Anti-inflammatory and anti-oxidant potential of the root extract and constituents of doronicum austriacum

Article abstract of DOI:10.3390/molecules22061003

Background: Doronicum austriacum Jacq., Asteraceae, is a plant which is used in traditional alpine medicine. Historical sources describe the medical use of the root, but up until now only a few studies evaluated its pharmacological properties. The evaluation of the dichloromethane extract, and its major compounds for their anti-inflammatory and anti-oxidant potential was performed in macrophages J774A.1 and C6 astrocytes. Nitric oxide (NO) and reactive oxygen species (ROS) release, as well as nitrotyrosine formation, were evaluated. Moreover, in order to evaluate the potential anti-proliferative activity, under the same experimental conditions, 3-(4,5-dimethyltiazol-2yl)-2,5-phenyl-2H-tetrazolium bromide (MTT) assay was also performed. Our results indicate that Doronicum austriacum has a significant effect in inhibiting both pro-inflammatory and pro-oxidative mediators. All isolated compounds were able to significantly inhibit NO and ROS release both in macrophage and in astrocytes cells, even if the effect was more pronounced in macrophage. In articular, among the tested compounds, 6,12-dihydroxy-(?)-2S-tremetone exerted stronger activity. Both extract and single compounds did not affect cellular viability. This study provides evidence for the pharmacological anti-inflammatory and anti-oxidant potential of Doronicum austriacum extract. These effects could be due to the activity of its major constituents and subsequent identification of benzofurans as a promising compound class to combat inflammation and related diseases.

Full text of DOI:10.3390/molecules22061003

molecules
Article
Anti-Inflammatory and Anti-Oxidant Potential
of the Root Extract and Constituents of
Doronicum austriacum
Stefania Marzocco 1, Simona Adesso 1,2, Mostafa Alilou 3, Hermann Stuppner 3
and Stefan Schwaiger 3,
*
1
Department of Pharmacy, University of Salerno, Via Giovanni Paolo II 132, I-84084 Fisciano SA, Italy;
smarzocco@unisa.it (S.M.); sadesso@unisa.it (S.A.)
PhD Program in Drug Discovery and Development, University of Salerno, Via Giovanni Paolo II 132,
I-84084 Fisciano SA, Italy
Institute of Pharmacy, Pharmacognosy, Member of the CMBI, University of Innsbruck, CCB, Innrain 80-82,
6020 Innsbruck, Austria; mostafa.alilou@student.uibk.ac.at (M.A.); hermann.stuppner@uibk.ac.at (H.S.)
Correspondence: stefan.schwaiger@uibk.ac.at; Tel.: +43-512-507-58409
2
3
*
Academic Editor: Thomas J. Schmidt
Received: 11 May 2017; Accepted: 12 June 2017; Published: 16 June 2017
Abstract: Background: Doronicum austriacum Jacq., Asteraceae, is a plant which is used in traditional
alpine medicine. Historical sources describe the medical use of the root, but up until now only a few
studies evaluated its pharmacological properties. The evaluation of the dichloromethane extract,
and its major compounds for their anti-inflammatory and anti-oxidant potential was performed in
macrophages J774A.1 and C6 astrocytes. Nitric oxide (NO) and reactive oxygen species (ROS) release,
as well as nitrotyrosine formation, were evaluated. Moreover, in order to evaluate the potential
anti-proliferative activity, under the same experimental conditions, 3-(4,5-dimethyltiazol-2yl)-2,5-
phenyl-2H-tetrazolium bromide (MTT) assay was also performed. Our results indicate that
Doronicum austriacum has a significant effect in inhibiting both pro-inflammatory and pro-oxidative
mediators. All isolated compounds were able to significantly inhibit NO and ROS release both
in macrophage and in astrocytes cells, even if the effect was more pronounced in macrophage.
In articular, among the tested compounds, 6,12-dihydroxy-( )-2S-tremetone exerted stronger activity.
−
Both extract and single compounds did not affect cellular viability. This study provides evidence for
the pharmacological anti-inflammatory and anti-oxidant potential of Doronicum austriacum extract.
These effects could be due to the activity of its major constituents and subsequent identification of
benzofurans as a promising compound class to combat inflammation and related diseases.
Keywords: Doronicum austriacum; benzofurans; macrophages; astrocytes; inflammation; oxidative
stress; NO; ROS; nitrotyrosine
1. Introduction
Inflammation is a response triggered by tissue injury, as well as by microbial infection, and
it can lead to the risk of sepsis, multiple organ failure, and death. Inflammation is necessary to
eliminate aggressors, but it can be detrimental if the initial reaction is not limited. In these cases,
anti-inflammatory compounds are administered to control this response and are therapeutically
useful. Macrophages play a pivotal role in defending the host during the inflammatory response.
However, their excessive activation may contribute to extensive tissue damage. The activation of
these cells by pro-inflammatory stimuli, such as lipopolysaccharide (LPS) from gram-negative bacteria
like E. coli, induces the production of large amounts of mediators involved in the inflammatory
Molecules 2017, 22, 1003; doi:10.3390/molecules22061003
www.mdpi.com/journal/molecules

Molecules 2017, 22, 1003
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onset such as nitric oxide (NO), pro-inflammatory enzymes, and cytokines [
1]. Reactive oxygen
species (ROS) regulate many physiological responses in human health. Like inflammation, if not
properly regulated, the oxidative response could also lead to a number of harmful effects mediating
many aspects of inflammatory-induced tissue damage and dysfunctions [
neuro-inflammation also contribute to the pathogenesis of neuronal degeneration [
cell membrane damage from lipid peroxidation, changes in protein structure and function due to
protein oxidation, and structural DNA damage hallmarks of several neurodegenerative diseases [ ].
The central nervous system is particularly vulnerable to oxidative stress, possibly due to its high
oxygen demand, polyunsaturated fatty acids, and low levels of glutathione (GSH) [ 10]. Increasing
2,
3]. Oxidative stress and
4] and can cause
5–8
9
,
ROS production can exacerbate the expression of inflammatory mediators as has been detected in
patients with neurodegenerative disease [11]. Nowadays, considering the role of inflammation and
oxidative stress in many chronic diseases, the study of natural compounds with anti-inflammatory and
anti-oxidant potential has been of growing interest among scientists. Positive results of an in-house
screening showing nitric oxide (NO) inhibitory activity of the dichloromethane (DCM) extract of the
roots of D. austriacum prompted a detailed phytochemical and pharmacological (re)investigation [12].
Doronicum austriacum Jacq., Asteraceae, is a plant which is used in traditional alpine medicine.
Historical sources describe the use of root extracts as a vermicidal and laxative agent, but also in heart
strengthening preparation [13]. Up to now, only a few pharmacological studies [14] have evaluated the
pharmacological properties of Doronicum extracts. One described activity is a cytostatic effect on mice
fibroblasts by a currently not further characterized alkaloid enriched fraction (positive reaction on TLC
with Dragendorff-, Diazo- and modified Ehrlichs-reagent) of the roots of D. austriacum. Due to the
positive chemical reactions, the alkaloid fraction was suspected to consist of pyrrolizidine alkaloids,
which are widely distributed in the sunflower family.
2. Results
2.1. Identification of the Major Compounds
HPLC analysis of the DCM extract of the roots of Doronicum austriacum showed the presence of
three major compounds when monitored at 205 nm (Figure 1).
Figure 1. Chromatogramm of the HPLC-DAD analysis of the investigated dichloromethane (DCM)
extract of the roots of Doronicum austriacum (2.5 mg/mL MeOH) at 205 nm. Analytical conditions:
◦
stationary phase: Phenomenex Synergi Max-RP 80 Å, 4
µ
m (4.6 mm
×
150 mm); temperature: 35 C;
mobile phase: A = water + 0.025% TFA, B = acetonitrile; flow rate: 1.00 mL/min; detection: 205 nm;
0
0
0
0
injection volume: 10
µ
L; solvent composition during analysis: 0 : 75% A; 10 : 60% A; 30 : 30% A; 35 :
0
0
2% A; 45 : stop; post time: 10 .
Isolation and structure elucidation by LC-MS, 1- and 2D-NMR spectroscopy and optical
activity determination enabled the identification of compound
1
as 6,12-dihydroxy-( )-2S-tremetone,
−
compound as (S)-2-(5-acetyl-6-hydroxy-2,3-dihydrobenzofuran-2-yl)allyl isobutyrate, and compound
2

Molecules 2017, 22, 1003
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3
as 2-((S)-5-acetyl-6-hydroxy-2,3-dihydrobenzofuran-2-yl)allyl (S)-2-methylbutanoate (see Figure 2).
Although the root material was already investigated in 1970 [12], only derivatives bearing an
12-hydroxy-, or an 4,12-dihydroxy-benzofuran skeleton, were identified. are described in a later
1–3
published work dealing with constituents of D. pradalianches [15], incorrectly stating that those were
already isolated from D. austriacum [12]. A third manuscript of the same authors describing the
investigation of D. marcrophyllum [16] contains for the first time 1H-NMR-data of
3. Since no further
NMR data of compounds 1–3 are published, the obtained values are summarized in Table 1.
11
CH3
CH3
CH3
7
4
CH3
HO
O
O
HO
7a
3a
O
(S)
2
OH
HO
O
O
6
10
(S)
12
8
O
3
O
O
O
O
5
1
2
3
9
CH3
CH3
CH3
Figure 2. Chemical structure of compounds 1 to 3.
The stereochemistry of the benzofurane skeleton was predictable by the measured optical rotation:
[α]D20 [α]D20 [α]D20
8.06 (c 0.124, MeOH); 21.96 (c 0.970, MeOH); 4.76 (c 0.124, MeOH)
and comparison with literature values of related compounds [17]. In order to prove this hypothesis,
a comparison of calculated and experimental ECD spectra was performed. Since the experimental
spectra (see Figure 3; Figures S11 and S17) showed no differences, except some noise in the lower
1
,
:
−
2,
:
−
3,
:
−
wavelengths (compound
2, see Figure S11) due to the presence of minor impurities, an identical
absolute configuration at C-2 of all three compounds was assumed. Although compound
3
comprises
an additional chiral center adjacent to a weak chromophore-carbonyl group, the transitions of this
carbonyl group was found to be obscured by the strong chromophore (benzene ring) present in the
analyzed scaffold. In order to reduce the calculation time, the structure of compound
a representative for further calculations.
1 was selected as
Figure 3. Measured (acetonitrile) and calculated ECD-spectra of compound 1.
Therefore, the 3D structure of was subjected to a conformational analysis in H2O by Optimized
1
Potential for Liquid Simulations (OPLS-3) force filed. Conformers in an energy window of 2 Kcal/mol
were selected and geometrical optimization in gas phase (DFT/B3LYP/6-31G**) was performed.
Calculation of free energies indicated the presence of eight conformers of compound
1 (see Figure S18).
The calculated ECD spectrum of compound was in good agreement with the experimental spectrum
1
especially CEs at 275 nm, 240 nm, and 215 nm (see Figure 3), while the corresponding enantiomer
showed the opposite ECD (not shown). From this, absolute configuration of C-2 in the isolated three
compounds was established as S. The absolute configuration of the second chiral center of compound
3
was determined by a hydrolysis experiment, which showed a positive optical rotation for the free acid
which enabled its identification as (S)-(+)-2-methylbutyric acid.

Molecules 2017, 22, 1003
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1
Table 1. H (300.13 MHz) and 13C (75.48 MHz) NMR data of compounds 1 to 3 (in CDCl3, referenced to 0.01% TMS; δ in ppm; multiplicity; J in Hz in parenthesis).
Position
1H-NMR Data of 1
13C-NMR Data of 1
1H-NMR Data of 2
13C-NMR Data of 2
1H-NMR Data of 3
13C-NMR Data of 3
2
3
5.40 t (8.6)
Ha 3.36 ddd (0.9, 9.4, 15.4)
Hb 3.09 ddd (1.3, 7.8, 15.3)
85.3 d
33.6 t
5.36 t (8.4)
Ha 3.36 ddd (0.8, 9.5, 15.2)
Hb 3.10 ddd (1.1, 7.5, 15.3)
84.9 d
33.6 t
5.36 t (8.4)
Ha 3.35 ddd (0.8, 9.5, 15.2)
Hb 3.10 ddd (1.2, 7.6, 15.4)
84.9 d
33.7 t
3a
4
5
6
7
7a
8
9
—
118.5 s
126.9 d
113.8 s
—
118.3 s
126.7 d
113.9 s
165.6 s
98.3 d
166.0 s
202.0 s
26.2 q
—
7.49 t (1.2)
—
—
6.37 s
—
—
2.53 s
118.7 s
126.7 d
114.0 s
166.1 s
98.3 d
166.5 s
202.1 s
26.1 q
7.49 t (1.3)
7.50 t (1.3)
—
—
6.36 s
—
—
2.53 s
—
—
6.37 s
—
—
165.3 1
98.3 d
s
165.3 1
s
201.9 s
26.1 q
2.53 s
10
—
146.8 s
—
142.5 s
—
142.6 s
Ha 5.27 dq (2.1, 0.7)
Hb 5.25 dt (1.7, 0.9)
4.25 s
Ha 5.33 br s
Hb 5.27 br s
4.67 dd (6.7, 13.6)
12.95 s
Ha 5.33 br s
Hb 5.28 br s
4.67 dd (7.8, 13.5)
12.95 s
11
112.8 t
114.6 t
114.8 t
12
62.8 t
—
63.4 t
—
63.2 t
—
OH at C6
12.96 s
1’
2’
—
176.8 s
33.9 d
—
176.0 s
41.0 d
2.56 hept (7.0)
2.38 hex (7.0)
Ha 1.67 m
Hb 1.47 hept (7.0)
0.89 t (7.4)
1.14 d (7.0)
3’
1.16 d (7.0)
1.16 d (7.0)
18.7 q
18.7 q
26.6 t
4’
5’
11.8 q
16.4 q
1
Signals overlapping.

Molecules 2017, 22, 1003
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2.2. Anti-Inflammatory and Anti-Oxidant Potential of the D. austriacum Extract and Isolated Compounds in
J774A.1 Macrophages and in C6 Astrocytes
In macrophages, LPS triggers an inflammatory response that culminates in the release of
pro-inflammatory mediators. NO is considered a pleiotropic mediator that acts in a variety of
physiological and pathophysiological processes. When macrophages are activated by LPS, massive
amounts of NO are produced to exert a nonspecific immune response, from inducible nitric oxide
synthase (iNOS). Induced NO is a “final common mediator” of inflammation, but is also essential for
the up-regulation of the inflammatory response. In our experiments, a dichloromethane root extract
(5–50
µg/mL) of D. austriacum significantly reduced NO release (p < 0.001 vs. LPS alone; Figure 4A),
and iNOS expression in macrophages at all tested concentrations (p < 0.001 vs. LPS alone; Figure 4B).
Figure 4. Effect of Doronicum austriacum DCM root extract on nitrite release, index of NO production
), measured by Griess reaction, iNOS expression, evaluated by cytofluorimetry ( ), and on ROS
production ( ), measured by DCHF assay, in J774A.1 macrophages ( ) and C6 cells ( ). Data
are expressed as mean S.E.M.; *** denotes p < 0.001 vs. LPS (1 g/mL) or LPS + IFN- g/mL;
100 U/mL). L-NAME (1 M) was used as positive control for inhibition of nitrite release and iNOS
expression and inhibited nitrite production with a percentage of 49.00 2.08% and with a percentage
of 59.00 0.74% iNOS expression in J774A.1 macrophages. In C6 cells, L-NAME inhibited nitrite
release with a percentage of 39.33 4.37% and iNOS expression with a percentage of 46.21 2.65%.
N-acetyl cysteine (NAC; 10 mM) was used as positive control for ROS release inhibition, and gave
(
A
,D
B,E
C
,
F
A
–C
D–F
±
µ
γ (1 µ
µ
±
±
±
±
a percentage of inhibition of ROS production of 34.50
C6 cells, respectively.
±
0.50% in J774A.1 and of 43.33
±
1.45% in

Molecules 2017, 22, 1003
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ROS, as well as NO, generation in the inflammatory site is typically induced as part of a defensive
reaction intended to clear infectious and environmental threats, including microbial agents and
particulate material. Alternatively, ROS activation could act as a significant and adverse participant
in inflammatory disease. LPS (1
µg/mL) induced a significant ROS production in macrophages after
24 h. Treatment with the extract, at all tested concentrations (5–50
µg/mL), significantly, and in a
concentration-related manner, reduced ROS production in macrophages (p < 0.001 vs. LPS alone;
Figure 4C), thus also indicating its antioxidant effects. Neurodegenerative diseases are considered
a growing group of disorders, and evidence indicates that neuroinflammation and oxidative stress
contribute to pathogenesis in these diseases. Therefore, the anti-inflammatory and anti-oxidant
potential of the root extract (5–50
and ROS levels in LPS + IFN-
µ
g/mL) was evaluated for the impact on NO release, iNOS expression,
γ
stimulated astrocytes primarily involved in central nervous system
(CNS) homeostasis [18]. Our results indicate that the D. austriacum root extract significantly, and in
a concentration-related manner, inhibited NO release, iNOS expression, and ROS production in C6
astrocytes, at all tested concentrations (p < 0.001 vs. LPS alone; Figure 4D–F).
NO produced during inflammation also rapidly reacts with superoxide anion generating the
toxic metabolite peroxynitrite. Peroxynitrite is able to nitrate tyrosine residues in proteins, resulting
in the formation of nitrotyrosine. Because nitration of tyrosine is an alternative to phosphorylation
at key residues, it can affect a protein’s enzymatic activity and interfere with intracellular signaling
processes [19]. By immunofluorescence assay, we are able to show that D. austriacum root extract (25 and
50
to cell control (Figure 5A,B). LPS or LPS + IFN-
induced an increase of nitrotyrosine formation. When D. austriacum root extract (25 and 50
was added 1 h before and simultaneously with LPS or LPS + IFN- followed by an incubation
µ
g/mL) alone does not induce nitrotyrosine formation in both J774A.1 and C6 cells with respect
added to macrophages and astrocytes respectively
g/mL)
γ
µ
γ
for 24 h, a reduction of nitrotyrosine formation was observed in both cellular lines (Figure 5A,B).
This suggests that D. austriacum root extract could reduce nitrotyrosine formation in both macrophages
and astrocytes cells.
Figure 5. Effect of Doronicum austriacum DCM root extract (25 and 50
evaluated by confocal microscopy in J774A.1 macrophages ( ) and C6 astrocytes (
combination with LPS (1 µg/mL) or LPS + IFN-γ (1 µg/mL; 100 U/mL) after 24 h.
µ
g/mL) on nitrotyrosine formation
A
B
) alone, or in
The phytochemical investigation of the dichloromethane extract revealed three tremetone-derivatives:
6,12-dihydroxy-( )-2S-tremetone ( ), (S)-2-(5-acetyl-6-hydroxy-2,3-dihydrobenzofuran-2-yl)allyl
−
1

Molecules 2017, 22, 1003
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isobutyrate (
). The evaluation of those compounds for their anti-inflammatory and anti-oxidant potential indicate
a significant inhibitory activity at all tested concentrations (5–50 M) both on NO release, iNOS
expression, and ROS production, and in both J774A.1 (Figure 6A–C) and C6 cells (Figure 6D–F).
In particular, 6,12-dihydroxy-( )-2S-tremetone ( ) showed the highest activity in the performed
experiments and reduced the ROS production in macrophages at 10 M by approximately 50% while
in C6 cells a concentration of approximately 45 M was needed for the same reduction. Comparable
results were observed for the measured NO-release, where ca. 15 M are needed for 50% reduction of
NO-release in macrophages and ca. 25 M for the same reduction in C6 cells and for measured iNOS
expression, where ca. <5 M are needed for 50% reduction of iNOS-expression in macrophages and
ca. <5 µM for the same reduction in C6 astrocytes.
2) and 2-((S)-5-acetyl-6-hydroxy-2,3-dihydrobenzofuran-2-yl)allyl (S)-2-methylbutanoate
(3
µ
−
1
µ
µ
µ
µ
µ
Figure 6. Effect of compounds
on ROS production ( ), in J774A.1 macrophages (
are expressed as mean S.E.M.; *** denotes p < 0.001 vs. LPS (1
denote p < 0.001, p < 0.01, p < 0.05 vs. . òòò, òò, ò denote p < 0.001, p < 0.01,
. L-NAME (1 M), used as positive control for NO and iNOS inhibition gave a percentage
of inhibition of 49.00 2.08% for nitrite production of and a percentage of inhibition of 59.00 0.74%
of iNOS expression in J774A.1 macrophages. In C6 astrocytes, L-NAME gave a percentage of inhibition
of 39.33 4.37% and 46.21 2.65% of nitrite production and iNOS expression, respectively. NAC
1–
3
(5–50
µ
M) on nitrite release (
) and C6 astrocytes (
g/mL) or LPS + IFN-
A
,
B
), iNOS expression (
) after 24 h. Data
(1 g/mL;
C,D), and
E
,F
A
,
C
,
E
B,D,F
±
µ
γ
µ
◦◦
◦
100 U/mL). ◦◦◦
p < 0.05 vs.
,
,
2
3
µ
±
±
±
±
(10 mM) was used as positive control for ROS release and gave a percentage of inhibition of ROS
production of 34.50 ± 0.50% in J774A.1 macrophages and 43.33 ± 1.45% in C6 astrocytes.

Molecules 2017, 22, 1003
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In our opinion, the compound class of benzofurans seems to be a promising substance group
for further investigations, although some related derivatives are suspected to be harmful. Those
compounds, tremetone, 6-hydroxy-trementone, and further derivatives, can be found in white
snake-root (Ageratina altissima syn. Eupatorium rugosum, Asteraceae) and are suspected to cause trembles
in livestock and the so-called “milk sickness syndrome” in humans. A recent in vivo study in goats [20
investigated, beside other preparations, the in vivo effect (intra-ruminal application) of an n-hexane
extract containing 2.10 g/mg, 0.96 g/mg, and 0.53 g/mg of tremetone, 6-hydroxytremetone,
]
µ
µ
µ
and dehydrotremetone. The extract group showed no clinical signs, exercise intolerance, serum
enzyme changes, or histological lesions as observed for the snake-root plant dosed group. Therefore,
the authors concluded that there may be another compound or group of compounds that act either
independently or synergistically with tremetone, or other benzofurans, to cause the myotoxic effect of
white snake-root in goats. This study supports the investigations of Bowen et al., who showed that
tremetone is non-toxic (short term, 7d) to mice, rabbits, lambs, or chickens, while the compound affected
goldfish and insects [21]. In accordance with these results, our experiments showed, in the investigated
time interval and dosages, no signs of toxic effects in the performed MTT assay, which showed no
effect on macrophages and astrocytes proliferation (extract and by tested compounds; data not shown).
Therefore, the potential anti-inflammatory and anti-oxidant potential can not be ascribed to an effect
on cell viability.
3. Materials and Methods
3.1. Reagents
Unless stated otherwise, all reagents and compounds were purchased from Sigma Chemicals
Company (Sigma, Milan, Italy).
3.2. Cell Cultures
3.2.1. J774A.1 Murine Macrophages Cell Line
J774A.1 murine monocyte macrophage cell line (American Type Culture Collection, Rockville,
MD, USA), was grown adjacent to Petri dishes with Dulbecco’s modified Eagle’s medium (DMEM),
supplemented with 10% fetal calf serum (FCS), 25 mM HEPES, 2 mM glutamine, 100 U/mL penicillin,
and 100 mg/mL streptomycin at 37 ◦C in a 5% CO2 atmosphere. Macrophages J774A.1 were plated
in 96 well plates (2.0
with pure fresh medium or containing serial dilutions of D. austriacum root extracts (5–50
or isolated compounds (5–50 M) for 1 h, and then co-exposed to a final concentration of LPS
(1 g/mL) for further 18 or 24 h for NO, and ROS detection. L-NAME (1 M) and NAC (10 mM) were
used as positive controls.
×
104) and allowed to adhere for 4 h. Thereafter, the medium was replaced
g/mL)
µ
1–
3
µ
µ
µ
3.2.2. C6 Astroglial Cell Line
Glioma cells (C6) were obtained from American Type Culture Collection (ATCC, Manassas, VA,
USA). Cells were grown in DMEM, 10% FBS (mL/L), 2 mM L-glutamine and penicillin/streptomycin
◦
(100 units/0.1 mg/mL) at 37 C in 5% CO2 atmosphere. Cells were passaged at confluence using
a solution of 0.025% trypsin and 0.01% EDTA. C6 astrocytes were plated in 96 well plates (2.0
and allowed to adhere for 24 h. Thereafter, the medium was replaced with fresh medium alone or
containing serial dilutions of isolated compounds (5–50 M) for 1 h, and then co-exposed to a final
concentration of LPS (1 g/mL) plus Interferon-y (IFN 100 U/mL) for further 24 h in order to evaluate
NO and ROS release. L-NAME (1 µM) and NAC (10 mM) were used as positive controls.
×
104),
1–
3
µ
µ

Molecules 2017, 22, 1003
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3.2.3. Antiproliferative Assay
J774A.1 macrophages (5
plates and allowed to adhere for 4 h and 24 h, respectively. Thereafter, the medium was replaced
with fresh medium alone or containing serial dilutions of the extract or of the isolated compounds
×
104/well) and C6 cells (2.0
×
104/well) were plated on 96-well
1
–
3
(5–50
Cell viability was assessed through 3-(4,5-dimethyltiazol-2yl)-2,5-phenyl-2H-tetrazolium bromide
(MTT) assay as previously reported [22]. Briefly, 25 L of MTT (5 mg/mL) was added and the cells
were incubated for an additional 3 h. Thereafter, cells were lysed and the dark blue crystals solubilized
with 100 L of a solution containing 50% (mL/L) N,N-dimethylformamide, 20% (mL/L) sodium
µg/mL and 5–50 µM respectively), and incubation was performed for 24, 48 and 72 h.
µ
µ
dodecyl sulfate (SDS) with an adjusted pH of 4.5. The optical density (OD) of each well was measured
with a microplate spectrophotometer Titertek (Dasit, Cornaredo, Milan, Italy), equipped with a 620 nm
filter. C6 viability in response to treatment was calculated as: % dead cells = 100
control) × 100.
−
(OD treated/OD
3.2.4. Measurement of Intracellular ROS
ROS formation was evaluated by means of the probe 20,70-dichlorofluorescin-diacetate
(H2DCF-DA). H2DCF-DA is a non-fluorescent permeant molecule that passively diffuses into cells,
where the acetates are cleaved by intracellular esterases to form H2DCF, and thereby traps it within
the0cell. In the presence of intracellular ROS, H2DCF is rapidly oxidized to the highly fluorescent
0
2 ,7 -dichlorofluorescein (DCF). After cell treatment, previously reported, cells were collected, washed
twice with phosphate buffer saline (PBS), and then incubated in PBS containing H2DCF-DA (10 µM)
at 37 ◦C. After 15 min, fluorescence was evaluated using a fluorescence-activated cell sorting
(BD FacsCalibur, Milan, Italy) and elaborated with Cell Quest software, as previously reported [23].
3.2.5. Measurement of NO Release
NO generation was measured as nitrite (NO2−), index of NO released by cells, in the culture
medium of J774A.1 macrophages and C6 astrocytes 24 h after LPS, or LPS + IFN-γ, stimulation by
Griess reaction, as previously reported [24]. Briefly, 100 mL of cell culture medium were mixed with
100 mL of Griess reagent, equal volumes of 1% (w:v) sulphanilamide in 5% (v:v) phosphoric acid and
0.1% (w:v) naphtylethylenediamine–hydrogen chloride and incubated at room temperature for 10 min,
and then the absorbance was measured at 550 nm in a microplate reader Titertek (Dasit, Cornaredo,
−
Milan, Italy). The amount of NO2 in−the measured samples is expressed as
µM concentration,
which was calculated via a sodium NO2 standard curve.
3.2.6. iNOS Detection by Cytofluorimetry
J774A.1 macrophages and C6 astrocytes were plated into 96-well plates (5
×
104 cells/well) and
were allowed to grow for 24 h at 37 ◦C in a 5% CO2 atmosphere before experiments. Thereafter, the
medium was replaced with fresh medium, and cells were treated with serial dilutions of the extract
(5–50
µg/mL) or of the isolated compounds (5–50
µM) added 1 h before and simultaneously to LPS or
LPS + IFN-
γ
, respectively, for 24 h. Cells were collected, washed twice with PBS, incubated in Fixing
Solution for 20 min at 4 ◦C, and then incubated in Fix Perm Solution for 30 min at 4 ◦C. Anti-iNOS
(BD Laboratories, Milan, Italy) was added to J774A.1 and C6 cells for a further 30 min. The secondary
antibody was added in Fix Perm solution and cells were evaluated using a fluorescence-activated
cell sorting (FACSscan; Becton Dickinson, Milan, Italy) and elaborated with Cell Quest software,
as previously reported [25].
3.2.7. Nitrotyrosine Detection by Confocal Microscopy
J774A.1 macrophages and C6 cells (3
treated with serial dilutions of the extract or of the isolated compounds (5–50
×
105/well) were seeded on coverslips in 12 well plate and
g/mL and 5–50 µM
µ

Molecules 2017, 22, 1003
10 of 13
respectively) added 1 h before and simultaneously to LPS or LPS + IFN-
γ
for 24 h. Then, cells were fixed
with 4% paraformaldehyde in PBS for 15 min and permeabilized with 0.1% saponin (Sigma #47036) in
PBS for 15 min. After addition of BSA and PBS and a waiting time of 1 h, cells were incubated with
rabbit anti-nitrotyrosine (Millipore, Billerica, MA, USA) for 1 h at room temperature. The slides were
then washed with PBS three times and fluorescein-conjugated secondary antibody (FITC) was added
and left for 1 h. DAPI was used for counterstaining of nuclei. Coverslips were finally mounted in
mounting medium and fluorescent images were taken under the Laser Confocal Microscope (Leica TCS
SP5) as previously reported [26].
3.2.8. Data Analysis
Data are reported as mean
±
standard error of the mean (S.E.M.) values of at least three
independent experiments. Statistical analysis was performed by analysis of variance test, and multiple
comparisons were made by Bonferroni’s test by using Prism 6 (GraphPad Software, San Diego, CA,
USA). p-values smaller than 0.05 were considered significant.
3.2.9. Plant Material
The plant material used in this study was collected at two locations close to Wels, Austria
(herbarium number, Pharmacognosy, University of Innsbruck: MD 00-1848, 282.0 g; MD-2050, 254.0
g). The plants were in flowering stage and were identified by Dr. Michael Dobner. In order to obtain
a higher amount of starting material, both batches were combined and processed together.
3.2.10. Extract Preparation
The milled plant material (536.0 g) was mixed with 2 L dichloromethane and macerated at room
temperature for 24 h. Before further processing, the mixture was sonicated for 10 min and filtered.
The remaining plant material was further extracted as described before (in total five times), and the
combined solutions were evaporated to dryness to yield 10.02 g of DCM-extract.
3.2.11. Isolation
A part of the obtained DCM-extract (7.17 g) was separated by Sephadex-LH20 CC (GE Healthcare,
Uppsala, Sweden; 1.5 cm
×
80 cm) using DCM/acetone (85/15; v/v) as mobile phase. Eluate was
collected in tubes in portions of 8 mL, which were combined according to their composition indicated
by TLC (Merck, Darmstadt, Germany, silica gel 60 on aluminum, layer 0.2 mm, F254; No. 5554; mobile
phase: dichloromethane + ethyl acetate + acetic acid (5 + 0.5 + 0.1; v/v/v) at UV 366 nm. Fraction
3 (tubes 35 to 49, 3.93 g) was further separated by silica gel CC (silica gel 60; 40–63
µm, Merck,
Darmstadt, Germany; 100 g, 3 45 cm) using DCM/acetone mixtures with small amounts of acetone
×
(<2.5 v %) for elution. The fraction eluted with 610–790 mL (421.91 mg) was further separated
by semi-preparative HPLC (Dionex-system, UltiMate 3000, Dionex Softron GmbH, Germering,
Germany); stationary phase: Phenomenex Aqua C18 column (5
80% methanol, 20% water, isocratic; flow 2.00 mL/min; 40 ◦C; 50
of 52.74 mg/mL methanol). Separation of 4.5 mL afforded (Rt: 17.56–18.35 min; 88.64 mg; (S)-2-(5-
acetyl-6-hydroxy-2,3-dihydrobenzofuran-2-yl)allyl isobutyrate and (Rt: 21.66–22.51 min; 39.20 mg;
2-((S)-5-acetyl-6-hydroxy-2,3-dihydrobenzofuran-2-yl)allyl (S)-2-methylbutanoate. Fraction 5 of the
µ
m; 250
×
10 mm); mobile phase:
µ
L injection volume of a solution
2
3
initial Sephadex CC (tube 67–120; 334.64 mg) was further separated by silica gel CC (4.3 g, 1.2 × 43 cm
using DCM/acetone mixtures with increasing amounts of acetone. A fraction eluted with 300–980 mL
(53.89 mg) was further purified by semi-preparative HPLC (stationary phase: Phenomenex Aqua C18
)
column (5
µ
m; 250
×
10 mm); mobile phase: solvent A: water solvent B: acetonitrile, gradient elution:
L injection
0 min: 45% A; 16 min: 2% A; 21 min: 45% A; 30 min: stop; flow 2.00 mL/min; 40 ◦C; 150
µ
volume of a solution of 18.00 mg/mL methanol). The collection of the eluate at Rt 12.00–13.89 min of
20 injections afforded 26.51 mg of compound 1, 6,12-dihydroxy-(−)-2S-tremetone.

Molecules 2017, 22, 1003
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HPLC-MS analyses were carried out on a 1100 Agilent system (Agilent, Waldbronn, Germany)
using the following analytical conditions: stationary phase: Phenomenex Synergi Max-RP 80 Å, 4
(4.6 mm
150 mm); temperature: 35 ◦C; mobile phase: A = water + 0.9% formic acid and 0.1% acetic
acid, B = acetonitrile; flow rate: 1.00 mL/min; detection: 205 nm; injection volume: 10
µm
×
µL;0solvent
0
0
0
0
0
composition during analysis:0 : 75% A; 10 : 60% A; 30 : 30% A; 35 : 2% A; 45 : stop; posttime 10 . HPLC
was hyphenated via 1:5 split to an Esquire 3000 plus (Bruker Daltonics, Bremen, Germany) ion trap
using ESI in alternating mode with the following settings: temperature: 350 ◦C; dry gas: 10.00 L/min;
nebulizer 40 psi N2; full scan mode: m/z 100–1500. The optical rotations were determined with a
Perkin-Elmer 341 polarimeter (Wellesley, MA, USA) at 20 ◦C. 1D- and 2D-NMR-experiments were
performed on a Bruker DRX 300 (Bruker Biospin Rheinstetten, Germany) operating at 300.13 MHz
(1H) and 75.48 MHz (13C) at 300 K; NMR solvent: CDCl3 with 0.03% TMS (Eurisotop, Gif-Sur-Yvette,
France), which was used as an internal standard. ECD spectra were simulated using the 3D structure
of
1 which was forwarded to Schrödinger MacroModel 9.1 (Schrödinger LLC., New York, NY, USA) to
perform conformational analysis under OPLS-3 molecular mechanic force field in H2O. Conformers
in an energy window of 2 Kcal/mol distance from global minima were subjected to geometrical
optimization and energy calculation using DFT with B3LYP/6-31G** level of theory in gas phase with
Gaussian 09 (Gaussian 09, Revision A.02, Gaussian, Inc., Wallingford, CT, USA, 2016). Calculation of
excitation energy (nm), rotatory strength dipole velocity (Rvel) and dipole length (Rlen) were performed
by TDDFT/CAM-b3LYP/6-31G** in acetonitrile using SCRF (self-consistent reaction field) method
with the CPCM (conductor-like polarizable continuum). ECD curves were simulated on the basis of
rotatory strengths using SpecDis v1.6 [27] with half-band of 0.25 eV. ECD spectra were recorded in
acetonitrile at a Jasco J715 instrument (Easton, MD, USA).
For the hydrolysis experiment, 2.75 mg of compound
trifluoroacetic acid and heated in a closed glass container for 2 h to 90 C. Then, the cooled hydrolysate
was mixed three times with 1.0 mL of diethyl ether to recover the free alcohol part. The combined
3
was dissolved in 0.50 mL of 3 M aqueous
◦
organic layer was evaporated to dryness (yield: 2.01 mg, 99.3% of theoretical yield) and optical rotation
578 nm
was measured ([α]
:
−
32.83; c 1.005, MeOH); while the remaining water fraction was filled up to
20◦C
◦
2.00 mL with 3 M aqueous trifluoroacetic acid and measured directly (measured value +0.015 ; specific
rotation, calculation based on the yield of the alcohol part: [α]52078◦Cnm: +34.25; c 0.044, 3 M aqueous
trifluoroacetic acid).
4. Conclusions
This study provides evidence for the pharmacological anti-inflammatory and anti-oxidant
potential of Doronicum austriacum extract. Phytochemical investigations identified the three major
compounds of the DCM root extract as 6,12-dihydroxy-( )-2S-tremetone, (S)-2-(5-acetyl-6-hydroxy-
−
2,3-dihydrobenzofuran-2-yl)allyl isobutyrate , and 2-((S)-5-acetyl-6-hydroxy-2,3-dihydrobenzofuran-
2-yl)allyl (S)-2-methylbutanoate, which were analyzed the first time for their ability to lower NO
and ROS release. Termetone derivatives were identified as a promising compound class to combat
inflammation and related diseases.
Supplementary Materials: The following are available online: Figures S1–S17 and structures of conformers of
compound 1 used for ECD calculations.
Acknowledgments: The authors want to thank Simone Reisenzein for the technical support by the isolation
of compounds and Nora Engels for proofreading. The work was financially supported by the Commission of
the European Community, Framework Program 7, (PEOPLE-Industry-Academia Partnerships and Pathways)
Grant agreement no. 286287 NATPROTEC, FARB 2014 (ORSA147579) and by the Progetto Campania Research in
Experimental Medicine “CREME” (POR Campania FSE 2007–2013).
Author Contributions: S.M. and S.S. conceived and designed the experiments; S.M., S.S. and S.A. M.A. performed
the experiments; S.M. and S.S. analyzed the data; S.M., S.S and H.S. wrote the paper.
Conflicts of Interest: The authors declare no conflict of interest.

Molecules 2017, 22, 1003
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Sample Availability: Samples of the compounds 1 to 3 are available from the authors.
©
2017 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access
article distributed under the terms and conditions of the Creative Commons Attribution
(CC BY) license (http://creativecommons.org/licenses/by/4.0/).