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Studies on the Anti-Inflammatory Activity of Phytopharmaceuticals Prepared from Arnica Flowers1

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Phytopharmaceuticals prepared from flowerheads of Arnica montana of Spanish origin and of the new type ”Arbo”, which can be easily and economically cultivated, were studied for their capability to impair activation of the transcription factors NF-κB and NF-AT. Both proteins are responsible for the transcription of genes encoding various inflammatory mediators. Additionally, their influence on the release of the cytokines IL-1 and TNF-α were examined. The inhibitory activities correlate with their quantitative and qualitative content of sesquiterpene lactones (Sls). Moreover, it was shown that the inhibitory potency of 11α,13-dihydrohelenalin derivatives being the main Sls in the Spanish flowers depend on their esterfication. Compounds with unsaturated acyl moieties, such as methacrylate and tiglinate, exhibited a stronger activity in the NF-κB EMSA as well as in the croton oil ear test in mice than the acetate derivative. Abbreviations Arbo tincture:tincture prepared from Arnica flowerheads type Arbo AP-1:activator protein 1 BSA:bovine serum albumen c.p.m.:counts per minute DTT:dithiothreitol EDTA:ethylenediaminetetraacetic acid EGTA:ethylene glycol-bis(β-aminoethyl ether)- N,N,N′,N′-tetraacetic acid EMSA:electrophoretic mobility shift assay FCS:fetal calf serum HEPES:N-[2-hydroxyethyl]-piperazine-N′- [2-ethanesulfonic acid] IL:interleukin NP-40:nonylphenoxypolyethoxyethanol NF-AT:nuclear factor of activated T cells BPS:phosphate buffered saline PMA:phorbol myristate acetate PMSF:phenylmethylsulfonyl fluoride Poly (dI-dC):polydeoxyinosinicdeoxycytidylic acid, double-stranded alternating copolymer Spanish tincture:tincture prepared from Spanish Arnica flowerheads Sl:sesquiterpene lactone TNF-α:tumor necrosis factor alpha
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Studies on the Anti-Inflammatory Activity of
Phytopharmaceuticals Prepared from Arnica Flowers
1
Christoph A. Klaas
1
Gerd Wagner
2
Stefan Laufer
2
Silvio Sosa
3
Roberto Della Loggia
3
Ulrich Bomme
4
Heike L. Pahl
5
Irmgard Merfort
1
Affiliation
1
Institute of Pharmaceutical Biology, University of Freiburg, Freiburg, Germany
2
Institute of Pharmacy, University of Tübingen, Germany
3
DEMREP, Università di Trieste, Italy
4
Bayerische Landesanstalt für Bodenkultur und Pflanzenbau, Freising-Weihenstephan, Germany
5
Department of Experimental Anaesthesiology, University Hospital, Freiburg, Germany
Correspondence
Prof. Dr. Irmgard Merfort ´ Institute of Pharmaceutical Biology ´ Albert-Ludwigs-University ´ Stefan-Meier-Str.
19 ´ 79104 Freiburg ´ Germany ´ Phone: +49±761±203±8373 ´ Fax: +49±761±203±8383 ´
E-Mail: merfort@uni-freiburg.de
Received June 27, 2001 ´ Accepted October 7, 2001
Bibliography
Planta Med 2002; 68: 385±391 ´ Georg Thieme Verlag Stuttgart ´ New York ´ ISSN 0032-0943
Abstract
Phytopharmaceuticals prepared from flowerheads of Arnica
montana of Spanish origin and of the new type ªArboº, which
can be easily and economically cultivated, were studied for their
capability to impair activation of the transcription factors NF-
k
B
and NF-AT. Both proteins are responsible for the transcription of
genes encoding various inflammatory mediators. Additionally,
their influence on the release of the cytokines IL-1 and TNF-
a
were examined. The inhibitory activities correlate with their
quantitative and qualitative content of sesquiterpene lactones
(Sls). Moreover, it was shown that the inhibitory potency of
11
a
,13-dihydrohelenalin derivatives being the main Sls in the
Spanish flowers depend on their esterfication. Compounds with
unsaturated acyl moieties, such as methacrylate and tiglinate,
exhibited a stronger activity in the NF-
k
B EMSA as well as in the
croton oil ear test in mice than the acetate derivative.
Key words
Arnica montana ´ Asteraceae ´ anti-inflammatory activity ´ sesqui-
terpene lactones ´ NF-
k
B ´ NF-AT ´ IL-1
b
´ TNF-
a
´ mouse ear edema
Abbreviations
Arbo tincture: tincture prepared from Arnica flowerheads
type Arbo
AP-1: activator protein 1
BSA: bovine serum albumen
c.p.m.: counts per minute
DTT: dithiothreitol
EDTA: ethylenediaminetetraacetic acid
EGTA: ethylene glycol-bis(
b
-aminoethyl ether)-
N,N,N
¢
,N
¢
-tetraacetic acid
EMSA: electrophoretic mobility shift assay
FCS: fetal calf serum
HEPES: N-[2-hydroxyethyl]-piperazine-N
¢
-
[2-ethanesulfonic acid]
IL: interleukin
NP-40: nonylphenoxypolyethoxyethanol
NF-AT: nuclear factor of activated T cells
BPS: phosphate buffered saline
PMA: phorbol myristate acetate
PMSF: phenylmethylsulfonyl fluoride
Poly (dI-dC): polydeoxyinosinicdeoxycytidylic acid,
double-stranded alternating copolymer
Spanish tincture: tincture prepared from Spanish Arnica
flowerheads
Sl: sesquiterpene lactone
TNF-
a
: tumor necrosis factor alpha
1
Partly presented at the 3
rd
International Congress on Phytomedicine,
2000, Munich, Germany.
Original Paper
385
Downloaded by: Roberto Della Loggia. Copyrighted material.
Introduction
Phytopharmaceuticals prepared from Arnica flowers are exter-
nally used to treat a variety of different ailments. These are relat-
ed to injuries and accidents, e. g., for haematomas, dislocations,
sprains, bruising, edema associated with fractures, rheumatic
muscle and joint complaints, inflammations of the mucous
membranes of the mouth and throat, furuncles, inflamed insect
bites, and surface phlebitis [1]. The secondary metabolites that
mediate the anti-inflammatory effects of Arnica flowers are
mainly sesquiterpene lactones (Sls) of the 10
a
-methylpseudo-
guaianolide type like helenalin, 11
a
,13-dihydrohelenalin and
their ester derivatives (see Fig.1) ([1], and lit. cited therein). Re-
cently, we made a contribution to the molecular mechanism by
which they exert their anti-inflammatory effect. We have dem-
onstrated that these Sls as well as Arnica tincture prepared from
flowerheads of A. montana (Spanish chemotype) effectively in-
terferes with the activation of NF-
k
B and that the total concen-
tration of Sls is high enough to explain this effect [2], [3], [4].
Moreover, we could show that Sls also inhibit the transcription
factor NF-AT [5].
The inducible transcription factors NF-
k
B and NF-AT are impor-
tant regulators of the human immune response. NF-
k
B regulates
the transcription of genes encoding, e. g., the cytokines IL-1
b
, IL-
2, TNF-
a
, adhesion molecules or enzymes like iNOS, cyclooxy-
genase-II and 5-lipoxygenase [6]. Components of the NF-AT family
bind cooperatively with those of the AP-1 family to composite
NF-AT:AP-1 sites [7] and play a major role in the control of acti-
vation and differentiation of T cells. These cells activate other
cells of the immune system, thus maintaining or enhancing in-
flammatory processes [7]. In addition to NF-
k
B, NF-AT is involved
in regulating the transcription of cytokines, such as TNF-
a
, IL-2
or IL-4 in lymphocytes.
In continuation of our studies on the anti-inflammatory activity
of sesquiterpene lactones, this paper is related to investigations
on preparations from Arnica flowerheads as well as of some
isolated Sls from these preparations. Two Arnica tinctures, are
compared for their ability to inhibit DNA binding of the tran-
scription factors NF-
k
B and NF-AT. One is prepared from flower-
heads of the Spanish chemotype, the other from those of type
ªArboº which has been recently introduced in the market and
has the advantage to be more easily and economically cultivated
than the species from Spain [8]. Additionally, a commercially
available Arnica preparation was studied as to the extent to
which NF-
k
B inhibition occurs. The inhibitory activities were
correlated with their qualitative and quantitative contents of
Sls. In order to evaluate whether the production of cytokines de-
creases in correlation with the NF-
k
B inhibition results both
Arnica tinctures were studied for their ability to influence the re-
lease of the cytokines IL-1
b
and TNF-
a
. Moreover, it was investi-
gated whether the type of esterification within the dihydrohele-
nalins influence the potency to inhibit NF-
k
B. Finally, the in vivo
mouse ear edema test was undertaken with two Sls in order to
ascertain whether a correlation exists between the ex vivo and
the in vivo results.
Materials and Methods
Test compounds
Arnica tincture (German Pharmacopoeia, DAB) and ArnikaGel
were a gift from the Kneipp Company, Würzburg, Germany.
Arnica tincture (ªArboº) was prepared from Arnica montana
flowers type ªArboº growing at Steinach (Bavaria, Germany) ac-
cording to the German Pharmacopoeia. A voucher specimen from
type ªArboº is deposited at the Bayerische Landesanstalt für Bo-
denkultur und Pflanzenbau, Freising-Weihenstephan, Germany.
11
a
,13-Dihydrohelenalin acetate, 11
a
,13-dihydrohelenalin tigli-
nate and 11
a
,13-dihydrohelenalin methacrylate were isolated
from flowerheads of Arnica montana, Spanish chemotype, as pre-
viously described [9]. Identity was confirmed by NMR and MS
analysis, purity was evaluated by GC analysis.
10 mM stock solutions of the respective Sl were prepared in
DMSO for the NF-
k
B assays. The tinctures as well as the Arnika-
Gel
were directly applied to the Jurkat T cells.
HPLC analysis
10 g of the respective Arnica tincture were diluted with 50 ml
H
2
O. Santonin (Sigma, Deisenhofen, Germany) was added as an
internal standard and the solution was heated for 30 min at
608C. Ethanol was removed by reduced pressure. The next steps
were carried out as previously described [10]. The obtained val-
ues in mg/g were converted to mg/ml by using the correction fac-
tor of 0.868 (0.868 g = 1 ml tincture).
10 g of ArnikaGel
were diluted with 50 ml H
2
O and santonin was
added as an internal standard. About 0.45 ml of aq. HCl (2.5%), to
a pH of 5.3, and 5 ml MeOH were added to destroy the gel struc-
ture. This solvent was heated 30 min at 608C and handled as de-
scribed in [10]. Finally the obtained values were converted to
mg/ml using the factor of 0.975 (0.975 g = 1 ml gel).
Cell culture
Jurkat T cells were maintained in RPMI 1640 medium supple-
mented with 10 % fetal calf serum, 100 IU/ml penicillin and
100
m
g/ml streptomycin (all Gibco-BRL, Groningen, Nether-
lands).
Fig. 1 Sesquiterpene lactones from flowerheads of A. montana and
structure of SB203580.
Klaas CA et al. Studies on the ¼ Planta Med 2002; 68: 385 ± 391
Original Paper
386
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NF-
kk
B EMSA
Total protein extracts from Jurkat T cells were prepared using a
high-salt detergent buffer (Totex: 20 mM Hepes, pH 7.9, 350
mM NaCl, 20 % (v/v) glycerol, 1 % (w/v) NP-40, 1 mM MgCl
2
, 0.5
mM EDTA, 0.1 mM EGTA, 0.5 mM DTT, 1 mM PMSF,1% aprotinin).
Cells were harvested by centrifugation, washed once in ice-cold
PBS (Sigma, Deisenhofen, Germany) and resuspended in four cell
volumes of Totex buffer. The cell lysate was incubated on ice for
30 min, then centrifuged for 5 min at 13,000 rpm at 4 8C. The pro-
tein content of the supernatant was determined and equal
amounts of protein (10± 20
m
g) added to a reaction mixture con-
taining 20
m
g BSA (Sigma, Deisenhofen, Germany), 2
m
g poly (dl-
dC) (Roche Diagnostics, Mannheim, Germany), 2
m
l buffer D+ (20
mM Hepes, pH 7.9; 20% glycerol, 100 mM KCl, 0.5 mM EDTA,
0.25% NP-40, 2 mM DTT, 0.1 % PMSF), 4
m
l buffer F (20 % Ficoll
400, 100 mM Hepes, 300 mM KCl, 10 mM DTT, 0.1% PMSF) and
100,000 c. p.m. (Cerenkov) of a
32
P-labeled oligonucleotide,
made up to a final volume of 20
m
l with distilled water. Samples
were incubated at room temperature for 25 min. NF-
k
B oligonuc-
leotide (Promega, Mannheim, Germany) was labeled using
g
-
[
32
P]-ATP (3000 Ci/mmol; Amersham Pharmacia Biotech, Frei-
burg, Germany) and T4 polynucleotide kinase (Promega, Mann-
heim, Germany).
NF-AT EMSA
Nuclear cell extracts were prepared as previously described [11].
The protein content was determined and equal amounts of pro-
tein (10
m
g) were added to the same reaction mixture as in the
NF-
k
B EMSA. A oligonucleotide spanning the distal NF-AT site of
the human GM-CSF promoter was used [12]. Sequence: 5
¢
- TTT
CTC ATG GAA AGA TGA CAT A - 3
¢
.
Cellular assay of cytokine release inhibition, enzyme linked
immunosorbent assay for TNF-
aa
and IL-1
bb
Arnica tinctures were serially diluted in 70 % ethanol (concentra-
tion range 10 ± 0.001
m
l/ml). Mononuclear cells were isolated
from whole blood of healthy human donors by density gradient
centrifugation. Cell samples were preincubated for 15 min (37 8C,
5%CO
2
) with various concentrations of Arnica tincture (test sam-
ples) or 70% ethanol (control samples). Biosynthesis of cytokines
was induced by cells stimulation with 1
m
g/ml LPS (from E. coli,
serotype 026:B6) for 4 h (378C, 5% CO
2
). Cell reaction was termi-
nated in an ice bath and cell samples were centrifuged. Concen-
trations of IL-1
b
and TNF-
a
were determined in supernatants
using commercially available ELISA kits (Beckman Coulter, Kre-
feld, Germany). The anti-cytokine activity of each extract was
calculated as percent reduction of cytokine concentration in test
samples compared to control samples. Results are given as IC
50
values (
m
l/ml).
Vitality test
The viability of cells which were treated with the respective sam-
ple for four hours was determined by trypan blue exclusion as
described in [13].
Croton oil-induced ear edema in mice
The in vivo anti-inflammatory activity of the sesquiterpene lac-
tones was measured as inhibition of the croton oil-induced ear
edema in mice, as previously described [14]. Cutaneous inflam-
mation was induced by applying 15
m
l of an acetonic solution
containing 80
m
g of croton oil to the inner surface of the right
ear of male CD-1 mice (28 ± 32 g, Harlan-Italy, S. Pietro al Nati-
sone, Italy), anesthetized with ketamine-HCl (145 mg/kg intra-
peritoneally; Virbac, Milano, Italy). The test substances were dis-
solved in the croton oil solution. The left ear remained untreated
since preliminary experiments had shown that acetone does not
interfere with the inflammatory response. All experiments were
uniformly started between 10 : 00 and 12: 00 a.m. in order to
avoid variations in the inflammatory response due to circadian
fluctuations in the levels of corticosteroids. Six hours after induc-
tion of the inflammation, mice were sacrificed and a plug (6 mm
é) was excised from both the treated and the untreated ears. The
edema formation was quantified as the difference in weight be-
tween the two ear plugs. The anti-inflammatory activity was
evaluated as percent inhibition of the edematous response in
the animals treated with the test substances in comparison to
the animals treated with the irritant alone. The experimental de-
sign was approved by the ethics committee of the University of
Trieste.
Statistical analysis
Values of HPLC analysis were obtained from three independent
measurements, those of the EMSAs from two measurements.
Graphical analysis on semi-logarithmic paper was used to calcu-
late IC
50
values for the Arnica tinctures from percent reduction of
cytokine release at various concentrations in one particular run.
Experiments were carried out three/four times and the overall
IC
50
value was calculated as the mean +/-SD of all three/four
IC
50
s. The in vivo data were evaluated by the Student's t-test
and significance was assumed for p values lower than 0.05.
Results
To compare the inhibitory activity on the NF-
k
B DNA binding of
different phytopharmaceuticals Jurkat T cells (5 ±6 10
5
cells/
ml) were incubated with various amounts of the respective
Arnica tincture or the ArnikaGel
for 1 h. Cells were stimulated
for 1 h with 200 U/ml TNF-
a
, and subsequently total cell extracts
were prepared and analyzed for NF-
k
B DNA binding activity (see
Fig. 2A and B). Stimulation with TNF-
a
induced one novel DNA
binding activity in Jurkat T cells (see Fig. 2, lane 2). Antibody re-
activity and competition assays identified this complex as an NF-
k
B p50/p65 heterodimer (data not shown). 5
m
l/ml of the Arbo
and 10
m
l/ml of the Spanish tincture completely inhibited NF-
k
B
DNA binding (see Fig. 2A). ArnikaGel
was less active since a con-
centration of 50
m
l/ml was necessary for a complete inhibition
(see Fig. 2B). In each case no cytotoxic effects could be observed.
The differences in the activity of the Arnica preparations corre-
late with their content and type of Sls (see Table 1). The qualita-
tive composition was determined by means of GC/MS analyses
and by direct GC comparison with authentic compounds as far
as available, the quantitative content was studied by HPLC anal-
ysis according to [10]. The Arbo tincture, yielding mostly helena-
lin derivatives was two fold more active in NF-
k
B inhibition than
the Spanish tincture containing 11
a
,13-dihydrohelenalin esters.
ArnikaGel
, with the lowest content of dihydrohelenalin deriva-
tives, was less active.
In contrast to the results with the tinctures, it was recently
shown that pure helenalin was about 20-fold more active than
Klaas CA et al. Studies on the ¼ Planta Med 2002; 68: 385 ± 391
Original Paper
387
Downloaded by: Roberto Della Loggia. Copyrighted material.
pure 11
a
,13-dihydrohelenalin [3]. Therefore, we elucidated
whether there might be differences in the inhibitory potency be-
tween dihydrohelenalin esters which possess different acyl mo-
ities and the unesterified compound. As shown in Fig. 3A and B,
11
a
,13-dihydrohelenalin acetate also impaired NF-
k
B DNA bind-
ing at a 200
m
M concentration and showed the same activity as
the respective unesterified compound. However, the methacry-
late and tiglinate, which are the dominant Sls in Spanish Arnica
flowerheads, only required the concentration of 100
m
M for com-
plete NF-
k
B inhibition without showing any cytotoxic effects
(see Fig. 3A and B).
NF-
k
B is required for maximal transcription of various cytokines.
Arnica preparations impair NF-
k
B DNA binding in an EMSA. To
investigate whether signal transduction steps downstream of
the transcription of cytokine genes are also inhibited, we eval-
uated whether the production of cytokines is also decreased by
Arnica tinctures. We focused on IL-1 and TNF-
a
. Mononuclear
cells from human blood were preincubated with the Spanish or
Arbo tincture at various concentrations and subsequently stimu-
lated with 1
m
g/ml LPS from Escherichia coli for 4 h. Supernatants
were collected and assayed in an ELISA. As cytokine release from
mononuclear cells is mediated via a p38 MAP kinase dependent
pathway, the pyridinylimidazole SB 203580, a potent and selec-
tive inhibitor of p38 MAP kinase, was used as a positive control
[15]. Both tinctures suppressed cytokine production in a concen-
tration-dependent manner at low concentrations (see Fig. 4). The
Arbo tincture was more active. A concentration of 0.12
m
l/ml was
required to induce a 50 % inhibition (IC
50
) of IL-1
b
. Higher con-
centrations were needed for suppression of TNF-
a
synthesis and
Table 1 Quantification of the phytopharmaceuticals for Sls by HPLC analysis (n = 3) using compound specific correction factors [10] and con-
centrations of phytopharmaceuticals from flowerheads of A. montana causing complete NF-
k
B or NF-AT inhibition. The amounts of the
main Sls are separately calculated. Values are given in mg/ml. The total amount is related to the main SI occurring, in Arnica tincture
from Spanish flowerheads and ArnikaGel
to 11
a
,13-dihydrohelenalin methacrylate, and in Arnica tincture from type Arbo flowerheads
to helenalin tiglinate
Arnica tincture
Spanish origin
ArnikaGel
Arnica tincture
Arbo
total content 0.70 mg/ml 0.22 mg/ml 0.56 mg/ml
DH-ac 0.04 0.01
DH-meacr 0.22 0.05 0.02
DH-tig 0.10 0.03 0.05
DH-i-val 0.06 0.02
H-ac 0.05
H-i-but 0.09
H-tig 0.08
complete NF-
k
B inhibition 10
m
l/ml 50
m
l/ml 5
m
l/ml
complete NF-AT inhibition 5
m
l/ml 2
m
l/ml
Fig. 2Aand B Effect of Arnica tinctures and ArnikaGel
on NF-
k
B
DNA binding. Lane 1 shows unstimulated control cells; lane 2, cells
treated with TNF-
a
alone. In the other lanes cells were pretreated for
1 h with the indicated amounts of Arnica tinctures or ArnikaGel
. Sub-
sequently, cells were stimulated with 200 U/ml TNF-
a
for 1 h. Equal
amounts of protein from cell extracts were analyzed for NF-
k
B activity
by EMSA. A filled arrowhead indicates the position of NF-
k
B DNA com-
plexes. The open circle denotes a non-specific activity binding to the
probe and the open arrowhead shows unbound oligonucleotide.
Fig. 3Aand B. Effects of 11
a
,13-dihydrohelenalin methacrylate, ti-
glinate and acetate, Sls isolated from flowerheads of A. montana,on
NF-
k
B DNA binding. Lane 1 shows unstimulated control cells; lane 2,
cells treated with TNF-
a
alone. In the other lanes cells were pretreated
for 1 h with various concentrations of the indicated SL. Subsequently,
cells were stimulated with 200 U/ml TNF-
a
for 1 h. Equal amounts of
protein from cell extracts were analyzed for NF-
k
B activity by EMSA. A
filled arrowhead indicates the position of NF-
k
B DNA complexes. The
open circle denotes a non-specific activity binding to the probe and
the open arrowhead shows unbound oligonucleotide.
Klaas CA et al. Studies on the ¼ Planta Med 2002; 68: 385 ± 391
Original Paper
388
Downloaded by: Roberto Della Loggia. Copyrighted material.
an IC
50
value of 0.38
m
l/ml was obtained. In contrast, concentra-
tions of 0.24 and 1.78
m
l/ml of the Spanish tincture were neces-
sary for a half maximal suppression of IL-1
b
and TNF-
a
, respec-
tively. The cell viability test by trypan blue exclusion was carried
out to determine whether the inhibitory effects of the Arnica
tinctures on cytokine release might be due to cytotoxic effects.
Up to a concentration of 1
m
l/ml no cytotoxicity was observed
with Arbo tincture whereas 0.1, 1.0 and 10.0
m
l/ml of the Spanish
tincture all caused a mortality rate of about 4%, which can be ne-
glected. Thus, it can be concluded that IC
50
values reported here
are not influenced by cytotoxicity.
Having shown that dihydrohelenalin esters with unsaturated
acyl moieties possess stronger inhibitory properties on NF-
k
B
DNA binding in EMSA than those compounds esterified with sa-
turated acids it was interesting to investigate whether this
behavior could also be observed in in vivo inflammatory models.
Therefore, the croton oil-induced mouse ear edema assay was
used as an established model of acute inflammatory response
[14]. The acetate and methacrylate of 11
a
,13-dihydrohelenalin
were tested at a dose of 1
m
mol/cm
2
, in comparison to the non-
steroidal anti-inflammatory drug indomethacin (0.2
m
mol/cm
2
)
which was used as a positive reference. The results obtained are
given in Table 2.11
a
,13-Dihydrohelenalin esterified with the un-
saturated methacrylic acid induced 77% edema inhibition and
exerted a stronger effect than the corresponding acetate deriva-
tive, which reduced the edematous response by 54 %. As expect-
ed, 0.2
m
mol/cm
2
of indomethacin reduced the edematous re-
sponse by 44 %.
Transcription of the cytokine TNF-
a
is regulated by the transcrip-
tion factor NF-
k
B and NF-AT [7]. Therefore, the tinctures were
also investigated for an influence on the activation of NF-AT. Jur-
kat T cells (5± 6 10
5
cells/ml) were incubated with tincture at
Fig. 4 Inhibitory effect of Arnica tinctures prepared from different Arnica flowers on IL-1
b
and TNF-
a
synthesis in human mononuclear cells stim-
ulated with LPS from Escherichia coli for 4 h. Supernatants were assayed in ELISAs. SB 203 580 was used as reference compound yielding IC
50
values
of 0.08 + 0.06
m
mol/l for IL-1
b
inhibition and 0.19 + 0.06
m
mol/l for that of TNF-
a
. All values are the mean of three/four independent measure-
ments.
Table 2 Effect of 11
a
,13-dihydrohelenalin acetate and methacrylate from Arnica flowers on the croton oil-induced mouse ear edema. Indome-
thacin was used as reference compound
Substance Dose
(
mm
mol/cm
2
)
No.
of animals
Edema
(in mg)
m S.E.
Edema
Inhibition (%)
control ± 10 7.0 0.3 ±
11
a
,13-dihydrohelenalin acetate 1.0 10 3.2 0.5* 54
11
a
,13-dihydrohelenalin methacrylate 1.0 10 1.6 0.3* 77
Indomethacin 0.2 10 3.9 + 0.4* 44
*p < 0.001 in the Student's t-test.
Klaas CA et al. Studies on the ¼ Planta Med 2002; 68: 385 ± 391
Original Paper
389
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various concentrations for 1 h prior to stimulation with ionomy-
cin and PMA for 3 h. Nuclear protein extracts were prepared and
analyzed for NF-AT DNA binding activity in an EMSA using an oli-
gonucleotide comprising the NF-AT binding sequence from the
GM-CSF promoter [12]. Stimulation with ionomycin and PMA in-
duced one novel DNA binding activity in Jurkat T cells (Fig. 5, lane
2). Competition assays identified this complex as NF-AT (data
not shown). NF-AT DNA binding was significantly reduced by
pretreatment of Jurkat T-cells with 2
m
l/ml of Arnica tincture
from Spanish flowerheads, while 5
m
l/ml completely prevented
NF-AT DNA binding (see Fig. 5, lanes 10 and 11). Arbo tincture
was nearly twice as active. Here no NF-AT could be detected at a
concentration of 2
m
l/ml (see Fig. 5, lane 5).
Discussion
Herbal remedies have become increasingly popular in recent
years. Therefore, it is not only important to prove their biological
activity, but also to identify their active components and eluci-
date their molecular mechanism of action. This can already be
applied to preparations from flowerheads of Arnica montana [2],
[3], [4], whose most active compounds are known to be sesqui-
terpene lactones [1].
Here an Arnica tincture was prepared from Arnica flowerheads
which originate from the easily and economically cultivated
type ªArboº. For the first time its content and composition of Sls
is described and correlated with its NF-
k
B inhibiting activity.
Moreover, these results are compared to those obtained from
the Spanish tincture. Each tincture should have its advantages.
While the Spanish one presumably causes fewer side effects like
contact dermatitis, for which compounds with an
a
-methylene-
g
-lactone ring are mainly responsible [1], the Arbo tincture pos-
sesses a stronger NF-
k
B inhibitory activity.
Moreover, three 11
a
,13-dihydrohelenalin esters being the main
occurring Sls in the Spanish tincture were studied for their inhi-
bitory NF-
k
B DNA binding activity and new insights in structure-
activity relationships were gained. Whereas we have previously
proposed that the most important structural elements for a po-
tent inhibitory activity are reactive centers, such as an exocyclic
methylene group and an
a
,û-unsaturated carbonyl group [16], a
further one, namely an
a
,û-unsaturated ester group has to be ad-
ded. The methacrylate and the tiglinate of dihydrohelenalin were
twice as active as dihydrohelenalin or its acetate. Studies are in
progress to elucidate the basis for this stronger activity.
Both Arnica tinctures were studied for the first time on their in-
fluence on cytokine production. It was shown that their content
and type of Sls correlate with their ability to inhibit cytokine re-
lease. Again, Arbo tincture inhibited IL-1 and TNF-
a
production
more effectively than the Spanish tincture. Both cytokines are
upregulated and/or disregulated in inflamed tissue and contrib-
ute directly or indirectly to the pathology in the synovial joint
tissue [17]. IL-1 causes activation of T- and B-lymphocytes and
induces chemotaxis of neutrophil granulocytes and other cells.
It stimulates proliferation of fibroblasts and upregulation of
COX-2, phospholipase A
2
and connective tissue degrading metal-
loproteinases, like collagenase. TNF-
a
has similar biological
functions to those of IL-1 [17]. Our results are in accordance
with recent reports demonstrating that Sls are able to inhibit
the cytokines TNF-
a
and IL-1 ([18], and lit. cited therein), which
are regulated by the transcription factor NF-
k
B.
A time-dependent cytokine production of IL-1 can also be ob-
served in the croton oil-induced mouse ear edema [19]. For this
reason, we compared the topical anti-inflammatory effect of
11
a
,13-dihydrohelenalin acetate and methacrylate, showing
that the latter compound was more active than the acetate deri-
vative (77 and 54 % oedema reduction, respectively, at a dosage of
1
m
mol/cm
2
). This result correlates well with that obtained in the
NF-
k
B EMSA and shows again the influence of the unsaturated
ester moiety.
TNF-
a
, which is believed to be one of the main proinflammatory
cytokines, has also binding sites in its promoter region for the
transcription factor NF-AT. Therefore, we extended our studies
on NF-AT and demonstrated that both tinctures were even more
active in the NF-AT than in the NF-
k
B assay. For both, no NF-AT
was detected at about half the concentration necessary for a
complete NF-
k
B inhibition. Studies are in progress to elucidate
how the effective ingredients of the tinctures, the Sls, inhibit the
DNA binding activity of NF-AT. Preliminary studies suggest a dif-
ferent mechanism than that proved for cyclosporin and FK506
(unpublished data). These immunosuppressive drugs inhibit the
phosphatase activity of calcium- and calmodulin-dependent cal-
cineurin [20]. However, they also inhibit calcineurin activity in
other tissues, which causes side effects, e. g. nephrotoxicity and
neurotoxicity. Therefore, drugs which act in another way as cy-
closporin and FK506 may be clinically preferable.
By inhibiting NF-
k
B and NF-AT and subsequently cytokine pro-
duction Arnica tinctures act in a similar way as glucocorticoids,
which also interfere with NF-AT and NF-
k
B±driven promoter ac-
tivity [21], [22]. For inhibition of NF-
k
B a model was proposed in
which glucocorticoids repress NF-
k
B-driven genes by interfering
with the mechanistic interaction of p65 with the basal transcrip-
tion machinery. Recently, we proved that Sls act directly on the
p65 subunit, too [3]. It has been suggested that therapeutic
agents directed at this protein might be useful, for instance in
Fig. 5 Effect of Arnica tinctures on NF-AT DNA binding. Lane 1 shows
unstimulated control cells. In lane 2 cells were treated with 2
m
M iono-
mycin and 100 ng/ml PMA for 3 h. In the other lanes cells were prein-
cubated with various amounts of Arnica tincture for 1 h. A filled arrow-
head indicates the position of the NF-AT DNA complex. The open circle
denotes a non-specific activity binding to the probe and the open ar-
rowhead shows unbound oligonucleotide.
Klaas CA et al. Studies on the ¼ Planta Med 2002; 68: 385 ± 391
Original Paper
390
Downloaded by: Roberto Della Loggia. Copyrighted material.
the treatment of inflammatory bowel or rheumatic disease [6].
Phytomedicines may also be used successfully for the treatment
of these inflammatory diseases, especially when their active
compounds can be defined as is the case with preparations from
Arnica montana flowers. However, it has also to be taken into ac-
count that the benefit of a therapy suppressing the activity of
central mediators of the immune response, such as NF-
k
B and
NF-AT, will depend on the delicate balance between suppressing
inflammation and interfering with normal cellular functions.
Acknowledgements
We thank Mrs. I. Hirschmüller-Ohmes for technical assistance in
HPLC analysis and isolation of sesquiterpene lactones and Mrs.
M. Weber for recording the GC/MS (both Institute of Pharmaceu-
tical Biology, Freiburg). We are grateful to the Kneipp-Werke,
Würzburg, Germany, for the supply of the Spanish Arnica tinc-
ture and ArnikaGel
.
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... Extracts prepared from the Arbo and Spanish varieties of Arnica montana L., rich in helenalin and dihydrohelenalin esters, inhibited IL-1β and TNF-α release in peripheral blood mononuclear cells (PBMCs), as well as the deoxyribonucleic acid (DNA)-binding of the transcription factors NF-κB and the nuclear factor of activated T-cells (NFAT) in Jurkat cells, both of which are responsible for the transcription of pro-inflammatory genes [23]. The Arbo variety was shown to be more effective than the Spanish one, a result attributed to the fact that the main SLs present in the former were helenalin-derivatives, as opposed to the predominant dihydrohelenalin-derivatives in the latter [23]. ...
... Extracts prepared from the Arbo and Spanish varieties of Arnica montana L., rich in helenalin and dihydrohelenalin esters, inhibited IL-1β and TNF-α release in peripheral blood mononuclear cells (PBMCs), as well as the deoxyribonucleic acid (DNA)-binding of the transcription factors NF-κB and the nuclear factor of activated T-cells (NFAT) in Jurkat cells, both of which are responsible for the transcription of pro-inflammatory genes [23]. The Arbo variety was shown to be more effective than the Spanish one, a result attributed to the fact that the main SLs present in the former were helenalin-derivatives, as opposed to the predominant dihydrohelenalin-derivatives in the latter [23]. These results underline the importance of the α-methylene-γ-lactone moiety in the bioactive potential of SLs. ...
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... These plant molecules therefore act against inflammation through their ability to inhibit the production of prostaglandins. Extracts of A.montana flowers have a long history of use in European traditional medicine as a remedy to treat several ailments (Willuhn,1998), including pain, injuries, hematomas, dislocations, sprains, bruising, edema associated with fractures, rheumatic muscle and joint complaints, inflammations of the mucous membranes of the mouth and throat, furuncles, inflamed insect bites and surface phlebitis (Klaas et al., 2002). (Klaas et al., 2002) have proved that the inhibition of the transcription factor NFkB (underline the importance of Arnica preparations as anti-inflammatory remedies. ...
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Arnica flowers from the European Compositae Arnica montana have been used in traditional medicine to treat a large variety of different ailments. It has been shown that the known diverse effects of the flowers correlate closely with the pharmacological and toxicological profile of their constituents helenalin, 11α,13-dihydrohelenalin and their esters, which therefore must be considered as their main active compounds. Methods for their qualitative and quantitative determination have been established and the variability in flowers from different geographical origin investigated. Arnica chamissonis ssp. foliosa is used as alternative source for the official "Arnicae flos." In addition to the sesquiterpene lactones of A. montana, they contain 2,3-dihydro- 2α-hydroxy- and 2,3-dihydro-2α-,4α-dihydroxy-helenanolide derivatives. The qualitative and quantitative variation is much larger than in the case of A. montana flowers, but populations exist equal to that of A. montana.
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The family of genuine NF-AT transcription factors consists of four members (NF-AT1 [or NF-ATp], NF-AT2 [or NF-ATc], NF-AT3 and NF-AT4 [or NF-ATx]) which are characterized by a highly conserved DNA binding domain (is designated as Rel similarity domain) and a calcineurin binding domain. The binding of the Ca2+-dependent phosphatase calcineurin to this region controls the nuclear import and exit of NF-ATs. This review deals (1) with the structure of NF-AT proteins, (2) the DNA binding of NF-AT factors and their interaction with AP-1, (3) NF-AT target genes, (4) signalling pathways leading to NF-AT activation: the role of protein kinases and calcineurin, (5) the nuclear entry and exit of NF-AT factors, (6) transcriptional transactivation by NF-AT factors, (7) the structure and expression of the chromosomal NF-AT2 gene, and (8) NF-AT factors in Th cell differentiation. The experimental data presented and discussed in the review show that NF-AT factors are major players in the control of T cell activation and differentiation and, in all likelihood, also of the cell cycle and apoptosis of T lymphocytes.
Article
Alcoholic extracts prepared form Arnicae flos, the collective name for flowerheads from Arnica montana and A. chamissonis ssp. foliosa, are used therapeutically as anti-inflammatory remedies. The active ingredients mediating the pharmacological effect are mainly sesquiterpene lactones, such as helenalin, 11 alpha,13-dihydrohelenalin, chamissonolid and their ester derivatives. While these compounds affect various cellular processes, current data do not fully explain how sesquiterpene lactones exert their anti-inflammatory effect. We show here that helenalin, and, to a much lesser degree, 11 alpha,13-dihydrohelenalin and chamissonolid, inhibit activation of transcription factor NF-kappa B. This difference in efficacy, which correlates with the compounds' anti-inflammatory potency in vivo, may be explained by differences in structure and conformation. NF-kappa B, which resides in an inactive, cytoplasmic complex in unstimulated cells, is activated by phosphorylation and degradation of its inhibitory subunit, I kappa B. Helenalin inhibits NF-kappa B activation in response to four different stimuli in T-cells, B-cells and epithelial cells and abrogates kappa B-driven gene expression. This inhibition is selective, as the activity of four other transcription factors, Oct-1, TBP, Spl and STAT 5 was not affected. We show that inhibition is not due to a direct modification of the active NF-kappa B heterodimer. Rather, helenalin modifies the NF-kappa B/I kappa B complex, preventing the release of I kappa B. These data suggest a molecular mechanism for the anti-inflammatory effect of sesquiterpene lactones, which differs from that of other nonsteroidal anti-inflammatory drugs (NSAIDs), indomethacin and acetyl salicylic acid.
Article
Many sesquiterpene lactones (SLs) possess considerable anti-inflammatory activity. They inhibit the transcription factor NF-κB by selectively alkylating its p65 subunit probably by reacting with cysteine residues. Here we assayed 28 sesquiterpene lactones for their ability to inhibit NF-κB. The majority of the potent NF-κB inhibitors possess two reactive centers in form of an α-methylene-γ-lactone group and an α,β- or α,β,γ,δ-unsaturated carbonyl group. Based on computer molecular modelling we propose a molecular mechanism of action, which is able to explain the p65 selectivity of the SLs and the observed correlation of high activity with alkylant bifunctionality. A single bifunctional SL molecule can alkylate the cysteine residue (Cys 38) in the DNA binding loop 1 (L1) and a further cysteine (Cys 120) in the nearby E’ region. This cross link alters the position of tyrosine 36 and additional amino acids in such a way that their specific interactions with the DNA become impossible. We also created a model for monofunctional SLs.