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Phytomedicine 14 (2007) 70–82

www.elsevier.de/phymed

Application of the ‘‘-Omic-’’ technologies in phytomedicine

G. Ulrich-Merzenicha,, H. Zeitlera, D. Jobstb, D. Paneka, H. Vettera, H. Wagnerc
a
Medical Policlinic of the Rheinische Friedrich-Wilhelms-University of Bonn, Wilhelmstr. 35-37, D-53111 Bonn, Germany
b
Heinrich Heine University of Düsseldorf, Rheinische Friedrich-Wilhelms-University of Bonn
c
Department of Pharmacy, University of Munich, Butenandtstr. 5-13, Haus B, D-81377 Munich

Received 10 December 2005; accepted 5 November 2006

Abstract
The proof of efficacy of phytopreparations and the determination of their mode of action are permanent challenges
for an evidence-based phytotherapy. The technology platform of genomics, proteomics and metabolomics (‘‘-omic-’’
technologies) are high-throughput technologies. They increase substantially the number of proteins/genes that can be
detected simultaneously and have the potential to relate complex mixtures to complex effects in the form of gene/
protein expression profiles. Provided that phytopreparation-specific signatures in the form of gene/protein expression
profiles can be developed, these technologies will be useful for the chemical and pharmacological standardization and
the proof of the toxicological potential of a plant extract. Over a long-term perspective they may economize the proof
of efficacy, the determination of the mode of action of phytomedicines and allow to investigate herbal extracts without
prominent active principle(s). The application of this genomics revealed already that gene expression profiles induced
by single drugs and the ones induced by the combination of the same drugs can be entirely different. These results
make the information of the mode of action of isolated ‘‘active principles/lead substances’’ of phytopreparations
questionable. The application of the ‘‘-omic-’’ technologies may lead to a change of paradigms towards the application
of complex mixtures in medicine and open the new field of phytogenomics, -proteomics and -metabolomics.
r 2006 Elsevier GmbH. All rights reserved.

Keywords: Phytomedicine; Genomics; Proteomics; Metabolomics; Synergy

Introduction qualitative and quantitative analysis of all low-molecu-

lar-weight metabolites of a cell or organism and their
The recent technical developments in genomics, dynamics in biological systems. These new platforms of
proteomics and metabolomics have created great the so-called – ‘‘-omic’’ technologies allow the analysis
excitement and optimism in the field of life science and characterization of biological systems in so far
research. Genomics aims at the comprehensive descrip- unknown details – tens of thousands of genes and
tion of the genetic information, proteomics at the proteins can be detected simultaneously. This represents
description of proteins and metabolomics at the a challenging complexity for scientific analysis and
will open new perspectives for ethnobotanical and
Corresponding author. Tel.: +49 228 2872 2674; phytomedical research purposes. Before highlighting
fax: +49 228 2872 2019. expected changes in plant research, it will be useful to
E-mail address: [email protected] briefly describe the technical basis for these ‘‘-omic’’
(G. Ulrich-Merzenich). technologies.

0944-7113/$ - see front matter r 2006 Elsevier GmbH. All rights reserved.
doi:10.1016/j.phymed.2006.11.011
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G. Ulrich-Merzenich et al. / Phytomedicine 14 (2007) 70–82 71

Genomics and transcriptomics

The definition of genomics is not precise. The term was

coined by Tom Roderick and originally meant analysis of
the whole genome. Now it commonly refers to large-
scale, high-throuput molecular analysis of multiple genes,
gene products or regions of genes (Cook-Deegan et al.,
2000). Transcriptomics, often included in the term
genomics, depicts the expression level of genes. For both,
the new tools are the microarrays. The term ‘‘micro-
array’’ itself, often called ‘‘biochip’’, simply describes that
a high number of molecules (oligonucleotides) are
arranged on an extremely small space, commonly on a
glass surface (up to 200.000 spots/cm2) (Fig. 1). The
interactions of RNA- or DNA extracts with these
biochips are investigated and allow a simultaneous
analysis of pleiotropic alterations at the genome and
transcriptome level. Based on the target sequences on the
glass surface, hundreds of genes can be targeted and
significant changes of their mRNA can be estimated
simultaneously. Identified functional gene clusters (Fig. 2)
with uniform change in expression levels allow predic-
tions of major changes in e.g. metabolic pathways leading
over to the next ‘‘omic’’ – technology – proteomics.

Fig. 2. Cluster Analysis groups relevant genes according to

their similar expression profiles. All genes are ordered in a
hierarchical tree. The shown cluster shows correlated groups of
genes and experiments. Neighboring genes or experiments in
the tree are similar, distant ones are different from each other.
For example gene 9 is induced in experiments 1, 3 and 5,
repressed in experiments 2, 4 and 6. It is most similar to gene
11. Red: induction; green: repression; black: no differential
regulation. Kindly provided by Miltenyi Biotec.

Proteomics

Fig. 1. Picoliterprobes are spotted on glass carriers. The spot The term proteome was proposed by Wilkins et al.
density can vary (3000–10,000 spots/cm2) and up to 20,0000 (1995, 1996) and depicts the entire PROTEin comple-
spots are brought on one glass carrier. Spots are organized in ment expressed by a genOME. Proteomics is the large-
grids and supergrids. The bare code at the bottom allows the scale study of proteins, particularly their structures and
correct identification. Kindly provided by Miltenyi Biotec. functions. The term was coined to create an analogy
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72 G. Ulrich-Merzenich et al. / Phytomedicine 14 (2007) 70–82

with genomics. Although it is often viewed as the ‘‘next complementary analytic methods; in particular, ‘‘hyphe-
step’’, proteomics is more complicated than genomics. nated’’ techniques of LC/MS, LC/MS and LC/NMR
The Human Genome Project revealed that there are are likely to have increased impact. A more detailed
fewer protein-coding genes in the human genome than description of each method is given in Metabolomics-
there are proteins in the human proteome (22,000 nrp (2006). These approaches rely on chromatographic
genes versus 400,000 proteins). This discrepancy separations, often coupled with well-developed calibra-
implies that protein diversity cannot be fully character- tions for specific analytes. The metabolic fingerprinting
ized by gene expression analysis alone, making proteo- analyses crude extracts without any separations step,
mics a useful tool for characterizing cells and tissues of using NMR, direct injection mass spectrometry (MS) or
interest. The key technologies used in proteomics are Fourier transform infrared spectroscopy.
one- and two-dimensional gel electrophoresis to identify
the relative mass of a protein and its isoelectric point
(Williams et al., 2003). Affinity chromatography,
Systems biology
fluorescence resonance energy transfer and surface
plasmon resonance are used to identify protein–protein
The combined information from genomics, proteo-
or protein–DNA interactions. X-ray tomography is used
mics and metabolomics will help us to obtain an
to determine the location of proteins or protein
integrated understanding of a cell or organism. How-
complexes in labeled cells. Further, fluorescent proteins
ever, these new analytic platforms are high-throughput
like green fluorescent protein (GFP), yellow FP, cyan
technologies which substantially increase the dynamic
FP or red FP are frequently used to study cellular events range and number of metabolites and genes that can be
such as localization of proteins to membranes and to
detected (Kell, 2006; Dunn and Ellis, 2005; Kell and
cellular organelles. They can mark homogenous popula-
Mendes, 2000). This has created an increasing need for
tions of specialized cells whose gene expression profiles
informatic tools to transform parallel information into
should be determined by DNA microarray analysis. In
real biological data and knowledge (Wang et al., 2005a).
fact, FPs like GFP are often used as direct transcrip-
One outcome of the development of informatic tools is
tional and translational reporters in living cells in a way
the advancement of systems biology. In systems biology,
linking transcriptomics and proteomics.
especially metabolomic data are presently organized
with the aim to create computer models simulating
biological system. Since the metabolic control analysis
1 and functional genomics share the same agenda
Metabolomics (Kell and Mendes, 2000), systems biology is expected
on the long term to predict both genomic activations
Metabolic analysis can be divided into four areas: (1)
and metabolite flows in complex systems. Their joint
target compound analysis – the quantification of specific
application is already now judged to be the ultimate
metabolites, (2) the metabolic profiling – the quantita-
phenotyping of a cell or plant and considered to
tive and qualitative determination of a group of related have the potential to revolutionize natural product
compounds or of specific metabolic pathways, (3)
research and to advance the development of scientific-
metabolomic – the qualitative and quantitative analysis
based herbal medicine (Wang et al., 2005a; Verpoorte,
of all metabolites and (4) metabolomic fingerprinting –
2005; Patwardhan et al., 2005b). For example these
sample classification by rapid global analysis.
technologies are likely to change and expedite the
The techniques used are multidisciplinary: for target
toxicological profiling of plants or drugs. In addition,
compound analysis and metabolic profiling, the main
the integration of these data into systems biology is
techniques are gas chromatography, high-performance
expected to enable the study and understanding of living
liquid chromatography and nuclear magnetic resonance systems from a holistic perspective and to become the
(NMR). Further, metabolomics makes use of several
adequate tool to analyze complex traditional systems of
1 medicine (TSM).
Definitions: Metabolic/ metabolite profile: determination of meta-
bolites of an organism without the demand of a complete analysis.
Commonly certain groups of metabolits like lipids or organic acids are
measured/. Sometimes used as synonym for metabolomics. Metabo-
lome: the totality of small molecules, which are formed by a cell, tissue Therapeutic approaches: ‘‘herbal shotgun versus
or organism under certain conditions, and under consideration of the silver bullet’’
concentration. Metabolomics: term was coined in the plant research. It
has the demand to determined the metabolom quantitatively and Phytobotanical and ethnobotanical research have
completely. Metabonomic: term was coined in the field medicine/
pharmacology and is primarily used here. It describes the measurement
focused for decades on the search for the mostly single
of the metabolic reaction towards medication, environment or ‘‘active principle’’ in plants, based on the assumption
diseases. It is comonly substituted by the term metabolomics. that a plant has one or a few ingredients which
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G. Ulrich-Merzenich et al. / Phytomedicine 14 (2007) 70–82 73

determine its therapeutic effect. However, European account for the unexpected synergetic effects often
phytomedicine and traditional Asian systems of medi- observed when different metabolic pathways or
cine like the TCM and Ayurveda generally assume drug targets are affected at the same time, both
synergy to be of vital importance with the application in theory (Cornish-Bowden et al., 1995; Fell and
of phytomedicine (Williamson, 2001). To complicate Thomas, 1995; Fell, 1998, Cascante et al., 2002)
matters further, herbalists use preparations and and in practice (McCafferty et al., 1999; Fan et al.,
mixtures which not necessarily intend to target a 2003; Borisy et al., 2003; Kell, 2006). Kell, (2006) and
particular organ, cell tissue or biochemical systems Verpoorte et al. (2005) even estimate that these results of
(Williamson, 2001). This kind of application has systems biology will lead to a replacement of the single
been described as the ‘‘herbal shotgun’’ approach, as compound or single target approach by multitarget
opposed to the ‘‘silver bullet’’ method of conventional approaches.
medicine (Ducke and Bogenschutz-Godwin, 1999; But even though multitarget treatment approaches
Williamson, 2001) to distinguish the multitargeted are generally more accepted, phytopharmaceuticals still
approach of herbals from the monotarget approach need to proof efficacy, provide a rational behind this
of synthetic drugs addressing specific enzymes or efficacy and demonstrate reproducibility through stan-
receptors. dardization. The standardization of the herbal extracts
This ‘‘shotgun approach’’ has so far been the major safeguards the pharmaceutical quality and forms the
complication of phytomedicine as summarized in a prerequisite for the reproducibility of the effect from
quotation of MacKenzie (2001) and Williamson (2001) batch to batch (Saller and Reichling, 2002). But
‘‘To market herbal derivatives with full patent protection, phytopharmaceuticals are biogenic products and can
there would have to be (they would have to do) clinical thus have fluctuating compositions of ingredients.
trials on the active ingredients, separately and together. According to the region or the harvest, season efficacy
Compared with testing a single magic bullet, this is and toxicity of the crude extracts may vary. In spite of
prohibitively expensive’’ and ‘‘without the support of the this somewhat ‘‘undefined’’ chemical state, phytochem-
pharmaceuticals industry, herbs are likely to remain mired icals have been used as therapeutic agents based on the
in uncertainty. What a waste.’’ chemical characterization of single ingredients which so
In recent years this ‘‘shotgun approach’’, understood far have been supposed to determine their mode of
as a therapeutic strategy which aims at multiple targets action (Saller and Reichling, 2002). In addition, the
in an organism, has gained an increasing acceptance. German (AMG3 y 26, paragraph 2) and the European
We even experience a paradigm shift (Wagner, 2001) law (Benedum, 1998) allow under certain circumstances
caused by the growing evidence of the multifactorial in accordance with the WHO guidelines, that written
nature of today’s disease challenges. Evidences increase historical material, if assessed with scientific methods,
that the side effects of a combination therapy are can be used as valid scientific source material or
not necessarily additive, but may be even less than substitute for a proof of efficacy. These regulations take
the ones of the single therapy and combined with a into consideration that many plants have a centuries-old
higher therapeutic efficacy. Well-established combina- traditional usage in their countries of origin. Never-
tion therapies are e.g. the cancer chemotherapies, or theless, in order to comply with the legal conditions with
the treatment of HIV and hypertension. The combina- respect to effectiveness, quality and safety, certain steps
tion of methotrexate (MTX) and TNF-a antagonists in of standardization are mandatory in Europe (Table 1).
rheumatoid arthritis was recently shown to be more A detailed review is available in Wagner (2001). The
efficient compared to single applications (Geletka and St standardization includes the correct taxonomic identifi-
Clair, 2005) while having less or not more than equal cation of the plant(s) and the identification of the major
side effects. We have recently demonstrated that the active principle(s) of plants, extract(s) or multiextract
application of a multimodal therapeutic strategy can mixtures. The identification of the active principle(s) is
successfully cure the autoimmune disease acquired followed by purification and concentration. The ‘‘un-
hemophilia (Zeitler et al., 2005). In the context of wanted’’ effects of accompanying plant ingredients are
systems biology, Kell (2006) demonstrated in a model eliminated and the effect through concentration of the
analysis how targeting a particular step in the signaling isolated component(s) is increased. In case of drug
pathway of the transcription factor NFkB can have development, a chemical synthesis of the identified
qualitatively (directionally) different effects depending single active principle may follow. The single isolated
on the actual state of the system. For some values of the chemically defined component(s) or its synthesized
two rate constants of the reaction, there was no derivate(s) are not termed phytopharmaceuticals
influence on the signaling pathway. Other combinations any more (Saller and Reichling, 2002). As examples
could lead to entirely opposite effects. This type of among the newest isolated and therapeutically used
systemic nonlinearity of biological systems makes compounds only taxol, camptothecine and artemisinin
designing safe drugs a challenging activity and can also are mentioned.
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Table 1. Quality assurance of phytopreparations and caffeic acid derivatives. They are used in the
treatment of arthritis (Klingelhöfer, 2001; Obertreis
1 Definitive authentication and taxonomic et al., 1996). Another classical example is the ‘‘Salix
assignment
extract 1520L’’ which inhibits cyclooxygenase-2-
e.g. through DNA-fingerprinting+DNA
barcoding mediated prostaglandin E2 release through compounds
2 Isolation and structural elucidation of all other than salicin or salicylate (Fiebich and Chrubasik,
major constituents of the herbal drug 2004). In the case of Harpagophytum procumbens much
3 Identification of the true bioactive higher concentrations of the ‘‘active principle’’ harpa-
constituents gosid are necessary for the downregulation of iNOS
4 Multiextract mixtures: standardization of expression in rat mesangial cells than available in an
the single extracts or 3D HPLC fingerprint effective concentration of the total extracts (Kaszkin
analysis of the multicompound extracts et al., 2004). Further, an activity-directed fractionation
5 Global harmonization of standardization of the herbal extract revealed a wide distribution of the
criteria under the umbrella of the desired activities in the plant with no clear separation
International Federation of Pharmaceutical
into polar and non-polar fractions (Gilani et al., 2005).
Manufacturers Associations (IFPMA)
Another classical example is St. John’s worth (Hyper-
icum perforatum), in which no single active compound
has been found that can explain the full proven clinical
activity (Mueller, 2005; Verpoorte et al., 2005).
Polyvalent pharmacological activity and synergy In these cases, the application of the refined herbal
extract rather than ‘‘isolated active principle(s)’’ may be
Many phytopharmaceuticals on the today’s drug favored in order to benefit from the broad therapeutical
market are crude extracts and thus complex mixtures and pharmacological action related to the special
of compounds. Detailed pharmacological experiments composition of the ingredients in the entire plant.
with single isolated compounds versus the original Clinical and experimental evidences on synergy are
extract or extract fractions have confirmed that many reviewed in Williamson (2001). They include the plants
plant constituents, among them primarily phenolic Ginkgo biloba, Piper methysticum, Glycyrrhiza glabra,
compounds and terpenoids, exert polyvalent pharmaco- Cannabis sativa, flavonoids and essential oils or mixed
logical effects (Wagner, 2001). This might explain some herb formulations as the Indian ‘‘Trikatu’’ or the
of the pharmacological synergetic effects and the combination of Urtica dioica and Pygeum africanum
phenomenon that very often an extract possesses a (Williamson, 2001).
much better therapeutic effect than single isolated The presence of synergistic effects has consequences
constituents. Examples for polyvalent pharmacological for the determination of the active principle(s) of plant
activities and proven synergistic effects are given in extracts as demanded in Table 1. Williamson has
Table 2. Hawthorn (Crataegus oxyacantha) e.g. is summarized this dilemma in the following quotation:
indicated for the treatment of heart insufficiency grades ‘‘If a combination of substances is needed for the effect,
I and II (NYHA I u. II). It was shown to posses a then the bioassay-led method of investigation, narrowing
positive inotrop activity and an ACE-inhibitory and activity down firstly to a fraction and eventually a
vessel dilatative activity in angina pectoris. The active compound is doomed to failure and this has led to the
compounds of Garlic (Allium sativum) ajoene and suggestion that the plants are in fact devoid of activity’’.
allicine have a cholesterol- and lipid lowering activity,
they act as antioxidants, inhibit NO formation and have
an antihypertensive action. It was also shown that
ajoene induces apoptosis in human leukemic cells Evidences from the ‘‘-transcriptomic’’
probably via the induction of reactive oxygen species; technology
however the clinical relevance of this has not yet been
investigated (Wagner, 2001; Dirsch et al., 1998a, b). Can the application of the transcriptomic technology
These examples demonstrate that active compounds of a support the identification of synergy and polyvalent
plant can act via different mechanisms of actions with a pharmacological activities?
relevance to entirely different diseases. The well- Recently, microarrays were applied for the investiga-
documented efficacy of the root of Urtica dioica in tion of the combination therapy of MTX and mercap-
prostate hyperplasia is probably based on a synergism of topurin. It was demonstrated that the combination
antiproliferative and antiinflammatory effects caused by therapy of both to treat human leukemia cells causes a
lectins and polysaccharides. Interestingly also the leaves different gene expression profile than the single applica-
of U. dioica possess antiinflammatory effects due to the tion of each drug. Only 14% of genes that changed when
content of oxylipines (13-hydroxy-octadecatrienoic acid) these medications were given as single agents also
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changed when they were given in combination. Thus, treatments (Cheok et al., 2003). Similarly Schulte et al.
86% of the previous upregulated genes did not respond. (2003) demonstrated that the combined treatment of
Discriminating genes that changed when these medica- neuroblastoma cells with cisplatin and hyperthermia
tions were given together included those involved in lead to the upregulation of 131 new genes which were
apoptosis, mismatch repair, cell cycle control and stress not expressed under treatment with either cisplatin or
response (Cheok et al., 2003). hyperthermia alone, confirming that multimodal treat-
These findings demonstrate that complex gene ex- ment approaches can apparently led to different effects
pression analysis by microarray can detect differences in on the level of gene expression.
cellular responses to drug combinations versus single
agents (Cheok et al., 2003). It further shows that drug
combinations can lead to the activation of entirely
different genes than those genes activated by the Role of the ‘‘active principle’’
individual single agents. Thus, the mode of action of
the combination is, based on the gene expression, Based on the above results, it should be questioned
entirely different from the mode of action of the single whether it makes sense to focus on a single mode of
agents. Although one may question whether the action of a plant or plant extract by searching and
discriminating genes for the treatments are the same isolating the active principle(s) or whether it may be
which are responsible for the main action of the single more meaningful to screen refined herbal extracts for
agent or not, the authors conclude that the method is their complex modes of action on a microarray directly.
highly suitable for the discrimination of different If a combination of constituents yields a new mode of

Table 2. Herbal drugs with evidences for synergistic effect/polyvalent activities

Herb/major constituents Effects/indications

a,b,c
Allium sativum
Allicin+Ajoene Inhibition-inducible nitric oxide synthase expression in activated
macrophages, inhibition of thrombocyte aggregation, antiinflammatory,
triglyceride- and cholesterol-decreasing, antioxidant, antimicrobial
Ajoene Induction of apoptosis in human leucemic cells, production of
intracellular peroxides
Cannabis sativad,e,f
Tetrahydrocannabinol Muscle-relaxant, appetite-stimulating, analgesic effects
Cannabidiol Amplification of the antispastic activity
Crataegus oxyacanthac,g
Procyanidines, flavon-C-glycosides Cardiotonic activity, angiotensin-converting enzyme inhibiting effect,
endothelin-dependent, smooth muscle-relaxing effect
Ginkgo bilobah,i
Ginkgolides A+B Synergy effects in a thrombocyte-aggregation inhibiting assay
j,k,l
Glycyrrhiza glabra
Glycyrrhizin/isoliquiritin Antitussiva, anti-inflammatory/amplification of activity of
hydrocortisone
Harpagophytum procumbens (Burch);
H. zeyheri (Decne)c,m
Harpagosid+components of total extract Stronger inhibition of leucotriene-and thromboxanebiosynthesis than
harpagoside alone
Hypericum (St. John’s wort)n,o
Hypericins, hyperforin, xanthones, flavonoids, Antidepressiva, no prominent lead substance
procyanidins
Piper methysticump,q
Yangonin, desmethoxy-yangonin dihydromethysticin Anxiolytic, sedative, anti-convulsant, spasmolytic, anti-inflammatory,
analgetic activity
Salix cortex L.r,s,t
Salicin+salicylalcohol derivates, flavonoids, tannins Stronger antiinflammatory and analgesic effect than salicin alone
h,u,v
Urtica dioica L. (radix)
Urtica dioica agglutinine (UDA)+ Competitive inhibition of EGF induced proliferation
Polysaccharides Immunostimulation
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Table 2. (continued )

Herb/major constituents Effects/indications

Urtica dioica L. (folium)

Oxylipine (13-Hydroxyoctadecatrienoic acid), caffeic Cyclooxygenase- and cytokine inhibition for the treatment of arthritis
acid derivatives and rheumatoid arthritis
Valeriana officinalisw
Valtrate, isovaltrate,valerenone, valerenic acid Sedation, increasing GABA concentrations
Viscum albumx,y
Mistletoe lectin I Induction of apoptosis of cancer cells, immunostimulating activity
Zingiber officinale (essential oil)c,z
a-Zingiberene, b-sesquiphellandrene Bisabolene, Whole ginger preparation with different effects than single components.
curcumene Antipyretic, analgetic, cardiac inotrop, sedative, antibiotic+others
a
Dirsch, V.M., Gerbes, A.T., Vollmar, A.M., 1998. Ajoene, a compound of garlic, induces apoptosis in human promyclo-leucemie species and
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Ginkgolide A [µM] 14

12
O ze O
10 ro
HO O -in HO O
OH 8 t er OH
OH ac OH
O O 6 tio O O
H
O sy n OH
4 ne O
rg
HO
O 2
ism HO
H O
O O H
0
0.5 1 1.5 2 2.5 3 3.5 Ginkgolide B [µM]

IC50 – values for various dose-combinations of PAF-induced thrombocyte aggregation

GA : GB IC50 [µg/ml] Ginkgolide A [µM] Ginkgolide B [µM]

3: 1 2.40 4.41 1.42

2: 1 2.20 3.60 1.72
1: 1 1.80 2.21 2.12
1: 2 1.55 1.27 2.43
1: 3 1.40 0.88 2.57
1 : 10 1.30 0.29 2.79

Fig. 3. Isobol curve for 50% inhibition of a Ginkgolide AB combination.

action, separate clinical trials on single active constitu- structures were isolated and characterized (Soejarto et
ents would be superfluous. This would tremendously al., 2005). Clinically significant cancer chemotherapeutic
accelerate and economize phytomedical research on time agents that emerged from this project included for
and work. The development of gene expression signa- example paclitaxel (Taxols), topotecan (Hycamtins)
tures for extracts would allow a fast screening and and CPT-11 (Taxman et al., 2003). The latter two
would simultaneously generate the potential to cover compounds are semisynthetic derivates of camptothecin
synergistic effects of the plant extracts. However, the from Camptotheca acuminata Decne., Nyssaceae (Soe-
development of these expression signatures with the jarto et al., 2005). The yield of these huge screening
microarray technology is nowadays highly cost intensive projects for the identification of single bioactive
with one microarray analysis costing easily 1200h. compounds appears moderate. A provision for syner-
The presently used method to proof synergy is the gistic effects might elevate the yield.
isobole method (reviewed in Williamson, 2001). This
method is independent of the mode of action and
measures the (end-) effect. Synergy takes place if the
dose–response curve demonstrates that the achievement Recent applications of DNA microarrays and
of a certain effect requires less dosages of two substances metabolomics to phytomedicine
than expected from their individual dose–response
curve. An example of such a dose–response curve is Microarray technology has so far not been used
given in Fig. 3 for the ginkgolides A and B as measured extensively in phytomedicine and is presently in the
in a thrombocyte aggregation inhibiting assay. stage of ‘‘proof of principle’’. Wang et al. examined the
The method, however, is still a demonstration of effect of so-called ‘‘herbal glycoside recipes’’ on the
‘‘additivity’’ or ‘‘superadditivity’’ – related to the effect ability of spatial learning memory in mice suffering from
measured. The microarray results of Cheok et al. (2003) cerebral ischemia/reperfusion. The herbal preparations
bring into synergy an entirely different quality. They were obviously derived from the roots of Scutellaria
demonstrate that the combination of two components baicalensis and Dioscorea spp. and contained baicalein
brings about a new mode of action resulting possibly (5,6,7-trihydroxyflavone) and dioscin (ratio 1:1). Using
also in new effects. a cDNA microarray system containing 1176 known
The necessity to look into synergistic effects in genes, Wang et al. (2004) showed a reproducible dose-
phytomedicine may also be underlined by the following dependent effect of these herbal preparations and
figures: the mass bioprospecting effort of the national suggested the usefulness of this methodology for
cancer institute of the United States screened about elucidating the mechanism of pharmacological functions
114,000 extracts from an estimated 35,000 plant samples of herbal preparations.
against a number of tumor systems (Cragg and Boyd, A very recent report describes the analysis of two soja
1996). A wide variety of compounds with different bean extracts. The gene expression profiles of the herbal
ARTICLE IN PRESS
78 G. Ulrich-Merzenich et al. / Phytomedicine 14 (2007) 70–82

extracts were compared with those of the single existing chemical compounds (Lettieri, 2006). In the
phytoestrogens. The profiles of the extracts correlated United States, the national institute of environmental
with those of the phytoestrogens, but gave quite health science has created the national center for
different R-values for each phytoestrogen (Ise et al., toxicogenomics to provide a reference system of
2005). Interestingly, the gene expression profiles induced genome-wide gene expression data and to develop a
by 10 mM of the phytoestrogen daidzein correlated with knowledge base of chemical effects in biological systems
those derived from the total extracts (R-values:0.73 and (Tennant, 2002; Lettieri, 2006). Studies here showed that
0.75), but the estimated concentrations of daidzein in it is possible to identify a signature of expressed gene
the extracts were much lower. They were roughly 1/100 patterns after exposure to a given toxicant (Tennant,
of 10 mM (Ise et al., 2005). 2002; Lettieri, 2006).
Mur et al. (2006) investigated the interaction of plant These reports are promising for the application of
components. Salicylic acid has been proposed to microarrays in phytoresearch and phytomedicine and they
antagonize jasmonic acid biosynthesis and signaling in are likely to change or develop our understanding of
plants. Microarray analysis demonstrated that the synergy. However, the standardization of herbal extracts
combination of both acted transiently synergistic on remains crucial. It may be simplified using those plant
certain gene expressions (defensin and thionin, beta- ingredients which show the maximum overlap in gene
glucuronidase) in tobacco plants when both were expression with the refined extract as shown in the
applied at low concentrations, but antagonism was example of the soja extract and daidzein (Ise et al., 2005).
observed at more prolonged treatment durations or at Also, Verpoorte has already hypothesized that by
higher concentrations. The authors concluded that there measuring the activity in a living organism for extracts
seems to be a greater sophistication in interactions than with different composition, one may possibly identify a
‘‘simple’’ antagonism or synergism (Mur et al., 2006). compound or a combination of compounds that
Instead, synergistic/antagonistic mechanisms may re- correlate with the activity. This means that activity
present positive and negative feedback loops of the same due to synergism and also activity of pro-drugs can be
molecule combination allowing the tailoring of the plant recognized (Verpoorte et al., 2005).
response to a particular situation.
First clinical studies in the application of a metabonic
strategy, utilizing high-resolution 1H NMR in conjunc-
tion with chemometric methods showed that a clear Proprietary drugs in the – ‘‘omic-technology’’ –
differentiation of metabolite profiles before and after Aera
Chamomile tea drinking can be obtained although
strong extrinsic physiological variations were observed. The so-called ‘‘proprietary drugs’’ prescribed in the
About 14 volunteers had ingested chamomile tea for a European, Chinese and Indian systems of medicine have
period of 2 weeks. Urine samples before, during and for long been used in the West as source material for the
after chamomile ingestion were analyzed. Chamomile tea development of new drugs. The potential of fixed
ingestion was shown to lead to an increased urinary combination formulae which are mostly applied in the
excretion of hippurate and glycine with depleted TSM could so far not be exploited. Their application is
creatinine concentrations. This study highlights the often already described with exact time tables and
potential for the metabonic technology in the assessment antidots. For example Ayurveda describes to counteract
of ‘‘small’’ interventions despite a high degree of the ‘‘side effect’’ of Semicarpus anacardium preparations
variation from genetic and environmental sources with Fructus Coriandri (Ulrich-Merzenich, 1998). In
(Wang et al., 2005b). Variations in diet or local addition, dosages during summer seasons are to be
environment can become important confounding factors reduced, with higher dosages necessary during winter
when metabolic responses to nutritional or minor seasons. These peculiar knowledge could so far not be
interventions are studied. To extract meaningful biolo- explored. The ‘‘omic-technologies’’ are tools, even
gical information from data confounded by such diverse though costly and laborious, which are likely to
extraneous physiological variations, data-filtering meth- provide a basis to investigate these complex phenomena
ods can be employed. One of the most frequently used and subsequently substantiate or dismiss such recom-
data-filtering methods is orthogonal signal correction mendations.
(OSC) which was also applied in the above study. A few drug formulations of TSM have recently gained
In the field of molecular toxicology, the high-quality interest – the ayurvedic formulation ‘‘Trikatu’’ or the
gene arrays commercially available have already allowed Chinese medicine ‘‘Iijen’’. Further details and additional
this technology to become a standard tool (Lettieri, examples are given in Table 3. More than half of
2006). Several national and international initiatives those herbal preparations were tested against synthetic
provided the proof-of principle tests for the application standard substances in clinical placebo-controlled double-
of gene expression for the study of toxicity and new blind studies. The fact that they have shown at minimum
ARTICLE IN PRESS
G. Ulrich-Merzenich et al. / Phytomedicine 14 (2007) 70–82 79

Table 3. Drugs from traditional medical systems of Asia, China and Europe intensively investigated

Antipsoriatic composition Argemone mexicanae (US-patent application)

Arthritis Boswellia serrata, Curcuma longa, Withania somnifera, Zingiber officinale (Artrexs,
US-Patent); Phytodolors (Fraxinus excelsior, Populus tremula, Solidago virgaurea)
Bowl syndrome (Colitis ulcerosa) Ayurmedica H-15 kaps (Boswellia serrata)
Gastric and abdominal disorders Piper longum, Zingiber officinale (Trikatu) Iberogasts (Iberis amara, Angelica
archangelica, Matricaria recutita, Carum carvi, Silybum marianum, Melissa
officinalis, Mentha piperita, Chelidonium majus, Glycyrrhiza glabra)
Hyperlipidemia, Atherosclerosis Commiphora wightii (Guglips, Cipla Ltd)
Non-steroidal anti-inflammatory drug Boswellia serrata gum resin (Sallakis Gufic)
Non-small lung cancer Coix lachryma-jobi (Phase II Trial US)
General tonic Astragalus membranaceus (Xue baoPG2)
Prevention and therapy of stroke Salvia miltiorrhiza, Paeonia rubra, Angelica pubescens, Stephania tetranda, Uncarial
c. Uncis, Gastrodia elata, Panax ginseng
Anticancer drugs Camptotheca acuminata, CPT11, Topotecan
Cognitive performance Panax ginseng, Ginkgo biloba, Panax ginseng extract (GK-501)+Ginkgo extract
(GK-511)
Insomnia Piper methysticum, Valeriana officinalis (Kava extract (LI-150)+Valerian extract
(LI-15))

Summarized from Patwardhan et al. (2005a, b), Schempp et al. (2006), Wagner (2001) and Williamson (2001).

a therapeutic equivalence with often less or no side toxicology will decide how easy and how soon we can
effects – results not necessarily to be expected – is implement these technologies in the routine standardi-
another indicator for [the likelihood of] synergistic zation process and how far legislation can provide
effects of phytopreparations. appropriate framework conditions for these new devel-
Patwardhan (2005a) and Patwardhan et al. (2005b) opments.
reviewed citation and patent data for Indian and Reproducibility of microarrays on plant extracts will
Chinese medicine. The Pubmed search revealed 1045 have consequences also for an often criticized aspect of
citations for Indian and 10,278 citations for Chinese phytomedicine: So far it is only accepted that chemically
medicine; so far 3 US patents have been granted for defined compounds cause a defined effect. If it is
ayurvedic medicines and 195 for Chinese medicines possible, however, to attribute a reproducible gene
(Patwardhan et al., 2005b). These figures demonstrate expression profile to a chemically not fully defined
the growing interest in fixed combinations, but also the complex herbal mixture, this paradigm needs to be
already-mentioned dilemma of patenting complex mix- questioned. Physicists already acknowledged earlier that
tures as force to develop phytomedicine. For patenting, ‘‘The breakdown of an aggregate as an entity into partial
novelty and innovation are required (Verpoorten, 2005). aggregates is, to be precise, not really possible. There is
The proof of a unique qualities of herbal medicine and only an entirety which somewhat can be perceived as an
the rationalization of therapeutic effects of complex entity of parts, but which is then more than the sum of its
mixtures of single drugs or of complex mixtures may parts.’’ (Dürr, 1989).
have consequences on the legislation and patenting. A Nevertheless, there are presently still a number of
gene expression signature of an extract represents the general limitations in the microarray methodology. We
documentation of a unique mode of action, presumed have so far not yet identified each and every gene, not to
that the gene profiles are reproducible. Gene profiles mention their function. Even though a broad spectrum
can, however, vary depending on the individual, the age of mRNAs can be determined simultaneously, we are
or the dosage. But results of Cheok et al. (2003) and presently still in the process to unravel the association of
Wang et al. (2005b) are promising. Cheok et al. (2003) upregulated mRNA and the protein formation. In
found treatment-specific gene expression profiles in addition, the limited knowledge of gene regulation, of
leukemia cells of 60 individuals for MTX and/or the interplay of protein networks and of the large
mercaptopurin whereas Wang et al. (2005b) demon- number of regulatory feedback loops makes the inter-
strated in a minor intervention treatment-specific pretation of micorarray- and or/proteome-based data
changes with Chamomile tea. The reproducibility of still difficult (Daniel and tom Diek, 2004). For plant
the new technologies, e.g., gene and protein arrays extracts, customized or focused DNA microarray and
considering extract variations due to season or batches the refinement of the statistical approach by adopting
still needs to be evaluated. Data on these experiments in the correlation analysis were proposed to improve the
combination with gene expression profiling in reliability of data (Ise et al., 2005). Data filtering
ARTICLE IN PRESS
80 G. Ulrich-Merzenich et al. / Phytomedicine 14 (2007) 70–82

methods like the OSC have been proposed and are lare Biotechnologie, Abteilung Biologie (IPMB), Heidel-
applied to extract meaningful biological information berg) for his valuable suggestions to the manuscript.
from data confounded by diverse extraneous physiolo-
gical variations like age, sex and genetic polymorphisms
in natural populations.
Presently, there is a strong demand and necessity to
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