BACKGROUND
herbal drug combinations have long been used in traditional medicinal concepts, and
multicomponent preparations, such as plant-derived pharmaceuticals, botanicals and
dietary supplements, are becoming increasingly important in modern medicine and
lifestyles.
the concept of effect potentiation by synergistic actions has opened new
approaches for phytopharmaceutical and nutraceutical product development, and
suggests the utilization of some traditional formulations for therapeutic
issues.
however, this development is hampered by the difficulties that accompany the
assessment of molecular mechanisms and pharmacological actions of multicomponent
preparations that contain an enormous chemical diversity of substances  <cit> .
one major beneficial effect of many multicomponent preparations is their antioxidant
activity.
reactive oxygen species  are involved in the development and the
progression of diseases such as cardiovascular disorders  <cit> , neurodegeneration  <cit>  and atherosclerosis  <cit> .
the capacity of dietary antioxidants to modulate the cellular redox
balance is discussed to be favourable in both the treatment and prevention of
ros-related disorders  <cit> .
an antioxidant capacity may not only originate from the direct radical
scavenging activity of a compound, but also from indirectly induced effects such as
interference with the endogenous antioxidant machinery, the transcription induction
of detoxifying and cytoprotective enzymes or the increase of immunoprotective
mechanisms  <cit> .
several of these actions are mediated by the nuclear-factor-e2-related
factor - <dig> that transactivates the transcription of protective genes upon
binding of antioxidant response element  promoter sequences to maintain
homeostasis during oxidative stress  <cit> .
however, an extensive input of antioxidants can also promote adverse
responses such as allergies or toxicity-related phenomena  <cit> .
studies into the interference of phytochemicals with cellular signaling pathways and
gene expression have revealed many examples of potential biological activities that
go far beyond an antioxidant action  <cit> .
further, the use of large-scale data acquisition strategies, such as
gene expression analysis, is suggested to be helpful in deciphering complex
molecular information flows  <cit> .
currently, dna microarray technology is the most widely used
genome-scale data in pharmacological and clinical research.
transcriptomics allows
the simultaneous detection of multiple transcriptional events in a non-biased
manner, enabling standards for analysis and interpretation to be continuously
developed  <cit> .
microarray expression data may provide key insights into gene function
and interactions within and across metabolic pathways.
however, it appears necessary
to go beyond simple clustering by integrating data into biological interaction
databases  <cit> .
in general, the therapeutic efficacy of botanical multicomponents is suggested to be
based on the interference of a multitude of phytochemicals with multiple cellular
targets  <cit> .
this enormous complexity of interactions represents a challenge for
activity monitoring.
often, the active principles of such remedies cannot be
identified, as the pharmacological effects are suggested to be generated from the
joint actions of several substances.
thus, reductionist and hypothesis-driven
research strategies may prove insufficient to deal with such pleiotropic activity
profiles.
the combination of pathway or molecule focused and large-scale data
assessment strategies, such as “omics” technologies, has proved useful
in monosubstance and drug combination activity assessment and is also considered
helpful for multicomponents  <cit> .
based on these assumptions, we attempted to explore the mode of action of the
indo-tibetan remedy padma  <dig> at the molecular and transcriptional level in human
hepatoma hepg <dig> cells  <cit> .
padma  <dig> is a polyherbal preparation containing a variety of secondary
plant substances with potential bioactivity, such as essential oils, flavonoids,
tanning agents and sesquiterpenes.
it contains  <dig> different powdered plants
including natural camphor and calcium sulphate.
the formulation has been subjected
to extensive in vitro and in vivo studies and has been approved by
the swiss drug authorities  for symptoms associated with
circulatory disorders such as tingling sensation, formication, feeling of heaviness
and tension in the legs and arms, numbness of the hands and feet and calf cramps  <cit> .
studies have been performed where padma  <dig> was applied in the treatment
of intermittent claudication, a hallmark of peripheral arterial occlusive disease
<cit> .
hepg <dig> cells were used because the liver is the main organ for drug
metabolism after the ingestion of compounds and because of their suitability for
studying the mechanism of action of drugs, dietary genotoxicants, as well as the
cytoprotective and antigenotoxic activities of agents  <cit> .
furthermore, this cell line is useful for the evaluation of pro- and
antioxidant agents as they express many related enzymes such as mn-superoxide
dismutase, catalase, glutathione reductase and thioredoxin reductase  <cit> .
a hepg <dig> derived reporter gene cell line, cellsensor® are-bla hepg <dig>
has been applied to investigate are-mediated transcriptional response.
RESULTS
hepg <dig> cell proliferation
to determine the effect of padma  <dig> on cell viability and to define optimal
treatment conditions for further cell culture experiments, hepg <dig> cells were
treated with increasing concentrations of the ethanolic extract  and the solvent control .
cell viability was
calculated in relation to the solvent control.
as shown in figure  <dig>  treatment of hepg <dig> cells with padma  <dig> for  <dig> h
dose-dependently decreased the number of viable cells with an ic50-value of
<dig>  ±  <dig>  μg/ml.
for  <dig> h and then treated with solvent  or padma  <dig> ethanolic
extract  for  <dig> h. the mean percentages of
cell growth relative to solvent-treated cells were plotted against the
concentrations of padma  <dig>  mean values ± s.e.m.
of four
independent experiments run in duplicates  are shown.
antioxidant capacity
to ensure potent biological activity of the ethanolic extract of padma  <dig>  its
antioxidant capacity was evaluated using the oxygen radical absorbance capacity
assay, which measures the direct capacity of chain-breaking antioxidants
based on the hydrogen atom transfer mechanism in a cell-free system.
the
polyherbal extract showed potent peroxyl-radical scavenging capacity in
vitro, with an orac value of  <dig> ±  <dig> μmol te/g .
accordingly,  <dig> μg/ml of padma  <dig> corresponds to the net protection
against peroxyl-radicals produced by about  <dig> μm or  <dig>  μg/ml of the
water-soluble vitamin e derivate trolox.
the antioxidant capacity of padma  <dig> in living cells was analysed by measurement
of peroxyl-radical-induced intracellular ros-levels in hepg <dig> cells pretreated
with increasing concentrations of ethanolic extract.
as shown in
figure  <dig>  exposure of hepg <dig> cells to padma  <dig>
at concentrations ranging from 50– <dig> μg/ml, significantly and
dose-dependently diminished  <dig> '-azobis dihydrochloride
-induced ros levels .
at a concentration of  <dig> μg/ml, a
significant reduction of aaph-stimulated ros levels to  <dig> ±  <dig> % could be
observed.
in comparison, treatment of cells with  <dig> μm quercetin, a
flavonoid with potent antioxidant properties, decreased aaph-induced ros
production to  <dig>  ±  <dig> %.
-induced formation of ros in hepg <dig> cells pretreated
with either quercetin , as a positive control, or increasing
concentrations of padma  <dig> .
the mean
percentages of dcf fluorescence, as a measure of ros formation, are
shown in relation to the aaph-treated etoh solvent control .
mean values ± s.e.m.
of three independent experiments run in
quadruplicates  are
shown.
gene expression changes
affymetrix genechip human genome u <dig> plus  <dig>  arrays were used to analyse padma
<dig> regulated gene expression in hepg <dig> cells.
an experimental approach using only
one microarray per treatment  was applied, in which the cells of three
biological replicates were pooled to obtain a “biological average”
of stimulated gene expression changes.
particular attention was paid to profound
validation of expression data by qpcr.
analysis of differential gene expression
was performed by comparison of gene expression patterns of hepg <dig> cells treated
for  <dig> h with an ethanolic extract of padma  <dig>  at doses corresponding to the
50% inhibitory concentration  after  <dig> h , and solvent
, respectively.
at this concentration, no effect on cell viability
was detectable after  <dig> h of incubation .
microarray analysis revealed a total of  <dig> genes whose expression was modulated
more than two-fold upon extract treatment:  <dig> genes were up-regulated and 225
down-regulated.
of these,  <dig> showed a four-fold or higher increase in gene
expression, whereas nine genes were down-regulated by a fold change of more than
four-fold .
selected modulated candidate
genes were validated in five independent experiments using qpcr .
as shown
in table  <dig>  nine of the  <dig> most up-regulated genes
and three of the nine most down-regulated genes were significantly positively
validated , thus confirming most findings of the microarray
analysis.
treatment with padma 28
*primers detect gsta1-a <dig> and a5; **primers detect akr1c1-c4a.
the p value calculated for the qpcr experiments indicates the
probability that the difference between sample and control group is
significant.
network and pathway analysis
a total of  <dig>  of the  <dig>  probe sets could be mapped in the ingenuity
knowledge base of the analysis software  to the corresponding molecule
record  <cit> .
using a cut-off value of  <dig>  , ipa revealed  <dig> genes eligible for network generation and 318
genes eligible for pathway analysis.
in the network analysis, differentially
regulated genes are inter-connected using information of known interactions and
associations between genes or proteins extracted from literature and databank
findings.
network analysis of >two-fold differentially expressed genes in hepg <dig> cells
after treatment with padma  <dig>  for  <dig> h, revealed  <dig> networks,
of which  <dig> displayed various degrees of interrelationship.
a list of networks
with a high score of > <dig> and the corresponding differentially expressed
molecules identified is provided as supplementary file .
these genes are associated with signaling
pathways essential for the metabolic processing of drugs, amino acids, lipids,
small molecules, vitamins and minerals.
besides affecting general mechanisms
such as molecular transport, cell-to-cell signaling and interaction, some
molecules are part of signaling cascades that are active during development and
morphogenesis or play a role in pathological conditions such as genetic
disorders, cancer and connective tissue disorders.
interestingly, network analysis revealed that padma  <dig> also modulates networks
associated with cardiovascular system development and function as well as with
inflammatory responses , which appear to be
central within the overlapping networks identified.
figure  <dig> shows the relationships of  <dig> overlapping networks, of which the
top seven , are highlighted
in grey.
obtained by ipa network analysis of microarray data of differential gene
expression in hepg <dig> cells after exposure to  <dig> μg/ml padma 28
ethanolic extract for  <dig> h.
connections extracted from the findings of altered gene expression in
hepg <dig> cells following padma  <dig> treatment.
ingenuity network analysis
identified  <dig> networks, of which  <dig> displayed various degrees of
interrelationship such as molecules influencing other components within
a network or molecules participating in more than one biological
process, thus appearing in more than one network.
the top seven networks
with a score > <dig> are highlighted and labelled.
both up- and
down-regulated identifiers were included in the analysis.
furthermore,  <dig> canonical pathways were identified from the ipa library, which
appeared with significance  in the dataset.
the top six canonical
pathways  are involved in the metabolism of xenobiotics by cytochrome
p <dig> , glutathione metabolism, aminoacyl-trna biosynthesis,
lipopolysaccharide /interleukin- <dig> -mediated inhibition of retinoid x
receptor  function, nrf2-mediated oxidative stress response and
aryl-hydrocarbon receptor  signaling .
qpcr analysis positively validated the differential expression of at least two
genes in each pathway in five independent experiments .
in hepg <dig> cells
metabolism of xenobiotics by cytochrome p450
lps/il- <dig> mediated inhibition of rxr function
nrf2-mediated oxidative stress response
aryl hydrocarbon receptor signaling
the p-value is a measure of the probability of the association of a
specific pathway and the dataset.
the ratio indicates the number of
molecules in a given pathway from the input dataset that meet the
cut-off criteria  divided by the number of
molecules that make up that pathway in the database.
effect of treatment on nrf2-mediated oxidative stress response
based on the observation that padma  <dig> up-regulates various genes of the
nrf2-mediated oxidative stress response in the microarray analysis and the fact
that nrf <dig> is a transcriptional activator of are-mediated gene expression, we
used the cellsensor® are-bla hepg <dig> cell system to verify padma 28-induced
transcriptional activation of are-driven reporter gene expression.
as shown in
figure  <dig>  treatment of hepg <dig> cells with padma 28
for  <dig> h dose-dependently stimulated are-driven β-lactamase expression.
within a concentration range of 25– <dig> μg/ml, padma  <dig> induced
are-mediated transcriptional activity  <dig>  ±  <dig>  to  <dig>  ±  <dig> -fold.
quercetin at  <dig> μm showed nearly the same transactivation potency  as padma  <dig> at doses of  <dig> μg/ml.
β-lactamase reporter gene expression.
cellsensor®
are-bla hepg <dig> cells were treated with quercetin , as a
positive control, and increasing concentrations of padma 28
for  <dig> h. the mean fold induction of
β-lactamase activity, as a measure of are-mediated transcriptional
activation, is shown relative to the solvent control .
mean
values ± s.e.m.
of four independent experiments run in
quadruplicates  are
shown.
effect on heme oxygenase  <dig> gene  and protein  expression
based on the finding that inducible hmox was up-regulated in the microarray
experiment , as validated by qpcr , western blot
analysis was performed under the same culture conditions to confirm the
up-regulation of ho- <dig> at the protein level.
as shown in figure  <dig>  expression of ho- <dig> was dose-dependently induced after
treatment of hepg <dig> cells with padma  <dig> ethanolic extract for  <dig> h, whereas ho-1
protein was undetectable in untreated and solvent-treated cells.
oxygenase- <dig>  expression after treatment of hepg <dig> cells with
increasing concentrations of padma  <dig>  for 18
h.  a representative western blot of ho- <dig> protein expression
is shown.
densitometric analysis of ho-1/gapdh expression.
mean values ± s.e.m.
of three independent experiments  are shown.
discussion
in traditional medical systems, combinations of herbs and natural materials are often
used in the treatment of multifactorial diseases  <cit> .
however, evaluation of the molecular mechanisms modulated by complex
multicomponent preparation in living cells is often limited because of the chemical
diversity and multiple effects on a variety of molecular targets.
functional
redundancy and regulatory loops add further complexity to the interaction networks
responsible for biological actions  <cit> .
re-establishment of a healthy state following disease development
requires interventions on multiple targets at different levels  <cit> .
here, we employed the effects of the well-studied indo-tibetan polyherbal remedy
padma  <dig> on hepg <dig> cells using a microarray approach to gain deeper insights into
multi-target effects of polyherbal preparations.
we used the orac method to reveal a peroxyl-radical scavenging activity of 2006
±  <dig> μmol te/g, which is much higher than that of other herbal
preparations.
liao et al.
reported an orac range of 40– <dig> μmol te/g for
<dig> herbs used in traditional chinese medicine, with a further eight showing
antioxidant capacities of 1000– <dig> μmol te/g and only two approaching
the level of padma  <dig>  <cit> .
of note, the antioxidant capacity of herbal preparations may depend not
only from the plant species used, but also from other factors e.g.
the extract
processing procedure or the storage under non oxidizing conditions.
also, in
cultivated hepg <dig> cells, padma  <dig> ethanolic extract showed a considerable antioxidant
capacity by dose-dependently decreasing peroxyl-radical-induced intracellular ros
levels.
these results are consistent with previous reports about the concentration
dependent antioxidant and pro-oxidant ability of padma  <dig>  <cit> .
beside peroxyl radical, other ros such as superoxide, hydrogen peroxide,
hydroxyl and alkoxyl radicals, as well as reactive nitrogen species  are
produced under physiological conditions and may be neutralized by
antioxidant compounds  <cit> .
to investigate genome-wide transcriptional alterations in treated hepg <dig> cells, we
used a concentration of the extract equal to the ic <dig> value of growth after  <dig> h
, but limited the incubation period to  <dig> h, where cell viability
was not yet affected .
at this concentration, aaph-induced ros level
in hepg <dig> cells was significantly decreased.
a total of  <dig> genes were more than
two-fold differentially regulated following padma  <dig> treatment compared to the
solvent control.
however, the extent of regulation was mostly moderate and only 24
genes showed a four-fold or higher increase in gene expression, whereas nine were
down-regulated at least four-fold.
notably, despite the weakness of regulation, the
gene expression change of  <dig> selected transcripts was confirmed by qpcr in five
independent experiments, implicating the robustness of the experimental design.
according to the biological response fingerprinting  approach of rong et al.
<cit> , this set of validated transcripts may be used as a signature pattern,
which may be specific for the biological response of padma  <dig> in hepg <dig> cells.
thus,
qpcr analysis of marker genes might serve as a sensitive and cost-effective method
for quality assessment and batch-to-batch comparison of polyherbal formulations.
however, these results should be confirmed by further experiments using different
incubation times, treatment concentrations and other cell models.
one of the major findings of this microarray analysis was the differential expression
of phase i and ii metabolic enzymes upon treatment of hepg <dig> cells with padma  <dig>  the
cyp superfamily plays an essential role in detoxification phase i metabolism
reactions of a wide range of xenobiotics including therapeutic agents, food
additives, cruciferous vegetables and herbal components  <cit> .
the majority of cyp-mediated drug biotransformation reactions are
catalysed by cyp1a <dig>  2c <dig>  2c <dig>  2d <dig> and 3a <dig>  and, to a limited extent, cyp2c <dig>  2b6
and 3a <dig>  <cit> .
other cyp isoenzymes such as cyp1a <dig>  1b <dig>  2a <dig>  and 2e <dig> are modulated by
only a few, if any, therapeutics  <cit> .
in the present study, the isoenzyme cyp1a <dig> was differentially expressed to the
greatest extent.
it is of a particular interest because of its implication in the
metabolic activation of some exogenous pro-carcinogens or endogenous molecules  <cit> .
interestingly, cyp1a <dig> also exerts important functions in the
detoxification of environmental carcinogens and the metabolic conversion of dietary
compounds into substances with cancer preventive properties  <cit> .
as many phytochemicals are known to interact with cyp1a <dig> and ahr
signaling, a deeper examination of the regulation of cyp1a <dig> by herbal components is
of pharmacological relevance  <cit> .
in general, cyp1a <dig> expression is mediated by the aryl hydrocarbon
receptor  transcription factor.
additionally, ahr signaling is involved in many
cellular processes such as cell growth, apoptosis, hypoxia signaling, cell adhesion
and cell matrix metabolism  <cit> .
ahr induces the expression of several phase ii xenobiotic metabolizing
enzymes, such as glutathione s-transferase alpha  <dig> , nadh: quinone
oxidoreductase  <dig> , microsomal glutathione s-transferase  <dig>  or aldehyde
dehydrogenase  <dig> family member l <dig> .
interestingly, in the present study,
several ahr regulated enzymes were up-regulated by padma  <dig> treatment in hepg <dig> cells
.
the effect of padma  <dig> on cell growth and apoptosis has been illustrated by the
>six-fold increase of nuclear protein  <dig> , which responds to proapoptotic
stimuli and promotes cellular growth to counteract diverse injuries  <cit> .
in addition, up-regulation of nqo <dig> by padma  <dig> indicates an involvement
of apoptosis and hypoxia signalling, since nqo <dig> is able to stabilize the p <dig> tumour
suppressor protein, especially under oxidative stress conditions  <cit> .
as expected from a pleiotropic acting antioxidant remedy, nrf2-regulated stress
response genes were also differentially expressed following padma  <dig> treatment
.
nrf <dig> is a transcriptional activator for
the are-mediated induction of antioxidant and phase ii-detoxifying genes such as
hmox or glutathione s-transferase , which protect cells and tissues from
oxidative damage  <cit> .
to validate this result, we showed that are-promoter driven
β-lactamase expression was dose-dependently induced in a reporter cell line
transactivation assay .
we also observed a
significant up-regulation of further antioxidant and detoxification genes such as
hmox, glutamate-cysteine ligase modifier  and catalytic  subunits,
aldo-keto reductase family  <dig>  member c <dig>  and member c <dig>  in the
microarray analysis .
the induction of heme
oxygenase expression upon treatment of hepg <dig> cells with padma  <dig> could be confirmed
at both the transcript  and protein level
.
the expression of heme oxygenase- <dig> is
associated with therapeutic benefits in a number of pathologic conditions such as
atherosclerotic vascular disease and inflammation  <cit> .
a number of natural and synthetic molecules have been reported to induce
ho- <dig> as additive mechanism responsible for their therapeutic effects  <cit> .
the network visualization of the interaction of differentially expressed genes
provides a link between transcriptional changes and their functional consequences in
biological processes.
we observed interference of the herbal preparation with
molecules relevant to cardiovascular system development and function, cell-to-cell
signaling and interaction and inflammatory responses
within a centrally arranged subnetwork.
within this subnetwork, tumor necrosis
factor  and interferon gamma  have a central position.
tnf is a principal
mediator of the acute inflammatory response and coordinates the proliferation and
protective functions of pathogen-reactive cells  <cit> .
tnf is engaged in the activation of various pathways under both
pathological and physiological conditions, and shows a remarkable functional
duality, thus being also responsible for many of the systemic complications of
severe infections  <cit> .
the pro-inflammatory cytokine interferon gamma  plays a
central role in the cellular immune response, as it induces several
immuno-regulatory pathways and cellular response signals e.g.
via the janus
activated kinase -signal transducer and activator of transcription
pathway  <cit> .
beside anti-microbial and inflammatory responses, ifnγ also
interferes with growth suppression, cell death, tumor immunity and autoimmunity  <cit> .
the strong interconnection of network  <dig> with other subnetworks might indicate that
the represented interactions are involved in the regulation of some important cell
behavior, as the removal of this part would affect the whole interaction network  <cit> .
however, it should be kept in mind that transcriptional signatures are
time and dosage dependent.
therefore, these results should be further validated
using additional experimental approaches in order to design a more comprehensive
activity model of padma  <dig> that reflects also low-dose and long term effects.
the
application of “omics” technologies in multicomponent activity
assessment is suggested to be most useful when applied in an iterative strategy in
combination with focused bioassay  <cit> .
moreover, the interaction databases used for network generation might
influence the result.
thus, our experimental findings must be interpreted with
caution and a broader experimental design is necessary to use such data in the
context of a comprehensive risk-benefit assessment.
CONCLUSIONS
in the present study, we show that the indo-tibetan polyherbal remedy padma 28
displays both direct and indirect antioxidant capacities and exerts a variety of
actions including the transcriptional activation of cytoprotective genes.
the
identification of a number of potential key molecules and signaling cascades that
are differentially regulated upon cell treatment in vitro, supports the
hypothesis for a pleiotropic mode of action.
the results agree with several previous
studies that described antioxidant, anti-inflammatory and anti-atherosclerotic
properties using different experimental approaches.
the combination of molecular and pathway-based bioassays with unbiased transcriptome
analysis has proved to be a valuable strategy in multicomponent activity assessment.
nevertheless, the selection of an appropriate experimental design, the cellular
model system and the verification of findings in an iterative manner is necessary to
generate valuable results.

 herbal drug combinations have long been used in traditional medicinal concepts, and multicomponent preparations, such as plant-derived pharmaceuticals, botanicals and dietary supplements, are becoming increasingly important in modern medicine and lifestyles. the concept of effect potentiation by synergistic actions has opened new approaches for phytopharmaceutical and nutraceutical product development, and suggests the utilization of some traditional formulations for therapeutic issues. however, this development is hampered by the difficulties that accompany the assessment of molecular mechanisms and pharmacological actions of multicomponent preparations that contain an enormous chemical diversity of substances. one major beneficial effect of many multicomponent preparations is their antioxidant activity. reactive oxygen species are involved in the development and the progression of diseases such as cardiovascular disorders, neurodegeneration and atherosclerosis. the capacity of dietary antioxidants to modulate the cellular redox balance is discussed to be favourable in both the treatment and prevention of ros-related disorders. an antioxidant capacity may not only originate from the direct radical scavenging activity of a compound, but also from indirectly induced effects such as interference with the endogenous antioxidant machinery, the transcription induction of detoxifying and cytoprotective enzymes or the increase of immunoprotective mechanisms. several of these actions are mediated by the nuclear-factor-e2-related factor - that transactivates the transcription of protective genes upon binding of antioxidant response element promoter sequences to maintain homeostasis during oxidative stress. however, an extensive input of antioxidants can also promote adverse responses such as allergies or toxicity-related phenomena. studies into the interference of phytochemicals with cellular signaling pathways and gene expression have revealed many examples of potential biological activities that go far beyond an antioxidant action. further, the use of large-scale data acquisition strategies, such as gene expression analysis, is suggested to be helpful in deciphering complex molecular information flows. currently, dna microarray technology is the most widely used genome-scale data in pharmacological and clinical research. transcriptomics allows the simultaneous detection of multiple transcriptional events in a non-biased manner, enabling standards for analysis and interpretation to be continuously developed. microarray expression data may provide key insights into gene function and interactions within and across metabolic pathways. however, it appears necessary to go beyond simple clustering by integrating data into biological interaction databases. in general, the therapeutic efficacy of botanical multicomponents is suggested to be based on the interference of a multitude of phytochemicals with multiple cellular targets. this enormous complexity of interactions represents a challenge for activity monitoring. often, the active principles of such remedies cannot be identified, as the pharmacological effects are suggested to be generated from the joint actions of several substances. thus, reductionist and hypothesis-driven research strategies may prove insufficient to deal with such pleiotropic activity profiles. the combination of pathway or molecule focused and large-scale data assessment strategies, such as “omics” technologies, has proved useful in monosubstance and drug combination activity assessment and is also considered helpful for multicomponents. based on these assumptions, we attempted to explore the mode of action of the indo-tibetan remedy padma at the molecular and transcriptional level in human hepatoma hepg cells. padma is a polyherbal preparation containing a variety of secondary plant substances with potential bioactivity, such as essential oils, flavonoids, tanning agents and sesquiterpenes. it contains different powdered plants including natural camphor and calcium sulphate. the formulation has been subjected to extensive in vitro and in vivo studies and has been approved by the swiss drug authorities for symptoms associated with circulatory disorders such as tingling sensation, formication, feeling of heaviness and tension in the legs and arms, numbness of the hands and feet and calf cramps. studies have been performed where padma was applied in the treatment of intermittent claudication, a hallmark of peripheral arterial occlusive disease. hepg cells were used because the liver is the main organ for drug metabolism after the ingestion of compounds and because of their suitability for studying the mechanism of action of drugs, dietary genotoxicants, as well as the cytoprotective and antigenotoxic activities of agents. furthermore, this cell line is useful for the evaluation of pro- and antioxidant agents as they express many related enzymes such as mn-superoxide dismutase, catalase, glutathione reductase and thioredoxin reductase. a hepg derived reporter gene cell line, cellsensor® are-bla hepg has been applied to investigate are-mediated transcriptional response.  hepg cell proliferation to determine the effect of padma on cell viability and to define optimal treatment conditions for further cell culture experiments, hepg cells were treated with increasing concentrations of the ethanolic extract and the solvent control. cell viability was calculated in relation to the solvent control. as shown in figure  treatment of hepg cells with padma for h dose-dependently decreased the number of viable cells with an ic50-value of ± μg/ml. for h and then treated with solvent or padma ethanolic extract for h. the mean percentages of cell growth relative to solvent-treated cells were plotted against the concentrations of padma mean values ± s.e.m. of four independent experiments run in duplicates are shown. antioxidant capacity to ensure potent biological activity of the ethanolic extract of padma its antioxidant capacity was evaluated using the oxygen radical absorbance capacity assay, which measures the direct capacity of chain-breaking antioxidants based on the hydrogen atom transfer mechanism in a cell-free system. the polyherbal extract showed potent peroxyl-radical scavenging capacity in vitro, with an orac value of ± μmol te/g. accordingly, μg/ml of padma corresponds to the net protection against peroxyl-radicals produced by about μm or μg/ml of the water-soluble vitamin e derivate trolox. the antioxidant capacity of padma in living cells was analysed by measurement of peroxyl-radical-induced intracellular ros-levels in hepg cells pretreated with increasing concentrations of ethanolic extract. as shown in figure  exposure of hepg cells to padma at concentrations ranging from 50– μg/ml, significantly and dose-dependently diminished'-azobis dihydrochloride -induced ros levels. at a concentration of μg/ml, a significant reduction of aaph-stimulated ros levels to ± % could be observed. in comparison, treatment of cells with μm quercetin, a flavonoid with potent antioxidant properties, decreased aaph-induced ros production to ± %. -induced formation of ros in hepg cells pretreated with either quercetin, as a positive control, or increasing concentrations of padma. the mean percentages of dcf fluorescence, as a measure of ros formation, are shown in relation to the aaph-treated etoh solvent control. mean values ± s.e.m. of three independent experiments run in quadruplicates are shown. gene expression changes affymetrix genechip human genome u plus arrays were used to analyse padma regulated gene expression in hepg cells. an experimental approach using only one microarray per treatment was applied, in which the cells of three biological replicates were pooled to obtain a “biological average” of stimulated gene expression changes. particular attention was paid to profound validation of expression data by qpcr. analysis of differential gene expression was performed by comparison of gene expression patterns of hepg cells treated for h with an ethanolic extract of padma at doses corresponding to the 50% inhibitory concentration after h, and solvent, respectively. at this concentration, no effect on cell viability was detectable after h of incubation. microarray analysis revealed a total of genes whose expression was modulated more than two-fold upon extract treatment: genes were up-regulated and 225 down-regulated. of these, showed a four-fold or higher increase in gene expression, whereas nine genes were down-regulated by a fold change of more than four-fold. selected modulated candidate genes were validated in five independent experiments using qpcr. as shown in table  nine of the most up-regulated genes and three of the nine most down-regulated genes were significantly positively validated, thus confirming most findings of the microarray analysis. treatment with padma 28 *primers detect gsta1-a and a5; **primers detect akr1c1-c4a. the p value calculated for the qpcr experiments indicates the probability that the difference between sample and control group is significant. network and pathway analysis a total of of the probe sets could be mapped in the ingenuity knowledge base of the analysis software to the corresponding molecule record. using a cut-off value of, ipa revealed genes eligible for network generation and 318 genes eligible for pathway analysis. in the network analysis, differentially regulated genes are inter-connected using information of known interactions and associations between genes or proteins extracted from literature and databank findings. network analysis of >two-fold differentially expressed genes in hepg cells after treatment with padma for h, revealed networks, of which displayed various degrees of interrelationship. a list of networks with a high score of > and the corresponding differentially expressed molecules identified is provided as supplementary file. these genes are associated with signaling pathways essential for the metabolic processing of drugs, amino acids, lipids, small molecules, vitamins and minerals. besides affecting general mechanisms such as molecular transport, cell-to-cell signaling and interaction, some molecules are part of signaling cascades that are active during development and morphogenesis or play a role in pathological conditions such as genetic disorders, cancer and connective tissue disorders. interestingly, network analysis revealed that padma also modulates networks associated with cardiovascular system development and function as well as with inflammatory responses, which appear to be central within the overlapping networks identified. figure  shows the relationships of overlapping networks, of which the top seven, are highlighted in grey. obtained by ipa network analysis of microarray data of differential gene expression in hepg cells after exposure to μg/ml padma 28 ethanolic extract for h. connections extracted from the findings of altered gene expression in hepg cells following padma treatment. ingenuity network analysis identified networks, of which displayed various degrees of interrelationship such as molecules influencing other components within a network or molecules participating in more than one biological process, thus appearing in more than one network. the top seven networks with a score > are highlighted and labelled. both up- and down-regulated identifiers were included in the analysis. furthermore, canonical pathways were identified from the ipa library, which appeared with significance in the dataset. the top six canonical pathways are involved in the metabolism of xenobiotics by cytochrome p, glutathione metabolism, aminoacyl-trna biosynthesis, lipopolysaccharide /interleukin- -mediated inhibition of retinoid x receptor function, nrf2-mediated oxidative stress response and aryl-hydrocarbon receptor signaling. qpcr analysis positively validated the differential expression of at least two genes in each pathway in five independent experiments. in hepg cells metabolism of xenobiotics by cytochrome p450 lps/il- mediated inhibition of rxr function nrf2-mediated oxidative stress response aryl hydrocarbon receptor signaling the p-value is a measure of the probability of the association of a specific pathway and the dataset. the ratio indicates the number of molecules in a given pathway from the input dataset that meet the cut-off criteria divided by the number of molecules that make up that pathway in the database. effect of treatment on nrf2-mediated oxidative stress response based on the observation that padma up-regulates various genes of the nrf2-mediated oxidative stress response in the microarray analysis and the fact that nrf is a transcriptional activator of are-mediated gene expression, we used the cellsensor® are-bla hepg cell system to verify padma 28-induced transcriptional activation of are-driven reporter gene expression. as shown in figure  treatment of hepg cells with padma 28 for h dose-dependently stimulated are-driven β-lactamase expression. within a concentration range of 25– μg/ml, padma induced are-mediated transcriptional activity ± to ± -fold. quercetin at μm showed nearly the same transactivation potency as padma at doses of μg/ml. β-lactamase reporter gene expression. cellsensor® are-bla hepg cells were treated with quercetin, as a positive control, and increasing concentrations of padma 28 for h. the mean fold induction of β-lactamase activity, as a measure of are-mediated transcriptional activation, is shown relative to the solvent control. mean values ± s.e.m. of four independent experiments run in quadruplicates are shown. effect on heme oxygenase gene and protein expression based on the finding that inducible hmox was up-regulated in the microarray experiment, as validated by qpcr, western blot analysis was performed under the same culture conditions to confirm the up-regulation of ho- at the protein level. as shown in figure  expression of ho- was dose-dependently induced after treatment of hepg cells with padma ethanolic extract for h, whereas ho-1 protein was undetectable in untreated and solvent-treated cells. oxygenase- expression after treatment of hepg cells with increasing concentrations of padma for 18 h. a representative western blot of ho- protein expression is shown. densitometric analysis of ho-1/gapdh expression. mean values ± s.e.m. of three independent experiments are shown. discussion in traditional medical systems, combinations of herbs and natural materials are often used in the treatment of multifactorial diseases. however, evaluation of the molecular mechanisms modulated by complex multicomponent preparation in living cells is often limited because of the chemical diversity and multiple effects on a variety of molecular targets. functional redundancy and regulatory loops add further complexity to the interaction networks responsible for biological actions. re-establishment of a healthy state following disease development requires interventions on multiple targets at different levels. here, we employed the effects of the well-studied indo-tibetan polyherbal remedy padma on hepg cells using a microarray approach to gain deeper insights into multi-target effects of polyherbal preparations. we used the orac method to reveal a peroxyl-radical scavenging activity of 2006 ± μmol te/g, which is much higher than that of other herbal preparations. liao et al. reported an orac range of 40– μmol te/g for herbs used in traditional chinese medicine, with a further eight showing antioxidant capacities of 1000– μmol te/g and only two approaching the level of padma. of note, the antioxidant capacity of herbal preparations may depend not only from the plant species used, but also from other factors e.g. the extract processing procedure or the storage under non oxidizing conditions. also, in cultivated hepg cells, padma ethanolic extract showed a considerable antioxidant capacity by dose-dependently decreasing peroxyl-radical-induced intracellular ros levels. these results are consistent with previous reports about the concentration dependent antioxidant and pro-oxidant ability of padma. beside peroxyl radical, other ros such as superoxide, hydrogen peroxide, hydroxyl and alkoxyl radicals, as well as reactive nitrogen species are produced under physiological conditions and may be neutralized by antioxidant compounds. to investigate genome-wide transcriptional alterations in treated hepg cells, we used a concentration of the extract equal to the ic value of growth after h, but limited the incubation period to h, where cell viability was not yet affected. at this concentration, aaph-induced ros level in hepg cells was significantly decreased. a total of genes were more than two-fold differentially regulated following padma treatment compared to the solvent control. however, the extent of regulation was mostly moderate and only 24 genes showed a four-fold or higher increase in gene expression, whereas nine were down-regulated at least four-fold. notably, despite the weakness of regulation, the gene expression change of selected transcripts was confirmed by qpcr in five independent experiments, implicating the robustness of the experimental design. according to the biological response fingerprinting approach of rong et al., this set of validated transcripts may be used as a signature pattern, which may be specific for the biological response of padma in hepg cells. thus, qpcr analysis of marker genes might serve as a sensitive and cost-effective method for quality assessment and batch-to-batch comparison of polyherbal formulations. however, these results should be confirmed by further experiments using different incubation times, treatment concentrations and other cell models. one of the major findings of this microarray analysis was the differential expression of phase i and ii metabolic enzymes upon treatment of hepg cells with padma the cyp superfamily plays an essential role in detoxification phase i metabolism reactions of a wide range of xenobiotics including therapeutic agents, food additives, cruciferous vegetables and herbal components. the majority of cyp-mediated drug biotransformation reactions are catalysed by cyp1a 2c 2c 2d and 3a and, to a limited extent, cyp2c 2b6 and 3a. other cyp isoenzymes such as cyp1a 1b 2a and 2e are modulated by only a few, if any, therapeutics. in the present study, the isoenzyme cyp1a was differentially expressed to the greatest extent. it is of a particular interest because of its implication in the metabolic activation of some exogenous pro-carcinogens or endogenous molecules. interestingly, cyp1a also exerts important functions in the detoxification of environmental carcinogens and the metabolic conversion of dietary compounds into substances with cancer preventive properties. as many phytochemicals are known to interact with cyp1a and ahr signaling, a deeper examination of the regulation of cyp1a by herbal components is of pharmacological relevance. in general, cyp1a expression is mediated by the aryl hydrocarbon receptor transcription factor. additionally, ahr signaling is involved in many cellular processes such as cell growth, apoptosis, hypoxia signaling, cell adhesion and cell matrix metabolism. ahr induces the expression of several phase ii xenobiotic metabolizing enzymes, such as glutathione s-transferase alpha, nadh: quinone oxidoreductase, microsomal glutathione s-transferase or aldehyde dehydrogenase family member l. interestingly, in the present study, several ahr regulated enzymes were up-regulated by padma treatment in hepg cells. the effect of padma on cell growth and apoptosis has been illustrated by the >six-fold increase of nuclear protein, which responds to proapoptotic stimuli and promotes cellular growth to counteract diverse injuries. in addition, up-regulation of nqo by padma indicates an involvement of apoptosis and hypoxia signalling, since nqo is able to stabilize the p tumour suppressor protein, especially under oxidative stress conditions. as expected from a pleiotropic acting antioxidant remedy, nrf2-regulated stress response genes were also differentially expressed following padma treatment. nrf is a transcriptional activator for the are-mediated induction of antioxidant and phase ii-detoxifying genes such as hmox or glutathione s-transferase, which protect cells and tissues from oxidative damage. to validate this result, we showed that are-promoter driven β-lactamase expression was dose-dependently induced in a reporter cell line transactivation assay. we also observed a significant up-regulation of further antioxidant and detoxification genes such as hmox, glutamate-cysteine ligase modifier and catalytic subunits, aldo-keto reductase family member c and member c in the microarray analysis. the induction of heme oxygenase expression upon treatment of hepg cells with padma could be confirmed at both the transcript and protein level. the expression of heme oxygenase- is associated with therapeutic benefits in a number of pathologic conditions such as atherosclerotic vascular disease and inflammation. a number of natural and synthetic molecules have been reported to induce ho- as additive mechanism responsible for their therapeutic effects. the network visualization of the interaction of differentially expressed genes provides a link between transcriptional changes and their functional consequences in biological processes. we observed interference of the herbal preparation with molecules relevant to cardiovascular system development and function, cell-to-cell signaling and interaction and inflammatory responses within a centrally arranged subnetwork. within this subnetwork, tumor necrosis factor and interferon gamma have a central position. tnf is a principal mediator of the acute inflammatory response and coordinates the proliferation and protective functions of pathogen-reactive cells. tnf is engaged in the activation of various pathways under both pathological and physiological conditions, and shows a remarkable functional duality, thus being also responsible for many of the systemic complications of severe infections. the pro-inflammatory cytokine interferon gamma plays a central role in the cellular immune response, as it induces several immuno-regulatory pathways and cellular response signals e.g. via the janus activated kinase -signal transducer and activator of transcription pathway. beside anti-microbial and inflammatory responses, ifnγ also interferes with growth suppression, cell death, tumor immunity and autoimmunity. the strong interconnection of network with other subnetworks might indicate that the represented interactions are involved in the regulation of some important cell behavior, as the removal of this part would affect the whole interaction network. however, it should be kept in mind that transcriptional signatures are time and dosage dependent. therefore, these results should be further validated using additional experimental approaches in order to design a more comprehensive activity model of padma that reflects also low-dose and long term effects. the application of “omics” technologies in multicomponent activity assessment is suggested to be most useful when applied in an iterative strategy in combination with focused bioassay. moreover, the interaction databases used for network generation might influence the result. thus, our experimental findings must be interpreted with caution and a broader experimental design is necessary to use such data in the context of a comprehensive risk-benefit assessment.  in the present study, we show that the indo-tibetan polyherbal remedy padma 28 displays both direct and indirect antioxidant capacities and exerts a variety of actions including the transcriptional activation of cytoprotective genes. the identification of a number of potential key molecules and signaling cascades that are differentially regulated upon cell treatment in vitro, supports the hypothesis for a pleiotropic mode of action. the results agree with several previous studies that described antioxidant, anti-inflammatory and anti-atherosclerotic properties using different experimental approaches. the combination of molecular and pathway-based bioassays with unbiased transcriptome analysis has proved to be a valuable strategy in multicomponent activity assessment. nevertheless, the selection of an appropriate experimental design, the cellular model system and the verification of findings in an iterative manner is necessary to generate valuable results. 
phase ii detoxification and the oxidative stress response. the results were validated using reporter results obtained might be the basis for further investigations and new functions such as drug and bio-molecule metabolism, molecular transport and radical absorbance capacity assay was employed, which resulted in a this formulation showed a considerable antioxidant capacity by transcriptome analysis in combination with pathway-focused bioassays is integrated transcriptome analysis revealed a major influence on phase i and genes, such as heme oxygenase were validated in qpcr experiments. significantly reducing ros levels in a dose-dependent manner. the identified molecular targets and pathways suggest several mechanisms that to reveal the direct radical scavenging effects of the ethanolic extract of assays were employed to evaluate the antioxidant capacity. furthermore, the antioxidant capacity of padma was analysed in living indo-tibetan preparation, padma approved by the swiss drug authorities the indo-tibetan polyherbal remedy padma an in vitro oxygen
 transcriptome analysis in combination with pathway-focused bioassays is suggested to be a helpful approach for gaining deeper insights into the complex mechanisms of action of herbal multicomponent preparations in living cells. the polyherbalism based concept of tibetan and ayurvedic medicine considers therapeutic efficacy through multi-target effects. a polyherbal indo-tibetan preparation, padma approved by the swiss drug authorities, was applied to a more detailed dissection of mechanism of action in human hepatoma hepg cells. cell-free and cell-based assays were employed to evaluate the antioxidant capacity. genome-wide expression profiling was done by applying human genome u plus affymetrix arrays. pathway- and network-oriented analysis elucidated the affected biological processes. the results were validated using reporter gene assays and quantitative real-time pcr.  to reveal the direct radical scavenging effects of the ethanolic extract of the indo-tibetan polyherbal remedy padma an in vitro oxygen radical absorbance capacity assay was employed, which resulted in a peroxyl-radical scavenging activity of ± μmol te/g. furthermore, the antioxidant capacity of padma was analysed in living hepg cells, by measuring its scavenging potential against radical induced ros. this formulation showed a considerable antioxidant capacity by significantly reducing ros levels in a dose-dependent manner. integrated transcriptome analysis revealed a major influence on phase i and phase ii detoxification and the oxidative stress response. selected target genes, such as heme oxygenase were validated in qpcr experiments. network analysis showed interrelated networks involved in important biological functions such as drug and bio-molecule metabolism, molecular transport and cellular communication. some molecules are part of signaling cascades that are active during development and morphogenesis or are involved in pathological conditions and inflammatory response.  the identified molecular targets and pathways suggest several mechanisms that underlie the biological activity of the preparation. although extrapolation of these findings to the in vivo situation is not possible, the results obtained might be the basis for further investigations and new hypotheses to be tested. this study demonstrates the potential of the combination of focused and unbiased research strategies in the mode of action analysis of multicomponent herbal mixtures. hepg2microarraymulticomponentpathway analysispolyherbalqpcr 