Arctium minus crude extract presents antinociceptive effect in a mice acute gout attack model

Article abstract of DOI:10.1007/s10787-017-0384-6

Abstract: Gout is a disorder that triggers a severe inflammatory reaction which generates episodes of intense pain and discomfort to the patient. Arctium minus (Hill) Bernh. (Asteraceae) is known as “burdock” and displays anti-inflammatory, antinociceptive, against rheumatic pain and radical-scavenging activities. Species of the genus Arctium have been used in assistant therapy of gout and other inflammatory processes. We investigated the antinociceptive and anti-edematogenic effects of the crude extract of A. minus seeds in an acute gout attack model induced by intra-articular injection of monosodium urate (MSU) crystals in adult male Swiss mice (25–30?g). The crude extract of A. minus (100?mg/kg, p.o.) reduced the mechanical allodynia induced by the injection of MSU (1.25 mg/site, i.a.) from 4 until 8?h after its administration. A. minus seeds crude extract prevented mechanical allodynia at doses of 30 and 100?mg/kg, but not 10?mg/kg. Allopurinol (10?μg/mL) and A. minus crude extract (10–300?μg/mL) inhibited the xanthine oxidase activity in vitro. The A. minus?seeds crude extract did not cause adverse effects since did not change the toxicological parameters evaluated. A. minus crude extract can be used as an assistant therapy of gout pain, supporting its traditional use, without causing adverse effects.

Full text of DOI:10.1007/s10787-017-0384-6

Inflammopharmacol
DOI 10.1007/s10787-017-0384-6
Inflammopharmacology
ORIGINAL ARTICLE
Arctium minus crude extract presents antinociceptive effect
in a mice acute gout attack model
1
,2
1,2
1,2
•
Susana Paula Moreira Fischer
3
•
Indiara Brusco
3
•
Camila Camponogara
3
Mariana Piana
,4
•
Henrique Faccin
Sara Marchesan Oliveira
•
Luciana Assis Gobo
1,2,5
•
Leandro Machado de
3
Carvalho
•
Received: 21 June 2017 / Accepted: 1 August 2017
Ó Springer International Publishing AG 2017
Abstract Gout is a disorder that triggers a severe inflam-
matory reaction which generates episodes of intense pain
and discomfort to the patient. Arctium minus (Hill) Bernh.
extract of A. minus (100 mg/kg, p.o.) reduced the
mechanical allodynia induced by the injection of MSU
(1.25 mg/site, i.a.) from 4 until 8 h after its administration.
A. minus seeds crude extract prevented mechanical allo-
dynia at doses of 30 and 100 mg/kg, but not 10 mg/kg.
Allopurinol (10 lg/mL) and A. minus crude extract
(10–300 lg/mL) inhibited the xanthine oxidase activity
in vitro. The A. minus seeds crude extract did not cause
adverse effects since did not change the toxicological
parameters evaluated. A. minus crude extract can be used as
an assistant therapy of gout pain, supporting its traditional
use, without causing adverse effects.
(
Asteraceae) is known as ‘‘burdock’’ and displays anti-in-
flammatory, antinociceptive, against rheumatic pain and
radical-scavenging activities. Species of the genus Arctium
have been used in assistant therapy of gout and other
inflammatory processes. We investigated the antinocicep-
tive and anti-edematogenic effects of the crude extract of
A. minus seeds in an acute gout attack model induced by
intra-articular injection of monosodium urate (MSU)
crystals in adult male Swiss mice (25–30 g). The crude
&
Sara Marchesan Oliveira
saramarchesan@hotmail.com
1
Neurotoxicity and Psychopharmacology Laboratory, Center
of Natural and Exact Sciences, Federal University of Santa
Maria, Santa Maria, RS, Brazil
2
3
Graduate Program in Biological Sciences: Biochemistry
Toxicology, Federal University of Santa Maria, Santa Maria,
RS, Brazil
Graduate Program in Pharmaceutical Sciences, Center of
Health Sciences, Federal University of Santa Maria (UFSM),
Santa Maria, RS, Brazil
4
5
Department of Chemistry, Federal University of Santa Maria
(UFSM), Santa Maria, RS, Brazil
Department of Biochemistry and Molecular Biology, Federal
University of Santa Maria, Av. Roraima 1000, Camobi,
Santa Maria, RS Zip Code: 97105-900, Brazil
123

S. P. M. Fischer et al.
Graphical Abstract
Keywords Joint Á MSU Á Xanthine oxidase Á Antioxidant Á
Pain Á Burdock
deposition occurs due to an excess of circulating uric acid
(greater than 6.8–7.0 mg/dL) by under-excretion or over-
production, which can be easily determined in most
patients and it is a predictive factor for the development of
gout (Khanna et al. 2012; Zamudio-Cuevas et al. 2015).
Previous disease, mechanical trauma, low temperatures,
and underlying osteoarthritis make the joint susceptible to
MSU crystals, whose repetitive accumulation forms tophi
deposition (called tophaceous gout) (Roddy et al. 2007).
The MSU crystals are originate from uric acid, an end-
product of purine metabolism by the action of the xanthine
oxidase enzyme (Richette and Bardin 2010).
Abbreviations
MSU
AST
ALT
Monosodium urate
Aspartate aminotransferase
Alanine aminotransferase
NSAIDs Non-steroidal anti-inflammatory drugs
DPPH
PWT
1,1-Diphenyl-2-picrylhydrazyl
Paw withdrawal threshold
The non-soluble MSU crystals, formed when urate
concentrations exceed its solubility limit, trigger the for-
mation of the interleukin-1b, which will activate their
receptors. This activation induces the production of
chemokines and cytokines and the recruitment of inflam-
matory cells, mainly neutrophils, which plays a crucial role
in inflammatory hypernociception (Richette and Bardin
Introduction
The gout is an arthritis form characterized by an intense
inflammatory reaction caused by the deposition of mono-
sodium urate (MSU) crystals in tissues such as the joints,
usually in the big toe or ankle (Choi et al. 2005). The MSU
1
23

Arctium minus crude extract presents antinociceptive effect in a mice acute gout attack model
2
010; Cronstein and Esserman 2013; Martinon et al. 2006).
January (2015), and a dried voucher specimen is preserved
in the herbarium of the Department of Biology at Federal
University of Santa Maria (register number, SMBD
16046). The seeds (50 g) were removed carefully from
flowers and placed to macerate in 70% ethanol for a week,
with a daily shake-up. After filtration, the solvent was
renewed each week during 1 month. The hydroalcoholic
extract was evaporated under reduced pressure to remove
the ethanol; the remaining aqueous was dried (temperature
below 40 °C) to give the extract.
Thus, an acute gout attack induces the development of
swelling, erythema, and temperature elevation of affected
joint, with severe pain perception, which is a significant
experience of patients with acute gouty arthritis and
responsible for the reduction of their quality of life
(
Richette and Bardin 2010; Terkeltaub 2010).
Among the first-line agents for the treatment of acute
attacks of gout are non-steroidal anti-inflammatory drugs
NSAIDs) or colchicine, an alkaloid derived from the
(
autumn crocus (Colchicum autumnale). However, the use
of NSAIDs is associated with gastrointestinal toxicity and
cardiovascular risk (Cronstein and Esserman 2013; Zhang
et al. 2006), while higher dosages of colchicine can cause
blood dyscrasias, neuromuscular toxicity, and gastroin-
testinal adverse effects (Cronstein and Esserman 2013;
Salvo et al. 2011). Allopurinol, a xanthine oxidase inhi-
bitor, is the main agent used to reduce the hyperuricemia,
although it also can cause adverse reactions including
pruritus and rash (Khanna et al. 2012; Stamp et al. 2015).
The search for new analgesic molecules that combine
high efficacy without inducing adverse effects is the main
objective of the research in the field of pain and analgesia
Drugs and reagents
Synthetic MSU crystals were prepared as described pre-
viously (Hoffmeister et al. 2011; Silva et al. 2013). First,
4 g of uric acid (Vetec, Brazil) was dissolved and heated in
800 mL of ultra-distilled H O (Milli-Q) adjusted to pH 8.9
2
with NaOH (9 mL, 0.5 N) at 60 °C, cooled overnight in a
refrigerator, and then filtered and dried. The needle-like
crystals were recovered and suspended in phosphate-buf-
fered solution (PBS, 10.71 mM K HPO , 6.78 mM
2
4
NaH PO , and 120.4 mM NaCl; pH 7.4). Polarized light
2
4
microscopy was used to confirm that the crystals were rod-
shaped and varied in length (12 ± 2 mm). The preparation
was endotoxin-free, as determined by an amebocyte cell
lysate assay (Sigma, St Louis, MO, USA). The crude
(
Cocco et al. 2003). Natural products have been used in the
process of discovery of new drugs with therapeutic rele-
vance, including analgesic molecules (Koehn and Carter
Ò
2
005; Calixto et al. 2000). Arctium minus (Hill) Bernh. is a
plant which belongs to the family Asteraceae, known as
‘burdock’’ or ‘‘lesser burdock’’, and is a species native to
extract of A. minus was diluted into Tween 80 (polysor-
bate 80, 5%) and saline (0.9% NaCl, 95%) for oral
administration (p.o.). Allopurinol was purchased from
Sigma (USA), dissolved in saline and was also adminis-
tered by oral route. Indomethacin was also purchased from
‘
Asia minor that has spread throughout North America and
Asia (Erdemoglu et al. 2009; Robbins 2013). The A. minus
leaves are used against rheumatic pain, fever, and sun-
stroke in Turkish folk medicine. Furthermore, A. minus
displays anti-inflammatory, antinociceptive, and radical-
scavenging activities (Erdemoglu et al. 2009; Fujita et al.
Ò
local suppliers, but diluted in DMSO (10%), Tween 80
(10%), and saline (0.9% NaCl, 80%), and it was also
administered by oral route. In addition, xanthine oxidase
(XO) from bovine milk and pterin was purchased from
Sigma (USA).
1
995). Species from Arctium genus has been used as an
assistant therapy for the treatment of gout, hypertension,
arteriosclerosis, and other inflammatory diseases (Chan
et al. 2011).
Acetonitrile and methanol were of Chromasolv LC–MS
grade and supplied by Sigma-Aldrich (St. Louis, MO,
United States). Acetic acid was obtained from Sigma-
Aldrich (St. Louis, USA).
Since natural products have been biologically evaluated
for their therapeutic potential, we verified the effect of the
oral treatment with preparations from the A. minus seed
crude extract on antinociceptive and anti-edematogenic
effects in a model of acute gout attack induced by intra-
articular injections of MSU.
The standards (?)-catechin, 3-acetyl coumarin, 3,6-di-
hydroxyflavone, 4-hydroxycoumarin, 6-hydroxycoumarin,
apigenin, chlorogenic acid, chrysin, fisetin, galangin, gallic
acid, kaempferol, luteolin, myricetin, p-coumaric acid,
quercetin, quercitrin, trans-resveratrol, rosmarinic acid,
rutin, trans-cinnamic acid, and vanillic acid were obtained
from Sigma-Aldrich (St. Louis, MO, USA). Caffeic and
ferulic acids were obtained from Fluka Analytical (Buchs,
Switzerland). All the standards were of analytical grade
with a minimum of 95% purity and were used as received.
Materials and methods
Plant collection and extraction
-
1
Stock solutions of the phenolic standards (1000 mg L
)
Flowers of A. minus were collected in Maximiliano de
were prepared by dissolution of appropriate amounts of
substances in LC–MS grade methanol.
Almeida (The Rio Grande do Sul State of Brazil) in
123

S. P. M. Fischer et al.
The triterpenoids such as arjunic acid, maslinic acid,
betulinic acid, ursolic acid, erythrodiol, lupeol, b-amyrin,
a-amyrin, friedelin, sitosterol, stigmasterol, and campes-
terol were purchased from Fluka and Sigma-Aldrich (St.
Louis, MO, United States).
2 mL of MeOH. Just before the chromatographic analysis,
the eluate obtained from the SPE procedure was diluted in an
MeOH:0.2% CH COOH (1:1, v/v) solution. The standard
3
addition method was used to quantify the samples. The
calibration curves were built with seven equally spaced
concentration levels, in addition to a blank extract. The data
points of the calibration curve were determined in triplicate.
The phenolic compounds were determined by the method
previously developed by Silveira et al. (2016). This method
uses a gradient elution composed of 0.1% acetic acid in
water (A) and acetonitrile (B) as the mobile phase at a
Arctium minus analyses
UHLC-MS Chromatograph
An Agilent 1260 Infinity UHLC–MS chromatograph
-
1
(
Santa Clara, CA, United States) with automatic injection
constant flow rate (0.8 mL min ) according to the fol-
lowing elution program: 8.0% (0.00–0.10 min);
8.0–25.8% (0.10–3.45 min); 25.8–54.0%
and an Agilent 6430 triple quadrupole mass spectrometer
was used. High-purity nitrogen (99.999%) obtained from
Linde (Munich, Germany) was used as the gas for inducing
collision at ESI source and as the drying gas. A Zorbax SB-
C18 Rapid Resolution HD column (2.1 9 50 mm, 1.8 lm,
Agilent) was used at a temperature of 40 °C. Ultrapure
water was obtained from a Milli-Q Synergy UV (Merck
Millipore, Darmstadt, Germany) system.
B
B
B
(3.45–6.90 min); 54.0–100.0% B (6.90–7.00 min); and
100.0% B (7.00–9.00 min). The injection volume was 5 lL,
and the injected aliquots were acidified to a final concen-
tration of 0.1% acetic acid (v/v).
Determination of triterpenic compounds
Instrumentation, reagents, and solution
Approximately 50 mg of the dried extract was resuspended
in 5 mL of 50% acetonitrile in H O (v/v) and vortexed for
2
The standard solutions of each triterpenic compound as
well the mixtures were prepared in acetonitrile as follows:
30 s followed by 10 min sonication. This 1% extract was
diluted 20 times in acetonitrile, filtered through a 0.2 lm
PTFE filter, and injected into the chromatographic system.
The triterpenic compounds were determined by the method
previously developed by Silveira et al. (2016) in which
each compound was individually injected into the mass
spectrometer to obtain its spectrum and also to optimize the
fragmentor and the energy of the collision cell. This
method uses APPI ion source and toluene as a dopant. The
determination of triterpenes was performed using a gradi-
ent elution that consisted initially of 70% acetonitrile/water
-
1
1
000 mg L for maslinic acid, arjunic acid, b-amyrin, a-
-
1
amyrin, and erythrodiol; 985 mg L
-
for ursolic acid;
-1
1
1
25 mg L
for campesterol; 196 mg L
for betulinic
-
acid; 378 mg L for sitosterol; 365 mg L for lupeol;
1
-1
-
1
-1
4
81 mg L
for stigmasterol; and 500 mg L
for
friedelin.
All the solutions were stored in ambar glass recipients at
-30 °C until their use. Working solutions of the studied
phenolic and terpenic compounds were prepared by dilu-
-
1
tion of the stock solutions in the respective solvents.
over 4.5 min. The flow rate was 0.6 mL min during the
-
1
first 4.5 min and was increased to up to 0.8 mL min in
5 min, and remained at this rate until the end of the run.
The injection volume was 5 lL. A post-run period of 4 min
was adopted to re-equilibrate the system.
Determination of phenolic compounds
Around 60 mg of this powdered extract was resuspended in
3
mL of methanol, sonicated until complete dissolution, and
diluted with 12 mL of ultrapure water and next acetic acid
was added to a final proportion of 0.1% (v/v). Then, the
extract was filtered through hydrophilic PTFE membranes
with a 0.2 lm pore size. The final extracts obtained were
submitted to a cleanup step using solid-phase extraction
Antioxidant capacity
The antioxidant capacity of the A. minus extract was
evaluated according to the spectrophotometric assay
described by Piana et al. (2016). The extract was evaluated
at six ethanol dilutions (7.81, 15.62, 31.25, 62.50, 125, and
250 lg/mL). Each dilution was mixed with 1.0 mL of
DPPH 0.3 mM in ethanol solution. After 30 min, the
absorption was assessed at 518 nm. Ascorbic acid was used
as positive control, and a solution of DPPH (1 mL,
0.3 mM) in ethanol (2.5 mL) was used as negative control.
The assay was performed in triplicate, and the evaluation
of the antioxidant capacity followed the equation:
(
SPE). The Strata C18-E cartridges (Phenomenex, Torrance,
USA), 500 mg, 3 mL, were conditioned with 6 mL of
MeOH:0.2% CH COOH (1:1, v/v) and equilibrated with
3
6
2
mL of 0.1% CH COOH (v/v) in water. A fixed volume of
3
mL of the obtained extract with a final MeOH:H O:CH
2
3-
COOH composition of 20:80:0.1 (v/v) was percolated with a
-
mL min flow rate, followed by washing with 2 mL of
1
2
0
.1% CH COOH. Finally, the cartridge was eluted with
3
1
23

Arctium minus crude extract presents antinociceptive effect in a mice acute gout attack model
glass boxes individually (7 9 9 9 11 cm) on an elevated
ðABSsampleÀABSblankÞ Â 100 ;
control
%
inhibition ¼ 100À
platform wire mesh to allow access to the plantar surface of
the hind paws. Von Frey calibrated filaments of increasing
stiffness (0.02–10.0 g) were applied to the plantar surface
of the left hind leg of mice. The absence of a paw with-
drawal after 5 s from the application of the filament led to
the use of a filament with increasing stiffness. Paw lifting
against the stimulus applied indicated a positive response
to paw withdrawal and led to the use of a filament of a
lesser stiffness. This paradigm continued until a total of six
measurements or even four consecutive negative or posi-
tive responses. The mechanical paw withdrawal threshold
Abs
where Abssample is absorbance of each fraction, Absblank is
absorbance of fractions without adding the DPPH, and
Abscontrol is absorbance the solution of ethanol in DPPH.
The scavenging concentration required to inhibit 50% of
the DPPH in the assay medium was expressed in SC50 (lg/
mL).
Animals
The experiments were conducted using adult male Swiss
mice (25–30) maintained under controlled temperature
(
PWT) was calculated as described by Dixon (1980),
expressed in grams (g) and evaluated before and several
times after injection of MSU or treatments. A significant
decrease in PWT compared to baseline values was con-
sidered as mechanical allodynia.
(22 ± 1 °C) in light/dark cycle of 12 h, with free access to
food and tap water. The experiments were performed in
accordance with national and international legislation
(
Guidelines of Brazilian Council of Animal Experimenta-
The basal mechanical threshold (baseline 1) of the ani-
mals was first evaluated with von Frey filaments. Then, one
group of animals received the crude extract of A. minus
tion—CONCEA—and of U.S. Public Health Service’s
Policy on Humane care and Use of Laboratory Animals—
PHS Policy). All animal experiments also complied with
the ARRIVE guidelines and the National Institutes of
Health guide for the care and use of Laboratory animals
(100 mg/kg, p.o.) or vehicle (10 mL/kg, p.o.) by the oral
route. One hour after oral treatment, the intra-articular
injection of MSU crystals was performed. The mechanical
threshold was assessed from 1 up to 24 h after MSU
injection (time–response curve). To calculate the 50%
effective dose (ED ) of oral treatment with A. minus
(
NIH Publications No. 8023, revised 1978). The protocols
were approved by Ethics Committee for Animal Research
of the Federal University of Santa Maria (process number
5
0
1
946180116/2016). The number of animals and noxious
extract was performed a dose–response curve (10, 30, or
00 mg/kg, p.o) at 8 h after MSU injection.
stimulus intensities used was the minimum necessary to
demonstrate consistent treatment effects.
1
MSU-induced spontaneous nociception The spontaneous
nociception behavior was observed immediately before
starting the measurements of mechanical allodynia, and
it was evaluated through of guarding behavior of the
hind paw (Silva et al. 2016). According to the position
of the hind paw at the time of the observation, a score of
0, 1, 2, or 3 was assigned. The score 1 was considered
when the paw pressure was slightly reduced, with the
paw completely on the floor, but the toes are not spread;
score 2 was considered when the paw pressure was
moderately reduced, with only some parts of the paw
slightly touching the floor, and finally, the score 3 was
used when the paw was completely removed from the
floor.
Induction of acute gout by MSU crystals injection
An arthritic gout model was induced by intra-articular
injection of MSU crystals according to Silva et al. (2016)
to study the effect of A. minus on acute gout attack. An
injection of MSU suspension (1.25 mg/site; 20 lL) or
vehicle (saline 0.9%; 20 lL) was performed into the tibial–
tarsal joint (ankle) left in animals anesthetized with
isoflurane. For verification of the analgesic and anti-ede-
matogenic effect of A. minus, the animals were treated
orally with vehicle (95% saline 0.9% ? 5% Tween 80;
1
0 mL/kg, p.o.) or the crude extract of A. minus (10, 30, or
1
00 mg/kg, p.o.). The edema and nociceptive parameters
were evaluated until when necessary to verify the consis-
tent effects of treatment with A. minus.
Edema measurement The formation of edema was eval-
uated as an increase in the injected ankle thickness [in
millimeters (mm)], and it was measured using a digital
caliper before and at several times after MSU injection
(Hoffmeister et al. 2011). The edema evaluation was
observed after the measurements of the mechanical allo-
dynia. The results were expressed as the difference
Nociceptive and inflammatory parameters
Mechanical allodynia The mechanical allodynia measure
was evaluated using the paradigm of up and down (Oliveira
et al. 2016). The mice were acclimated first in transparent
123

S. P. M. Fischer et al.
between the value of the ankle thickness after the injection
of MSU and the baseline measurement.
variation in body temperature of the animals about basal
temperature was assessed.
Activity of xanthine oxidase
Biochemical markers
The activity of xanthine oxidase enzyme was determined in
according to Silva et al. (2016). Xanthine oxidase (0.5 U/mL)
was incubated with its substrate, pterin (20 mM) and A. minus
crude extract (10–300 lg/mL), allopurinol (10 lg/mL) or
vehicle, boiled, and centrifuged. The supernatant was diluted
in sodium acetate buffer (0.1 M, pH 5.5). The xanthine oxi-
dase activity was assessed using a spectrofluorometer, and the
fluorescence was monitored at an excitation wavelength of
To evaluate the possible biochemical changes caused by
the A. minus crude extract, the animals were treated with
the crude extract (100 or 2000 mg/kg, p.o.), indomethacin
(100 mg/kg, p.o.), or vehicle (10 mL/kg, p.o.). After 8 h or
14 days of the oral treatments, the animals were eutha-
nized, and the serum was collected for the biochemical
analysis. The AST (aspartate aminotransferase) and ALT
(alanine aminotransferase) activities and urea, creatinine,
and glucose levels were evaluated spectrophotometrically
according to the standard procedures provided by com-
mercially available kits (Labtest, Red Lake, MG, Brazil).
3
47 nm and an emission wavelength of 405 nm.
Adverse effect evaluation
We evaluated the possible adverse effects caused by A. minus
Gastric lesion assessment
seed crude extract at 8 h after its administration at the dose of
1
00 mg/kg (p.o.) and in other group of animals at 14 days
For the gastric lesion test, the animals received oral
administration of the A. minus seed crude extract (100 or
2000 mg/kg, p.o.), indomethacin (100 mg/kg, p.o.), or
vehicle (10 mL/kg, p.o.). After 8 h or 14 days of treatment,
they were euthanized and their stomachs removed. The
quantification of the lesions was evaluated with the aid of a
magnifying glass and was carried out by assigning a score
according to the number and size of lesions on a scale from
0 up to 6 points (Oliveira et al. 2009).
after its single administration at the dose of 2000 mg/kg as
preconizedbytheOECD Guideline423(2001).Indomethacin
(
100 mg/kg, p.o.) was used as a positive control.
Open-field and rotarod tests
To check the possible adverse effects (sedation, hyperactivity,
and loss of motor coordination) of the A. minus seed crude
extract, the animals were subjected to evaluationofforced and
spontaneous locomotor activity (Trevisan et al. 2012). These
tests were evaluated at 8 h or 14 days after the oral treatment
with A. minus crude extract (100 or 2000 mg/kg, p.o.,
respectively), indomethacin (100 mg/kg, p.o.) or vehicle
Statistical analyses
The results are expressed as the mean ± standard error of the
mean (SEM), except for the inhibitory doses (ID ) or inhi-
5
0
(
10 mL/kg). For the forced locomotor activity was used the
bitory concentration (IC ) values, which were expressed as
50
rotarod test, and the animals were trained to remain in the
revolving cylinder (3.7 cm in diameter, 8 rpm) for 60 s
withoutfalling,1 daybeforethe treatment. Onthe testday,the
animals were evaluated for 4 min, and the number of falls
during this periodwas monitored. The spontaneous locomotor
activity was evaluate using the open field test, consisting of
glass box measuring 40 9 60 9 50 cm, wherein the floor of
theboxwasdividedinto12equalsquares.Thus, thenumberof
areas crossed with all paws, and the number of rearing was
counted during 5 min.
geometric means accompanied by their respective 95% con-
fidence limits. Gastric lesion and spontaneous nociception
scores were reported as medians followed by their 25th and
75th percentiles. All data were analyzed by Student’s t test,
one-wayortwo-wayanalysesofvariance(ANOVA)followed
by Bonferroni’s post hoc test, Tukey’s post hoc, Kruskal–
Wallis test, or Student Newman–Keuls test when appropriate,
using the GraphPad Prism6.0 software (San Diego California,
USA). P \ 0.05was consideredasstatistically significant. No
statistical methods were used to predetermine group sizes, but
our group sizes are similar to those reported in previous
publications in the field.
Body temperature
To evaluate the phytochemical composition was used a
calibration curve, and the experimental values were
expressed as mean ± SEM (n = 3). Antioxidant activity
values of SC50 (scavenging concentration required for to
inhibit the DPPH radical or decrease carbonyl protein
levels in 50%) were obtained by linear regression and
expressed as mean ± SEM.
The body temperature of the animals was evaluated with a
digital thermometer (Oliveira et al. 2009) and recorded
before of oral administration (baseline 1) of the A. minus
crude extract (100 or 2000 mg/kg, p.o.) or vehicle (10 mL/
kg, p.o.) as well as at 8 h and 14 days after treatments. The
1
23

Arctium minus crude extract presents antinociceptive effect in a mice acute gout attack model
Fig. 1 Extracted ion chromatograms obtained from the LC–MS/MS
analysis of the crude hydroethanolic extract of A. minus; a phenolic
compounds: (1) chlorogenic acid, (2) vanillic acid, (3) caffeic acid,
acid, (8) luteolin, and (9) apigenin; b triterpenic compounds: (1)
ursolic acid, (2) lupeol, (3) b-amyrin, (4) a-amyrin, and (5) sitosterol.
LC–MS/MS and sample conditions as described in ‘‘Materials and
methods’’ section (Silveira et al. 2016)
(
4) q-coumaric acid, (5) ferulic acid, (6) quercitrin, (7) trans-cinnamic
Results
acid, ferulic acid, trans-cinnamic acid, quercitrin, luteolin,
apigenin, ursolic acid, lupeol, sitosterol, a-amyrin, and b-
amyrin. As can be seen in Table 1, chlorogenic, caffeic,
and vanillic acids were found to be the majority phenolic
compounds within the screened compounds by the LC–
MS/MS method. Furthermore, a-amyrin, b-amyrin, ursolic
acid, lupeol, and sitosterol were the more abundant triter-
penic compounds found as active markers within this
Presence of phenolic and triterpenic compounds
in the A. minus
Chromatograms in Fig. 1a, b show the majority phenolic
and triterpenic compounds found in crude extracts:
chlorogenic acid, vanillic acid, caffeic acid, q-coumaric
123

S. P. M. Fischer et al.
Table 1 Phenolic and triterpenic chemical markers identified and
quantified in extracts of A. minus by LC–MS/MS
The ID value (and the 95% confidence intervals) calculated
50
was 265 (50–1399) mg/kg (Fig. 3b).
We also carried out a dose–response analysis (10–100 mg/
kg, p.o.) toassesstheeffectofA.minuscrudeextractonedema
and spontaneous nociception at 8 h after MSU injection. The
animals that received an intra-articular injection of MSU
crystals (1.25 mg/site) showed an increase in the score of
spontaneous nociception when compared to the vehicle intra-
articular injected group (Fig. 4a). The injection of MSU
crystals was also able to induce an increase in ankle thickness
indicating the development of edema when compared to the
vehicle intra-articular injected group (Fig. 4b). However, A.
minus crude extract neither alters the spontaneous nociception
nor alters the MSU-induced edema formation in any of the
doses tested when compared to the oral vehicle treated plus
MSU intra-articular injected group (Fig. 4a, b).
Chemical compound
Concentration in crude extract (mg/kg)
Chlorogenic acid
Vanillic acid
Caffeic acid
q-Coumaric acid
Ferulic acid
trans-Cinnamic acid
Quercitrin
727.3 ± 8.20
115.2 ± 7.00
225.0 ± 5.10
24.8 ± 0.90
11.4 ± 1.90
11.4 ± 2.10
0.8 ± 0.30
Luteolin
29.6 ± 0.20
Apigenin
2.3 ± 0.40
Ursolic acid
Lupeol
162.21 ± 3.73
158.21 ± 3.62
143.29 ± 4.59
331.35 ± 2.92
1465.26 ± 6.87
Sitosterol
a-Amyrin
b-Amyrin
In vitro xanthine oxidase assay
screened class. Furthermore, the majority compounds
which were found in A. minus crude extract are structurally
represented in Fig. 2.
To verify if the A. minus crude extract exerts its antinoci-
ceptive effect through inhibition of xanthine oxidase
enzyme, we evaluated the xanthine oxidase activity using
pterin as a substrate in vitro. The allopurinol (10 lg/mL;
used as a positive control) inhibited the xanthine oxidase
activity by 30 ± 1%. Furthermore, the A. minus crude
extract inhibited the xanthine oxidase activity in a con-
centration-dependent manner. The concentration of
300 lg/mL showed an inhibition of 26 ± 1%, similar to
allopurinol, while the doses of 100 and 10 lg/mL showed
inhibitions of 13 ± 1 and 8 ± 1%, respectively (Fig. 5).
The IC50 value (and the 95% confidence intervals) calcu-
lated was 116.6 (88.86–153.1) lg/mL.
Antioxidant capacity of A. minus
The A. minus seed crude extract presented an interesting
antioxidant capacity, evaluated by DPPH test, when com-
pared to positive control (ascorbic acid). The SC50 value of
the A. minus seed crude extract was 49.17 ± 1.83 lg/ml,
while the SC50 value of the ascorbic acid was
1
6.05 ± 2.72 lg/mL.
Effects of the A. minus crude extract on MSU-
induced nociception and edema
Adverse effects assessment caused by A. minus
treatment
The intra-articular injection of MSU-induced mechanical
allodynia characterized by a reduction in the PWT that
started from 4 h up to 8 h and disappeared 24 h after MSU
injection when compared to the baseline (B). However, the
intra-articular injection of the vehicle did not reduce the
PWT when compared to the baseline (B) (Fig. 3a). The oral
treatment with A. minus crude extract (100 mg/kg, p.o.) was
able to prevent the mechanical allodynia induced by MSU
from 4 to 8 h after its administration when compared to the
oral vehicle-treated group, with a maximum inhibition of
Open-field and rotarod tests
The treatment with A. minus crude extract (100 or
2000 mg/kg, p.o.) or indomethacin (100 mg/kg, p.o., used
as a control) neither altered the number of crossings or
rearing responses in the open-field test, nor the number of
falls in the rotarod test at 8 h or 14 days after administra-
tions, respectively, when compared with the vehicle group
(Table 2). Thus, the A. minus crude extract caused no
change in the spontaneous or forced locomotor activity of
the animals, respectively.
7
6.2 ± 9% at 8 h (Fig. 3a). Therefore, the time of 8 h was
selected (10–100 mg/kg, p.o.) to perform a dose-response
analysis. Thus, we observed that the dose of 30 and 100 mg/
kg of the A. minus crude extract was effective in preventing
the mechanical allodynia with a maximum inhibition of
Body temperature
7
8.7 ± 7% (100 mg/kg, p.o). In contrast, the lower dose
10 mg/kg, p.o.) did not alter the MSU-induced mechanical
allodynia when compared to the oral vehicle-treated group.
(
Arctium minus crude extract (100 or 2000 mg/kg, p.o.) did
not alter the body temperature of the animals at 8 h or
1
23

Arctium minus crude extract presents antinociceptive effect in a mice acute gout attack model
Fig. 2 Chemical structures of majority compounds found in the A. minus seed crude extract. Phenolic acids: chlorogenic acid (1), caffeic acid
(
2), vanillic acid (3), triterpenes: a-amyrin (4), b-amyrin (5), ursolic acid (6), lupeol (7), and sitosterol (8)
1
4 days after administrations, respectively, when compared
at 8 h after treatments were 115 ± 6.8 and
114.9 ± 4.2 mg/dL to the vehicle or A. minus (100 mg/
kg)-treated groups, respectively. Furthermore, after
14 days of treatments, serum levels of glucose of
with vehicle-treated animals (data not shown).
Biochemical markers
104.1 ± 9.6 mg/dL to the vehicle and 98.72 ± 3.6 mg/dL
The hepatic alteration indicator serum activity (AST and
ALT) and renal alteration indicator serum level (urea and
creatinine) were not altered by treatment with the crude
extract from A. minus seeds (100 mg/kg, p.o.) or (2000 mg/
kg, p.o.) at 8 h or 14 days after administrations, respec-
tively, when compared to vehicle-treated animals
to the A. minus (2000 mg/kg)-treated group were verified.
Gastric lesion assessment
The treatment with the crude extract from A. minus seeds at
the doses of 100 or 2000 mg/kg did not induce gastric
lesions when compared with vehicle-treated animals. The
previous treatment (8 h or 14 days) with indomethacin
(100 mg/kg, p.o.) induced gastric lesions when compared
with vehicle-treated animals. The score of stomach lesions
observed after 8 h of treatments was 1 (0.75–1.75) to the
(
Table 2). The indomethacin (100 mg/kg, p.o., used as a
positive control) increased the ALT levels when compared
with the vehicle-treated group at 14 days after treatment.
Moreover, the A. minus crude extract treated group did not
alter the glucose serum level. The serum levels of glucose
123

S. P. M. Fischer et al.
Fig. 3 Effect of the oral
administration of the A. minus
crude extract on the mechanical
allodynia induced by intra-
articular MSU injection
(
(
(
1.25 mg/site). Time–response
a) and dose–response
b) curves of the anti-allodynic
effect of A. minus. The dose–
response curve was performed
at 8 h after the MSU injection.
Data are expressed as the
mean ? SEM (n = 5–9 per
group). B denotes
baseline threshold before MSU
##
injection. P \ 0.01 and
#
P \ 0.05 when compared with
the baseline; one-way ANOVA
followed by Tukey’s post hoc
test. ***P \ 0.001, **P \ 0.01,
and *P \ 0.05 when compared
with the oral vehicle-treated
group plus MSU intra-articular
injected; two-way ANOVA
followed by Bonferroni’s post
hoc test
vehicle-treated group, 1 (1–4) to the A. minus crude extract
we observed that the A. minus seeds crude extract pre-
vented the mechanical allodynia, but it was ineffective in
reducing the spontaneous nociception and edema induced
by MSU injections. Thus, A. minus crude extract seems to
not have anti-edematogenic activity on the MSU-induced
gout model. However, the antinociceptive effect observed
is an interesting finding, since the pain is the most frequent
and unpleasant sign of acute gout attacks (Martinon et al.
2006; Terkeltaub 2010). This antinociceptive effect found
can be attributed to the active constituent presents in the A.
minus.
(
100 mg/kg)-treated group, and 4.5 (3.25–6.75) to the
group treated with indomethacin (100 mg/kg, p.o.). The
score of stomach lesions observed after 14 days of treat-
ments was 1 (0.75–1.75) to the vehicle-treated group, 1
(
1–4) to the A. minus crude extract (2000 mg/kg)-treated
group, and 4 (3.75–6.25) to the group treated with indo-
methacin (100 mg/kg, p.o.). Gastric lesions score was
reported as median followed by 25–75% quartiles.
Discussion
Active constituents are considered unique components
that contribute to the therapeutic effects of herbal medi-
cine, including primary and secondary metabolites. Thus,
the identification and quantitation of constituents that occur
in majority quantities are a very important step in phyto-
chemical studies to correlate therapeutic effects with
potentially active chemical structures. Phenolic and triter-
penic compounds are secondary metabolites, which belong
to a group of naturally occurring compounds, showing a
broad spectrum of biological activities (Weng and Yen
2012). Thus, within the possible active markers that can be
determined in A. minus extracts, this study focused on the
Gout is caused by deposition of MSU crystals mainly in
joints, which triggers excruciating pain that can be reported
as the most important clinical symptom in patients affected
by this disease. Pain experienced by patients can be a
consequence of inflammatory process induced by crystal
deposition, which prompts the release of pro-nociceptive
mediators (Terkeltaub 2010; Ramonda et al. 2015). In the
folk medicine, A. minus is empirically used as anti-in-
flammatory, antinociceptive, and in the rheumatic pain
treatment (Erdemoglu et al. 2009; Fujita et al. 1995). Here,
1
23

Arctium minus crude extract presents antinociceptive effect in a mice acute gout attack model
Fig. 4 Dose–response curve of
the A. minus crude extract effect
on the score of spontaneous
nociception (a) and edema
(
b) induced by intra-articular
MSU injection (1.25 mg/site) at
h after the MSU injection.
n = 5–8 per group). Data are
8
(
expressed as the median and
interquartile ranges for score;
#
P \ 0.05 when compared with
the vehicle intra-articular
injected group; one-way
ANOVA followed by Kruskal–
Wallis test. Data are expressed
as the mean ? SEM for edema;
###
P \ 0.001 when compared
with the vehicle intra-articular
injected group; one-way
ANOVA followed by
Bonferroni’s post hoc test
possible phenolic and triterpenic antioxidants extracted in a
hydroethanolic extract. Among the 14 chemical markers
identified in the crude extract, one can see that a- and b-
amyrin and chlorogenic acid are the more concentrated
compounds and can be related to the biological activity of
the A. minus. Furthermore, other compounds found in a
smaller proportion (ursolic acid, lupeol, sitosterol, caffeic
acid, and vanillic acid) can also contribute to the
antinociceptive effect of the A. minus.
abscess, carbuncles, and sores (But et al. 1997). A. lappa is
also used to treat various chronic skin affections, rheuma-
tism, and gout and it is also claimed to have diuretic and
blood purifier properties (Lorenzi and Matos 2002; De Smet
et al. 1993).
Terpenes such as a- and b-amyrin or plant extracts con-
taining a- and b-amyrin exhibit a wide range of biological
effects, including antinociceptive activity. The previous
study demonstrated that a- and b-amyrin exhibit antinoci-
ceptive effect in screening tests for new analgesics and
persistent nociception models (arthritic and neuropathic
pain) besides present anti-inflammatory activity (Otuki et al.
Previous studies demonstrated that a- and b-amyrin
found in the A. lappa could be responsible for its biological
activity, for example, in the treatment for erysipelas,
123

S. P. M. Fischer et al.
Table 2 Effect of the crude extract of A. minus or indomethacin on spontaneous or forced locomotor activity, on AST and ALT activities and
urea and creatinine levels 8 h or 14 days after treatments
8
h after treatments
14 days after treatments
Vehicle (10 mL/ A. minus (100 mg/ Indomethacin
kg, p.o.)
Vehicle (10 mL/ A. minus (2000 mg/ Indomethacin
(100 mg/kg, p.o.)
kg, p.o.)
(100 mg/kg, p.o.)
kg, p.o.)
kg, p.o.)
Number of
crossing
44.83 ± 5
36.83 ± 6
36.33 ± 3
51.67 ± 9
62.8 ± 8
46.75 ± 16
Number of
rearing
21.83 ± 3
0 ± 0
16.33 ± 4
0.5 ± 0.3
22.8 ± 4
16.44 ± 5
18.5 ± 2
0.16 ± 0.1
19.35 ± 2
19.21 ± 4
27.83 ± 4
2.5 ± 0.4
20.8 ± 6.1
22.4 ± 4
33.4 ± 4
23.5 ± 11
0.8 ± 0.5
Number of
falls
1.6 ± 0.5
17.2 ± 3.9
35.3 ± 4.3
AST activity 15.36 ± 2
U/L)
ALT activity 12.44 ± 3
U/L)
Urea (mg/dL) 51.00 ± 7
Creatinine 2.33 ± 0.24
mg/dL)
17.35 ± 2.3
36.48 ± 3*
(
(
34.42 ± 6
33.04 ± 4
26.6 ± 2.2
1.82 ± 0.1
29.59 ± 5
2.34 ± 0.7
33.6 ± 6.3
1.33 ± 0.3
1.75 ± 0.25
1.58 ± 0.35
(
Effect of the crude extract of A. minus (100–2000 mg/kg, p.o.), indomethacin (100 mg/kg, p.o.), or vehicle (10 mL/kg, p.o.) on spontaneous
open-field test) or forced (rotarod test) locomotor activity, on AST and ALT activities and urea and creatinine levels 8 h or 14 days after
treatments. The data are presented as mean ± SEM (n = 4–6 per group)
(
*
P \ 0.05 when compared with the vehicle-treated group; one-way ANOVA followed by Tukey’s test
amyrin may function as a non-selective TRPV1 antagonist,
and this finding can be relevant on gout, since the MSU can
produce nociception by activating TRPV1 receptor
(Hoffmeister et al. 2011; 2013). In addition to a- and b-
amyrin, other terpenes such as ursolic acid, lupeol, and
sitosterol can also contribute to the antinociceptive effect of
the A. minus, since these compounds have analgesic action in
pre-clinical pain models, including inhibition TRPV1
channels involved in arthritic pain, in the case of ursolic acid
(Abdul-Wahab et al. 2012; Alonso-Castro et al. 2016; De
lima et al. 2013, Rodrigues et al. 2012; Verano et al. 2013).
In addition to terpenes, A. minus presents high amounts
of phenolic compounds which also exhibit antinociceptive
activity. The phenolic compound which was found in
higher concentration in the A. minus was the chlorogenic
acid. Literature data show that chlorogenic acid presents
antinociceptive effects in screening tests for new analgesics
and neuropathic pain models (Bagdas et al. 2013; Qu et al.
Fig. 5 Effect of different concentrations of the crude extract of A.
minus (10, 100, and 300 lg/mL), allopurinol (10 lg/mL), or vehicle
on xanthine oxidase activity in vitro. Data are expressed as the
mean ? SEM (n = 5 per group). **P \ 0.01 and ***P \ 0.001
when compared with the vehicle group; one-way ANOVA followed
by the Student–Newman–Keuls’ test
2014; Hara et al. 2014). Thus, chlorogenic acid can be a
responsible, at least in part, by antinociceptive effect pro-
moted by A. minus. Other phenolic compounds found in the
A. minus crude extract were caffeic and vanillic acids. Both
also possess antinociceptive properties (Morucci et al.
2012).
2
005; Lima-J u´ nior et al. 2006). This antinociceptive effect of
In addition to the analgesic activity, phenolic and fla-
a- and b-amyrin seems to involve PKA and PKC pathways,
cannabinoids receptors, as well as opioid receptor and tran-
sient receptor potential vanilloid 1 (TRPV1) (Lima-J u´ nior
et al. 2006; Chicca et al. 2012, Sim a˜ o da Silva et al. 2011).
Lima-J u´ nior et al. (2006) showed that the mixture a, b-
vonoid compounds also present antioxidant activity, which
may be related to the analgesic action of the A. minus. It
has been reported that A. minus is a rich source of natural
antioxidant compound (Erdemoglu et al. 2009). In addition,
the burdock has high phenolic compound levels in seed
1
23

Arctium minus crude extract presents antinociceptive effect in a mice acute gout attack model
extracts (Ferracane et al. 2010). These information are
consistent with our results, since the phytochemical
screening revealed that the A. minus seed extract contains
high quantity of phenolic compounds and a great amount of
flavonoids which seem to be responsible for their antioxi-
dant capacity as verified by DPPH test.
These results support the use of this plant to the treatment
of acute gout attack.
Acknowledgements The authors would like to thank Professor
Juliano Ferreira for donated reagents. This study was supported by the
Funda c¸ a˜ o de Amparo a` Pesquisa do Estado do Rio Grande do Sul—
FAPERGS (process number 16/2551-0000281-9) and by the Con-
selho Nacional de Desenvolvimento Cient ´ı fico e Tecnol o´ gico—
CNPq. We thank CNPq and CAPES for their fellowship support.
The precipitation of urate crystals can be an indirect result
of hyperuricemia, which has been considered the most
important factor for the development of gout (Zamudio-
Cuevas et al. 2015; Busso and So 2010; Zhu et al. 2011).
Allopurinol is the first xanthine oxidase inhibitor discovered,
and ithasbeenthe most frequently useddrugathyperuricemia
control (Nukiand Simkin 2006; Rundlesetal. 1963). Previous
studies have shown that the xanthine oxidase inhibition and
hypouricemic activities of some medicinal plants may be
attributed to the presence of flavonoids and other phenolic
compounds, due to their anti-inflammatory and antioxidant
activities (Umamaheswari et al. 2007; Irondi et al. 2016). The
detection of some secondary metabolites as flavonoids and
triterpenes may be related toanti-hyperuricemic effects (Lima
et al. 2015). Recent studies demonstrated that b-amyrin
Author contributions All authors had full access to all the data in
the study and take responsibility for the integrity and accuracy of the
data analysis. Study concept and design: SPMF, IB, and SMO.
Acquisition of data: SPMF, IB, CC, MP, HF, LAG, LMC, and SMO.
Analysis and interpretation of data: SPMF, IB, CC, MP, HF, LAG,
LMC, and SMO. Drafting of the manuscript: SPMF, IB, MP, and
SMO. Study supervision: SMO.
Compliance with ethical standards
Conflict of interest The authors declare that there are no conflicts of
interest.
Ethical approval All procedures performed were in accordance with
international and national and were approved by Ethics Committee
for Animal Research of the Federal University of Santa Maria (pro-
cess number 1946180116/2016).
(
Ferraz-Filha et al. 2016; Lin et al. 2011), chlorogenic acid
(
Meng et al. 2014; Chen et al. 2011), and caffeic acid (Chen
et al. 2011) are capable of inhibiting xanthine oxidase. On this
study, A. minus crude extract was effective in inhibiting
xanthine oxidase activity in vitro, an ability that can be asso-
ciated with substances contained in this extract with different
potential to inhibit this enzyme as the b-amyrin, chlorogenic,
and caffeic acids. Thus, the antinociceptive effect of A. minus
may have occurred by xanthine oxidase inhibition. This
finding suggests that the A. minus could be useful to treat gout
attacks acting in a similar way to allopurinol.
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