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Phytomedicine 19 (2012) 930939

Contents lists available at SciVerse ScienceDirect

Phytomedicine

journal homepage: www.e lsev ier .de /phymed

Novel neurological and immunological targets for salicylate-based

phytopharmaceuticals and for the anti-depressant imipramine

G. Ulrich-Merzenicha,, O. Kelberb, A. Koptinaa,h, A. Freischmidt c,J. Heilmann c,J. Mllerb, H. Zeitlerd, M.F. Seidel e, M. Ludwig f, E.U. Heinrichb, H. Winterhoffg,1

aMedizinische Poliklinik, Universittsklinikum, Rheinische Friedrich-Wilhelms-UniversittBonn, Germanyb SteigerwaldArzneimittelwerk GmbH, Darmstadt, Germanyc PharmazeutischeBiologie, UniversittRegensburg, Germany

dMedizinischeKlinik I, Universittsklinikum, Rheinische Friedrich-Wilhelms-UniversittBonn, GermanyeMedizinischeKlinik I, RheumatologyUnit, Universittsklinikum, Rheinische Friedrich-Wilhelms-UniversittBonn, GermanyfDepartmentofClinical Chemistry and Clinical Pharmacology, Universittsklinikum, Rheinische Friedrich-WilhelmsUniversittBonn, Germanyg InstitutfrPharmakologie und Toxikologie, WestflischeWilhelms-Universitt,Mnster, GermanyhMari State Technical University, YoshkarOla, Russia

a r t i c l e i n f o

Keywords:

Inflammation

Neurologicaldiseases

Multitargeting

Microarray

Polyphenols

Salicylates

a b s t r a c t

Inflammatory processes are increasingly recognised to contribute to neurological and neuropsychatric

disorders suchas depression. Thuswe investigatedwhethera standardizedwillowbark preparation (WB)

which contains among other constituents salicin, the forerunner ofnon-steroidal antiphlogistic drugs,

would have an effect in a standardmodel ofdepression, the forced swimming test (FST), compared to the

antidepressant imipramine. Studies were accompanied by gene expression analyses. In order to allocate

potential effects to the different constituents ofWB, fractions ofthe extract with different compositions

ofsalicyl alcohol derivative and polyphenols were also investigated.

Male Sprague Dawley rats (n= 12/group) were treated for 14 days (p.o.) with the WB preparationSTW 33-I (group A) and its fractions (FR) (groups FR-B to E) in concentrations of30mg/kg. The FRs were

characterizedby a high content offlavoneand chalconeglycosides (FR-B), flavonoid glycosides and salicyl

alcohol derivatives (FR-C), salicin and related salicyl alcohol derivatives (FR-D) and proanthocyanidines

(FR-E). The tricyclic antidepressant imipramine (20mg/kg) (F) was used as positive control. The FST was

performed on day 15. The cumulative immobility time was significantly (p2 fold,p< 0.01), affected byWB and/or FR-D and imipramine, included both inflammatory (e.g.

IL-3, IL-10) and neurologically relevant targets. Common genes regulated by WB, FR-D and imipramine

were GRIA 2 , SRP54 , CYP26B , DNM1L and KITLG . In addition, the hippocampus ofrats treated

(27d) withWB (1560mg/kgWB) or imipramine (15mg/kg bw) showed a slower serotonin turnover (5-

hydroxyindol acetic acid/serotonin (p< 0.05)) dependingon the dosage. ThusWB(30mg/kg), its ethanolic

fraction rich in salicyl alcohol derivatives (FR-D) (30mg/kg) and imipramine, by being effective in the

FST, modulated known and new targets relevant for neuro- and immunofunctions in rats. These findingscontribute to our understanding ofthe link between inflammation and neurological functions and may

also support the scope for the development ofco-medications from salicylate-containing phytopharma-

ceuticals as multicomponent mixtures with single component synthetic drugs.

2012 Elsevier GmbH. All rights reserved.

Abbreviations: 5-HT, serotonin; 5-HIAA, 5-hydroxyindol acetic acid; AD, Alzheimers disease; AMPA, alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid; ASA,

acetylsalicylic acid; CAT, catalase; CD, Sprague-Dawley; CNS, central nervous system; COX, cyclooxygenases; CRH, corticotrophin-releasinghormone; CSF, cerebral spinal

fluid;CYP26B1,cytochrome P450protein26B1;DNM1L, dynamin likeprotein1; EDNRB, endothelin B receptorgene; ER, endoplasmaticreticulum;EtOH-FR,ethanolfraction;

FR, fraction; FST, Porsolt-Swimming Test; GR, glutathionereductase;GST, glutathioneS-transferase;GTPase, guanosine triphosphatase;HGF, haematopoieticgrowth factor;

HPA-axis, hypothalamic-pituitary-adrenocorticalaxis; MS, multiple sclerosis; NMDA, N-methyl-d-aspartate; RA, retinoic acid; SCF, stem cell factor; SNP, single nucleotide

polymorphism; SNRIs, serotonin and noradrenalin-reuptake inhibitors; SOD, superoxidedismutase; SRP, signal recognition protein; SSRIs, selective serotonin-reuptake

inhibitors;TNFRSF1A, TNF-receptor superfamilymember 1 A; WB,willow bark. Corresponding author. Tel.: +49 22828722674; fax: +49 22828722019.

E-mailaddress:[email protected] (G. Ulrich-Merzenich).1 Deceased.

0944-7113/$ see frontmatter 2012 Elsevier GmbH. All rights reserved.http://dx.doi.org/10.1016/j.phymed.2012.05.004


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G. Ulrich-Merzenich etal./Phytomedicine 19 (2012) 930939 931

Introduction

Willow bark (WB) was already used by Hippocrates as an anti-

inflammatory agent against fever and pain. It contains salicin and

other salicylic acid derivatives which were designated as active

principles since 1831. These compounds are prodrugs which aremetabolised in the gut and the liver via salicylic alcohol to salicylic

acid the active compound. To improve tolerability compared to

isolated salicyclic acid, Felix Hoffmann synthesised acetylsalicylic

acid (ASA) in 1897, today one ofthemostly consumedanalgetic and

anti-inflammatory drugs worldwide.

Salicylic acid is known to inhibit the cyclooxygenases (COX)

1 and 2 (Aronoff et al. 2003). Therefore, COX-inhibition was first

regarded the main mechanism ofthe anti-inflammatory activity of

WB. In themeanwhile it was shown thatWB can alsomodulate rel-

evant pro- and anti-inflammatory cytokines like TNF-, IL-6, IL-1,

IL-10,and IL-8 (Bonaterra etal. 2009;Fiebichetal. 2005; Fiebichand

Chrubasik 2004) and that not only salicyl alcohol derivatives, but

also the polyphenols ofWB contribute to this modulation (Khayyal

et al. 2005; Nahrstedt et al. 2007). In addition, polyphenols are

known to possess antioxidant and neuroprotective effects whichcan also interfere with inflammatory events (Kannappan et al.

2011). Very recently also a contribution of catechol to an antiin-

flammatory effect ofWB was discussed (Freischmidt et al. 2012;

Knuth et al. 2011).

Recent clinical data propose that proinflammatory cytokines

also influence the pathogenesis of depression. It was shown that

the IL-1RA (interleukin-1 receptor antagonist) is increased in the

plasma ofmiddle aged and elderly patients (>65 years) suffering

from depression (Milaneschi et al. 2009; Ovaskainen et al. 2009).

Lindqvist et al. (2009) demonstrated increased IL-6 concentrations

in the CSF ofpatients with depression. A recentmeta-analysis sum-

marising med-line studies on depression between 1967 and 2008

concluded thatmajormarkersofinflammation like CRP, IL-6and IL-

1 are significantly associatedwith depression (Howren et al. 2009).

Even though the causal relationship is presently not clear, the link

between the immune systemvia cytokines and the pathogenesis of

psychiatric disorders led to the understanding that immunomodu-

latory drugsmay also be beneficial for the treatment ofpsychiatric

disorders (Berthold-Losleben et al. 2009).

In addition, it was observed that in clinical studies with wil-

low bark extract the treatment groups had a lower number of

adverse events (AEs) than in the placebo group (e.g. Schmid et al.

2000). Thus, it had been hypothesised by Winterhoff that willow

barkmight have an anxiolytic effect and could thereby be useful in

depression. This was first evidenced by her working group (Hegger

et al. 2005;Winterhoffet al. 2008) using the forced swimming test

(FST) as an animal model ofdepression.

We now queried which molecular targets are affected by WB

compared to the antidepressant imipramine and which compo-

nents oftheWB extract are active, especially considering that plant

constituents have been repeatedly proposed for a possible future

application in co-medications (Wagner 2011).

Materials and methods

Willow bark extract

The driedwillow bark preparation STW33-I (WB) was obtained

from Steigerwald Arzneimittelwerk GmbH, Darmstadt, Germany.

The extract was prepared from willow bark according to PhEur.

6.1, with a DEVnativof1623:1, total salicin content 2326% (m/m)

Europe C (2008). Imipramine hydrochloride was obtained from

Sigma (Deisenhofen, Germany).

Fig. 1. HPLC-fingerprints ofwillow bark (WB) and the different isolated fractions

(FR).

Preparation and characterization ofthe testedfractions

The investigated fractions were prepared as described in

Freischmidt (2011) by application of partition and precipitation

processes with ethyl acetate (Fr-B), butanol (Fr-C), ethanol (Fr-D)and water (FR-E) (Fig. 1).


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932 G. Ulrich-Merzenichetal./Phytomedicine19 (2012) 930939

Table 1

Characterization ofWBE and its fractions (% SD, n= 3).

WB FR-B FR-C FR-D FR-E

A

Total polyphenols 19.7 0.3 41.7 0.1 16.3 0.3 6.7 0.03 4.4 0.02

Tannin polyphenols 6.8 0.2 8.7 0.1 4.3 0.2 2.7 0.01 2.8 0.06Rest phenols 12.9 0.1 33.0 0.1 11.9 0.2 4.0 0.03 1.6 0.07

B

Flavonoids a 8.4 0.2 21.8 0.1 7.3 0.5 0.3 0.02 n.d.b

C

Salicin 12.8 0.1 5.2 0.2 18.2 1.0 19.7 0.3 3.9 0.1

Salicyl alcohol derivatives 23.0 0.8 14.2 0.2 34.7 1.8 27.3 0.9 5.4 0.3

a Determined as naringenin derivatives.b Not detectable by TLC and thus not determined.

As only for Fr-B an exact phytochemical characterization is

described, revealing different flavanone and chalcone glycosides

as well as catechol as most abundant phenols (Freischmidt 2011),

a quantitative determination of different classes of compounds in

the WB and the resulting fractions was done. The salicin and sali-

cyl alcohol content was determined according Ph.Eur 6.4 (Europe C

2008), total polyphenol, tannin and rest phenol content was quanti-

fied according to Glasl (1983). As the flavonoid spectrum ofwillow

bark mainly consists offlavanone and chalcone glycosides the com-

mon PhEur methods for determination ofoverall flavonoid content

is not applicable. Thus, it was determined according a newly devel-

oped method (Freischmidt 2011).

The WB is rich in salicin, salicin derivatives and polyphenols;

the ethyl acetate fraction contains a high amount of polyphenols,

whereas the ethanol fraction contains a high amount ofsalicin and

salicin derivatives while having a comparatively low polyphenol

content (Table 1).

Animals

Male Sprague-Dawley (CD) rats (150170 g, Charles River Labo-ratories, Sulzfeld, Germany) were housed in groups oftwo and kept

in climatised rooms (24 1 C, lightdark cycle 12/12 h). Animals

had free access to food (Altromin 1324, Altromin, Lage, Germany)

and tap water. The procedures used comply with the European

Communitys Council Directive of24 November 1986 (86/609/EEC)

and were officially approved by the local committee on animal care

(Regierungsprsident Mnster, AC/2004).

Testsubstances

Animals (n= 12 per group) received the test solutions (WB, its

fractions, or imipramine, suspended in water, 10 ml/kg bw) p.o.

once daily. The positive control drug imipramine was first dissolved

in 160l ethanol and then diluted with deionised water to a final

volume of 10 ml (Butterweck et al. 2002). As negative control thesolvent was used.

Forced swimmingtest

The forced swimming test (FST) was performed with 72 male

CD-rats according to Porsolt et al. (1977, 1978). The animals (n= 12

per group), received the test solutions (p.o. 30 mg/kg bw/d over

a period of 14 days) of WB (A), its ethyl acetate fraction (FR-

B), its n-butanol fraction (FR-C), its ethanol fraction (FR-D), its

aqueous fraction (E), 30 mg/kg bw/d each, or the positive control

imipramine, 20 mg/kg bw/d. One day before the test, the rats expe-

rienced a 15 min pre-test session, in which the animals were singly

placed in a plexiglas cylinder that contained a 1721 cm (depending

on the body weight) high column ofwater at 25

1

C. Experimen-tal testing was conducted 24 h later by exposing the rats to the

same test conditions for 5 min 12 h after last drug administration.

Behaviour was recorded by video camera. The cumulative time rats

persisted in an immobile position (despair behaviour) was eval-

uated afterwards by a blinded observer. A rat was judged to be

immobile whenever it remained floating in the water in contrast to

motor activities that represented attempts to escape from the sit-

uation. The time rats spent making small movements to keep the

head above the water was assigned to time ofimmobility. A short-

ened immobility time indicates an antidepressant like effect. The

FST was performed between 9.00 a.m. and 1.00 p.m.

Determination ofserotonin and 5-hydroxyindol aceticacid in the

hippocampus

CD-rats (n = 12 per group) received WB (15 mg, 30 mg, 60 mg and

125 mg/kg bw/d) or imipramine (15 mg/kg bw/d) over a period of

27 days. Animals were sacrificed by decapitation between 9.00 a.m.

and 11.00 a.m. at the day after the last drug administration. Sero-

tonin (5-hydroxytryptamine, 5-HT) and 5-hydroxyindol acetic acid

(5-HIAA) concentrations were estimated in the hippocampus of72male CD-rats as described earlier (Butterweck et al. 2002).

Statistics

For the statistical analysis of the results of the FST and the

serotonin and 5-HIAA determinations, the STATVIEW statistical

software package, version 5.0 (SAS, USA) was used. Data anal-

ysis was performed by analysis of variance (ANOVA) with the

StudentNewmanKeuls post hoc test for multiple comparisons.

PCR-data were analysed by Students t-test with Sigma Stat Vers.1

statistical software. Data are expressed as means S.E.M. Statistical

significance was set at p< 0.05.

Gene microarrays and dataprocessing

Blood samples (3 ml) of treated and untreated rats were col-

lected and prepared as described (Ulrich-Merzenich et al. 2012).

Only RNA oftreatment groups showing significant responses in the

FST compared to the untreated group were selected for detailed

microarray analysis. For analysis single colour hybridization of

the rat RNA on the Rat Agilent Whole Genome Oligo Micorarrays

(41,013 genes) was performed (Miltenyi Biotec, Bergisch Gladbach,

Germany). The ratios were computed using a common artificial

reference (4 control samples combined). This common reference

was used as baseline for all samples. The computed ratios were used

for further analyses by Ingenuity systems Inc., Redwood City, USA

as described (Ulrich-Merzenich et al. 2012).


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Table 2

The sequences ofthe PCRprimers.

Gene Nucleotide sequence Product length, bp Literature

-Actin F: 5-AACCGCGAGAAGATGACCCAGATCATGTTT-3

350 Park et al. (1997)R: 5-AGCAGCCGTGGCCATCTCTTGCTCGAACTG-3

Cyp26b1 F: 5

-CAGCTAGTGAGCACGGAGTG-3

345 Wang et al. (2010)R: 5-CGGCAGAGAGAAGACATTCTC-3

Gria 2R: 5-GCTCGAGAGAGGGTGATTG-3

144 Designed

(NM 017261.2)F: 5-CTGTTGATGCCTTGCGTC-3

Il-3F: 5-GAGTTTGATTCTCAGGACAC

360 Designed

(NC 005109)R: 5-GTCATCCAGATCTTTATTGTAG

Lox-1 F: 5-GACTGGATCTGGCATAAAGA-3

361 Nagase et al. (2000)R: 5-CCTTCTTCTGACATATGCTG-3

Srp54 F: 5-CTTGTAGACCCTGGAGTTAAAG-3

188 Designed

(NM 053871.1)R: 5-AGCTGGTCAAAAGCTCCTGCTC-3

Reverse transcription andgene specificpolymerase chain reaction

In order to validate the data obtained by microarray, genes that

were highly affected by the treatment and/or belonged to different

biological pathways were selected for RT-PCR(glutaminergic sys-

tem: Gria2; protein secretory pathway: signal recognition protein(SRP) 54; neurogenesis: cytochrome P450 protein 26B1 (Cyp26B1);

cytokines: IL-3; redox system: Lox-1). -Actin was used as inter-nal reference control. In each group 4 samples were analysed ifnot

mentioned otherwise. In brief, cDNA was synthesised from 1000 ng

of RNA by Transcriptor High Fidelity cDNA Synthesis Kit (Roche).

The gene specific primers are listed in Table 2. PCRs were performed

on Perkin Elmer DNA Thermal Cycler 480 (-actin, Cyp26b1, Il-3,Lox-1) and Eppendorf Mastercycler (Srp54, Gria2). The thermocy-

cler conditions were as follows: -actin: initial denaturing step:94 C for 7 min, amplification: 35 cycles ofdenaturation at 94 C for

1 min, annealing at 64 C for 1 min and elongation at 74 C for 3 min;

and final elongation step: 74 C for 7 min; Cyp26b1: initial denatur-

ing step: 95 C for 5 min, amplification: 30 cycles of denaturation

at 94 C for 1 min, annealing at 54 C for 1 min and elongation at

74 C for 2 min; and final elongation step: 74 C for 12 min, Gria2: initial denaturing step: 95 C for 2 min, amplification: 35 cycles

of denaturation at 94 C for 30 s, annealing at 51 C for 30 s and

elongation at 72 C for 1 min, and final elongation step: 72 C for

10 min; Il-3: initial denaturing step: 94 C for 7 min, amplification

30 cycles of denaturation at 94 C for 30 s, annealing at 52 C for

90 s and elongation at 72 C for 1 min, and final elongation step:

72 C for 10 min, Lox-1: initial denaturing step: 95 C for 4 min,

amplification: 30 cycles of denaturation at 94 C for 45 s, anneal-

ing at 47 C for 1 min and elongation at 74 C for 2 min, and final

elongation step: 74 C for 7 min; Srp54: initial denaturing step:

95 C for 5 min, amplification: 35 cycles of denaturation at 94 C

for 1 min, annealing at 56 C for 1 min and elongation at 72 C for

40 s; and final elongation step: 72 C for 5 min; Semi quantitative

analysis was performed by densitometry employing the BioRAD

GelDoc 100 system using the software Multi-Analyst as described

earlier (Ulrich-Merzenich et al. 2007). Data were expressed as

ratios to -actin. PCR-products were confirmed as the expectedtarget genes via purification (Wizard PCRPreps DNA Purification

system, Promega) and product sequencing (data not shown) per-

formed using standard techniques and the 3130/x Genetic Analyzer

(Applied Biosystems).

Results

Forced swimmingtest(FST)

Table 1 shows a characterization ofthe WB and its different frac-

tions with regard to the content ofdifferent polyphenols and salicylalcohol derivatives. Besides WB (A) especially FR-D, a fraction with

a low content offlavonoids and polyphenols but rich in salicin and

other salicyl alcohol derivatives, proved to be active.

As shown in Fig. 2 the cumulative immobility time was sig-

nificantly (p < 0.05) reduced in groups A (36%), FR-D (44%) and

imipramine (16%). Other fractions did not show a significant activ-

ity in a dosage of30 mg/kg bw (Fig. 2).

Concentrations of5-HT(serotonin) and 5-hydroxyindol acetic

acid in the hippocampus

The four weeks treatment ofCD-rats with WB (15 and 60 mg/kg

bw) increased the serotonin concentrations in the hippocampus

significantly, whereas the concentrations of 5-HIAA decreased

(WB: 15 and 30 mg/kg bw) as depicted in Fig. 3. The turnover of5-

HT, calculated as the ratio of5-HIAA/5-HT was significantly reduced

in a concentration range between 15 and 60 mg/kg. The tricyclic

antidepressant imipramine (15 mg/kg bw) as reference drug also

reduced the ratio of5-HIAA/5-HT, but did neither increase the sero-

tonin concentrations nor decrease the 5-HIAA concentrations in the

hippocampus significantly in the chosen dose of15 mg/kg bw.

Wholegenome micro-arrays

The analysis of the blood samples revealed that each group

showed a distinct expression profile (data not shown). Data of

groups, which had shown a significant response in the FST (group

A, FR-D, imipramine) were further analysed. The top up- and down-

regulated genes are given in the supplementary data.

Gene expression which was commonly modulated in the groups

with a significant response in the FST is shown in Table 3. Four of

these seven genes were commonly down-regulated. Gria2 encodes

a subunit of the group of AMPA-glutamate receptors. They are

Fig. 2. 12 rats per group were treated with imipramine, WB (A) or FR-B-E per os

for 14 days (see Material and methods). The FST was performed on day 15: groups

treated with imipramine (A), WB or FR-D did show significant decreases of theirimmobility time compared to controls, (*p< 0.05).


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934 G. Ulrich-Merzenichetal./Phytomedicine 19 (2012) 930939

Fig. 3. 12 rats per group were treated with increasing concentrations of WB

(15125 mg/kg). Serotonin and 5-hydroxyindol acetic acid (HIAA) concentrations

were measured in the hippocampus (for details see: Materials and methods).

Serotonin concentrations increased significantly for 15 and 60 mg/kg, 5-HIAA con-

centrations decreased for 15 and 30 mg/kg. The ratio HIAA/serotonin as indicator of

the serotoninturnover decreased dose dependentlyafter treatment of1560 mg/kg

ofWB and imipramine, (*p < 0.05).

Table 3

Genes and fold changes regulated in all experiments in comparison to the control

group.

Molecule Group/fold changes

Willow bark Ethanol fraction Imipramine

SRP54 19.3 25.5 22.9

CYP26B1 12.6 20.3 16.1

GRIA2 6.4 10.6 8.3

KITLG 4.9 9.1 7.0

DNM1L 3.6 2.8 3.1

AGAP1 4.6 2.2 2.9

EDNRB 2.5 6.1 2.3

All values represent statistically significant results (p < 0.01); Bold: different regula-

tion comparedto the other groups.SRP: signalrecognitionparticle,CYP: Cytochrome

P450, GRIA: glutamate receptor ionotropic AMPA, KITLG: stem cell factor; DNM1L:

dynamin like protein, AGAP: GTPase activating protein, EDNRB: endothelin B recep-tor gene.

activated in a variety of normal neurophysiological processes.

Cyp26b1 codes for a protein belonging to the P450 family. Srp54

encodes a signal recognition particle (SRP) and Kitlg, a stem cell

factor. In contrast, mRNA ofDnm1l (dynamin like protein 1), a reg-

ulator ofmitochondrial fission and distribution, was up-regulated

in all three groups.

The mRNA ofAgap1, which belongs to the GTPase-activating pro-

tein family, was up-regulated by WB and down-regulated by FR-D

Table 5

Fold changes ofinflammation associated genes.

Molecule Group/fold changes

Willow bark Ethanol fraction Imipramine

IL3

4.3

1.1

3.4IL10 2.2 1.1 4.3

IL15 1.2 1.1 2.0

IL13RA1 2.6 1.8 2.5

IL13RA2 4.8

IL17RA 2.7 1.4 1.5

IL1 2.3 2.1 1.4

IL1RL2 9.1

IL20RB 4.7 3.3

IL21R 2.1 1.2 1.5

IL22RA2 1.9 6.1 2.0

IL4R 1.9 1.1 2.7

IL7R 3.1 1.3 2.3

TNFAIP2 2.2

TNFRSF1A 2.7 1.1 1.5

TNFSF12 1.5 1.7 2.1

TNFSF14 3.0 1.6 1.0

Bold: statistically significant values (p < 0.01), IL: interleukine, R: receptor, TNFSF:

tumor necrosis factor-superfamily, TNFAIP: tumor necrosis factor, alpha-inducedprotein.

and imipramine. Similarly, Ednrb, an endothelin B receptor gene,

was differentially regulated up-regulated by FR-D and down-

regulated by WB and imipramine. The modulation ofglutamatergic

ionotropic or metabotropic receptors is shown in Table 4.

Cytokines, which were significantly (p < 0.01) and more than

twofold modulated in at least one of the groups are shown in

Table 5. The treatment response was not uniform in the differ-

ent treatment groups and considering the top fold changes in the

groups A, FR-D and imipramine, the cytokine response was low.

The imipramine treatment provoked in the interleukin- and TNF-

families still the strongest response (changes between 1.5 and

9.1 fold). Interestingly, the lowest response with respect to thecytokine families was seen in FR-D (salicin rich ethanol fraction).

This group differed from group A (WB) and group F (imipramine).

It is noteworthy, that the gene expression of one major

proinflammatory mediator, Il-6 was down-regulated by WB and

imipramine (1.4 and 1.3 fold respectively,p < 0.05). Equally, Nfb1

was down-regulated by WB and imipramine (1.9 and 1.5 fold,

p < 0.05). Genes encoding the extracellular Sod3 as well as catalase

(Cat) were also down-regulated by WB (1.8 and 1.7 fold respec-

tively) and Sod3 alone by imipramine (1.2 fold). Even though the

magnitude of these regulations did not meet our initial selection

criteria (2 fold/p < 0.01), it was still significant. The mitochondrial

Table 4

Fold changes ofgenes coding for the group ofglutamate receptors.

Molecule Official full name Group/fold changes

Willow bark EtOH-Fr. Imipramine

GRIA2 Glutamate receptor, ionotropic, AMPA 2 6.4 10.6 8.3

GRID2 Glutamate receptor, ionotropic, delta 2 3.5 1.1 2.3

GRID2IP Glutamate receptor, ionotropic, delta 2 (Grid2) interacting protein 3.0 3.6

GRIK4 Glutamate receptor, ionotropic, kainate 4 2.8 1.6 1.2

GRIK5 Glutamate receptor, ionotropic, kainate 5 2.0 1.1 3.1

GRIN2B Glutamate receptor, ionotropic, N-methyld-aspartate 2B 2.2 1.1 3.9

GRIN3B Glutamate receptor, ionotropic, N-methyl-d-aspartate 3B 2.1 1.0 1.3

GRINA Glutamate receptor, ionotropic, N-methyld-aspartate-associated

protein 1 (glutamate binding)

3.8 1.7 1.5

GRIP1 Glutamate receptor interacting protein 1 2.4 1.3 2.8

GRIP2 Glutamate receptor interacting protein 2 2.4

GRM1 Glutamate receptor, metabotropic 1 3.3 1.2 1.7




Bold: statistically significant values (p < 0.01).


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-30.00

-20.00

-10.00

0.00

EthF Salix IM

fold

changes

Srp 54 regulaon

PCR

Microarray

-15.00

-10.00

-5.00

0.00

EthF Salix IM

fold

changes

Gria2 regulaon

PCR

Microarray

-30.00

-20.00

-10.00

0.00

EthF

Salix IM

fold

changes

Cyp26b1 regulaon

PCR

Microarray

-10

-5

0

EthF Salix IM

fold

changes

Lox1 regulaon

PCR

Microarray

-6.0

-4.0

-2.0

0.0

EthF Salix IM

fold

changes

Interleukin 3 regulaon

PCR

Microarray

***

******

***

**

*

***

***

**

*

**

**

**

***

**

*

****

*

* *

***

***

0.06

0.057

Fig. 4. Fold-changes ofthe gene expression measured by microarray and RT-PCR.

RT-PCRdata for the different genes are expressed as ratios to -actin (n=4 for each

gene fromeach group). Microarraydata represent the computed ratios to one com-

monreference(4 controlsamplescombined).FordetailsseeMaterialsandmethods.

Srp: signal recognition particle, Cyp: cytochrome P450, Gria: glutamate receptor

ionotropic AMPA, Il-3: interleukine3, Lox-1: oxidizedlowdensitylipoproteinrecep-

tor 1. Salix: willow bark, (***p


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936 G. Ulrich-Merzenichetal./Phytomedicine 19 (2012) 930939

Glutaminergicsystem

WB, FR-D as well as imipramine targeted the group of

peripheral glutamate receptors, including Gria2. Gria2 codes

for a subunit of the alpha-amino-3-hydroxy-5-methyl-4-

isoxazolepropionic acid (AMPA)-receptors, which belong tothe group of glutamate receptors. They are the predominant

excitatory neurotransmitter receptors in the mammalian brain

and are activated in a variety ofnormal neurophysiologic processes

(http://www.ncbi.nlm.nih.gov/gene/29627). Diseases which have

been associated with an altered gene function of Gria2 includ-

ing major depression and Alzheimers disease are reviewed in

Machado-Vieira et al. (2009).

In the brain, the tricyclic antidepressants, selective serotonin-

reuptake inhibitors (SSRIs) as well as serotonin and noradrenalin-

reuptake inhibitors (SNRIs) were shown to down-regulate the

N-methyl-d-aspartate (NMDA) glutamate receptors and to poten-

tiate the AMPA glutamate receptors (Novak et al. 1993; Paul et al.

1994; Skolnick et al. 1996; Machado-Vieira et al. 2009). Our data

however, show a 6- to 10-fold peripheral down-regulation ofGria2

for imipramine as well as for WB treatment and a broad modula-tion ofmembers of the glutamate receptor family. In the context

of recent discoveries that platelets express AMPA-receptors for

the excitatory neurotransmitter glutamate and influence platelet

activation (Morrell et al. 2008), this peripheral down-regulation

appears to be noteworthy. Amodifiedplatelet GLUR1 phosphoryla-

tion inmajor depressivedisorders (MDD) has beenhypothesised to

contribute to the comorbidity ofMDD and cardiovascular disorders

(Chen 2009).

Further studies ofthese effects may increase the understanding

ofa contribution ofthe peripheral GluR family to the comorbidity

ofMDD and CVD and support improved drug developments.

Protein secretorypathways

One of the strongest regulated genes in the three groups is

the signal regulating protein 54 (Srp54) gene (1925-fold) encod-

ing a protein involved in the secretory pathway to guide proteins

through the endoplasmatic reticulum (ER). Protein translocation

across the ER-membrane requires signal sequence binding to Srp54

(Grudnik et al. 2009). As one ofthe two central elements ofthe so

called SRP-system, human SRP54 has a guanosine triphosphatase

(GTPase) activity and is universally conserved (Grudnik et al. 2009).

A single nucleotide polymorphism (SNP) in SRP54 has been associ-

ated with bipolar affective disorder (Baum et al. 2008). Watanuki

et al. (2008) showed that the mRNA of SRP20, but not of SRP54

was increased in patients suffering from major depressive disor-

ders. However, authors acknowledge limitations of their findings

since the treatment ofpatients was not standardized.We can show

here that in our animal model, WB, FR-D and imipramine down-

regulate the central element ofthe SRP-system, Srp54, suggesting

that protein secretion is decreased.

DNM1L (DLP1) is known to constrict and tubulate membranes

and to divide mitochondria and peroxisomes. Recent data indi-

cate that at the Golgi complex DNM1L is a component of the

sorting/targeting machinery en route to the plasma membrane

(Bonekamp et al. 2010). Mitochondrial dysfunction and synaptic

loss are among the earliest events linked to Alzheimers disease

(AD) and might play a causative role in disease onset and pro-

gression (Bossy et al. 2010). Dnm1l was up-regulated in all three

groups suggesting that the regulation ofmitochondrial fission and

distribution will positively influence protein targeting and sorting.

Neurogenesis/immune system

Another strongly down-regulated gene (12 to 20 fold) in all

groups was Cyp26b1, encoding a cytochrome-P450 enzyme that

catabolises retinoic acid (RA) (Maclean et al. 2009). All-trans-RA

stimulates neurogenesis, dendritic growth of hippocampal neu-rons and higher cognitive functions. Genetic disruption ofmouse

Cyp26b1 affects the development of neural crest-derived central

nervous system structures, but does not compromise hindbrain

patterning (Maclean et al. 2009). A down-regulation of CYP26B1

mRNA in brain motor cortex has been shown to be involved in

Huntingtons disease (Hodges et al. 2006). Brain CYP26 activity

is considered a key effector inhibiting neuronal differentiations

(Gonzalez-Quevedo et al. 2010). The relevance of a peripheral

down-regulation is not known so far. However, we detected that

imipramine as tricyclic antidepressant as well asWB and FR-D tar-

get this gene in the context ofan antidepressant like activity in the

FST. A very recent report also links CYP26B1 to the regulation of

RA-dependant signals in activated T-cells and thus to the immune

system (Takeuchi et al. 2011).

The mRNA of Kitlg or stem cell factor (SCF) was 4- to 9-folddown-regulated in all three groups. SCF activates the c-kit ligand

ofthe tyrosine-kinase receptor. It is also known as a haematopoi-

etic growth factor (HGF) which promotes neuroprotective effects

and supports neurogenesis in the brain (Laske et al. 2008; Zhao

et al. 2007). Transcription ofSCF is up-regulated in inflammatory

conditions (Reber et al. 2009). Therefore, a down-regulation ofSCF

suggests an anti-inflammatory effect.

Modulation ofcytokines in depression

WB is well known for its anti-inflammatory action. A down-

regulation of the proinflammatory cytokines TNF- and IL-6, aswell as the proinflammatory transcription regulator NF-B, hasbeen shown in human cellular (Bonaterra et al. 2009; Fiebich

and Chrubasik 2004; Khayyal et al. 2005) and animal models of

inflammation (Khayyal et al. 2005) earlier. We stated under our

experimental conditions aminor down-regulationofIl-6 andNf-btranscription throughWB in agreementwith these earlier findings.

Interestingly, also imipramine as serotonin-uptake inhibitor did

mildly down-regulate both molecules. However, before discussing

the cytokine modulation, the relevance ofa peripheral regulation

ofcytokines in the context ofdepression needs to be addressed.

Initial clinical data indicate that peripherally administered

cytokines (IFN-) can activate a CNS inflammatory response inhumans which interacts with the 5-HT metabolism (Raison et al.

2009). Changes included increases in MCP-1 and IL-6 in the CSF.

Further, several cytokines were reported to trigger the transcrip-

tion of genes at cells of the blood-brain barrier including NF-B

and COX-2, and these proteins then promoted signalling within the

CNS (Laflamme and Rivest 1999; Seguin et al. 2009). Himmerich

et al. (2009) proposed that TNF- and its soluble receptors mightcontribute to the pathogenesis of neurological diseases through

an activation of the HPA axis, of neuronal 5-HT transporters and

the stimulation of the indoleamine 2,3-dioxygenase leading to

a tryptophan depletion, through an immunologically mediated

destruction of neurons, or through the neurotoxic release of glu-

tamate (Himmerich et al. 2009). It was further proposed that

peripheral rather than central actions ofTNF- will influence thebirth ofnew hippocampal dentate gyrus cells. Considering the rel-

evance ofthe individual cytokines, other experiments ofthis group

showed that IL-1 generally producedmore profound behavioural,neuroendocrine and neurotransmitter effects than TNF- (Seguinet al. 2009).

In our gene expression analyses of peripheral blood cells Il-3-mRNA showed a down-regulation through WB and imipramine


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(4.3 fold and 3.4 fold respectively). IL-3 is a growth promot-

ing cytokine, supporting the proliferation of a broad range of

haematopoietic cell types. It is proposed to be associatedwith neu-

rological disorders (HGNC) andwas shown to induce the activation

of the JAK-STAT and MAP kinase pathways in microglial cells, the

residentmacrophages ofthe brain (Natarajan et al. 2004). A down-regulation, as seen in our experiments, could be favourable since

the stress inducedJAK-STAT pathway would not be activated.

Other down-regulated mRNA levels of cytokine receptors (>2

fold) were those ofIl-17ra and Il-20rb. Human IL-17RAwas shown

to beexpressed in the central nervous systemglia andup-regulated

in experimental autoimmune encephalomyelitis (Das Sarma et al.

2009), whereas not much is known so far on the IL-20 receptor

beta except that the cytokine IL-20 is regarded to be a pleiotropic

cytokine with potent inflammatory, angiogenic and chemoattrac-

tive characteristics (Wei et al. 2006).

The Il-13-receptor, slightly down-regulated by WB, is present

in human glioma cells and appears to be over expressed in solid

brain tumors (Shimamura et al. 2006). However, the relevance of

this finding is unclear.

WB up-regulated also the rat Il-21 receptor and Il-7r mRNAlevels. In human, IL-21Rhas been attributed a critical role in the

development of substantial memory B-cells (Rankin et al. 2011).

Its presence on neurons has been demonstrated leading to the pro-

posal that it plays a role inboth, acute and chronic stagesofmultiple

sclerosis (MS) via direct effects on T and B lymphocytes (Tzartos

et al. 2011). Similarly IL-7Rwas shown to be involved in MS (Haas

et al. 2011). Here, T-cells showed a reducedexpressionofthe recep-

tor and since IL-7 appears to play a critical role in Treg maturation

(Haas et al. 2011), a relevance for the Treg homeostasis in MS was

proposed.

TheTNF-receptor superfamilymember1A (TNFRSF1A)has been

found to work in cancer cells as trigger molecule for the apoptotic

cascade (Karabulut et al. 2010). WB lead to its down-regulation

in the rat, whereas TNFSF14 (LIGAND), which was shown to be

particularly involved in apoptosis and inflammation was slightly

up-regulated here.

The proinflammatory cytokine transcript of Il-1 was slightlyup-regulated (2.3 fold) byWB. This is somewhat surprising, since in

severalmodels ofinflammation the COX-inhibitorWBalso reduced

IL-1 (Fiebich and Chrubasik 2004; Khayyal et al. 2005). In addi-tion, mRNA of other proinflammatory cytokines like Il-3 or Il-6

was down-regulated in our experiments. In depression a periph-

eral up-regulation of IL-1 has been implicated (Milaneschi et al.

2009) and in the central nervous system IL-1was shown to induceCOX-2, thereby contributing to inflammatory pain hypersensitiv-

ity (Howren et al. 2009). The explanation for this at first glance

contradictory result may lay in the interplay of the cytokine net-

work which still requires further investigations. The estimated

expression profile of WB may represent just one profile for an

anti-inflammatory activity.

Plasticity

There is an increasing evidence that mood disorders are asso-

ciated with impairments in neuroplasticity and cellular resilience

(Machado-Vieira et al. 2009). Alterations ofthe glutamatergic sys-

tem (Machado-Vieira et al. 2009) aswell as cytokines are attributed

a major role in these impairments (Bret-Dibat et al. 1995; Rivest

et al. 2000; Banks et al. 2001; Seguin et al. 2009). We show a low

down-regulation ofIl-3, Il-6 and Tnf- associated receptor RNA incombination with a low up-regulation of Il-1. At the same time,the diversity of glutamatergic receptors is targeted by all three

groups (Table4). IL-1andTNF-were reportedto alterhippocam-

pal long-term potentiation and, together with IL-6, also influenceddendritic branching ofhippocampal neurons. Seguin et al. (2009)

proposed that they may differentially regulate hippocampal plas-

ticity rather than proliferation. Thus, an effect on the plasticity can

be expected for the three effective treatment groups.

Antioxidantstatus and depression

Recently, Zafir et al. (2009) proposed the antioxidant system as

target for an antidepressive treatment. They showed that in rats

identified by FST as depressive-like phenotypes the Sod, Cat, glu-

tathione S-transferase (Gst) and glutathione reductase (Gr) were

increased. These parameters declined following antidepressant

treatment (fluoxetine as selective serotonin reuptake inhibitor,

imipramine as tricyclic antidepressant; venlafaxine as dual sero-

tonin/norepinephrine reuptake inhibitor (Zafir et al. 2009). In line

with these findings, we observed a down-regulation ofthe mRNAs

encoding extracellular Sod3 and the mitochondrial Sod2 under

imipramine treatment. In addition, Cat and Sod3 were down-

regulated by WB, supporting earlier studies that WB preparations

have an antioxidant activity (Bonaterra et al. 2009; Khayyal et al.

2005). These data support the idea that regulation of the redox-

state plays a role in depression.

Shortterm and longterm administration

The time of administration will influence the mode of action

especiallywith respect to the cytokine networkand theneurotrans-

mitter levels. For example, it was shown that a single infusion of

IL-6 or IL-1was not a sufficiently potent challenge to influencethe hippocampus (Seguin et al. 2009). Our cytokine data pertain to

a treatment of14 days which led to a positive FST and a measur-

able effect in the peripheral cytokine network for the treatments

withWB, FR-D and imipramine (20mg/kg). The 5-HT levels as well

as its turn over were significantly affected by a WB treatment over

4 weeks, whereas imipramine changed the turn over, but not the

serotonin or the 5-HIAA levels. These data for imipramine corre-

spond to earlier studies ofthis group (Butterweck et al. 2002).

Interestingly, the native extract of Hypericum perforatum L.

yielded in comparable experiments (FST) undertaken by this group

significant results in concentrations as high as 125mg/kg bw and

more (Winterhoff et al. 1995). The ratio of 5-HT/5-HIAA in the

hippocampus was reduced after a two weeks and an eight weeks

treatment with this preparation in concentrations of 500mg/kg

bw. Isolated fractions applied in combinations of procyanidines

with naphtodianthrones, hypericin and pseudohypericin were,

however, already significantly active in the FST at much lower

concentrations (0.028mg/kg and 0.166mg/kg) (Butterweck et al.

2003). Also the biflavones ofHypericumperforatum L. are regarded

useful for the treatment ofinflammation and depression (Michler

et al. 2011). A positive response in the FSTand increased5-HT levels

in the hippocampus caused by a treatment with the WB extract in

concentrations of30mg/kg and 15mg/kg bwallows the question of

the relevance ofinflammatory processes for the neurotransmitter

metabolism.

A combinatorial treatment approach is supported by results

of a recent double blind placebo controlled trial in patients with

major depression. The treatment of celecoxib (COX-2 inhibitor)

and fluoxetine had a significant superiority over fluoxetine alone

in the treatment ofsymptoms ofmajor depression (Akhondzadeh

et al. 2009). Since both drugs have a good bioavailability, a phar-

macokinetic interaction may not be the reason for this effect.

We analysed our microarray data also for the prediction of

AEs. The model analysis revealed less potential AEs for WB as

multicomponent mixture compared to imipramine as single com-

ponent drug (Ulrich-Merzenich et al. 2012).

In summary, these investigations identified novel neuro-and immunological targets associated with salicylate-containing


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