less
aggressive than commonly thought and thus also allows the survival of the other group of
sensitive molecules, the polyacetylenes.
107
Polyacetylenes, especially panaxynol, are commonly assumed to be instable in heat treatment
and so we supposed that the effect of the treated red ginseng should be reduced compared to
the untreated root. However, the results showed an enhanced activity both against HeLa cells
and T. b. brucei of the hexane extract of red ginseng. Additionally, our GLC-MS data showed
that panaxynol and other polyacetylenes are present in all four hexane extracts (Fig. 3,4). This
demonstrates that no significant loss of activity occurred by the treatment to transform the
fresh ginseng into the red ginseng.
P. notoginseng is valued as an important drug with similar medicinal properties as P. ginseng
in Chinese Medicine. Our experiments showed comparable results, suggesting that it can be
used under certain circumstances as a substitute.
No differences were seen in the cytotoxicity of the methanol and dichloromethane extracts
between all four ginseng variations. The activity of the methanol extracts was with IC50 values
of around 1300 µg/ml comparable to that of the bidesmosidic ginsenosides of group I, which
are also the main compounds detected by mass spectrometry in these extracts (Tab. 4-6).
While the ginsenosides and methanol extracts showed no selectivity and no remarkable
cytotoxicity towards T. b. brucei, panaxynol and the hexane extracts were not only highly
cytotoxic, but also extremely selective towards T. b. brucei. Panaxynol was superior to all
licensed drugs applied in the treatment of African
trypanosomiasis (Tab. 6). We conclude that the
polyacetylenes, especially panaxynol are the
active principle in ginseng against T. b. brucei.
The general cytotoxic mechanism of panaxynol is
well understood. The hydrophobic molecule can
easily penetrate the cells. There, the loss of water
at the C3 hydroxyl group forms an extremely
stable carbocation. This carbocation is a very
reactive alkylating agent towards SH and amino
groups in proteins and other biomolecules
Fig. 14: Glutathione disulfide (a) and
trypanothione disulfide (b) after Bond et al.,
1999 [117]
[21,40,41,112]. One example is the inactivation of
Na+ channels [113].
108
The reason for the selectivity of panaxynol against T. b. brucei seems to be based on several
mechanisms working together. The most important target seems to be the inhibition of the
trypanothione reductase. This enzyme is specific for trypanosomastids, while mammalian
cells use the glutathione reductase instead. These enzymes are members of the FADdependent NADPH oxidoreductase family. They are dimeric molecules and protect the cell
against reactive oxygen species formed during the aerobic metabolism [110,114-116]. The
two enzymes are able to distinguish their substrates trypanothione disulfide and glutathione
disulfide by the factor 1000 because of differences in size and charge. Glutathione disulfide is
smaller and -2 charged at physiological pH, while trypanothione disulfide is +1 charged with
a hydrophobic body of seven methylene groups (Fig. 14). The trypanothione disulfide binding
site in the trypanothione reductase is larger with a negatively charged glutamate side chain
and a hydrophobic cleft for the polyamine moiety (Fig. 15). Contrary to this, the glutathione
reductase has a positively charged, smaller active centre [110,118].
The panaxynol carbocation
can enter the active centre of
the trypanothione reductase
due to its positive charge and
lipophilic chain. There, it can
bind to the active centre and
block the enzyme. This is not
possible with the glutathione
reductase where it is repelled
because of its positive charge.
Fig. 15: Trypanothione reductase with binding site after Bond et al.,
1999 [117]
There, the CC triple bonds of the carbocation can form irreversible bonds with the active
centre.
Additionally, panaxynol is known to modify AMP levels in mammalian cells by direct
inhibition of the mitochondrial respiration resulting in a significant reduction of ATP in the
cells [75,118,119]. The immediate consequence is a cell cycle arrest [42,120,121]. Thus, fast
proliferating cells are especially sensitive to panaxynol, as was demonstrated by several
investigations [69,112,122-124]. This is probably happening in rapidly proliferating T. b.
brucei as well.
Trypanosomes require specific sterols for their cell viability and are extremely sensitive to
ergosterol biosynthesis inhibitors [125-127]. Polyacetylenes, on the other side, have been
109
shown to inhibit the cholesterol acyltransferase (ACAT) [128-130]. We suggest that the
observed selectivity of panaxynol is based on these three mechanisms, while the most
important one seems to be the inhibition of the trypanothione reductase.
For future drug developments, the bioavailability of panaxynol is crucial. This was already
established by Christensen and Brandt [40], who demonstrated that the oral uptake of 300,
600 and 900 ml carrot juice with 13.3 mg panaxynol/l resulted in a maximum serum
concentration of 2.5 ng/ml. The amount of panaxynol in P. ginseng is 5 times higher than in
carrots, lowering the necessary amount of drug to obtain sufficient serum concentration levels
[53,131].
This concentration is already in the active range demonstrated in our experiments, making
panaxynol and other polyacetylenes in general very promising compounds for the treatment of
African trypanosomiasis.
110
Fig. 3: GLC-profiles of hexane extracts (TIC)
Hexane extracts:
A: Panax ginseng (Korea) B: Panax ginseng (China) C: Panax notoginseng D: Panax ginseng
(Red)
Major peaks:
1: Panaxynol 2: unidentified polyacetylene of panaxynol type 3: unidentified polyacetylene
111
Fig. 4: MS-spectra of polyacetylenes
1: MS of panaxynol
2: MS of unidentified polyacetylene of panaxynol type
3: MS of unidentified polyacetylene
112
Fig. 5: Cytotoxic effects of the hexane and methanol extracts of P. ginseng (Korea) against T.
b. brucei and HeLa cells.
150
Hexane
Hexane
Methanol
Methanol
5000
2500
1250
625
313
156
9.8
Concentration [µg/ml]
78
0
5000
2500
625
1250
313
78
156
39
9.8
19.5
4.9
2.4
1.2
0.6
0.3
0.15
0.075
0
50
39
50
100
19.5
100
Survival [%]
Survival [%]
150
Concentration [µg/ml]
A: Cytotoxic effect of P. ginseng (Korea) extracts against T. b. brucei
B: Cytotoxic effect of P. ginseng (Korea) extracts against HeLa cells
Fig. 6: Cytotoxic effects of the hexane and methanol extracts of P. ginseng (China) against T.
b. brucei and HeLa cells.
150
Hexane
Hexane
Methanol
Methanol
Survival [%]
5000
2500
1250
625
313
9.8
Concentration [µg/ml]
156
0
5000
2500
1250
625
313
156
78
39
19.5
9.8
4.9
2.4
1.2
0.6
0.3
0.15
0.075
0
50
78
50
100
39
100
19.5
Survival [%]
150
Concentration [µg/ml]
A: Cytotoxic effect of P. ginseng (China) extracts against T. b. brucei
B: Cytotoxic effect of P. ginseng (China) extracts against HeLa cells
Fig. 7: Cytotoxic effects of the hexane and methanol extracts of P. notoginseng against T. b.
brucei and HeLa cells.
150
Hexane
Hexane
Methanol
Methanol
Concentration [µg/ml]
A: Cytotoxic effect of P. notoginseng extracts against T. b. brucei
B: Cytotoxic effect of P. notoginseng extracts against HeLa cells
113
5000
2500
1250
625
313
156
9.8
Concentration [µg/ml]
78
0
5000
2500
1250
625
313
156
78
39
19.5
9.8
4.9
2.4
1.2
0.6
0.3
0.15
0.075
0
50
39
50
100
19.5
100
Survival [%]
Survival [%]
150
Fig. 8: Cytotoxic effects of the hexane and methanol extracts of P. ginseng (Red) against T. b.
brucei and HeLa cells.
150
Hexane
Hexane
Methanol
Methanol
5000
2500
1250
625
313
156
9.8
Concentration [µg/ml]
78
0
5000
2500
625
1250
313
78
156
39
9.8
19.5
4.9
2.4
1.2
0.6
0.3
0.15
0.075
0
50
39
50
100
19.5
100
Survival [%]
Survival [%]
150
Concentration [µg/ml]
A: Cytotoxic effect of P. ginseng (Red) extracts against T. b. brucei
B: Cytotoxic effect of P. ginseng (Red) extracts against HeLa cells
Fig. 9: Panaxynol
150
150
Panaxynol
100
Survival [%]
50
50
Concentration [µg/ml]
50
25
12.5
6.4
3.2
1.6
0.8
0.1
0.4
0
0.5
0.25
0.125
0.064
0.032
0.016
0.008
0.004
0.002
0.001
0
100
0.2
Survival [%]
Panaxynol
Concentration [µg/ml]
A: Cytotoxic effect of panaxynol against T. b. brucei
B: Cytotoxic effect of panaxynol against HeLa cells
Fig. 10: Ginsenosides group I
Ginsenoside Rb1
Ginsenoside Rc
Ginsenoside Rd
Ginsenoside Re
Ginsenoside Rg1
150
100
Survival [%]
50
100
50
Concentration [µg/ml]
A: Cytotoxic effect of the ginsenoside group I against T. b. brucei
B: Cytotoxic effect of the ginsenoside group I against HeLa cells
114
2500
1250
625
312
156
78
39
20
10
5
0.63
Concentration [µg/ml]
2.5
0
2500
1250
625
312
156
78
39
20
10
5
2.5
1.25
0.63
0
Ginsenoside Rb1
Ginsenoside Rc
Ginsenoside Rd
Ginsenoside Re
Ginsenoside Rg1
1.25
Survival [%]
150
Fig. 11: Ginsenosides group II
150
Ginsenoside Rg3
Ginsenoside Rh2
Ginsenoside Rg3
Ginsenoside Rh2
Survival [%]
100
50
100
50
312
156
78
39
20
10
5
0.63
312
156
78
39
20
10
5
2.5
1.25
0.63
2.5
0
0
1.25
Survival [%]
150
Concentration [µg/ml]
Concentration [µg/ml]
A: Cytotoxic effect of the ginsenoside group II against T. b. brucei
B: Cytotoxic effect of the ginsenoside group II against HeLa cells
Fig. 12: Ginsenosides group III
150
Ginsenoside Rg2
Ginsenoside Rh1
Pseudoginsenoside F11
Ginsenoside Rg2
Ginsenoside Rh1
Pseudoginsenoside F11
Survival [%]
5000
2500
1250
625
312
10
156
0
5000
2500
1250
625
312.5
156.25
78.12
39.06
19.53
9.76
0
50
78
50
100
39
100
20
Survival [%]
150
Concentration [µg/ml]
Concentration [µg/ml]
A: Cytotoxic effect of the ginsenoside group III against T. b. brucei
B: Cytotoxic effect of the ginsenoside group III against HeLa cells
Fig. 13: Ginsenoside group IV
150
Protopanaxatriol
Protopanaxadiol
Protopanaxatriol
Protopanaxadiol
Survival [%]
100
50
100
50
Concentration [µg/ml]
Concentration [µg/ml]
A: Cytotoxic effect of the ginsenoside group IV against T. b. brucei
B: Cytotoxic effect of the ginsenoside group IV against HeLa cells
115
312
156
78
39
20
10
5
0.63
312
156
78
39
20
10
5
2.5
1.25
0.63
2.5
0
0
1.25
Survival [%]
150
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6. Carlina oxide – a natural polyacetylene from Carlina acaulis
(Asteraceae) with potent antitrypanosomal and antimicrobial
properties!
6.1 Abstract
Carlina acaulis (Asteraceae) has a long history of medicinal use in Europe due to its
antimicrobial properties. The strong activity of Carlina oxide, the main compound of the
essential oil of C. acaulis against two MRSA strains, Streptococcus pyogenes, Pseudomonas
aeruginosa, Candida albicans and C. glabrata was confirmed. A strong and selective activity
against Trypanosoma brucei brucei with an IC50 of 1.0 µg/ mL and a SI of 446 compared to
human HeLa cells was recorded. The selective toxicity of Carlina oxide makes it a promising
lead compound for the development of drugs to treat African trypanosomiasis and
multiresistant gram positive bacteria.
6.2 Introduction
Parasites are still a major cause of diseases in most tropical countries. Especially protozoa and
helminths are responsible for great damage to the health of people and their domestic animals
[1]. However, suitable drugs are rare: Between 1975 and 1999, only 13 drugs were developed
against tropical diseases, compared to 1300 new drugs in total against other health disorders
[2]. The situation improved slightly since 2000; yet, a reliable cure for tropical diseases is
often missing.
African trypanosomiasis is a parasitic disease in sub-Saharan Africa causing major health and
economic problems. The parasites are protozoa of the genus Trypanosoma: Trypanosoma
brucei rhodesiense and T. b. gambiense are causing human sleeping sickness while T. b.
brucei is responsible for the cattle disease nagana. T. b. brucei is commonly used as a model
for human sleeping sickness due to its morphological and biochemical similarity but lack of
infectivity for the human researcher [1,3,4].
Since 1970 sleeping sickness is increasing due to various factors. Nowadays, it is endemic in
36 countries and 60 million people are threatened. The WHO estimates that 300 000 to 500
000 cases of human trypanosomiasis occur per year [5,6]. 46 million cattle are exposed to
trypanosomes, resulting in annual costs of 1340 billion USD [7].
126
Currently, only four drugs are approved: Suramin, pentamidine, melarsoprol and eflornithine.
Diminazene is approved for the treatment of animals but not humans [3]. Unfortunately, these
drugs have strong side effects and several cases of resistance were reported recently [8-10].
Thus, the discovery of new drugs is urgently required [11-13].
Carlina acaulis (Asteraceae) is originated in
central Europe and has a long history as a
medicinal
plant
with
diuretic,
stomachic,
antimicrobial and antihelminthic properties [1416]. The same effects have been reported for C.
acanthifolia, a close relative and legal substitute
for C. acaulis. The main compounds of Carlina
spp. are inulin (18-20%), flavonoids and an
essential oil (2%) with 80% Carlina oxide [1719]. We selected Carlina oxide (Fig.1), the main
F ig 1: Carlina oxide
compound of the essential oil of C. acaulis
(Asteraceae) and tested it for its antitrypanosomal activity in comparison with known
antibacterial and antifungal properties.
6.3 Material and Methods
Chemicals
All chemicals were purchased from Gibco Invitrogen, Germany.
The positive controls diminazene (D7770, ≥90% purity), DL-α-difluoromethylornithine
(D193, ≥97% purity), metronidazole (M3761, analytical purity), ornidazole (O5879,
analytical purity), suramin (S2671, ≥95% purity) were purchased from Sigma-Aldrich,
Germany.
Plant material
Dried C. acaulis root was purchased from Caesar & Loretz GmbH, Hilden, Germany (669a
Radix Carlinae, Lot: 81805079). Voucher specimen (P8082) was stored at the IPMB,
Heidelberg. Identity was confirmed according to visual and microscopic characteristics.
127
Extract preparation8
100 g of dried C. acaulis root powder was exhaustively extracted with first hexane, then
dichloromethane and finally methanol under moderate heat using a reflux condenser. 100 g
root powder gave 1.39 g dry hexane extract, 0.84 g dry dichloromethane extract and 6.9 g dry
methanol extract. Extracts were concentrated using a rotation evaporator. The highly
condensed extracts were stored at -40 °C under exclusion of light. Prior to the experiments the
extracts were vacuum dried, powdered and dissolved in DMSO.
Test organisms
Trypanosomes:
T. b. brucei TC 221 derived from stock 427 and was originally obtained from Prof. Peter
Overath (Max-Plank Institut für Biologie, Tübingen) to Dr. D. Steverding; they were cultured
at the IPMB since 1999.
Gram- positive bacteria9:
Methicillin-resistant Staphylococcus aureus (MRSA ATCC 10442), vancomycine resistant
Enterococcus (VRE VanB ATCC 31299) and Streptococcus pyogenes (ATCC12344) in
addition to two clinical isolates; MRSA 1000/93 and VRE 902291 were used in antimicrobial
tests.
Gram-negative bacteria9:
Escherichia coli (ATCC 25922), Klebsiella pneumonia (ATCC 700603) and Pseudomonas
aeruginosa (ATCC 27853) were employed as representatives of gram-negative bacteria.
Fungi9:
Candida albicans (ATCC 90028) and Candida glabrata (ATCC MYA 2950) were selected as
representatives of fungi with pathogen potential.
All microorganism cultures were supplied by Medical Microbiology Lab., Hygiene Institute,
Heidelberg University, Germany.
8
Experiments performed by Institute of Pharmacy and Molecular Biotechnology, Heidelberg University,
Heidelberg, Germany: Frank Sporer
9
Experiments performed by Institute of Pharmacy and Molecular Biotechnology, Heidelberg University,
Heidelberg, Germany: Razan Hamoud
128
Methods
HeLa and T. b. brucei culture
Human HeLa cells were grown at 37 °C in a humidified atmosphere of 5 % CO2 in DMEM
complete media (L-glutamine supplemented with 10 % heat-inactivated fetal bovine serum,
5% penicillin/ streptomycin and 5% non-essential amino acids). T. b. brucei cells were
cultured in BALTZ medium [20]. The experiments were performed during the logarithmic
growth phase of the cells.
MTT assay
Cell
viability
was
determined
according
to
the
3-(4.5-dimethylthiazol-2-yl)-2.5-
diphenyltetrazolium bromide (MTT) cell viability assay [21]. Extracts and Carlina oxide were
dissolved prior to the experiment in dimethylsulfoxide (DMSO). Ten different concentrations
were obtained by serial dilution in medium, so that the highest concentration of DMSO did
not exceed 0.05%. Cells were (2 x 104 cells/well) were seeded in 96-well plates (Greiner
Labortechnik), cultured for 24 h till 80% confluence was reached before the test samples were
applied. The medium was replaced after 24 h incubation with fresh medium containing 0.5
mg/mL MTT. 4 h later, the medium was replaced by 100 µL DMSO to dissolve the formazan
crystals. The absorbance was detected at 570 nm with a Tecan Safire II Reader. Diminazene,
DL-α-difluoromethylornithine, metronidazole, ornidazole and suramin were used as positive
control; cell viability of T. b. brucei was additionally evaluated using microscopical
techniques.
Screening
Cytotoxicity in T. b. brucei was compared to HeLa cells and an SI index was calculated.
SI index: ratio of the IC50 value of mammalian cells divided by the IC50 value of
trypanosomes.
Statistical analysis
The results are shown as means and standard errors of at least three times repeated triplicates
for each measurement. The IC50 values were calculated using a four parameter logistic curve
(SigmaPlot® 11.0) representing 50% reduction of viability compared to the positive control.
129
Bacterial and fungal cultures10
Columbia with 5% sheep blood (BD) was used for bacterial subculturing and MBC
determination. Mueller-Hinton Broth (MHB) and Brain Heart Infusion (BHI) (Fluka) were
used for bacterial inoculum dilutions and MIC determination. All bacterial cultures were
incubated at 37 °C for 24 h. CHROMagar Candida (BD) was used for fungal sub-culturing
and MMC determination. Sabouraud Dextrose Broth (SDB) (Oxid) was used for fungal
inoculum dilutions and MIC determination. All fungal cultures were incubated at 25 °C for 48
h.
Inoculum preparations10
One or two bacterial or fungal colonies from an 18-24 h agar plate were suspended in saline to
a turbidity matching 0.5 McFarland ≈ 1x 108 CFU/mL (bacteria) and ≈ 1x 106 CFU/mL
(fungi), 100 µL of the bacterial suspension was diluted 1:100 with 9900 µL broth to 1x106
CFU/mL and 1000 µL of the fungal suspension was diluted 1:10 with 9000 µL broth to 1x105
CFU/mL [22].
Well Diffusion Test11
Colombia medium with 5% sheep blood and CHROMagar Candida were inoculated with 1 x
106 CFU/mL of bacterial and fungal suspensions, respectively. Wells with a diameter of 6 mm
were cut out and filled with 40 µL of the extract. DMSO was used as a negative control;
ampicillin, vancomycin and nystatin were used as positive controls. Zones of inhibition were
recorded at 37 °C after 24 h (bacteria) and at 25 °C after 48 h (fungi).
Determination of Minimum Inhibitory concentration (MIC) and Minimum Microbiocidal
concentration (MMC)11
Micro dilution method was used to determine MIC as described by CLSI [22]. The plant
extract was first dissolved in 5% DMSO to the concentration of 8 mg/mL, and then diluted
two fold with MHB (bacteria) and SDB (fungi) in 96-well plates to obtain a range of
concentrations between 0.015 and 8 mg/mL. Carlina oxide was dissolved in DMSO to a
concentration 1.25 mg/mL and diluted two fold to obtain a range of concentration between 7
and 125 µg/mL. The bacterial and fungal suspensions of 1 x 106 CFU/mL were then added
10
Experiments performed by Institute of Pharmacy and Molecular Biotechnology, Heidelberg University,
Heidelberg, Germany: Razan Hamoud
11
Experiments performed by Institute of Pharmacy and Molecular Biotechnology, Heidelberg University,
Heidelberg, Germany: Razan Hamoud
130
and the plates were incubated at 37 °C for 24 h (bacteria) and at 25 °C for 48 h (fungi). The
first well that showed no visible turbidity matching with a negative control was defined as
MIC. Each test was performed in duplicate. 3 µL of each clear well (with no visible turbidity)
were incubated in appropriate agar media and incubated under the appropriate conditions for
bacteria and fungi. MMC was determined as the concentration that did not result in growth on
agar.
Isolation and identification of Carlina oxide12
As starting material a hexane extract of the milled drug was used. Carlina oxide was isolated
via column chromatography on silica gel (70-230 mesh, Merck KGaA, Darmstadt, Germany).
Eluation was achieved by increasing polarity of a hexane-ethylacetate gradient. GLC-MS
analysis was carried out on a Hewlett-Packard gas chromatograph (GLC 5890 II), coupled to
a Finnigan SSQ 7000 Quadrupol Mass Spectrometer (Thermo-Finnigan, Bremen, Germany).
The head pressure of the GLC column was 15 kPa Helium, the temperature program 40300 °C at a rate of 4 °C/ min. For injection the sample was solved in hexane. 2 µL samples
were injected with the carrier gas helium at a flow rate of 2 mL/ min in split mode (split ratio
1:30) at the injector temperature of 250°C. The MS spectrum was recorded at the electron
energy 70 eV operated in EI mode with the ion source at 175°C. The purity and identity of
isolated Carlina oxide were confirmed by GLC-MS. The mass fragmentation pattern of the
isolated pure compound was identical with that of the previously published data of Carlina
oxide (benzyl-2-furylacetylene) [17]. The published retention index for Carlina oxide
(RI=1613.1) on a HP-5MS phase was in good correlation with our retention index (RI=1614)
obtained by using an OV-5 column with the same dimensions (30 m x 0.25 mm ID, film
thickness 0.25 µm; Ohio Valley Specialty, Marietta, OH, USA).
LC-MS analysis of C. acaulis extracts13
Mass spectrometry was performed on a VG Quattro II system from Micromass provided with
ESI source at positive scan mode under the parameters: Capillary: 3.5 kV, HV Lens: 0.5 kV,
Cone: 30 V. Drying and nebulising gas N2. Source temperature 120 oC. Scan range: 100-700.
HPLC was performed on a Merck-Hitachi L-6200A system with 1 mL/min flow rate under a
water/0.5% formic acid/ ACN gradient; split 10%. The stationary phase was an RP18e column
12
Experiments performed by Institute of Pharmacy and Molecular Biotechnology, Heidelberg University,
Heidelberg, Germany: Frank Sporer
13
Experiments performed by Institute of Pharmacy and Molecular Biotechnology, Heidelberg University,
Heidelberg, Germany: Ahmad Tahrani
131
(LiChrospher, 250-4mm, 5 µm, Darmstadt, Germany). Data were processed using MassLynx
4.0 software.
6.4 Results
Table 1: Cytotoxicity of Carlina acaulis extracts and Carlina oxide
Carlina acaulis
Hexane extract
DCM extract
MeOH extract
Carlina oxide
Controls
Diminazene
DL-αDifluoromethylornithine
Metronidazole
Ornidazole
Suramin
T. b. brucei
IC50 [µg/mL]
3.7
4.5
698.1
1.0
HeLa
IC50 [µg/mL]
1722.4
1995.0
1236.0
446.0
Ratio (HeLa/ T. b.
brucei)
465.5
443.3
1.7
446.0
0.1
11.7
170.6
1502.6
1706.0
128.4
18.8
18.8
4.7
1180.5
849.8
1317.1
62.7
45.2
280.2
Fig 2: Cytotoxic effect of C. acaulis and Carlina oxide
120
HeLa
T. b. brucei
100
100
80
80
Survival [%]
Survival [%]
120
60
40
HeLa
T. b. brucei
60
40
20
20
0
0
0.37 0.75
1.5
3
6
12
24
49
98
195
390
0.75
780 1560 3130 6250 12500
1.5
3
6
12
24
49
98
195
390
780 1560 3130 6250 1250025000
-20
-20
Concentration [µg/mL]
Concentration [µg/mL]
A: Cytotoxic effect of Carlina oxide
B: Cytotoxic effect of a hexane extract of C. acaulis
120
120
HeLa
T. b. brucei
Survival [%]
Survival [%]
100
80
60
40
HeLa
T. b. brucei
100
80
60
40
20
20
0
0
0.75
1.5
3
6
12
24
49
98
195
390
780
0.098
1560 3130 6250 12500
0.195
0.39
0.78
1.56
3.13
6.25
-20
-20
Concentration [mg/mL]
Concentration [µg/mL]
C: Cytotoxic effect of a dichloromethane extract of C. acaulis
D: Cytotoxic effect of a methanol extract of C. acaulis
132
12.5
25
50
The isolated Carlina oxide had 100% purity. The hexane extract contained seven times more
Carlina oxide than the dichloromethane extract while no Carlina oxide could be detected in
the methanol extract (Fig. 3, 4).
The cytotoxic effects of the hexane, dichloromethane and methanol extracts of C. acaulis
were tested against the human cell line HeLa and T. b. brucei. In addition, cytotoxicity of the
main compound of the essential oil of C. acaulis, Carlina oxide, was determined. The
lipophilic hexane and dichloromethane extracts showed similar cytotoxicity as Carlina oxide
while the polar methanol extract was significantly less active (Tab. 1; Fig. 2). The IC50 values
for both hexane and dichloromethane extract in T. b. brucei are 3.7 µg/mL and 4.5 µg/mL
while they are 1722.4 µg/mL and 1995.0 µg/mL for HeLa. The resulting SI is 465 and 443
respectively. Pure Carlina oxide has an IC50 value for T. b. brucei of 1.0 µg/mL while the IC50
value for HeLa cells is 446.0 µg/mL, giving a SI of 446. In comparison to the positive control
substance suramin, one of four licensed drugs against African human trypanosomiasis, the SI
of Carlina oxide is approximately twice as high. Only diminazene, which is licensed only for
the treatment of animals, has a higher SI than Carlina oxide (Tab. 1).
The antimicrobial activity of extracts from C. acaulis is illustrated in table 2. The hexane
extract of C. acaulis has a strong activity against both strains of fungi with MIC values of
0.25 mg/mL and 0.5 mg/mL. It achieved an MIC between 0.125 mg/mL against Streptococcus
pyogenes and 4 mg/mL against the clinical isolate VRE 902291; a weak antimicrobial activity
was found against gram negative bacteria with an MIC between 4 and 8 mg/mL. Carlina
oxide exhibited strong antimicrobial activity against all the strains of bacteria and fungi even
against the multiresistant strains and the clinical isolates, MIC range was between 15 and 60
µg/mL.
6.5 Discussion
The essential oil of C. acanthifolia with Carlina oxide as major compound has antimicrobial,
anti-inflammatory, anti-ulcer and antioxidant activities [14]. Strong effects against the gram
positive bacteria Bacillus subtilis, Enterococcus faecalis and Staphylococcus aureus, against
the gram negative bacteria Klebsiella pneumoniae and against the fungi Candida albicans and
Aspergillus niger have been reported [14]. The main compound of the essential oil, Carlina
oxide, was isolated for the first time in 1889, making it one of the oldest known
polyacetylenes [23].
133
Table 2: Bacterial and fungal test results
Carlina acaulis
hexane extract
Microorganism
Carlina oxide
Ampicilline
Vancomycine
Nystatine
Inhibition
zone
(mm)
80 mg/mL
MIC
mg/mL
MMC
mg/mL
MIC
µg/mL
MMC
µg/mL
Inhibition
zone (mm)
1 mg/mL
MIC
µg/mL
MMC
µg/mL
Inhibition
zone
(mm)
MIC
µg/mL
MMC
µg/mL
Inhibition
zone
(mm)
MIC
µg/mL
MMC
µg/mL
G+
MRSA
ATCC 10442
5.1±0.1
2
4
15
60
14.5±0.5
25
>25
10.0±0.2
0.8
12.5
NT
NT
NT
G+
MRSA
1000/93
4.8±0.1
0.5
1
15
60
13.5±0.5
50
>50
NT
7
12.5
NT
NT
NT
G+
VRE
ATCC 31299
4.8±0.1
2
8
60
125
15
1
7
NT
25
>50
NT
NT
NT
G+
VRE
902291
4.8±0.1
4
>4
60
125
NA
NA
NA
NA
NA
NA
NT
NT
NT
G+
Streptococcus pyogenes
ATCC 12344
6.1±0.1
0.125
0.2
15
30
25.0±1.0
0.05
0.1
15.0±1.0
0.1
0.4
NT
NT
NT
G-
Escherichia coli
ATCC 25922
4.8±0.1
8
>8
60
>60
14
12.5
25
NA
NA
NA
NT
NT
NT
G-
Klebsiella pneumonia
ATCC 700603
3.1±0.1
4
>4
60
>60
-
25
25
NT
25
50
NT
NT
NT
G-
Pseudomonas aeruginosa
ATCC 27853
4.1±0.1
4
>4
60
>60
NA
NA
NA
NA
NA
NA
NT
NT
NT
Fungi
Candida albicans
ATCC 90028
7.7±0.1
0.25
0.5
15
30
NT
NT
NT
NT
NT
NT
10±1.2
0.2
0.4
Fungi
Candida glabrata
ATCC MYA 2950
10
0.5
1
15
30
NT
NT
NT
NT
NT
NT
12±1
0.2
0.2
NT; Not tested, NA; Not active
134
The exact chemical constitution of Carlina oxide was established in 1933 [24]. Other
polyacetylenes and the biogenesis of Carlina oxide were studied in the 1960s [25,26].
Our results confirm the previous findings that the essential oil of Carlina has an important
potential to inhibit and kill bacteria and fungi as compared with standard antimicrobial drugs.
Carlina oxide was more active against all the strains than the oil. The highest antimicrobial
activity of Carlina extract and Carlina oxide was recorded against S. pyogenes, Candida
albicans and C. glabrata, respectively.
Carlina oxide showed significant activity against the gram positive MRSA strains ATCC
10442 and 1000/93. The MIC was in both cases with 15 µg/ ml below that of Ampicilline
with 25 and 50 µg/mL, respectively, while vancomycine showed stronger activities with 0.8
µg/mL and 7 µg/mL, respectively.
Only a weak activity was exerted by Carlina extract and Carlina oxide against Gram negative
strains of bacteria, especially Pseudomonas aeruginosa, that are well known to be insensitive
to essential oils in general and a wide range of antibiotics [27,28]. The previous results lead us
to the conclusion that Carlina has a significant antimicrobial activity compared to the
antimicrobial drugs used in this study. In view of the low cytotoxicity for human cells Carlina
oxide might be an interesting candidate for the treatment of multiresistant bacteria.
For the first time the high activity of both Carlina extracts and Carlina oxide against T. b.
brucei was recorded. Carlina oxide was active against T. b. brucei at IC50 1.0 µg/mL, while
being cytotoxic to HeLa at IC50 446.0 µg/mL. The hexane and dichloromethane extracts also
showed significant cytotoxicity against T. b. brucei at IC50 3.7 µg/mL and 4.5 µg/mL,
respectively while the activity against HeLa was significantly less toxic at IC50 1722.4 µg/mL
and 1995.0 µg/mL, respectively. The high SI of 465 to 443 of the extracts and the SI of 446 of
Carlina oxide make it a very promising lead structure for the development of
antitrypanosomal drugs. The SI of Carlina oxide is with 446 even higher than the one of
suramin with a SI of 280, one of four licensed drugs for the treatment of African
trypanosomiasis.
We suggest that Carlina oxide inhibits trypanothione reductase, an enzyme specific for
trypanosomastids. Mammalian cells use the glutathione reductase instead, which could
135
explain the different sensitivity to Carlina oxide. Both enzymes belong to the FAD-dependent
NADPH oxidoreductase family which are essential for the protection of the cell against
reactive oxygen species [29-32]. The enzymes are highly selective regarding their substrates
trypanothione disulfide and glutathione disulfide. The larger trypanothione disulfide is +1
charged, while the smaller glutathione disulfide is -2 charged. The binding site of the
trypanothione reductase contains a negatively charged glutamate side chain together with a
hydrophobic cleft for the polyamine moiety, while the active centre of the glutathione
reductase is smaller and positively charged [30,33].
Carlina oxide seems to fit into the active centre of the trypanothione reductase because of its
lipophilic side chain and the electropositive C-atom of the furan ring. Inside of the cleft, the
CC triple bond can form irreversible links to the SH groups of the active centre, thus blocking
the enzyme. Due to its size and electropositively charged C-atom, Carlina oxide seems to be
unable to enter the active centre of the glutathione reductase. This proposed mechanism needs
to be experimentally confirmed in future studies.
136
Fig 3: GLC- profile of a hexane extract of C. acaulis and isolated Carlina oxide analysed by
GLC-MS
Fig 4: Carlina oxide [1] in hexane, dichloromethane and methanol extracts of C. acaulis
analysed by LC-MS
137
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140
7. Synergistic Interactions of Saponins and Monoterpenes in HeLa
cells, Cos7 cells and in Erythrocytes
7.1 Abstract
In phytomedicine complex extracts consisting of phenolics, monoterpenes or saponins are
traditionally used. It is often impossible to attribute the biological activity of an extract to one
or few compounds. As an explanation of the superior activity of extracts, a synergistic effect
of combinations of active compounds has been suggested. Since lipophilic monoterpenes or
saponins targeting the biomembrane usually accompany polar polyphenols in phytomedical
preparations, we decided to investigate their effect as single substances and in combination to
gain further insight into potential synergistic effects of herbal medicine. Combinations of the
monoterpenes α-pinene, thymol and menthol with the monodesmosidic saponins digitonin,
aescin, glycyrrhizic acid and Quillaja saponin demonstrated strong synergistic activity. The
IC50 of haemolysis was lowered by a factor of 10 to 100 from 316 µg/ml to 2 µg/ml for aescin,
157 µg/ml to 11 µg/ml for Quillaja saponins and 20 µg/ml to 3 µg/ml for digitonin when
combined with thymol. A similar significant synergistic cytotoxicity occurred both in HeLa
and Cos7 cells by combining the α-pinene, thymol and menthol with the saponins. The IC50 of
glycyrrhizic acid was lowered by a factor 100 from around 300 µg/ml to around 1 to 10 µg/ml
and the IC50 of aescin, digitonin and Quillaja saponins about the factor 10. Monoterpenes and
monodesmosidic saponins have a common target, the biomembrane, which is present in all
animal, fungal and bacterial cells. Disturbance of membrane fluidity and permeability is the
mode of action. This activity is non-specific which makes it extremely difficult for bacteria
and fungi to develop resistance. This explains the overall success of these molecules as
defence chemicals in the plant kingdom. The synergistic effect of combinations of saponins
with monoterpenes opens a complete new field of possible applications in medicine to
overcome resistance in multidrug resistant microbial and human cell.
7.2 Introduction
In approximately 90% of medicinal plants used in phytomedicine a single active compound
which could explain the activity of the extract could not be detected. The most common
groups of natural products found in plants are polyphenols, flavonoids and terpenoids ranging
from monoterpenes to saponins [1]. While phenolic compounds are the most common natural
products, saponins are still produced by more than 50% of the plants [2,3].
141
Fig. 1: Saponins
Fig. 2: Monoterpenes
Because the mode of action of complex mixtures from herbal medicines cannot be attributed
to a single compound in most cases, additive or even synergistic interactions of combinations
are postulated [4]. Synergy is present if the “effect seen by a combination of substances is
greater than would have been expected from a consideration of individual contributions” [5].
In this context it is important to distinguish between polyvalence and synergy. Synergy means
that several compounds acting on one target have an enhanced effect, while in polyvalence a
range of activity on several targets results in an overall effect [6].
In Western medicine, research has traditionally been focused on single compounds to treat a
specific disease (mono-target therapy), while many traditional systems apply a mixture of
herbs to treat the patient. In traditionally used herbal infusions available in European
pharmacies, mixtures of several herbs are still common. This is also true for the two important
medicinal systems of Asia, the Ayurvedic medicine and the Traditional Chinese medicine
(TCM). Especially in TCM, herbal mixtures of 10 or more herbs are common [7,8]. It has
been assumed that these mixtures act synergistically to enhance the healing effect while
minimizing the side effects. Several studies have been conducted to show this mode of action
142
of TCM [9,10]. High throughput screening of these traditional medicine combinations offers a
new approach to potentially effective drug formulations [11].
Synergy is important to explain the efficacy and mode of action of herbal medicine and
should not be neglected in the discovery of new treatments [12-14]. In recent years a change
of attitude towards complex extracts and mixtures of drugs (multi-target therapies) both in
phytomedicine and in modern drug therapy has taken place [15,16]. Examples include the use
of drug combinations in the treatment of HIV or in cancer therapy [17].
Several recent publications treat the phenomenon of synergy in phytomedicine; mainly
focusing on the effect of extracts but hardly explaining the detailed mode of action [18-20].
Several scientifically proven examples of synergy exist however. In this context, two
examples are especially interesting. The effect of the essential oil of Zingiber officinalis in the
treatment of ulcer could be related to four molecules which in combination are 66 times more
effective than the sum of the individual compounds [21]. The other example is a double-blind,
crossover trial of 20 volunteers treated with Panax ginseng and Ginkgo biloba. A stronger
improvement of the cognitive performance was detected in combination as compared to the
single treatments [22]. The influence of the saponins of P. ginseng is most likely responsible
for an increased uptake of flavonoids from G. biloba, explaining the detected effect.
It is striking that most publications on synergy either deal with the combination of anti cancer
drugs with saponins or the combination of monoterpenes with antibiotics. Saponins are well
known to modify the cell membrane and thus facilitate the uptake of chemotherapeutics.
Several well documented cases exist where this combination leads to synergistic effects
effective against tumour cells [23-26]. The second, equally important synergistic combination
between therapeutics and natural products is between antibiotics and monoterpenes. Here as
well the synergy is explained by the modification of the cell membrane by monoterpenes to
increase the uptake of antibiotics into the bacteria cell. Several studies document this
synergistic interaction [27-32].
Combinations of monoterpenes and saponins have not been analysed for a potential
synergistic effect. Biomembranes, which are present in animals, fungal and bacterial cells are
the main target of both monoterpenes and saponins. Monoterpenes interact with the lipophilic
side chain of phospholipids or cholesterol and thus increase the membrane fluidity and
143
permeability. Another target for monoterpenes is the lipophilic core of proteins. The lipophilic
moiety of monodesmosidic saponins forms complexes with cholesterol while at the same time
the hydrophilic part interacts with membrane proteins, thus modifying the membrane fluidity
and the conformation of membrane proteins [14,33]. At higher concentrations saponins act as
detergents which can lyse cell membranes.
To gain further insight into the potential synergistic effect in herbal medicine, we decided to
investigate the effects of combinations of monoterpenes and saponins on cancer cells and
biomembranes. We used haemolysis of erythrocytes as a model for the physical membrane
effects and cytotoxicity against the two cancer cell lines HeLa and Cos7 to monitor the effects
on living cells.
7.3 Material and methods
Chemicals
Aescin (E1378; ≥95%), digitonin (D141, pure), glycyrrhizic acid (50531; ≥95%), saponin
from Quillaja bark (S4521; 20-35% sapogenin), menthol (M2772; ≥99%), thymol (T0501;
≥99.5%), α-pinene (268070; ≥99%) and 3- (4, 5 dimethylthiazol-2-yl)-2, 5-diphenyltetrazolium bromide (MTT; M2128; ≥98%) were purchased from Sigma-Aldrich GmbH,
Germany. Defibrinated sheep blood was purchased from Oxoid Deutschland GmbH,
Germany. DMEM media, supplements, fetal bovine serum (FBS), trypsin-EDTA and
dimethyl sulfoxide (DMSO) were purchased from Gibco Invitrogen; Germany.
Methods
In the haemolysis assay 1 ml sample in isotonic sodium chloride solution was mixed carefully
with 1 ml defibrinated sheep blood, incubated for 30 min at 37 °C and centrifuged at 10000
rpm for 5 min. The supernatant which contains haemoglobin after haemolysis was detected
photometrically at 554 nm. Controls included water and isotonic sodium chloride solution.
Cos7 (African green monkey epithelial kidney cells) and HeLa (cervical cancer) cell lines
were maintained in DMEM complete media (L-glutamine supplemented with 10 % heatinactivated fetal bovine serum, 5% penicillin/ streptomycin and 5% non-essential amino
acids). Cells were grown at 37 °C in a humidified atmosphere of 5 % CO2. All experiments
were performed with cells in the logarithmic growth phase.
144
The 3-(4.5-dimethylthiazol-2-yl)-2.5-diphenyltetrazolium bromide (MTT) cell viability assay
[34] was used to determine the cytotoxicity of monoterpenes and saponins in the cancer cell
lines HeLa and Cos7. The pure compounds were dissolved in dimethylsulfoxide (DMSO) and
serially diluted in medium into ten different concentrations. A 100 µl sample containing
medium was dispensed into each well. The concentration of DMSO did not exceed 0.05% in
the medium that contained the highest concentration of compound tested. Cells (2 x 104 cells/
well) were seeded in 96-well plates (Greiner Labortechnik) and cultured for 24 h to 80%
confluence before treating them with the test samples. The test samples were replaced with
fresh medium containing 0.5 mg/ml MTT after 24 h incubation. 4 h later the formed formazan
crystals were dissolved in 100 µl DMSO; the absorbance was detected at 570 nm with a
Tecan Safire II Reader. Experiments were repeated at least three times with triplicates of each
measurement point. Controls included wells with not treated cells, wells without cells and the
cytotoxic compound doxorubicin (IC50 0.6 µg/ ml).
IC50 values were calculated using a four parameter logistic curve (SigmaPlot® 11.0)
representing 50% reduction of viability or 50% haemolysis compared to the positive control.
Results were expressed as mean and standard error.
Williamson [35] compared the different methods to distinguish synergy from other effects
with the result that the FIC index, usually represented in the isobole method of Berenbaum
[36] is the method of choice to demonstrate synergy.
Drug interaction was classified as either synergistic, additive, indifferent, or antagonistic
based on the fractional inhibitory concentration (FIC) index. The fractional effect (FE) of two
compounds is calculated from the effect caused by two compounds in combination in relation
to the effect of one compound alone: FEa = IC50 a+b / IC50 a; FEb = IC50 a+b / IC50 b. By
plotting these values against each other the isobologram showing the areas of synergy is
obtained. The FIC index is the sum of both FE indexes. According to Berenbaum [36], FIC ≤
1.0 signifies synergy, FIC = 1.0 an additive effect and FIC ≥ 1.0 antagonism. Schelz [37]
regards FIC ≤ 0.5 as synergy, FIC > 0.5 to 1.0 as additive, FIC = 1.0 to 4.0 as indifferent and
FIC ≥ 4.0 as antagonism. We follow the second perspective.
145
7.4 Results
Haemolysis
The three saponins aescin, digitonin and Quillaja saponin have substantial haemolytic
properties with IC50 values between 316, 20 and 157 µg/ml, respectively. The monoterpene
thymol exhibits a much lower haemolytic activity with an IC50 value of 763 µg/ml (Tab. 1).
These three saponins were combined with thymol to investigate an influence on the
haemolytic efficacy of saponins (Fig. 3). The combination of ascending concentrations of
saponins with a low constant concentration of thymol corresponding to its IC30 value
increased the haemolytic activity dramatically. All three combinations produced FIC values
between 0.01 and 0.1, indicating a very strong synergistic effect (Tab. 1). The monoterpenes
α-pinene and menthol could not be tested in the haemolytic assay since their solubility was
too poor to reach the concentrations necessary for a haemolytic effect.
Table 1: Synergistic haemolytic activities of saponins and monoterpenes.
*The concentration of thymol was kept constant at IC30, while the saponins were serially
diluted.
Compound
FIC
Aescin
Digitonin
Quillaja saponin
Thymol
Erythrocytes
IC50 [µg/ ml]
316.5
20.3
157.5
763.3
Combination
Aescin + thymol*
Digitonin + thymol *
Quillaja saponin + thymol *
2.3
3.6
11.5
0.011
0.184
0.088
Cytotoxicity
Cos7 and HeLa cells were treated with a dilution series of the saponins aescin, digitonin,
glycyrrhizic acid and Quillaja saponin (containing 20-35 % quillajic acid) (Fig. 1) and the
monoterpenes α-pinene, menthol and thymol (Fig. 2). The cytotoxic activities of aescin,
digitonin and Quillaja saponin were similar with IC50 values between 12.2 µg/ml and 41.3
µg/ml (Tab. 2). Glycyrrhizic acid was an exception with a 10 times lower cytotoxicity of
311.8 and 313.4 µg/ml respectively (Tab. 2). The monoterpenes α-pinene and menthol
showed a medium cytotoxicity with IC50 values between 243.0 µg/ml and 357.9 µg/ml, while
146
thymol was four times more toxic with 82.5 µg/ml and 83.6 µg/ml respectively in Cos7 and
HeLa cells (Tab. 2).
Table 2: Cytotoxic activities of saponins and monoterpenes
Compound
Aescin
Digitonin
Glycyrrhizic acid
Quillaja saponin
α-Pinene
Menthol
Thymol
HeLa
IC50 [µg/ ml]
41.3
12.2
313.4
33.5
357.9
243.0
83.6
Cos7
IC50 [µg/ ml]
37.4
14.7
311.8
23.4
337.5
319.9
82.5
In a consecutive experiment, Cos7 cells were treated with a dilution series of the saponins
aescin, digitonin, glycyrrhizic acid and Quillaja saponin in combination with α-pinene and
thymol. In HeLa cells menthol was employed as a combination partner. Cos7 cells were
extremely sensitive to the combinations aescin, digitonin, glycyrrhizic acid and Quillaja
saponins with thymol with IC50 values between 0.1 µg/ml and 1.0 µg/ml; while the
combinations of the saponins with α-pinene were 10 times less active (Tab. 3, Fig. 4). In
HeLa cells the combinations aescin, digitonin, glycyrrhizic acid and Quillaja saponins with
thymol and menthol had IC50 values between 4.9 µg/ml and 35.7 µg/ml, while the
combinations of the saponins with α-pinene were again up to 10 times less active (Tab. 3).
Table 3: Synergistic cytotoxic effects of saponins and monoterpenes; monoterpenes were
applied at their corresponding IC50 concentration. ND = not determined
Compound
HeLa
IC50
[µg/ ml]
51.3
ND
7.7
4.9
7.6
213.6
ND
25.4
35.7
Aescin + α-pinene
Aescin + thymol
Digitonin + α-pinene
Digitonin + thymol
Digitonin + menthol
Glycyrrhizic acid + α-pinene
Glycyrrhizic acid + thymol
Quillaja saponin + α-pinene
Quillaja saponin + thymol
147
FIC
1.387
0.651
0.465
0.657
1.278
0.828
1.491
Cos7
IC50
[µg/ ml]
11.1
1.0
1.8
0.1
ND
18.6
0.7
0.7
0.7
FIC
0.330
0.039
0.788
0.013
0.115
0.001
0.033
0.039
For Cos7 cells FIC values between 0.001 and 0.3 were found while HeLa cells showed FIC
values between 0.4 and 1.
Menthol, thymol and α-pinene were combined with each other in varying concentrations in
HeLa cells. These combinations were not synergistic and FIC values of the different
combinations ranged between 0.5 and 1.0, signifying an additive effect (Tab. 4, Fig. 5).
Table 4: Synergistic cytotoxic effects of monoterpenes combined at their corresponding IC50
concentrations
Compound
HeLa
IC50 [µg/ ml]
150.0
38.5
89.1
115.1
19.2
62.1
Menthol + α-pinene
Menthol + thymol
α-Pinene + menthol
α-Pinene + thymol
Thymol + menthol
Thymol + α-pinene
FIC
1.036
0.619
0.615
1.697
0.309
0.916
7.5 Discussion
Saponins and monoterpenes are a highly variable class of molecules found all over the plant
kingdom. So far, approximately 2500 monoterpenes and 5000 saponins are known today [33].
They play an important role in plant defence against bacteria, fungi and herbivores. Mankind
has long learned to use these plants for the treatment of various diseases, such as the common
cold and infections.
Several studies were conducted showing the synergistic effect of monoterpenes on bacterial
membranes [31,32,38-40], but so far no combination with saponins has been tested. Saponins
on the other hand have been studied for their supportive effect in cancer chemotherapy where
they seem to facilitate the absorption of cytotoxic drugs [25,41,42]. By enhancing the
membrane permeability they also facilitate the absorption of proteins [43] and antibiotics [44],
often with a synergistic effect.
Our haemolysis experiment revealed a synergistic effect of the combination of monoterpenes
and saponins (Tab. 1). The enhanced haemolysis is primarily a physical membrane effect by
making membranes leaky. The combinations of thymol with saponins were far more active
than one substance alone. Sheep erythrocytes are an excellent model for studying the
148
haemolytic effect of natural products on natural biomembranes. Their biomembranes are not a
simple lipid bilayer but contain also proteins and a sugar-coat on their surface. The major
difference to common cells however is the absence of a nucleus, which excludes apoptosis as
explanation for the shown effects.
In a second set of experiments the cell lines HeLa and Cos7 were treated with saponins in
combination with monoterpenes. Interestingly, HeLa and Cos7 were up to 10 times more
sensitive than erythrocytes. Combinations of monoterpenes and saponins exhibited synergistic
cytotoxic effects (Tab. 2, 3).
Fig.6: Biomembrane as target for saponins and monoterpenes after Wink 2008 [14]
Both saponins and monoterpenes act on the same biological target, the biomembrane (Fig. 6).
Due to their completely different chemical structures, their mode of action differs. Saponins,
especially the biologically active monodesmosides with only one sugar moiety intercalate
with their lipophilic part in the lipid bilayer of the membrane while their hydrophilic part, the
sugar chain, remains outside. Accordingly, saponins are able to change the membrane fluidity
and make it leaky.
149
Monoterpenes, on the other hand, are small, lipophilic molecules that can easily penetrate the
lipid bilayer of the biomembrane. Because of their lipophily they can accumulate within the
bilayer and increase its fluidity. Thymol is a phenol which can dissociate to a phenolate ion
under physiological conditions. In this case the polar part will drive the molecule to the
membrane borders, whereas the lipopophilic parts will dive into the bilayer. This may explain
the different activity of thymol as compared to the less polar menthol and α-pinene.
When saponins and monoterpenes are combined, however, the effect is triggered to new
levels of synergistic interactions. While monoterpenes are weakening the biomembrane,
saponins form little pores that eventually cause the rupture of the membrane. The exact nature
of the saponins does not seem to be of primary importance as long as they are
monodesmosides. The more molecules of monoterpenes are incorporated into the membrane,
the less stable it becomes, potentiating the effect of low doses of saponins.
It is not necessary for a plant to produce a specific saponin in combination with a specific
monoterpene to evoke lytic action against biomembranes. Although the physicochemical
mechanism is relatively simple and nonspecific it is highly efficient. Furthermore it is nearly
impossible for bacteria and fungi to develop resistances against this synergetic interaction.
This explains the overall success and abundance of these molecules in the plant kingdom as
defence compounds [45].
The synergistic effect of saponins with monoterpenes opens a complete new field of possible
applications. The efficacy of the treatment of antibiotic resistant bacterial infections could be
improved by combining antibiotics with monoterpenes and saponins. A combination therapy
of both monoterpenes and saponins together with cancer chemotherapeutics could also be
another option worth of consideration.
150
Fig. 3: Haemolytic effect of saponins in combination with monoterpenes. The concentration
of thymol was kept constant at IC30, while the saponin was serially diluted.
120
Aescin
Thymol
Thymol + Aescin
100
Haemolysis [%]
80
60
40
2000
500
1000
425
400
375
350
325
300
275
250
225
200
125
62.5
31.25
7.81
15.63
3
3.905
2.5
2.75
2
2.25
1.5
1.75
0.75
0.19
0.375
0.09
0.02
0.047
0.01
0
0.005
20
-20
Concentration [µg/ ml]
A: Haemolytic effect of aescin in combination with thymol
120
Digitonin
Thymol
Thymol + Digitonin
100
Haemolysis [%]
80
60
40
20
-20
Concentration [µg/ ml]
B: Haemolytic effect of digitonin in combination with thymol
151
2000
1000
500
425
400
375
350
325
300
275
250
225
200
100
50
25
20
22.5
17.5
15
12.5
6.25
3.13
1.6
0.7
0.35
0.25
0.125
0.06
0
120
Quillaja saponin
Thymol
Thymol + Quillaja saponin
100
Haemolysis [%]
80
60
40
20
-20
Concentration [µg/ ml]
C: Haemolytic effect of Quillaja saponin in combination with thymol
152
2000
950
1000
900
850
800
750
700
650
600
550
500
450
400
350
300
250
200
150
125
62.5
31.25
15.63
7.81
2.3
3.905
1.2
0.6
0
Fig. 4: Cytotoxicity of saponins in combination with monoterpenes in Cos7 cells. The
concentration of the monoterpene was kept constant at IC50, while the saponin was serially
diluted.
120
Aescin
Thymol
Thymol + Aescin
100
Survival [%]
80
60
40
20
0
0.781
1.562
3.125
6.25
12.5
25
50
100
200
400
-20
Concentration [µg/ ml]
A: Effect of aescin in combination with thymol against Cos7 cells
120
Digitonin
Thymol
Thymol + Digitonin
100
Survival [%]
80
60
40
20
0
0.117 0.234 0.468 0.937 1.875
3.75
7.5
15
30
60
-20
Concentration [µg/ ml]
B: Effect of digitonin in combination with thymol against Cos7 cells
153
100
200
400
140
Glycyrrhizic Acid
Thymol
Thymol + Glycyrrhizic Acid
120
Survival [%]
100
80
60
40
20
0
0.781
1.562
3.125
6.25
12.5
25
50
100
200
400
Concentration [µg/ ml]
C: Effect of glycyrrhizic acid in combination with thymol against Cos7 cells
140
Quillaja saponin
Thymol
Thymol + Quillaja saponin
120
Survival [%]
100
80
60
40
20
0
0.781
1.562
3.125
6.25
12.5
25
50
100
-20
Concentration [µg/ ml]
D: Effect of Quillaja saponin in combination with thymol against Cos7 cells
154
200
400
Fig. 5: Cytotoxicity of monoterpene combinations in HeLa cells. One monoterpene was kept
constant at IC50, while the other was serially diluted.
120
α-Pinene
Menthol
Menthol + α-Pinene
100
Survival [%]
80
60
40
20
0
1.171
2.441
4.882
9.765
19.531
39.062
78.125
156.25
312.5
625
1250
-20
Concentration [µg/ ml]
A: Effect of α-pinene in combination with menthol against HeLa cells
120
α-Pinene
Thymol
Thymol + α-Pinene
100
Survival [%]
80
60
40
20
0
0.781
1.562
3.125
6.25
12.5
25
50
100
200
-20
Concentration [µg/ ml]
B: Effect of α-pinene in combination with thymol against HeLa cells
155
400
625
1250
120
Thymol
Menthol
Menthol + Thymol
100
Survival [%]
80
60
40
20
0
0.781
1.171
2.343
4.687
9.375
18.75
37.5
75
-20
Concentration [µg/ ml]
C: Effect of thymol in combination with menthol against HeLa cells
156
150
300
600
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8. General conclusion and outlook
The acceptance and availability of Traditional Chinese Medicine has increased tremendously
in Europe and North America in recent years. However, the identity of these drugs is not
always guaranteed. The correct authentication is accordingly a necessity prior to all serious
research on this topic. We successfully used DNA barcoding to authenticate 66 TCM drugs
tested in our study. Only one adulteration could be detected where Arctium lappa was
wrongly labelled Fraxinus rhynchophylla. In the process of our project, we became aware of
both the advantages and the limitations the use of the rbcL gene offers for authentication of
herbal medicine drugs. We could show that DNA barcoding offers a fast, reliable way of
authentication and is superior to chemical profiling because of its practicability even for
unidentified plant species.
Adding to our survey several important European medicinal plants, we screened the
dichloromethane, methanol and water extracts of altogether 82 plants used in phytomedicine
for their antiviral, antitrypanosomal and anticancer properties. Several plants showed
extremely exciting results in their activities, so that we decided to focus in our further
research on the antitrypanosomal properties of Panax ginseng where our screening results
were especially promising.
Being widely regarded as an important medicinal drug with a broad range of pharmacological
properties, we decided to take a closer look at the natural products known from P. ginseng.
The pharmacological effects are mostly attributed to the ginsenosides, a highly variable group
of saponins. Accordingly, we investigated the individual effects of 12 pure ginsenosides to
gain a better understanding of their mode of action. We could show that their cytotoxic effect
is linked to membrane disturbance. The position of the sugar moieties and the resulting ability
to be incorporated into the lipid bilayer of the cell membrane are responsible for the huge
differences we could detect in their cytotoxic effects. The monodesmosidic ginsenosides Rg3
and Rh2 with the sugar moiety attached to C3 demonstrated a far higher cytotoxicity than
monodesmosidic ginsenosides with the sugar moiety attached to C6 or bidesmosidic
ginsenosides.
The antitrypanosomal activity of P. ginseng however was not due to the ginsenosides, and so
we decided to investigate the other important group of natural products present in P. ginseng,
160
the polyacetylenes. This proved to be an extremely exciting research field. We detected that
the hexane extracts and one of their major compounds, panaxynol, expressed an extreme
selectivity in their cytotoxicity against trypanosomes. Panaxynol was cytotoxic against
Trypanosoma brucei brucei at the IC50 concentration of 0.01 µg/ml with a selectivity index of
858. This selectivity is even superior to the established antitrypanosomal drugs like suramin
with a selectivity index of only 280. We suggest that the inhibition of the trypanothione
reductase, essential for the resistance of the parasite against oxidative stress and unique to
trypanosomes, is the primary principle of this selectivity.
Being aware of the great potential of polyacetylenes like panaxynol against trypanosomes, we
decided to investigate another polyacetylene plant, Carlina acaulis. The main compound of
the essential oil of C. acaulis, Carlina oxide, differs fundamentally in its chemical structure
from panaxynol. However, we could again detect a strong and selective activity against T. b.
brucei with an IC50 of 1.0 µg/ ml and a selectivity index of 446. We suggest a similar mode of
action as in panaxynol as explanation for this selectivity.
Our previous results obtained from the activity of ginsenosides had shown us the efficacy, but
also the structure dependency of the membrane effects of saponins. Thus, we decided to
explore this effect further and to investigate possible synergistic interactions found in the
multiple compound mixtures that form the natural defence system of plants. Saponins and
monoterpenes are the two groups of natural products whose activity is based on their effect on
the cell membrane. Since they are almost omnipresent in those plants commonly applied in
phytomedicine, we wanted to know if there is more to it than just a coincidence. We
combined several monoterpenes with different saponins and explored their effect on
biomembranes modelled by erythrocytes and living cells. Our results were truly astonishing.
The combination of a monoterpene with a saponin lowered the dosis necessary for haemolysis
up to the factor 100 compared to the single substances. The same effect could be detected in
living cells. Since both groups of natural products target the same structure of the cell, the
biomembrane, but are completely different in their structure, they work together
synergistically by enhancing their activity far above a simple additive effect.
We conclude that the potential of traditional medicinal systems such as traditional Chinese
Medicine as a source for novel lead compounds is far from being exhausted. If even a well
161
know drug like ginseng can offer such astonishing results, how much more can be detected in
unexplored or forgotten medicinal plants?
Being part of the so-called neglected diseases, the research on and discovery of new drugs
against sleeping sickness is of international importance. We are glad to have been able to
contribute to the research in this highly relevant field that might eventually influence the lives
of millions of people in Africa.
162
9. Acknowledgements
I am indebted to all people in the Institute of Pharmacy and Molecular Biotechnology,
University of Heidelberg, who have contributed with their time, valuable advice and
laboratory experience to the successful completion of this thesis.
I am most grateful to my supervisor Prof. Dr. M. Wink for giving me the opportunity to do
my dissertation in his laboratory. He always took the time to listen to my ideas, gave valuable
advice and provided the necessary materials and space for the completion of my experiments.
I am especially thankful that he was willing to let me pursue my ideas and gave me all liberty
to follow those that appeared most promising and most interesting to me. To give the PhD
student the chance to follow his own ideas while at the same time directing him with valuable
advice is a rare trait I am most fortunate to have encountered in him. His broad knowledge in
so different fields such as phytomedicine, traditional medicinal systems like TCM, evolution
and zoology was a great inspiration to me and enabled me to include a broader range of
exciting topics in my thesis than commonly possible.
I am also very grateful to Prof. Dr. T. Efferth for his constant interest and friendly support of
my research and his kind cooperation in several projects we could work at together. His
strong interest in traditional Chinese Medicine was a great inspiration and influence in my
thesis.
I also want to thank Prof. Dr. J. Reichling whose friendly approach and whose constructive
ideas were helpful to look at angles of my project that might have otherwise passed unnoticed.
I am very much indebted to Prof. Dr. J.J.G. Marin and his co-workers Dr. M. R. Romero and
Dr. A.G. Blazquez of the Laboratory of Experimental Hepatology and Drug Targeting
(HEVEFARM), University of Salamanca, CIBERehd, Spain, for their cooperation in our large
screening project where they performed all the viral and some of the cancer assays. In the
course of this project I am also thankful to Dorothea Kaufmann for performing the cytotoxic
screening in Cos7 cells.
I am very much indebted to the group of Prof. Dr. U. Müller for giving me the opportunity to
measure my cytotoxic assays with their plate reader. They were always cheerful no matter
how many 96 well plates I carried into their laboratory each week to measure.
I am especially thankful to Frank Sporer who performed the GLC-MS analysis in our projects
on Carlina acaulis and Panax ginseng. Without him the successful conclusion of these
projects would not have been possible.
I am indebted to several people in our institute who helped me with the daily laboratory work.
First of all I want to thank Hedi Sauer-Gürth who taught me all the basics of DNA barcoding
163
and how to overcome the daily frustration when the secondary metabolites in the isolated
DNA yet again made a successful PCR impossible. I also want to thank Heidi Stauder who
was always cheerful and very helpful in obtaining plant samples from the botanical garden as
references for the DNA barcoding. Here I also want to thank Michael Braun and Dr. Javier
Gonzalez for their support with the phylogenetic analysis of my samples.
Our cell culture laboratory would not have been such a good working place without Dorothea
Kaufmann who tireless fought to keep it in order and to organize a schedule that allowed all
of us to use the sterile benches. She was also most helpful in ordering the daily chemicals we
needed to perform our research. Last, but not least made her friendship and good humor the
many hours in front of the sterile bench an agreeable time!
My thanks go also to Razan Hamoud who performed the antimicrobial and antifungal tests in
some of our projects. It was always great to work with her and she often shared my frustration
when things were not going as smoothly as hoped.
I also want to thank Dr. M. Ashour for his support with the extract preparation of our TCM
plants when we spent months with extracting and concentrating the TCM plants that formed
the basis of many of the projects now being performed in our institute. At this place I would
also want to thank Astrid Backhaus for her friendly support whenever we had new extracts to
prepare in the course of our experiments.
My thanks go also to Endalk Nibret for his friendly cooperation in our cultivation of the
trypanosomes.
I want to thank as well Ted Cole for his friendly advice regarding formal procedure occuring
during the final phase of my dissertation.
At last I want to thank Sami Abbas, Andreas Bauer, Amin Eimanifar, Petra Fellhauer, Ina
Groß, Pille Link, Alexandre Mendes Fernandes, Dr. Holger Schäfer, Nina Strychalski, Ikhwan
Resmala Sudji, Chen Wei and all the others in the lab for their friendship and help.
Not part of our institute but equally important for the successful termination of my thesis is
my family. I am most grateful for their constant support and love. Without them, this project
would not have been possible.
164
10. Appendix
10.1 List of plants used in the screening with IPMB and GenBank accession number
Species
Andrographis paniculata
Celosia cristata
Bupleurum chinense
Bupleurum marginatum
Centella asiatica
Cnidium monnieri
Saposhnikovia divaricata
Eleutherococcus senticosus
Araliaceae
Panax ginseng China
Panax ginseng Korea
Panax notoginseng
Areca catechu
Arecaceae
Asclepiadaceae Cynanchum paniculatum
Artemisia annua
Asteraceae
Artemisia capillaris
Arctium lappa
Centipeda minima
Chrysanthemum indicum
Chrysanthemum morifolium
Eclipta prostata
Senecio scandens
Siegesbeckia orientalis
Taraxacum officinale
Berberis bealei
Berberidaceae
Dysosma versipellis
Epimedium koreanum
Capsella bursa-pastoris
Brassicaceae
Isatis indigotica (root)
Isatis indigotica (leaf)
Lonicera confusa
Caprifoliaceae
Convallariaceae Polygonatum kingianum
Rhodiola rosea
Crassulaceae
Platycladus orientalis
Cupressaceae
Dryopteridaceae Cyrtomium fortunei
Ephedra sinica
Ephedraceae
Equisetum hiemale
Equisetaceae
Croton tiglium
Euphorbiaceae
Family
Acanthaceae
Amaranthaceae
Apiaceae
165
IPMB / No
P6838 / 04
P6848 / 14
P6844 / 10
P6845 / 11
P6849 / 15
P6854 / 20
P6902 / 68
P6919 / 79
P8088 / 81
P8086 / 81
P6887 / 53
P6840 / 06
P6858 / 24
P6841 / 07
P6842 / 08
P6839 / 05
P6850 / 16
P6851 / 17
P6852 / 18
P6863 / 29
P6905 / 71
P6906 / 72
P6908 / 74
P6883 / 49
P6862 / 28
P6865 / 31
P6846 / 12
P6877 / 43
P6878 / 44
P6880 / 46
P6892 / 58
P6920 / 84
P6891 / 57
P6859 / 25
P6864 / 30
P6866 / 32
P6856 / 22
GenBank
JF949965
JF949970
JF950021
JF949968
JF950022
JF949973
JF949988
JF950028
JF950029
JF950030
JF949975
JF949966
JF949967
JF949994
JF949971
JF949972
JF950000
JF949989
JF949990
JF950019
JF949996
JF950002
JF949997
JF949981
JF949981
JF949982
JF950027
JF950011
JF949998
JF950001
JF950003
-
Fabaceae
Geraniaceae
Ginkgoaceae
Hypericaceae
Iridaceae
Lamiaceae
Lauraceae
Loranthaceae
Lythraceae
Magnoliaceae
Melanthiaceae
Myrsinaceae
Myrtaceae
Ophioglossaceae
Orchidaceae
Paeoniaceae
Pedaliaceae
Poaceae
Polygonaceae
Ranunculaceae
Rosaceae
Rubiaceae
Rutaceae
Saururaceae
Schisandraceae
Abrus cantoniensis
Acacia catechu
Cassia tora
Desmodium styracifolium
Glycyrrhiza inflata
Spatholobus suberectus
Sutherlandia frutescens
Geranium wilfordii
Pelargonium sidoides
Ginkgo biloba
Hypericum japonicum
Belamcanda chinensis
Mentha haplocalyx
Prunella vulgaris
Scutellaria baicalensis
Cinnamomum cassia
Taxillus chinensis
Punica granatum
Magnolia officinalis
Paris polyphylla
Lysimachia christinae
Eucalyptus robusta
Ophioglossum vulgatum
Dendrobium loddigesii
Paeonia lactiflora
Harpagophytum procumbens
Cymbopogon distans
Fallopia japonica (syn.
Polygonum cuspidatum)
Fallopia multiflora (syn.
Polygonum multiflorum)
Polygonum aviculare
Rheum officinale
Coptis chinensis
Rosa chinensis
Rosa laevigata
Sanguisorba officinalis
Hedyotis diffusa
Evodia lepta
Evodia rutaecarpa
Phellodendron chinense
Houttuynia cordata
Kadsura longipedunculata
166
P6835 / 01
P6836 / 02
P6847 / 13
P6861 / 27
P6873 / 39
P6907 / 73
tba / 83
P6867 / 33
tba / 82
P6872 / 38
P6876 / 42
P6843 / 09
P6884 / 50
P6896 / 62
P6903 / 69
P6853 / 19
P6909 / 75
P6897 / 63
P6882 / 48
P6888 / 54
P6881 / 47
P6868 / 34
P6885 / 51
P6860 / 26
P6886 / 52
tba / 80
P6857 / 23
JF949964
JF949969
JF949976
JF950025
JF949991
JF949977
JF950005
JF949980
JF949995
JF949984
JF950013
JF950017
JF950023
JF949992
JF950014
JF950008
JF950010
JF949983
JF950009
JF949999
JF950026
JF949974
P6894 / 60
JF950004
P6895 / 61
P6893 / 59
P6898 / 64
P6855 / 21
P6899 / 65
P6900 / 66
P6901 / 67
P6874 / 40
P6869 / 35
P6870 / 36
P6890 / 56
P6875 / 41
P6879 / 45
JF949987
JF950012
JF950015
JF950024
JF950016
JF949979
JF949978
JF949986
JF950006
JF950007
Selaginellaceae
Valerianaceae
Verbenaceae
Violaceae
Zingiberaceae
Selaginella tamariscina
Patrinia scabiosaefolia
Verbena officinalis
Viola yezoensis
Alpinia galanga
Alpinia oxyphylla
167
P6904 / 70
P6889 / 55
P6910 / 76
P6911 / 77
P6837 / 03
P6917 / 78
JF950018
JF949985
JF950020
JF949993
-
10.2 TCM plants with photos and Chinese name
Family
Species
Acanthaceae
Andrographis
paniculata
Chinese Name
Chuanxinlian
穿 心 莲
Jiguanhua
Amaranthaceae
Celosia cristata
鸡 冠 花
Chaihu
Apiaceae
Bupleurum chinense
柴 胡
Apiaceae
Nanchaihu
Bupleurum
marginatum
南 柴 胡
Leigonggen
Apiaceae
Centella asiatica
雷 公 根
Shechuangzi
Apiaceae
Cnidium monnieri
蛇 床 子
Fangfeng
Apiaceae
Saposhnikovia
divaricata
防 风
168
Renshen
Araliaceae
Panax ginseng China
人 参
Renshen
Araliaceae
Panax ginseng Korea
人 参
Sanqi
Araliaceae
Panax notoginseng
三 七
Binglang
Arecaceae
Areca catechu
槟 榔
Liaodiaozhu
Asclepiadaceae
Cynanchum
paniculatum
了 刁 竹
Huanghuahao
Asteraceae
Artemisia annua
黄 花 蒿
Yinchenhao
Asteraceae
Artemisia capillaris
茵 陈 蒿
169
Niubang
Asteraceae
Arctium lappa
牛 蒡
Ebushicao
Asteraceae
Centipeda minima
鹅 不 食 草
Asteraceae
Yejuhua
Chrysanthemum
indicum
野 菊 花
Juhua
Asteraceae
Chrysanthemum
morifolium
菊 花
Hanliancao
Asteraceae
Eclipta prostata
旱 莲 草
Qianliguang
Asteraceae
Senecio scandens
千 里 光
Xixiancao
Asteraceae
Siegesbeckia
orientalis
稀
170
莶
草
Pugongying
Asteraceae
Taraxacum officinale
蒲 公 英
Shidagonglao
Berberidaceae
Berberis bealei
十 大 功 劳
Bajiaolian
Berberidaceae
Dysosma versipellis
八 角
莲
Yinyanghuo
Berberidaceae
Epimedium koreanum
淫 羊 霍
Jicai
Brassicaceae
Capsella bursapastoris
荠 菜
Banlangen
Brassicaceae
Isatis indigotica (root)
板 蓝 根
Daqingye
Brassicaceae
Isatis indigotica (leaf)
大 青 叶
171
Rendongteng
Caprifoliaceae
Lonicera confusa
忍 冬 藤
Convallariaceae
Huangjing
Polygonatum
kingianum
黄 精
Hongjingtian
Crassulaceae
Rhodiola rosea
红 景 天
Cebaiye
Cupressaceae
Platycladus orientalis
侧 柏 叶
Guanzhong
Dryopteridaceae
Cyrtomium fortunei
贯 众
Mahuang
Ephedraceae
Ephedra sinica
麻 黄
Muzei
Equisetaceae
Equisetum hiemale
木 贼
172
Badou
Euphorbiaceae
Croton tiglium
巴 豆
Jigucao
Fabaceae
Abrus cantoniensis
鸡 骨 草
Ercha
Fabaceae
Acacia catechu
儿 茶
Juemingzi
Fabaceae
Cassia tora
决 明 子
Guangjinqiancao
Fabaceae
Desmodium
styracifolium
广 金 钱 草
Gancao
Fabaceae
Glycyrrhiza inflata
甘 草
Jixueteng
Fabaceae
Spatholobus
suberectus
鸡 血 藤
173
Laoguancao
Geraniaceae
Geranium wilfordii
老 鹳 草
Yinxing
Ginkgoaceae
Ginkgo biloba
银 杏
Tianjihuang
Hypericaceae
Hypericum japonicum
田 基 黄
Shegan
Iridaceae
Belamcanda chinensis
射 干
Bohe
Lamiaceae
Mentha haplocalyx
薄 荷
Xiakucao
Lamiaceae
Prunella vulgaris
夏 枯 草
Huangqin
Lamiaceae
Scutellaria baicalensis
黄 芩
174
Guizhi
Lauraceae
Cinnamomum cassia
桂 枝
Sangjisheng
Loranthaceae
Taxillus chinensis
桑 寄 生
Shiliu
Lythraceae
Punica granatum
石 榴
Houpu
Magnoliaceae
Magnolia officinalis
厚 朴
Qiyeyizhihua
Melanthiaceae
Paris polyphylla
七 叶 一 枝 花
Jinqiancao
Myrsinaceae
Lysimachia christinae
金 钱 草
Anshu
Myrtaceae
Eucalyptus robusta
桉 树
175
Yizhijian
Ophioglossaceae
Ophioglossum
vulgatum
一 支 箭
Shihu
Orchidaceae
Dendrobium
loddigesii
石 斛
Chishao
Paeoniaceae
Paeonia lactiflora
赤 芍
Yunxiancao
Poaceae
Cymbopogon distans
芸 香 草
Polygonaceae
Polygonaceae
Huzhang
Fallopia japonica
(syn. Polygonum
cuspidatum)
虎 杖
Heshouwu
Fallopia multiflora
(syn. Polygonum
multiflorum)
何 首 乌
Pianxu
Polygonaceae
Polygonum aviculare
扁 蓄
176
Dahuang
Polygonaceae
Rheum officinale
大 黄
Huanglian
Ranunculaceae
Coptis chinensis
黄 连
Yuejihua
Rosaceae
Rosa chinensis
月 季 花
Jinyingzi
Rosaceae
Rosa laevigata
金 樱 子
Diyu
Rosaceae
Sanguisorba
officinalis
地 榆
Baihuasheshecao
Rubiaceae
Hedyotis diffusa
紫 花 地 丁 草
Sanchaku
Rutaceae
Evodia lepta
三 叉 苦
177
Wuzhuyu
Rutaceae
Evodia rutaecarpa
吴 茱 萸
Huangbai
Rutaceae
Phellodendron
chinense
黄 柏
Yuxingcao
Saururaceae
Houttuynia cordata
鱼 腥 草
Zigingpi
Schisandraceae
Kadsura
longipedunculata
紫 荆 皮
Juanbai
Selaginellaceae
Selaginella
tamariscina
卷 柏
Baijiang
Valerianaceae
Patrinia
scabiosaefolia
败 酱
Mabiancao
Verbenaceae
Verbena officinalis
马 鞭 草
178
Zihuadidingcao
Violaceae
Viola yezoensis
紫 花 地 丁 草
Hongdoukou
Zingiberaceae
Alpinia galanga
红 豆 蔻
Yizheren
Zingiberaceae
Alpinia oxyphylla
益 智 仁
179
10.3 DNA Barcoding Sequences
Documentation of the authentication of the TCM drugs:
DNA sequence and GenBank accession number of the rbcL gene sequences of the TCM
drugs aligned with species obtained from GenBank.
Andrographis paniculata
P6838 / 04
JF949965
Aligned with
Andrographis paniculata
GQ436496
ribulose-1,5-bisphosphate carboxylase/oxygenase large subunit (rbcL) gene
Acanthaceae
TTCAAAGCGGGTGTTAAAGAGTACAAATTGACTTATTATACTCCTGAATACGAAACCAAAGATACTGATATCTTGGCAGCATT
TTCAAAGCGGGTGTTAAAGAGTACAAATTGACTTATTATACTCCTGAATACGAAACCAAAGATACTGATATCTTGGCAGCATT
CCGAGTAACTCCTCAACCCGGAGTTCCGCCTGAAGAAGCAGGGGCAGCGGTAGCTGCCGAATCTTCCACTGGTACATGGACA
CCGAGTAACTCCTCAACCCGGAGTTCCGCCTGAAGAAGCAGGGGCAGCGGTAGCTGCCGAATCTTCCACTGGTACATGGACA
ACCGTGTGGACTGATGGACTTACCAGCCTTGATCGTTACAAAGGGCGATGCTACAACATCGAGCCCGTTCTTGGCGAAACAG
ACCGTGTGGACTGATGGACTTACCAGCCTTGATCGTTACAAAGGGCGATGCTACAACATCGAGCCCGTTCTTGGCGAAACAG
ATCAATATATTTGTTATGTAGCTTACCCTTTAGACCTTTTTGAAGAAGGTTCTGTTACCAACATGTTTACTTCCATTGTAGGAA
ATCAATATATTTGTTATGTAGCTTACCCTTTAGACCTTTTTGAAGAAGGTTCTGTTACCAACATGTTTACTTCCATTGTAGGAA
ATGTATTTGGATTCAAAGCCCTGCGTGCTCTACGCCTGGAAGATCTGCGAATCCCTACTGCTTATATTAAAACTTTCCAAGGTC
ATGTATTTGGATTCAAAGCCCTGCGTGCTCTACGCCTGGAAGATCTGCGAATCCCTACTGCTTATATTAAAACTTTCCAAGGTC
CGCCTCATGGGATCCAAGTTGAGAGAGATAAATTGAACAAATATGGTCGTCCTCTGCTGGGATGTACTATTAAACCTAAATTG
CGCCTCATGGGATCCAAGTTGAGAGAGATAAATTGAACAAATATGGTCGTCCTCTGCTGGGATGTACTATTAAACCTAAATTG
GGATTATCCGCTAAAAACTACGGCAGAGCATGTTATGAATGTCTTCGCGGTGGACTTGATTTTACCAAAGATGATGAGAACGT
GGATTATCCGCTAAAAACTACGGCAGAGCATGTTATGAATGTCTTCGCGGTGGACTTGATTTTACCAAAGATGATGAGAACGT
GAACTCCCAGCCATTTATGCGTTGGAGAGATCGTT
GAACTCCCAGCCATTTATGCGTTG GAGAGATCGTT
Celosia cristata
P6848 / 14
JF949970
Aligned with
Celosia argentea
GQ436716
ribulose-1,5-bisphosphate carboxylase/oxygenase large subunit (rbcL) gene
Amaranthaceae
CTGACCTATTATACCCCGGAATATGAAACCCTGGATACCGATATTCTGGCGGCGTTTCGTGTGACCCCGCAGCCGGGCGTGCC
CTGACCTATTATACCCCGGAATATGAAACCCTGGATACCGATATTCTGGCGGCGTTTCGTGTGACCCCGCAGCCGGGCGTGCC
GCCGGAAGAAGCGGGCGCGGCGGTGGCGGCGGAAAGCAGCACCGGCACCTGGACCACCGTGTGGACCGATGGCCTGACCAG
GCCGGAAGAAGCGGGCGCGGCGGTGGCGGCGGAAAGCAGCACCGGCACCTGGACCACCGTGTGGACCGATGGCCTGACCAG
CCTGGATCGTTATAAAGGCCGTTGCTATCATATTGAACCGGTGGCGGGCGAAGAAAACCAGTATATTTGCTATGTGGCGTATC
CCTGGATCGTTATAAAGGCCGTTGCTATCATATTGAACCGGTGGCGGGCGAAGAAAACCAGTATATTTGCTATGTGGCGTATC
CGCTGGATCTGTTTGAAGAAGGCAGCGTGACCAACATGTTTACCAGCATTGTGGGCAACGTGTTTGGCTTTAAAGCGCTGCGT
CGCTGGATCTGTTTGAAGAAGGCAGCGTGACCAACATGTTTACCAGCATTGTGGGCAACGTGTTTGGCTTTAAAGCGCTGCGT
GCGCTGCGTCTGGAAGATCTGCGTATTCCGGTGGCGTATATTAAAACCTTTCAGGGCCCGCCGCATGGCATTCAGGTGGAACG
GCGCTGCGTCTGGAAGATCTGCGTATTCCGGTGGCGTATATTAAAACCTTTCAGGGCCCGCCGCATGGCATTCAGGTGGAACG
TGATAAACTGAACAAATATGGCCGTCCGCTGCTGGGCTGCACCATTAAACCGAAACTGGGCCTGAGCGCGAAAAACTATGGC
TGATAAACTGAACAAATATGGCCGTCCGCTGCTGGGCTGCACCATTAAACCGAAACTGGGCCTGAGCGCGAAAAACTATGGC
CGTGCGTGCTATGAATGCCTG
CGTGCGTGCTATGAATGCCTG
Bupleurum chinense
P6844 / 10
JF950021
Aligned with
Bupleurum falcatum
U50224
ribulose-1,5-bisphosphate carboxylase/oxygenase large subunit (rbcL) gene
Apiaceae
GATTACAAATTGACTTATTATACTCCTGAATATGAAACCAAAGATACTGATATCTTGGCAGCATTCAGAGTAACTCCTCAACC
GATTACAAATTGACTTATTATACTCCTGAATATGAAACCAAAGATACTGATATCTTGGCAGCATTCAGAGTAACTCCTCAACC
180
AGGAGTTCCACCTGAAGAAGCGGGGGCCGCGGTAGCTGCCGAATCTTCTACTGGTACATGGACCACTGTGTGGACCGATGGA
AGGAGTTCCACCTGAAGAAGCGGGGGCCGCGGTAGCTGCCGAATCTTCTACTGGTACATGGACCACTGTGTGGACCGATGGA
CTTACCAGCCTTGATCGTTACAAAGGGCGATGCTACGGAATCGAGCCCGTTCCTGGAGAAGAAAATCAATTTATCGCTTATGT
CTTACCAGCCTTGATCGTTACAAAGGGCGATGCTACGGAATCGAGCCCGTTCCTGGAGAAGAAAATCAATTTATCGCTTATGT
AGCTTACCCATTAGATCTTTTTGAAGAAGGTTCTGTTACTAACATGTTTACTTCCATCGTAGGTAATGTATTTGGGTTCAAAGC
AGCTTACCCATTAGATCTTTTTGAAGAAGGTTCTGTTACTAACATGTTTACTTCCATCGTAGGTAATGTATTTGGGTTCAAAGC
CCTGCGCGCTCTACGTCTGGAAGATCTGCGAATCCCCGTTGCTTATGTTAAAACTTTCCAAGGACCGCCTCATGGCATCCAAG
CCTGCGCGCTCTACGTCTGGAAGATCTGCGAATCCCCGTTGCTTATGTTAAAACTTTCCAAGGACCGCCTCATGGCATCCAAG
TTGAGAGAGATAAATTGAACAAGTATGGTCGTCCCCTGTTGGGATGTACTATTAAACCTAAATTGGGGTTATCCGCTAAAAAC
TTGAGAGAGATAAATTGAACAAGTATGGTCGTCCCCTGTTGGGATGTACTATTAAACCTAAATTGGGGTTATCCGCTAAAAAC
TACGGTAGAGCGGTTTATGAATGTCTCCGCGGTGGACTTGATTTTACCAAAGACGATGAGAATGTGAATTCCCAACCATTTAT
TACGGTAGAGCGGTTTATGAATGTCTCCGCGGTGGACTTGATTTTACCAAAGACGATGAGAATGTGAATTCCCAACCATTTAT
GCGTTGGAGAGACCGTTTCTTATTTTGTGCCGAAGCACTTTATAAAGCCCAAGCCGAAACTGGTGAAATCAAAGGGCATTACT
GCGTTGGAGAGACCGTTTCTTATTTTGTGCCGAAGCACTTTATAAAGCCCAAGCCGAAACTGGTGAAATCAAAGGGCATTACT
TGAACGCCACTGCGGGTACATGTGAA
TGAACGCCACTGCGGGTACATGTGAA
Bupleurum marginatum
P6845 / 11
JF949968
Aligned with
Bupleurum falcatum
U50224
ribulose-1,5-bisphosphate carboxylase/oxygenase large subunit (rbcL) gene
Apiaceae
GATTACAAATTGACTTATTATACTCCTGAATATGAAACCAAAGATACTGATATCTTGGCAGCATTCAGAGTAACTCCTCAACC
GATTACAAATTGACTTATTATACTCCTGAATATGAAACCAAAGATACTGATATCTTGGCAGCATTCAGAGTAACTCCTCAACC
AGGAGTTCCACCTGAAGAAGCGGGGGCCGCGGTAGCTGCCGAATCTTCTACTGGTACATGGACCACTGTGTGGACCGATGGA
AGGAGTTCCACCTGAAGAAGCGGGGGCCGCGGTAGCTGCCGAATCTTCTACTGGTACATGGACCACTGTGTGGACCGATGGA
CTTACCAGCCTTGATCGTTACAAAGGGCGATGCTACGGAATCGAGCCCGTTCCTGGAGAAGAAAATCAATTTATCGCTTATGT
CTTACCAGCCTTGATCGTTACAAAGGGCGATGCTACGGAATCGAGCCCGTTCCTGGAGAAGAAAATCAATTTATCGCTTATGT
AGCTTACCCATTAGATCTTTTTGAAGAAGGTTCTGTTACTAACATGTTTACTTCCATCGTAGGTAATGTATTTGGGTTCAAAGC
AGCTTACCCATTAGATCTTTTTGAAGAAGGTTCTGTTACTAACATGTTTACTTCCATCGTAGGTAATGTATTTGGGTTCAAAGC
CCTGCGCGCTCTACGTCTGGAAGATCTGCGAATCCCCATTGCTTATGTTAAAACTTTCCAAGGACCGCCTCATGGCATCCAAG
CCTGCGCGCTCTACGTCTGGAAGATCTGCGAATCCCCGTTGCTTATGTTAAAACTTTCCAAGGACCGCCTCATGGCATCCAAG
TTGAGAGAGATAAATTGAACAAGTATGGTCGTCCTCTGTTGGGATGTACTATTAAACCTAAATTGGGGTTATCCGCTAAAAAC
TTGAGAGAGATAAATTGAACAAGTATGGTCGTCCCCTGTTGGGATGTACTATTAAACCTAAATTGGGGTTATCCGCTAAAAAC
TACGGTAGAGCGGTTTATGAATGTCTCCGCGGTGGACTTGATTTTACCAAAGACGATGAGAATGTGAATTCCCAACCATTTAT
TACGGTAGAGCGGTTTATGAATGTCTCCGCGGTGGACTTGATTTTACCAAAGACGATGAGAATGTGAATTCCCAACCATTTAT
GCGTTGGAGAGACCGTTTCTTATTTTGTGCCGAAGCACTTTATAAAGCCCAAGCCGAAACTGGTGAAATCAAAGGGCATTACT
GCGTTGGAGAGACCGTTTCTTATTTTGTGCCGAAGCACTTTATAAAGCCCAAGCCGAAACTGGTGAAATCAAAGGGCATTACT
TGA
TGA
Centella asiatica
P6849 / 15
JF950022
Aligned with
Centella asiatica
GQ436636
ribulose-1,5-bisphosphate carboxylase/oxygenase large subunit (rbcL) gene
Apiaceae
TAAAGATTACAAATTGACTTATTATACTCCTGACTATGAAACCAAAGATACTGATATCTTGGCAGCATTCCGAGTAACTCCTC
TAAAGATTACAAATTGACTTATTATACTCCTGACTATGAAACCAAAGATACTGATATCTTGGCAGCATTCCGAGTAACTCCTC
AACCTGGAGTTCCACCTGAAGAAGCAGGGGCCGCGGTAGCTGCCGAATCTTCTACTGGTACATGGACAACTGTGTGGACCGA
AACCTGGAGTTCCACCTGAAGAAGCAGGGGCCGCGGTAGCTGCCGAATCTTCTACTGGTACATGGACAACTGTGTGGACCGA
CGGACTTACCAGCCTTGATCGTTACAAAGGGCGATGCTACGGAATCGAGCCCGTTCCTGGAGAAGAAAATCAATTTATTGCTT
CGGACTTACCAGCCTTGATCGTTACAAAGGGCGATGCTACGGAATCGAGCCCGTTCCTGGAGAAGAAAATCAATTTATTGCTT
ATGTAGCTTACCCATTAGACCTTTTTGAAGAAGGTTCTGTTACTAACATGTTTACTTCCATTGTTGGTAATGTATTTGGGTTCA
ATGTAGCTTACCCATTAGACCTTTTTGAAGAAGGTTCTGTTACTAACATGTTTACTTCCATTGTTGGTAATGTATTTGGGTTCA
181
AAGCCCTGCGTGCCCTACGTCTGGAAGATCTGCGAATCCCTGTTGCTTATGTGAAAACTTTCCAAGGCCCGCCTCATGGTATC
AAGCCCTGCGTGCCCTACGTCTGGAAGATCTGCGAATCCCTGTTGCTTATGTGAAAACTTTCCAAGGCCCGCCTCATGGTATC
CAAGTTGAAAGAGATAAATTGAACAAGTATGGTCGTCCCCTGTTGGGATGTACTATTAAACCTAAATTGGGGTTATCCGCTAA
CAAGTTGAAAGAGATAAATTGAACAAGTATGGTCGTCCCCTGTTGGGATGTACTATTAAACCTAAATTGGGGTTATCCGCTAA
AAACTACGGTAGAGCGGTTTATGAATGTCTCCGCGGTGGACTTGATTTTACCAAAGACGATGAGAACGTGAACTC
AAACTACGGTAGAGCGGTTTATGAATGTCTCCGCGGTGGACTTGATTTTACCAAAGACGATGAGAACGTGAACTC
Cnidium monnieri
P6854 / 20
JF949973
Aligned with
Cnidium japonicum
D44562
ribulose-1,5-bisphosphate carboxylase/oxygenase large subunit (rbcL) gene
Apiaceae
TAAAGATTACAAATTGACTTATTATACTCCTGACTATGAAACCAAAGATACTGATATCTTGGCAGCATTCCGAGTAACTCCTC
TAAAGATTACAAATTGACTTATTATACTCCTGACTATGAAACCAAAGATACTGATATCTTGGCAGCATTCCGAGTAACTCCTC
AACCTGGAGTTCCACCTGAAGAAGCGGGGGCCGCGGTAGCTGCCGAATCTTCTACTGGTACATGGACCACTGTGTGGACCGA
AACCTGGAGTTCCACCTGAAGAAGCGGGGGCCGCGGTAGCTGCCGAATCTTCTACTGGTACATGGACCACTGTGTGGACCGA
TGGACTTACCAGCCTTGATCGTTACAAAGGGCGCTGCTACGAAATCGAGCCCGTTGCTGGAGAAGAAAATCAATTTATCGCTT
TGGACTTACCAGCCTTGATCGTTACAAAGGGCGCTGCTACGAAATCGAGCCCGTTGCTGGAGAAGAAAATCAATTTATCGCTT
ATGTAGCTTACCCATTAGACCTTTTTGAAGAAGGTTCTGTTACTAACATGTTTACTTCCATTGTAGGTAATGTATTTGGGTTCA
ATGTAGCTTACCCATTAGACCTTTTTGAAGAAGGTTCTGTTACTAACATGTTTACTTCCATTGTAGGTAATGTATTTGGGTTCA
AAGCCCTGCGCGCTCTACGTCTGGAAGATCTGCGAATCCCCGTTGCTTATGTTAAAACTTTCCAAGGACCGCCTCATGGCATC
AAGCCCTGCGTGCTCTACGTCTGGAAGATCTGCGAATCCCCGTTGCTTATGTTAAAACTTTCCAAGGACCGCCTCATGGCATC
CAAGTTGAGAGAGATAAATTGAACAAGTACGGTCGTCCCCTGTTGGGATGTACTATTAAACCTAAATTGGGGTTATCCGCTAA
CAAGTTGAGAGAGATAAATTGAACAAGTACGGTCGTCCCCTGTTGGGATGTACTATTAAACCTAAATTGGGGTTATCCGCTAA
AAACTACGGTAGAGCGGTTTATGAATGTCTCCGCGGTGGACTTGATTTTACCAAAGACGATGAGAATGTGAACTCCCAACCAT
AAACTACGGTAGAGCGGTTTATGAATGTCTCCGCGGTGGACTTGATTTTACCAAAGACGATGAGAATGTGAACTCCCAACCAT
TTATGCGTTGGAGAGATCGTTTCTTATTTTGTGCCGAAGCAATTTATAAAGCACAGGCTGAAACTGGTGAAATCAAAGGGCAT
TTATGCGTTGGAGAGATCGTTTCTTATTTTGTGCCGAAGCAATTTATAAAGCACAGGCTGAAACTGGTGAAATCAAAGGGCAT
TACTTGAATGCTACTGCGGGTACATG
TACTTGAATGCTACTGCGGGTACATG
Saposhnikovia divaricata
P6902 / 68
JF949988
Aligned with
Saposhnikovia divaricata
GQ436633
ribulose-1,5-bisphosphate carboxylase/oxygenase large subunit (rbcL) gene
Apiaceae
TAAAGATTACAAATTGACTTATTATACTCCTGACTATGAAACCAAAGATACTGATATCTTGGCAGCATTCCGAGTAACTCCTC
TAAAGATTACAAATTGACTTATTATACTCCTGACTATGAAACCAAAGATACTGATATCTTGGCAGCATTCCGAGTAACTCCTC
AACCTGGAGTTCCACCTGAAGAAGCGGGGGCCGCGGTAGCTGCCGAATCTTCTACTGGTACATGGACCACTGTGTGGACCGA
AACCTGGAGTTCCACCCGAAGAAGCGGGGGCCGCGGTAGCTGCCGAATCTTCTACTGGTACATGGACCACTGTGTGGACCGA
TGGACTTACCAGCCTTGATCGTTACAAAGGGCGCTGCTACGGAATCGAGCCCGTTGCTGGAGAAGAAAATCAATATATCGCTT
TGGACTTACCAGCCTTGATCGTTACAAAGGGCGCTGCTACGGAATCGAGCCCGTTGCTGGAGAAGAAAATCAATATATCGCTT
ATGTAGCTTACCCATTAGACCTTTTTGAAGAAGGTTCTGTTACTAACATGTTTACTTCCATTGTAGGTAATGTATTTGGGTTCA
ATGTAGCTTACCCATTAGACCTTTTTGAAGAAGGTTCTGTTACTAACATGTTTACTTCCATTGTAGGTAATGTATTTGGGTTCA
AAGCCCTGCGCGCTCTACGTCTGGAAGATCTGCGAATCCCCGTTGCTTATGTTAAAACTTTCCAAGGACCGCCTCATGGCATC
AAGCCCTGCGCGCTCTACGTCTAGAAGATCTGCGAATCCCCGTTGCTTATGTTAAAACTTTCCAAGGACCGCCTCATGGCATC
CAAGTTGAGAGAGATAAATTGAACAAGTATGGTCGTCCCCTGTTGGGATGTACTATTAAACCTAAATTGGGGTTATCCGCTAA
CAAGTTGAGAGAGATAAATTGAACAAGTACGGTCGTCCCCTGTTGGGATGTACTATTAAACCTAAATTGGGGTTATCCGCTAA
AAACTACGGTAGAGCGGTTTATGAATGTCTCCGCGGTGGACTTGATTTTACCAAAGACGATGAGAATGTGAACTCCCAACCAT
AAACTACGGTAGAGCGGTTTATGAATGTCTCCGCGGTGGACTTGATTTTACCAAAGACGATGAGAATGTGAACTCCCAACCAT
TTATGCGTTGGAGAGATCGTTTCTTATTTTGTGCCGAAGCACTTTATAAAGCACAGGCTGAAACTGGTGAAATCAAAGGGCAT
TTATGCGTTGGAGAGATCGTTTCGTATTTTGTGCCGAAGCAATTTATAAAGCACAGGCTGAAACTGGTGAAATCAAAGGGCAT
TACTTGAAT
TACTTGAAT
182
Panax ginseng China
P8088 / 81
JF950028
Aligned with
Panax ginseng
NC_006290
ribulose-1,5-bisphosphate carboxylase/oxygenase large subunit (rbcL) gene
Araliaceae
AAAGATTACAAATTGACTTATTATACTCCTGACTATGATCCCAAAGATACTGATATCTTGGCAGCATTCCGAGTAACTCCTCA
AAAGATTACAAATTGACTTATTATACTCCTGACTATGATCCCAAAGATACTGATATCTTGGCAGCATTCCGAGTAACTCCTCA
ACCTGGAGTTCCAGCTGAAGAAGCAGGGGCCGCGGTAGCTGCCGAATCTTCTACTGGTACATGGACAACTGTGTGGACCGAT
ACCTGGAGTTCCAGCTGAAGAAGCAGGGGCCGCGGTAGCTGCCGAATCTTCTACTGGTACATGGACAACTGTGTGGACCGAT
GGACTTACTAGCCTTGATCGTTACAAAGGGCGATGCTACAAAATAGAGCCCGTTGCTGGAGAAGAAACTCAATTTATTGCTTA
GGACTTACTAGCCTTGATCGTTACAAAGGGCGATGCTACAAAATAGAGCCCGTTGCTGGAGAAGAAACTCAATTTATTGCTTA
TGTAGCTTACCCATTAGACCTTTTTGAAGAAGGTTCTGTTACTAACATGTTTACTTCCATTGTAGGTAATGTATTTGGGTTCAA
TGTAGCTTACCCATTAGACCTTTTTGAAGAAGGTTCTGTTACTAACATGTTTACTTCCATTGTAGGTAATGTATTTGGGTTCAA
AGCCCTGCGTGCTCTACGTCTGGAAGATCTGCGAATCCCTGTTGCTTATGTTAAAACTTTCCAAGGCCCGCCTCATGGCATCCA
AGCCCTGCGTGCTCTACGTCTGGAAGATCTGCGAATCCCTGTTGCTTATGTTAAAACTTTCCAAGGCCCGCCTCATGGCATCCA
AGTTGAGAGAGATAAATTGAACAAGTATGGTCGTCCCCTGTTGGGATGTACTATTAAACCTAAATTGGGGTTATCTGCTAAAA
AGTTGAGAGAGATAAATTGAACAAGTATGGTCGTCCCCTGTTGGGATGTACTATTAAACCTAAATTGGGGTTATCTGCTAAAA
ACTACGGTAGAGCGGTTTATGAATGTCTCCGGGGTGGACTTGATTTTACCAAAGACGATGAGAACGTGAACTCACAACCATTT
ACTACGGTAGAGCGGTTTATGAATGTCTCCGGGGTGGACTTGATTTTACCAAAGACGATGAGAACGTGAACTCACAACCATTT
ATGCGTTGGAGAGATCGTTTCTTATTTTGTGCCGAAGCACTTTATAAAGCACAGGCTGAAACAGGTGAAATCAAAGGGCATTA
ATGCGTTGGAGAGATCGTTTCTTATTTTGTGCCGAAGCACTTTATAAAGCACAGGCTGAAACAGGTGAAATCAAAGGGCATTA
CTTGAATGCTACTG
CTTGAATGCTACTG
Panax ginseng Korea
P8086 / 81
JF950029
Aligned with
Panax ginseng
NC_006290
ribulose-1,5-bisphosphate carboxylase/oxygenase large subunit (rbcL) gene
Araliaceae
AAAGATTACAAATTGACTTATTATACTCCTGACTATGATCCCAAAGATACTGATATCTTGGCAGCATTCCGAGTAACTCCTCA
AAAGATTACAAATTGACTTATTATACTCCTGACTATGATCCCAAAGATACTGATATCTTGGCAGCATTCCGAGTAACTCCTCA
ACCTGGAGTTCCAGCTGAAGAAGCAGGGGCCGCGGTAGCTGCCGAATCTTCTACTGGTACATGGACAACTGTGTGGACCGAT
ACCTGGAGTTCCAGCTGAAGAAGCAGGGGCCGCGGTAGCTGCCGAATCTTCTACTGGTACATGGACAACTGTGTGGACCGAT
GGACTTACTAGCCTTGATCGTTACAAAGGGCGATGCTACAAAATAGAGCCCGTTGCTGGAGAAGAAACTCAATTTATTGCTTA
GGACTTACTAGCCTTGATCGTTACAAAGGGCGATGCTACAAAATAGAGCCCGTTGCTGGAGAAGAAACTCAATTTATTGCTTA
TGTAGCTTACCCATTAGACCTTTTTGAAGAAGGTTCTGTTACTAACATGTTTACTTCCATTGTAGGTAATGTATTTGGGTTCAA
TGTAGCTTACCCATTAGACCTTTTTGAAGAAGGTTCTGTTACTAACATGTTTACTTCCATTGTAGGTAATGTATTTGGGTTCAA
AGCCCTGCGTGCTCTACGTCTGGAAGATCTGCGAATCCCTGTTGCTTATGTTAAAACTTTCCAAGGCCCGCCTCATGGCATCCA
AGCCCTGCGTGCTCTACGTCTGGAAGATCTGCGAATCCCTGTTGCTTATGTTAAAACTTTCCAAGGCCCGCCTCATGGCATCCA
AGTTGAGAGAGATAAATTGAACAAGTATGGTCGTCCCCTGTTGGGATGTACTATTAAACCTAAATTGGGGTTATCTGCTAAAA
AGTTGAGAGAGATAAATTGAACAAGTATGGTCGTCCCCTGTTGGGATGTACTATTAAACCTAAATTGGGGTTATCTGCTAAAA
ACTACGGTAGAGCGGTTTATGAATGTCTCCGGGGTGGACTTGATTTTACCAAAGACGATGAGAACGTGAACTCACAACCATTT
ACTACGGTAGAGCGGTTTATGAATGTCTCCGGGGTGGACTTGATTTTACCAAAGACGATGAGAACGTGAACTCACAACCATTT
ATGCGTTGGAGAGATCGTTTCTTATTTTGTGCCGAAGCACTTTATAAAGCACAGGCTGAAACAGGTGAAATCAAAGGGCATTA
ATGCGTTGGAGAGATCGTTTCTTATTTTGTGCCGAAGCACTTTATAAAGCACAGGCTGAAACAGGTGAAATCAAAGGGCATTA
CTTGAATGCTACTG
CTTGAATGCTACTG
Panax notoginseng
P6887 / 53
JF950030
Aligned with
Panax notoginseng
GQ436707
ribulose-1,5-bisphosphate carboxylase/oxygenase large subunit (rbcL) gene
Araliaceae
TAAAGATTACAAATTGACTTATTATACTCCTGACTATGACCCCAAAGATACTGATATCTTGGCAGCATTCCGAGTAACTCCTC
TAAAGATTACAAATTGACTTATTATACTCCTGACTATGACCCCAAAGATACTGATATCTTGGCAGCATTCCGAGTAACTCCTC
183
AACCTGGAGTTCCAGCTGAAGAAGCAGGGGCCGCGGTAGCTGCCGAATCTTCTACTGGTACATGGACAACTGTGTGGACCGA
AACCTGGAGTTCCAGCTGAAGAAGCAGGGGCCGCGGTAGCTGCCGAATCTTCTACTGGTACATGGACAACTGTGTGGACCGA
TGGACTTACCAGCCTTGATCGTTACAAAGGGCGATGCTACAAAATAGAGCCCGTTGCTGGAGAAGAAACTCAATTTATTGCTT
TGGACTTACCAGCCTTGATCGTTACAAAGGGCGATGCTACAAAATAGAGCCCGTTGCTGGAGAAGAAACTCAATTTATTGCTT
ATGTAGCTTACCCATTAGACCTTTTTGAAGAAGGTTCTGTTACTAACATGTTTACTTCCATTGTAGGTAATGTATTTGGGTTCA
ATGTAGCTTACCCATTAGACCTTTTTGAAGAAGGTTCTGTTACTAACATGTTTACTTCCATTGTAGGTAATGTATTTGGGTTCA
AAGCCCTGCGTGCTCTACGTCTGGAAGATCTGCGAATCCCTGTTGCTTATGTTAAAACTTTCCAAGGCCCGCCTCATGGCATCC
AAGCCCTGCGTGCTCTACGTCTGGAAGATCTGCGAATCCCTGTTGCTTATGTTAAAACTTTCCAAGGCCCGCCTCATGGCATCC
AAGTTGAGAGAGATAAATTGAACAAGTATGGTCGTCCCCTGTTGGGATGTACTATTAAACCTAAATTGGGGTTATCTGCTAAA
AAGTTGAGAGAGATAAATTGAACAAGTATGGTCGTCCCCTGTTGGGATGTACTATTAAACCTAAATTGGGGTTATCTGCTAAA
AACTACGGTAGGGCGGTTTATGAATGTCTCCGTGGTGGACTTGATTTTACCAAAGACGATGAGAACGTGAACTCACAACCATT
AACTACGGTAGGGCGGTTTATGAATGTCTCCGTGGTGGACTTGATTTTACCAAAGACGATGAGAACGTGAACTCACAACCATT
TATGCGTTGGAGAGATCGTTTCTTATTTTGTGCCGAAGCACTTTATAAAGCACAGGCTGAAACAGGTGAAATCAAAGGGCATT
TATGCGTTGGAGAGATCGTTTCTTATTTTGTGCCGAAGCACTTTATAAAGCACAGGCTGAAACAGGTGAAATCAAAGGGCATT
ACTTGAAT
ACTTGAAT
Panax pseudoschinseng
P6887 / 53
JF950030
Aligned with
Panax ginseng
NC_006290
ribulose-1,5-bisphosphate carboxylase/oxygenase large subunit (rbcL) gene
Araliaceae
TTAAGATTACAAATTGACTTATTATACTCCTGACTATGACCCCAAAGATACTGATACCTTGGCAGCATTCCGAGTAACTCCTC
TAAAGATTACAAATTGACTTATTATACTCCTGACTATGATCCCAAAGATACTGATATCTTGGCAGCATTCCGAGTAACTCCTC
AACCTGGAGTTCCACCTGAAGAAGCAGGGGCCGCGGTAGCTGCCGAATCTTCTACTGGTACATGGACAACTGTGTGGACCGA
AACCTGGAGTTCCAGCTGAAGAAGCAGGGGCCGCGGTAGCTGCCGAATCTTCTACTGGTACATGGACAACTGTGTGGACCGA
TGGACTTACCAGCCTTGATCGTTACAAAGGGCGATGCTACAAAATCGAGCCCGTTGCTGGAGAAGAAACTCAATTTATTGCTT
TGGACTTACTAGCCTTGATCGTTACAAAGGGCGATGCTACAAAATAGAGCCCGTTGCTGGAGAAGAAACTCAATTTATTGCTT
ATGTAGCTTACCCATTAGACCTTTTTGAAGAAGGTTCTGTTACTAACATGTTTACTTCCATTGTAGGTAATGTATTTGGGTTCA
ATGTAGCTTACCCATTAGACCTTTTTGAAGAAGGTTCTGTTACTAACATGTTTACTTCCATTGTAGGTAATGTATTTGGGTTCA
AAGCCCTGCGTGCTCTACGTCTGGAAGATCTGCGAATCCCTGTTGCTTATGTTAAAACTTTCCAAGGCCCGCCTCATGGCATCC
AAGCCCTGCGTGCTCTACGTCTGGAAGATCTGCGAATCCCTGTTGCTTATGTTAAAACTTTCCAAGGCCCGCCTCATGGCATCC
AAGTTGAGAGAGATAAATTGAACAAGTATGGTCGTCCCCTGTTGGGATGTACTATTAAACCTAAATTGGGGTTATCTGCTAAA
AAGTTGAGAGAGATAAATTGAACAAGTATGGTCGTCCCCTGTTGGGATGTACTATTAAACCTAAATTGGGGTTATCTGCTAAA
AACTACGGTAGAGCGGTTTATGAATGTCTCCGTGGTGGACTTGATTTTACCAAAGACGATGAGAACGTGAACTCCCAACCAT
AACTACGGTAGAGCGGTTTATGAATGTCTCCGGGGTGGACTTGATTTTACCAAAGACGATGAGAACGTGAACTCACAACCAT
TTATGCGTTGGAGAGACCGTTTCTTATTTT
TTATGCGTTGGAGAGATCGTTTCTTATTTT
Cynanchum paniculatum
P6858 / 24
JF949975
Aligned with
Cynanchum mongolicum
GQ436512
ribulose-1,5-bisphosphate carboxylase/oxygenase large subunit (rbcL) gene
Asclepiadaceae
GATATCTTGGCAGCATTCCGAGTAACTCCTCAACCCGGAGTTCCACCTGAAGAAGCAGGGGCCGCGGTAGCTGCCGAATCTTC
GATATCTTGGCAGCATTCCGAGTAACTCCTCAACCCGGAGTTCCACCTGAAGAAGCAGGGGCCGCGGTAGCTGCCGAATCTTC
TACTGGTACATGGACAACTGTTTGGACCGATGGACTTACCAGCCTTGATCGTTACAAAGGGCGATGCTACCACATCGAGGCCG
TACTGGTACATGGACAACTGTTTGGACCGATGGACTTACCAGCCTTGATCGTTACAAAGGGCGATGCTACCACATCGAGCCCG
TTCCTGGAGAAGAAGATCAATTTATTGCTTATGTAGCTTACCCTTTAGACCTTTTTGAAGAAGGTTCTGTTACTAACATGCTTA
TTCCTGGAGAAGAAGATCAATTTATTGCTTATGTAGCTTACCCTTTAGACCTTTTTGAAGAAGGTTCTGTTACTAACATGCTTA
CTTCCATTGTAGGTAATGTATTTGGGTTCAAAGCCCTACGCGCTCTACGTCTGGAAGATTTGCGAATCCCTCCGGCTTATATTA
CTTCCATTGTAGGTAATGTATTTGGGTTCAAAGCCCTACGCGCTCTACGTCTGGAAGATTTGCGAATCCCTCAGGCTTATGTTA
AAACCTTCCAAGGCCCGCCGCATGGCATCCAGGTTGAGAGAGATAAATTGAACAAATATGGTCGTCCCCTGTTGGGATGTAC
AAACCTTCCAAGGCCCGCCACATGGCATCCAGGTTGAGAGAGATAAATTGAACAAATATGGTCGTCCCCTGTTGGGATGTAC
184
TATTAAACCAAAATTGGGGTTATCAGCTAAAAACTATGGTAGGGCGGTTTATGAATGTCTTCGTGGTGGACTTGATTTTACCA
TATTAAACCAAAATTGGGGTTATCAGCTAAAAACTATGGTAGGGCGGTTTATGAATGTCTTCGTGGTGGACTTGATTTTACCA
AAGATGATGAAAACGTGAACTCCCAACCGTTTATGCGTTGGAGAGATCGTTTCTTGTTTTGTGCCGAAGCAATTTTTAAATCA
AAGATGATGAAAACGTGAACTCCCAACCGTTTATGCGTTGGAGAGATCGTTTCTTGTTTTGTGCCGAAGCAATTTTTAAATCA
CAGGCTGAAACCGGCGAAATCAAAGGGCATTACTTGAAT
CAGGCTGAAACCGGCGAAATCAAAGGGCATTACTTGAAT
Artemisia annua
P6841 / 07
JF949966
Aligned with
Artemisia annua
DQ006057
ribulose-1,5-bisphosphate carboxylase/oxygenase large subunit (rbcL) gene
Asteraceae
GGGGTTAAAGATTATAAATTGACTTATTATACTCCTGAGTATGAAACCAAGGATACTGATATCTTGGCAGCATTTCGAGTAAC
GGGGTTAAAGATTATAAATTGACTTATTATACTCCTGAGTATGAAACCAAGGATACTGATATCTTGGCAGCATTTCGAGTAAC
TCCTCAACCTGGAGTTCCGCCTGAAGAAGCAGGGGCCGCAGTAGCTGCCGAATCTTCTACTGGTACATGGACAACTGTGTGG
TCCTCAACCTGGAGTTCCGCCTGAAGAAGCAGGGGCCGCAGTAGCTGCCGAATCTTCTACTGGTACATGGACAACTGTGTGG
ACCGATGGACTTACGAGCCTTGATCGTTACAAAGGGCGATGCTATGGAATTGAGCCTGTTCCTGGAGAAGAGAATCAATATA
ACCGATGGACTTACGAGCCTTGATCGTTACAAAGGGCGATGCTATGGAATTGAGCCTGTTCCTGGAGAAGAGAATCAATATA
TTTGCTATGTAGCTTACCCATTAGACCTTTTTGAAGAAGGTTCTGTTACTAACATGTTTACTTCCATTGTAGGTAACGTATTTG
TTTGCTATGTAGCTTACCCATTAGACCTTTTTGAAGAAGGTTCTGTTACTAACATGTTTACTTCCATTGTAGGTAACGTATTTG
GTTTCAAAGCCCTGCGTGCTCTACGTCTGGAAGATTTGCGAATTCCTACTGCGTATGTTAAAACTTTCCAAGGTCCGCCTCACG
GTTTCAAAGCCCTGCGTGCTCTACGTCTGGAAGATTTGCGAATTCCTACTGCGTATGTTAAAACTTTCCAAGGTCCGCCTCACG
GTATCCAAGTTGAAAGAGATAAATTGAACAAGTATGGTCGTCCTCTGTTGGGATGTACTATTAAACCTAAATTGGGGTTATCT
GTATCCAAGTTGAAAGAGATAAATTGAACAAGTATGGTCGTCCTCTGTTGGGATGTACTATTAAACCTAAATTGGGGTTATCT
GCTAAAAACTACGGTAGAGCTGTTTATGAATGTCTTCGTGGTGGCCTTGATTTTACTAAAGATGATGAGAACGTGAACTCCCA
GCTAAAAACTACGGTAGAGCTGTTTATGAATGTCTTCGTGGTGGCCTTGATTTTACTAAAGATGATGAGAACGTGAACTCCCA
ACCATTTATGCGTTGGAGAGACCGTTTCTTATTTTGTGCCGAAGCTATTTTTAAATCACAAGCTGAAACAGGTGAAATCAAAG
ACCATTTATGCGTTGGAGAGACCGTTTCTTATTTTGTGCCGAAGCTATTTTTAAATCACAAGCTGAAACAGGTGAAATCAAAG
Artemisia capillaris
P6842 / 08
JF949967
Aligned with
Artemisia annua
DQ006057
ribulose-1,5-bisphosphate carboxylase/oxygenase large subunit (rbcL) gene
Asteraceae
TGGGGTTAAAGATTATAAATTGACTTATTATACTCCTGAGTATGAAACCAAGGATACTGATATCTTGGCAGCATTTCGAGTAA
TGGGGTTAAAGATTATAAATTGACTTATTATACTCCTGAGTATGAAACCAAGGATACTGATATCTTGGCAGCATTTCGAGTAA
CTCCTCAACCTGGAGTTCCGCCTGAAGAAGCAGGGGCCGCAGTAGCTGCCGAATCTTCTACTGGTACATGGACAACTGTGTGG
CTCCTCAACCTGGAGTTCCGCCTGAAGAAGCAGGGGCCGCAGTAGCTGCCGAATCTTCTACTGGTACATGGACAACTGTGTGG
ACCGATGGACTTACGAGCCTTGATCGTTACAAAGGGCGATGCTATGGAATTGAGCCTGTTCCTGGAGAAGAGAATCAATATA
ACCGATGGACTTACGAGCCTTGATCGTTACAAAGGGCGATGCTATGGAATTGAGCCTGTTCCTGGAGAAGAGAATCAATATA
TTTGCTATGTAGCTTACCCATTAGACCTTTTTGAAGAAGGTTCTGTTACTAACATGTTTACTTCCATTGTAGGTAACGTATTTG
TTTGCTATGTAGCTTACCCATTAGACCTTTTTGAAGAAGGTTCTGTTACTAACATGTTTACTTCCATTGTAGGTAACGTATTTG
GTTTCAAAGCCCTGCGTGCTCTACGTCTGGAAGATTTGCGAATTCCTACTGCGTATGTTAAAACTTTCCAAGGTCCGCCTCACG
GTTTCAAAGCCCTGCGTGCTCTACGTCTGGAAGATTTGCGAATTCCTACTGCGTATGTTAAAACTTTCCAAGGTCCGCCTCACG
GTATCCAAGTTGAAAGAGATAAATTGAACAAGTATGGTCGTCCTCTGTTGGGATGTACTATTAAACCTAAATTGGGGTTATCC
GTATCCAAGTTGAAAGAGATAAATTGAACAAGTATGGTCGTCCTCTGTTGGGATGTACTATTAAACCTAAATTGGGGTTATCT
GCTAAAAACTACGGTAGAGCTGTTTATGAATGTCTTCGTGGTGGCCTTGATTTTACTAAAGATGATGAGAACGTGAACTCCCA
GCTAAAAACTACGGTAGAGCTGTTTATGAATGTCTTCGTGGTGGCCTTGATTTTACTAAAGATGATGAGAACGTGAACTCCCA
ACCATTTATGCGTTGGAGAGACCGTTTCTTATTTTGTGCCGAAGCTATTTTTAAATCACAAGCTGAAACAGGTGAAATCAAAG
ACCATTTATGCGTTGGAGAGACCGTTTCTTATTTTGTGCCGAAGCTATTTTTAAATCACAAGCTGAAACAGGTGAAATCAAAG
GGCA
GGCA
185
Arctium lappa
P6839 / 05
JF949994
Aligned with
Arctium lappa
GU724233
ribulose-1,5-bisphosphate carboxylase/oxygenase large subunit (rbcL) gene
Asteraceae
GGTGTTAAAGATTATAAATTGACTTATTATACTCCTGACTATCAAACCAAGGATACTGATATCTTGGCAGCATTTCGAGTAAC
GGTGTTAAAGATTATAAATTGACTTATTATACTCCTGACTATCAAACCAAGGATACTGATATCTTGGCAGCATTTCGAGTAAC
TCCTCAACCTGGAGTTCCGCCTGAAGAAGCAGGGGCCGCAGTAGCTGCCGAATCTTCTACTGGTACATGGACAACTGTGTGG
TCCTCAACCTGGAGTTCCGCCTGAAGAAGCAGGGGCCGCAGTAGCTGCCGAATCTTCTACTGGTACATGGACAACTGTGTGG
ACCGATGGACTTACGAGCCTTGATCGTTACAAGGGGCGATGCTATGGAATCGAGCCTGTTCCTGGAGAAGAAAGTCAATTTA
ACCGATGGACTTACGAGCCTTGATCGTTACAAGGGGCGATGCTATGGAATCGAGCCTGTTCCTGGAGAAGAAAGTCAATTTA
TTGCTTATGTAGCTTACCCATTAGACCTTTTTGAAGAAGGTTCTGTTACTAACATGTTTACTTCCATTGTAGGTAATGTATTTGG
TTGCTTATGTAGCTTACCCATTAGACCTTTTTGAAGAAGGTTCTGTTACTAACATGTTTACTTCCATTGTAGGTAATGTATTTGG
GTTCAAAGCCCTGCGTGCTCTACGTCTGGAAGATTTGCGAATCCCTACTGCGTATGTTAAAACTTTCCAAGGTCCGCCTCACG
GTTCAAAGCCCTGCGTGCTCTACGTCTGGAAGATTTGCGAATCCCTACTGCGTATGTTAAAACTTTCCAAGGTCCGCCTCACG
GCATCCAAGTTGAGAGAGATAAATTGAACAAGTATGGTCGTCCCCTGTTGGGATGTACTATTAAACCTAAATTGGGGTTATC
GCATCCAAGTTGAGAGAGATAAATTGAACAAGTATGGTCGTCCCCTGTTGGGATGTACTATTAAACCTAAATTGGGGTTATC
CGCTAAAAACTACGGTAGAGCTGTTTATGAATGTCTTCGTGGTGGCCTTGATTTTACTAAAGATGATGAGAACGTGAACTCCC
CGCTAAAAACTACGGTAGAGCTGTTTATGAATGTCTTCGTGGTGGCCTTGATTTTACTAAAGATGATGAGAACGTGAACTCCC
AACCATTTATGCGTTGGAGAGACCGTTTCTTATTTTGTGCCGAAGCTATTTATAAAGCACAAGCTGAAACAGGTGAAATCAAA
AACCATTTATGCGTTGGAGAGACCGTTTCTTATTTTGTGCCGAAGCTATTTATAAAGCACAAGCTGAAACAGGTGAAATCAAA
GGGCATTACTTGAAT
GGGCATTACTTGAAT
Chrysanthemum indicum
P6851 / 17
Aligned with
Chrysanthemum lavandulifolium
ribulose-1,5-bisphosphate carboxylase/oxygenase large subunit (rbcL) gene
Asteraceae
JF949971
GQ436485
TATAAATTGACTTATTATACTCCTGAGTATGAAACCAAGGATACTGATATCTTGGCAGCATTTCGAGTAACTCCTCAACCTGG
TATAAATTGACTTATTATACTCCTGAGTATGAAACCAAGGATACTGATATCTTGGCAGCATTTCGAGTAACTCCTCAACCTGG
AGTTCCGCCTGAAGAAGCAGGGGCCGCGGTAGCTGCCGAATCTTCTACTGGTACCTGGACAACTGTGTGGACCGATGGACTT
AGTTCCGCCTGAAGAAGCAGGGGCCGCAGTAGCTGCCGAATCTTCTACTGGTACATGGACAACTGTGTGGACCGATGGACTT
ACGAGCCTTGATCGTTACAAAGGGCGATGCTATGGAATTGAGCCTGTTCTTGGAGAAGAGAATCAATATATTTGCTATGTAGC
ACGAGCCTTGATCGTTACAAAGGGCGATGCTATGGAATTGAGCCTGTTCTTGGAGAAGAGAATCAATATATTTGCTATGTAGC
TTACCCATTAGACCTTTTTGAAGAAGGTTCTGTTACTAACATGTTTACTTCCATTGTAGGTAACGTATTTGGTTTCAAAGCCCT
TTACCCATTAGACCTTTTTGAAGAAGGTTCTGTTACTAACATGTTTACTTCCATTGTAGGTAACGTATTTGGTTTCAAAGCCCT
GCGTGCTCTACGTCTGGAAGATTTGCGAATTCCTACCGCGTATGTTAAAACTTTCCAAGGTCCGCCTCACGGTATCCAAGTTG
GCGTGCTCTACGTCTGGAAGATTTGCGAATTCCTACTGCGTATGTTAAAACTTTCCAAGGTCCGCCTCACGGTATCCAAGTTG
AAAGAGATAAATTGAACAAGTATGGTCGTCCTCTGTTGGGATGTACTATTAAACCTAAATTGGGGTTATCCGCTAAAAACTAC
AAAGAGATAAATTGAACAAGTATGGTCGTCCTCTGTTGGGATGTACTATTAAACCTAAATTGGGGTTATCCGCTAAAAACTAC
GGTAGAGCTGTTTATGAATGTCTTCGTGGTGGCCTTGATTTTACTAAAGATGATGAAAACGTGAACTCCCAACCGTTTATG
GGTAGAGCTGTTTATGAATGTCTTCGTGGTGGCCTTGATTTTACTAAAGATGATGAGAACGTGAACTCCCAACCATTTATG
Chrysanthemum morifolium
P6852 / 18
Aligned with
Chrysanthemum lavandulifolium
ribulose-1,5-bisphosphate carboxylase/oxygenase large subunit (rbcL) gene
Asteraceae
JF949972
GQ436485
TATAAATTGACTTATTATACTCCTGAGTATGAAACCAAGGATACTGATATCTTGGCAGCATTTCGAGTAACTCCTCAACCTGG
TATAAATTGACTTATTATACTCCTGAGTATGAAACCAAGGATACTGATATCTTGGCAGCATTTCGAGTAACTCCTCAACCTGG
AGTTCCGCCTGAAGAAGCAGGGGCCGCAGTAGCTGCCGAATCTTCTACTGGTACATGGACAACTGTGTGGACCGATGGACTT
AGTTCCGCCTGAAGAAGCAGGGGCCGCAGTAGCTGCCGAATCTTCTACTGGTACATGGACAACTGTGTGGACCGATGGACTT
ACGAGCCTTGATCGTTACAAAGGGCGATGCTATGGAATTGAGCCTGTTCTTGGAGAAGAGAATCAATATATTTGCTATGTAGC
ACGAGCCTTGATCGTTACAAAGGGCGATGCTATGGAATTGAGCCTGTTCTTGGAGAAGAGAATCAATATATTTGCTATGTAGC
186
TTACCCATTAGACCTTTTTGAAGAAGGTTCTGTTACTAACATGTTTACTTCCATTGTAGGTAACGTATTTGGTTTCAAAGCCCT
TTACCCATTAGACCTTTTTGAAGAAGGTTCTGTTACTAACATGTTTACTTCCATTGTAGGTAACGTATTTGGTTTCAAAGCCCT
GCGTGCTCTACGTCTGGAAGATTTGCGAATTCCTACTGCGTATGTTAAAACTTTCCAAGGTCCGCCTCACGGTATCCAAGTTG
GCGTGCTCTACGTCTGGAAGATTTGCGAATTCCTACTGCGTATGTTAAAACTTTCCAAGGTCCGCCTCACGGTATCCAAGTTG
AAAGAGATAAATTGAACAAGTATGGTCGTCCTCTGTTGGGATGTACTATTAAACCTAAATTGGGGTTATCCGCTAAAAACTAC
AAAGAGATAAATTGAACAAGTATGGTCGTCCTCTGTTGGGATGTACTATTAAACCTAAATTGGGGTTATCCGCTAAAAACTAC
GGTAGAGCTGTTTATGAATGTCTTCGTGGTGGCCTTGATTTTACTAAAGATGATGAGAACGTGAACTCCCAACCATTTATG
GGTAGAGCTGTTTATGAATGTCTTCGTGGTGGCCTTGATTTTACTAAAGATGATGAGAACGTGAACTCCCAACCATTTATG
Eclipta prostrata
P6863 / 29
JF950000
Aligned with
Eclipta prostrata
GQ436455
ribulose-1,5-bisphosphate carboxylase/oxygenase large subunit (rbcL) gene
Asteraceae
GGTGTTAAAGATTATAAATTGACTTATTATACTCCTGAATATGAAACCAAGGATACTGATATCTTGGCAGCATTTCGAGTAAC
GGTGTTAAAGATTATAAATTGACTTATTATACTCCTGAATATGAAACCAAGGATACTGATATCTTGGCAGCATTTCGAGTAAC
TCCTCAACCTGGAGTTCCGCCTGAAGAAGCAGGGGCCGCAGTAGCTGCCGAATCTTCTACTGGTACATGGACAACTGTATGG
TCCTCAACCTGGAGTTCCGCCTGAAGAAGCAGGGGCCGCAGTAGCTGCCGAATCTTCTACTGGTACATGGACAACTGTATGG
ACCGATGGACTTACGAGCCTTGATCGTTACAAAGGCCGATGCTATGGAATCGAGCCTGTTCCTGGAGAAGAAAATCAATATA
ACCGATGGACTTACGAGCCTTGATCGTTACAAAGGCCGATGCTATGGAATCGAGCCTGTTCCTGGAGAAGAAAATCAATATA
TTGCTTATGTAGCTTACCCATTAGACCTTTTTGAAGAAGGTTCTGTTACTAACATGTTTACTTCCATTGTAGGTAATGTATTTGG
TTGCTTATGTAGCTTACCCATTAGACCTTTTTGAAGAAGGTTCTGTTACTAACATGTTTACTTCCATTGTAGGTAATGTATTTGG
GTTCAAAGCCCTGCGTGCTCTACGTCTGGAAGATTTGCGAATCCCTACTGCGTATATTAAAACTTTCGAGGGTCCGCCTCACG
GTTCAAAGCCCTGCGTGCTCTACGTCTGGAAGATTTGCGAATCCCTACTGCGTATATTAAAACTTTCGAGGGTCCGCCTCACG
GTATCCAAGTTGAGAGAGATAAATTGAACAAGTATGGTCGTCCTCTGTTGGGATGTACTATTAAACCTAAATTGGGGTTATCC
GTATCCAAGTTGAGAGAGATAAATTGAACAAGTATGGTCGTCCTCTGTTGGGATGTACTATTAAACCTAAATTGGGGTTATCC
GCTAAAAACTACGGTAGAGCTTGTTATGAATGTCTTCGTGGTGGCCTTGATTTTACTAAAGATGATGAGAACGTGAACTCCCA
GCTAAAAACTACGGTAGAGCTTGTTATGAATGTCTTCGTGGTGGCCTTGATTTTACTAAAGATGATGAGAACGTGAACTCCCA
ACCATTTATGCGTTGGAGAGACCGTTTCGTATTTTGTGCCGAAGCTATTTATAAAGCACAAGCTGAAACAGGTGAAATCAAAG
ACCATTTATGCGTTGGAGAGACCGTTTCGTATTTTGTGCCGAAGCTATTTATAAAGCACAAGCTGAAACAGGTGAAATCAAAG
Senecio scandens
P6905 / 71
JF949989
Aligned with
Senecio scandens
GQ436472
ribulose-1,5-bisphosphate carboxylase/oxygenase large subunit (rbcL) gene
Asteraceae
GGTGTTAAAGATTATAAATTGACTTATTATACTCCTGATTATGAAACCAAGGATACTGATATCTTGGCAGCATTTCGAGTAAC
GGTGTTAAAGATTATAAATTGACTTATTATACTCCTGATTATGAAACCAAGGATACTGATATCTTGGCAGCATTTCGAGTAAC
TCCTCAACCAGGAGTTCCGCCTGAAGAAGCAGGGGCCGCAGTAGCTGCCGAATCTTCTACTGGTACATGGACAACTGTGTGG
TCCTCAACCAGGAGTTCCGCCTGAAGAAGCAGGGGCCGCAGTAGCTGCCGAATCTTCTACTGGTACATGGACAACTGTGTGG
ACCGATGGACTTACGAGCCTTGATCGTTACAAAGGGCGATGCTATGGAATCGAGCCTGTTCTTGGAGAAGAAAATCAATTTAT
ACCGATGGACTTACGAGCCTTGATCGTTACAAAGGGCGATGCTATGGAATCGAGCCTGTTCTTGGAGAAGAAAATCAATTTAT
TGCTTATGTAGCTTACCCATTAGACCTTTTTGAAGAAGGTTCTGTTACTAACATGTTTACTTCCATTGTAGGTAATGTATTTGG
TGCTTATGTAGCTTACCCATTAGACCTTTTTGAAGAAGGTTCTGTTACTAACATGTTTACTTCCATTGTAGGTAATGTATTTGG
GTTCAAAGCCCTGCGTGCTCTACGTCTGGAAGATTTGCGAATCCCTACTGCGTATGTTAAAACTTTCCAAGGTCCGCCTCACG
GTTCAAAGCCCTGCGTGCTCTACGTCTGGAAGATTTGCGAATCCCTACTGCGTATGTTAAAACTTTCCAAGGTCCGCCTCACG
GCATCCAAGTTGAGAGAGATAAATTGAACAAGTATGGTCGTCCTCTATTGGGATGTACTATTAAACCTAAATTGGGGCTATCC
GCATCCAAGTTGAGAGAGATAAATTGAACAAGTATGGTCGTCCTCTATTGGGATGTACTATTAAACCTAAATTGGGGCTATCC
GCTAAAAACTACGGTAGAGCTGTTTATGAGTGTCTTCGTGGTGGCCTTGATTTTACTAAAGATGATGAAAACGTGAACTCCCA
GCTAAAAACTACGGTAGAGCTGTTTATGAGTGTCTTCGTGGTGGCCTTGATTTTACTAAAGATGATGAAAACGTGAACTCCCA
ACCATTTATGCGTTGGAGAGACCGTTTCTTATTTTGTGCCGAAGCTATTTATAAATCACAAGCTGAAACAGGTGAAATCAAAG
ACCATTTATGCGTTGGAGAGACCGTTTCTTATTTTGTGCCGAAGCTATTTATAAATCACAAGCTGAAACAGGTGAAATCAAAG
GGCATTACTTGAAT
GGCATTACTTGAAT
187
Siegesbeckia orientalis
P6906 / 72
JF949990
Aligned with
Siegesbeckia orientalis
GQ436447
ribulose-1,5-bisphosphate carboxylase/oxygenase large subunit (rbcL) gene
Asteraceae
GGTGTTAAAGATTATAAATTGACTTATTATACTCCTGAATATGAAACCAAGGATACTGATATCTTGGCAGCATTTCGAGTAAC
GGTGTTAAAGATTATAAATTGACTTATTATACTCCTGAATATGAAACCAAGGATACTGATATCTTGGCAGCATTTCGAGTAAC
TCCTCAACCTGGAGTTCCGCCTGAAGAAGCAGGGGCCGCAGTAGCTGCCGAATCTTCTACTGGTACATGGACAACTGTATGG
TCCTCAACCTGGAGTTCCGCCTGAAGAAGCAGGGGCCGCAGTAGCTGCCGAATCTTCTACTGGTACATGGACAACTGTATGG
ACCGATGGACTTACGAGCCTTGATCGTTACAAAGGCCGATGCTATGGAATCGAGCCTGTTCCTGGAGAAGAAAATCAATTTAT
ACCGATGGACTTACGAGCCTTGATCGTTACAAAGGCCGATGCTATGGAATCGAGCCTGTTCCTGGAGAAGAAAATCAATTTAT
TGCTTATGTAGCTTACCCATTAGACCTTTTTGAAGAAGGTTCTGTTACTAACATGTTTACTTCCATTGTAGGTAATGTATTTGG
TGCTTATGTAGCTTACCCATTAGACCTTTTTGAAGAAGGTTCTGTTACTAACATGTTTACTTCCATTGTAGGTAATGTATTTGG
GTTCAAAGCCCTGCGTGCTCTACGTCTGGAAGATTTGCGAATCCCTACTGCGTATGTTAAAACTTTCGATGGTCCGCCTCACG
GTTCAAAGCCCTGCGTGCTCTACGTCTGGAAGATTTGCGAATCCCTACTGCGTATGTTAAAACTTTCGATGGTCCGCCTCACG
GTATCCAAGTTGAGAGAGATAAATTGAACAAGTATGGTCGTCCCCTGTTGGGATGTACTATTAAACCTAAATTGGGGTTATCC
GTATCCAAGTTGAGAGAGATAAATTGAACAAGTATGGTCGTCCCCTGTTGGGATGTACTATTAAACCTAAATTGGGGTTATCC
GCTAAAAACTACGGTAGAGCTTGTTATGAATGTCTTCGTGGTGGCCTTGATTTTACTAAAGATGATGAGAACGTGAACTCCCA
GCTAAAAACTACGGTAGAGCTTGTTATGAATGTCTTCGTGGTGGCCTTGATTTTACTAAAGATGATGAGAACGTGAACTCCCA
ACCATTTATGCGTTGGAGAGACCGTTTCTTATTTTGTGCCGAAGCTATTTATAAAGCACAAGCTGAAACAGGTGAAATCAAAG
ACCATTTATGCGTTGGAGAGACCGTTTCTTATTTTGTGCCGAAGCTATTTATAAAGCACAAGCTGAAACAGGTGAAATCAAAG
GGCATTACTTGAAT
GGCATTACTTGAAT
Taraxacum officinale
P6908 / 74
JF950019
Aligned with
Taraxacum officinale
AY395562
ribulose-1,5-bisphosphate carboxylase/oxygenase large subunit (rbcL) gene
Asteraceae
GGTGTTAAAGATTATAAATTGACTTATTATACTCCTGACTATGAAACCAAGGATACTGATATTTTGGCAGCATTTCGAGTAAC
GGTGTTAAAGATTATAAATTGACTTATTATACTCCTGACTATGAAACCAAGGATACTGATATTTTGGCAGCATTTCGAGTAAC
TCCTCAACCTGGAGTTCCGCCTGAAGAAGCAGGGGCCGCAGTAGCTGCCGAATCTTCTACTGGTACATGGACAACTGTGTGG
TCCTCAACCTGGAGTTCCGCCTGAAGAAGCAGGGGCCGCAGTAGCTGCCGAATCTTCTACTGGTACATGGACAACTGTGTGG
ACCGATGGACTTACGAGCCTTGATCGTTACAAAGGGCGATGCTATGGAATTGAGCCTGTTCCTGGAGAAGAAAGTCAATTTAT
ACCGATGGACTTACGAGCCTTGATCGTTACAAAGGGCGATGCTATGGAATTGAGCCTGTTCCTGGAGAAGAAAGTCAATTTAT
TGCTTATGTAGCTTACCCATTAGACCTTTTTGAAGAAGGTTCTGTTACTAACATGTTTACTTCCATTGTAGGTAATGTATTTGG
TGCTTATGTAGCTTACCCATTAGACCTTTTTGAAGAAGGTTCTGTTACTAACATGTTTACTTCCATTGTAGGTAATGTATTTGG
GTTCAAAGCCCTGCGTGCTCTACGTCTGGAAGATTTGCGAATCCCTGTTGCGTATGTTAAAACTTTCCAAGGTCCGCCTCACG
GTTCAAAGCCCTGCGTGCTCTACGTCTGGAAGATTTGCGAATCCCTGTTGCGTATGTTAAAACTTTCCAAGGTCCGCCTCACG
GCATCCAAGTTGAGAGAGATAAATTGAACAAGTATGGTCGTCCTCTGTTGGGATGTACTATTAAACCTAAATTGGGGTTATCC
GCATCCAAGTTGAGAGAGATAAATTGAACAAGTATGGTCGTCCTCTGTTGGGATGTACTATTAAACCTAAATTGGGGTTATCC
GCTAAAAACTACGGTAGAGCTGTTTATGAATGTCTTCGTGGTGGCCTTGATTTT
GCTAAAAACTACGGTAGAGCTGTTTATGAATGTCTTCGTGGTGGCCTTGATTTT
Berberis bealei
P6883 / 49
JF949996
Aligned with
Berberis bealei
FJ449858
ribulose-1,5-bisphosphate carboxylase/oxygenase large subunit (rbcL) gene
Berberidaceae
AATTATTATACTCCTGACTATGTAACCAAAGATACTGATATCTTGGCAGCATTCCGAGTAACTCCTCAACCCGGAGTTCCACC
AATTATTATACTCCTGACTATGTAACCAAAGATACTGATATCTTGGCAGCATTCCGAGTAACTCCTCAACCCGGAGTTCCACC
GGAAGAAGCAGGGGCCGCTGTAGCTGCCGAATCTTCTACGGGTACATGGACAACTGTGTGGACCGATGGACTTACCAGTCTT
GGAAGAAGCAGGGGCCGCTGTAGCTGCCGAATCTTCTACGGGTACATGGACAACTGTGTGGACCGATGGACTTACCAGTCTT
GATCGTTACAAAGGACGATGCTACGACATTGAGCCCGTTGCTGGAGAAGACAATCAATATATTTGTTATGTAGCCTATCCTTT
GATCGTTACAAAGGACGATGCTACGACATTGAGCCCGTTGCTGGAGAAGACAATCAATATATTTGTTATGTAGCCTATCCTTT
188
AGACCTTTTTGAAGAAGGTTCTGTTACTAACATGTTGACCTCTATTGTGGGTAATGTTTTTGGGTTCAAAGCGCTGCGCGCTCT
AGACCTTTTTGAAGAAGGTTCTGTTACTAACATGTTGACCTCTATTGTGGGTAATGTTTTTGGGTTCAAAGCGCTGCGCGCTCT
ACGTCTGGAGGATCTGCGAATTCCTACTGCTTATGTTAAAACTTTCCAAGGCCCGCCTCATGGCATCCAAGTTGAGAGAGATA
ACGTCTGGAGGATCTGCGAATTCCTACTGCTTATGTTAAAACTTTCCAAGGCCCGCCTCATGGCATCCAAGTTGAGAGAGATA
AATTGAACAAGTATGGTCGTCCCCTATTAGGATGTACTATTAAACCAAAATTGGGATTATCCGCTAAGAACTATGGTAGAGCG
AATTGAACAAGTATGGTCGTCCCCTATTAGGATGTACTATTAAACCAAAATTGGGATTATCCGCTAAGAACTATGGTAGAGCG
GTTTATGAATGTCTCCGTGGTGGACTTGATTTTACCAAAGATGATGAGAACGTGAACTCCCAACCATTTATGCGTTGGCGCGA
GTTTATGAATGTCTCCGTGGTGGACTTGATTTTACCAAAGATGATGAGAACGTGAACTCCCAACCATTTATGCGTTGGCGCGA
CCGTTTCCTATTTTGTGCCGAAGCACTTTTTAAAGCACAGGCTGAAACAGGTGAAATCAAAGGACATTACTTAAATGCTACT
CCGTTTCCTATTTTGTGCCGAAGCACTTTTTAAAGCACAGGCTGAAACAGGTGAAATCAAAGGACATTACTTAAATGCTACT
Epimedium koreanum
P6865 / 31
JF950002
Aligned with
Epimedium koreanum
L75869
ribulose-1,5-bisphosphate carboxylase/oxygenase large subunit (rbcL) gene
Berberidaceae
TACAAATTGACTTATTATACTCCTGACTATGTAACGAAGGATACTGATATCTTGGCAGCATTCCGCGTCACTCCTCAACCTGG
TACAAATTGACTTATTATACTCCTGACTATGTAACGAAGGATACTGATATCTTGGCAGCATTCCGCGTCACTCCTCAACCTGG
AGTTCCACCTGAAGAAGCAGGGGCCGCTGTAGCTGCCGAATCTTCTACAGGTACATGGACAACNGTGTGGACCGATGGACTT
AGTTCCACCTGAAGAAGCAGGGGCCGCTGTAGCTGCCGAATCTTCTACAGGTACATGGACAACNGTGTGGACCGATGGACTT
ACCAGTCTTGATCGTTACAAAGGACGRTGCTACCACATTGAGCCTGTTGCTGGAGAAGACAATCAATATATTTGTTACGTAGC
ACCAGTCTTGATCGTTACAAAGGACGRTGCTACCACATTGAGCCTGTTGCTGGAGAAGACAATCAATATATTTGTTACGTAGC
CTATCCTTTAGACCTTTTTNAAGAGGGTTCTGTTACTAACATGTTTACTTCTATTGTGGGTAATGTATTTGGGTTCAAAGCGCT
CTATCCTTTAGACCTTTTTNAAGAGGGTTCTGTTACTAACATGTTTACTTCTATTGTGGGTAATGTATTTGGGTTCAAAGCGCT
GCGCGCTCTACGTCTGGAGGATCTGCGAATTCCTCTTGCTTATGTTAAAACTTTCCAAGGCCCGCCTCATGGNATCCAAGTTG
GCGCGCTCTACGTCTGGAGGATCTGCGAATTCCTCTTGCTTATGTTAAAACTTTCCAAGGCCCGCCTCATGGNATCCAAGTTG
AGAGAGATAAATTGAACAAGTATGGTCGTCCTCTATTAGGATGTACTATTAAACCAAAATTGGGATTATCCGCTAAGAACTAT
AGAGAGATAAATTGAACAAGTATGGTCGTCCTCTATTAGGATGTACTATTAAACCAAAATTGGGATTATCCGCTAAGAACTAT
GGTAGAGCGGTTTATGAATGTCTCCGCGGTGGRCTTGATTTTACCAAGGATGATGAGAACGTGAACTCCCAGCCATTTATGCG
GGTAGAGCGGTTTATGAATGTCTCCGCGGTGGRCTTGATTTTACCAAGGATGATGAGAACGTGAACTCCCAGCCATTTATGCG
TTGGAGAGANCGTTTCCTATTTTGTGCCGAAGCTATTTATAAATCACAGGCGGAAACAGGTGAAATCAAAGGACATTACTTGA
TTGGAGAGANCGTTTCCTATTTTGTGCCGAAGCTATTTATAAATCACAGGCGGAAACAGGTGAAATCAAAGGACATTACTTGA
ATGCTACT
ATGCTACT
Capsella bursa-pastoris
P6846 / 12
JF949997
Aligned with
Capsella bursa-pastoris
FN594844
ribulose-1,5-bisphosphate carboxylase/oxygenase large subunit (rbcL) gene
Brassicaceae
TATAAATTGACTTATTATACTCCTGAATATGAAACCAAGGATACTGATATCTTGGCAGCATTCCGAGTAACTCCTCAACCCGG
TATAAATTGACTTATTATACTCCTGAATATGAAACCAAGGATACTGATATCTTGGCAGCATTCCGAGTAACTCCTCAACCCGG
AGTTCCACCTGAAGAAGCAGGGGCTGCGGTAGCTGCTGAATCTTCTACTGGTACATGGACAACTGTGTGGACCGATGGGCTT
AGTTCCACCTGAAGAAGCAGGGGCTGCGGTAGCTGCTGAATCTTCTACTGGTACATGGACAACTGTGTGGACCGATGGGCTT
ACCAGCCTTGATCGTTACAAAGGACGATGCTACCACATCGAGCCCGTTCCAGGAGAAGAAACTCAATTTATTGCGTATGTAGC
ACCAGCCTTGATCGTTACAAAGGACGATGCTACCACATCGAGCCCGTTCCAGGAGAAGAAACTCAATTTATTGCGTATGTAGC
TTACCCCTTAGACCTTTTTGAAGAAGGTTCGGTTACTAACATGTTTACTTCGATTGTGGGTAATGTATTTGGGTTCAAAGCCCT
TTACCCCTTAGACCTTTTTGAAGAAGGTTCGGTTACTAACATGTTTACTTCGATTGTGGGTAATGTATTTGGGTTCAAAGCCCT
GGCTGCCCTACGTCTAGAGGATCTGCGAATCCCTCCTGCTTATACTAAAACTTTCCAAGGACCACCTCATGGTATCCAAGTTG
GGCTGCCCTACGTCTAGAGGATCTGCGAATCCCTCCTGCTTATACTAAAACTTTCCAAGGACCACCTCATGGTATCCAAGTTG
AAAGAGATAAATTGAACAAGTATGGACGTCCCCTATTAGGATGTACTATTAAACCAAAATTGGGGTTATCCGCGAAGAACTA
AAAGAGATAAATTGAACAAGTATGGACGTCCCCTATTAGGATGTACTATTAAACCAAAATTGGGGTTATCCGCGAAGAACTA
TGGTAGAGCGGTTTATGAATGTCTACGTGGTGGACTTGATTTTACCAAAGATGATGAGAATGTGAACTCCCAACCATTTATGC
TGGTAGAGCGGTTTATGAATGTCTACGTGGTGGACTTGATTTTACCAAAGATGATGAGAATGTGAACTCCCAACCATTTATGC
189
GTTGGAGAGACCGTTTCTTATTTTGTGCCGAAGCTATTTATAAATCACAGGCTGAAACAGGTGAAATCAAAGGGCATTATTTG
GTTGGAGAGACCGTTTCTTATTTTGTGCCGAAGCTATTTATAAATCACAGGCTGAAACAGGTGAAATCAAAGGGCATTATTTG
AATGCTACTGC
AATGCTACTGC
Isatis indigotica
P6877, P6878
JF949981
Aligned with
Isatis pachycarpa
FN594830
ribulose-1,5-bisphosphate carboxylase/oxygenase large subunit (rbcL) gene
Brassicaceae
TATAAATTGACTTATTATACTCCTGAATATGAAACCAAGGATACTGATATCTTGGCAGCATTCCGAGTAACTCCTCAACCCGG
TATAAATTGACTTATTATACTCCTGAATATGAAACCAAGGATACTGATATCTTGGCAGCATTCCGAGTAACTCCTCAACCCGG
AGTTCCACCTGAAGAAGCAGGGGCTGCGGTAGCTGCTGAATCTTCTACTGGTACATGGACAACTGTGTGGACCGATGGGCTT
AGTTCCACCTGAAGAAGCAGGGGCTGCGGTAGCTGCTGAATCTTCTACTGGTACATGGACAACTGTGTGGACCGATGGGCTT
ACCAGCCTTGATCGTTACAAAGGAAGATGCTACCACATCGAGCCCGTTCCAGGAGAAGAAACTCAATTTATTGCGTATGTAG
ACCAGCCTTGATCGTTACAAAGGAAGATGCTACCACATCGAGCCCGTTCCAGGAGAAGAAACTCAATTTATTGCATATGTAG
CTTACCCCTTAGACCTTTTTGAAGAAGGGTCGGTTACTAACATGTTTACCTCGATTGTGGGTAATGTATTTGGGTTCAAAGCCC
CTTACCCCTTAGACCTTTTTGAAGAAGGGTCGGTTACTAACATGTTTACCTCGATTGTGGGTAATGTATTTGGGTTCAAAGCCC
TGGCTGCTCTACGTCTAGAGGATCTGCGAATCCCTCCTGCTTATACTAAAACTTTCCAGGGACCACCTCATGGTATCCAAGTTG
TGGCTGCTCTACGTCTAGAGGATCTGCGAATCCCTCCTGCTTATACTAAAACTTTCCAGGGACCACCTCATGGTATCCAAGTTG
AAAGAGATAAATTGAACAAGTACGGACGTCCCCTATTAGGATGTACTATTAAACCTAAATTGGGGTTATCCGCGAAGAATTA
AAAGAGATAAATTGAACAAGTACGGACGTCCCCTATTAGGATGTACTATTAAACCTAAATTGGGGTTATCCGCGAAGAATTA
TGGTAGAGCAGTTTATGAATGTCTACGTGGTGGACTTGATTTTACCAAAGATGATGAGAATGTGAACTCCCAACCATTTATGC
TGGTAGAGCAGTTTATGAATGTCTACGTGGTGGACTTGATTTTACCAAAGATGATGAGAATGTGAACTCCCAACCATTTATGC
GTTGGAGAGACCGTTTCTTATTTTGTGCCGAAGCTATTTATAAATCACAGGCTGAAACAGGTGAAATCAAAGGGCATTATTTG
GTTGGAGAGACCGTTTCTTATTTTGTGCCGAAGCTATTTATAAATCACAGGCTGAAACAGGTGAAATCAAAGGGCATTATTTG
AATGCTACTGC
AATGCTACTGC
Lonicera confusa
P6880 / 46
JF949982
Aligned with
Lonicera confusa
HM228484
ribulose-1,5-bisphosphate carboxylase/oxygenase large subunit (rbcL) gene
Caprifoliaceae
AGTGTTGGATTCAAAGCGGGTGTTAAAGATTACAAATTGACTTATTATACTCCTGACTATGAAACCAAAGATACTGATATCTT
AGTGTTGGATTCAAAGCGGGTGTTAAAGATTACAAATTGACTTATTATACTCCTGACTATGAAACCAAAGATACTGATATCTT
GGCAGCATTCCGAGTAACTCCTCAACCCGGAGTTCCGCCTGAAGAAGCGGGGGCCGCGGTAGCTGCTGAATCTTCAACCGGT
GGCAGCATTCCGAGTAACTCCTCAACCCGGAGTTCCGCCTGAAGAAGCGGGGGCCGCGGTAGCTGCTGAATCTTCAACCGGT
ACATGGACAACTGTGTGGACCGATGGACTTACCAGCCTTGATCGTTACAAAGGGCGATGCTACCACATCGAGCCCGTTGCTG
ACATGGACAACTGTGTGGACCGATGGACTTACCAGCCTTGATCGTTACAAAGGGCGATGCTACCACATCGAGCCCGTTGCTG
GAGAAGAAAATCAATTTATTGCTTATGTAGCTTACCCATTAGACCTTTTTGAAGAAGGTTCTGTTACTAACATGTTTACTTCTA
GAGAAGAAAATCAATTTATTGCTTATGTAGCTTACCCATTAGACCTTTTTGAAGAAGGTTCTGTTACTAACATGTTTACTTCTA
TTGTGGGTAATGTATTTGGGTTCAAAGCCCTGCGCGCTCTACGTCTGGAAGATCTGCGAATCCCTGTCTCTTATGTTAAAACTT
TTGTGGGTAATGTATTTGGGTTCAAAGCCCTGCGCGCTCTACGTCTGGAAGATCTGCGAATCCCTGTCTCTTATGTTAAAACTT
TCCAAGGCCCGCCTCATGGTATCCAAGTTGAGAGAGATAAATTGAACAAGTACGGCCGCCCCCTGTTGGGATGTACTATTAA
TCCAAGGCCCGCCTCATGGTATCCAAGTTGAGAGAGATAAATTGAACAAGTACGGCCGCCCCCTGTTGGGATGTACTATTAA
ACCTAAATTGGGGTTATCTGCTAAAAACTATGGTAGGGCGGTTTATGAATGTCTACGTGGTGGACTTGATTTTACCAAAGATG
ACCTAAATTGGGGTTATCTGCTAAAAACTATGGTAGGGCGGTTTATGAATGTCTACGTGGTGGACTTGATTTTACCAAAGATG
ATGAGAACGTGAACTCCCAACCATTTATGCGTTGGAGAGATCGTTTCTTATTTTGTGCCGAAGCCCTTTTTAAAGCACAAGTT
ATGAGAACGTGAACTCCCAACCATTTATGCGTTGGAGAGATCGTTTCTTATTTTGTGCCGAAGCCCTTTTTAAAGCACAAGTT
GAAACAGGTGAAATCAAAGGGCATTACTTGAAT
GAAACAGGTGAAATCAAAGGGCATTACTTGAAT
190
Polygonatum kingianum
P6892 / 58
JF950027
Aligned with
Polygonatum humile
AB009947
ribulose-1,5-bisphosphate carboxylase/oxygenase large subunit (rbcL) gene
Convallariaceae
AGCTGGTGTTAAAGATTACAGATTGACTTATTATACTCCTGATTACGAAACCAAAGCTACTGATATCTTGGCAGCATTCCGAG
AGCTGGTGTTAAAGATTACAGATTGACTTATTATACTCCTGATTACGAAACCAAAGCTACTGATATCTTGGCAGCATTCCGAG
TAACTCCTCAACCCGGAGTTCCCGCTGAAGAAGCAGGGGCTGCGGTAGCTGCCGAATCCTCTACTGGTACATGGACAACTGT
TAACTCCTCAACCCGGAGTTCCCGCTGAAGAAGCAGGGGCTGCGGTAGCTGCCGAATCCTCTACTGGTACATGGACAACTGT
GTGGACTGATGGACTTACCAGTCTTGATCGTTACAAAGGACGATGCTACCACATTGAGGCCGTTATTGGGGAAGAAAATCAA
GTGGACTGATGGACTTACCAGTCTTGATCGTTACAAAGGACGATGCTACCACATTGAGGCCGTTATTGGGGAAGAAAATCAA
TATATTTGTTATGTAGCTTATCCTTTAGACCTTTTTGAAGAAGGTTCTGTTACTAACATGTTTACTTCCATTGTGGGTAATGTAT
TATATTTGTTATGTAGCTTATCCTTTAGACCTTTTTGAAGAAGGTTCTGTTACTAACATGTTTACTTCCATTGTGGGTAATGTAT
TTGGTTTCAAAGCCCTACGAGCTCTACGTCTGGAGGATCTGCGAATTCCCCCTGCTTATTCCAAAACTTTCCAAGGCCCGCCTC
TTGGTTTCAAAGCCCTACGAGCTCTACGTCTGGAGGATCTGCGAATTCCCCCTGCTTATTCCAAAACTTTCCAAGGCCCGCCTC
ATGGCATTCAAGTTGAAAGAGATAAATTGAACAAGTATGGTCGTCCCCTATTGGGATGTACTATTAAACCAAAATTGGGATTA
ATGGCATTCAAGTTGAAAGAGATAAATTGAACAAGTATGGTCGTCCCCTATTGGGATGTACTATTAAACCAAAATTGGGATTA
TCCGCAAAAAACTACGGTAGAGCGGTTTATGAATGTCTACGCGGTGGACTTGATTTTACCAAGGATGATGAA
TCCGCAAAAAACTACGGTAGAGCGGTTTATGAATGTCTACGCGGTGGACTTGATTTTACCAAGGATGATGAA
Platycladus orientalis
P6891 / 57
JF950011
Aligned with
Platycladus orientalis
HM024345
ribulose-1,5-bisphosphate carboxylase/oxygenase large subunit (rbcL) gene
Cupressaceae
TTATTATACTCCGGAATATCAGACCAAAGATACTGATATCTTGGCAGCATTCCGAGTCACTCCTCAACCTGGAGTGCCCCCCG
TTATTATACTCCGGAATATCAGACCAAAGATACTGATATCTTGGCAGCATTCCGAGTCACTCCTCAACCTGGAGTGCCCCCCG
AAGAAGCGGGAGCAGCAGTAGCTGCCGAATCTTCCACTGGTACGTGGACCACTGTTTGGACCGATGGACTTACCAGTCTTGA
AAGAAGCGGGAGCAGCAGTAGCTGCCGAATCTTCCACTGGTACGTGGACCACTGTTTGGACCGATGGACTTACCAGTCTTGA
TCGCTACAAGGGGCGATGCTATGATATTGAACCCGTTCCTGGAGAAGAAACTCAATTTATTGCCTATGTAGCTTACCCTTTAG
TCGCTACAAGGGGCGATGCTATGATATTGAACCCGTTCCTGGAGAAGAAACTCAATTTATTGCCTATGTAGCTTACCCTTTAG
ATCTTTTTGAAGAAGGCTCTGTGACTAACCTGTTTACTTCTATTGTAGGTAATGTATTTGGATTCAAAGCTTTACGGGCTCTAC
ATCTTTTTGAAGAAGGCTCTGTGACTAACCTGTTTACTTCTATTGTAGGTAATGTATTTGGATTCAAAGCTTTACGGGCTCTAC
GTCTGGAAGATTTACGAATTCCTCCTGCTTATTCAAAAACTTTTCAAGGCCCACCACATGGTATTCAAGTAGAAAGGGATAAA
GTCTGGAAGATTTACGAATTCCTCCTGCTTATTCAAAAACTTTTCAAGGCCCACCACATGGTATTCAAGTAGAAAGGGATAAA
TTAAATAAATATGGTCGTCCTTTGTTGGGATGTACTATCAAACCAAAATTGGGTCTATCTGCCAAGAATTATGGTAGAGCGGT
TTAAATAAATATGGTCGTCCTTTGTTGGGATGTACTATCAAACCAAAATTGGGTCTATCTGCCAAGAATTATGGTAGAGCGGT
TTATGAATGTCTCCGTGGTGGACTTGATTTTACCAAGGATGATGAAAACGTGAATTCCCAACCATTTATGCGCTGGAGAGATC
TTATGAATGTCTCCGTGGTGGACTTGATTTTACCAAGGATGATGAAAACGTGAATTCCCAACCATTTATGCGCTGGAGAGATC
GTTTCTGCTTTTGTGCAGAAGCACTTTATAAAGCTCAGGCTGAGACGGGTGAGATTAAGGGACATTACCTGAATGC
GTTTCTGCTTTTGTGCAGAAGCACTTTATAAAGCTCAGGCTGAGACGGGTGAGATTAAGGGACATTACCTGAATGC
Dryopteridaceae Cyrtomium fortunei
P6859 / 25
JF949998
Aligned with
Cyrtomium fortunei
AF537227
ribulose-1,5-bisphosphate carboxylase/oxygenase large subunit (rbcL) gene
ATACAAGACCAAAGATACCGATATCTTAGCAGCCTTCAGAATGACCCCACAACCCGGAGTACCGGCTGAGGAAGCCGGAGCT
ATACAAGACCAAAGATACCGATATCTTAGCAGCCTTCAGAATGACCCCACAACCCGGAGTACCGGCTGAGGAAGCCGGAGCT
GCGGTAGCTGCGGAATCCTCAACGGGTACGTGGACCACTGTATGGACAGATGGGTTGACCAGTCTTGACCGTTACAAGGGCC
GCGGTAGCTGCGGAATCCTCAACGGGTACGTGGACCACTGTATGGACAGATGGGTTGACCAGTCTTGACCGTTACAAGGGCC
GATGCTACGATATCGAACCCGTCGCCGGGGAAGAAAACCAATATATCGCGTATGTAGCTTATCCTTTGGATCTATTCGAAGAA
GATGCTACGATATCGAACCCGTCGCCGGGGAAGAAAACCAATATATCGCGTATGTAGCTTATCCTTTGGATCTATTCGAAGAA
GGTTCCGTCACTAATTTGTTTACCTCTATCGTAGGTAATGTTTTTGGATTTAAGGCTCTACGCGCTTTACGCCTGGAAGACCTT
GGTTCCGTCACTAATTTGTTTACCTCTATCGTAGGTAATGTTTTTGGATTTAAGGCTCTACGCGCTTTACGCCTGGAAGACCTT
191
CGAATTCCTCCTGCTTATTCTAAAACTTTCATTGGACCGCCTCATGGTATTCAGGTCGAAAGGGATAAACTGAACAAATATGG
CGAATTCCTCCTGCTTATTCTAAAACTTTCATTGGACCGCCTCATGGTATTCAGGTCGAAAGGGATAAACTGAACAAATATGG
ACGTCCTTTATTGGGATGTACAATCAAGCCAAAATTAGGTCTGTCTGCTAAAAATTATGGTAGAGCCGTCTACGAATGCCTTC
ACGTCCTTTATTGGGATGTACAATCAAGCCAAAATTAGGTCTGTCTGCTAAAAATTATGGTAGAGCCGTCTACGAATGCCTTC
GCGGTGGACTTGATTTCACAAAAGATGATGAAAATGTGAATTCTCAGCCATTCATGCGTTGGAGAGATCGCTTCCTATTCGTA
GCGGTGGACTTGATTTCACAAAAGATGATGAAAATGTGAATTCTCAGCCATTCATGCGTTGGAGAGATCGCTTCCTATTCGTA
GCAGAAGCTCTCTTCAAATCCCAGGCTGAAACAGGAGAAATCAAAGGGCATTACTTAAATGCTACTGCAGGTAC
GCAGAAGCTCTCTTCAAATCCCAGGCTGAAACAGGAGAAATCAAAGGGCATTACTTAAATGCTACTGCAGGTAC
Ephedra sinica
P6864 / 30
JF950001
Aligned with
Ephedra sinica
AY492046
ribulose-1,5-bisphosphate carboxylase/oxygenase large subunit (rbcL) gene
Ephedraceae
GATTATAGATTAACTTATTATACTCCAGAGTATCAGACTAAGAACACTGATATCTTGGCAGCATTCCGAGTAACTCCTCAACC
GATTATAGATTAACTTATTATACTCCAGAGTATCAGACTAAGAACACTGATATCTTGGCAGCATTCCGAGTAACTCCTCAACC
CGGAGTACCGGCCGAGGAAGCAGGCGCAGCGGTAGCTGCTGAATCTTCTACAGGTACATGGACCACAGTTTGGACTGATGGT
CGGAGTACCGGCCGAGGAAGCAGGCGCAGCGGTAGCTGCTGAATCTTCTACAGGTACATGGACCACAGTTTGGACTGATGGT
CTTACTAGTCTTGATCGTTACAAAGGACGATGCTATGATATCGAACCTGTTCCGGGAGAAGACAATCAATTTATTGCTTATGT
CTTACTAGTCTTGATCGTTACAAAGGACGATGCTATGATATCGAACCTGTTCCGGGAGAAGACAATCAATTTATTGCTTATGT
AGCATATCCTTTGGACCTTTTTGAAGAAGGTTCTGTTACTAACATGTTTACTTCTATTGTGGGGAATGTTTTTGGTTTTAAAGCT
AGCATATCCTTTGGACCTTTTTGAAGAAGGTTCTGTTACTAACATGTTTACTTCTATTGTGGGGAATGTTTTTGGTTTTAAAGCT
CTACGAGCTCTACGCCTAGAAGATTTGCGAATTCCTACTGCTTATATCAAAACATTTCAAGGTCCACCTCATGGGATCCAAGT
CTACGAGCTCTACGCCTAGAAGATTTGCGAATTCCTACTGCTTATATCAAAACATTTCAAGGTCCACCTCATGGGATCCAAGT
AGAAAGAGATAAATTAAACAAATACGGACGTCCTTTGTTGGGATGTACCATCAAACCTAAATTAGGTCTATCTGCTAAAAATT
AGAAAGAGATAAATTAAACAAATACGGACGTCCTTTGTTGGGATGTACCATCAAACCTAAATTAGGTCTATCTGCTAAAAATT
ACGGTAGAGCAGTTTATGAATGTCTTCGTGGCGGACTAGATTTTACTAAAGATGATGAAAATGTAAATTCTCAACCCTTTATG
ACGGTAGAGCAGTTTATGAATGTCTTCGTGGCGGACTAGATTTTACTAAAGATGATGAAAATGTAAATTCTCAACCCTTTATG
CGTTGGAGAGATCGCTTTGTTTTTTGCGCGGAAGCTATTTATAAAGCTCAAGCTGAAACAGGTGAAATTAAGGGACATTACTT
CGTTGGAGAGATCGCTTTGTTTTTTGCGCGGAAGCTATTTATAAAGCTCAAGCTGAAACAGGTGAAATTAAGGGACATTACTT
AAATGCTACTGC
AAATGCTACTGC
Equisetum hyemale
P6866 / 32
JF950003
Aligned with
Equisetum hyemale
AB574685
ribulose-1,5-bisphosphate carboxylase/oxygenase large subunit (rbcL) gene
Equisetaceae
TTTACTCCAGATTATGAAACCAAAGATACCGATATTTTAGCAGCATTTCGTATGACTCCGCAGCCGGGGGTACCACCGGAAGA
TTTACTCCAGATTATGAAACCAAAGATACCGATATTTTAGCAGCATTTCGTATGACTCCGCAGCCGGGGGTACCACCGGAAGA
AGCAGGAGCAGCTGTAGCTGCTGAATCCTCCACGGGCACCTGGACTACCGTATGGACAGATGGACTTACTAGTCTTGATCGAT
AGCAGGAGCAGCTGTAGCTGCTGAATCCTCCACGGGCACCTGGACTACCGTATGGACAGATGGACTTACTAGTCTTGATCGAT
ATAAAGGTCGCTGCTATAATATTGAGCCTGTTGCTGGAGAAGATAACCAATTCATAGCTTATGTAGCCTATCCTTTAGATCTTT
ATAAAGGTCGCTGCTATAATATTGAGCCTGTTGCTGGAGAAGATAACCAATTCATAGCTTATGTAGCCTATCCTTTAGATCTTT
TTGAAGAAGGTTCTGTTACCAATCTGTTTACTTCAATTGTTGGTAATGTTTTCGGCTTCAAAGCTCTACGTGCTTTACGTTTAG
TTGAAGAAGGTTCTGTTACCAATCTGTTTACTTCAATTGTTGGTAATGTTTTCGGCTTCAAAGCTCTACGTGCTTTACGTTTAG
AAGATTTACGAATTCCTCCTGCTTATTCTAAAACTTTTATAGGACCACCCCATGGTATCCAGGTTGAAAGAGATAAGTTAAAC
AAGATTTACGAATTCCTCCTGCTTATTCTAAAACTTTTATAGGACCACCCCATGGTATCCAGGTTGAAAGAGATAAGTTAAAC
AAATATGGTCGTCCGTTATTAGGTTGTACAATTAAACCAAAATTGGGACTTTCTGCTAAAAACTATGGTAGAGCTGTTTATGA
AAATATGGTCGTCCGTTATTAGGTTGTACAATTAAACCAAAGTTGGGACTTTCTGCTAAAAACTATGGTAGAGCTGTTTATGA
ATGTCTTCGTGGTGGACTTGACTTCACCAAAGATGATGAGAATGTAAACTCTCAACCTTTTATGCGTTGGA
ATGTCTTCGTGGTGGACTTGACTTCACCAAAGATGATGAGAATGTAAACTCTCAACCTTTTATGCGTTGGA
192
Abrus cantoniensis
P6835 / 01
JF949964
Aligned with
Abrus precatorius
U74224
ribulose-1,5-bisphosphate carboxylase/oxygenase large subunit (rbcL) gene
Fabaceae
GTTGGGTTCAAAGCTGGTGTTAAAGATTATAAATTGAATTATTATACTCCTGACTATGAAACCAAAGATACTGATATCTTGGC
GTTGGGTTCAAAGCTGGTGTTAAAGATTATAAATTGAATTATTATACTCCTGACTATGAAACCAAAGATACTGATATCTTGGC
AGCATTCCGAGTAACTCCTCAACCCGGAGTTCCGCCTGAAGAAGCAGGTGCCGCGGTAGCTGCCGAATCTTCTACTGGTACAT
AGCATTCCGAGTAACTCCTCAACCCGGAGTTCCGCCTGAAGAAGCAGGTGCCGCGGTAGCTGCCGAATCTTCTACAGGTACAT
GGACAACTGTGTGGACCGATGGGCTTACCAGTCTTGATCGTTACAAAGGACGATGCTACCACATCGAACCCGTTGCTGGAGA
GGACAACTGTGTGGACCGATGGGCTTACCAGTCTTGATCGTTACAAAGGACGATGCTACCACATCGAACCCGTTGCTGGAGA
AGAAAATCAATATATTGCTTATGTAGCTTATCCCTTAGACCTTTTTGAAGAAGGTTCTGTTACTAACATGTTTACTTCCATTGT
AGAAAATCAATATATTGCTTATGTAGCTTATCCCTTAGACCTTTTTGAAGAAGGTTCTGTTACTAACATGTTTACTTCCATTGT
AGGTAATGTATTTGGGTTCAAGGCCCTGCGCGCTCTACGTTTGGAGGATTTGCGAATCCCTACTTCTTATATTAAAACTTTCCA
AGGTAATGTATTTGGGTTCAAGGCCCTGCGCGCTCTACGTTTGGAGGATTTGCGAATCCCTACTTCTTATATTAAAACTTTCCA
AGGTCCGCCTCATGGCATCCAAGTTGAGAGAGATAAATTGAACAAGTATGGTCGTCCCCTATTGGGATGTACTATTAAACCGA
AGGTCCGCCTCATGGCATCCAAGTTGAGAGAGATAAATTGAACAAGTATGGCCGTCCCCTATTGGGATGTACTATTAAACCTA
AATTGGGATTATCCGCTAAGAATTACGGTAGAGCGGTTTATGAATGTCTCCGCGGGGGGCTTGATTTTACCAAAGATGATGAA
AATTGGGATTATCCGCTAAGAATTACGGTAGAGCGGTTTATGAATGTCTCCGCGGGGGGCTTGATTTTACCAAAGATGATGAA
AATGTGAATTCCCAACCATTTATGCGTTGGAGAGACCGTTTCTTATTTTGTGCCGAAGCACTTTATAAAGCACAGGCTGAAAC
AATGTGAATTCCCAACCATTTATGCGTTGGAGAGACCGTTTCTTATTTTGTGCCGAAGCACTTTATAAAGCACAGACTGAAAC
GGGTGAAATCAAAGGGCATTACTTAAATGCTACTGCAGGTACATGCGAA
GGGTGAAATCAAAGGGCATTACTTAAATGCTACTGCAGGTACATGCGAA
Cassia tora
P6847 / 13
JF949969
Aligned with
Cassia grandis
AM234244
ribulose-1,5-bisphosphate carboxylase/oxygenase large subunit (rbcL) gene
Fabaceae
GTTGGGTTCAAAGCTGGTGTTAAAGATTATAAATTGACTTATTATACTCCTGACTATGAAACCAAAGATACTGATATCTTGGC
GTTGGGTTCAAAGCTGGTGTTAAAGATTATAAATTGACTTATTATACTCCTGACTATGAAACCAAAGATACTGATATCTTGGC
AGCATTCCGAGTAACTCCTCAACCTGGAGTTCCGCCTGAAGAAGCAGGTGCCGCGGTAGCTGCTGAATCTTCTACTGGTACAT
AGCATTCCGAGTAACTCCTCAACCTGGAGTTCCGCCTGAAGAAGCAGGTGCCGCGGTAGCTGCTGAATCTTCTACTGGTACAT
GGACAACTGTGTGGACCGATGGGCTTACCAGTCTTGATCGTTACAAAGGACGATGCTACCACATCGAGCCCGTTGCTGGAGA
GGACAACTGTGTGGACCGATGGGCTTACCAGTCTTGATCGTTACAAAGGACGATGCTACCACATCGAGCCCGTTGCTGGAGA
AGAAAATCAATATATTGCTTATGTAGCTTATCCCTTAGACCTTTTTGAAGAAGGTTCTGTTACTAACATGTTTACTTCCATTGT
AGAAAATCAATATATTGCTTATGTAGCTTATCCCTTAGACCTTTTTGAAGAAGGTTCTGTTACTAACATGTTTACTTCCATTGT
GGGTAATGTATTTGGGTTCAAGGCCCTGCGCGCTCTACGTCTGGAGGATTTGCGAATCCCTACTTCTTATACTAAAACTTTCCA
GGGTAATGTATTTGGGTTCAAGGCCCTGCGCGCTCTACGTCTGGAGGATTTGCGAATCCCTATTTCTTATATTAAAACTTTCCA
AGGTCCGCCTCACGGCATCCAAGTTGAGAGAGATAAATTGAACAAGTACGGCCGTCCCCTATTGGGATGTACTATTAAACCT
AGGTCCGCCCCACGGCATCCAAGTTGAGAGAGATAAATTGAACAAGTATGGCCGTCCCCTATTGGGATGTACTATTAAACCT
AAATTGGGGTTATCCGCTAAGAATTACGGTAGAGCAGTTTATGAATGTCTCCGCGGTGGACTTGATTTTACCAAAGATGATGA
AAATTGGGGTTATCCGCTAAGAATTACGGTAGAGCAGTTTATGAATGTCTCCGCGGTGGACTTGATTTTACCAAAGATGATGA
GAATGTGAATTCCCAACCATTTATGCGTTGGAGAGACCGTTTCTTATTTTGTGCCGAAGCCCTTTTTAAAGCACAGGCCGAAA
GAATGTGAATTCCCAACCATTTATGCGTTGGAGAGACCGTTTCTGCTTTTGTGCCGAAGCACTTTATAAAGCACAGGCCGAAA
CAGGTGAAATCAAAGGGCATTACTTGAATGCTACTGCAGG
CAGGTGAAATCAAAGGGCATTACTTGAATGCTACGGCAGG
Desmodium styracifolium
P6861 / 27
JF949976
Aligned with
Desmodium styracifolium
GQ436338
ribulose-1,5-bisphosphate carboxylase/oxygenase large subunit (rbcL) gene
Fabaceae
GTTGGGTTCAAAGCTGGTGTTAAAGATTATAAATTGACTTATTATACTCCTGAGTATCAAACCAAAGATACTGATATCTTGGC
GTTGGGTTCAAAGCTGGTGTTAAAGATTATAAATTGACTTATTATACTCCTGAGTATCAAACCAAAGATACTGATATCTTGGC
193
AGCCTTCCGAGTAACTCCTCAACCGGGAGTTCCACCTGAAGAAGCAGGTGCTGCGGTAGCTGCGGAATCTTCTACTGGTACAT
AGCCTTCCGAGTAACTCCTCAACCGGGAGTTCCACCTGAAGAAGCAGGTGCTGCGGTAGCTGCGGAATCTTCTACTGGTACAT
GGACAACTGTGTGGACCGATGGGCTTACCAGTCTTGATCGTTACAAAGGACGATGCTACGGCCTCGAACCTGTTGCTGGAGA
GGACAACTGTGTGGACCGATGGGCTTACCAGTCTTGATCGTTACAAAGGACGATGCTACGGCCTCGAACCTGTTGCTGGAGA
AGAAAATCAATATATTGCTTATGTAGCGTATCCCTTAGACCTTTTTGAAGAAGGTTCTGTTACTAACATGTTTACTTCCATTGT
AGAAAATCAATATATTGCTTATGTAGCGTATCCCTTAGACCTTTTTGAAGAAGGTTCTGTTACTAACATGTTTACTTCCATTGT
CGGTAATGTATTTGGGTTCAAGGCACTGCGCGCCCTACGTCTGGAGGATTTGCGAATCCCTAAGGCTTATATTAAAACTTTTG
CGGTAATGTATTTGGGTTCAAGGCACTGCGCGCCCTACGTCTGGAGGATTTGCGAATCCCTAAGGCTTATATTAAAACTTTTG
AAGGTCCACCTCATGGCATCCAAGTTGAAGAGAGATAAATTGAACAAGTATGGCCGTCCTCTATTAGGATGTACTATTAAACT
AAGGTCCACCTCATGGCATCCAAGTTGAAGAGAGATAAATTGAACAAGTATGGCCGTCCTCTATTAGGATGTACTATTAAACT
AAATTGGGGTTATCCGCTAAGAATTACGGTAGAGCAGTTTACGAATGTCTTCGTGGGGGACTTGATTTTACCAAAGATGATGA
AAATTGGGGTTATCCGCTAAGAATTACGGTAGAGCAGTTTACGAATGTCTTCGTGGGGGACTTGATTTTACCAAAGATGATGA
AAATGTGAATTCCCAACCATTTATGCGTTGGAGAGACCGTTTCTTATTTTGTGCCGAAGCCCTTTTTAAAGCACAGGCTGAAA
AAATGTGAATTCCCAACCATTTATGCGTTGGAGAGACCGTTTCTTATTTTGTGCCGAAGCCCTTTTTAAAGCACAGGCTGAAA
CAGGTGAAATCAAAGGGCATTACTTGAAT
CAGGTGAAATCAAAGGGCATTACTTGAAT
Glycyrrhiza inflata
P6873 / 39
JF950025
Aligned with
Glycyrrhiza inflata
AB012127
ribulose-1,5-bisphosphate carboxylase/oxygenase large subunit (rbcL) gene
Fabaceae
GTTGGGTTCAAAGCTGGTGTTAAAGATTATAAATTGACTTATTATACTCCTGAATATGAAACCAAGGATACTGATATCTTGGC
GTTGGGTTCAAAGCTGGTGTTAAAGATTATAAATTGACTTATTATACTCCTGAATATGAAACCAAGGATACTGATATCTTGGC
AGCATTCCGAGTAACTCCTCAACCAGGAGTTCCGCCGGAAGAAGCAGGTGCAGCGGTAGCAGCCGAATCTTCCACTGGGACA
AGCATTCCGAGTAACTCCTCAACCAGGAGTTCCGCCGGAAGAAGCAGGTGCAGCGGTAGCAGCCGAATCTTCCACTGGGACA
TGGACAACTGTGTGGACCGATGGTCTTACCAGTCTTGATCGTTATAAAGGACGATGCTACGGGCTAGAGCCCGTTGCTGGAGA
TGGACAACTGTGTGGACCGATGGTCTTACCAGTCTTGATCGTTATAAAGGACGATGCTACGGGCTAGAGCCCGTTGCTGGAGA
AGAAAATCAATATATTGCTTATGTAGCTTATCCTTTAGACCTTTTTGAAGAAGGTTCTGTTACTAACATGTTTACCTCCATTGT
AGAAAATCAATATATTGCTTATGTAGCTTATCCTTTAGACCTTTTTGAAGAAGGTTCTGTTACTAACATGTTTACCTCCATTGT
AGGTAATGTATTTGGTTTCAAGGCCTTGCGCGCTCTACGTCTTGAGGATTTGCGAATTCCTGTTTCTTACATTAAAACTTTCCA
AGGTAATGTATTTGGTTTCAAGGCCTTGCGCGCTCTACGTCTTGAGGATTTGCGAATTCCTGTTTCTTACATTAAAACTTTCCA
AGGTCCGCCTCACGGAATCCAAGTTGAAAGAGATAAATTGAACAAGTATGGCCGCCCTCTATTGGGATGTACTATTAAACCT
AGGTCCGCCTCACGGAATCCAAGTTGAAAGAGATAAATTGAACAAGTATGGCCGCCCTCTATTGGGATGTACTATTAAACCT
AAATTGGGGTTATCTGCTAAGAATTATGGTAGAGCAGTTTATGAATGTCTTCGCGGGGGACTTGATTTTACCAAAGATGATGA
AAATTGGGGTTATCTGCTAAGAATTATGGTAGAGCAGTTTATGAATGTCTTCGCGGGGGACTTGATTTTACCAAAGATGATGA
AAATGTGAACTCCCAACCATTTATGCGTTGGAGAGACCGTTTCTTATTTTGTGCCGAAGCAATTTATAAAGCACAGGCCGAAA
AAATGTGAACTCCCAACCATTTATGCGTTGGAGAGACCGTTTCTTATTTTGTGCCGAAGCAATTTATAAAGCACAGGCCGAAA
CTGGTGAAATCAAAGGGCATTATTTGAATGCTAC
CTGGTGAAATCAAAGGGCATTATTTGAATGCTAC
Spatholobus suberectus
P6907 / 73
JF949991
Aligned with
Spatholobus parviflorus
AB045825
ribulose-1,5-bisphosphate carboxylase/oxygenase large subunit (rbcL) gene
Fabaceae
ATGAAACCAAAGATACTGATATCTTGGCAGCATTCCGAGTAACTCCTCAACCTGGAGTTCCGCCTGAAGAAGCAGGTGCCGC
ATGAAACCAAAGATACTGATATCTTGGCAGCATTCCGAGTAACTCCTCAACCTGGAGTTCCGCCTGAAGAAGCAGGTGCCGC
GGTAGCTGCCGAATCTTCTACTGGTACATGGACAACTGTGTGGACCGATGGGCTTACCAGTCTTGATCGTTACAAAGGACGAT
GGTAGCTGCCGAATCTTCTACTGGTACATGGACAACTGTGTGGACCGATGGGCTTACCAGTCTTGATCGTTACAAAGGACGAT
GCTATCACATCGAACCTGTTGCTGGAGAAGAAAATCAATTTATTGCTTATGTAGCTTATCCCTTAGACCTTTTTGAAGAAGGTT
GCTATAACATCGAACCTGTTGCTGGAGAAGAAAATCAATTTATTGCTTATGTAGCTTATCCCTTAGACCTTTTTGAAGAAGGTT
CTGTTACTAACATGTTTACTTCCATTGTCGGTAATGTATTTGGGTTCAAGGCCCTTCGCGCTCTACGTCTGGAGGATTTGCGAA
CTGTTACTAACATGTTTACTTCCATTGTCGGTAATGTATTTGGGTTCAAGGCACTACGCGCTCTACGTCTGGAGGATTTGCGAA
194
TCCCTATTTCTTATGTTAAAACTTTCCAAGGTCCGCCTCATGGCATCCAAGTTGAGAGAGATAAATTGAACAAGTATGGCCGT
TCCCTATTTCTTATGTTAAAACTTTCCAAGGTCCGCCTCATGGCATCCAAGTTGAGAGAGATAAATTGAACAAGTATGGCCGT
CCCCTATTAGGATGTACTATTAAACCTAAATTGGGGTTATCCGCTAAGAATTACGGTAGAGCAGTTTATGAATGTCTTCGTGG
CCCCTATTAGGATGTACTATTAAACCTAAATTGGGGTTATCCGCTAAGAATTACGGTAGAGCAGTTTATGAATGTCTTCGTGG
GGGACTTGATTTTACCAAAGATGATGAAAATGTGAATTCCCAACCATTTATGCGTTGGAGAGACCGTTTCTTATTTTGTGCCG
GGGACTTGATTTTACCAAAGATGATGAAAATGTGAATTCCCAACCATTTATGCGTTGGAGAGACCGTTTCTTATTTTGTGCCG
AAGCGCTTTATAAAGCACAGGCTGAA
ATGCGCTTTATAAAGCACAGGCTGAA
Geranium wilfordii
P6867 / 33
JF949977
Aligned with
Geranium grandiflorum
L01920
ribulose-1,5-bisphosphate carboxylase/oxygenase large subunit (rbcL) gene
Geraniaceae
TAGTGTTGGATTTAAAGCGGGTGTTAAAGACTATAAATTGACTTATTATACTCCTGATTATGAAACCAAGGATACTGATATCT
TAGTGTTGGATTTAAAGCGGGTGTTAAAGACTATAAATTGACTTATTATACTCCTGATTATGAAACCAAGGATACTGATATCT
TGGCAGCATTCCGAGTAACTCCTCAACCCGGAGTTCCACCTGAGGAAGCGGGGGCCGCGGTAGCTGCTGAATCTTCTACCGG
TGGCAGCATTCCGAGTAACTCCTCAACCCGGAGTTCCACCTGAGGAAGCAGGGGGCGCGGTAGCTGCTGAATCTTCTACCGG
TACATGGACAACCGTGTGGACCGATGGGCTTACTAGTCTGGATCGTTACAAAGGACGCTGCTATCACATCGAGCCCGTTGCTG
TACATGGACAACCGTGTGGACCGATGGGCTTACTAGTCTGGATCGTTACAAAGGACGCTGCTATCACATCGAGCCCGTTGCTG
GAGAAGAAAATCAATTTATTGCTTATGTAGCTTACCCTTTAGACCTTTTTGAAGAAGGTTCCGTTACTAATATGTTTACTTCCA
GAGAAGAAAATCAATATATTGCTTATGTAGCTTACCCTTTAGACCTTTTTGAAGAAGGTTCCGTTACTAATATGTTTACTTCCA
TCGTGGGTAATGTATTTGGGTTCAAAGCCCTTCGCGCTCTGCGTCTGGAGGATCTGCGAATCCCTCCTGCTTATGTGAAAACTT
TCGTGGGTAATGTATTTGGGTTCAAAGCCCTTCGCGCTTTGCGTCTGGAGGATCTGCGAATCCCTCCTGCTTATGTGAAAACTT
TCCAGGGCCCGCCTCACGGCATCCAAGTTGAGAGAGATAAATTGAACAAGTATGGACGTCCCCTATTGGGATGTACTATTAA
TCCAGGGCCCGCCTCACGGCATCCAAGTTGAGAGAGATAAATTGAACAAGTATGGACGTCCCCTATTGGGATGTACTATTAA
ACCGAAATTGGGATTGTCCGCTAAGAACTACGGTCGAGCGGTTTTTGAATGTCTTCGCGGTGGACTTGATTTTACTAAAGATG
ACCGAAATTGGGATTGTCCGCTAAGAACTACGGTCGAGCAGTTTTTGAATGTCTTCGCGGTGGACTTGATTTTACTAAAGATG
ATGAGAACGTGAACTCCCAACCGTTTATGCGTTGGAGAGACCGTTTCTTATTTTGTGCCGAAGCGCTTTATAAAGCACAGGCC
ATGAGAACGTGAACTCTCAACCGTTTATGCGTTGGAGAGACCGTTTCTTATTTTGTGCCGAAGCTATTTATAAAGCACAGGCT
GAAACAGGTGAAATCAAGGGGCATTACTTGAATGCTACTGCAGG
GAAACAGGTGAAATCAAGGGGCATTACTTGAATGCTACTGCAGG
Ginkgo biloba
P6872 / 38
JF950005
Aligned with
Ginkgo biloba
DQ069500
ribulose-1,5-bisphosphate carboxylase/oxygenase large subunit (rbcL) gene
Ginkgoaceae
ATTACAGATTGACTTATTATACTCCTGAATATCAAACCAAAGATACTGATATCTTGGCAGCATTCCGAGTAACTCCTCAACCT
ATTACAGATTGACTTATTATACTCCTGAATATCAAACCAAAGATACTGATATCTTGGCAGCATTCCGAGTAACTCCTCAACCT
GGAGTGCCACCTGAGGAAGCGGGAGCTGCAGTAGCTGCCGAATCTTCCACTGGTACATGGACCACTGTTTGGACCGATGGAC
GGAGTGCCACCTGAGGAAGCGGGAGCTGCAGTAGCTGCCGAATCTTCCACTGGTACATGGACCACTGTTTGGACCGATGGAC
TTACCAGTCTTGATCGTTACAAGGGAAGATGCTATGACATCGAGCCCGTTCCTGGGGAGGAAAATCAATTTATTGCCTATGTA
TTACCAGTCTTGATCGTTACAAGGGAAGATGCTATGACATCGAGCCCGTTCCTGGGGAGGAAAATCAATTTATTGCCTATGTA
GCTTACCCTTTGGATCTTTTCGAGGAAGGTTCTGTTACTAACCTGTTCACTTCCATTGTAGGGAATGTATTTGGATTCAAAGCC
GCTTACCCTTTGGATCTTTTCGAGGAAGGTTCTGTTACTAACCTGTTCACTTCCATTGTAGGGAATGTATTTGGATTCAAAGCC
CTACGAGCTCTACGTCTGGAAGATCTGCGAATTCCTCCTGCTTATTCCAAAACTTTCCAGGGTCCACCTCATGGTATCCAAGTT
CTACGAGCTCTACGTCTGGAAGATCTGCGAATTCCTCCTGCTTATTCCAAAACTTTCCAGGGTCCACCTCATGGTATCCAAGTT
GAAAGGGATAAATTGAATAAATATGGCCGTCCCCTATTGGGATGTACTATCAAGCCAAAATTGGGTTTATCTGCCAAAAATTA
GAAAGGGATAAATTGAATAAATATGGCCGTCCCCTATTGGGATGTACTATCAAGCCAAAATTGGGTTTATCTGCCAAAAATTA
TGGTAGAGCAGTTTACGAATGTCTTCGTGGTGGACTTGATTTTACTAAAGATGATGAGAACGTAAATTCCCAACCATTCATGC
TGGTAGAGCAGTTTACGAATGTCTTCGTGGTGGACTTGATTTTACTAAAGATGATGAGAACGTAAATTCCCAACCATTCATGC
GCTGGAGAGATCGTTTCTTGTTTTGTGCAGAAGCAATTTATAAAGCTCAGACCGAGACGGGTGAAATTAAGGGACATT
GCTGGAGAGATCGTTTCTTGTTTTGTGCAGAAGCAATTTATAAAGCTCAGACCGAGACGGGTGAAATTAAGGGACATT
195
Hypericum japonicum
P6876 / 42
JF949980
Aligned with
Hypericum japonicum
GQ436683
ribulose-1,5-bisphosphate carboxylase/oxygenase large subunit (rbcL) gene
Hypericaceae
GTTGGATTCAAGGCCGGCGTTAAAGAGTATAAATTGACTTATTATACTCCTGAATATGAAACCAAAGATACTGATATCTTGGC
GTTGGATTCAAGGCCGGCGTTAAAGAGTATAAATTGACTTATTATACTCCTGAATATGAAACCAAAGATACTGATATCTTGGC
AGCATTTCGAGTAACTCCTCAACCTGGGGTTCCCCCGGAGGAAGCAGGAGCAGCGGTAGCTGCGGAATCTTCTACTGGTACCT
AGCATTTCGAGTAACTCCTCAACCTGGGGTTCCCCCGGAGGAAGCAGGAGCAGCGGTAGCTGCGGAATCTTCTACTGGTACCT
GGACAACTGTCTGGACGGATGGACTGACAAGTCTTGATCGTTATAAAGGACGATGCTACCACATTGAGCCTGTTGCTGGAGA
GGACAACTGTCTGGACGGATGGACTGACAAGTCTTGATCGTTATAAAGGACGATGCTACCACATTGAGCCTGTTGCTGGAGA
GGAAAATCAATATATATGTTATGTAGCTTACCCCTTAGACCTTTTTGAAGAAGGTTCTGTTACTAACATGTTTACTTCCATTGT
GGAAAATCAATATATATGTTATGTAGCTTACCCCTTAGACCTTTTTGAAGAAGGTTCTGTTACTAACATGTTTACTTCCATTGT
AGGGAATGTATTTGGATTCAAAGCCCTGCGTGCTCTACGCTTGGAGGATTTGCGAATCCCTACTGCTTATACGAAAACTTTCC
AGGGAATGTATTTGGATTCAAAGCCCTGCGTGCTCTACGCTTGGAGGATTTGCGAATCCCTACTGCTTATACGAAAACTTTCC
AAGGTCCGCCTCATGGGATCCAAGTTGAAAGAGATAAATTGAACAAGTATGGCCGCCCGCTATTAGGTTGTACTATTAAACCT
AAGGTCCGCCTCATGGGATCCAAGTTGAAAGAGATAAATTGAACAAGTATGGCCGCCCGCTATTAGGTTGTACTATTAAACCT
AAATTGGGGTTATCCGCTAAGAATTACGGTCGAGCAGTTTATGAATGTCTGCGTGGTGGGCTTGATTTTACCAAAGATGATGA
AAATTGGGGTTATCCGCTAAGAATTACGGTCGAGCAGTTTATGAATGTCTGCGTGGTGGGCTTGATTTTACCAAAGATGATGA
GAACGTGAACTCCCAACCATTTAT
GAACGTGAACTCCCAACCATTTAT
Belamcanda chinensis
P6843 / 09
JF949995
Aligned with
Belamcanda chinensis
AJ309694
ribulose-1,5-bisphosphate carboxylase/oxygenase large subunit (rbcL) gene
Iridaceae
TAAAGCAAGTGTTGGATTTAAAGCTGGTGTTAAAGATTACAGATTGACTTATTATACTCCTGATTACGAAACCAAAGATACTG
TAAAGCAAGTGTTGGATTTAAAGCTGGTGTTAAAGATTACAGATTGACTTATTATACTCCTGATTACGAAACCAAAGATACTG
ATATCTTGGCAGCATTTCGAGTAACTCCTCAACCCGGAGTTCCTGCTGAAGAAGCGGGGGCCGCGGTAGCTGCCGAATCTTCT
ATATCTTGGCAGCATTTCGAGTAACTCCTCAACCCGGAGTTCCTGCTGAAGAAGCGGGGGCCGCGGTAGCTGCCGAATCTTCT
ACTGGTACATGGACAACAGTGTGGACTGATGGACTTACCAGTCTTGATCGTTACAAAGGACGATGCTACCACATCGAGGCCG
ACTGGTACATGGACAACAGTGTGGACTGATGGACTTACCAGTCTTGATCGTTACAAAGGACGATGCTACCACATCGAGGCCG
TTGTTGGGGAGGAAAATCAATATATTGCTTATGTAGCTTATCCTTTAGACCTTTTTGAAGAAGGTTCTGTTACTAACATGTTTA
TTGTTGGGGAGGAAAATCAATATATTGCTTATGTAGCTTATCCTTTAGACCTTTTTGAAGAAGGTTCTGTTACTAACATGTTTA
CTTCCATTGTGGGTAACGTATTTGGTTTCAAAGCCCTACGAGCTCTACGTCTGGAAGATCTGCGAATTCCTCCTGCTTATTCCA
CTTCCATTGTGGGTAACGTATTTGGTTTCAAAGCCCTACGAGCTCTACGTCTGGAAGATCTGCGAATTCCTCCTGCTTATTCCA
AAACTTTCCAAGGCCCGCCTCATGGCATCCAGGTTGAAAGAGATAAATTGAACAAGTATGGTCGTCCCCTATTGGGATGTACT
AAACTTTCCAAGGCCCGCCTCATGGCATCCAGGTTGAAAGAGATAAATTGAACAAGTATGGTCGTCCCCTATTGGGATGTACT
ATTAAACCTAAATTGGGATTATCCGCAAAAAACTACGGTAGGGCGGTTTATGAATGTCTACGCGGTGGGCTTGATTTTACCAA
ATTAAACCTAAATTGGGATTATCCGCAAAAAACTACGGTAGGGCGGTTTATGAATGTCTACGCGGTGGGCTTGATTTTACCAA
GGATGATGAAAACGTGAACTCACAACCTTTTATGCGTTGGAGAGACCGTTTCGTATTTTGCGCTGAAGCAATTTATAAAGCGC
GGATGATGAAAACGTGAACTCACAACCTTTTATGCGTTGGAGAGACCGTTTCGTATTTTGCGCTGAAGCAATTTATAAAGCGC
AAGCCGAAACAGGTGAAATCAAAGGACATTACTTGAATGC
AAGCCGAAACAGGTGAAATCAAAGGACATTACTTGAATGC
Mentha haplocalyx
P6884 / 50
JF949984
Aligned with
Mentha longifolia
AY570390
ribulose-1,5-bisphosphate carboxylase/oxygenase large subunit (rbcL) gene
Lamiaceae
TTCAAAGCGGGTGTTAAAGAGTACAAATTGACTTATTATACTCCTGAATACGAAACCAAAGATACTGATATCTTGGCAGCATT
TTCAAAGCGGGTGTTAAAGAGTACAAATTGACTTATTATACTCCTGAATACGAAACCAAAGATACTGATATCTTGGCAGCATT
CCGAGTAACTCCTCAACCCGGAGTTCCGCCTGAAGAAGCAGGGGCCGCGGTAGCTGCCGAATCTTCTACTGGTACATGGACA
CCGAGTAACTCCTCAACCTGGAGTTCCGCCTGAAGAAGCAGGGGCCGCGGTAGCTGCCGAATCTTCTACTGGTACATGGACA
196
ACTGTGTGGACCGATGGACTTACCAGCCTTGATCGTTACAAAGGGCGATGCTACCACATTGAGCCCGTTCCTGGAGAAAAAG
ACTGTGTGGACCGATGGACTTACCAGCCTTGATCGTTACAAAGGGCGATGCTACCACATTGAGCCCGTTCCTGGAGAAAAAG
ATCAATATATCTGTTATGTAGCTTACCCTTTAGACCTTTTTGAAGAAGGTTCTGTTACTAACATGTTTACTTCCATTGTAGGAA
ATCAATATATCTGTTATGTAGCTTACCCTTTAGACCTTTTTGAAGAAGGTTCTGTTACTAACATGTTTACTTCCATTGTAGGAA
ATGTATTTGGATTCAAAGCCCTACGTGCTCTACGTCTGGAAGATCTGCGAATTCCTGTTGCTTATGTTAAAACTTTCCAAGGCC
GTGTATTTGGATTCAAAGCCCTACGTGCTCTACGTCTGGAAGATCTGCGAATTCCTGTTGCTTATGTTAAAACTTTCCAAGGCC
CGCCTCATGGGATCCAAGTTGAGAGAGATAAATTGAACAAGTATGGTCGTCCTCTGCTGGGATGTACTATTAAACCTAAATTG
CGCCTCATGGGATCCAAGTTGAGAGAGATAAATTGAACAAGTATGGTCGTCCTCTGCTGGGATGTACTATTAAACCTAAATTG
GGGTTATCTGCTAAAAACTACGGTAGAGCGGTTTATGAATGTCTTCGCGGTGGACTTGATTTTACCAAAGATGATGAGAACGT
GGGTTATCTGCTAAAAACTACGGTAGAGCGGTTTATGAATGTCTTCGCGGTGGACTTGATTTTACCAAAGATGATGAGAACGT
GAACTCCCAGCCATTTATGCGTTGGAGAGACCGCT
GAACTCCCAGCCATTTATGCGTTGGAGAGACCGCT
Prunella vulgaris
P6896 / 62
JF950013
Aligned with
Prunella vulgaris
AY395556
ribulose-1,5-bisphosphate carboxylase/oxygenase large subunit (rbcL) gene
Lamiaceae
TTCAAAGCGGGTGTTAAAGAGTACAAATTGACTTATTATACTCCTGAATATGAAACCAAAGATACTGATATCTTGGCAGCATT
TTCAAAGCGGGTGTTAAAGAGTACAAATTGACTTATTATACTCCTGAATATGAAACCAAAGATACTGATATCTTGGCAGCATT
CCGAGTAACTCCTCAACCTGGAGTTCCGCCTGAAGAAGCAGGGGCCGCAGTAGCTGCGGAATCTTCTACTGGTACATGGACA
CCGAGTAACTCCTCAACCTGGAGTTCCGCCTGAAGAAGCAGGGGCCGCAGTAGCTGCGGAATCTTCTACTGGTACATGGACA
ACTGTGTGGACCGATGGACTTACCAGCCTTGATCGTTACAAAGGGCGATGCTACCACATTGAGCCCGTTCCTGGAGAAAAAG
ACTGTGTGGACCGATGGACTTACCAGCCTTGATCGTTACAAAGGGCGATGCTACCACATTGAGCCCGTTCCTGGAGAAAAAG
ATCAATATATCTGTTATGTAGCTTACCCTTTAGACCTTTTTGAAGAAGGTTCTGTTACTAACATGTTTACTTCCATTGTAGGAA
ATCAATATATCTGTTATGTAGCTTACCCTTTAGACCTTTTTGAAGAAGGTTCTGTTACTAACATGTTTACTTCCATTGTAGGAA
ATGTATTTGGATTCAAAGCCCTACGTGCTCTACGTCTGGAAGATCTGCGAATTCCTCCTGCTTATATTAAAACTTTCCAAGGCC
ATGTATTTGGATTCAAAGCCCTACGTGCTCTACGTCTGGAAGATCTGCGAATTCCTCCTGCTTATATTAAAACTTTCCAAGGCC
CGCCTCATGGGATCCAAGTTGAGAGAGATAAATTGAACAAGTATGGTCGTCCTCTGCTGGGATGTACTATTAAACCTAAATTG
CGCCTCATGGGATCCAAGTTGAGAGAGATAAATTGAACAAGTATGGTCGTCCTCTGCTGGGATGTACTATTAAACCTAAATTG
GGATTATCTGCTAAAAACTATGGTAGAGCGGTTTATGAATGTCTTCGCGGTGGACTTGATTTTACCAAAGATGATGAGAACGT
GGATTATCTGCTAAAAACTATGGTAGAGCGGTTTATGAATGTCTTCGCGGTGGACTTGATTTTACCAAAGATGATGAGAACGT
GAACTCCCAGCCATTTATGCGTTGGAGAGACCGCT
GAACTCCCAGCCATTTATGCGTTGGAGAGACCGCT
Scutellaria baicalensis
P6903 / 69
JF950017
Aligned with
Scutellaria baicalensis
FJ513152
ribulose-1,5-bisphosphate carboxylase/oxygenase large subunit (rbcL) gene
Lamiaceae
TTCAAAGCGGGTGTTAAAGAGTACAAATTGACTTATTATACCCCTGAATATGAAACCAAGGATACTGATATCTTGGCAGCATT
TTCAAAGCGGGTGTTAAAGAGTACAAATTGACTTATTATACCCCTGAATATGAAACCAAGGATACTGATATCTTGGCAGCATT
CCGAGTAACTCCTCAACCTGGAGTTCCGCCTGAAGAAGCAGGGGCCGCGGTAGCTGCCGAATCTTCTACTGGTACATGGACA
CCGAGTAACTCCTCAACCTGGAGTTCCGCCTGAAGAAGCAGGGGCCGCGGTAGCTGCCGAATCTTCTACTGGTACATGGACA
ACTGTGTGGACCGATGGACTTACCAGCCTTGATCGTTACAAAGGGCGATGCTACCACATTGAGCCCGTTATTGGAGAAAAAG
ACTGTGTGGACCGATGGACTTACCAGCCTTGATCGTTACAAAGGGCGATGCTACCACATTGAGCCCGTTATTGGAGAAAAAG
ATCAATATATATGTTATGTAGCTTACCCCTTAGACCTTTTTGAAGAAGGTTCTGTTACTAACATGTTTACTTCCATTGTAGGAA
ATCAATATATATGTTATGTAGCTTACCCCTTAGACCTTTTTGAAGAAGGTTCTGTTACTAACATGTTTACTTCCATTGTAGGAA
ATGTATTTGGATTCAAAGCCCTACGTGCTCTACGTCTGGAAGATCTGCGAATCCCTCCTGCTTATGTTAAAACTTTCCAAGGCC
ATGTATTTGGATTCAAAGCCCTACGTGCTCTACGTCTGGAAGATCTGCGAATCCCTCCTGCTTATGTTAAAACTTTCCAAGGCC
CGCCTCATGGGATCCAAGTTGAGAGAGATAAATTGAACAAGTATGGTCGTCCTCTGTTGGGATGTACTATTAAACCAAAATTG
CGCCTCATGGGATCCAAGTTGAGAGAGATAAATTGAACAAGTATGGTCGTCCTCTGTTGGGATGTACTATTAAACCAAAATTG
GGGTTATCTGCTAAAAACTATGGTAGAGCAGTTTATGAATGTCTTCGCGGTGGACTTGATTTTACCAAAGATGATGAAAACGT
GGGTTATCTGCTAAAAACTATGGTAGAGCAGTTTATGAATGTCTTCGCGGTGGACTTGATTTTACCAAAGATGATGAAAACGT
197
GAACTCCCAGCCATTTATGCGTTGGAGAGATCGCT
GAACTCCCAGCCATTTATGCGTTGGAGAGATCGCT
Cinnamomum cassia
P6853 / 19
JF950023
Aligned with
Cinnamomum aromaticum
HM019459
ribulose-1,5-bisphosphate carboxylase/oxygenase large subunit (rbcL) gene
Lauraceae
GACTATGAAACCAAAGATACTGATATTTTGGCAGCATTTCGAGTAACTCCTCAACCCGGAGTTCCACCTGAGGAAGCAGGGG
GACTATGAAACCAAAGATACTGATATTTTGGCAGCATTTCGAGTAACTCCTCAACCCGGAGTTCCACCTGAGGAAGCAGGGG
CTGCGGTAGCTGCCGAATCTTCTACTGGTACATGGACAACTGTGTGGACCGATGGACTTACCAGCCTTGATCGTTACAAAGGA
CTGCGGTAGCTGCCGAATCTTCTACTGGTACATGGACAACTGTGTGGACCGATGGACTTACCAGCCTTGATCGTTACAAAGGA
CGATGCTACCACATCGAGCCCGTTCCTGGGGAGGAAACTCAATTTATTGCCTATGTAGCTTACCCTTTAGACCTTTTTGAAGA
CGATGCTACCACATCGAGCCCGTTCCTGGGGAGGAAACTCAATTTATTGCCTATGTAGCTTACCCCTTAGACCTTTTTGAAGA
AGGTTCTGTTACGAACATGTTTACTTCTATTGTGGGTAATGTATTTGGGTTCAAAGCTCTACGAGCTCTACGTCTGGAGGATCT
AGGTTCTGTTACGAACATGTTTACTTCTATTGTGGGTAATGTATTTGGGTTCAAAGCTCTACGAGCTCTACGTCTGGAGGATCT
GCGAATTCCTCCTGCTTATTCCAAAACTTTCCAAGGCCCGCCCCATGGCATCCAAGTTGAGAGAGATAAATTGAACAAGTATG
GCGAATTCCTCCTGCTTATTCCAAAACTTTCCAAGGCCCGCCCCATGGCATCCAAGTTGAGAGAGATAAATTGAACAAGTATG
GTCGTCCCCTATTGGGATGTACTATTAAACCAAAATTGGGGTTATCCGCCAAGAACTACGGTAGAGCGGTTTATGAATGTCT
GTCGTCCCCTATTGGGATGTACTATTAAACCAAAATTGGGGTTATCCGCCAAGAACTACGGTAGAGCGGTTTATGAATGTCT
Taxillus chinensis
P6909 / 75
JF949992
Aligned with
Atkinsonia ligustrina
EF464526
ribulose-1,5-bisphosphate carboxylase/oxygenase large subunit (rbcL) gene
Loranthaceae
GAGTACAAATTGACTTATTATACTCCTGATTATGAAACTAAAGATACTGATATCTTGGCAGCATTCCGAGTAACTCCTCAACC
GAGTACAAATTGACTTATTATACTCCTGATTATGAAACTAAAGATACTGATATCTTGGCAGCATTCCGAGTAACTCCTCAACC
TGGAGTTCCACCCGAGGAAGCAGGGGCCGCGGTAGCTGCCGAATCTTCTACTGGTACATGGACCACTGTGTGGACCGATGGA
TGGAGTTCCACCCGAGGAAGCGGGGGCCGCGGTAGCTGCCGAATCTTCTACTGGTACATGGACCACTGTGTGGACCGATGGA
CTTACCAGCCTTGATCGTTACAAAGGACGATGCTATCACATCGAGCCCGTTGCTGGAGAAGAAAATCAATATATTGCTTATGT
CTTACTAGCCTTGATCGTTACAAAGGACGATGCTATCACATCGAGCCCGTTGCTGGAGAAGAAAATCAATATATTGCTTATGT
AGCTTACCCCTTAGACCTTTTTGAAGAAGGTTCTGTTACTAACATGTTTACTTCCATTGTGGGTAATGTATTTGGGTTTAAAGC
AGCTTACCCTTTAGACCTTTTTGAAGAAGGTTCTGTTACTAACATGTTTACTTCCATTGTGGGTAATGTATTTGGGTTTAAAGC
CCTACGTGCTCTACGTCTGGAGGATCTTCGAATCCCTCCTGCTTATTCTAAAACTTTCCAAGGCCCGCCTCATGGTATCCAAGT
CCTACGTGCTCTACGTCTGGAGGATCTTCGAATCCCCCCTGCTTATTCTAAAACTTTCCAAGGCCCGCCTCATGGTATCCAAGT
TGAGAGAGATAAATTGAACAAGTATGGTCGTCCTCTATTGGGATGTACTATTAAACCTAAATTGGGGTTATCCGCTAAGAACT
TGAGAGAGATAAATTGAACAAGTATGGTCGTCCTCTATTGGGATGTACTATTAAACCCAAATTGGGGTTATCCGCTAAGAACT
ACGGTCGAGCGGTTTATGAGTGTCTTCGCGGTGGACTTGATTTTACCAAAGATGATGAGAACGTAAACTCCCAACCATTTATG
ACGGTCGAGCGGTTTATGAGTGTCTTCGCGGTGGACTTGATTTTACCAAAGATGATGAGAACGTAAACTCCCAACCATTTATG
CGTTGGAGAGACCGTTTCTTATTTTGTGCCGAAGCTCTTTATAAAGCACAGGCCGAAACTGGCGAAATCAAAGGGCATTATTT
CGTTGGAGAGACCGTTTCTTATTTTGTGCCGAAGCTCTTTATAAAGCACAGGCCGAAACTGGCGAAATCAAAGGGCATTATTT
GAATGCTACTGCAG
GAATGCTACTGCAG
Punica granatum
P6897 / 63
JF950014
Aligned with
Punica granatum
GQ436656
ribulose-1,5-bisphosphate carboxylase/oxygenase large subunit (rbcL) gene
Lythraceae
TAAAGCAAGTGTTGGATTCAAAGCCGGTGTTAAAGATTATAAACTGACTTATTATACTCCTGAATATGAAACCAAAGATACTG
TAAAGCAAGTGTTGGATTCAAAGCCGGTGTTAAAGATTATAAACTGACTTATTATACTCCTGAATATGAAACCAAAGATACTG
ATATCTTGGCAGCATTCCGAGTAACTCCTCAACCTGGAGTTCCGCCTGAGGAAGCAGGGGCTGCAGTAGCCGCTGAATCTTCT
ATATCTTGGCAGCATTCCGAGTAACTCCTCAACCTGGAGTTCCGCCTGAGGAAGCAGGGGCTGCAGTAGCCGCTGAATCTTCT
ACTGGTACCTGGACAACTGTGTGGACCGATGGGCTTACCAGCCTTGATCGTTATAAAGGAAGATGCTACCACATCGAGCCTGT
ACTGGTACCTGGACAACTGTGTGGACCGATGGGCTTACCAGCCTTGATCGTTATAAAGGAAGATGCTACCACATCGAGCCTGT
198
TGCTGGAGAAGAAAATCAATATATATGTTATGTAGCTTACCCTTTAGACCTTTTTGAAGAAGGTTCTGTTACTAATATGTTTAC
TGCTGGAGAAGAAAATCAATATATATGTTATGTAGCTTACCCTTTAGACCTTTTTGAAGAAGGTTCTGTTACTAATATGTTTAC
TTCCATTGTGGGTAATGTATTTGGGTTCAAAGCCCTGCGCGCTCTACGTCTGGAGGATCTGAGAATCCCTACTGCATATACTA
TTCCATTGTGGGTAATGTATTTGGGTTCAAAGCCCTGCGCGCTCTACGTCTGGAGGATCTGAGAATCCCTACTGCATATACTA
AAACTTTCCAAGGCCCGCCTCATGGTATCCAAGTTGAGAGAGATAAATTGAACAAGTATGGTCGTCCCCTATTGGGATGTACT
AAACTTTCCAAGGCCCGCCTCATGGTATCCAAGTTGAGAGAGATAAATTGAACAAGTATGGTCGTCCCCTATTGGGATGTACT
ATTAAACCTAAATTGGGGTTATCCGCTAAGAACTACGGTAGAGCGGTTTATGAATGTCTTCGTGGTGGACTTGATTTTACGAA
ATTAAACCTAAATTGGGGTTATCCGCTAAGAACTACGGTAGAGCGGTTTATGAATGTCTTCGTGGTGGACTTGATTTTACGAA
GGATGATGAGAACGTGAACTCACAACCATTTATGCGTTGGAGAGACCGTTTCTTATTTTGTGCCGAAGCACTTTATAAAGCAC
GGATGATGAGAACGTGAACTCACAACCATTTATGCGTTGGAGAGACCGTTTCTTATTTTGTGCCGAAGCACTTTATAAAGCAC
AGGCTGAAACTGGTGAAATCAAGGGGCATTACTTGAAT
AGGCTGAAACTGGTGAAATCAAGGGGCATTACTTGAAT
Magnolia officinalis
P6882 / 48
JF950008
Aligned with
Magnolia officinalis
AY008933
ribulose-1,5-bisphosphate carboxylase/oxygenase large subunit (rbcL) gene
Magnoliaceae
TTGACTTATTATACTCCTGAATATGAAACCAAAGATACTGATATCTTGGCAGCATTCCGAGTAACTCCTCAACCCGGAGTTCC
TTGACTTATTATACTCCTGAATATGAAACCAAAGATACTGATATCTTGGCAGCATTCCGAGTAACTCCTCAACCCGGAGTTCC
ACCTGAGGAAGCAGGGGCTGCGGTAGCTGCCGAATCTTCTACTGGTACATGGACAACTGTGTGGACCGATGGACTTACCAGC
ACCTGAGGAAGCAGGGGCTGCGGTAGCTGCCGAATCTTCTACTGGTACATGGACAACTGTGTGGACCGATGGACTTACCAGC
CTTGATCGTTACAAAGGACGATGCTACCACATCGAGCCCGTTCCTGGGGAGGAAAGTCAATTTATTGCTTATGTAGCTTACCC
CTTGATCGTTACAAAGGACGATGCTACCACATCGAGCCCGTTCCTGGGGAGGAAAGTCAATTTATTGCTTATGTAGCTTACCC
TTTAGACCTTTTTGAAGAAGGTTCTGTTACTAACATGTTTACTTCCATTGTAGGTAATGTATTTGGGTTCAAAGCCCTACGAGC
TTTAGACCTTTTTGAAGAAGGTTCTGTTACTAACATGTTTACTTCCATTGTAGGTAATGTATTTGGGTTCAAAGCCCTACGAGC
TCTACGTCTGGAGGATCTGCGAATTCCTACTGCTTATGTCAAAACTTTCCAAGGCCCGCCCCATGGCATCCAAGTTGAGAGAG
TCTACGTCTGGAGGATCTGCGAATTCCTACTGCTTATGTCAAAACTTTCCAAGGCCCGCCCCATGGCATCCAAGTTGAGAGAG
ATAAATTGAACAAGTATGGTCGTCCACTATTGGGATGTACTATTAAACCAAAATTGGGGTTATCCGCCAAGAACTACGGTAG
ATAAATTGAACAAGTATGGTCGTCCACTATTGGGATGTACTATTAAACCAAAATTGGGGTTATCCGCCAAGAACTACGGTAG
GGCGGTTTATGAATGTCTCCGTGGTGGACTTGATTTTACCAAGGATGATGAGAACGTGAACTCCCAACCATTTATGCGTTGGA
GGCGGTTTATGAATGTCTCCGTGGTGGACTTGATTTTACCAAGGATGATGAGAACGTGAACTCCCAACCATTTATGCGTTGGA
GAGACCGTTTCTTATTTTGTGCCGAAGCACTTTATAAAGCGCAGGCCGAAACAGGTGAAATCAAAGGACATTACTTGAATGCT
GAGACCGTTTCTTATTTTGTGCCGAAGCACTTTATAAAGCGCAGGCCGAAACAGGTGAAATCAAAGGACATTACTTGAATGCT
ACTGCAGGTACATG
ACTGCAGGTACATG
Paris polyphylla
P6888 / 54
JF950010
Aligned with
Paris polyphylla
D28155
ribulose-1,5-bisphosphate carboxylase/oxygenase large subunit (rbcL) gene
Melanthiaceae
TGCTGGATTCAAAGCTGGTGTTAAAGATTACAAATTGAATTATTATACTCCTGACTACACACCCAAAGATACTGATATCTTGG
TGCTGGATTCAAAGCTGGTGTTAAAGATTACAAATTGAATTATTATACTCCTGACTACACACCCAAAGATACTGATATCTTGG
CAGCATTCCGAGTAGCTCCTCAACCCGGAGTTCCGGCTGAAGAAGCGGGCGCTGCGGTAGCAGCCGAATCTTCTACTGGTAC
CAGCATTCCGAGTAGCTCCTCAACCCGGAGTTCCGGCTGAAGAAGCGGGCGCTGCGGTAGCAGCCGAATCTTCTACTGGTAC
ATGGACAACTGTGTGGACTGATGGACTTACCAGTCTTGATCGTTACAAAGGACGATGCTACCAGATCGAGAGCGTTGCTGGG
ATGGACAACTGTGTGGACTGATGGACTTACCAGTCTTGATCGTTACAAAGGACGATGCTACCAGATCGAGAGCGTTGCTGGG
GAGGAAAATCAATATATTGCTTATGTAGCTTATCCTTTAGACCTTTTTGAAGAAGGTTCTGTTACTAATATGTTTACTTCCATT
GAGGAAAATCAATATATTGCTTATGTAGCTTATCCTTTAGACCTTTTTGAAGAAGGTTCTGTTACTAATATGTTTACTTCCATT
GTGGGTAATGTATTTGGGTTCAAAGCCCTACGAGCTCTACGTCTGGAGGATCTGCGAATTCCTCCTGCTTATTCAAAAACTTTT
GTGGGTAATGTATTTGGGTTCAAAGCCCTACGAGCTCTACGTCTGGAGGATCTGCGAATTCCTCCTGCTTATTCAAAAACTTTT
CAAGGCCCGCCCCATGGTATCCAGGTTGAAAGAGATAAATTGAACAAATACGGTCGTCCCCTATTGGGATGTACAATTAAAC
CAAGGCCCGCCCCATGGTATCCAGGTTGAAAGAGATAAATTGAACAAATACGGTCGTCCCCTATTGGGATGTACAATTAAAC
199
CAAAATTGGGATTATCCGCAAAGAACTACGGTAGAGCAGTTTATGAGTGTCTACGCGGTGGACTTGATTTTACTAAGGATGAT
CAAAATTGGGATTATCCGCAAAGAACTACGGTAGAGCAGTTTATGAGTGTCTACGCGGTGGACTTGATTTTACTAAGGATGAT
GAAAACGTGAACTCACAACCATTTATGCGTTGGAGAGACCGTTTCTTATTTTGTGCCGAAGCACTTTATAAGGCGCAGGCCGA
GAAAACGTGAACTCACAACCATTTATGCGTTGGAGAGACCGTTTCTTATTTTGTGCCGAAGCACTTTATAAGGCGCAGGCCGA
AACGGGTGAAATCAAAGGGCATTACTTGAATGC
AACGGGTGAAATCAAAGGGCATTACTTGAATGC
Lysimachia christinae
P6881 / 47
JF949983
Aligned with
Lysimachia vulgaris
AF421095
ribulose-1,5-bisphosphate carboxylase/oxygenase large subunit (rbcL) gene
Myrsinaceae
GTTAAAGATTACAAATTGACTTATTATACTCCTGAGTATGCAACCAAGGATACTGATATCTTGGCAGCATTCCGAGTAACTCC
GTTAAAGATTACAAATTGACTTATTATACTCCTGAGTATGTAACCAAGGATACTGATATCTTGGCAGCATTCCGAGTAACTCC
TCAACCAGGAGTTCCGCCCGAAGAAGCAGGGGCCGCAGTAGCTGCCGAATCTTCTACTGGTACATGGACAACCGTGTGGACC
TCAACCTGGAGTTCCGCCCGAAGAAGCAGGGGCCGCAGTAGCTGCCGAATCTTCTACTGGTACATGGACAACCGTGTGGACC
GATGGACTTACTAGCCTTGATCGTTACAAAGGGCGATGCTACAACATCGAGCCTGTTGCTGGAGAAGAAAATCAATATATTTG
GATGGACTTACTAGCCTTGATCGTTACAAAGGGCGATGCTACGGCATCGAGCCTGTTGCTGGAGAAGAAAATCAATTTATTGC
TTATGTAGCTTATCCTTTAGACCTTTTTGAAGAAGGTTCTGTTACTAACATGTTTACTTCTATTGTTGGTAATGTATTTGGGTTC
TTATGTAGCTTATCCTTTAGACCTTTTTGAAGAAGGTTCTGTTACTAACATGTTTACTTCTATTGTGGGTAATGTATTTGGGTTC
AAAGCCCTGCGCGCTCTACGTCTGGAAGATCTTCGAATCCCGCCTGCGTATGTTAAAACTTTCCAAGGACCGCCTCATGGTAT
AAAGCCCTGCGCGCTCTACGTCTGGAAGATCTGCGAATCCCGCCTGCGTATGTTAAAACTTTCCAAGGACCGCCTCATGGTAT
CCAAGTTGAAAGAGATAAATTGAATAAATATGGTCGTCCACTGTTGGGATGTACTATTAAACCTAAATTGGGGTTATCCGCTA
CCAAGTTGAAAGAGATAAATTGAATAAATATGGTCGTCCACTGTTGGGATGTACTATTAAACCTAAATTGGGGTTATCCGCTA
AAAACTACGGTAGGGCTGTTTATGAATGTCTTCGTGGTGGACTTGATTTTACCAAAGATGATGAGAACGTGAACTCCCAACCA
AAAACTACGGTAGGGCTGTTTATGAATGTCTTCGTGGTGGACTTGATTTTACCAAAGATGATGAGAACGTGAACTCCCAACCA
TTTATGCGTTGGCGAGACCGTTTCTTATTTTGTGCCGAAGCTCTTTATAAAGCACAGGCTGAAACTGGTGAAATCAAAGGTCA
TTTATGCGTTGGCGAGACCGTTTCGTATTTTGTGCCGAAGCTATTTATAAAGCACAGTCTGAAACTGGTGAAATCAAAGGTCA
TTACTTGAATGCTACTGCAG
TTACTTGAATGCTACTGCAG
Ophioglossaceae Ophioglossum vulgatum
P6885 / 51
JF950009
Aligned with
Ophioglossum vulgatum
HQ676505
ribulose-1,5-bisphosphate carboxylase/oxygenase large subunit (rbcL) gene
GAGCTGCAGTAGCTGCTGAATCCTCCACGGGTACGTGGACTACCGTATGGACCGATGGGCTTACCAGTCTCGATCGTTACAAA
GAGCTGCAGTAGCTGCTGAATCCTCCACGGGTACGTGGACTACCGTATGGACCGATGGGCTTACCAGTCTCGATCGTTACAAA
GGTCGATGCTATGAAATCGAACCTGTTGCTGGGGAGGAAGATCAATACATTGCTTATGTAGCGTATCCCTTAGACCTTTTTGA
GGTCGATGCTATGAAATCGAACCTGTTGCTGGGGAGGAAGATCAATACATTGCTTATGTAGCGTATCCCTTAGACCTTTTTGA
AGAAGGTTCCGTCACCAACATGTTCACCTCCATTGTAGGTAACGTATTCGGATTCAAGGCATTGAAAGCTCTACGGTTGGAGG
AGAAGGTTCCGTCACCAACATGTTCACCTCCATTGTAGGTAACGTATTCGGATTCAAGGCATTGAAAGCTCTACGGTTGGAGG
ATTTACGAATTCCTCCTGCTTATTCTAAGACTTTCATGGGCCCTCCCCACGGTATCCAAGTCGAAAGGGATAAATTAAACAAA
ATTTACGAATTCCTCCTGCTTATTCTAAGACTTTCATGGGCCCTCCCCACGGTATCCAAGTCGAAAGGGATAAATTAAACAAA
TATGGTCGTCCCTTACTGGGGTGTACCATCAAACCCAAATTGGGATTATCTGCCAAGAATTATGGTAGAGCCGTTTATGAATG
TATGGTCGTCCCTTACTGGGGTGTACCATCAAACCCAAATTGGGATTATCTGCCAAGAATTATGGTAGAGCCGTTTATGAATG
CTTACGCGGTGGGCTCGACTTCACCAAAGATGATGAAAACGTAAATTCTCAACCATTTATGCGTTGGAGAGATCGTTTCGTAT
CTTACGCGGTGGGCTCGACTTCACCAAAGATGATGAAAACGTAAATTCTCAACCATTTATGCGTTGGAGAGATCGTTTCGTAT
TTGTGGCGGAAGCTCTTTTCAAATCTCAGGCGGAAACGGGAGAGATTAAGGGGCATTACTTAAATGCTAC
TTGTGGCGGAAGCTCTTTTCAAATCTCAGGCGGAAACGGGAGAGATTAAGGGGCATTACTTAAATGCTAC
200
Dendrobium loddigesii
P6860 / 26
JF949999
Aligned with
Dendrobium loddigesii
FJ216573
ribulose-1,5-bisphosphate carboxylase/oxygenase large subunit (rbcL) gene
Orchidaceae
TTGGATTTAAAGCTGGTGTTAAAGATTACAAATTGACTTATTATACTCCTGACTACGAAACCAAAGATACTGATATCTTGGCA
TTGGATTTAAAGCTGGTGTTAAAGATTACAAATTGACTTATTATACTCCTGACTACGAAACCAAAGATACTGATATCTTGGCA
GCATTCCGAGTAACTCCTCAACCGGGAGTTCCGCCTGAAGAAGCGGGGGCTGCGGTAGCTGCCGAATCTTCTACTGGTACATG
GCATTCCGAGTAACTCCTCAACCGGGAGTTCCGCCTGAAGAAGCGGGGGCTGCGGTAGCTGCCGAATCTTCTACTGGTACATG
GACAACTGTGTGGACTGATGGACTTACCAGTCTTGATCGTTACAAAGGACGATGCTACCACATCGAGGTCGTTGTTGGGGAG
GACAACTGTGTGGACTGATGGACTTACCAGTCTTGATCGTTACAAAGGACGATGCTACCACATCGAGGTCGTTGTTGGGGAG
GAAAATCAATATATTGCTTATGTAGCTTATCCTTTAGACCTTTTTGAAGAAGGTTCTGTTACTAACATGTTTACTTCCATTGTG
GAAAATCAATATATTGCTTATGTAGCTTATCCTTTAGACCTTTTTGAAGAAGGTTCTGTTACTAACATGTTTACTTCCATTGTG
GGTAATGTATTTGGTTTCAAAGCCCTGCGAGCTCTACGTCTGGAAGATCTGCGAATTCCTACTTCTTATTCCAAAACTTTCCAA
GGTAATGTATTTGGTTTCAAAGCCCTGCGAGCTCTACGTCTGGAAGATCTGCGAATTCCTACTTCTTATTCCAAAACTTTCCAA
GGTCCGCCTCATGGCATCCAAGTTGAAAGAGATAAATTGAACAAGTATGGTCGTCCCCTATTGGGATGTACTATTAAACCAAA
GGTCCGCCTCATGGCATCCAAGTTGAAAGAGATAAATTGAACAAGTATGGTCGTCCCCTATTGGGATGTACTATTAAACCAAA
ATTGGGATTATCCGCAAAAAACTACGGTAGAGCGGTTTATGAATGTCTACGGGGTGGACTTGATTTTACTAAGGATGATGAA
ATTGGGATTATCCGCAAAAAACTACGGTAGAGCGGTTTATGAATGTCTACGGGGTGGACTTGATTTTACTAAGGATGATGAA
AACGTAAATTCACAACCATTTATGCGTTGGAGAGATCGTTTCTTATTTTGTGCCGAAGCAATCTTTAAAGCGCAAGCCGAAAC
AACGTAAATTCACAACCATTTATGCGTTGGAGAGATCGTTTCTTATTTTGTGCCGAAGCAATCTTTAAAGCGCAAGCCGAAAC
GGGTGAAATTAAAGG
GGGTGAAATTAAAGG
Paeonia lactiflora
P6886 / 52
JF950026
Aligned with
Paeonia lactiflora
GQ436534
ribulose-1,5-bisphosphate carboxylase/oxygenase large subunit (rbcL) gene
Paeoniaceae
TATTATACTCCTGACTATAAAACCAAGGATACTGATATCTTGGCAGCATTCCGAGTAACTCCTCAACCCGGAGTTCCACCGGA
TATTATACTCCTGACTATAAAACCAAGGATACTGATATCTTGGCAGCATTCCGAGTAACTCCTCAACCCGGAGTTCCACCGGA
GGAAGCGGGGGCCGCGGTAGCTGCCGAATCTTCTACGGGTACTTGGACAACTGTGTGGACCGATGGACTTACCAGCCTTGAT
GGAAGCGGGGGCCGCGGTAGCTGCCGAATCTTCTACGGGTACTTGGACAACTGTGTGGACCGATGGACTTACCAGCCTTGAT
CGTTACAAAGGACGATGCTATCACATTGAGCCTGTTGCTGGAGAAGAAAATCAATTTATTGCTTATGTAGCCTACCCACTCGA
CGTTACAAAGGACGATGCTATCACATTGAGCCTGTTGCTGGAGAAGAAACTCAATTTATTGCTTATGTAGCCTACCCACTCGA
CCTTTTTGAAGAAGGTTCTGTTACTAACATGTTTACTTCCATTGTGGGTAATGTATTTGGGTTCAAAGCCTTGCGCGCCCTACG
CCTTTTTGAAGAAGGTTCTGTTACTAACATGTTTACTTCCATTGTGGGTAATGTATTTGGGTTCAAAGCCTTGCGCGCCCTACG
TCTGGAGGATCTGCGAATTCCTCCTGCTTATGTTAAAACTTTTCAAGGTCCGCCTCACGGCATCCAGGTTGAGAGAGATAAAT
TCTGGAGGATCTGCGAATTCCTACTGCTTATGTTAAAACTTTTCAAGGTCCGCCTCACGGCATCCAGGTTGAGAGAGATAAAT
TGAACAAGTATGGCCGTCCCCTATTGGGATGTACTATTAAACCTAAATTGGGATTATCCGCTAAAAACTACGGTAGAGCAGTT
TGAACAAGTATGGCCGTCCCCTATTGGGATGTACTATTAAACCTAAATTGGGATTATCCGCTAAAAACTACGGTAGAGCAGTT
TATGAATGTCTTCGCGGTGGGCTTGATTTTACCAAAGATGATGAAAACGTGAATTCCCAACCATTTATGCGTTGGAGAGATCG
TATGAATGTCTTCGCGGTGGGCTTGATTTTACCAAAGATGATGAAAACGTGAATTCCCAACCATTTATGCGTTGGAGAGATCG
TTTCTTATTTTGTGCAGAAGCTATTTATAAAGCACAAGCCGAAACAGGCGAAATCAAGGGGCATTACTTAAAT
TTTCTTATTTTGTGCAGAAGCTCTTTATAAAGCACAAGCCGAAACAGGCGAAATCAAGGGGCATTACTTAAAT
Cymbopogon distans
P6857 / 23
JF949974
Aligned with
Cymbopogon citratus
AM887872
ribulose-1,5-bisphosphate carboxylase/oxygenase large subunit (rbcL) gene
Poaceae
TTTAAAGCTGGTGTTAAGGATTATAAATTGACTTACTACACCCCGGAGTACGAAACCAAGGATACTGATATCTTGGCAGCATT
TTTAAAGCTGGTGTTAAGGATTATAAATTGACTTACTACACCCCGGAGTACGAAACCAAGGATACTGATATCTTGGCAGCATT
CCGAGTAACTCCTCAGCTCGGGGTTCCGCCTGAAGAAGCAGGAGCTGCAGTAGCTGCGGAATCTTCTACTGGTACATGGACA
CCGAGTAACTCCTCAGCTCGGGGTTCCGCCTGAAGAAGCAGGAGCTGCAGTAGCTGCGGAATCTTCTACTGGTACATGGACA
201
ACTGTTTGGACTGATGGACTTACCAGTCTTGATCGTTACAAAGGACGATGCTATCACATCGAGCCCGTTCCTGGGGACCCAGA
ACTGTTTGGACTGATGGACTTACCAGTCTTGATCGTTACAAAGGACGATGCTATCACATCGAGCCCGTTCCTGGGGACCCAGA
TCAATATATCTGTTATGTAGCTTATCCATTAGACCTATTTGAAGAGGGTTCTGTTACTAACATGTTTACTTCCATTGTGGGTAA
TCAATATATCTGTTATGTAGCTTATCCATTAGACCTATTTGAAGAGGGTTCTGTTACTAACATGTTTACTTCCATTGTGGGTAA
CGTATTTGGTTTCAAAGCCTTACGCGCTCTACGTTTGGAGGATCTACGAATTCCCCCTGCTTATGCAAAAACTTTCCAAGGTCC
CGTATTTGGTTTCAAAGCCTTACGCGCTCTACGTTTGGAGGATCTACGAATTCCCCCTGCTTATGCAAAAACTTTCCAAGGTCC
GCCTCACGGTATCCAAGTTGAAAGGGATAAGTTGAACAAGTACGGTCGTCCTTTATTGGGATGTACTATTAAACCAAAATTGG
GCCTCACGGTATCCAAGTTGAAAGGGATAAGTTGAACAAGTACGGTCGTCCTTTATTGGGATGTACTATTAAACCAAAATTGG
GATTATCCGCAAAAAATTATGGTAGAGCGTGTTATGAGTGTCTACGCGGTGGACTTGATTTTACCAAAGATGATGAAAACGTA
GATTATCCGCAAAAAATTATGGTAGAGCGTGTTATGAGTGTCTACGCGGTGGACTTGATTTTACCAAAGATGATGAAAACGTA
AACTCACAACCATTTATGCGCTGGAGAGACCGTTTTGTCTTTTGTGCCGAAGCAATTTATAAAGCACAAGCCGAAACCGGTGA
AACTCACAACCATTTATGCGCTGGAGAGACCGTTTTGTCTTTTGTGCCGAAGCAATTTATAAAGCACAAGCCGAAACCGGTGA
AATCAAGGGGCATTACTTGAATGCTACTGCAGGTACATGCGAA
AATCAAGGGGCATTACTTGAATGCGACTGCAGGTACATGCGAA
Fallopia japonica
P6894 / 60
JF950004
Aligned with
Fallopia japonica
AF297131
ribulose-1,5-bisphosphate carboxylase/oxygenase large subunit (rbcL) gene
Polygonaceae
GGATTCAAAGCTGGTGTTAAAGAATACAAATTGACTTATTATACTCCTGAGTATGAACCCCATGATCATGATATCTTGGCAGC
GGATTCAAAGCTGGTGTTAAAGAATACAAATTGACTTATTATACTCCTGAGTATGAACCCCATGATCATGATATCTTGGCAGC
ATTTCGAGTAACTCCTCAACCTGGAGTTCCGCCAGAAGAAGCAGGGGCCGCGGTAGCTGCCGAATCTTCTACTGGTACATGG
ATTTCGAGTAACTCCTCAACCTGGAGTTCCGCCAGAAGAAGCAGGGGCCGCGGTAGCTGCCGAATCTTCTACTGGTACATGG
ACAACCGTGTGGACCGATGGACTTACCAGCCTTGATCGTTACAAAGGACGATGCTACGACATCGAGCCTGTTCCTGGAGAAG
ACAACCGTGTGGACCGATGGACTTACCAGCCTTGATCGTTACAAAGGACGATGCTACGACATCGAGCCTGTTCCTGGAGAAG
AAAATCAATTTATTGCTTATGTAGCTTACCCATTAGACCTTTTTGAAGAGGGTTCTGTTACTAACATGTTTACTTCCATTGTGG
AAAATCAATTTATTGCTTATGTAGCTTACCCATTAGACCTTTTTGAAGAGGGTTCTGTTACTAACATGTTTACTTCCATTGTGG
GTAATGTATTTGGGTTCAAAGCCCTGCGTGCTCTTCGTTTGGAGGATTTGCGAATCCCTCCTGCTTATACGAAAACTTTCCAAG
GTAATGTATTTGGGTTCAAAGCCCTGCGTGCTCTTCGTTTGGAGGATTTGCGAATCCCTCCTGCTTATACGAAAACTTTCCAAG
GCCCGCCTCACGGTATCCAAGTTGAAAGAGATAAATTGAACAAATATGGACGTCCCCTATTGGGATGTACTATTAAACCGAA
GCCCGCCTCACGGTATCCAAGTTGAAAGAGATAAATTGAACAAATATGGACGTCCCCTATTGGGATGTACTATTAAACCGAA
ATTGGGGTTGTCCGCTAAGAACTACGGTCGGGCAGTTTATGAATGTCTTCGTGGCGGACTTGATTTTACCAAAGATGATGAAA
ATTGGGGTTGTCCGCTAAGAACTACGGTCGGGCAGTTTATGAATGTCTTCGTGGCGGACTTGATTTTACCAAAGATGATGAAA
ACGTGAACTCCCAACCATTTATGCGTTGGAGAGACCGTTTCTTATTCTGTGCCGAAGCTATTTTTAAATCACAGTGCGAAACA
ACGTGAACTCCCAACCATTTATGCGTTGGAGAGACCGTTTCTTATTCTGTGCCGAAGCTATTTTTAAATCACAGTGCGAAACA
GGTGAAATCAAAGGACATTACTTGAAT
GGTGAAATCAAAGGACATTACTTGAAT
Fallopia multiflora
P6895 / 61
JF949987
Aligned with
Fallopia multiflora
HM357901
ribulose-1,5-bisphosphate carboxylase/oxygenase large subunit (rbcL) gene
Polygonaceae
TTTCGAGTAACTCCTCAACCTGGAGTTCCGCCAGAAGAAGCAGGGGCCGCGGTAGCTGCCGAATCTTCTACTGGTACATGGAC
TTTCGAGTAACTCCTCAACCTGGAGTTCCGCCAGAAGAAGCAGGGGCCGCGGTAGCTGCCGAATCTTCTACTGGTACATGGAC
AACCGTGTGGACCGATGGACTTACCAGCCTTGATCGTTACAAAGGACGATGCTACGACATCGAGCCTGTTCCTGGAGAAGAA
AACCGTGTGGACCGATGGACTTACCAGCCTTGATCGTTACAAAGGACGATGCTACGACATCGAGCCTGTTCCTGGAGAAGAA
AATCAATTTATTGCTTATGTAGCTTACCCATTAGACCTTTTTGAAGAAGGTTCTGTTACTAACATGTTTACTTCCATTGTGGGT
AATCAATTTATTGCTTATGTAGCTTACCCATTAGACCTTTTTGAAGAAGGTTCTGTTACTAACATGTTTACTTCCATTGTGGGT
AATGTATTTGGGTTCAAAGCCCTGCGTGCTCTTCGTTTGGAGGATTTGCGAATCCCTCCTGCTTATACGAAAACTTTCCAAGGC
AATGTATTTGGGTTCAAAGCCCTGCGTGCTCTTCGTTTGGAGGATTTGCGAATCCCTCCTGCTTATACGAAAACTTTCCAAGGC
CCGCCTCATGGTATCCAAGTTGAGAGAGATAAATTGAACAAATATGGACGTCCCCTATTGGGATGTACTATTAAACCGAAATT
CCGCCTCATGGTATCCAAGTTGAGAGAGATAAATTGAACAAATATGGACGTCCCCTATTGGGATGTACTATTAAACCGAAATT
202
GGGGTTGTCCGCTAAGAACTACGGTCGGGCAGTTTATGAATGTCTTCGTGGCGGACTTGATTTTACCAAAGATGATGAAAACG
GGGGTTGTCCGCTAAGAACTACGGTCGGGCAGTTTATGAATGTCTTCGTGGCGGACTTGATTTTACCAAAGATGATGAAAACG
TGAACTCCCAACCATTTATGCGTTGGAGAGACCGTTTCTTATTCTGTGCCGAAGCTATTTTTAAATCACAGTGCGAAACAGGT
TGAACTCCCAACCATTTATGCGTTGGAGAGACCGTTTCTTATTCTGTGCCGAAGCTATTTTTAAATCACAGTGCGAAACAGGT
GAAATCAAAGGACATTACTTGAAT
GAAATCAAAGGACATTACTTGAAT
Polygonum aviculare
P6893 / 59
JF950012
Aligned with
Polygonum aviculare
EU554012
ribulose-1,5-bisphosphate carboxylase/oxygenase large subunit (rbcL) gene
Polygonaceae
GGATTCAAAGCTGGTGTTAAAGAATACAAATTGACTTATTATACTCCTGACTATGAACCGCATGATCATGATATCTTGGCAGC
GGATTCAAAGCTGGTGTTAAAGAATACAAATTGACTTATTATACTCCTGACTATGAACCGCATGATCATGATATCTTGGCAGC
ATTTCGAGTAACTCCTCAACCTGGAGTTCCGCCAGAAGAAGCAGGGGCCGCGGTAGCTGCCGAATCTTCTACTGGTACATGG
ATTTCGAGTAACTCCTCAACCTGGAGTTCCGCCAGAAGAAGCAGGGGCCGCGGTAGCTGCCGAATCTTCTACTGGTACATGG
ACAACCGTGTGGACCGATGGACTTACCAGCCTTGATCGTTACAAAGGACGATGCTACGACATCGAGCCTGTTCCTGGCGAAG
ACAACCGTGTGGACCGATGGACTTACCAGCCTTGATCGTTACAAAGGACGATGCTACGACATCGAGCCTGTTCCTGGCGAAG
AAAGTCAATTTATTGCTTATGTAGCTTACCCATTAGACCTTTTTGAAGAAGGTTCTGTTACTAACATGTTTACTTCCATTGTGG
AAAGTCAATTTATTGCTTATGTAGCTTACCCATTAGACCTTTTTGAAGAAGGTTCTGTTACTAACATGTTTACTTCCATTGTGG
GTAATGTATTTGGGTTCAAAGCCTTGCGTGCTCTTCGTTTGGAGGATTTGCGAATCCCTCCTGCTTATACGAAAACTTTCCAAG
GTAATGTATTTGGGTTCAAAGCCTTGCGTGCTCTTCGTTTGGAGGATTTGCGAATCCCTCCTGCTTATACGAAAACTTTCCAAG
GCCCGCCTCATGGTATCCAAGTTGAAAGAGATAAATTGAACAAATATGGACGTCCCCTATTGGGATGTACTATTAAACCGAA
GCCCGCCTCATGGTATCCAAGTTGAAAGAGATAAATTGAACAAATATGGACGTCCCCTATTGGGATGTACTATTAAACCGAA
ATTGGGATTGTCCGCTAAGAACTACGGTCGGGCAGTTTATGAATGTCTTCGTGGCGGACTTGATTTTACCAAAGATGATGAAA
ATTGGGATTGTCCGCTAAGAACTACGGTCGGGCAGTTTATGAATGTCTTCGTGGCGGACTTGATTTTACCAAAGATGATGAAA
ACGTGAACTCCCAACCATTTATGCGTTGGAGAGACCGTTTCTTATTCTGTGCCGAAGCTATTTTTAAATCACAGTCCGAAACA
ACGTGAACTCCCAACCATTTATGCGTTGGAGAGACCGTTTCTTATTCTGTGCCGAAGCTATTTTTAAATCACAGTCCGAAACA
GGTGAAATCAAAGGACATTACTTGAAT
GGTGAAATCAAAGGACATTACTTGAAT
Rheum officinale
P6898 / 64
JF950015
Aligned with
Rheum officinale
GQ436767
ribulose-1,5-bisphosphate carboxylase/oxygenase large subunit (rbcL) gene
Polygonaceae
CTGGTGTTAAAGAATACAAATTGACTTATTATACTCCTGACTATGAACCCCATGACCATGATATCTTGGCAGCATTTCGCGTA
CTGGTGTTAAAGAATACAAATTGACTTATTATACTCCTGACTATGAACCCCATGACCATGATATCTTGGCAGCATTTCGCGTA
ACTCCTCAACCTGGAGTTCCACCAGAAGAAGCAGGGGCCGCGGTAGCTGCCGAATCTTCTACTGGTACATGGACAACTGTGT
ACTCCTCAACCTGGAGTTCCACCAGAAGAAGCAGGGGCCGCGGTAGCTGCCGAATCTTCTACTGGTACATGGACAACTGTGT
GGACCGATGGACTTACCAGCCTTGATCGTTACAAAGGACGATGCTACCACATCGAGCCTGTTCCTGGAGAAGAAAATCAATT
GGACCGATGGACTTACCAGCCTTGATCGTTACAAAGGACGATGCTACCACATCGAGCCTGTTCCTGGAGAAGAAAATCAATT
TATTGCTTATGTAGCTTACCCATTAGACCTTTTTGAAGAAGGTTCTGTTACTAACATGTTTACTTCCATTGTGGGTAATGTATTT
TATTGCTTATGTAGCTTACCCATTAGACCTTTTTGAAGAAGGTTCTGTTACTAACATGTTTACTTCCATTGTGGGTAATGTATTT
GGGTTCAAAGCCCTGCGTGCTCTACGTTTGGAGGATTTGCGAATCCCTCCTGCTTATACGAAAACTTTCCAAGGCCCGCCTCA
GGGTTCAAAGCCCTGCGTGCTCTACGTTTGGAGGATTTGCGAATCCCTCCTGCTTATACGAAAACTTTCCAAGGCCCGCCTCA
TGGTATCCAAGTTGAGAGAGATAAATTGAACAAATATGGACGTCCCCTATTGGGATGTACTATTAAACCGAAATTGGGGTTGT
TGGTATCCAAGTTGAGAGAGATAAATTGAACAAATATGGACGTCCCCTATTGGGATGTACTATTAAACCGAAATTGGGGTTGT
CCGCTAAGAACTACGGCCGAGCAGTTTATGAATGTCTTCGTGGCGGACTTGATTTTACCAAAGATGATGAAAACGTGAACTCC
CCGCTAAGAACTACGGCCGAGCAGTTTATGAATGTCTTCGTGGCGGACTTGATTTTACCAAAGATGATGAAAACGTGAACTCC
CAACCATTTATGCGTTGGAGAGACCGTTTCTTATTCTGTGCCGAAGCTCTTTTTAAAGCACAGTCTGAAACAGGTGAAATCAA
CAACCATTTATGCGTTGGAGAGACCGTTTCTTATTCTGTGCCGAAGCTCTTTTTAAAGCACAGTCTGAAACAGGTGAAATCAA
AGGACATTACTTGAAT
AGGACATTACTTGAAT
203
Coptis chinensis
P6855 / 21
JF950024
Aligned with
Coptis chinensis
FJ449856
ribulose-1,5-bisphosphate carboxylase/oxygenase large subunit (rbcL) gene
Ranunculaceae
TACAAATTGACTTATTATACTCCTCAATATACACCCAAAGATACTGATACCCTGGCAGCATTCCGAGTAACTCCTCAACCTGG
TACAAATTGACTTATTATACTCCTCAATATACACCCAAAGATACTGATACCCTGGCAGCATTCCGAGTAACTCCTCAACCTGG
AGTTCCACCGGAAGAAGCGGGGGCTGCTGTAGCTGCCGAATCTTCTACAGGTACATGGACAACTGTGTGGACCGATGGACTT
AGTTCCACCGGAAGAAGCGGGGGCTGCTGTAGCTGCCGAATCTTCTACAGGTACATGGACAACTGTGTGGACCGATGGACTT
ACCAGCCTTGATCGTTACAAAGGACGATGCTACGACATCGAGCCCGTTGCTGGAGAAGAAAATCAATATATTTGTTATGTAGC
ACCAGCCTTGATCGTTACAAAGGACGATGCTACGACATCGAGCCCGTTGCTGGAGAAGAAAATCAATATATTTGTTATGTAGC
CTATCCTTTAGACCTTTTTGAAGAGGGTTCTGTTACTAACATGTTTACTTCCATTGTGGGTAATGTTTTTGGGTTCAAAGCGCT
CTATCCTTTAGACCTTTTTGAAGAGGGTTCTGTTACTAACATGTTTACTTCCATTGTGGGTAATGTTTTTGGGTTCAAAGCGCT
ACGCGCTCTACGTTTGGAGGATCTGCGAATTCCTGTTGCTTATGTTAAAACTTTCCAGGGGCCGCCCCATGGTATCCAAGTTG
ACGCGCTCTACGTTTGGAGGATCTGCGAATTCCTGTTGCTTATGTTAAAACTTTCCAGGGGCCGCCCCATGGTATCCAAGTTG
AGAGAGATAAATTGAACAAGTATGGTCGTCCCCTATTGGGATGTACTATTAAACCAAAATTGGGATTATCTGCTAAGAACTAC
AGAGAGATAAATTGAACAAGTATGGTCGTCCCCTATTGGGATGTACTATTAAACCAAAATTGGGATTATCTGCTAAGAACTAC
GGTAGAGCGGTTTATGAATGTCTCCGCGGTGGACTTGATTTTACCAAGGATGATGAGAACGTGAACTCCCAACCATTTATGCG
GGTAGAGCGGTTTATGAATGTCTCCGCGGTGGACTTGATTTTACCAAGGATGATGAGAACGTGAACTCCCAACCATTTATGCG
TTGGAGAGACCGTTTCCTATTTTGTGCTGAAGCAATTTATAAAGCACAAGCCGAAACCGGTGAAATCAAAGGACATTACTTGA
TTGGAGAGACCGTTTCCTATTTTGTGCTGAAGCAATTTATAAAGCACAAGCCGAAACCGGTGAAATCAAAGGACATTACTTGA
ATGCTACTGC
ATGCTACTGC
Sanguisorba officinalis
P6901 / 67
JF950016
Aligned with
Sanguisorba officinalis
AY395560
ribulose-1,5-bisphosphate carboxylase/oxygenase large subunit (rbcL) gene
Rosaceae
GTTGGATTCAAAGCTGGTGTTAAAGATTATAAATTGACTTATTATACTCCGGACTATGAAACCAAAGATACTGATATCTTGGC
GTTGGATTCAAAGCTGGTGTTAAAGATTATAAATTGACTTATTATACTCCGGACTATGAAACCAAAGATACTGATATCTTGGC
AGCATTTCGAGTAACTCCTCAACCTGGAGTTCCGCCTGAGGAAGCAGGGGCGGCGGTAGCTGCGGAATCTTCTACTGGTACAT
AGCATTTCGAGTAACTCCTCAACCTGGAGTTCCGCCTGAGGAAGCAGGGGCGGCGGTAGCTGCGGAATCTTCTACTGGTACAT
GGACAACTGTATGGACTGACGGGCTTACCAGTCTTGATCGTTACAAAGGGCGCTGCTACCATATTGAACCTGTTGCTGGAGAA
GGACAACTGTATGGACTGACGGGCTTACCAGTCTTGATCGTTACAAAGGGCGCTGCTACCATATTGAACCTGTTGCTGGAGAA
GAAAATCAATATATTGCTTATGTAGCTTACCCCTTAGACCTTTTTGAAGAGGGTTCGGTTACTAACATGTTTACTTCCATTGTA
GAAAATCAATATATTGCTTATGTAGCTTACCCCTTAGACCTTTTTGAAGAGGGTTCGGTTACTAACATGTTTACTTCCATTGTA
GGTAATGTATTTGGGTTCAAGGCCTTGCGCGCTCTACGTCTGGAGGATTTACGAATTCCTCCTGCTTATGTTAAAACTTTCCAA
GGTAATGTATTTGGGTTCAAGGCCTTGCGCGCTCTACGTCTGGAGGATTTACGAATTCCTCCTGCTTATGTTAAAACTTTCCAA
GGCCCGCCTCACGGGATCCAAGTTGAAAGAGATAAATTGAACAAGTACGGCCGCCCGCTATTGGGATGCACTATTAAACCTA
GGCCCGCCTCACGGGATCCAAGTTGAAAGAGATAAATTGAACAAGTACGGCCGCCCGCTATTGGGATGCACTATTAAACCTA
AATTGGGGTTATCCGCTAAGAATTACGGTAGAGCAGTTTATGAATGTCTCCGCGGTGGACTTGATTTTACCAAAGATGATGAG
AATTGGGGTTATCCGCTAAGAATTACGGTAGAGCAGTTTATGAATGTCTCCGCGGTGGACTTGATTTTACCAAAGATGATGAG
AATGTTAATTCCCAACCATTTATGCGTTGGAGAGACCGTTTCTTATTTTGTGTCGAAGCAATTAATAAAGCGCAGGCTGAAAC
AATGTTAATTCCCAACCATTTATGCGTTGGAGAGACCGTTTCTTATTTTGTGTCGAAGCAATTAATAAAGCGCAGGCTGAAAC
AGGTGAAATCAAAGGGCATTACTTGAATGCTACTGCAGGTACATGCGAA
AGGTGAAATCAAAGGGCATTACTTGAATGCTACTGCAGGTACATGCGAA
Hedyotis diffusa
P6874 / 40
JF949979
Aligned with
Hedyotis nigricans
AJ288606
ribulose-1,5-bisphosphate carboxylase/oxygenase large subunit (rbcL) gene
Rubiaceae
AGTGTTGGATTCAAAGCTGGTGTTAAAGAGTACAAATTAACTTATTATACTCCTGAATACGAAACCAAAGATACTGATATCTT
AGTGTTGGATTTAAAGCTGGTGTTAAAGAGTACAAATTAACTTATTATACTCCTGAATACGAAACCAAAGATACTGATATCTT
204
GGCAGCATTCCGAGTAACTCCTCAACCCGGAGTTCCACCGGAAGAAGCAGGGGCCGCGGTAGCTGCCGAGTCTTCTACTGGT
GGCAGCATTCCGAGTAACTCCTCAACCCGGAGTTCCACCGGAAGAAGCCGGGGCTGCGGTAGCTGCCGAGTCTTCTACTGGT
ACATGGACAACTGTATGGACGGATGGACTTACCAGTCTTGACCGTTACAAAGGACGATGCTACCACATCGAGCCAGTTCCTG
ACATGGACAACTGTATGGACGGATGGACTTACCAGTCTTGACCGTTACAAAGGACGATGCTACCACATCGAGCCAGTTCCTG
GAGAAGAAGATCAATTTATTGCTTATGTAGCTTACCCTTTAGACCTTTTTGAAGAAGGTTCTGTTACTAACATGTTTACTTCCA
GAGAAGAAGATCAATTTATTGCTTATGTAGCGTACCCTTTAGACCTTTTTGAAGAAGGTTCTGTTACTAACATGTTTACTTCCA
TCGTAGGTAATGTATTTGGGTTCAAAGCCCTGCGCGCCCTACGTCTGGAAGATTTGCGAATTCCCATTGCTTATGTTAAAACCT
TCGTAGGTAATGTATTTGGGTTCAAAGCCTTGCGCGCCCTACGTCTGGAAGATTTGCGAATTCCCATTGCTTATGTTAAAACCT
TCGAAGGTCCACCTCACGGCATTCAGGTCGAGAGAGATAAATTGAACAAGTATGGTCGTCCCCTATTGGGATGTACTATTAAA
TCGAAGGTCCACCTCACGGCATTCAGGTCGAGAGAGATAAATTGAACAAGTATGGTCGTCCCCTATTGGGATGTACTATTAAA
CCTAAATTAGGTTTATCTGCTAAAAACTACGGTAGAGCATGTTATGAATGTCTTCGTGGTGGACTTGATTTTACTAAAGATGA
CCTAAATTAGGTTTATCTGCTAAAAACTACGGTAGAGCATGTTATGAATGTCTTCGTGGTGGACTTGATTTTACTAAAGATGA
TGAAAACGTGAACTCCCAACCATTTATGCGTTGGAGAGATCGTTTCTTATTTTGTGCCGAAGCAATTTATAAAGCGCAGGCTG
TGAAAACGTGAACTCCCAACCATTTATGCGTTGGAGAGATCGTTTCTTATTTTGTGCCGAAGCAATTTATAAAGCGCAGGCTG
AAACTGGTGAAATCAAAGGGCATTACTTGAATGCTACTGCAGGT
AAACTGGTGAAATCAAAGGGCATTACTTGAATGCTACTGCAGGT
Evodia lepta
P6869 / 35
JF949978
Aligned with
Zanthoxylum monophyllum
U39282
ribulose-1,5-bisphosphate carboxylase/oxygenase large subunit (rbcL) gene
Rutaceae
TCAAAGCCGGTGTTAAAGATTATAAATTGACTTATTATACTCCTGACTATGTAACCAAAGATACTGATATCTTGGCAGCATTC
TCAAAGCCGGTGTTAAAGATTATAAATTGACTTATTATACTCCTGACTATGTAACCAAAGATACTGATATCTTGGCAGCATTC
CGAGTAACTCCTCAACCCGGAGTTCCACCCGAGGAAGCGGGGGCTGCAGTAGCTGCGGAATCTTCTACTGGTACATGGACAA
CGAGTAACTCCTCAACCCGGAGTTCCACCCGAGGAAGCGGGGGCTGCGGTAGCTGCGGAATCTTCTACTGGTACATGGACAA
CTGTGTGGACCGATGGGCTTACCAGCCTTGATCGTTACAAAGGGCGATGCTACAACATTGAGCCCGTTGCTGGAGAAGAAAA
CTGTGTGGACCGACGGGCTTACCAGCCTTGATCGTTACAAAGGGCGATGCTACAACATTGAGCCCGTTGCTGGAGAAGAAAA
TCAATATATATGTTATGTAGCTTACCCGTTAGACCTTTTTGAAGAAGGTTCTGTTACTAACATGTTTACTTCCATTGTGGGTAA
TCAATATATATGTTATGTAGCTTACCCGTTAGACCTTTTTGAAGAAGGTTCTGTTACTAACATGTTTACTTCCATTGTGGGTAA
TGTATTTGGTTTCAAAGCCCTGCGCGCTCTACGTCTAGAGGATCTACGAATCCCTCCCGCGTATTCTAAAACTTTTCAAGGCC
TGTATTTGGTTTCAAAGCCCTGCGCGCTCTACGTCTAGAGGATCTACGAATCCCTACCGCGTATACTAAAACTTTCCAAGGCC
CGCCTCATGGTATCCAAGTTGAAAGAGATAAATTGAACAAGTATGGACGTCCCCTGTTGGGATGTACTATTAAACCGAAATT
CGCCTCACGGCATCCAAGTTGAGAGAGATAAATTGAACAAGTATGGACGTCCCCTGTTGGGATGTACTATTAAACCGAAATT
GGGGTTATCCGCTAAGAATTACGGTAGGGCGGTTTATGAATGTCTACGCGGTGGACTTGACTTTACCAAAGATGATGAGAAC
GGGGTTATCCGCTAAGAATTACGGTAGGGCAGTTTATGAATGTCTACGCGGTGGACTTGACTTTACCAAAGATGATGAGAAC
GTGAACTCCCAACCATTTATGCGTTGGAGGGACCGTTTCGTATTTTGTGCGGAAGCACTTTATAAAGCGCAAGCTGAAACAGG
GTGAACTCCCAACCATTTATGCGTTGGAGGGACCGTTTCTTATTTTGTGCGGAAGCACTTTATAAAGCGCAAGCTGAAACAGG
TGAAATCAAAGGTCATTACTTGAATGCTACTGCGGGTACATGCGAA
TGAAATCAAAGGTCATTACTTGAATGCTACTGCAGGTACATGCGAA
Phellodendron chinense
P6890 / 56
JF949986
Aligned with
Phellodendron chinense
GQ436735
ribulose-1,5-bisphosphate carboxylase/oxygenase large subunit (rbcL) gene
Rutaceae
CTTATTATACTCCTGACTATGCAACCAAAGATACTGATATCTTGGCAGCATTCCGAGTAAGTCCTCAACCCGGAGTTCCACCC
CTTATTATACTCCTGACTATGCAACCAAAGATACTGATATCTTGGCAGCATTCCGAGTAAGTCCTCAACCCGGAGTTCCACCC
GAGGAAGCGGGGGCTGCGGTAGCTGCGGAATCTTCTACTGGTACATGGACAACTGTGTGGACCGATGGGCTTACCAGCCTTG
GAGGAAGCGGGGGCTGCGGTAGCTGCGGAATCTTCTACTGGTACATGGACAACTGTGTGGACCGATGGGCTTACCAGCCTTG
ATCGTTACAAAGGGCGATGCTACAACATTGAGCCCGTTGCTGGAGAAGAAAATCAATATATATGTTATGTAGCTTACCCGTTA
ATCGTTACAAAGGGCGATGCTACAACATTGAGCCCGTTGCTGGAGAAGAAAATCAATATATATGTTATGTAGCTTACCCGTTA
GACCTTTTTGAAGAAGGTTCTGTTACTAACATGTTTACTTCCATTGTGGGTAATGTATTTGGTTTCAAAGCCCTGCGCGCTCTA
GACCTTTTTGAAGAAGGTTCTGTTACTAACATGTTTACTTCCATTGTGGGTAATGTATTTGGTTTCAAAGCCCTGCGCGCTCTA
205
CGTCTAGAGGATCTACGAATCCCTACCGCGTATACTAAAACTTTCCAAGGCCCGCCTCACGGCATCCAAGTTGAGAGAGATA
CGTCTAGAGGATCTACGAATCCCTACCGCGTATACTAAAACTTTCCAAGGCCCGCCTCACGGCATCCAAGTTGAGAGAGATA
AATTGAATAAGTATGGACGTCCCCTGTTGGGATGTACTATTAAACCTAAATTGGGGTTATCCGCTAAGAATTACGGTAGGGCA
AATTGAATAAGTATGGACGTCCCCTGTTGGGATGTACTATTAAACCTAAATTGGGGTTATCCGCTAAGAATTACGGTAGGGCA
GTTTATGAATGTCTACGCGGTGGACTTGACTTTACCAAAGATGATGAGAACGTGAACTCCCAACCATTTATGCGTTGG
GTTTATGAATGTCTACGCGGTGGACTTGACTTTACCAAAGATGATGAGAACGTGAACTCCCAACCATTTATGCGTTGG
Houttuynia cordata
P6875 / 41
JF950006
Aligned with
Houttuynia cordata
L08762
ribulose-1,5-bisphosphate carboxylase/oxygenase large subunit (rbcL) gene
Saururaceae
GTTGGATTCAAAGCTGGTGTTAAAGATTACAAATTAACTTATTATACTCCTGACTATGAAACCAAAGATACTGATATCTTGGC
GTTGGATTCAAAGCTGGTGTTAAAGATTACAAATTAACTTATTATACTCCTGACTATGAAACCAAAGATACTGATATCTTGGC
AGCATTCCGAGTAACTCCGCAACCCGGAGTTCCACCCGAGGAAGCAGGGGCTGCGGTACGTGCCGAATCCTCTACTGGTACA
AGCATTCCGAGTAACTCCGCAACCCGGAGTTCCACCCGAGGAAGCAGGGGCTGCGGTACGTGCCGAATCCTCTACTGGTACA
TGGACAACTGTGTGGACCGATGGACTTACCAGCCTTGATCGTTACAAAGGTCGATGCTACCACATCGAGCCCGTTGCTGGAGA
TGGACAACTGTGTGGACCGATGGACTTACCAGCCTTGATCGTTACAAAGGTCGATGCTACCACATCGAGCCCGTTGCTGGAGA
GGAAAGTCAATTTATTGCTTATGTAGCTTATCCTTTAGACCTTTTTGAAGAAGGTTCTGTTACTAACATGTTTACTTCCATTGTG
GGAAAGTCAATTTATTGCTTATGTAGCTTATCCTTTAGACCTTTTTGAAGAAGGTTCTGTTACTAACATGTTTACTTCCATTGTG
GGTAATGTATTTGGGTTCAAAGCCCTACGAGCTCTACGTCTGGAAGATCTACGAATTCCTCCTGCTTATTCCAAAACTTTCCAA
GGTAATGTATTTGGGTTCAAAGCCCTACGAGCTCTACGTCTGGAAGATCTACGAATTCCTCCTGCTTATTCCAAAACTTTCCAA
GGCCCACCCCATGGAATCCAAGTTGAAAGAGATAAATTGAACAAGTATGGTCGTCCTCTATTGGGATGTACTATTAAACCTAA
GGCCCACCCCATGGAATCCAAGTTGAAAGAGATAAATTGAACAAGTATGGTCGTCCTCTATTGGGATGTACTATTAAACCTAA
ATTGGGGTTATCGGCTAAGAACTACGGTAGGGCGGTTTATGAATGTCTCCGCGGTGGGCTTGATTTCACCAAGGATGATGAAA
ATTGGGGTTATCGGCTAAGAACTACGGTAGGGCGGTTTATGAATGTCTCCGCGGTGGGCTTGATTTCACCAAGGATGATGAAA
ATGTGAACTCACAACCATTTATGCGTTGGAGAGACCGTTTCCTATTTTGTGCCGAAGCTATTTACAAAGCGCAGGCCGAAACA
ATGTGAACTCACAACCATTTATGCGTTGGAGAGACCGTTTCCTATTTTGTGCCGAAGCTATTTACAAAGCGCAGGCCGAAACA
GGTGAAATTAAAGGACATTACTTAAATGCTACTGCAGGTAC
GGTGAAATTAAAGGACATTACTTAAATGCTACTGCAGGTAC
Kadsura longipedunculata
P6879 / 45
JF950007
Aligned with
Kadsura japonica
AF193969
ribulose-1,5-bisphosphate carboxylase/oxygenase large subunit (rbcL) gene
Schisandraceae
ATTCAAGGCTGGTGTTAAAGATTACAGATTGACTTATTATACTCCTGACTATGAAACGAAAGATACTGATATCTTGGCAGCAT
ATTCAAGGCTGGTGTTAAAGATTACAGATTGACTTATTATACTCCTGACTATGAAACGAAAGATACTGATATCTTGGCAGCAT
TCCGAGTAACTCCTCAACCTGGAGTTCCACCCGAGGAAGCGGGAGCTGCGGTAGCTGCGGAATCTTCTACTGGTACCTGGACT
TCCGAGTAACTCCTCAACCTGGAGTTCCACCCGAGGAAGCGGGAGCTGCGGTAGCTGCGGAATCTTCTACTGGTACCTGGACT
ACTGTGTGGACTGATGGACTTACCAGCCTCGATCGTTATAAAGGGCGATGCTACCACATTGAGCCCGTTGCTGGGGAGGAAA
ACTGTGTGGACTGATGGACTTACCAGCCTCGATCGTTATAAAGGGCGATGCTACCACATTGAGCCCGTTGCTGGGGAGGAAA
ATCAATTTATTGCTTATGTAGCTTACCCTTTAGACCTTTTTGAAGAAGGTTCTGTTACTAACATGTTTACTTCTATTGTAGGTAA
ATCAATATATTGCTTATGTAGCTTACCCTTTAGACCTTTTTGAAGAAGGTTCTGTTACTAACATGTTTACTTCTATTGTAGGTAA
TGTGTTTGGGTTCAAAGCCCTACGAGCTCTGCGTCTGGAAGATTTGCGAATTCCTCCTGCTTATTCCAAAACTTTCCAAGGCCC
TGTGTTTGGGTTCAAAGCCCTACGAGCTCTGCGTCTGGAAGATTTGCGAATTCCTCCTGCTTATTCCAAAACTTTCCAAGGCCC
ACCCCATGGCATCCAAGTTGAGAGAGATAAATTGAACAAGTATGGTCGTCCCCTATTGGGATGTACTATTAAACCAAAATTA
ACCCCATGGCATCCAAGTTGAGAGAGATAAATTGAACAAGTATGGTCGTCCCCTATTGGGATGTACTATTAAACCAAAATTA
GGGTTATCTGCCAAGAACTACGGTAGAGCGGTTTATGAATGTCTCCGCGGTGGACTTGATTTTACCAAGGATGAT
GGGTTATCTGCCAAGAACTACGGTAGAGCGGTTTATGAATGTCTCCGCGGTGGACTTGATTTTACCAAGGATGAT
206
Selaginella tamariscina
P6904 / 70
JF950018
Aligned with
Selaginella tamariscina
AJ295861
ribulose-1,5-bisphosphate carboxylase/oxygenase large subunit (rbcL) gene
Selaginellaceae
TACAAGACCAAAAACAGCGATATCCTGGCAGCGTTCCGAATGACTCCGCAACCCGGAGTGCCCGCCGAAGAGGCAGGAGCC
TACAAGACCAAAAACAGCGATATCCTGGCAGCGTTCCGAATGACTCCGCAACCCGGAGTGCCCGCCGAAGAGGCAGGAGCC
GCAGTAGCTGCGGAATCCTCTACTGGCACATGGACCACCGTTTGGACCGACGGGCTTACCAATCTTGACCGTTATAAAGGTCG
GCAGTAGCTGCGGAATCCTCTACTGGCACATGGACCACCGTTTGGACCGACGGGCTTACCAATCTTGACCGTTATAAAGGTCG
GTGCTACGATATCGAACCCGTCCCTGGGGAAAAAGATCAATACATTGCCTATGTAGCCTATCCTTTGGATTTGTTTGAGGAAG
GTGCTACGATATCGAACCCGTCCCTGGGGAAAAAGATCAATACATTGCCTATGTAGCCTATCCTTTGGATTTGTTTGAGGAAG
GTTCCGTTACCAACATGTTTACTTCCATTGTGGGTAATGTTTTTGGATTCAAGGCCTTACGAGCCCTGCGTTTGGAAGATCTGC
GTTCCGTTACCAACATGTTTACTTCCATTGTGGGTAATGTTTTTGGATTCAAGGCCTTACGAGCCCTGCGTTTGGAAGATCTGC
GAATCCCCCCCGCTTATTCCAAAACCTTCGAGGGCCCGCCTCATGGTATCCAAGTCGAAAGGGATAAATCAAATAAATACGG
GAATCCCCCCCGCTTATTCCAAAACCTTCGAGGGCCCGCCTCATGGTATCCAAGTCGAAAGGGATAAATCAAATAAATACGG
CCGCCCCCTGCTGGGATGCACTATCAAACCCAAGTTAGGTTTATCTGCCAAAAACTATGGCAGAGCGTGCTATGAATGTCTTC
CCGCCCCCTGCTGGGATGCACTATCAAACCCAAGTTAGGTTTATCTGCCAAAAACTATGGCAGAGCGTGCTATGAATGTCTTC
GTGG
GTGG
Patrinia scabiosaefolia
P6889 / 55
JF949985
Aligned with
Patrinia triloba
AF446951
ribulose-1,5-bisphosphate carboxylase/oxygenase large subunit (rbcL) gene
Valerianaceae
AGTGCTGGATTCAAAGCGGGCGTTAAAGATTACAAATTGACTTATTATACTCCTGACTATGAAACCAAAGATACTGATATCTT
AGTGCTGGATTCAAAGCGGGCGTTAAAGATTACAAATTGACTTATTATACTCCTGACTATGAAACCAAAGATACTGATATCTT
GGCAGCATTCCGAGTAACTCCTCAACCTGGAGTTCCACCTGAAGAAGCAGGGGCCGCAGTAGCTGCCGAATCTTCAACTGGT
GGCAGCATTCCGAGTAACTCCTCAACCTGGAGTTCCTGCTGAAGAAGCAGGGGCCGCAGTAGCTGCCGAATCTTCAACTGGT
ACATGGACAACTGTGTGGACCGATGGACTTACCAGCCTTGATCGTTACAAAGGGCGATGCTACCACATCGAGCCCGTTGCTG
ACATGGACAACTGTGTGGACCGATGGACTTACCAGCCTTGATCGTTACAAAGGGCGATGCTACCACATCGAGCCCGTTGCTG
GAGAAGAAAGTCAATTTATTGCTTATGTAGCTTACCCATTAGACCTTTTTGAAGAAGGTTCTGTTACTAACATGTTTACTTCTA
GAGAAGAAAGTCAATTTATTGCTTATGTAGCTTACCCATTAGACCTTTTTGAAGAAGGTTCTGTTACTAACATGTTTACTTCTA
TTGTGGGTAATGTATTTGGGTTCAAAGCCCTGCGCGCTCTACGTCTGGAAGATCTGCGAATCCCTGTCGCTTATGTTAAAACTT
TTGTGGGTAATGTATTTGGGTTCAAAGCCCTGCGCGCTCTACGTCTGGAAGATCTGCGAATCCCTGTCGCTTATGTTAAAACTT
TCCAAGGCCCGCCTCATGGTATCCAAGTTGAGAGAGATAAATTGAACAAGTACGGTCGCCCCCTGTTGGGATGTACTATTAAA
TCCAAGGCCCGCCTCATGGTATCCAAGTTGAGAGAGATAAATTGAACAAGTACGGTCGCCCCCTGTTGGGATGTACTATTAAA
CCCAAATTGGGGTTGTCTGCTAAAAACTATGGTAGAGCGGTTTATGAATGTCTACGTGGTGGACTTGATTTTACCAAAGATGA
CCCAAATTGGGGTTGTCTGCTAAAAACTATGGTAGAGCGGTTTATGAATGTCTACGTGGTGGACTTGATTTTACCAAAGATGA
TGAGAACGTGAACTCCCAACCATTTATGCGTTGGAGAGATCGTTTCTTATTTTGTGCCGAAGCCCTTTATAAAGCACAAACTG
TGAGAACGTGAACTCCCAACCATTTATGCGTTGGAGAGATCGTTTCTTATTTTGTGCCGAAGCCCTTTATAAAGCACAAACTG
AAACAGGTGAAATCAAAGGGCATTACTTGAATGCTACTGCAGG
AAACAGGTGAAATCAAAGGGCATTACTTGAATGCGACTGCAGG
Verbena officinalis
P6910 / 76
JF950020
Aligned with
Verbena officinalis
GQ436523
ribulose-1,5-bisphosphate carboxylase/oxygenase large subunit (rbcL) gene
Verbenaceae
GTTGGATTCAAAGCGGGTGTTAAAGAGTACAAGTTGACTTATTATACTCCTGAATACGAAACCAAAGATACTGATATCTTGGC
GTTGGATTCAAAGCGGGTGTTAAAGAGTACAAGTTGACTTATTATACTCCTGAATACGAAACCAAAGATACTGATATCTTGGC
AGCATTCCGAGTAACTCCTCAACCTGGAGTTCCGCCTGAAGAAGCAGGGGCTGCGGTAGCTGCCGAATCTTCTACTGGTACAT
AGCATTCCGAGTAACTCCTCAACCTGGAGTTCCGCCTGAAGAAGCAGGGGCTGCGGTAGCTGCCGAATCTTCTACTGGTACAT
GGACAACTGTGTGGACCGATGGACTTACCAGCCTTGATCGTTACAAAGGGCGATGCTACCACATCGAGCCCGTTCCTGGAGA
GGACAACTGTGTGGACCGATGGACTTACCAGCCTTGATCGTTACAAAGGGCGATGCTACCACATCGAGCCCGTTCCTGGAGA
207
CCCAGATCAATATATTTGTTATGTAGCTTACCCTTTAGACCTTTTTGAAGAAGGTTCTGTTACGAACATGTTTACTTCCATTGT
CCCAGATCAATATATTTGTTATGTAGCTTACCCTTTAGACCTTTTTGAAGAAGGTTCTGTTACGAACATGTTTACTTCCATTGT
AGGAAATGTATTTGGATTCAAAGCCCTACGGGCTCTACGTCTGGAAGATCTGCGAATCCCCGTTGCCTATACTAAAACTTTCC
AGGAAATGTATTTGGATTCAAAGCCCTACGGGCTCTACGTCTGGAAGATCTGCGAATCCCCGTTGCCTATACTAAAACTTTCC
AAGGCCCGCCTCATGGGATCCAAGTTGAGAGAGATAAATTGAACAAGTATGGTCGTCCTCTGTTGGGATGTACTATTAAACCT
AAGGCCCGCCTCATGGGATCCAAGTTGAGAGAGATAAATTGAACAAGTATGGTCGTCCCCTGTTGGGATGTACTATTAAACCT
AAATTGGGTTTATCTGCTAAAAACTATGGTAGAGCAGTTTATGAATGTCTTCGCGGTGGACTTGATTTTACCAAAGATGATGA
AAATTGGGTTTATCTGCTAAAAACTATGGTAGAGCAGTTTATGAATGTCTTCGCGGTGGACTTGATTTTACCAAAGATGATGA
GAACGTGAACTCTCAGCCATTTATGCGTTGGAGAGATCGTTTCTTATTTTGTGCCGAAGCAATTTATAAAGCACAGGCTGAAA
GAACGTGAACTCTCAGCCATTTATGCGTTGGAGAGATCGTTTCTTATTTTGTGCCGAAGCAATTTATAAAGCACAGGCTGAAA
CAGGTGAAATCAAAGGGCA
CAGGTGAAATCAAAGGGCA
Viola yezoensis
P6911 / 77
JF949993
Aligned with
Viola pubescens
DQ006129
ribulose-1,5-bisphosphate carboxylase/oxygenase large subunit (rbcL) gene
Violaceae
GTTGGATTCAAAGCTGGGGTTAAAGATTATAAATTGACTTATTATACTCCTGACTATGAAACCAAAGATACTGATATCTTGGC
GTTGGATTCAAAGCTGGGGTTAAAGATTATAAATTGACTTATTATACTCCTGACTATGAAACCAAGGATACTGATATCTTGGC
AGCATTCCGAGTAACTCCGCAACCCGGAGTTCCGCCTGAGGAAGCAGGGGCTGCGGTAGCTGCTGAATCTTCTACTGGTACAT
AGCATTCCGAGTAACTCCGCAACCCGGAGTTCCGCCTGAGGAAGCAGGGGCTGCGGTAGCTGCTGAATCTTCTACTGGTACAT
GGACAACTGTGTGGACCGATGGGCTTACCTCCCTTGATCGTTATAAAGGACGATGCTACGACATCGAGCCCGTTCCTGGAGAA
GGACAACTGTGTGGACCGATGGGCTTACTTCCCTTGATCGTTATAAAGGACGATGCTACGACATCGAGCCCGTTCCTGGAGAA
GAAAGTCAATTTATTGCTTATGTAGCTTACCCTTTAGACCTTTTTGAAGAAGGTTCTGTTACGAACATGTTTACTTCCATTGTG
GAAAGTCAATTTATTGCTTATGTAGCTTACCCTTTAGACCTTTTTGAAGAAGGTTCTGTTACGAACATGTTTACTTCCATTGTG
GGTAATGTATTTGGGTTCAAAGCCCTACGGGCTTTACGTTTGGAGGATTTGCGAATCCCTCCTGCTTATACTAAAACTTTCCAA
GGTAATGTATTTGGGTTCAAAGCCCTACGGGCTTTACGTTTGGAGGATTTGCGAATCCCTCCTGCTTATACTAAAACTTTCCAA
GGTCCGCCTCATGGTATCCAAGTTGAGAGAGATAAATTGAACAAGTACGGTCGTCCCCTATTGGGCTGTACTATTAAACCTAA
GGTCCGCCTCATGGTATCCAAGTTGAGAGAGATAAATTGAACAAGTACGGTCGTCCCCTATTGGGCTGTACTATTAAACCGAA
ATTGGGGTTATCCGCTAAGAATTATGGGAGAGCTGTTTATGAGTGTCTCCGCGGTGGACTTGATTTTACTAAAGATGATGAGA
ATTGGGGTTATCCGCTAAGAATTATGGGAGAGCTGTTTATGAGTGTCTCCGCGGTGGACTTGATTTTACTAAAGATGATGAGA
ATGTGAACTCCCAACCATTTAT
ATGTGAACTCCCAACCATTTAT
208
10.4 Documentation of screening results
10.4.1 Dichloromethane extracts: HeLa
Acanthaceae
Andrographis paniculata
P6838 / 04
JF949965
IC50 188.4 µg/ml
P6848 / 14
JF949970
IC50 472.0 µg/ml
P6844 / 10
P6845 / 11
P6849 / 15
P6854 / 20
P6902 / 68
JF950021
JF949968
JF950022
JF949973
JF949988
IC50 235.2 µg/ml
IC50 176.0 µg/ml
IC50 175.0 µg/ml
IC50 127.1 µg/ml
IC50 410.1 µg/ml
Andrographis paniculata
150
Survival
[%]
100
50
0
9.8
19.5
39
78
156
313
625
1250
2500
5000
Concentration
[µg/ml]
Amaranthaceae Celosia cristata
Celosia cristata
150
Survival
[%]
100
50
0
9.8
19.5
39
78
156
313
625
1250
2500
5000
Concentration
[µg/ml]
Apiaceae
Bupleurum chinense
Bupleurum marginatum
Centella asiatica
Cnidium monnieri
Saposhnikovia divaricata
Bupleurum chinense
Bupleurum marginatum
100
100
Survival
[%]
150
Survival
[%]
150
50
50
0
0
9.8
19.5
39
78
156
313
625
1250
2500
5000
9.8
Concentration
[µg/ml]
19.5
39
78
156
313
Concentration
[µg/ml]
209
625
1250
2500
5000
Centella asiatica
Cnidium monnieri
100
100
Survival
[%]
150
Survival
[%]
150
50
50
0
0
9.8
19.5
39
78
156
313
625
1250
2500
5000
9.8
19.5
39
78
Concentration
[µg/ml]
156
313
625
1250
2500
5000
Concentration
[µg/ml]
Saposhnikovia divaricata
150
Survival
[%]
100
50
0
9.8
19.5
39
78
156
313
625
1250
2500
5000
Concentration
[µg/ml]
Araliaceae
Eleutherococcus senticosus
Panax ginseng China
Panax ginseng Korea
Panax notoginseng
P6919 / 79
P8088 / 81
P8086 / 81
P6887 / 53
Eleutherococcus senticosus
JF950028
JF950029
JF950030
IC50 300.0 µg/ml
IC50 152.4 µg/ml
IC50 173.1 µg/ml
IC50 263.0 µg/ml
Panax ginseng China
100
100
Survival
[%]
150
Survival
[%]
150
50
50
0
0
9.8
19.5
39
78
156
313
625
1250
2500
5000
9.8
19.5
39
78
Concentration
[µg/ml]
156
313
625
1250
2500
5000
625
1250
2500
5000
Concentration
[µg/ml]
Panax ginseng Korea
Panax notoginseng
100
100
Survival
[%]
150
Survival
[%]
150
50
50
0
0
9.8
19.5
39
78
156
313
625
1250
2500
5000
9.8
Concentration
[µg/ml]
19.5
39
78
156
313
Concentration
[µg/ml]
210
Arecaceae
Areca catechu
P6840 / 06
-
IC50 1023.3 µg/ml
Areca catechu
150
Survival
[%]
100
50
0
9.8
19.5
39
78
156
313
625
1250
2500
5000
Concentration
[µg/ml]
Asclepiadaceae Cynanchum paniculatum
P6858 / 24
JF949975 IC50 395.6 µg/ml
P6841 / 07
P6842 / 08
P6839 / 05
P6850 / 16
P6851 / 17
JF949966
JF949967
JF949994
JF949971
P6852 / 18
JF949972 IC50 129.4 µg/ml
P6863 / 29
P6905 / 71
P6906 / 72
P6908 / 74
JF950000
JF949989
JF949990
JF950019
Cynanchum paniculatum
150
Survival
[%]
100
50
0
9.8
19.5
39
78
156
313
625
1250
2500
5000
Concentration
[µg/ml]
Asteraceae
Artemisia annua
Artemisia capillaris
Arctium lappa
Centipeda minima
Chrysanthemum indicum
Chrysanthemum
morifolium
Eclipta prostrata
Senecio scandens
Siegesbeckia orientalis
Taraxacum officinale
Artemisia annua
IC50 107.9 µg/ml
IC50 93.5 µg/ml
IC50 345.0 µg/ml
IC50 63.3 µg/ml
IC50 152.1 µg/ml
IC50 266.4 µg/ml
IC50 268.6 µg/ml
IC50 101.5 µg/ml
IC50 232.8 µg/ml
Artemisia capillaris
100
100
Survival
[%]
150
Survival
[%]
150
50
50
0
0
9.8
19.5
39
78
156
313
625
1250
2500
5000
9.8
Concentration
[µg/ml]
19.5
39
78
156
313
Concentration
[µg/ml]
211
625
1250
2500
5000
Arctium lappa
Centipeda minima
100
100
Survival
[%]
150
Survival
[%]
150
50
50
0
0
9.8
19.5
39
78
156
313
625
1250
2500
5000
9.8
19.5
39
78
Concentration
[µg/ml]
156
313
625
1250
2500
5000
625
1250
2500
5000
625
1250
2500
5000
625
1250
2500
5000
Concentration
[µg/ml]
Chrysanthemum indicum
Chrysanthemum morifolium
100
100
Survival
[%]
150
Survival
[%]
150
50
50
0
0
9.8
19.5
39
78
156
313
625
1250
2500
5000
9.8
19.5
39
78
Concentration
[µg/ml]
156
313
Concentration
[µg/ml]
Eclipta prostrata
Senecio scandens
100
100
Survival
[%]
150
Survival
[%]
150
50
50
0
0
9.8
19.5
39
78
156
313
625
1250
2500
5000
9.8
19.5
39
78
Concentration
[µg/ml]
156
313
Concentration
[µg/ml]
Siegesbeckia orientalis
Taraxacum officinale
100
100
Survival
[%]
150
Survival
[%]
150
50
50
0
0
9.8
19.5
39
78
156
313
625
1250
2500
5000
9.8
Concentration
[µg/ml]
19.5
39
78
156
313
Concentration
[µg/ml]
212
Berberidaceae
Berberis bealei
Dysosma versipellis
Epimedium koreanum
P6883 / 49
P6862 / 28
P6865 / 31
JF949996 IC50 93.8 µg/ml
IC50 213.9 µg/ml
JF950002 IC50 48.7 µg/ml
Berberis bealei
Dysosma versipellis
100
100
Survival
[%]
150
Survival
[%]
150
50
50
0
0
9.8
19.5
39
78
156
313
625
1250
2500
5000
9.8
19.5
39
78
Concentration
[µg/ml]
156
313
625
1250
2500
5000
Concentration
[µg/ml]
Epimedium koreanum
150
Survival
[%]
100
50
0
9.8
19.5
39
78
156
313
625
1250
2500
5000
Concentration
[µg/ml]
Brassicaceae
Capsella bursa-pastoris
Isatis indigotica (root)
Isatis indigotica (leaf)
P6846 / 12
P6877 / 43
P6878 / 44
Capsella bursa-pastoris
JF949997 IC50 196.4 µg/ml
JF949981 IC50 321.5 µg/ml
JF949981 IC50 226.5 µg/ml
Isatis indigotica (root)
100
100
Survival
[%]
150
Survival
[%]
150
50
50
0
0
9.8
19.5
39
78
156
313
625
1250
2500
5000
9.8
Concentration
[µg/ml]
Isatis indigotica (leaf)
Survival
[%]
100
50
0
19.5
39
78
156
313
39
78
156
313
Concentration
[µg/ml]
150
9.8
19.5
625
1250
2500
5000
Concentration
[µg/ml]
213
625
1250
2500
5000
Caprifoliaceae
Lonicera confusa
P6880 / 46
JF949982 IC50 226.5 µg/ml
P6892 / 58
JF950027 IC50 279.6 µg/ml
Lonicera confusa
150
Survival
[%]
100
50
0
9.8
19.5
39
78
156
313
625
1250
2500
5000
Concentration
[µg/ml]
Convallariaceae Polygonatum kingianum
Polygonatum kingianum
150
Survival
[%]
100
50
0
9.8
19.5
39
78
156
313
625
1250
2500
5000
Concentration
[µg/ml]
Crassulaceae
Rhodiola rosea
P6920 / 84
-
IC50 164.1 µg/ml
Rhodiola rosea
150
Survival
[%]
100
50
0
9.8
19.5
39
78
156
313
625
1250
2500
5000
Concentration
[µg/ml]
Cupressaceae
Platycladus orientalis
P6891 / 57
Platycladus orientalis
150
Survival
[%]
100
50
0
9.8
19.5
39
78
156
313
625
1250
2500
5000
Concentration
[µg/ml]
214
JF950011 IC50 121.7 µg/ml
Dryopteridaceae Cyrtomium fortunei
P6859 / 25
JF949998 IC50 572.4 µg/ml
P6864 / 30
JF950001 IC50 95.3 µg/ml
P6866 / 32
JF950003 IC50 125.6 µg/ml
Cyrtomium fortunei
150
Survival
[%]
100
50
0
9.8
19.5
39
78
156
313
625
1250
2500
5000
Concentration
[µg/ml]
Ephedraceae
Ephedra sinica
Ephedra sinica
150
Survival
[%]
100
50
0
9.8
19.5
39
78
156
313
625
1250
2500
5000
Concentration
[µg/ml]
Equisetaceae
Equisetum hiemale
Equisetum hiemale
150
Survival
[%]
100
50
0
9.8
19.5
39
78
156
313
625
1250
2500
5000
Concentration
[µg/ml]
Euphorbiaceae Croton tiglium
P6856 / 22
Croton tiglium
150
Survival
[%]
100
50
0
9.8
19.5
39
78
156
313
625
1250
2500
5000
Concentration
[µg/ml]
215
-
IC50 422.9 µg/ml
Fabaceae
Abrus cantoniensis
Acacia catechu
Cassia tora
Desmodium styracifolium
Glycyrrhiza inflata
Spatholobus suberectus
Sutherlandia frutescens
P6835 / 01
P6836 / 02
P6847 / 13
P6861 / 27
P6873 / 39
P6907 / 73
tba / 83
JF949964
JF949969
JF949976
JF950025
JF949991
-
Abrus cantoniensis
IC50 494.4 µg/ml
IC50 164.1 µg/ml
IC50 1335.4 µg/ml
IC50 156.0 µg/ml
IC50 26.4 µg/ml
IC50 299.1 µg/ml
IC50 367.7 µg/ml
Acacia catechu
100
100
Survival
[%]
150
Survival
[%]
150
50
50
0
0
9.8
19.5
39
78
156
313
625
1250
2500
5000
9.8
19.5
39
78
Concentration
[µg/ml]
156
313
625
1250
2500
5000
625
1250
2500
5000
625
1250
2500
5000
Concentration
[µg/ml]
Cassia tora
Desmodium styracifolium
100
100
Survival
[%]
150
Survival
[%]
150
50
50
0
0
9.8
19.5
39
78
156
313
625
1250
2500
5000
9.8
19.5
39
78
Concentration
[µg/ml]
156
313
Concentration
[µg/ml]
Glycyrrhiza inflata
Spatholobus suberectus
100
100
Survival
[%]
150
Survival
[%]
150
50
50
0
0
9.8
19.5
39
78
156
313
625
1250
2500
5000
9.8
Concentration
[µg/ml]
19.5
39
78
156
313
Concentration
[µg/ml]
216
Sutherlandia frutescens
150
Survival
[%]
100
50
0
9.8
19.5
39
78
156
313
625
1250
2500
5000
Concentration
[µg/ml]
Geraniaceae
Geranium wilfordii
Pelargonium sidoides
P6867 / 33
tba / 82
Geranium wilfordii
JF949977 IC50 99.1 µg/ml
IC50 488.2 µg/ml
Pelargonium sidoides
100
100
Survival
[%]
150
Survival
[%]
150
50
50
0
0
9.8
19.5
39
78
156
313
625
1250
2500
5000
9.8
19.5
Concentration
[µg/ml]
Ginkgoaceae
39
78
156
313
625
1250
2500
5000
Concentration
[µg/ml]
Ginkgo biloba
P6872 / 38
JF950005 IC50 768.3 µg/ml
P6876 / 42
JF949980 IC50 163.3 µg/ml
Ginkgo biloba
150
Survival
[%]
100
50
0
9.8
19.5
39
78
156
313
625
1250
2500
5000
Concentration
[µg/ml]
Hypericaceae
Hypericum japonicum
Hypericum japonicum
150
Survival
[%]
100
50
0
9.8
19.5
39
78
156
313
625
1250
2500
5000
Concentration
[µg/ml]
217
Iridaceae
Belamcanda chinensis
P6843 / 09
JF949995 IC50 324.4 µg/ml
P6884 / 50
P6896 / 62
P6903 / 69
JF949984 IC50 108.5 µg/ml
JF950013 IC50 282.1 µg/ml
JF950017 IC50 90.9 µg/ml
Belamcanda chinensis
150
Survival
[%]
100
50
0
9.8
19.5
39
78
156
313
625
1250
2500
5000
Concentration
[µg/ml]
Lamiaceae
Mentha haplocalyx
Prunella vulgaris
Scutellaria baicalensis
Mentha haplocalyx
Prunella vulgaris
100
100
Survival
[%]
150
Survival
[%]
150
50
50
0
0
9.8
19.5
39
78
156
313
625
1250
2500
5000
9.8
19.5
Concentration
[µg/ml]
39
78
156
313
625
1250
2500
5000
Concentration
[µg/ml]
Scutellaria baicalensis
150
Survival
[%]
100
50
0
9.8
19.5
39
78
156
313
625
1250
2500
5000
Concentration
[µg/ml]
Lauraceae
Cinnamomum cassia
P6853 / 19
Cinnamomum cassia
150
Survival
[%]
100
50
0
9.8
19.5
39
78
156
313
625
1250
2500
5000
Concentration
[µg/ml]
218
JF950023 IC50 138.9 µg/ml
Loranthaceae
Taxillus chinensis
P6909 / 75
JF949992 IC50 417.8 µg/ml
P6897 / 63
JF950014 IC50 583.3 µg/ml
P6882 / 48
JF950008 IC50 23.6 µg/ml
P6888 / 54
JF950010 IC50 952.6 µg/ml
Taxillus chinensis
150
Survival
[%]
100
50
0
9.8
19.5
39
78
156
313
625
1250
2500
5000
Concentration
[µg/ml]
Lythraceae
Punica granatum
Punica granatum
150
Survival
[%]
100
50
0
9.8
19.5
39
78
156
313
625
1250
2500
5000
Concentration
[µg/ml]
Magnoliaceae
Magnolia officinalis
Magnolia officinalis
150
Survival
[%]
100
50
0
9.8
19.5
39
78
156
313
625
1250
2500
5000
Concentration
[µg/ml]
Melanthiaceae
Paris polyphylla
Paris polyphylla
150
Survival
[%]
100
50
0
9.8
19.5
39
78
156
313
625
1250
2500
5000
Concentration
[µg/ml]
219
Myrsinaceae
Lysimachia christinae
P6881 / 47
JF949983 IC50 53.4 µg/ml
P6885 / 51
JF950009 IC50 188.9 µg/ml
P6860 / 26
JF949999 IC50 83.0 µg/ml
P6886 / 52
JF950026 IC50 166.9 µg/ml
Lysimachia christinae
150
Survival
[%]
100
50
0
9.8
19.5
39
78
156
313
625
1250
2500
5000
Concentration
[µg/ml]
Ophioglossaceae Ophioglossum vulgatum
Ophioglossum vulgatum
150
Survival
[%]
100
50
0
9.8
19.5
39
78
156
313
625
1250
2500
5000
Concentration
[µg/ml]
Orchidaceae
Dendrobium loddigesii
Dendrobium loddigesii
150
Survival
[%]
100
50
0
9.8
19.5
39
78
156
313
625
1250
2500
5000
Concentration
[µg/ml]
Paeoniaceae
Paeonia lactiflora
Paeonia lactiflora
150
Survival
[%]
100
50
0
9.8
19.5
39
78
156
313
625
1250
2500
5000
Concentration
[µg/ml]
220
Pedaliaceae
Harpagophytum procumbens
tba / 80
-
IC50 36.2 µg/ml
Harpagophytum procumbens
150
Survival
[%]
100
50
0
9.8
19.5
39
78
156
313
625
1250
2500
5000
Concentration
[µg/ml]
Poaceae
Cymbopogon distans
P6857 / 23
JF949974 IC50 425.9 µg/ml
P6894 / 60
JF950004 IC50 88.0 µg/ml
P6893 / 59
P6895 / 61
P6898 / 64
JF950012 IC50 118.5 µg/ml
JF949987 IC50 469.4 µg/ml
JF950015 IC50 22.5 µg/ml
Cymbopogon distans
150
Survival
[%]
100
50
0
9.8
19.5
39
78
156
313
625
1250
2500
5000
Concentration
[µg/ml]
Fallopia japonica (syn.
Polygonum cuspidatum)
Polygonum aviculare
Polygonum multiflorum
Rheum officinale
Polygonaceae
Fallopia japonica
Polygonum aviculare
100
100
Survival
[%]
150
Survival
[%]
150
50
50
0
0
9.8
19.5
39
78
156
313
625
1250
2500
5000
9.8
19.5
39
78
Concentration
[µg/ml]
156
313
625
1250
2500
5000
625
1250
2500
5000
Concentration
[µg/ml]
Polygonum multiflorum
Rheum officinale
100
100
Survival
[%]
150
Survival
[%]
150
50
50
0
0
9.8
19.5
39
78
156
313
625
1250
2500
5000
9.8
Concentration
[µg/ml]
19.5
39
78
156
313
Concentration
[µg/ml]
221
Ranunculaceae Coptis chinensis
P6855 / 21
JF950024 IC50 100.0 µg/ml
P6899 / 65
P6900 / 66
P6901 / 67
IC50 559.4 µg/ml
IC50 712.3 µg/ml
JF950016 IC50 66.5 µg/ml
Coptis chinensis
150
Survival
[%]
100
50
0
9.8
19.5
39
78
156
313
625
1250
2500
5000
Concentration
[µg/ml]
Rosaceae
Rosa chinensis
Rosa laevigata
Sanguisorba officinalis
Rosa chinensis
Rosa laevigata
100
100
Survival
[%]
150
Survival
[%]
150
50
50
0
0
9.8
19.5
39
78
156
313
625
1250
2500
5000
9.8
19.5
Concentration
[µg/ml]
39
78
156
313
625
1250
2500
5000
Concentration
[µg/ml]
Sanguisorba officinalis
150
Survival
[%]
100
50
0
9.8
19.5
39
78
156
313
625
1250
2500
5000
Concentration
[µg/ml]
Rubiaceae
Hedyotis diffusa
P6874 / 40
Hedyotis diffusa
150
Survival
[%]
100
50
0
9.8
19.5
39
78
156
313
625
1250
2500
5000
Concentration
[µg/ml]
222
JF949979 IC50 147.8 µg/ml
Rutaceae
Evodia lepta
Evodia rutaecarpa
Phellodendron chinense
P6869 / 35
P6870 / 36
P6890 / 56
JF949978 IC50 232.0 µg/ml
IC50 50.4 µg/ml
JF949986 IC50 370.1 µg/ml
Evodia lepta
Evodia rutaecarpa
100
100
Survival
[%]
150
Survival
[%]
150
50
50
0
0
9.8
19.5
39
78
156
313
625
1250
2500
5000
9.8
19.5
Concentration
[µg/ml]
39
78
156
313
625
1250
2500
5000
Concentration
[µg/ml]
Phellodendron chinense
150
Survival
[%]
100
50
0
9.8
19.5
39
78
156
313
625
1250
2500
5000
Concentration
[µg/ml]
Saururaceae
Houttuynia cordata
P6875 / 41
JF950006 IC50 279.9 µg/ml
Houttuynia cordata
150
Survival
[%]
100
50
0
9.8
19.5
39
78
156
313
625
1250
2500
5000
Concentration
[µg/ml]
Schisandraceae Kadsura longipedunculata
Kadsura longipedunculata
150
Survival
[%]
100
50
0
9.8
19.5
39
78
156
313
625
1250
2500
5000
Concentration
[µg/ml]
223
P6879 / 45 JF950007 IC50 9.9 µg/ml
Selaginellaceae Selaginella tamariscina
P6904 / 70
JF950018 IC50 339.2 µg/ml
P6889 / 55
JF949985 IC50 140.5 µg/ml
P6910 / 76
JF950020 IC50 298.1 µg/ml
P6911 / 77
JF949993 IC50 59.6 µg/ml
Selaginella tamariscina
150
Survival
[%]
100
50
0
9.8
19.5
39
78
156
313
625
1250
2500
5000
Concentration
[µg/ml]
Valerianaceae
Patrinia scabiosaefolia
Patrinia scabiosaefolia
150
Survival
[%]
100
50
0
9.8
19.5
39
78
156
313
625
1250
2500
5000
Concentration
[µg/ml]
Verbenaceae
Verbena officinalis
Verbena officinalis
150
Survival
[%]
100
50
0
9.8
19.5
39
78
156
313
625
1250
2500
5000
Concentration
[µg/ml]
Violaceae
Viola yezoensis
Viola yezoensis
150
Survival
[%]
100
50
0
9.8
19.5
39
78
156
313
625
1250
2500
5000
Concentration
[µg/ml]
224
Zingiberaceae
Alpinia galanga
Alpinia oxyphylla
P6837 / 03
P6917 / 78
-
Alpinia galanga
IC50 55.7 µg/ml
IC50 119.6 µg/ml
Alpinia oxyphylla
100
100
Survival
[%]
150
Survival
[%]
150
50
50
0
0
9.8
19.5
39
78
156
313
625
1250
2500
5000
9.8
Concentration
[µg/ml]
19.5
39
78
156
313
Concentration
[µg/ml]
225
625
1250
2500
5000
10.4.2 Dichloromethane extracts: T. b. brucei
Acanthaceae
Andrographis paniculata
P6838 / 04
JF949965 IC50 16.8 µg/ml
P6848 / 14
JF949970 IC50 55.2 µg/ml
P6844 / 10
P6845 / 11
P6849 / 15
P6854 / 20
P6902 / 68
JF950021
JF949968
JF950022
JF949973
JF949988
Andrographis paniculata
150
Survival
[%]
100
50
0
0.49
0.98
1.95
3.91
7.81 15.63 31.25 62.5
125
313
625
1250 2500 5000
Concentration
[µg/ml]
Amaranthaceae Celosia cristata
Celosia cristata
150
Survival
[%]
100
50
0
0.49
0.98
1.95
3.91
7.81 15.63 31.25 62.5
125
313
625
1250 2500 5000
Concentration
[µg/ml]
Apiaceae
Bupleurum chinense
Bupleurum marginatum
Centella asiatica
Cnidium monnieri
Saposhnikovia divaricata
Bupleurum chinense
IC50 17.0 µg/ml
IC50 16.2 µg/ml
IC50 14.0 µg/ml
IC50 14.9 µg/ml
IC50 5.1 µg/ml
Bupleurum marginatum
100
100
Survival
[%]
150
Survival
[%]
150
50
50
0
0
0.49
0.98
1.95
3.91
7.81 15.63 31.25 62.5
125
313
625
1250 2500 5000
Concentration
[µg/ml]
0.49
0.98
1.95
3.91
7.81 15.63 31.25 62.5
Concentration
[µg/ml]
226
125
313
625
1250 2500 5000
Centella asiatica
Cnidium monnieri
100
100
Survival
[%]
150
Survival
[%]
150
50
50
0
0
0.49
0.98
1.95
3.91
7.81 15.63 31.25 62.5
125
313
625
1250 2500 5000
0.49
0.98
1.95
3.91
7.81 15.63 31.25 62.5
Concentration
[µg/ml]
125
313
625
1250 2500 5000
Concentration
[µg/ml]
Saposhnikovia divaricata
150
Survival
[%]
100
50
0
0.49
0.98
1.95
3.91
7.81 15.63 31.25 62.5
125
313
625
1250 2500 5000
Concentration
[µg/ml]
Araliaceae
Eleutherococcus senticosus
Panax ginseng China
Panax ginseng Korea
Panax notoginseng
P6919 / 79
P8088 / 81
P8086 / 81
P6887 / 53
Eleutherococcus senticosus
JF950028
JF950029
JF950030
IC50 13.5 µg/ml
IC50 0.9 µg/ml
IC50 3.3 µg/ml
IC50 0.9 µg/ml
Panax ginseng China
150
100
100
Survival
[%]
Survival
[%]
150
50
50
0
0
0.49
0.98
1.95
3.91
7.81 15.63 31.25 62.5
125
313
625
1250 2500 5000
0.075 0.15
0.3
0.6
1.2
2.4
Concentration
[µg/ml]
4.9
9.8
19.5
39
78
156
313
625
39
78
156
313
625
Concentration
[µg/ml]
Panax ginseng Korea
Panax notoginseng
150
100
100
Survival
[%]
Survival
[%]
150
50
50
0
0
0.075 0.15
0.3
0.6
1.2
2.4
4.9
9.8
19.5
39
78
156
313
625
Concentration
[µg/ml]
0.075 0.15
0.3
0.6
1.2
2.4
4.9
9.8
19.5
Concentration
[µg/ml]
227
Arecaceae
Areca catechu
P6840 / 06
-
IC50 22.5 µg/ml
Areca catechu
150
Survival
[%]
100
50
0
0.49
0.98
1.95
3.91
7.81 15.63 31.25 62.5
125
313
625
1250 2500 5000
Concentration
[µg/ml]
Asclepiadaceae Cynanchum paniculatum
P6858 / 24
JF949975 IC50 53.1 µg/ml
P6841 / 07
P6842 / 08
P6839 / 05
P6850 / 16
P6851 / 17
P6852 / 18
P6863 / 29
P6905 / 71
P6906 / 72
P6908 / 74
JF949966
JF949967
JF949994
JF949971
JF949972
JF950000
JF949989
JF949990
JF950019
Cynanchum paniculatum
150
Survival
[%]
100
50
0
0.49
0.98
1.95
3.91
7.81 15.63 31.25 62.5
125
313
625
1250 2500 5000
Concentration
[µg/ml]
Asteraceae
Artemisia annua
Artemisia capillaris
Arctium lappa
Centipeda minima
Chrysanthemum indicum
Chrysanthemum morifolium
Eclipta prostrata
Senecio scandens
Siegesbeckia orientalis
Taraxacum officinale
Artemisia annua
IC50 8.1 µg/ml
IC50 10.6 µg/ml
IC50 3.6 µg/ml
IC50 2.2 µg/ml
IC50 16.0 µg/ml
IC50 19.3 µg/ml
IC50 38.1 µg/ml
IC50 13.1 µg/ml
IC50 7.9 µg/ml
IC50 17.5 µg/ml
Artemisia capillaris
100
100
Survival
[%]
150
Survival
[%]
150
50
50
0
0
0.49
0.98
1.95
3.91
7.81 15.63 31.25 62.5
125
313
625
1250 2500 5000
Concentration
[µg/ml]
0.49
0.98
1.95
3.91
7.81 15.63 31.25 62.5
Concentration
[µg/ml]
228
125
313
625
1250 2500 5000
Arctium lappa
Centipeda minima
100
100
Survival
[%]
150
Survival
[%]
150
50
50
0
0
0.49
0.98
1.95
3.91
7.81 15.63 31.25 62.5
125
313
625
1250 2500 5000
0.49
0.98
1.95
3.91
Concentration
[µg/ml]
7.81 15.63 31.25 62.5
125
313
625
1250 2500 5000
125
313
625
1250 2500 5000
125
313
625
1250 2500 5000
125
313
625
1250 2500 5000
Concentration
[µg/ml]
Chrysanthemum indicum
Chrysanthemum morifolium
100
100
Survival
[%]
150
Survival
[%]
150
50
50
0
0
0.49
0.98
1.95
3.91
7.81 15.63 31.25 62.5
125
313
625
1250 2500 5000
0.49
0.98
1.95
3.91
Concentration
[µg/ml]
7.81 15.63 31.25 62.5
Concentration
[µg/ml]
Eclipta prostrata
Senecio scandens
100
100
Survival
[%]
150
Survival
[%]
150
50
50
0
0
0.49
0.98
1.95
3.91
7.81 15.63 31.25 62.5
125
313
625
1250 2500 5000
0.49
0.98
1.95
3.91
Concentration
[µg/ml]
7.81 15.63 31.25 62.5
Concentration
[µg/ml]
Siegesbeckia orientalis
Taraxacum officinale
100
100
Survival
[%]
150
Survival
[%]
150
50
50
0
0
0.49
0.98
1.95
3.91
7.81 15.63 31.25 62.5
125
313
625
1250 2500 5000
Concentration
[µg/ml]
0.49
0.98
1.95
3.91
7.81 15.63 31.25 62.5
Concentration
[µg/ml]
229
Berberidaceae
Berberis bealei
Dysosma versipellis
Epimedium koreanum
P6883 / 49
P6862 / 28
P6865 / 31
Berberis bealei
JF949996 IC50 5.9 µg/ml
IC50 39.5 µg/ml
JF950002 IC50 4.2 µg/ml
Dysosma versipellis
100
100
Survival
[%]
150
Survival
[%]
150
50
50
0
0
0.49
0.98
1.95
3.91
7.81 15.63 31.25 62.5
125
313
625
1250 2500 5000
0.49
0.98
1.95
3.91
Concentration
[µg/ml]
7.81 15.63 31.25 62.5
125
313
625
1250 2500 5000
Concentration
[µg/ml]
Epimedium koreanum
150
Survival
[%]
100
50
0
0.49
0.98
1.95
3.91
7.81 15.63 31.25 62.5
125
313
625
1250 2500 5000
Concentration
[µg/ml]
Brassicaceae
Capsella bursa-pastoris
Isatis indigotica (root)
Isatis indigotica (leaf)
P6846 / 12
P6877 / 43
P6878 / 44
Capsella bursa-pastoris
JF949997 IC50 13.7 µg/ml
JF949981 IC50 2.9 µg/ml
JF949981 IC50 45.3 µg/ml
Isatis indigotica (root)
150
100
100
Survival
[%]
Survival
[%]
150
50
50
0
0
0.49
0.98
1.95
3.91
7.81 15.63 31.25 62.5
125
313
625
1250 2500 5000
Concentration
[µg/ml]
Isatis indigotica (leaf)
Survival
[%]
100
50
0
0.98
1.95
3.91
7.81 15.63 31.25 62.5
0.98
1.95
3.91
7.81 15.63 31.25 62.5
Concentration
[µg/ml]
150
0.49
0.49
125
313
625
1250 2500 5000
Concentration
[µg/ml]
230
125
313
625
1250 2500 5000
Caprifoliaceae
Lonicera confusa
P6880 / 46
JF949982 IC50 16.2 µg/ml
P6892 / 58
JF950027 IC50 52.6 µg/ml
Lonicera confusa
150
Survival
[%]
100
50
0
0.49
0.98
1.95
3.91
7.81 15.63 31.25 62.5
125
313
625
1250 2500 5000
Concentration
[µg/ml]
Convallariaceae Polygonatum kingianum
Polygonatum kingianum
150
Survival
[%]
100
50
0
0.49
0.98
1.95
3.91
7.81 15.63 31.25 62.5
125
313
625
1250 2500 5000
Concentration
[µg/ml]
Crassulaceae
Rhodiola rosea
P6920 / 84
-
IC50 43.9 µg/ml
Rhodiola rosea
150
Survival
[%]
100
50
0
0.49
0.98
1.95
3.91
7.81 15.63 31.25 62.5
125
313
625
1250 2500 5000
Concentration
[µg/ml]
Cupressaceae
Platycladus orientalis
P6891 / 57
Platycladus orientalis
150
Survival
[%]
100
50
0
0.49
0.98
1.95
3.91
7.81 15.63 31.25 62.5
125
313
625
1250 2500 5000
Concentration
[µg/ml]
231
JF950011 IC50 17.7 µg/ml
Dryopteridaceae Cyrtomium fortunei
P6859 / 25
JF949998 IC50 37.1 µg/ml
P6864 / 30
JF950001 IC50 20.9 µg/ml
P6866 / 32
JF950003 IC50 30.9 µg/ml
Cyrtomium fortunei
150
Survival
[%]
100
50
0
0.49
0.98
1.95
3.91
7.81 15.63 31.25 62.5
125
313
625
1250 2500 5000
Concentration
[µg/ml]
Ephedraceae
Ephedra sinica
Ephedra sinica
150
Survival
[%]
100
50
0
0.49
0.98
1.95
3.91
7.81 15.63 31.25 62.5
125
313
625
1250 2500 5000
Concentration
[µg/ml]
Equisetaceae
Equisetum hiemale
Equisetum hiemale
150
Survival
[%]
100
50
0
0.49
0.98
1.95
3.91
7.81 15.63 31.25 62.5
125
313
625
1250 2500 5000
Concentration
[µg/ml]
Euphorbiaceae Croton tiglium
P6856 / 22
Croton tiglium
150
Survival
[%]
100
50
0
0.49
0.98
1.95
3.91
7.81 15.63 31.25 62.5
125
313
625
1250 2500 5000
Concentration
[µg/ml]
232
-
IC50 86.5 µg/ml
Fabaceae
Abrus cantoniensis
Acacia catechu
Cassia tora
Desmodium styracifolium
Glycyrrhiza inflata
Spatholobus suberectus
Sutherlandia frutescens
P6835 / 01
P6836 / 02
P6847 / 13
P6861 / 27
P6873 / 39
P6907 / 73
tba / 83
JF949964
JF949969
JF949976
JF950025
JF949991
-
Abrus cantoniensis
IC50 14.5 µg/ml
IC50 13.1 µg/ml
IC50 185.9 µg/ml
IC50 16.3 µg/ml
IC50 6.4 µg/ml
IC50 25.4 µg/ml
IC50 41.8 µg/ml
Acacia catechu
100
100
Survival
[%]
150
Survival
[%]
150
50
50
0
0
0.49
0.98
1.95
3.91
7.81 15.63 31.25 62.5
125
313
625
1250 2500 5000
0.49
0.98
1.95
3.91
Concentration
[µg/ml]
7.81 15.63 31.25 62.5
125
313
625
1250 2500 5000
313
625
1250 2500 5000
313
625
1250 2500 5000
Concentration
[µg/ml]
Cassia tora
Desmodium styracifolium
100
100
Survival
[%]
150
Survival
[%]
150
50
50
0
0
0.49
0.98
1.95
3.91
7.81 15.63 31.25 62.5
125
313
625
1250 2500 5000
0.49
0.98
1.95
3.91
Concentration
[µg/ml]
7.81 15.63 31.25 62.5
125
Concentration
[µg/ml]
Glycyrrhiza inflata
Spatholobus suberectus
100
100
Survival
[%]
150
Survival
[%]
150
50
50
0
0
0.49
0.98
1.95
3.91
7.81 15.63 31.25 62.5
125
313
625
1250 2500 5000
Concentration
[µg/ml]
0.49
0.98
1.95
3.91
7.81 15.63 31.25 62.5
Concentration
[µg/ml]
233
125
Sutherlandia frutescens
150
Survival
[%]
100
50
0
0.49
0.98
1.95
3.91
7.81 15.63 31.25 62.5
125
313
625
1250 2500 5000
Concentration
[µg/ml]
Geraniaceae
Geranium wilfordii
Pelargonium sidoides
P6867 / 33
tba / 82
Geranium wilfordii
JF949977 IC50 23.0 µg/ml
IC50 52.1 µg/ml
Pelargonium sidoides
100
100
Survival
[%]
150
Survival
[%]
150
50
50
0
0
0.49
0.98
1.95
3.91
7.81 15.63 31.25 62.5
125
313
625
1250 2500 5000
0.49
0.98
1.95
Concentration
[µg/ml]
Ginkgoaceae
3.91
7.81 15.63 31.25 62.5
125
313
625
1250 2500 5000
Concentration
[µg/ml]
Ginkgo biloba
P6872 / 38
JF950005 IC50 71.9 µg/ml
P6876 / 42
JF949980 IC50 21.3 µg/ml
Ginkgo biloba
150
Survival
[%]
100
50
0
0.49
0.98
1.95
3.91
7.81 15.63 31.25 62.5
125
313
625
1250 2500 5000
Concentration
[µg/ml]
Hypericaceae
Hypericum japonicum
Hypericum japonicum
150
Survival
[%]
100
50
0
0.49
0.98
1.95
3.91
7.81 15.63 31.25 62.5
125
313
625
1250 2500 5000
Concentration
[µg/ml]
234
Iridaceae
Belamcanda chinensis
P6843 / 09
JF949995 IC50 22.3 µg/ml
P6884 / 50
P6896 / 62
P6903 / 69
JF949984 IC50 14.7 µg/ml
JF950013 IC50 13.2 µg/ml
JF950017 IC50 7.4 µg/ml
Belamcanda chinensis
150
Survival
[%]
100
50
0
0.49
0.98
1.95
3.91
7.81 15.63 31.25 62.5
125
313
625
1250 2500 5000
Concentration
[µg/ml]
Lamiaceae
Mentha haplocalyx
Prunella vulgaris
Scutellaria baicalensis
Mentha haplocalyx
Prunella vulgaris
100
100
Survival
[%]
150
Survival
[%]
150
50
50
0
0
0.49
0.98
1.95
3.91
7.81 15.63 31.25 62.5
125
313
625
1250 2500 5000
0.49
0.98
1.95
Concentration
[µg/ml]
3.91
7.81 15.63 31.25 62.5
125
313
625
1250 2500 5000
Concentration
[µg/ml]
Scutellaria baicalensis
150
Survival
[%]
100
50
0
0.49
0.98
1.95
3.91
7.81 15.63 31.25 62.5
125
313
625
1250 2500 5000
Concentration
[µg/ml]
Lauraceae
Cinnamomum cassia
P6853 / 19
Cinnamomum cassia
150
Survival
[%]
100
50
0
0.49
0.98
1.95
3.91
7.81 15.63 31.25 62.5
125
313
625
1250 2500 5000
Concentration
[µg/ml]
235
JF950023 IC50 11.0 µg/ml
Loranthaceae
Taxillus chinensis
P6909 / 75
JF949992 IC50 27.2 µg/ml
P6897 / 63
JF950014 IC50 14.6 µg/ml
P6882 / 48
JF950008 IC50 0.9 µg/ml
P6888 / 54
JF950010 IC50 73.6 µg/ml
Taxillus chinensis
150
Survival
[%]
100
50
0
0.49
0.98
1.95
3.91
7.81 15.63 31.25 62.5
125
313
625
1250 2500 5000
Concentration
[µg/ml]
Lythraceae
Punica granatum
Punica granatum
150
Survival
[%]
100
50
0
0.49
0.98
1.95
3.91
7.81 15.63 31.25 62.5
125
313
625
1250 2500 5000
Concentration
[µg/ml]
Magnoliaceae
Magnolia officinalis
Magnolia officinalis
150
Survival
[%]
100
50
0
0.49
0.98
1.95
3.91
7.81 15.63 31.25 62.5
125
313
625
1250 2500 5000
Concentration
[µg/ml]
Melanthiaceae
Paris polyphylla
Paris polyphylla
150
Survival
[%]
100
50
0
0.49
0.98
1.95
3.91
7.81 15.63 31.25 62.5
125
313
625
1250 2500 5000
Concentration
[µg/ml]
236
Myrsinaceae
Lysimachia christinae
P6881 / 47
JF949983 IC50 20.6 µg/ml
P6885 / 51
JF950009 IC50 19.8 µg/ml
P6860 / 26
JF949999 IC50 13.5 µg/ml
P6886 / 52
JF950026 IC50 9.1 µg/ml
Lysimachia christinae
150
Survival
[%]
100
50
0
0.49
0.98
1.95
3.91
7.81 15.63 31.25 62.5
125
313
625
1250 2500 5000
Concentration
[µg/ml]
Ophioglossaceae Ophioglossum vulgatum
Ophioglossum vulgatum
150
Survival
[%]
100
50
0
0.49
0.98
1.95
3.91
7.81 15.63 31.25 62.5
125
313
625
1250 2500 5000
Concentration
[µg/ml]
Orchidaceae
Dendrobium loddigesii
Dendrobium loddigesii
150
Survival
[%]
100
50
0
0.49
0.98
1.95
3.91
7.81 15.63 31.25 62.5
125
313
625
1250 2500 5000
Concentration
[µg/ml]
Paeoniaceae
Paeonia lactiflora
Paeonia lactiflora
150
Survival
[%]
100
50
0
0.49
0.98
1.95
3.91
7.81 15.63 31.25 62.5
125
313
625
1250 2500 5000
Concentration
[µg/ml]
237
Pedaliaceae
Harpagophytum procumbens
tba / 80
-
IC50 0.9 µg/ml
Harpagophytum procumbens
150
Survival
[%]
100
50
0
0.49
0.98
1.95
3.91
7.81 15.63 31.25 62.5
125
313
625
1250 2500 5000
Concentration
[µg/ml]
Poaceae
Cymbopogon distans
P6857 / 23
JF949974 IC50 31.1 µg/ml
P6894 / 60
JF950004 IC50 13.1 µg/ml
P6893 / 59
P6895 / 61
P6898 / 64
JF950012 IC50 18.2 µg/ml
JF949987 IC50 98.6 µg/ml
JF950015 IC50 34.0 µg/ml
Cymbopogon distans
150
Survival
[%]
100
50
0
0.49
0.98
1.95
3.91
7.81 15.63 31.25 62.5
125
313
625
1250 2500 5000
Concentration
[µg/ml]
Polygonaceae
Fallopia japonica (syn.
Polygonum cuspidatum)
Polygonum aviculare
Polygonum multiflorum
Rheum officinale
Fallopia japonica
Polygonum aviculare
100
100
Survival
[%]
150
Survival
[%]
150
50
50
0
0
0.49
0.98
1.95
3.91
7.81 15.63 31.25 62.5
125
313
625
1250 2500 5000
0.49
0.98
1.95
3.91
Concentration
[µg/ml]
7.81 15.63 31.25 62.5
125
313
625
1250 2500 5000
125
313
625
1250 2500 5000
Concentration
[µg/ml]
Polygonum multiflorum
Rheum officinale
100
100
Survival
[%]
150
Survival
[%]
150
50
50
0
0
0.49
0.98
1.95
3.91
7.81 15.63 31.25 62.5
125
313
625
1250 2500 5000
Concentration
[µg/ml]
0.49
0.98
1.95
3.91
7.81 15.63 31.25 62.5
Concentration
[µg/ml]
238
Ranunculaceae Coptis chinensis
P6855 / 21
JF950024 IC50 12.9 µg/ml
P6899 / 65
P6900 / 66
P6901 / 67
IC50 20.1 µg/ml
IC50 20.6 µg/ml
JF950016 IC50 12.3 µg/ml
Coptis chinensis
150
Survival
[%]
100
50
0
0.49
0.98
1.95
3.91
7.81 15.63 31.25 62.5
125
313
625
1250 2500 5000
Concentration
[µg/ml]
Rosaceae
Rosa chinensis
Rosa laevigata
Sanguisorba officinalis
Rosa chinensis
Rosa laevigata
100
100
Survival
[%]
150
Survival
[%]
150
50
50
0
0
0.49
0.98
1.95
3.91
7.81 15.63 31.25 62.5
125
313
625
1250 2500 5000
0.49
0.98
1.95
Concentration
[µg/ml]
3.91
7.81 15.63 31.25 62.5
125
313
625
1250 2500 5000
Concentration
[µg/ml]
Sanguisorba officinalis
150
Survival
[%]
100
50
0
0.49
0.98
1.95
3.91
7.81 15.63 31.25 62.5
125
313
625
1250 2500 5000
Concentration
[µg/ml]
Rubiaceae
Hedyotis diffusa
P6874 / 40
Hedyotis diffusa
150
Survival
[%]
100
50
0
0.49
0.98
1.95
3.91
7.81 15.63 31.25 62.5
125
313
625
1250 2500 5000
Concentration
[µg/ml]
239
JF949979 IC50 13.3 µg/ml
Rutaceae
Evodia lepta
Evodia rutaecarpa
Phellodendron chinense
P6869 / 35
P6870 / 36
P6890 / 56
JF949978 IC50 13.9 µg/ml
IC50 16.8 µg/ml
JF949986 IC50 15.6 µg/ml
Evodia lepta
Evodia rutaecarpa
100
100
Survival
[%]
150
Survival
[%]
150
50
50
0
0
0.49
0.98
1.95
3.91
7.81 15.63 31.25 62.5
125
313
625
1250 2500 5000
0.49
0.98
1.95
3.91
Concentration
[µg/ml]
7.81 15.63 31.25 62.5
125
313
625
1250 2500 5000
Concentration
[µg/ml]
Phellodendron chinense
150
Survival
[%]
100
50
0
0.49
0.98
1.95
3.91
7.81 15.63 31.25 62.5
125
313
625
1250 2500 5000
Concentration
[µg/ml]
Saururaceae
Houttuynia cordata
P6875 / 41
JF950006 IC50 68.3 µg/ml
Houttuynia cordata
150
Survival
[%]
100
50
0
0.49
0.98
1.95
3.91
7.81 15.63 31.25 62.5
125
313
625
1250 2500 5000
Concentration
[µg/ml]
Schisandraceae Kadsura longipedunculata
Kadsura longipedunculata
150
Survival
[%]
100
50
0
0.06
0.12
0.24
0.49
0.98
1.95
3.91
7.81 15.63 31.25 62.5
125
313
625
Concentration
[µg/ml]
240
P6879 / 45
JF950007 IC50 0.1 µg/ml
Selaginellaceae Selaginella tamariscina
P6904 / 70
JF950018 IC50 13.6 µg/ml
P6889 / 55
JF949985 IC50 13.7 µg/ml
P6910 / 76
JF950020 IC50 16.5 µg/ml
P6911 / 77
JF949993 IC50 3.3 µg/ml
Selaginella tamariscina
150
Survival
[%]
100
50
0
0.49
0.98
1.95
3.91
7.81 15.63 31.25 62.5
125
313
625
1250 2500 5000
Concentration
[µg/ml]
Valerianaceae Patrinia scabiosaefolia
Patrinia scabiosaefolia
150
Survival
[%]
100
50
0
0.49
0.98
1.95
3.91
7.81 15.63 31.25 62.5
125
313
625
1250 2500 5000
Concentration
[µg/ml]
Verbenaceae
Verbena officinalis
Verbena officinalis
150
Survival
[%]
100
50
0
0.49
0.98
1.95
3.91
7.81 15.63 31.25 62.5
125
313
625
1250 2500 5000
Concentration
[µg/ml]
Violaceae
Viola yezoensis
Viola yezoensis
150
Survival
[%]
100
50
0
0.49
0.98
1.95
3.91
7.81 15.63 31.25 62.5
125
313
625
1250 2500 5000
Concentration
[µg/ml]
241
Zingiberaceae Alpinia galanga
P6837 / 03
P6917 / 78
Alpinia oxyphylla
-
Alpinia galanga
IC50 1.4 µg/ml
IC50 0.7 µg/ml
Alpinia oxyphylla
100
100
Survival
[%]
150
Survival
[%]
150
50
50
0
0
0.49
0.98
1.95
3.91
7.81 15.63 31.25 62.5
125
313
625
1250 2500 5000
Concentration
[µg/ml]
0.49
0.98
1.95
3.91
7.81 15.63 31.25 62.5
Concentration
[µg/ml]
242
125
313
625
1250 2500 5000
10.4.3 Methanol extracts: HeLa
Acanthaceae
Andrographis paniculata
P6838 / 04
JF949965 IC50 323.3 µg/ml
P6848 / 14
JF949970 IC50 499.8 µg/ml
P6844 / 10
P6845 / 11
P6849 / 15
P6854 / 20
P6902 / 68
JF950021
JF949968
JF950022
JF949973
JF949988
Andrographis paniculata
150
Survival
[%]
100
50
0
9.8
19.5
39
78
156
313
625
1250
2500
5000
Concentration
[µg/ml]
Amaranthaceae Celosia cristata
Celosia cristata
150
Survival
[%]
100
50
0
9.8
19.5
39
78
156
313
625
1250
2500
5000
Concentration
[µg/ml]
Apiaceae
Bupleurum chinense
Bupleurum marginatum
Centella asiatica
Cnidium monnieri
Saposhnikovia divaricata
Bupleurum chinense
IC50 646.4 µg/ml
IC50 1147.0 µg/ml
IC50 773.0 µg/ml
IC50 251.1 µg/ml
IC50 1515.6 µg/ml
Bupleurum marginatum
100
100
Survival
[%]
150
Survival
[%]
150
50
50
0
0
9.8
19.5
39
78
156
313
625
1250
2500
5000
9.8
Concentration
[µg/ml]
19.5
39
78
156
313
Concentration
[µg/ml]
243
625
1250
2500
5000
Centella asiatica
Cnidium monnieri
100
100
Survival
[%]
150
Survival
[%]
150
50
50
0
0
9.8
19.5
39
78
156
313
625
1250
2500
5000
9.8
19.5
39
78
Concentration
[µg/ml]
156
313
625
1250
2500
5000
Concentration
[µg/ml]
Saposhnikovia divaricata
150
Survival
[%]
100
50
0
9.8
19.5
39
78
156
313
625
1250
2500
5000
Concentration
[µg/ml]
Araliaceae
Eleutherococcus senticosus
Panax ginseng China
Panax ginseng Korea
Panax notoginseng
P6919 / 79
P8088 / 81
P8086 / 81
P6887 / 53
Eleutherococcus senticosus
JF950028
JF950029
JF950030
IC50 692.0 µg/ml
IC50 1427.9 µg/ml
IC50 1317.9 µg/ml
IC50 1241.6 µg/ml
Panax ginseng China
100
100
Survival
[%]
150
Survival
[%]
150
50
50
0
0
9.8
19.5
39
78
156
313
625
1250
2500
5000
9.8
19.5
39
78
Concentration
[µg/ml]
156
313
625
1250
2500
5000
625
1250
2500
5000
Concentration
[µg/ml]
Panax ginseng Korea
Panax notoginseng
100
100
Survival
[%]
150
Survival
[%]
150
50
50
0
0
9.8
19.5
39
78
156
313
625
1250
2500
5000
9.8
Concentration
[µg/ml]
19.5
39
78
156
313
Concentration
[µg/ml]
244
Arecaceae
Areca catechu
P6840 / 06
-
IC50 414.2 µg/ml
Areca catechu
150
Survival
[%]
100
50
0
9.8
19.5
39
78
156
313
625
1250
2500
5000
Concentration
[µg/ml]
Asclepiadaceae Cynanchum paniculatum
P6858 / 24 JF949975 IC50 500.5 µg/ml
Cynanchum paniculatum
150
Survival
[%]
100
50
0
9.8
19.5
39
78
156
313
625
1250
2500
5000
Concentration
[µg/ml]
Asteraceae
Artemisia annua
Artemisia capillaris
Arctium lappa
Centipeda minima
Chrysanthemum indicum
Chrysanthemum morifolium
Eclipta prostrata
Senecio scandens
Siegesbeckia orientalis
Taraxacum officinale
P6841 / 07
P6842 / 08
P6839 / 05
P6850 / 16
P6851 / 17
P6852 / 18
P6863 / 29
P6905 / 71
P6906 / 72
P6908 / 74
JF949966
JF949967
JF949994
JF949971
JF949972
JF950000
JF949989
JF949990
JF950019
Artemisia annua
IC50 287.2 µg/ml
IC50 314.9 µg/ml
IC50 1467.7 µg/ml
IC50 219.1 µg/ml
IC50 355.7 µg/ml
IC50 349.2 µg/ml
IC50 329.7 µg/ml
IC50 299.3 µg/ml
IC50 237.5 µg/ml
IC50 636.7 µg/ml
Artemisia capillaris
100
100
Survival
[%]
150
Survival
[%]
150
50
50
0
0
9.8
19.5
39
78
156
313
625
1250
2500
5000
9.8
Concentration
[µg/ml]
19.5
39
78
156
313
Concentration
[µg/ml]
245
625
1250
2500
5000
Arctium lappa
Centipeda minima
100
100
Survival
[%]
150
Survival
[%]
150
50
50
0
0
9.8
19.5
39
78
156
313
625
1250
2500
5000
9.8
19.5
39
78
Concentration
[µg/ml]
156
313
625
1250
2500
5000
625
1250
2500
5000
625
1250
2500
5000
625
1250
2500
5000
Concentration
[µg/ml]
Chrysanthemum indicum
Chrysanthemum morifolium
100
100
Survival
[%]
150
Survival
[%]
150
50
50
0
0
9.8
19.5
39
78
156
313
625
1250
2500
5000
9.8
19.5
39
78
Concentration
[µg/ml]
156
313
Concentration
[µg/ml]
Eclipta prostrata
Senecio scandens
100
100
Survival
[%]
150
Survival
[%]
150
50
50
0
0
9.8
19.5
39
78
156
313
625
1250
2500
5000
9.8
19.5
39
78
Concentration
[µg/ml]
156
313
Concentration
[µg/ml]
Siegesbeckia orientalis
Taraxacum officinale
100
100
Survival
[%]
150
Survival
[%]
150
50
50
0
0
9.8
19.5
39
78
156
313
625
1250
2500
5000
9.8
Concentration
[µg/ml]
19.5
39
78
156
313
Concentration
[µg/ml]
246
Berberidaceae
Berberis bealei
Dysosma versipellis
Epimedium koreanum
P6883 / 49 JF949996 IC50 149.7 µg/ml
P6862 / 28
IC50 385.2 µg/ml
P6865 / 31 JF950002 IC50 257.5 µg/ml
Berberis bealei
Dysosma versipellis
100
100
Survival
[%]
150
Survival
[%]
150
50
50
0
0
9.8
19.5
39
78
156
313
625
1250
2500
5000
9.8
19.5
39
78
Concentration
[µg/ml]
156
313
625
1250
2500
5000
Concentration
[µg/ml]
Epimedium koreanum
150
Survival
[%]
100
50
0
9.8
19.5
39
78
156
313
625
1250
2500
5000
Concentration
[µg/ml]
Brassicaceae
Capsella bursa-pastoris
Isatis indigotica (root)
Isatis indigotica (leaf)
P6846 / 12 JF949997 IC50 141.0 µg/ml
P6877 / 43 JF949981 IC50 674.3 µg/ml
P6878 / 44 JF949981 IC50 274.2 µg/ml
Capsella bursa-pastoris
Isatis indigotica (root)
100
100
Survival
[%]
150
Survival
[%]
150
50
50
0
0
9.8
19.5
39
78
156
313
625
1250
2500
5000
9.8
Concentration
[µg/ml]
Isatis indigotica (leaf)
Survival
[%]
100
50
0
19.5
39
78
156
313
39
78
156
313
Concentration
[µg/ml]
150
9.8
19.5
625
1250
2500
5000
Concentration
[µg/ml]
247
625
1250
2500
5000
Caprifoliaceae
Lonicera confusa
P6880 / 46 JF949982 IC50 923.5 µg/ml
Lonicera confusa
150
Survival
[%]
100
50
0
9.8
19.5
39
78
156
313
625
1250
2500
5000
Concentration
[µg/ml]
Convallariaceae Polygonatum kingianum
P6892 / 58 JF950027 IC50 1517.9 µg/ml
Polygonatum kingianum
150
Survival
[%]
100
50
0
9.8
19.5
39
78
156
313
625
1250
2500
5000
Concentration
[µg/ml]
Cupressaceae
Platycladus orientalis
P6891 / 57 JF950011 IC50 705.5 µg/ml
Platycladus orientalis
150
Survival
[%]
100
50
0
9.8
19.5
39
78
156
313
625
1250
2500
5000
Concentration
[µg/ml]
Dryopteridaceae Cyrtomium fortunei
P6859 / 25 JF949998 IC50 722.0 µg/ml
Cyrtomium fortunei
150
Survival
[%]
100
50
0
9.8
19.5
39
78
156
313
625
1250
2500
5000
Concentration
[µg/ml]
248
Ephedraceae
Ephedra sinica
P6864 / 30 JF950001 IC50 163.5 µg/ml
Ephedra sinica
150
Survival
[%]
100
50
0
9.8
19.5
39
78
156
313
625
1250
2500
5000
Concentration
[µg/ml]
Equisetaceae
Equisetum hiemale
P6866 / 32 JF950003 IC50 241.2 µg/ml
Equisetum hiemale
150
Survival
[%]
100
50
0
9.8
19.5
39
78
156
313
625
1250
2500
5000
Concentration
[µg/ml]
Euphorbiaceae Croton tiglium
P6856 / 22
Croton tiglium
150
Survival
[%]
100
50
0
9.8
19.5
39
78
156
313
625
1250
2500
5000
Concentration
[µg/ml]
249
-
IC50 297.0 µg/ml
Abrus cantoniensis
Acacia catechu
Cassia tora
Desmodium styracifolium
Glycyrrhiza inflata
Spatholobus suberectus
Sutherlandia frutescens
Fabaceae
P6835 / 01
P6836 / 02
P6847 / 13
P6861 / 27
P6873 / 39
P6907 / 73
tba / 83
IC50 612.4 µg/ml
IC50 318.0 µg/ml
IC50 670.9 µg/ml
IC50 324.3 µg/ml
IC50 528.3 µg/ml
IC50 237.5 µg/ml
IC50 586.6 µg/ml
JF949964
JF949969
JF949976
JF950025
JF949991
-
Abrus cantoniensis
Acacia catechu
100
100
Survival
[%]
150
Survival
[%]
150
50
50
0
0
9.8
19.5
39
78
156
313
625
1250
2500
5000
9.8
19.5
39
78
Concentration
[µg/ml]
156
313
625
1250
2500
5000
625
1250
2500
5000
625
1250
2500
5000
Concentration
[µg/ml]
Cassia tora
Desmodium styracifolium
100
100
Survival
[%]
150
Survival
[%]
150
50
50
0
0
9.8
19.5
39
78
156
313
625
1250
2500
5000
9.8
19.5
39
78
Concentration
[µg/ml]
156
313
Concentration
[µg/ml]
Glycyrrhiza inflata
Spatholobus suberectus
100
100
Survival
[%]
150
Survival
[%]
150
50
50
0
0
9.8
19.5
39
78
156
313
625
1250
2500
5000
9.8
Concentration
[µg/ml]
19.5
39
78
156
313
Concentration
[µg/ml]
250
Sutherlandia frutescens
150
Survival
[%]
100
50
0
9.8
19.5
39
78
156
313
625
1250
2500
5000
Concentration
[µg/ml]
Geraniaceae
Geranium wilfordii
Pelargonium sidoides
P6867 / 33 JF949977 IC50 236.0 µg/ml
tba / 82
IC50 112.3 µg/ml
Geranium wilfordii
Pelargonium sidoides
100
100
Survival
[%]
150
Survival
[%]
150
50
50
0
0
9.8
19.5
39
78
156
313
625
1250
2500
5000
9.8
Concentration
[µg/ml]
Ginkgoaceae
19.5
39
78
156
313
625
1250
2500
5000
Concentration
[µg/ml]
Ginkgo biloba
P6872 / 38 JF950005 IC50 302.9 µg/ml
Ginkgo biloba
150
Survival
[%]
100
50
0
9.8
19.5
39
78
156
313
625
1250
2500
5000
Concentration
[µg/ml]
Hypericaceae
Hypericum japonicum
P6876 / 42 JF949980 IC50 177.5 µg/ml
Hypericum japonicum
150
Survival
[%]
100
50
0
9.8
19.5
39
78
156
313
625
1250
2500
5000
Concentration
[µg/ml]
251
Iridaceae
Belamcanda chinensis
P6843 / 09 JF949995 IC50 522.6 µg/ml
Belamcanda chinensis
150
Survival
[%]
100
50
0
9.8
19.5
39
78
156
313
625
1250
2500
5000
Concentration
[µg/ml]
Lamiaceae
Mentha haplocalyx
Prunella vulgaris
Scutellaria baicalensis
P6884 / 50 JF949984 IC50 375.0 µg/ml
P6896 / 62 JF950013 IC50 475.4 µg/ml
P6903 / 69 JF950017 IC50 367.6 µg/ml
Mentha haplocalyx
Prunella vulgaris
100
100
Survival
[%]
150
Survival
[%]
150
50
50
0
0
9.8
19.5
39
78
156
313
625
1250
2500
5000
9.8
Concentration
[µg/ml]
19.5
39
78
156
313
625
1250
2500
5000
Concentration
[µg/ml]
Scutellaria baicalensis
150
Survival
[%]
100
50
0
9.8
19.5
39
78
156
313
625
1250
2500
5000
Concentration
[µg/ml]
Lauraceae
Cinnamomum cassia
P6853 / 19 JF950023 IC50 272.4 µg/ml
Cinnamomum cassia
150
Survival
[%]
100
50
0
9.8
19.5
39
78
156
313
625
1250
2500
5000
Concentration
[µg/ml]
252
Loranthaceae
Taxillus chinensis
P6909 / 75 JF949992 IC50 1213.4 µg/ml
Taxillus chinensis
150
Survival
[%]
100
50
0
9.8
19.5
39
78
156
313
625
1250
2500
5000
Concentration
[µg/ml]
Lythraceae
Punica granatum
P6897 / 63 JF950014 IC50 211.2 µg/ml
Punica granatum
150
Survival
[%]
100
50
0
9.8
19.5
39
78
156
313
625
1250
2500
5000
Concentration
[µg/ml]
Magnoliaceae
Magnolia officinalis
P6882 / 48 JF950008 IC50 49.1 µg/ml
Magnolia officinalis
150
Survival
[%]
100
50
0
9.8
19.5
39
78
156
313
625
1250
2500
5000
Concentration
[µg/ml]
Melanthiaceae
Paris polyphylla
P6888 / 54 JF950010 IC50 35.0 µg/ml
Paris polyphylla
150
Survival
[%]
100
50
0
9.8
19.5
39
78
156
313
625
1250
2500
5000
Concentration
[µg/ml]
253
Myrsinaceae
Lysimachia christinae
P6881 / 47 JF949983 IC50 1752.6 µg/ml
Lysimachia christinae
150
Survival
[%]
100
50
0
9.8
19.5
39
78
156
313
625
1250
2500
5000
Concentration
[µg/ml]
Myrtaceae
Eucalyptus robusta
P6868 / 34
-
IC50 181.4 µg/ml
Eucalyptus robusta
150
Survival
[%]
100
50
0
9.8
19.5
39
78
156
313
625
1250
2500
5000
Concentration
[µg/ml]
Ophioglossaceae Ophioglossum vulgatum
P6885 / 51 JF950009 IC50 469.0 µg/ml
Ophioglossum vulgatum
150
Survival
[%]
100
50
0
9.8
19.5
39
78
156
313
625
1250
2500
5000
Concentration
[µg/ml]
Orchidaceae
Dendrobium loddigesii
P6860 / 26 JF949999 IC50 232.8 µg/ml
Dendrobium loddigesii
150
Survival
[%]
100
50
0
9.8
19.5
39
78
156
313
625
1250
2500
5000
Concentration
[µg/ml]
254
Paeoniaceae
Paeonia lactiflora
P6886 / 52 JF950026 IC50 294.6 µg/ml
Paeonia lactiflora
150
Survival
[%]
100
50
0
9.8
19.5
39
78
156
313
625
1250
2500
5000
Concentration
[µg/ml]
Pedaliaceae
Harpagophytum procumbens
tba / 80
-
IC50 692.6 µg/ml
Harpagophytum procumbens
150
Survival
[%]
100
50
0
9.8
19.5
39
78
156
313
625
1250
2500
5000
Concentration
[µg/ml]
Poaceae
Cymbopogon distans
P6857 / 23 JF949974 IC50 98.8 µg/ml
Cymbopogon distans
150
Survival
[%]
100
50
0
9.8
19.5
39
78
156
313
625
1250
2500
5000
Concentration
[µg/ml]
Fallopia japonica (syn.
Polygonum cuspidatum)
Polygonum aviculare
Polygonum multiflorum
Rheum officinale
Polygonaceae
P6894 / 60 JF950004 IC50 317.3 µg/ml
P6893 / 59 JF950012 IC50 342.3 µg/ml
P6895 / 61 JF949987 IC50 437.4 µg/ml
P6898 / 64 JF950015 IC50 270.9 µg/ml
Fallopia japonica
Polygonum aviculare
100
100
Survival
[%]
150
Survival
[%]
150
50
50
0
0
9.8
19.5
39
78
156
313
625
1250
2500
5000
9.8
Concentration
[µg/ml]
19.5
39
78
156
313
Concentration
[µg/ml]
255
625
1250
2500
5000
Polygonum multiflorum
Rheum officinale
100
100
Survival
[%]
150
Survival
[%]
150
50
50
0
0
9.8
19.5
39
78
156
313
625
1250
2500
5000
9.8
19.5
39
78
Concentration
[µg/ml]
156
313
625
1250
2500
5000
Concentration
[µg/ml]
Ranunculaceae Coptis chinensis
P6855 / 21 JF950024 IC50 81.8 µg/ml
Coptis chinensis
150
Survival
[%]
100
50
0
9.8
19.5
39
78
156
313
625
1250
2500
5000
Concentration
[µg/ml]
Rosaceae
Rosa chinensis
Rosa laevigata
Sanguisorba officinalis
P6899 / 65
IC50 266.6 µg/ml
P6900 / 66
IC50 1855.4 µg/ml
P6901 / 67 JF950016 IC50 158.5 µg/ml
Rosa chinensis
Rosa laevigata
100
100
Survival
[%]
150
Survival
[%]
150
50
50
0
0
9.8
19.5
39
78
156
313
625
1250
2500
5000
9.8
Concentration
[µg/ml]
Sanguisorba officinalis
Survival
[%]
100
50
0
19.5
39
78
156
313
39
78
156
313
Concentration
[µg/ml]
150
9.8
19.5
625
1250
2500
5000
Concentration
[µg/ml]
256
625
1250
2500
5000
Rubiaceae
Hedyotis diffusa
P6874 / 40 JF949979 IC50 796.1 µg/ml
Hedyotis diffusa
150
Survival
[%]
100
50
0
9.8
19.5
39
78
156
313
625
1250
2500
5000
Concentration
[µg/ml]
Rutaceae
Evodia lepta
Evodia rutaecarpa
Phellodendron chinense
P6869 / 35 JF949978 IC50 350.7 µg/ml
P6870 / 36
IC50 297.4 µg/ml
P6890 / 56 JF949986 IC50 487.6 µg/ml
Evodia lepta
Evodia rutaecarpa
100
100
Survival
[%]
150
Survival
[%]
150
50
50
0
0
9.8
19.5
39
78
156
313
625
1250
2500
5000
9.8
Concentration
[µg/ml]
19.5
39
78
156
313
625
1250
2500
5000
Concentration
[µg/ml]
Phellodendron chinense
150
Survival
[%]
100
50
0
9.8
19.5
39
78
156
313
625
1250
2500
5000
Concentration
[µg/ml]
Saururaceae
Houttuynia cordata
P6875 / 41 JF950006 IC50 575.2 µg/ml
Houttuynia cordata
150
Survival
[%]
100
50
0
9.8
19.5
39
78
156
313
625
1250
2500
5000
Concentration
[µg/ml]
257
Schisandraceae Kadsura longipedunculata
P6879 / 45 JF950007 IC50 86.1 µg/ml
Kadsura longipedunculata
150
Survival
[%]
100
50
0
9.8
19.5
39
78
156
313
625
1250
2500
5000
Concentration
[µg/ml]
Selaginellaceae Selaginella tamariscina
P6904 / 70 JF950018 IC50 393.9 µg/ml
Selaginella tamariscina
150
Survival
[%]
100
50
0
9.8
19.5
39
78
156
313
625
1250
2500
5000
Concentration
[µg/ml]
Valerianaceae
Patrinia scabiosaefolia
P6889 / 55 JF949985 IC50 159.4 µg/ml
Patrinia scabiosaefolia
150
Survival
[%]
100
50
0
9.8
19.5
39
78
156
313
625
1250
2500
5000
Concentration
[µg/ml]
Verbenaceae
Verbena officinalis
P6910 / 76 JF950020 IC50 334.7 µg/ml
Verbena officinalis
150
Survival
[%]
100
50
0
9.8
19.5
39
78
156
313
625
1250
2500
5000
Concentration
[µg/ml]
258
Violaceae
Viola yezoensis
P6911 / 77 JF949993 IC50 297.5 µg/ml
Viola yezoensis
150
Survival
[%]
100
50
0
9.8
19.5
39
78
156
313
625
1250
2500
5000
Concentration
[µg/ml]
Zingiberaceae
Alpinia galanga
Alpinia oxyphylla
P6837 / 03
P6917 / 78
-
Alpinia galanga
IC50 111.7 µg/ml
IC50 213.8 µg/ml
Alpinia oxyphylla
100
100
Survival
[%]
150
Survival
[%]
150
50
50
0
0
9.8
19.5
39
78
156
313
625
1250
2500
5000
9.8
Concentration
[µg/ml]
19.5
39
78
156
313
Concentration
[µg/ml]
259
625
1250
2500
5000
10.4.4 Methanol extracts: T. b. brucei
Andrographis paniculata
Acanthaceae
P6838 / 04
JF949965 IC50 28.8 µg/ml
P6848 / 14
JF949970 IC50 77.2 µg/ml
P6844 / 10
P6845 / 11
P6849 / 15
P6854 / 20
P6902 / 68
JF950021
JF949968
JF950022
JF949973
JF949988
Andrographis paniculata
150
Survival
[%]
100
50
0
0.49
0.98
1.95
3.91
7.81 15.63 31.25 62.5
125
313
625
1250 2500 5000
Concentration
[µg/ml]
Amaranthaceae Celosia cristata
Celosia cristata
150
Survival
[%]
100
50
0
0.49
0.98
1.95
3.91
7.81 15.63 31.25 62.5
125
313
625
1250 2500 5000
Concentration
[µg/ml]
Apiaceae
Bupleurum chinense
Bupleurum marginatum
Centella asiatica
Cnidium monnieri
Saposhnikovia divaricata
Bupleurum chinense
IC50 120.8 µg/ml
IC50 111.9 µg/ml
IC50 44.7 µg/ml
IC50 17.9 µg/ml
IC50 999.5 µg/ml
Bupleurum marginatum
100
100
Survival
[%]
150
Survival
[%]
150
50
50
0
0
0.49
0.98
1.95
3.91
7.81 15.63 31.25 62.5
125
313
625
1250 2500 5000
Concentration
[µg/ml]
0.49
0.98
1.95
3.91
7.81 15.63 31.25 62.5
Concentration
[µg/ml]
260
125
313
625
1250 2500 5000
Centella asiatica
Cnidium monnieri
100
100
Survival
[%]
150
Survival
[%]
150
50
50
0
0
0.49
0.98
1.95
3.91
7.81 15.63 31.25 62.5
125
313
625
1250 2500 5000
0.49
0.98
1.95
3.91
7.81 15.63 31.25 62.5
Concentration
[µg/ml]
125
313
625
1250 2500 5000
Concentration
[µg/ml]
Saposhnikovia divaricata
150
Survival
[%]
100
50
0
0.49
0.98
1.95
3.91
7.81 15.63 31.25 62.5
125
313
625
1250 2500 5000
Concentration
[µg/ml]
Araliaceae
Eleutherococcus senticosus
Panax ginseng China
Panax ginseng Korea
Panax notoginseng
P6919 / 79
P8088 / 81
P8086 / 81
P6887 / 53
Eleutherococcus senticosus
JF950028
JF950029
JF950030
IC50 17.3 µg/ml
IC50 319.0 µg/ml
IC50 1075.0 µg/ml
IC50 469.6 µg/ml
Panax ginseng China
150
100
100
Survival
[%]
Survival
[%]
150
50
50
0
0
0.49
0.98
1.95
3.91
7.81 15.63 31.25 62.5
125
313
625
1250 2500 5000
9.8
19.5
39
78
Concentration
[µg/ml]
156
313
625
1250
2500
5000
625
1250
2500
5000
Concentration
[µg/ml]
Panax ginseng Korea
Panax notoginseng
150
100
100
Survival
[%]
Survival
[%]
150
50
50
0
0
9.8
19.5
39
78
156
313
625
1250
2500
5000
9.8
Concentration
[µg/ml]
19.5
39
78
156
313
Concentration
[µg/ml]
261
Arecaceae
Areca catechu
P6840 / 06
-
IC50 118.1 µg/ml
Areca catechu
150
Survival
[%]
100
50
0
0.49
0.98
1.95
3.91
7.81 15.63 31.25 62.5
125
313
625
1250 2500 5000
Concentration
[µg/ml]
Asclepiadaceae Cynanchum paniculatum
P6858 / 24
JF949975 IC50 39.3 µg/ml
P6841 / 07
P6842 / 08
P6839 / 05
P6850 / 16
P6851 / 17
P6852 / 18
P6863 / 29
P6905 / 71
P6906 / 72
P6908 / 74
JF949966
JF949967
JF949994
JF949971
JF949972
JF950000
JF949989
JF949990
JF950019
Cynanchum paniculatum
150
Survival
[%]
100
50
0
0.49
0.98
1.95
3.91
7.81 15.63 31.25 62.5
125
313
625
1250 2500 5000
Concentration
[µg/ml]
Asteraceae
Artemisia annua
Artemisia capillaris
Arctium lappa
Centipeda minima
Chrysanthemum indicum
Chrysanthemum morifolium
Eclipta prostata
Senecio scandens
Siegesbeckia orientalis
Taraxacum officinale
Artemisia annua
IC50 51.2 µg/ml
IC50 51.9 µg/ml
IC50 2229.0 µg/ml
IC50 13.3 µg/ml
IC50 15.3 µg/ml
IC50 24.9 µg/ml
IC50 39.6 µg/ml
IC50 18.6 µg/ml
IC50 12.3 µg/ml
IC50 64.9 µg/ml
Artemisia capillaris
100
100
Survival
[%]
150
Survival
[%]
150
50
50
0
0
0.49
0.98
1.95
3.91
7.81 15.63 31.25 62.5
125
313
625
1250 2500 5000
Concentration
[µg/ml]
0.49
0.98
1.95
3.91
7.81 15.63 31.25 62.5
Concentration
[µg/ml]
262
125
313
625
1250 2500 5000
Arctium lappa
Centipeda minima
100
100
Survival
[%]
150
Survival
[%]
150
50
50
0
0
0.49
0.98
1.95
3.91
7.81 15.63 31.25 62.5
125
313
625
1250 2500 5000
0.49
0.98
1.95
3.91
Concentration
[µg/ml]
7.81 15.63 31.25 62.5
125
313
625
1250 2500 5000
125
313
625
1250 2500 5000
125
313
625
1250 2500 5000
125
313
625
1250 2500 5000
Concentration
[µg/ml]
Chrysanthemum indicum
Chrysanthemum morifolium
100
100
Survival
[%]
150
Survival
[%]
150
50
50
0
0
0.49
0.98
1.95
3.91
7.81 15.63 31.25 62.5
125
313
625
1250 2500 5000
0.49
0.98
1.95
3.91
Concentration
[µg/ml]
7.81 15.63 31.25 62.5
Concentration
[µg/ml]
Eclipta prostrata
Senecio scandens
100
100
Survival
[%]
150
Survival
[%]
150
50
50
0
0
0.49
0.98
1.95
3.91
7.81 15.63 31.25 62.5
125
313
625
1250 2500 5000
0.49
0.98
1.95
3.91
Concentration
[µg/ml]
7.81 15.63 31.25 62.5
Concentration
[µg/ml]
Siegesbeckia orientalis
Taraxacum officinale
100
100
Survival
[%]
150
Survival
[%]
150
50
50
0
0
0.49
0.98
1.95
3.91
7.81 15.63 31.25 62.5
125
313
625
1250 2500 5000
Concentration
[µg/ml]
0.49
0.98
1.95
3.91
7.81 15.63 31.25 62.5
Concentration
[µg/ml]
263
Berberidaceae Berberis bealei
P6883 / 49
P6862 / 28
P6865 / 31
Dysosma versipellis
Epimedium koreanum
Berberis bealei
JF949996 IC50 7.8 µg/ml
IC50 53.2 µg/ml
JF950002 IC50 12.6 µg/ml
Dysosma versipellis
100
100
Survival
[%]
150
Survival
[%]
150
50
50
0
0
0.49
0.98
1.95
3.91
7.81 15.63 31.25 62.5
125
313
625
1250 2500 5000
0.49
0.98
1.95
3.91
Concentration
[µg/ml]
7.81 15.63 31.25 62.5
125
313
625
1250 2500 5000
Concentration
[µg/ml]
Epimedium koreanum
150
Survival
[%]
100
50
0
0.49
0.98
1.95
3.91
7.81 15.63 31.25 62.5
125
313
625
1250 2500 5000
Concentration
[µg/ml]
Brassicaceae
Capsella bursa-pastoris
Isatis indigotica (root)
Isatis indigotica (leaf)
P6846 / 12
P6877 / 43
P6878 / 44
Capsella bursa-pastoris
JF949997 IC50 60.9 µg/ml
JF949981 IC50 94.6 µg/ml
JF949981 IC50 14.6 µg/ml
Isatis indigotica (root)
150
100
100
Survival
[%]
Survival
[%]
150
50
50
0
0
0.49
0.98
1.95
3.91
7.81 15.63 31.25 62.5
125
313
625
1250 2500 5000
Concentration
[µg/ml]
Isatis indigotica (leaf)
Survival
[%]
100
50
0
0.98
1.95
3.91
7.81 15.63 31.25 62.5
0.98
1.95
3.91
7.81 15.63 31.25 62.5
Concentration
[µg/ml]
150
0.49
0.49
125
313
625
1250 2500 5000
Concentration
[µg/ml]
264
125
313
625
1250 2500 5000
Caprifoliaceae Lonicera confusa
P6880 / 46
JF949982 IC50 38.0 µg/ml
P6892 / 58
JF950027 IC50 119.5 µg/ml
P6891 / 57
JF950011 IC50 84.2 µg/ml
P6859 / 25
JF949998 IC50 61.0 µg/ml
Lonicera confusa
150
Survival
[%]
100
50
0
0.49
0.98
1.95
3.91
7.81 15.63 31.25 62.5
125
313
625
1250 2500 5000
Concentration
[µg/ml]
Convallariaceae Polygonatum kingianum
Polygonatum kingianum
150
Survival
[%]
100
50
0
0.49
0.98
1.95
3.91
7.81 15.63 31.25 62.5
125
313
625
1250 2500 5000
Concentration
[µg/ml]
Cupressaceae
Platycladus orientalis
Platycladus orientalis
150
Survival
[%]
100
50
0
0.49
0.98
1.95
3.91
7.81 15.63 31.25 62.5
125
313
625
1250 2500 5000
Concentration
[µg/ml]
Dryopteridaceae Cyrtomium fortunei
Cyrtomium fortunei
150
Survival
[%]
100
50
0
0.49
0.98
1.95
3.91
7.81 15.63 31.25 62.5
125
313
625
1250 2500 5000
Concentration
[µg/ml]
265
Ephedraceae
Ephedra sinica
P6864 / 30
JF950001 IC50 23.4 µg/ml
P6866 / 32
JF950003 IC50 51.6 µg/ml
Ephedra sinica
150
Survival
[%]
100
50
0
0.49
0.98
1.95
3.91
7.81 15.63 31.25 62.5
125
313
625
1250 2500 5000
Concentration
[µg/ml]
Equisetaceae
Equisetum hiemale
Equisetum hiemale
150
Survival
[%]
100
50
0
0.49
0.98
1.95
3.91
7.81 15.63 31.25 62.5
125
313
625
1250 2500 5000
Concentration
[µg/ml]
Euphorbiaceae Croton tiglium
P6856 / 22
Croton tiglium
150
Survival
[%]
100
50
0
0.49
0.98
1.95
3.91
7.81 15.63 31.25 62.5
125
313
625
1250 2500 5000
Concentration
[µg/ml]
266
-
IC50 150.4 µg/ml
Fabaceae
Abrus cantoniensis
Acacia catechu
Cassia tora
Desmodium styracifolium
Glycyrrhiza inflata
Spatholobus suberectus
Sutherlandia frutescens
P6835 / 01
P6836 / 02
P6847 / 13
P6861 / 27
P6873 / 39
P6907 / 73
tba / 83
JF949964
JF949969
JF949976
JF950025
JF949991
-
Abrus cantoniensis
IC50 73.5 µg/ml
IC50 50.8 µg/ml
IC50 276.9 µg/ml
IC50 40.1 µg/ml
IC50 39.0 µg/ml
IC50 67.8 µg/ml
IC50 87.4 µg/ml
Acacia catechu
100
100
Survival
[%]
150
Survival
[%]
150
50
50
0
0
0.49
0.98
1.95
3.91
7.81 15.63 31.25 62.5
125
313
625
1250 2500 5000
0.49
0.98
1.95
3.91
Concentration
[µg/ml]
7.81 15.63 31.25 62.5
125
313
625
1250 2500 5000
313
625
1250 2500 5000
313
625
1250 2500 5000
Concentration
[µg/ml]
Cassia tora
Desmodium styracifolium
100
100
Survival
[%]
150
Survival
[%]
150
50
50
0
0
0.49
0.98
1.95
3.91
7.81 15.63 31.25 62.5
125
313
625
1250 2500 5000
0.49
0.98
1.95
3.91
Concentration
[µg/ml]
7.81 15.63 31.25 62.5
125
Concentration
[µg/ml]
Glycyrrhiza inflata
Spatholobus suberectus
100
100
Survival
[%]
150
Survival
[%]
150
50
50
0
0
0.49
0.98
1.95
3.91
7.81 15.63 31.25 62.5
125
313
625
1250 2500 5000
Concentration
[µg/ml]
0.49
0.98
1.95
3.91
7.81 15.63 31.25 62.5
Concentration
[µg/ml]
267
125
Sutherlandia frutescens
150
Survival
[%]
100
50
0
0.49
0.98
1.95
3.91
7.81 15.63 31.25 62.5
125
313
625
1250 2500 5000
Concentration
[µg/ml]
Geraniaceae
Geranium wilfordii
Pelargonium sidoides
P6867 / 33
tba / 82
Geranium wilfordii
JF949977 IC50 13.3 µg/ml
IC50 18.3 µg/ml
Pelargonium sidoides
100
100
Survival
[%]
150
Survival
[%]
150
50
50
0
0
0.49
0.98
1.95
3.91
7.81 15.63 31.25 62.5
125
313
625
1250 2500 5000
0.49
0.98
1.95
Concentration
[µg/ml]
Ginkgoaceae
3.91
7.81 15.63 31.25 62.5
125
313
625
1250 2500 5000
Concentration
[µg/ml]
Ginkgo biloba
P6872 / 38
JF950005 IC50 39.3 µg/ml
P6876 / 42
JF949980 IC50 23.6 µg/ml
Ginkgo biloba
150
Survival
[%]
100
50
0
0.49
0.98
1.95
3.91
7.81 15.63 31.25 62.5
125
313
625
1250 2500 5000
Concentration
[µg/ml]
Hypericaceae
Hypericum japonicum
Hypericum japonicum
150
Survival
[%]
100
50
0
0.49
0.98
1.95
3.91
7.81 15.63 31.25 62.5
125
313
625
1250 2500 5000
Concentration
[µg/ml]
268
Iridaceae
Belamcanda chinensis
P6843 / 09
JF949995 IC50 80.2 µg/ml
P6884 / 50
P6896 / 62
P6903 / 69
JF949984 IC50 16.2 µg/ml
JF950013 IC50 25.1 µg/ml
JF950017 IC50 86.2 µg/ml
Belamcanda chinensis
150
Survival
[%]
100
50
0
0.49
0.98
1.95
3.91
7.81 15.63 31.25 62.5
125
313
625
1250 2500 5000
Concentration
[µg/ml]
Lamiaceae
Mentha haplocalyx
Prunella vulgaris
Scutellaria baicalensis
Mentha haplocalyx
Prunella vulgaris
100
100
Survival
[%]
150
Survival
[%]
150
50
50
0
0
0.49
0.98
1.95
3.91
7.81 15.63 31.25 62.5
125
313
625
1250 2500 5000
0.49
0.98
1.95
Concentration
[µg/ml]
3.91
7.81 15.63 31.25 62.5
125
313
625
1250 2500 5000
Concentration
[µg/ml]
Scutellaria baicalensis
150
Survival
[%]
100
50
0
0.49
0.98
1.95
3.91
7.81 15.63 31.25 62.5
125
313
625
1250 2500 5000
Concentration
[µg/ml]
Lauraceae
Cinnamomum cassia
P6853 / 19
Cinnamomum cassia
150
Survival
[%]
100
50
0
0.49
0.98
1.95
3.91
7.81 15.63 31.25 62.5
125
313
625
1250 2500 5000
Concentration
[µg/ml]
269
JF950023 IC50 13.4 µg/ml
Loranthaceae
Taxillus chinensis
P6909 / 75
JF949992 IC50 59.2 µg/ml
P6897 / 63
JF950014 IC50 8.1 µg/ml
P6882 / 48
JF950008 IC50 4.3 µg/ml
P6888 / 54
JF950010 IC50 11.8 µg/ml
Taxillus chinensis
150
Survival
[%]
100
50
0
0.49
0.98
1.95
3.91
7.81 15.63 31.25 62.5
125
313
625
1250 2500 5000
Concentration
[µg/ml]
Lythraceae
Punica granatum
Punica granatum
150
Survival
[%]
100
50
0
0.49
0.98
1.95
3.91
7.81 15.63 31.25 62.5
125
313
625
1250 2500 5000
Concentration
[µg/ml]
Magnoliaceae
Magnolia officinalis
Magnolia officinalis
150
Survival
[%]
100
50
0
0.49
0.98
1.95
3.91
7.81 15.63 31.25 62.5
125
313
625
1250 2500 5000
Concentration
[µg/ml]
Melanthiaceae
Paris polyphylla
Paris polyphylla
150
Survival
[%]
100
50
0
0.49
0.98
1.95
3.91
7.81 15.63 31.25 62.5
125
313
625
1250 2500 5000
Concentration
[µg/ml]
270
Myrsinaceae
Lysimachia christinae
P6881 / 47
JF949983 IC50 52.1 µg/ml
Lysimachia christinae
150
Survival
[%]
100
50
0
0.49
0.98
1.95
3.91
7.81 15.63 31.25 62.5
125
313
625
1250 2500 5000
Concentration
[µg/ml]
Myrtaceae
Eucalyptus robusta
P6868 / 34
-
IC50 16.3 µg/ml
Eucalyptus robusta
150
Survival
[%]
100
50
0
0.49
0.98
1.95
3.91
7.81 15.63 31.25 62.5
125
313
625
1250 2500 5000
Concentration
[µg/ml]
Ophioglossaceae Ophioglossum vulgatum
P6885 / 51
JF950009 IC50 33.2 µg/ml
P6860 / 26
JF949999 IC50 27.6 µg/ml
Ophioglossum vulgatum
150
Survival
[%]
100
50
0
0.49
0.98
1.95
3.91
7.81 15.63 31.25 62.5
125
313
625
1250 2500 5000
Concentration
[µg/ml]
Orchidaceae
Dendrobium loddigesii
Dendrobium loddigesii
150
Survival
[%]
100
50
0
0.49
0.98
1.95
3.91
7.81 15.63 31.25 62.5
125
313
625
1250 2500 5000
Concentration
[µg/ml]
271
Paeoniaceae
Paeonia lactiflora
P6886 / 52
JF950026 IC50 11.7 µg/ml
Paeonia lactiflora
150
Survival
[%]
100
50
0
0.49
0.98
1.95
3.91
7.81 15.63 31.25 62.5
125
313
625
1250 2500 5000
Concentration
[µg/ml]
Pedaliaceae
Harpagophytum procumbens
tba / 80
-
IC50 21.4 µg/ml
Harpagophytum procumbens
150
Survival
[%]
100
50
0
0.49
0.98
1.95
3.91
7.81 15.63 31.25 62.5
125
313
625
1250 2500 5000
Concentration
[µg/ml]
Poaceae
Cymbopogon distans
P6857 / 23
JF949974 IC50 18.9 µg/ml
P6894 / 60
JF950004 IC50 19.0 µg/ml
P6893 / 59
P6895 / 61
P6898 / 64
JF950012 IC50 49.1 µg/ml
JF949987 IC50 62.1 µg/ml
JF950015 IC50 24.5 µg/ml
Cymbopogon distans
150
Survival
[%]
100
50
0
0.49
0.98
1.95
3.91
7.81 15.63 31.25 62.5
125
313
625
1250 2500 5000
Concentration
[µg/ml]
Polygonaceae
Fallopia japonica (syn.
Polygonum cuspidatum)
Polygonum aviculare
Polygonum multiflorum
Rheum officinale
Fallopia japonica
Polygonum aviculare
100
100
Survival
[%]
150
Survival
[%]
150
50
50
0
0
0.49
0.98
1.95
3.91
7.81 15.63 31.25 62.5
125
313
625
1250 2500 5000
Concentration
[µg/ml]
0.49
0.98
1.95
3.91
7.81 15.63 31.25 62.5
Concentration
[µg/ml]
272
125
313
625
1250 2500 5000
Polygonum multiflorum
Rheum officinale
100
100
Survival
[%]
150
Survival
[%]
150
50
50
0
0
0.49
0.98
1.95
3.91
7.81 15.63 31.25 62.5
125
313
625
1250 2500 5000
0.49
0.98
1.95
3.91
Concentration
[µg/ml]
7.81 15.63 31.25 62.5
125
313
625
1250 2500 5000
Concentration
[µg/ml]
Ranunculaceae Coptis chinensis
P6855 / 21
JF950024 IC50 0.4 µg/ml
P6899 / 65
P6900 / 66
P6901 / 67
IC50 12.5 µg/ml
IC50 102.9 µg/ml
JF950016 IC50 4.0 µg/ml
Coptis chinensis
150
Survival
[%]
100
50
0
0.49
0.98
1.95
3.91
7.81 15.63 31.25 62.5
125
313
625
1250 2500 5000
Concentration
[µg/ml]
Rosaceae
Rosa chinensis
Rosa laevigata
Sanguisorba officinalis
Rosa chinensis
Rosa laevigata
100
100
Survival
[%]
150
Survival
[%]
150
50
50
0
0
0.49
0.98
1.95
3.91
7.81 15.63 31.25 62.5
125
313
625
1250 2500 5000
Concentration
[µg/ml]
Sanguisorba officinalis
Survival
[%]
100
50
0
0.98
1.95
3.91
7.81 15.63 31.25 62.5
0.98
1.95
3.91
7.81 15.63 31.25 62.5
Concentration
[µg/ml]
150
0.49
0.49
125
313
625
1250 2500 5000
Concentration
[µg/ml]
273
125
313
625
1250 2500 5000
Rubiaceae
Hedyotis diffusa
P6874 / 40
JF949979 IC50 24.9 µg/ml
P6869 / 35
P6870 / 36
P6890 / 56
JF949978 IC50 44.4 µg/ml
IC50 29.5 µg/ml
JF949986 IC50 14.1 µg/ml
Hedyotis diffusa
150
Survival
[%]
100
50
0
0.49
0.98
1.95
3.91
7.81 15.63 31.25 62.5
125
313
625
1250 2500 5000
Concentration
[µg/ml]
Rutaceae
Evodia lepta
Evodia rutaecarpa
Phellodendron chinense
Evodia lepta
Evodia rutaecarpa
100
100
Survival
[%]
150
Survival
[%]
150
50
50
0
0
0.49
0.98
1.95
3.91
7.81 15.63 31.25 62.5
125
313
625
1250 2500 5000
0.49
0.98
1.95
Concentration
[µg/ml]
3.91
7.81 15.63 31.25 62.5
125
313
625
1250 2500 5000
Concentration
[µg/ml]
Phellodendron chinense
150
Survival
[%]
100
50
0
0.49
0.98
1.95
3.91
7.81 15.63 31.25 62.5
125
313
625
1250 2500 5000
Concentration
[µg/ml]
Saururaceae
Houttuynia cordata
P6875 / 41
Houttuynia cordata
150
Survival
[%]
100
50
0
0.49
0.98
1.95
3.91
7.81 15.63 31.25 62.5
125
313
625
1250 2500 5000
Concentration
[µg/ml]
274
JF950006 IC50 97.6 µg/ml
Schisandraceae Kadsura longipedunculata
P6879 / 45 JF950007 IC50 11.8 µg/ml
Kadsura longipedunculata
150
Survival
[%]
100
50
0
0.49
0.98
1.95
3.91
7.81 15.63 31.25 62.5
125
313
625
1250 2500 5000
Concentration
[µg/ml]
Selaginellaceae Selaginella tamariscina
P6904 / 70 JF950018 IC50 33.4 µg/ml
Selaginella tamariscina
150
Survival
[%]
100
50
0
0.49
0.98
1.95
3.91
7.81 15.63 31.25 62.5
125
313
625
1250 2500 5000
Concentration
[µg/ml]
Valerianaceae
Patrinia scabiosaefolia
P6889 / 55 JF949985 IC50 19.0 µg/ml
Patrinia scabiosaefolia
150
Survival
[%]
100
50
0
0.49
0.98
1.95
3.91
7.81 15.63 31.25 62.5
125
313
625
1250 2500 5000
Concentration
[µg/ml]
Verbenaceae
Verbena officinalis
P6910 / 76 JF950020 IC50 20.5 µg/ml
Verbena officinalis
150
Survival
[%]
100
50
0
0.49
0.98
1.95
3.91
7.81 15.63 31.25 62.5
125
313
625
1250 2500 5000
Concentration
[µg/ml]
275
Violaceae
Viola yezoensis
P6911 / 77 JF949993 IC50 24.7 µg/ml
Viola yezoensis
150
Survival
[%]
100
50
0
0.49
0.98
1.95
3.91
7.81 15.63 31.25 62.5
125
313
625
1250 2500 5000
Concentration
[µg/ml]
Zingiberaceae
Alpinia galanga
Alpinia oxyphylla
P6837 / 03
P6917 / 78
Alpinia galanga
-
IC50 15.4 µg/ml
IC50 2.0 µg/ml
Alpinia oxyphylla
100
100
Survival
[%]
150
Survival
[%]
150
50
50
0
0
0.49
0.98
1.95
3.91
7.81 15.63 31.25 62.5
125
313
625
1250 2500 5000
Concentration
[µg/ml]
0.49
0.98
1.95
3.91
7.81 15.63 31.25 62.5
Concentration
[µg/ml]
276
125
313
625
1250 2500 5000
10.4.5 Water extracts: HeLa
Andrographis paniculata
Acanthaceae
P6838 / 04 JF949965 IC50 576.0 µg/ml
Andrographis paniculata
150
Survival
[%]
100
50
0
9.8
19.5
39
78
156
313
625
1250
2500
5000
Concentration
[µg/ml]
Amaranthaceae Celosia cristata
P6848 / 14 JF949970 IC50 2773.5 µg/ml
Celosia cristata
150
Survival
[%]
100
50
0
9.8
19.5
39
78
156
313
625
1250
2500
5000
Concentration
[µg/ml]
Apiaceae
Bupleurum chinense
Bupleurum marginatum
Centella asiatica
Cnidium monnieri
Saposhnikovia divaricata
P6844 / 10
P6845 / 11
P6849 / 15
P6854 / 20
P6902 / 68
Bupleurum chinense
JF950021
JF949968
JF950022
JF949973
JF949988
IC50 339.3 µg/ml
IC50 838.1 µg/ml
IC50 1436.8 µg/ml
IC50 775.5 µg/ml
IC50 1024.7 µg/ml
Bupleurum marginatum
100
100
Survival
[%]
150
Survival
[%]
150
50
50
0
0
9.8
19.5
39
78
156
313
625
1250
2500
5000
9.8
Concentration
[µg/ml]
19.5
39
78
156
313
Concentration
[µg/ml]
277
625
1250
2500
5000
Centella asiatica
Cnidium monnieri
100
100
Survival
[%]
150
Survival
[%]
150
50
50
0
0
9.8
19.5
39
78
156
313
625
1250
2500
5000
9.8
19.5
39
78
Concentration
[µg/ml]
156
313
625
1250
2500
5000
Concentration
[µg/ml]
Saposhnikovia divaricata
150
Survival
[%]
100
50
0
9.8
19.5
39
78
156
313
625
1250
2500
5000
Concentration
[µg/ml]
Araliaceae
Eleutherococcus senticosus
Panax ginseng China
Panax ginseng Korea
Panax notoginseng
P6919 / 79
IC50 430.0 µg/ml
P8088 / 81 JF950028 IC50 2594.6 µg/ml
P8086 / 81 JF950029 IC50 2594.6 µg/ml
P6887 / 53 JF950030 IC50 1574.9 µg/ml
Eleutherococcus senticosus
Panax ginseng China
100
100
Survival
[%]
150
Survival
[%]
150
50
50
0
0
9.8
19.5
39
78
156
313
625
1250
2500
5000
9.8
19.5
39
78
Concentration
[µg/ml]
156
313
625
1250
2500
5000
625
1250
2500
5000
Concentration
[µg/ml]
Panax ginseng Korea
Panax notoginseng
100
100
Survival
[%]
150
Survival
[%]
150
50
50
0
0
9.8
19.5
39
78
156
313
625
1250
2500
5000
9.8
Concentration
[µg/ml]
19.5
39
78
156
313
Concentration
[µg/ml]
278
Arecaceae
Areca catechu
P6840 / 06
-
IC50 378.1 µg/ml
Areca catechu
150
Survival
[%]
100
50
0
9.8
19.5
39
78
156
313
625
1250
2500
5000
Concentration
[µg/ml]
Asclepiadaceae Cynanchum paniculatum
P6858 / 24 JF949975 IC50 693.9 µg/ml
Cynanchum paniculatum
150
Survival
[%]
100
50
0
9.8
19.5
39
78
156
313
625
1250
2500
5000
Concentration
[µg/ml]
Asteraceae
Artemisia annua
Artemisia capillaris
Arctium lappa
Centipeda minima
Chrysanthemum indicum
Chrysanthemum morifolium
Eclipta prostrata
Senecio scandens
Siegesbeckia orientalis
Taraxacum officinale
P6841 / 07
P6842 / 08
P6839 / 05
P6850 / 16
P6851 / 17
P6852 / 18
P6863 / 29
P6905 / 71
P6906 / 72
P6908 / 74
JF949966
JF949967
JF949994
JF949971
JF949972
JF950000
JF949989
JF949990
JF950019
Artemisia annua
IC50 775.5 µg/ml
IC50 561.7 µg/ml
IC50 516.3 µg/ml
IC50 207.2 µg/ml
IC50 583.4 µg/ml
IC50 1045.8 µg/ml
IC50 667.0 µg/ml
IC50 607.9 µg/ml
IC50 542.8 µg/ml
IC50 708.5 µg/ml
Artemisia capillaris
100
100
Survival
[%]
150
Survival
[%]
150
50
50
0
0
9.8
19.5
39
78
156
313
625
1250
2500
5000
9.8
Concentration
[µg/ml]
19.5
39
78
156
313
Concentration
[µg/ml]
279
625
1250
2500
5000
Arctium lappa
Centipeda minima
100
100
Survival
[%]
150
Survival
[%]
150
50
50
0
0
9.8
19.5
39
78
156
313
625
1250
2500
5000
9.8
19.5
39
78
Concentration
[µg/ml]
156
313
625
1250
2500
5000
625
1250
2500
5000
625
1250
2500
5000
625
1250
2500
5000
Concentration
[µg/ml]
Chrysanthemum indicum
Chrysanthemum morifolium
100
100
Survival
[%]
150
Survival
[%]
150
50
50
0
0
9.8
19.5
39
78
156
313
625
1250
2500
5000
9.8
19.5
39
78
Concentration
[µg/ml]
156
313
Concentration
[µg/ml]
Eclipta prostrata
Senecio scandens
100
100
Survival
[%]
150
Survival
[%]
150
50
50
0
0
9.8
19.5
39
78
156
313
625
1250
2500
5000
9.8
19.5
39
78
Concentration
[µg/ml]
156
313
Concentration
[µg/ml]
Siegesbeckia orientalis
Taraxacum officinale
100
100
Survival
[%]
150
Survival
[%]
150
50
50
0
0
9.8
19.5
39
78
156
313
625
1250
2500
5000
9.8
Concentration
[µg/ml]
19.5
39
78
156
313
Concentration
[µg/ml]
280
Berberidaceae
Berberis bealei
Dysosma versipellis
Epimedium koreanum
P6883 / 49 JF949996 IC50 659.4 µg/ml
P6862 / 28
IC50 1274.8 µg/ml
P6865 / 31 JF950002 IC50 280.2 µg/ml
Berberis bealei
Dysosma versipellis
100
100
Survival
[%]
150
Survival
[%]
150
50
50
0
0
9.8
19.5
39
78
156
313
625
1250
2500
5000
9.8
19.5
39
78
Concentration
[µg/ml]
156
313
625
1250
2500
5000
Concentration
[µg/ml]
Epimedium koreanum
150
Survival
[%]
100
50
0
9.8
19.5
39
78
156
313
625
1250
2500
5000
Concentration
[µg/ml]
Brassicaceae
Capsella bursa-pastoris
Isatis indigotica (root)
Isatis indigotica (leaf)
P6846 / 12 JF949997 IC50 2406.4 µg/ml
P6877 / 43 JF949981 IC50 2427.4 µg/ml
P6878 / 44 JF949981 IC50 1223.5 µg/ml
Capsella bursa-pastoris
Isatis indigotica (root)
100
100
Survival
[%]
150
Survival
[%]
150
50
50
0
0
9.8
19.5
39
78
156
313
625
1250
2500
5000
9.8
Concentration
[µg/ml]
Isatis indigotica (leaf)
Survival
[%]
100
50
0
19.5
39
78
156
313
39
78
156
313
Concentration
[µg/ml]
150
9.8
19.5
625
1250
2500
5000
Concentration
[µg/ml]
281
625
1250
2500
5000
Caprifoliaceae
Lonicera confusa
P6880 / 46 JF949982 IC50 812.2 µg/ml
Lonicera confusa
150
Survival
[%]
100
50
0
9.8
19.5
39
78
156
313
625
1250
2500
5000
Concentration
[µg/ml]
Convallariaceae Polygonatum kingianum
P6892 / 58 JF950027 IC50 2321.7 µg/ml
Polygonatum kingianum
150
Survival
[%]
100
50
0
9.8
19.5
39
78
156
313
625
1250
2500
5000
Concentration
[µg/ml]
Crassulaceae
Rhodiola rosea
P6920 / 84
-
IC50 144.4 µg/ml
Rhodiola rosea
150
Survival
[%]
100
50
0
9.8
19.5
39
78
156
313
625
1250
2500
5000
Concentration
[µg/ml]
Cupressaceae
Platycladus orientalis
P6891 / 57 JF950011 IC50 428.2 µg/ml
Platycladus orientalis
150
Survival
[%]
100
50
0
9.8
19.5
39
78
156
313
625
1250
2500
5000
Concentration
[µg/ml]
282
Dryopteridaceae Cyrtomium fortunei
P6859 / 25 JF949998 IC50 567.4 µg/ml
Cyrtomium fortunei
150
Survival
[%]
100
50
0
9.8
19.5
39
78
156
313
625
1250
2500
5000
Concentration
[µg/ml]
Ephedraceae
Ephedra sinica
P6864 / 30 JF950001 IC50 193.1 µg/ml
Ephedra sinica
150
Survival
[%]
100
50
0
9.8
19.5
39
78
156
313
625
1250
2500
5000
Concentration
[µg/ml]
Equisetaceae
Equisetum hiemale
P6866 / 32 JF950003 IC50 1058.2 µg/ml
Equisetum hiemale
150
Survival
[%]
100
50
0
9.8
19.5
39
78
156
313
625
1250
2500
5000
Concentration
[µg/ml]
Euphorbiaceae Croton tiglium
P6856 / 22
Croton tiglium
150
Survival
[%]
100
50
0
9.8
19.5
39
78
156
313
625
1250
2500
5000
Concentration
[µg/ml]
283
-
IC50 1052.7 µg/ml
Fabaceae
Abrus cantoniensis
Acacia catechu
Cassia tora
Desmodium styracifolium
Glycyrrhiza inflata
Spatholobus suberectus
Sutherlandia frutescens
P6835 / 01
P6836 / 02
P6847 / 13
P6861 / 27
P6873 / 39
P6907 / 73
tba / 83
JF949964
JF949969
JF949976
JF950025
JF949991
-
Abrus cantoniensis
IC50 587.1 µg/ml
IC50 157.5 µg/ml
IC50 1519.3 µg/ml
IC50 651.4 µg/ml
IC50 2288.0 µg/ml
IC50 174.1 µg/ml
IC50 1670.7 µg/ml
Acacia catechu
100
100
Survival
[%]
150
Survival
[%]
150
50
50
0
0
9.8
19.5
39
78
156
313
625
1250
2500
5000
9.8
19.5
39
78
Concentration
[µg/ml]
156
313
625
1250
2500
5000
625
1250
2500
5000
625
1250
2500
5000
Concentration
[µg/ml]
Cassia tora
Desmodium styracifolium
100
100
Survival
[%]
150
Survival
[%]
150
50
50
0
0
9.8
19.5
39
78
156
313
625
1250
2500
5000
9.8
19.5
39
78
Concentration
[µg/ml]
156
313
Concentration
[µg/ml]
Glycyrrhiza inflata
Spatholobus suberectus
100
100
Survival
[%]
150
Survival
[%]
150
50
50
0
0
9.8
19.5
39
78
156
313
625
1250
2500
5000
9.8
Concentration
[µg/ml]
19.5
39
78
156
313
Concentration
[µg/ml]
284
Sutherlandia frutescens
150
Survival
[%]
100
50
0
9.8
19.5
39
78
156
313
625
1250
2500
5000
Concentration
[µg/ml]
Geraniaceae
Geranium wilfordii
Pelargonium sidoides
P6867 / 33 JF949977 IC50 62.1 µg/ml
tba / 82
IC50 62.2 µg/ml
Geranium wilfordii
Pelargonium sidoides
100
100
Survival
[%]
150
Survival
[%]
150
50
50
0
0
9.8
19.5
39
78
156
313
625
1250
2500
5000
9.8
Concentration
[µg/ml]
Ginkgoaceae
19.5
39
78
156
313
625
1250
2500
5000
Concentration
[µg/ml]
Ginkgo biloba
P6872 / 38 JF950005 IC50 1717.0 µg/ml
Ginkgo biloba
150
Survival
[%]
100
50
0
9.8
19.5
39
78
156
313
625
1250
2500
5000
Concentration
[µg/ml]
Hypericaceae
Hypericum japonicum
P6876 / 42 JF949980 IC50 445.5 µg/ml
Hypericum japonicum
150
Survival
[%]
100
50
0
9.8
19.5
39
78
156
313
625
1250
2500
5000
Concentration
[µg/ml]
285
Iridaceae
Belamcanda chinensis
P6843 / 09 JF949995 IC50 1378.8 µg/ml
Belamcanda chinensis
150
Survival
[%]
100
50
0
9.8
19.5
39
78
156
313
625
1250
2500
5000
Concentration
[µg/ml]
Lamiaceae
Mentha haplocalyx
Prunella vulgaris
Scutellaria baicalensis
P6884 / 50 JF949984 IC50 519.1 µg/ml
P6896 / 62 JF950013 IC50 341.3 µg/ml
P6903 / 69 JF950017 IC50 150.0 µg/ml
Mentha haplocalyx
Prunella vulgaris
100
100
Survival
[%]
150
Survival
[%]
150
50
50
0
0
9.8
19.5
39
78
156
313
625
1250
2500
5000
9.8
Concentration
[µg/ml]
19.5
39
78
156
313
625
1250
2500
5000
Concentration
[µg/ml]
Scutellaria baicalensis
150
Survival
[%]
100
50
0
9.8
19.5
39
78
156
313
625
1250
2500
5000
Concentration
[µg/ml]
Lauraceae
Cinnamomum cassia
P6853 / 19 JF950023 IC50 713.6 µg/ml
Cinnamomum cassia
150
Survival
[%]
100
50
0
9.8
19.5
39
78
156
313
625
1250
2500
5000
Concentration
[µg/ml]
286
Loranthaceae
Taxillus chinensis
P6909 / 75 JF949992 IC50 1023.2 µg/ml
Taxillus chinensis
150
Survival
[%]
100
50
0
9.8
19.5
39
78
156
313
625
1250
2500
5000
Concentration
[µg/ml]
Lythraceae
Punica granatum
P6897 / 63 JF950014 IC50 152.4 µg/ml
Punica granatum
150
Survival
[%]
100
50
0
9.8
19.5
39
78
156
313
625
1250
2500
5000
Concentration
[µg/ml]
Magnoliaceae
Magnolia officinalis
P6882 / 48 JF950008 IC50 451.5 µg/ml
Magnolia officinalis
150
Survival
[%]
100
50
0
9.8
19.5
39
78
156
313
625
1250
2500
5000
Concentration
[µg/ml]
Melanthiaceae
Paris polyphylla
P6888 / 54 JF950010 IC50 42.6 µg/ml
Paris polyphylla
150
Survival
[%]
100
50
0
9.8
19.5
39
78
156
313
625
1250
2500
5000
Concentration
[µg/ml]
287
Myrsinaceae
Lysimachia christinae
P6881 / 47 JF949983 IC50 431.4 µg/ml
Lysimachia christinae
150
Survival
[%]
100
50
0
9.8
19.5
39
78
156
313
625
1250
2500
5000
Concentration
[µg/ml]
Myrtaceae
Eucalyptus robusta
P6868 / 34
-
IC50 15.8 µg/ml
Eucalyptus robusta
150
Survival
[%]
100
50
0
9.8
19.5
39
78
156
313
625
1250
2500
5000
Concentration
[µg/ml]
Ophioglossaceae Ophioglossum vulgatum
P6885 / 51 JF950009 IC50 1780.1 µg/ml
Ophioglossum vulgatum
150
Survival
[%]
100
50
0
9.8
19.5
39
78
156
313
625
1250
2500
5000
Concentration
[µg/ml]
Orchidaceae
Dendrobium loddigesii
P6860 / 26 JF949999 IC50 294.4 µg/ml
Dendrobium loddigesii
150
Survival
[%]
100
50
0
9.8
19.5
39
78
156
313
625
1250
2500
5000
Concentration
[µg/ml]
288
Paeoniaceae
Paeonia lactiflora
P6886 / 52 JF950026 IC50 287.3 µg/ml
Paeonia lactiflora
150
Survival
[%]
100
50
0
9.8
19.5
39
78
156
313
625
1250
2500
5000
Concentration
[µg/ml]
Pedaliaceae
Harpagophytum procumbens
tba / 80
-
IC50 733.4 µg/ml
Harpagophytum procumbens
150
Survival
[%]
100
50
0
9.8
19.5
39
78
156
313
625
1250
2500
5000
Concentration
[µg/ml]
Poaceae
Cymbopogon distans
P6857 / 23 JF949974 IC50 486.1 µg/ml
Cymbopogon distans
150
Survival
[%]
100
50
0
9.8
19.5
39
78
156
313
625
1250
2500
5000
Concentration
[µg/ml]
Fallopia japonica (syn.
Polygonum cuspidatum)
Polygonum aviculare
Polygonum multiflorum
Rheum officinale
Polygonaceae
P6894 / 60 JF950004 IC50 596.4 µg/ml
P6893 / 59 JF950012 IC50 488.6 µg/ml
P6895 / 61 JF949987 IC50 928.0 µg/ml
P6898 / 64 JF950015 IC50 670.9 µg/ml
Fallopia japonica
Polygonum aviculare
100
100
Survival
[%]
150
Survival
[%]
150
50
50
0
0
9.8
19.5
39
78
156
313
625
1250
2500
5000
9.8
Concentration
[µg/ml]
19.5
39
78
156
313
Concentration
[µg/ml]
289
625
1250
2500
5000
Polygonum multiflorum
Rheum officinale
100
100
Survival
[%]
150
Survival
[%]
150
50
50
0
0
9.8
19.5
39
78
156
313
625
1250
2500
5000
9.8
19.5
39
78
Concentration
[µg/ml]
156
313
625
1250
2500
5000
Concentration
[µg/ml]
Ranunculaceae Coptis chinensis
P6855 / 21 JF950024 IC50 101.0 µg/ml
Coptis chinensis
150
Survival
[%]
100
50
0
9.8
19.5
39
78
156
313
625
1250
2500
5000
Concentration
[µg/ml]
Rosaceae
Rosa chinensis
Rosa laevigata
Sanguisorba officinalis
P6899 / 65
IC50 135.8 µg/ml
P6900 / 66
IC50 781.7 µg/ml
P6901 / 67 JF950016 IC50 87.0 µg/ml
Rosa chinensis
Rosa laevigata
100
100
Survival
[%]
150
Survival
[%]
150
50
50
0
0
9.8
19.5
39
78
156
313
625
1250
2500
5000
9.8
Concentration
[µg/ml]
Sanguisorba officinalis
Survival
[%]
100
50
0
19.5
39
78
156
313
39
78
156
313
Concentration
[µg/ml]
150
9.8
19.5
625
1250
2500
5000
Concentration
[µg/ml]
290
625
1250
2500
5000
Rubiaceae
Hedyotis diffusa
P6874 / 40 JF949979 IC50 1542.7 µg/ml
Hedyotis diffusa
150
Survival
[%]
100
50
0
9.8
19.5
39
78
156
313
625
1250
2500
5000
Concentration
[µg/ml]
Rutaceae
Evodia lepta
Evodia rutaecarpa
Phellodendron chinense
P6869 / 35 JF949978 IC50 971.0 µg/ml
P6870 / 36
IC50 185.6 µg/ml
P6890 / 56 JF949986 IC50 750.3 µg/ml
Evodia lepta
Evodia rutaecarpa
100
100
Survival
[%]
150
Survival
[%]
150
50
50
0
0
9.8
19.5
39
78
156
313
625
1250
2500
5000
9.8
Concentration
[µg/ml]
19.5
39
78
156
313
625
1250
2500
5000
Concentration
[µg/ml]
Phellodendron chinense
150
Survival
[%]
100
50
0
9.8
19.5
39
78
156
313
625
1250
2500
5000
Concentration
[µg/ml]
Saururaceae
Houttuynia cordata
P6875 / 41 JF950006 IC50 2835.9 µg/ml
Houttuynia cordata
150
Survival
[%]
100
50
0
9.8
19.5
39
78
156
313
625
1250
2500
5000
Concentration
[µg/ml]
291
Schisandraceae Kadsura longipedunculata
P6879 / 45 JF950007 IC50 167.6 µg/ml
Kadsura longipedunculata
150
Survival
[%]
100
50
0
9.8
19.5
39
78
156
313
625
1250
2500
5000
Concentration
[µg/ml]
Selaginellaceae Selaginella tamariscina
P6904 / 70 JF950018 IC50 703.4 µg/ml
Selaginella tamariscina
150
Survival
[%]
100
50
0
9.8
19.5
39
78
156
313
625
1250
2500
5000
Concentration
[µg/ml]
Valerianaceae
Patrinia scabiosaefolia
P6889 / 55 JF949985 IC50 525.5 µg/ml
Patrinia scabiosaefolia
150
Survival
[%]
100
50
0
9.8
19.5
39
78
156
313
625
1250
2500
5000
Concentration
[µg/ml]
Verbenaceae
Verbena officinalis
P6910 / 76 JF950020 IC50 416.9 µg/ml
Verbena officinalis
150
Survival
[%]
100
50
0
9.8
19.5
39
78
156
313
625
1250
2500
5000
Concentration
[µg/ml]
292
Violaceae
Viola yezoensis
P6911 / 77 JF949993 IC50 1459.2 µg/ml
Viola yezoensis
150
Survival
[%]
100
50
0
9.8
19.5
39
78
156
313
625
1250
2500
5000
Concentration
[µg/ml]
Zingiberaceae
Alpinia galanga
Alpinia oxyphylla
P6837 / 03
P6917 / 78
-
Alpinia galanga
IC50 2357.3 µg/ml
IC50 1802.2 µg/ml
Alpinia oxyphylla
100
100
Survival
[%]
150
Survival
[%]
150
50
50
0
0
9.8
19.5
39
78
156
313
625
1250
2500
5000
9.8
Concentration
[µg/ml]
19.5
39
78
156
313
Concentration
[µg/ml]
293
625
1250
2500
5000
294
295