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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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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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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
GRM2 Glutamate receptor, metabotropic 2 2.6 1.0 1.9
GRM3 Glutamate receptor, metabotropic 3 2.0 1.2 1.7
GRM4 Glutamate receptor, metabotropic 4 2.5 1.2 7.0
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
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*
****
*
* *
***
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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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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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G. Ulrich-Merzenich etal./Phytomedicine 19 (2012) 930939 937
(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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