The application of DNA micro-arrays (gene arrays) to the study of herbal medicines

Journal of Ethnopharmacology 108 (2006) 2–15

Review

The application of DNA micro-arrays (gene arrays) to thestudy of herbal medicines

Jim Hudson ∗, Manuel AltamiranoDepartment of Pathology & Laboratory Medicine, University of British Columbia, Vancouver, BC, Canada V5Z 1M9

Received 8 June 2006; received in revised form 7 August 2006; accepted 8 August 2006Available online 18 August 2006

Abstract

DNA micro-arrays (gene arrays) have become a popular and useful tool with which to study the effects of various agents and treatments ongene expression in cells and tissues. In theory one can simultaneously evaluate, in a single experiment, changes in gene expression (at the level oftranscription) of the entire genome of the organism under study. Consequently these techniques have been used by many investigators interestedin cancer research, differentiation and development, toxicology, and the effects of pharmaceuticals on cells and animals. In addition, recentstudies have shown the capacity of the technique for revealing the importance of genes not previously implicated in a given response. However,relatively few attempts have been made so far to evaluate herbal medicines, although the potential to answer a number of relevant questions isthere.

In this review we first discuss the fundamental principles of the gene array technology, focusing on the individual steps in the process andtheir problems and pitfalls, and we discuss the analysis and interpretation of the data, the discipline of bio-informatics, without which meaningfulevaluation of gene expression changes would be impossible.

We next analyze specific studies, which utilized gene array technology, aimed at evaluating the effects of certain herbal medicine formulas andbioactive ingredients in animal tissues and in cell cultures. We also include a brief description of our own evaluation of Echinacea, which we havebeen studying for several years, to indicate possible mechanisms of action of this herbal, and also to illustrate how the techniques, especially thebio-informatics, continue to evolve.

We believe, on the basis of experience acquired by us and other investigators to date, that the technology of gene array analysis can make
significant contributions to understanding how herbal medicines work, and therefore can validate their applications in medicine.© 2006 Elsevier Ireland Ltd. All rights reserved.
Keywords: DNA micro-array; Gene array; Bio-informatics; Bioassays; Gene expression; Herbal medicines; Echinacea; Immune responses; Inflammatory diseases

Contents

1. Introduction. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 32. Methodology: procedures, pitfalls and problems . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3

2.1. RNA extraction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 42.2. Preparation of probes . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 52.3. Hybridization of probes to arrays . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5
2.4. Analysis and interpretation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5
3. Studies involving herbal medicines . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 64. Animal studies . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 6

4.1. Gingko biloba . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 6

Abbreviations: cDNA (cRNA), complementary DNA (RNA); chip, DNA array; Cy3, cyanin dye #3; Cy5, cyanin dye #5; EST, expressed sequence tag; IL-6,intrleukin-6; IL-8, interleukin-8; oligo, oligonucleotide; TNFalpha, tumor necrosis factor alpha

∗ Corresponding author.E-mail address: [email protected] (J. Hudson).

0378-8741/$ – see front matter © 2006 Elsevier Ireland Ltd. All rights reserved.doi:10.1016/j.jep.2006.08.013

mailto:[email protected]

dx.doi.org/10.1016/j.jep.2006.08.013

6. Echinacea and gene array analysis: our experience with a system in evolution . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 11. .. . .

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7. Discussion . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

. Introduction

The DNA array (micro-array; gene array) is a new methodol-gy developed for the purpose of analyzing the effects of variousgents on cellular gene expression. Thousands of genes can benalyzed in a single experiment. The application of this tech-ique to the fields of cancer research, inflammatory diseases, andthers, has led to significant advances in our understanding of theechanisms underlying disease and its treatment (eg., Calvano

t al., 2005; Raetz et al., 2006). Consequently we believe thatimilar advances could be made in undestanding how herbaledicines work.In this review we shall attempt to explain the methodology, as

ell as its limitations and pitfalls, and to critically evaluate thettempts made so far to apply the technique to the study of herbaledicines, including our own experience with Echinacea. Weust keep in mind, however, that the technique is new and is

till evolving, especially in connection with the methods usedor data correlation and analysis (bio-informatics).

The term DNA (gene) array refers to a solid surface to whichs attached numerous small pieces of DNA representing shortequences of specific genes. The arrays may be macro-arrays,hich are usually fabricated membranes of nylon, or other rel-

tively inert material, impregnated with scores or hundreds ofieces of DNA in the form of discrete spots. In contrast theicro-array is usually a siliconized glass slide to which thou-

ands of pieces of DNA fragments or oligonucleotides are pre-isely spotted by a robot (yes, robots can indeed play a role intudies on herbal and other traditional medicines!).

The macro-arrays, available in a number of commercial kits,re perhaps more amenable to routine laboratory use, but areestricted by the relatively small number of genes representedn a membrane, whereas up to 30,000 genes can be placed onsingle micro-array slide. However the latter requires sophis-

icated equipment for analysis. In practice there is probably noignificant difference in cost to a laboratory that has access tohe appropriate expertise and equipment. Needless to say, the

ore commercialization involved, the greater the total costs.A disadvantage of the macro-array, in our opinion, is the

act that restricting the number of genes for analysis keepss in the reductionist mental framework, in which we become

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nterested in examining only a small number of related genes,ccording to pre-conceived hypotheses, whereas the micro-arraypens up the possibility of looking at the entire genome, anderhaps revealing novel genes and pathways not previouslyhought to be involved in the actions of our test agent. In fact,ecent studies with viruses and other microbes have revealedhat in some infections hundreds of host genes, representing

multitude of different functions, can be switched on or offGeiss et al., 2001; Tian et al., 2002; Calvano et al., 2005),nd our own experience with Echinacea (discussed below)as indicated numerous interactions between virus-inducedhanges and herbal modulation, many of which had not beenre-conceived.

We believe that it is worthwhile to explore the potential ofhis new tool, to give it a trial run so to speak, and see what it caneveal. We do not believe that it is ready to replace traditionalthnopharmacology research; but it could at least augment it, anderhaps help to guide its progress. In addition, of even greaterossible significance and impact, this approach might mollifyhe critics who claim that there is no scientific basis for claims

ade about herbal medicines.

. Methodology: procedures, pitfalls and problems

The objective of the DNA array procedure is to compare,ualitatively and quantitatively, mRNA populations of “control”untreated) cells or tissues with corresponding mRNAs fromtreated” cells or tissues. In other words, we analyze and com-are the transcription programs (patterns, profiles). The controlssually comprise “normal” cell cultures, or tissues obtained fromormal animals. “Treated” refers to cell cultures exposed to aest material, which may be a herbal mixture, drug, infectiousrganism, chemical, etc. or to selected tissues obtained fromnimals inoculated or exposed to the test materials. Treated maylso refer to cancer cells which are compared with correspond-ng non-cancer cells or tissues (Clarke et al., 2001; Nelson etl., 2001). The technique is also used to compare transcriptional

J. Hudson, M. Altamirano / Journal of Ethnopharmacology 108 (2006) 2–15 3

4.2. St. John’s wort (Hypericum perforatum, SJW) and hypericin . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 74.3. Cannabinoids . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 74.4. Catalposide (Catalpa ovata), Boswellia serata, and inflammatory bowel disease (IBD) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 84.5. Herbal glycosides . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 9

5. Cell culture studies . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 95.1. Genistein-treated prostate carcinoma cells . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 95.2. PC-SPES and prostate carcinoma . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 95.3. Paeoniae radix and apoptosis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 105.4. Coptidis rhizoma and berberine . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 105.5. Tripterygium alkaloids and apoptosis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 105.6. Anoectochilus formosanus and plumbagin . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 105.7. Propolis and differentiation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 11

rograms of cells or tissues at different stages of developmentnd differentiation (Iyer et al., 1999). Thus we are not measur-ng absolute numbers of RNA molecules, but rather making aomparison, which we hope is quantitative and meaningful.

4 of Ethnopharmacology 108 (2006) 2–15

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In essence, the technique involves four steps:

1) Extracting and purifying total RNA (including all themRNAs) from the test cells.

2) Converting these RNAs to labelled “probes”, e.g. cDNA’s.3) The probes are then “hybridized” to representative

sequences of specific cellular genes embedded on the arrayslides or membranes.

4) The resulting arrays, one for control and one for each treatedpreparation, are then analyzed and compared in commercialscanners, and the data interpreted and displayed by means ofvarious software programs, including appropriate statisticalanalyses. A more recent modification involves comparisonof both treated and control preparations with a referencepreparation, to overcome some technical problems inherentin the earlier analyses.

Figs. 1 and 2 illustrate these steps diagrammatically.Behind each of these steps lies a voluminous literature of

asic studies, as well as many potential problems and pitfalls.ost of the steps have now been commercialized (and some

ven robotized). This has resulted in some advantages, such ashe use of commercial kits in several steps, giving greater con-istency, although one unfortunate disadvantage is that traineesntering this field may not be aware of the respective roles of allhe components of the system, and consequently may not knowow to fix or adjust the system when things go wrong. In otherords they may not know what they are doing if they simply fol-

ow the kit instructions. Of course the easiest remedy for suchproblem is to contact the local company technical rep. Unfor-

unately these individuals are usually young graduates who arequally unaware of the basis for most of the techniques, andho will likely suggest a replacement kit to “fix” the problem.evertheless many collaborative research groups will proba-

ictr

Fig. 2. Modified strategy. U-RNA is the

ig. 1. Original strategy. Cy3 and Cy5 are the cyanin dyes (green and red) usedo label the cDNA’s.

ly have at least one expert with background experience andnderstanding.

.1. RNA extraction

There are several well tested commercial kits for extract-ng intact mRNA populations from cells, in such a way as toive us consistent reliable preparations free from contaminat-
ng DNA and protein. The overall integrity of the preparationsan be checked (and should be checked) by denaturing gel elec-rophoresis techniques and measuring relative amounts of intactibosomal RNAs.
universal human reference RNA.

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.2. Preparation of probes

This step relies heavily on commercial kits, since it dependsn the use of reliable consistent enzymes to convert our RNAsnto a form, which can be labelled and measured. There arewo basically distinct systems for doing this. The commer-ial systems such as Affymetrix make use of an enzyme-ediated transcription system which converts all the mRNAolecules into cRNAs which are labelled with a biotinylated

ucleotide or radioctive nucleotide, although for various rea-ons the use of radioactive materials has fallen out of favourecently.

In the alternative system, each mRNA in the population isnzymatically reverse-transcribed into its corresponding cDNA,hich is labelled with a fluorescent nucleotide, usually one of

he cyanin dyes, abbreviated as Cy3 and Cy5 (green and red,espectively). Often one dye is used for control cDNA and thether dye for the treated cDNA, or both preparations can beimultaneously labelled with the same dye and independentlyybridized with the reference probe labelled with the other dyesee next section).

One of the major pitfalls of the entire methodology occurst this stage, if it is assumed that the rate of incorporation ofabelled nucleotide into the probes is equivalent for the two dyesor radioactive label) and equivalent among all the probes. Thiss however not necessarily the case, and more recent analysesave attempted to correct this problem by various means (seeelow).

.3. Hybridization of probes to arrays

The hybridization of complementary single stranded RNAnd DNA molecules, in which one of the molecules isabelled and therefore measurable, is carried out by meansf standard buffered solutions (usually based on standardaline citrate, “SSC”) containing hybrid-forming and hybrid-issociating chemicals (e.g. formamide) at specific tempera-ures. These solutions can be purchased from reliable suppli-rs who guarantee that they will work in specific conditions.mplicit in this phase of the work is the assumption that theybridization process is comparable between preparations underomparison.

In a more recent modification, the different preparationsntended for comparison are each labelled with the sameye, and are individually compared with a so-called “Uni-ersal Human Reference RNA” (labelled with the other dye),commercial reference collection of 10 mRNA populationsade from different human tumor cell lines. In practice each

est preparation is mixed with the reference and hybridizedogether.

The other component in the hybridization procedure is therray itself, containing sequences of DNA representing the genesf the organism under study. The arrays may be specially pre-
ared siliconized glass slides, or nylon membranes, which arempregnated with as many DNA “spots” as possible, usually inuplicate. This process is usually done with the aid of a robot.he principles and methods, as well as some pitfalls, have been
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nopharmacology 108 (2006) 2–15 5

escribed in detail in several reviews (Clarke et al., 2001; Nelsont al., 2001).

For most of us, we simply purchase prepared arrays as weeed them, from reliable suppliers (in our case the Prostateesearch Centre in Vancouver). Originally the “DNA” spotsere largely incompletely characterized pieces of cloned DNAs,

DNAs, or ESTs (expressed sequence tags), representing knownr unknown genes. Currently it is possible to obtain arraysomprising synthetic oligonucleotides representing well char-ctierized genes. These have the advantage that all the “oligos”n the spots are the same size, which should ensure a uniformybridization process.

.4. Analysis and interpretation

The arrays, following the hybridization and washing proto-ols, need to be scanned in an optical scanner, in order to quantifyhe intensities of each DNA spot using specific image analysisoftware such as the IMAGENE Version 6.0. This program canuantify the intensities of all the spots, subtract appropriate back-rounds, establish mean intensities for duplicate spots, allowor extraneous errors, such as those caused by minute piecesf dust or particles on the slides, and make corrections for dif-erent labelling intensities in different regions of a slide causedy non-uniform hybridisation, etc. There are numerous otheroftware programs, which can be incorporated to include sta-istical calculations, and comparisons between replicate experi-

ents (we used the GeneSpring program from Silicone Genet-cs, now Agilent Technologies). All of this can be includedithin the general category of bio-informatics, which has nowecome a vital discipline in itself for the purpose of interpre-ation of masses of related bio-data. The expressed genes cane analyzed for the establishment of interconnections betweenhe different genes by using the program Ingenuity Pathwaynalysis.Human input is still required however in order to establish

tatistical requirements and limitations, and to determine exactlyow to make the comparisons between controls and treatedamples. For example, it was customary in the early studies toimply calculate ratios of intensity between control and treatedor each gene on the array, and this is in fact what was pre-ented in most of the published studies to be reviewed in thisrticle. It was generally assumed that this was a reliable way toompare the populations. But we are faced with a decision aso what constitutes a significant change. Should we accept anyatio of 2.0 or greater as a real change? Or could we even thinkf going below 2.0, as some early investigators did? Maybee should set the limit to greater than 5.0. We really do notnow since it is quite likely that some genes may change theirxpression rates by only 1.5 while others may change by 20-foldr more.

Of course we must allow for significant decreases as wells increases. This is particularly pertinent when we realize that
e are examining all the genes on the array, perhaps as many
s 30,000, and for most herbal medicines we have previouslynly really thought about a few genes or metabolic pathways,ince we have been constricted by our reductionist concepts.

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In addition, since most herbal medicines are mixtures of bio-active compounds, each with its own potential effect on genesand pathways, as well as possible synergistic and antagonisticeffects, there could be numerous interactions at the gene level.

What is really happening is that we are finding our waythrough this conceptual maze, and beginning to realize that,in response to chemical mixtures and infectious organisms forexample, the genome and its transcriptome (transcription pro-file) is remarkably flexible. Nevertheless, optimism for mean-ingful interpretation is supported by recent studies in cancerresearch (Raetz et al., 2006) and analysis of the endotoxin-induced inflammatory response in humans (Calvano et al.,2005).

Reproducibility of the results is also essential, although therehas been no consensus on what constitutes a reproducible result.This is related to the issue of reproducible experimental con-ditions. The entire array procedure may be valid for a givenset of circumstances in which a cell line or animal has beenexposed to a given herbal preparation under specific conditions.Consequently the hybridization and analytical processes can berepeated on the same preparations as many times as we wish. Butif the whole experiment is repeated (as it should be of course)can we be sure that the experimental conditions are preciselyreproduced? This is doubtful, mainly because cell cultures andanimals vary from one occasion to another and consequently itis likely that some of the genes affected will tend to show some-what different quantitative (and perhaps qualitative) changes ondifferent occasions. To overcome this “problem” some investi-gators average out the results between two or three experiments(with the help of software programs). This will probably leadto reinforcement of the results for a number of changes in geneexpression, but may miss out on other significant changes thatmight not always be observed. Ideally, one would use a com-bination of technical replicates, in which three experiments aremade with the same RNA, and biological replicates in which theanalyses are made with RNA extracted from three independentexperiments

Recently, more innovative programs have become avail-able, such as the Ingenuity Pathway Analysis Program, whichallows the investigator to apply sophisticated statistical analy-sis together with network linking to related genes involved incommon pathways.

3. Studies involving herbal medicines

For this review we have selected a total of 18 studies (pub-lished in the period 2001–2005) in which some form of herbalmedicine has been evaluated in cell cultures or in animals, bymeans of DNA arrays. There have been important studies ongene-expression analysis, in response to herbal preparations,which involved examination of selected genes, but the presentreview is restricted to the use of gene arrays containing thou-
sands of genes. Many of the array studies utilized Affymetrixor other commercial form of array, while the others (includingours’) employed custom prepared micro-arrays on glass slides,with a wide variety of genes represented.
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nopharmacology 108 (2006) 2–15

. Animal studies

.1. Gingko biloba

Leaf extracts of Gingko biloba have been advocated for manyears as dietary supplements to ameliorate symptoms associatedith various brain and circulatory disorders, including mem-ry problems, and are currently among the most popular itemsold in health food stores. Some specific bioactive componentsave been examined, such as the gingkolides and flavonoids,lthough it has often been assumed that the whole leaf extracts more useful because of synergistic actions of many bioactiveonstituents.

Watanabe et al. (2001) decided to examine the effect ofingko biloba leaf extract (a standardized commercial extract,Gb 761, which has been used in clinical trials) for neuro-odulatory effects on gene expression in mouse cortex and hip-

ocampus. Mice were maintained for a month on a low flavonoidiet, or one supplemented with Gingko biloba extract, 300 mg/kgf food pellets. They were then sacrificed, and the cortex andippocampi separately removed and frozen. RNA was extractedrom pooled tissues by standard “Trizol” methodology, ampli-ed and converted into biotinylated probes for hybridization

o Affymetrix chips, containing oligonucleotides representing000 discrete mouse genes plus 6000 ESTs (expressed sequenceags).

They presumably used standard Affymetrix array analysisnd interpretation equipment and software, and they recordedeproducible changes of a least two-fold between control andreated tissues. Forty-three genes were significantly affected inhe cortex and 13 in the hippocampus (<1%), although only fourere activated in both tissues (Gohil, 2002). Various functionalroups were represented, including growth factors, transcriptionactors and signaling pathway components, and a few specificon channels and cytoskeletal proteins. Some of the more con-picuous changes were verified by RT-PCR measurements onhe same RNA preparations. The most impressive change wasn hippocampal transthyretin (increased 16-fold), which is con-idered to play an important function in hormone transport inhe brain, and possibly in amyloid protein production.

Obviously there are numerous limitations in the model used,uch as the use of pooled heterogeneous tissues, and particularlyhe choice of a single time point during the 1-month adminis-ration of extract. However a more detailed analysis of genehanges over time would require a lot more work, in addition tohe substantial expense incurred in using numerous Affymetrixhips.

Gohil (2002) subsequently described results from a studyf the effect of the same Gingko biloba extract on culturesf human bladder carcinoma cells, the line T24. Following a2 h incubation with extract, treated and control cells were ana-yzed for changes in gene expression by means of a 2000 genehip. Presumably the protocols were the same as in the previ-
us report (described above). One hundred and fifty-five genesere affected by two-fold or greater (approximately 8% of totalenes tested), mostly increases, and these represented a varietyf functional groups, including some that could be important

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n anti-oxidant defences. These could be significant to the longerm human consumption of the herb.

.2. St. John’s wort (Hypericum perforatum, SJW) andypericin

SJW is one of the most popular herbal medicines in Europend North America. Its principal application is for people whouffer from seasonal mood disorders and other types of depres-ion. Clinical studies have been conducted, with both posi-ive and negative outcomes. As is the case for most herbal

edicines, the nature of the SJW preparation has probably con-ributed significantly to its apparent success or lack of. Alcoholicxtracts/tinctures, and water-based teas, have been promoted asnti-stress and relaxation drinks.

Among the many bio-active ingredients, hypericin and itselated analogues, and hyperforin, have been incriminated in itseneficial actions (Agostinis, 2002), although extracts usuallyontain many other phenolic compounds, and the compositionaries between different Hypericum species (Rabanal et al.,005). Some of the bio-active compounds, such as hypericin andts anlogues, are also photosensitizers (Towers et al., 1997; Xie etl., 2001), a fact which has not always been taken into account inaboratory studies. Hypericin itself, and some extracts, possessntiviral and antibiotic activities, which might have accountedor some of the uses of this plant in Ayurvedic medicine (Hudsonnd Towers, 1999).

A potential drawback in the use of SJW has been popularisedecently, namely the fact that its interaction with CYP’s couldead to interference with the metablolism of drugs that are con-umed simultaneously (Wang et al., 2000; Schultz, 2001). Suchlaims have led to a negative press on SJW, although one couldurn the argument around and suggest that people taking SJW orypericin should avoid certain synthetic drugs that require CYPetabolism.Wong et al. (2004) used DNA arrays to study the effects of

JW extract, in comparison with the synthetic anti-depressantrug imipramine, on gene expression in the hypothalamus ofats. Hypothalamus was chosen because of its known importancen mood altering functions, and it was suggested that the SJWnd imipramine could be expected to share some common path-ays leading to their similar beneficial effects. Previous studiesad indicated that anti-stress effects in rats required a period ofeveral weeks for the test materials to show their effects. Accord-ngly, following administration of a partly characterized SJW (byaily gavage) or imipramine (daily intra-peritoneal injection),nd appropriate controls, rats were sacrificed after 8 weeks andheir hypothalami frozen. Subsequently RNAs were extractedrom these tissues (Trizol followed by “RNeasy”), one hypotha-amus for each array, and the RNAs reverse-transcribed and usedo prepare biotylinated RNA for use with Affymetrix chips.

The arrays contained 8799 rat genes plus EST’s. Followingybridization and processing, the chips were scanned and ana-
yzed by means of Affymetrix software.
The results were disappointing: SJW differentially regulatedtotal of only 66 genes/EST’s, representing <1%, in compar-

son to imipramine, which affected 74 genes/EST’s. A variety

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f different pathways were represented by these genes, but theurprising finding was that only six genes were common to thereatments, in spite of the similar physiological effects on thenimals. In addition to these small numbers, the actual magni-ude of the changes were generally small, less than two-fold,lthough they were claimed to be significant.

These relatively minor effects could have been due to the usef heterogeneous tissues (unavoidable in animal experiments) inhich real significant effects in certain cell types could have beenasked by non-responding (or differently-responding) cells. It

s also conceivable that the animals may have adapted theiretabolism during 8 weeks of daily exposure to the test mate-

ials. Another drawback was the use of a pure compound toompare with a crude extract, since the latter may have inducedifferent responses from different ingredients.

It would be useful to examine the effects of SJW/hypericinn appropriate cell lines, where one could anticipate a more sig-ificant response, although the choice of appropriate cell type isbviously open to discussion.

.3. Cannabinoids

The subject of cannabis, and its well known major con-tituents the cannabinoids, need no introduction. According tohe myriad of anecdotal observations and studies, based onoth traditional and modern experiencies, various preparationserived from the plant Cannabis sativa (Fam. Cannabaceae) aresed primarily for two purposes, the psycho-active one, due prin-ipally to delta-9-THC (tetrahydrocannabinol), and with whiche personally cannot profess to be experts, and the applicationsf so-called “medical marijuana”, which has been claimed toossess benefits to many patients suffering from different kindsf chronic disorders, such as cancer, AIDS, pain, multiple scle-osis, epilepsy, and others (Grinspoon and Balakar, 1997). Ineneral the psycho-active preparations are rich in delta-9-THC,hereas the medically applied preparations are low in THC but

ontain substantial quantities of cannabindiol and cannabinol,lus other cannabinoids. However both types of preparation alsoontain numerous other phytochemicals, some of which prob-bly contribute to the overall bioactivities (Turner et al., 1980;lein et al., 1998; Zurier, 2003).Studies performed to date with pure compounds have

evealed the presence of receptors, CB1, CB2, and likely addi-ional ones, that bind different cannabinoids to different degrees,nd thereby activate various signaling pathways, with a varietyf gene expression changes, depending upon the nature of theannabinoid and the cell type (Klein et al., 1998; Zurier, 2003).t has also become clear that there are several endogenous com-ounds, chemically unrelated to the cannabinoids, which sharehese receptors and which can mimic some of the bioactivitiesf the cannabinoids.

Since the cannabinoids, even individual compounds, clearlyossess multifunctional attributes, then a DNA micro-array anal-
sis might prove fruitful. Kittler et al. (2000) and Parmentier-atteur et al. (2002) attempted this in rats and mice, respectively.t the outset there are potential problems associated with such an
pproach. First of all is the obvious one that mice and rats are not

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ecessarily good models for humans, and we really cannot say ifnimals respond to THC in the same way that humans do; evenndividual humans differ widely in their responses (Grinspoonnd Balakar, 1997). Secondly there is the problem of tissueeterogeneity, as mentioned already; consequently if there isspecific target site (e.g. cell type) in the brain for a compound

uch as THC, then its effects would be diluted by inclusion ofnaffected tissues/cells. Furthermore different cell types mightespond in opposite ways to the compound, and analysis of thehole tissue could not reveal this. On the positive side, the studyas at least in vivo, which should make it theoretically more rel-

vant, although the method of administration of THC in animaltudies is unlikely to reflect human usage; we are not aware ofny reports of intra-peritoneal inoculations of cannabinoids orxtracts in humans.

Kittler et al. used rats, which were given daily intraperitonealnjections of delta-9-THC. Animals were sacrificed after 1, 7,r 21 days, and hypothalami were removed and frozen at −80 Cor subsequent RNA extractions. Commercial kits were usedo extract and purify the mRNAs, which were then reverse-ranscribed to yield 32P-labelled cDNAs. They produced rat generrays in - house from a library of more than 24,000 clonedDNAs, and spotted these on two sets of nylon membranes.ollowing standard hybridization procedures, they analyzed theembranes by means of scanning and software programs. Aboutquarter of the cloned cDNAs hybridized to test RNA, but only8 of them (<1%) showed alterations in response to delta-9-HC. However the significance of these changes is open touestion since most of them were less than two-fold and theuthors did not indicate if they made allowance for differential2P-labelling between preparations.

Parmentier-Batteur et al. (2002) made their analysis in therain tissues of THC treated mice. They administered delta-9-HC, or the DMSO vehicle alone, or in some cases a syntheticgonist, intraperitoneally in mice, and after 12 h killed the mice,emoved brains and extracted RNA. The cDNAs were labelledith Cy3 or Cy5, for controls and test compound respectively,

nd hybridized to slides containing 11,200 ESTs derived frommouse brain library. The data were scanned and analyzed by

ommercial software to give ratios of Cy3:Cy5. Averages wereaken from three separate experiments, which in retrospect mighte considered dubious, although to qualify for acceptance theatios had to exceed 1.8 in all three experiments.

A total of 86 genes (<1%) were affected by one or both agents,ostly decreases by two to three-fold, but only 20 genes in

ommon. A variety of functionally different genes were affected,lthough they pointed out that their results were not comparableo those of Kittler et al. (above). This might be explicable byhe use of different animal species and experimental protocols,ut it could just as easily reflect differences in the array systemssed and their processing and analyses.

Parmentier-Batteur et al., also mentioned that their data wereot comparable to those of Derocq et al. (2000), who had previ-
usly studied the effect of a cannabinoid CB2 receptor agonistn a human promyelocytic cell line, HL-60. This cell line haseen the object of many investigations in connection with cellifferentiation and signaling pathways in the immune system. It
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as the theoretical advantage of a “homogeneous” cell culture,n which significant changes in gene expression should be morebvious than in animal tissue models. Nevertheless this apparenthomogeneity” could be less real in a cell line that undergoesontinuous differentiation in culture. In other words, its attrac-ion as a model of differentiation mitigates against its value inhe analysis of gene expression changes induced by an agent.

Derocq et al., amplified the likelihood of seeing receptor-ediated changes by transfecting multiple copies of the receptor

ene into these cells. They then treated the transfected cellsith a synthetic cannabinoid agonist (CP 55,940), or synthetic

ntagonist (SR 144,528), for various times. RNA was extractedrom these different cell populations, converted into biotinylatedRNAs and used with standard Affymetrix DNA arrays (huGeneL chips containing 6800 genes). These were processed and ana-

yzed by means of the usual Affymetrix prescribed equipmentnd software.

Ten genes (�1%) were concluded to be significantly alteredfold ratios, treated to control, in the range 2.2–4.7), mostlytimulated, following a 1 h exposure to the receptor agonist, andhese represented genes involved in cytokine, transcription, andell cycle processes. The changes were confirmed by combina-ions of Northern blots and ELISAs. The authors were able toelate these changes to a demonstrable translocation of activatedFkB transcription factor, frequently implicated in immune acti-ation and differentiation related events. They suggested that thisnding could reflect an involvement of the receptor in normalifferentiation; but clearly the system bears no relationship to thenimal studies described above. However, considering the abun-ance of genes involved in differentiation processes and immunectivation, it is surprising that so few genes in the HL-60 cellsere altered.

.4. Catalposide (Catalpa ovata), Boswellia serata, andnflammatory bowel disease (IBD)

Extracts of both of these plants have been used traditionallyn Korea and India, respectively, to treat inflammatory con-itions. People with IBD often have raised levels of certainro-inflammatory cytokines, such as IL-6, IL-8, and TNFa, inolonic tissues. This has led to the prospect of therapy by meansf anti-inflammatory compounds and herbals. Kim et al. (2004),sed a combination of cell culture and mouse models to studyhe effect of catalposide, an iridoid isolated from Catalpa ovata,n gene expression. They had previously shown that this iri-oid inhibited the activation of NFkB, and hence the secretionf several cytokines, in a line of mouse macrophage-like cells.

In this study, they incubated the HT-29 human intestinal celline with TNFa (to stimulate pro-inflammatory cytokine secre-ion) ±catalposide (500 ng/mL) for 16 h, extracted RNA, andonverted the RNAs into Cy3 and Cy5 labelled cDNAs, byeans of a commercial kit. These were hybridized to cDNAs oncommercial human 8000 cDNA chip, and the results were ana-

yzed by means of standard imaging and processing programs.he data were not presented, although the authors stated that aumber of cytokine and chemokine-related genes were down-egulated, as expected in view of the anti-inflammatory effects.

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n some instances individual transcripts or proteins were mea-ured, for confirmation, by means of various Northern blots,estern blots and histochemical staining procedures.The chemically induced mouse IBD model was also ana-

yzed, by a similar combination of techniques. Mice receivedwo rectal doses of trinitrobenzene sulfonic acid (TNBS), withr without intra-colonic catalposide given before and after theNBS. However it appears that DNA micro-array studies wereot done on these tissues. The authors stressed the inhibition ofctivated NFkB as the focus of the anti-inflammatory effect ofatalposide.

The Boswellia serrata tree gives rise to a resinous gum, whichas been used traditionally as an anti-inflammatory agent inyurvedic medicine. Among the potential active ingredients isclass of terpenoids called boswellic acids, which have them-

elves been shown to possess anti-inflammatory activity in cellulture and animal models.

In the study reported by Kiela et al. (2005), colitis wasnduced in mice by either dextran sulfate in drinking water orrinitrobenzene sulfonic acid administered rectally. Two differ-nt kinds of Boswellia extract were tested for protective effects,hexane extract and a methanol extract. But no protective effectsere observed. Liver RNA was isolated from the various treatedice and converted into biotin-labelled cRNA for hybridization

o Affymetrix mouse array chips, containing 13,672 genes. Theyearched for genes repoducibly affected by three-fold or more. Aotal of 58 genes (<1%) were modulated only by the hexane frac-ion, 20 genes only by the methanol fraction; and in addition 24ere common to both. The differences could be anticipated since

he fractions were chemically distinct and probably containedvariety of different bioactive compounds.Among the genes

ffected were several CYP genes and glutathione S-transferase,uggestive of hepatotoxic rections, and this conclusion accordedith the histologic findings, i.e., rather than protection, the

xtracts were in fact hepatotoxic.In the human intestinal cell line CaCo2, the six individual

oswellic acids either inhibited experimentally induced NFkBevels, or had no effect on them. Perhaps the boswellic acidsperate through the mediation of alternative transcription factorsnd pathways. It is unfortunate that investigators in general haveecome pre-occupied with explaining inflammatory responsesy means of effects solely on NFkB.

.5. Herbal glycosides

Various recipes of herbal glycosides, including baicalein andioscin, have been used for a long time in Chinese medicine tonhance memory problems in stroke victims. Wang et al. (2004)sed a mouse model of surgically induced cerebral ischemia tonvestigate the spatial memory abilities of mice with and withoutlycoside treatment, which was given regularly before and afterurgery until the animals were sacrificed. Their hippocampi wererozen, and subsequently used for the extraction and purification
f RNAs. 32P-labelled cDNAs were produced and hybridizedo arrays of 1176 mouse genes in the Clontech Atlas sys-em. About half of the array genes reacted with transcripts inhe different RNA preparations, and of these 33–46 (approx-
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mately 8%) showed significant (>1.8-fold change) increasesr decreases compared with sham or vehicle-treated animals.owever, although these gene changes correlated with the per-

ormance of the animals in spatial memory tests, there was littleverlap among the nature of the genes affected by three differ-nt doses of the glycoside formulations, which makes completenterpretation of the data difficult.

. Cell culture studies

.1. Genistein-treated prostate carcinoma cells (Li andarkar, 2002)

Genistein, a major isoflavone isolated from soybeans, waslready known to inhibit the growth of cultured PC3 prostate car-inoma cells (human origin), and to inhibit NFkB and AKT sig-aling pathways. The investigators wanted to determine whichenes were involved in this process. They therefore treated theells with 50 umol/L of genistein for 6, 36, or 72 h (to examinearly and delayed responding genes, an important parameter toonsider), extracted RNA by means of standard commercial kitsnd prepared the labelled probes according to the Affymetrixrotocols, followed by hybridization to chips containing morehan 12,500 human genes (cDNAs). Results were obtained bysing the prescribed software and tools. Unfortunately it is notlear if the relative cell numbers of treated and untreated culturesere taken into account; presumably they were.They found what they considered to be significant alterations,

.e. a change by two-fold or more, in 832 genes (about 6.7%), ofhich 774 were decreased and 58 were increased. The major-

ty of these changes were observed at the 6 h time point, withurther decreases noticeable at the later times. Alterations inranscription levels were confirmed in 26 cases by means ofT-PCR measurements. A large number of the genes affected

nvolved players in signaling pathways, transcription factors,rotein kinases, apoptosis, and cell cycle functions, which is noturprising in view of the original rationale of the study.

This study is interesting because of the large number of genesffected, in contrast to some of the others described below, andlso this represents the activities of a single component of soy-ean extracts. It would be interesting to learn whether thesectivities are modulated by other components of soybean. Thiss clearly relevant to prospective applications of such extracts,r genistein itself, in the treatment of prostate and other cancers.

.2. PC-SPES and prostate carcinoma (Bonham et al.,002a,b)

This crude mix of eight different herbs has been advocated forome years in the prevention and treatment of cancer, especiallyor prostate carcinoma. The usual formulation comprises Scu-allaria baicalensis, Glycyrrhiza glabra, Ganoderma lucidum,satis indigotica, Panax pseudo-ginseng, Dendranthema mori-
olium, Rabdosia rebescens, and Serenoa repens. Needless toay, many members of the medical and scientific establishmentsre suspicious of such a “witches’ brew”, although many ofhese individual extracts are known to contain bioactive ingredi-

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nts, and in fact some trials have shown decreases in blood PSAprostate-specific antigen) levels over time with daily consump-ion of the mixture.

It would seem to be an ambitious task to analyze geneesponses to such a mixture of uncharacterized phytochemicals;evertheless this group has attempted to do this, and the data arepread over the two manuscripts cited. Of the three prostate car-inoma cell lines available to them, PC-3, DU-145 and LNCaP,he latter was selected for the gene array analysis. They usedlass micro-arrays spotted with 3000 cDNAs obtained from theiribrary of prostate cDNA clones. They compared each RNAreparation (PC-SPES-treated or solvent control treated) with aeference prostate RNA, one labelled with Cy3 the other withy5. They also did comparisons with the dye labels reversed,ssential to compensate for possible differences in dye-dUTPncorporation rates.

They used their own combination of processing and analyticalools, and searched for genes that were reproducibly increasedr decreased by a factor of 1.5, which is lower than most investi-ators would want to use. The number and intensity of changesncreased with time up to 48 h of treatment, resulting in 144enes increased and 175 genes decreased, approximately 10%f the genes represented. A variety of genes with different func-ions were affected, including numerous cytoskeletal functions,hich they followed up with further studies. Other functions

ncluded apoptosis, stress, cell cycle and proliferation, as wells androgen-regulated genes, all of which could be anticipatedrom the objectives of the study.

.3. Paeoniae radix and apoptosis (Lee et al., 2002)

The root of Paeoniae lactiflora pallas has been used tradition-lly in China to treat liver diseases, and various other disorders.his plant is often found as a constituent of multi-herb formu-

ations, and extracts of PRE (P. radix extract) have been showno induce apoptosis in two human hepatoma cell lines.

In this study Lee et al., prepared a hot water extract of PR andsed the filtered and lyophilized preparation, in 5–10 mg/mLoncentrations, to treat HepG2 cells. RNA was used to makey3 and Cy5 labelled cDNAs, which were hybridized to 374ligomer spots on the Operon human apoptosis array, with thesual collection of control spots. The most remarkable thingbout this study is that only four genes were affected (onencreased, three decreased, a total of ∼1%), even though therray was heavily biased toward apoptosis genes. On this basis,part from the confirmation by other tests that apoptosis wasnduced by such treatment, it would be difficult to concludenything from the array analysis.

.4. Coptidis rhizoma and berberine (Iizuka et al., 2003)

This plant, and one of its major constituents berberine, haseen shown to possess anti-proliferative effects on pancreatic
arcinoma cell lines. The authors attempted to compare ID50 val-es for both extract and berberine against the eight cell lines withorresponding effects on specific gene expression. The extractedNAs (from cells treated with berberine or hot water extract)
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ere purified and processed according to Affymetrix protocols,nd hybridized to 11,000 oligomers on the huU95A chips, alongith various controls.Comparisons between ID50 and a number of specific genes

as made, although it was not really clear what exactly waseing measured in terms of gene expression. Out of 33 genespparently altered (ratios or levels of significant changes wereot given), some correlated with both extract and berberine ID50,ut others only correlated with one or the other ID50. Among theenes allegedly altered were the usual mixture of functionallyiverse entities such as signaling, transcription, DNA repair, cellycle genes.

In a more recent publication (Hara et al., 2005) the same groupefined their analyses, using the original data, by concentratingn 27 specific genes affected by several specific compounds,cluding berberine, isolated from the extract.

.5. Tripterygium alkaloids and apoptosis (Zhuang et al.,004)

Tripterygium hypoglaucum root has been used traditionallyn Chinese medicine to treat a number of inflammatory dis-ases. An alkaloid fraction, prepared by organic solvent extrac-ions, has been shown to cause apoptosis in some culturedells.

In this study, the human promyelocytic leukemia cell line,L-60, was treated for 8 h with 40 ug/mL of the alkaloid frac-

ion, and the RNA extracted and processed to produce Cy3nd Cy5 (control and treated) labelled probes, which wereybridized to 3000 spots derived from a commercial humaneukocyte cDNA library. Unfortunately it was not clear whatontrols were used, whether background values were subtracted,r whether the labels were reversed. However 16 (<1%) genesere reported to show ratios of more than two-fold change, and

hese included apoptosis, signaling pathways, cell cycle and dif-erentiation functions. Thus the array data really did not addo the results obtained by the other techniques described in theeport.

.6. Anoectochilus formosanus and plumbagin (Yang et al.,004)

The anti-cancer extract from Anoectochilus formosanus andhe pure phytochemical plumbagin (a naphthoquinone fromlumbago rosea) were compared with respect to their effectsn gene expression in MCF-7 cells, a line of breast adenocarci-oma origin.

RNA extracted from the various treated and control cellsas converted into biotinylated cDNA probes, which wereybridized to a collection of 9600 genes on nylon membranes.arious equipment and software programs were used in the anal-sis and interpretation of the results.

Data for individual genes were not shown, although the
uthors concluded that 59 genes (only <1%) were significantlyffected (>3-fold change) by the extract, whereas plumbaginodulated 80 genes. It was not clear to what extent the effects
f the two agents had related effects, if at all.

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.7. Propolis and differentiation (Mishima et al., 2005)

Propolis has been advocated for innumerable purposes, inealth and disease. Its major drawback is the very nature of theroduct, which, because of its high variability in composition, islmost impossible to standardize in terms of phytochemistry andioactivities. Consequently a meaningful DNA array analysis ofts effects on gene expression is, to say the least, ambitious.

In this study, the scenario was made even more complex byvaluating two different extracts of propolis in a cell line under-oing differentiation, the promyelocytic leukemia HL-60 cells.he investigators were then faced with the challenge of decid-

ng which genes are affected by the extracts themselves, andhich are affected secondarily as a consequence of the processf differentiation. The results were compared with the effects ofTRA (all-trans retinoic acid), which is known to induce differ-ntiation in these cells.

The RNAs extracted from the various treated cells wereonverted to Cy3 and Cy5 labelled cDNAs and hybridized toceGene Human oligo chips, containing 10,000 genes (all in the

orm of 50-mers). Appropriate controls and dye-reversal reac-ions were carried out. One hundred and eighteen genes (∼1%)ere affected >2-fold by ATRA (all-trans retinoic acid), 79 by

queous extract of propolis, and only 6 by an ethanolic extract. Ineneral there were more decreases than increases, which mighte expected in the face of a differentiation process. Howeverorrelations were difficult because of the different degrees ofifferentiation (and possibly the type of differentiation?) showny the different agents.

. Echinacea and gene array analysis: our experienceith a system in evolution

Different species and parts of Echinacea (Asteraceae) haveeen used traditionally in North America for the treatment ofarious symptoms of “colds” and “flu”, as well as other skinpplications such as wound healing (Barrett, 2003). The taxon-my of the genus has been revised recently, based on new dataerived from genetic and phytochemical profiling (Binns et al.,002a). A number of well known marker compounds have beenharacterized, including polysaccharides, specific caffeic aciderivatives and alkylamides (Bauer, 1998; Binns et al., 2002b),nd these have all demonstrated biological activities in variousioassay tests, such as immune-modulating activities, anti-viralctivities, anti-fungal activities (Bauer, 1998; Rininger et al.,000; Goel et al., 2002; Merali et al., 2003; Randolph et al.,003; Brovelli et al., 2005; Hudson et al., 2005; Vimalanathant al., 2005; Sharma et al., 2006). Some of these activities haveeen attributed to photosensitizers, which are common amongembers of the Asteraceae, a fact that could explain some of the

fficacy of extracts in skin applications (Towers et al., 1997).Many clinical trials have been conducted, in individuals suf-

ering from natural or experimentally induced rhinovirus infec-
ions, but with variable results (Barrett et al., 2003; Turner et al.,005), many of which were undoubtedly due to the unfortunatese of source materials that had not been adequately charac-erized. Thus the question of clinical efficacy remains unre-
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olved. Meanwhile additional studies have been conducted withhe objectives of identifying the principal immuno-modulatingompounds and anti-viral compounds, and their mechanisms ofction (Hudson et al., 2005; Vimalanathan et al., 2005; Sharmat al., 2006).

There is also controversy about the timing of Echinacea con-umption, in relation to cold or ‘flu symptoms, and what kind oformulation should be consumed. Thus, should it be consumedt the “first sign of a cold”, or as a preventative measure? Shouldne take an ethanolic tincture/extract, or a water based formula-ion? These same issues are of course relevant to any “anti-cold”erbal or drug preparation, since, contrary to commercial adver-ising claims, we have a priori no idea how these formulationsre supposed to work.

In order to shed light on these problems, while at the sameime testing Echinacea as a model for evaluating the novel DNArray approach, we decided to analyze host gene expression in aine of human bronchial epithelial cells (BEAS-2B), infectedith rhinovirus type 14, or uninfected, and with or withoutne of two characterized preparations of Echinacea purpureaEchinacea purpurea (L.) Moench (Heliantheae: Asteraceae)).hinovirus was chosen because of its frequent association witholds and other upper respiratory infections, and because it hasften been implicated in exacerbation of asthmatic and aller-ic symptoms (Message and Johnston, 2001; Gwaltney, 2002;essage and Johnston, 2004; Johnston, 2005). The Echinacea

reparations used were an aqueous extract of mixed Echi-acea purpurea aerial plant parts, enriched in polysaccharideslabelled E1), and a 55% ethanol extract of the roots of Echi-acea purpurea (E2), which contained alkylamides and variousaffeic acid derivatives. Both preparations have been showno have pronounced effects on cytokine and chemokine secre-ion in uninfected and rhinovirus-infected cells (Sharma et al.,006).

Previous studies had shown that rhinovirus infection in cul-ured epithelial cells, and in nasal epithelial tissues in vivo,esulted in low levels of virus replication and cytopathology, butubstantial induction of secretion of certain pro-inflammatoryytokines and chemokines, including IL-6, IL-8, and TNFareviewed in Message and Johnston, 2004; Sharma et al., 2006).hus the typical symptoms of a common cold, such as sneezing,oughing, runny nose, stuffed nasal passages, etc., are not theirect result of viral pathology, but rather the indirect stimulationf pro-inflammatory cytokines and chemokines.

We extracted RNA from the six different types of cul-ure (uninfected cells with or without extract E1 or E2, andhinovirus-infected cells with or without E1 or E2), reverseranscribed the RNAs into Cy3 or Cy5 labelled cDNAs, andybridized them with arrays containing a total of 13,500 humanene-specific oligomers.

We made use of a commercially available Universal Ref-rence RNA, prepared from a collection of 10 different humanumor cell lines with various transcription programs. The advan-
age of this commercial product is that it is available readily tonvestigators and is a consistent product. We compared eachabelled test preparation with the reference preparation, as indi-ated schematically in Fig. 2.

12 J. Hudson, M. Altamirano / Journal of Ethnopharmacology 108 (2006) 2–15

Fig. 3. Gene tree showing gene expression patterns according to treatment. The six panels from left to right represent respectively, control untreated cells (C), cellst 2 (C( o relao ve the

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The arrays were scanned after hybridization by means of thecanArray Analyzer (Perkin-Elmer), and image intensities andackground correction were quantitated using the computer pro-ram Imagene 6. We selected only the experiments in which theatio of signal to noise was higher than 3.0 in at least 40% of theotal number of spots.

Gene expression was evaluated with the aid of Gene Spring.2 Program. Every spot intensity in the micro-array from thexperimental sample was compared against the intensity of theniversal control reference (two color experiments) in order tobtain a signal ratio as working value. Normalization was usedo adjust the individual hybridization intensities to balance themppropriately so that meaningful biological comparisons can beade. There are a number of reasons why data must be normal-

zed, including unequal quantities of starting RNA, differencesn labeling or detection efficiencies between the fluorescentyes used, and systematic biases in the measured expressionevels.

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maximum expression observed and represent genes of the interferon family.

Normalized data were subjected to statistical analysis of vari-nce (ANOVA), which statistically compares mean expressionevels between two or more groups of samples. The object waso find the set of genes for which the specified comparisonhowed statistically significant differences in the mean normal-zed expression levels. Clustering algorithms were used to divideenes into groups with similar expression patterns.

Additional processing of the data (details to be published)esulted in a table of ratios expressing differential expressionetween any two treatments and controls for each individualene. We constructed these so as to show the effects of eachxtract (E1, E2) on control uninfected cells, the effects of rhi-ovirus infection on the cells, and the modulating effects of E1nd E2 on the infected cells. Examples are shown for illustra-
ion in Fig. 3 (which represents a tree showing related genes) andig. 4, which shows the effects of infection and treatment on onepecific interferon-induced gene, the Mx1 gene, along with cor-esponding Northern blots. The stimulatory effects of the virus,

J. Hudson, M. Altamirano / Journal of Ethnopharmacology 108 (2006) 2–15 13

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ig. 4. Expression levels of myxovirus resistance gene (Mx1), an interferon-indevels are reflected in the corresponding Northern blots. C = control, uninfectedxtract E2; RV = virus-infected cells, no extract; RVE1 = infected cells treated w

nd the modulatory effect of both Echinacea extracts, are clearlyvident in these figures. Similar charts can be prepared for anyther specific gene on the array.

Analysis by means of the Ingenuity Pathway Network per-its the clustering of functionally related genes, as illustrated

n Fig. 3. This procedure has emphasized the importance ofmmune function genes in responses to the virus, and has alsoevealed other groups of genes modulated in uninfected cells byither one or both Echinacea extracts.

. Discussion

What have we learned from our analysis of the individualtudies described? It is not possible to draw general conclu-ions because the plant sources and the methods used, includinghe array techniques, differed so much between the studies. In

any cases a relatively small number of genes appeared to beffected by treatment. This is not surprising in the animal studies,n which heterogeneous whole tissues were compared betweenreated and control animals. Therefore important and dramatichanges in specific cell types could have been obscured. But inome of the cell culture studies also only small numbers of genesere affected, and in these cases the magnitude of the changesas often unimpressive. In contrast, in our Echinacea studies,e observed significant changes in hundreds of genes as a con-

equence of rhinovirus infection, in line with other DNA arraytudies showing the effects of viruses on cell gene expressionGeiss et al., 2001; Tian et al., 2002). Furthermore, the Echi-acea preparations modulated these virus-induced changes forany genes, and in addition showed equally impressive changes

n uninfected cells.
These latter observations accord with the general concept of
he cell transcriptome being a very flexible entity, responsiveo numerous challenges from the micro-environment. It wasemonstrated in the pioneering studies on DNA micro-arrays

tvht

gene (one of the red genes in Fig. 3, RV: infected cells). The relative expressionE1 = uninfected cells treated with extract E1; E2 = uninfected cells treated withtract E1; RVE2 = infected cells treated with extract E2.

hat a relatively simple manipulation of cultured cells (mam-alian or yeast) such as addition of fresh serum and medium

ould result in profound changes in the transcription profile,oth qualitatively and quantitativley (Iyer et al., 1999). This isbviously relevant to the use of herbal medicines, since suchixtures comprise numerous potentially bio-active molecules,

o all of which individual cells of superficial tissues might bexposed and hence modulated. Thus an apparently simple exer-ise of consuming orally an alcoholic tincture of Echinaceaould likely result in mucosal epithelial cells being exposed to ayriad of compounds, such as polysaccharides, caffeic acids andavonoids and many other phenolic compounds, alkylamides,olyacetylenes (Bauer, 1998; Binns et al., 2002b), and many ofhese could interact with numerous receptors on the cell surfaces,nd thereby activate various signaling pathways, transcriptionactors and eventually changes in specific gene transcription.here could also be more immediate effects on various path-ays not involving transcription. In addition one cannot ignore

he potential bioactive contribution of the ethanol, which is fre-uently present in concentrations of up to 50–60%.

The point of this argument is to illustrate the potential com-lexity of such interactions. Yet at the same time, if we anticipatepecificity in the actions of different herbal preparations, then weave to presume that consumption of a different herbal mixture,.g. St. John’s wort or a ginseng formulation for example, wouldroduce equally complex but different interactions because thehemical composition of the preparation would be substantiallyifferent from Echinacea. Some common chemicals would beresent, and this could account for a certain amount of overlap inossible activities, such that several apparently unrelated herbsould help alleviate some common symptoms, but the total pic-
ure, i.e. the resulting transciption profile, would be different. Iniew of this we are surprised that some of the studies reportedere observed so few changes. What could be the explanation forhis apparent disparity? We have no answer, other than the obvi-

14 J. Hudson, M. Altamirano / Journal of Eth

ous differences in technical details due to the use of differentarray analyses. We may find that the newer and more sophis-ticated bio-informatic analyses that are in development couldovercome these limitations.

Of course changes in transcription do not necessarily cor-relate with changes in corresponding proteins. Transcriptionis normally followed by many translation and post-translationsteps (Shay and Banz, 2005), and some of these can act as regula-tors. The science of proteomics is rapidly approaching the samelevel of analytical capability as genomics, and soon we will beable to correlate transcription changes with protein changes fora given herbal preparation.

In regard to the type of changes observed, there is moreagreement between the studies. In many cases, alterations intranscription factors, signaling pathway components, apoptosisfactors, cell cycle genes, and cyto-skeletal products, were seen,not surprisingly, but nevertheless clear indications of profoundchanges in the cells.

Is it really necessary for Readers of this Journal to learn aboutthis technique? We would say yes, if only because more inves-tigators are likely to attempt the kinds of analyses that we havedescibed. Experts in Ethnopharmacology will have to examinethe results of such studies in order to assess their relevance andimpact. We may conclude in another decade or so that all theseendeavours will have been a complete waste of time and money;alternatively, we might be on the verge of a real breakthrough inunravelling the detailed mechanisms of action of specific herbalmedicines.

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lossary

rray (chip, DNA array, gene array): a solid surface, usually a glass slide ora membrane, on which many spots of DNA or oligonucleotide have beenembedded and fixed

DNA, cRNA: a sequence of DNA or RNA complementary to a section of a geneunder study

y3, Cy5: nucleotides labelled with either green (Cy3) or red (Cy5) cyanin dyeswhich, when incorporated into DNA, will label that DNA

ST: expressed sequence tag. A cloned sequence of DNA derived from RNAof an unknown gene. No longer very popular since the advent of knownsequences

ybridization: the process of interaction between complementary pieces ofDNA or RNA.

L-6: interleukin-6, a cytokineL-8: interleukin-8, a chemokine

acroarray: an array containing relatively few spots of DNA on a membraneicro-array: an array containing numerous spots of DNA or oligonucleotides,

usually on a glass slide.ligonucleotide: a sequence of nucleotides, usually fewer than 100.robe: the cDNA or cRNA used to detect and measure a complementary

sequence in DNA or RNA preparations.
NFa: tumor necrosis factor alpha, a cytokineniversal reference RNA: a mixed collection of mRNA molecules derived from
numerous cell lines of one species; used as a standard in array analyses tocompare with test samples of RNA

The application of DNA micro-arrays (gene arrays) to the study of herbal medicines

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