In vitro binding affinities of a series of flavonoids for m-opioid receptors. Antinociceptive effect of the synthetic flavonoid 3,3-dibromoflavanone in mice

Article abstract of DOI:10.1016/j.neuropharm.2013.04.020

The pharmacotherapy for the treatment of pain is an active area of investigation. There are effective drugs to treat this problem, but there is also a need to find alternative treatments free of undesirable side effects. In the present work the capacity o

Full text of DOI:10.1016/j.neuropharm.2013.04.020

Neuropharmacology 72 (2013) 9e19
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Neuropharmacology
journal homepage: www.elsevier.com/locate/neuropharm
In vitro binding affinities of a series of flavonoids for m-opioid
receptors. Antinociceptive effect of the synthetic flavonoid
3,3-dibromoflavanone in mice
*
Josefina Higgs, Cristina Wasowski, Leonardo M. Loscalzo, Mariel Marder
Instituto de Química y Fisicoquímica Biológicas, Facultad de Farmacia y Bioquímica, Universidad de Buenos Aires, Junín 956 (C1113AAD), 1113 Buenos Aires,
Argentina
a r t i c l e i n f o
a b s t r a c t
Article history:
The pharmacotherapy for the treatment of pain is an active area of investigation. There are effective
drugs to treat this problem, but there is also a need to find alternative treatments free of undesirable side
Received 15 November 2012
Received in revised form
6 March 2013
effects. In the present work the capacity of a series of flavonoids to bind to the
evaluated. The most active compound, 3,3-dibromoflavanone (31), a synthetic flavonoid, presented a
significant inhibition of the binding of the selective
opioid ligand [3H]DAMGO, with a Ki of
0.846 ꢀ 0.263 M. Flavanone 31 was further synthesized using a simple and cheap procedure with good
m opioid receptor was
Accepted 8 April 2013
m
m
Keywords:
yield. Its in vivo effects in mice, after acute treatments, were studied using antinociceptive and behavioral
assays. It showed no sedative, anxiolytic, motor incoordination effects or inhibition of the gastrointes-
tinal transit in mice at the doses tested. It evidenced antinociceptive activity on the acetic acid-induced
nociception, hot plate and formalin tests (at 10 mg/kg and 30 mg/kg). The results showed that the 5-HT2
receptor and the adrenoceptors seem unlikely to be involved in its antinociceptive effects. Naltrexone, a
nonselective opioid receptors antagonist, totally blocked compound 31 antinociceptive effects on the hot
Natural and synthetic flavonoids
3,3-Dibromoflavanone
Opioid receptors
Antinociceptive effects
Acute treatment
plate test, but naltrindole (d opioid antagonist) and nor-binaltorphimine (k opioid antagonist) did not.
These findings demonstrated that 3,3-dibromoflavanone (31), at doses that did not interfere with the
motor performance, exerted clear dose dependent antinociception when assessed in the chemical and
thermal models of nociception in mice and it seems that its action is related to the activation of the
opioid receptor.
m
Ó 2013 Elsevier Ltd. All rights reserved.
1. Introduction
The endogenous opioid system is critical for many physiological
and behavioral effects. Opioid receptor activation by endogenous
and exogenous ligands results in a multitude of effects, which
include analgesia, respiratory depression, euphoria, inhibition of
gastrointestinal transit, effects on anxiety, etc. (Kieffer et al., 2009).
There are three known ‘classical’ types of opioid receptors, origi-
nally defined according to their selectivity for certain kinds of
Pain constitutes a major public health problem and it is the
primary reason why people seek medical care and one of the most
prevalent conditions that limits productivity and diminishes qual-
ity of life. Although adequate pain relief is achieved with the
currently available analgesic like opioids or nonsteroidal anti-
inflammatory drugs (NSAIDs), some of their serious side effects
are major limitations to their routine use in therapy. It is well
known that pharmaceutical companies around the world are
interested in developing safer and more effective drugs to treat pain
(Milano et al., 2008).
opioids,
m
(for morphine),
k
(for ketocyclazocine), and
d (first
identified in mouse vas deferens). Of them, the
m
receptor is
thought to be primarily responsible for the mediation of opioid
antinociception and tolerance.
Flavonoids form a large family of natural products which are
widely distributed in the plant kingdom. In the course of our survey
of substances exerting pharmacological effects on the central ner-
vous system (CNS) a range of available flavonoids were explored.
Many flavone aglycones were described as ligands for the benzo-
diazepine binding site (BDZ-bs) of the gamma aminobutyric acid
* Corresponding author. Tel.: þ54 11 4964 8289; fax: þ54 11 4962 5457.
E-mail addresses: jhiggs@qb.ffyb.uba.ar (J. Higgs), criswasowski@qb.ffyb.uba.ar
(C. Wasowski), leoloscalzo@hotmail.com (L.M. Loscalzo), mmarder@qb.ffyb.uba.ar
(M. Marder).
0028-3908/$ e see front matter Ó 2013 Elsevier Ltd. All rights reserved.
http://dx.doi.org/10.1016/j.neuropharm.2013.04.020

10
J. Higgs et al. / Neuropharmacology 72 (2013) 9e19
was purchased from PerkinElmer, USA; indomethacin from Montpellier, Argentina
and naltrindole hydrochloride from Tocris Bioscience, France. Diosmin (4), gossypin
(6), flavanone (7) and chalcone (32) were obtained from Extrasynthese, France.
Synthetic flavonoids aglycones were previously obtained in our laboratory (Marder
and Paladini, 2002). 3,3-Dibromoflavanone (31) (Fig. 1) was further synthesized as
indicated bellow. Chemical purity of the synthetic flavonoids was above 95%, esti-
mated by us based on analytical HPLC experiments.
Compound 31 was dissolved by the sequential addition of: dimethylsulfoxide up
to a final concentration of 5%, a solution of 0.25% Tween 80 up to a final concen-
tration of 20%, and saline to complete 100% volume. Morphine, naltrexone, ketan-
serin, naltrindole, nor-binaltorphimine and indomethacin were dissolved in saline
solution.
The rodents were intraperitoneally (i.p.) injected 20 min or 30 min before per-
forming the pharmacological tests, as indicated. In each session, a control group
receiving only vehicle was tested in parallel with those animals receiving drug
treatment. Vehicle control mice showed no significant differences in any of the tests
assayed compared to mice treated with saline (data not shown).
type A (GABAA) receptors in the CNS, mediating anxiolytic effects
in vivo (Jäger and Lasse, 2011; Wasowski and Marder, 2012). Several
flavonoid glycosides, present in plants used as tranquillizers, were
found to have CNS depressant effects in mice, but the behavioral
effects induced by these compounds do not involved classical
GABAA receptors (Fernández et al., 2006).
Although the mechanisms of action of a variety of biological
effects of flavonoids, such as its antioxidant and anti-
inflammatory properties have been studied extensively
(Harbone and Williams, 2000; Havsteen, 2002; Rathee et al.,
2009), little is known about the action of these compounds on
the modulation of pain transmission. Some glycosylated flavo-
noids, such as myricitrin and baicalin, produced systemic anti-
nociception when assessed in chemical models of nociception in
mice, but the opioid system seemed unlikely to participate in
them (Chou et al., 2003; Meotti et al., 2006). On the other hand,
other flavonoid glycosides with analgesic activities were reported
as involving an opiate like mechanism, since their activities were
reversed by naloxone pre-treatments, an opioid receptor antag-
onist; such is the case of gossypin (Viswanathan et al., 1984) and
the flavone glycoside linarin (Martinez-Vazquez et al., 1996).
Furthermore, several hydroxylated derivatives of flavones possess
antinociceptive properties that showed a mechanism mediated by
opioid receptors (Umamaheswari et al., 2006; Vidyalakshmi et al.,
2010).
The volume of i.p. injections was 0.15 mL/30 g of body weight.
2.2. Animals
Adult male Swiss mice weighing 25e30 g were used in the pharmacological
assays and adult male rats (200e300 g) Wistar strain for biochemical studies, both
were obtained from the Central Animal House of the School of Pharmacy and
Biochemistry, University of Buenos Aires. For behavioral assays mice were housed in
groups of five in a controlled environment (20e23 ꢁC), with free access to food and
water and maintained on a 12 h/12 h day/night cycle, light on at 06:00 AM. Housing,
handling, and experimental procedures complied with the recommendations set
forth by the National Institutes of Health Guide for Care and Use of Laboratory
Animals (Publication No. 85-23, revised 1985) and the Institutional Committee for
the Care and Use of Laboratory Animals, University of Buenos Aires, Argentina. All
efforts were taken in order to minimize animal suffering. The number of animals
used was the minimum number consistent with obtaining significant data. The
animals were randomly assigned to any treatment groups and were used only once.
The behavioral tests were evaluated by experimenters who were kept unaware of
the treatment administered and were performed between 10:00 AM and 2:00 PM.
Previous studies from our laboratory demonstrated that opioid
receptors were involved in the sedative and antinociceptive effects
of hesperidin, a widely consumed flavanone glycoside (Loscalzo
et al., 2008, 2011). Binding assays showed that hesperidin did not
bind to the
m opioid receptor, as it was not able to displace the
specific binding of [3H]DAMGO ([D-Ala2, N-MePhe4, Gly-ol]-
enkephalin) from synaptosomal membranes of rat’s forebrains.
Otherwise the aglycone of hesperidin, hesperetin, inhibited the
specific binding of this peptide but did not show any in vivo activity
at the doses tested (Fernández et al., 2006; Guzmán-Gutiérrez and
Navarrete, 2009; Loscalzo et al., 2008, 2011; Marder et al., 2003).
In this work the capacities of natural flavonoid glycosides and
aglycones, synthetic flavonoids, previously obtained in our labo-
ratory (Marder and Paladini, 2002), and related compounds to bind
2.3. Chemistry
The synthesis of compound 31 was performed as follows: to a solution of
flavanone (2.2 mmol) in CH3Cl (10 mL), at 25 ꢁC, an excess of bromine (7.8 mmol)
was added dropwise. The mixture was stirred for 2 h at 50 ꢁC. After cooling, the
reaction mixture was washed with two 10 mL portions of a saturated aqueous so-
lution of Na2S2O5 and then with water, dried over Na2SO4 and concentrated to
dryness in a rotary evaporator (De Diesbach and Kramer, 1945).
The reaction product was analyzed by TLC on silica gel on polyester sheets, with
254 nm fluorescent indicator (Sigma, USA) and by HPLC performed using C18
reversed phase Vydac columns (The Separation Group, Hesperia, CAL, USA) and
elutions carried out with a lineal gradient of 30e80% ACN in water, in 30 min, at a
flow rate of 1 mL/min.
The reaction product was then recrystalized twice from ethanol to water and
used for identification and assay (yield 66%). EIMS were measured in a Shimadzu
QP-1000 quadrupole mass spectrometer. NMR spectra were recorded in a Bruker
AMX 400 spectrometer. Reagents and solvents were of analytical reagent grade and
were purchased from SigmaeAldrich and Fluka.
to
m opioid receptors present in rat’s forebrain membranes were
evaluated. As 3,3-dibromoflavanone (31) emerged as the most
active compound of this series, an efficient syntheses of this novel
compound and its pharmacological effects in acute treatment in
mice were developed.
2. Materials and methods
3,3-dibromoflavanone (31) (Fig. 1) white crystals from ethanol-water. 1H NMR
(CDCl3, 300 MHz) dH 8.09 (dd, J ¼ 1.4 Hz, 6.7 Hz, H-5), 7.76 (m, H-20, H-60), 7.61 (td,
J ¼ 1.8 Hz, 7.4 Hz, H-7), 7.49 (m, H-30, H-40, H-50), 7.20 (t, J ¼ 7.5 Hz, H-6), 7.11 (d,
J ¼ 8.5 Hz, H-8), 5.30 (s, H-2). 13C NMR (CDCl3, 300 Mhz) dC 86.3 (C-2), 69.4 (C-3),
180.7 (C-4), 115.6 (C-4a), 123.3 (C-5), 129.6 (C-6), 137.0 (C-7), 117.9 (C-8), 160.0 (C-8a),
133.5 (C-10), 129.5 (C-20/C-60), 127.7 (C-30/C-50), 129.9 (C-40). EIMS m/z 380/382/384
(rel. 1/2/1) Mþ, 301/303 (rel. 1/1), 260/262/264 (rel. 1/2/1), 221, 120.
2.1. Drugs and injection procedures
Morphine hydrochloride was purchased from Gramon, Argentina. Naltrexone
hydrochloride, ketanserin tartrate salt, yohimbine hydrochloride, nor-
binaltorphimine dihydrochloride, hesperidin (1), neohesperidin (2), naringin (3),
rutin (5), hesperetin (8), naringenin (9), flavone (10), diosmetin (11), quercetin (12),
apigenin (13), chrysin (14), 6-methylflavone (15), hesperidin methyl chalcone (33),
2.4. Biochemical assay ([3H]-DAMGO binding assay)
cinnamic acid (34), caffeic acid (35), chromone (36), b-naphthoflavone (39), ipri-
flavone (37) and
a-naphthoflavone (38) were obtained from SigmaeAldrich
Chemical Company, USA. [3H]-DAMGO ([D-Ala2, N-Me-Phe4, Gly-ol5] enkephalin)
A crude membrane fraction was prepared from male Wistar rat forebrains as
previously described (Ioja et al., 2007) with small modifications. Rats were hu-
manely killed by decapitation and the brains without cerebellum were rapidly
removed and washed several times in ice-cold 50 mM TriseHCl buffer pH 7.4. Af-
terward they were homogenized in 10 volumes/weight of 0.32 M sucrose at 0 ꢁC and
the homogenate was centrifuged at 900 ꢂ g for 10 min at 4 ꢁC. The supernatant was
decanted and centrifuged at 100,000 ꢂ g for 30 min at 4 ꢁC, the resulting pellets
were resuspended in 30 volumes/weight of 50 mM TriseHCl buffer pH 7.4 and
incubated at 37 ꢁC for 30 min to remove any endogenous opioid peptides. The last
centrifugation step was repeated under the same conditions as described above and
the final pellets were resuspended in 10 volumes/weight of the same buffer and
stored at ꢃ80 ꢁC until use. Protein concentration was determined by the method of
Bradford using bovine serum albumin as standard (Bradford, 1976).
Fig. 1. Molecular structure of 3,3-dibromoflavanone (31).

J. Higgs et al. / Neuropharmacology 72 (2013) 9e19
11
For the binding assay, membranes were thawed and suspended in 50 mM Trise
HCl pH 7.4 to a final protein concentration of 0.25e0.35 mg/mL. The incubation was
carried out at 25 ꢁC for 1 h in a final volume of 1 mL of membrane suspension (in
duplicate) in the presence of the sample assayed and with 1 nM of [3H]DAMGO
(56.8 Ci/mmol). Non specific binding was determined in parallel incubations in the
dimensions, with free access to all arms from the crossing point. The closed arms
had walls 15 cm high all around. The maze was suspended 50 cm from the room
floor. Mice were placed on the central part of the cross facing an open arm. The
number of entries and the time spent going into open arms were counted during
5 min under red dim light. The total exploratory activity (number of entries in both
arms) was also determined (Lister, 1987).
presence of 10
mM naltrexone. In the screening assays each compound was tested at
300 M in triplicate. In the competition assays, the incubations were done with a
m
The test was performed 20 min after the i.p. injection of 3 mg/kg, 10 mg/kg and
30 mg/kg of compound 31 or vehicle.
range of concentrations of compounds 8, 9,10,12,15, 31 and 32. Naltrexone was used
as positive control in concentrations between 0.01 and 30 nM. The assays were
terminated by filtration under vacuum through Whatman GF/A glassfiber filters and
three washes with 3 mL each of incubation medium. Filters were counted after
addition of Optiphase “Hisafe” 3 (Wallac, Turku, Finland) liquid scintillation cocktail.
2.5.2.4. Inverted screen test. The inverted screen test was used to assess the motor
toxicity of the drug (Coughenour et al., 1977). Mice were placed on a 13 cm ꢂ 13 cm
wire mesh screen elevated 40 cm above the ground. After slowly inverting the
screen through an angle of 180ꢁ the mice were tested for their ability to climb to the
top. Mice were i.p. injected with vehicle or compound 31 (30 mg/kg) 30 min before
the assay. Mice not climbing to the top (all four paws on upper surface) were
counted as failures.
2.5. Pharmacological studies
2.5.1. Antinociceptive assays
2.5.1.1. Writhing test. The writhing test is a visceral pain model widely used for the
evaluation of peripheral antinociceptive activity of new agents. The test was carried
out according to the method described earlier (Koster et al., 1959); 0.75% acetic acid
aqueous solution was injected i.p. (0.2 mL/30 g body weight) and the animals were
placed in a glass observation chamber (15 cm ꢂ 15 cm ꢂ 30 cm). The number of
writhing responses (abdominal cramps) was counted for 15 min after 5 min of the
injection of the acetic acid solution.
2.5.3. Blockade experiments
The involvement of various brain receptors on 3,3-dibromoflavanone (31)
antinociceptive effects in mice were investigated by measuring its blockade with
different specific antagonists in the hot plate test.
Several doses of each antagonist were tested to find the maximal dose devoid of
intrinsic action to be used in the blockade experiments and were based on our
previous studies (Loscalzo et al., 2008) and other literature data (Kaur et al., 2005;
Jürgensen et al., 2005).
To assess the participation of the opioid system, mice were pretreated with
naltrexone (5 mg/kg, i.p., a nonselective opioid receptor antagonist), naltrindole
(5 mg/kg, i.p., a potent and highly selective
The writhing test was performed 30 min after the i.p. injections of 3 mg/kg,
10 mg/kg and 30 mg/kg of compound 31, morphine (6 mg/kg) or vehicle.
2.5.1.2. Hot plate test. The hot plate assay is one of the most commonly used tests
for determining the antinociceptive efficacy of experimental drugs in rodents
(Carter, 1991). It is suitable for the study of central drugs by assessing the acute pain
sensitivity to a thermal stimulus. Each mouse was placed into a transparent beaker
made of Plexiglass with a height of 18 cm and a diameter of 10 cm to avoid the
animals escaping from the plate which temperature was set at 55 ꢁC ꢀ 0.1 ꢁC by
using a thermoregulated water-circulating pump (Schreiber et al., 1999).
The time (in seconds) between the placement of the animal and the first
response: hind paw licking/fanning or jumping was measured as latency. A 45 s cut-
off was used to prevent tissue damage.
d opioid receptor antagonist) or nor-
binaltorphimine (10 mg/kg, subcutaneously, s.c., a potent and highly selective
k
opioid receptor antagonist), and after 20 min or 24 hr (for nor-binaltorphimine), the
animals received an injection of compound 31 (30 mg/kg, i.p.) or vehicle. The hot
plate assay was performed 30 min later.
To examine the possible participation of the serotonin receptors and the adre-
noceptors on the antinociceptive effect of compound 31, mice were pretreated with
ketanserin (0.5 mg/kg, i.p., a selective antagonist for the 5-HT2A/C receptor), prazosin
(0.2 mg/kg, i.p., an a1-adrenoceptor antagonist) or yohimbine (2 mg/kg, i.p., an a2
-
adrenoceptor antagonist). After 20 min, the animals received an injection of com-
pound 31 (30 mg/kg) or vehicle and mice were evaluated on the hot plate assay
30 min later.
This test was performed 30 min after the i.p. injection of 3 mg/kg, 10 mg/kg and
30 mg/kg of compound 31, morphine (6 mg/kg) or vehicle. A time course of the effect
of compound 31 at 30 mg/kg was also determined.
2.5.4. Gastrointestinal transit
2.5.1.3. Formalin test. The formalin test measures the response to a long-lasting
nociceptive stimulus and thus may have a closer resemblance to clinical pain
(Abbott et al., 1982; Murray et al.,1988) due to its tonic nature. It is not only used as a
tonic pain model but also as an inflammatory pain model.
Animals were first habituated for 10 min in an observation chamber made of
transparent acrylic. 20 ml of 1% formalin solution, made up in phosphate buffered
solution (pH 7.4), were injected into the dorsal surface of the right hind paw of the
mice with a microsyringe and a 30-gauge needle, 30 min after the i.p. administration
of compound 31 (10 mg/kg and 30 mg/kg), morphine (6 mg/kg), indomethacin
(10 mg/kg) or vehicle. Immediately after the formalin injection animals were placed
individually in the chamber; beneath the floor, a mirror was mounted at a 45ꢁ angle
to allow clear observation of the animal’s paws. The amount of time the animal spent
licking the injected paw was considered as indicative of pain. The nociceptive
response was recorded in two phases: first or early phase (5 min after formalin
injection) and second or late phase (15e50 min after formalin injection).
Gastrointestinal transit was assessed using the charcoal meal test (Manara et al.,
1986; Schulz et al., 1979). Mice were fasted, with water available ad libitum, for 18 h
before the experiments. Thirty min after the i.p. injection of compound 31 (30 mg/
kg), morphine (6 mg/kg), or vehicle a suspension of a charcoal meal (10% vegetable
charcoal in 5% arabic gum) was orally administered at a volume of 0.25 mL/mouse.
Thirty minutes after administration of the charcoal meal animals were sacrificed by
cervical dislocation and their stomach and small intestine were carefully removed.
Both the length of the small intestine from pyloric sphincter to the ileocoecal
junction and the farthest distance to which the charcoal meal had traveled were
measured. For each animal, the gastrointestinal transit was calculated as the per-
centage of distance traveled by the charcoal meal relative to the total length of the
small intestine. The inhibition of gastrointestinal transit (%) was calculated as: In-
hibition of gastrointestinal transit (%)
gastrointestinal transit)/(vehicle gastrointestinal transit)] ꢂ 100.
¼
[(vehicle gastrointestinal transit-drug
The percentages of the reduction of the licking time were calculated as:
% reduction of licking time : 100% ꢃ ðA ꢂ 100%=BÞ
A: licking time of mice injected with compound 31
2.6. Statistical analyses
B: licking time of control mice
The effects of compounds in mice were analyzed by one-way analysis of variance
(ANOVA) and post-hoc comparisons between treatments and vehicle were made
using Dunnett multiple comparison test. The blockade experiments were analyzed
by two-way ANOVA (pre-treatment vs. treatment) and post-hoc comparison was
made using Bonferroni post test. When a significant interaction between pre-
treatment and treatment was observed, subsequent one-way ANOVAs and New-
maneKeuls Multiple Comparison post-hoc test was applied.
Data from the inverted screen tests were analyzed with Chi-square test and
unpaired t-test. The time course of the effect of compound 31 in the hot plate test
was analyzed using unpaired t-test.
2.5.2. Behavioral studies
2.5.2.1. Hole board assay. This assay was conducted in a walled black Plexiglass
arena with a floor of 60 cm ꢂ 60 cm and 30 cm high walls, with four centered and
equally spaced holes in the floor, 2 cm in diameter each as previously described
(Fernández et al., 2006) and illuminated by an indirect and dimly light. Each animal
was placed in the center of the hole board and allowed to freely explore the appa-
ratus for 5 min and the number of head dips, the time spent head dipping and the
number of rearings were measured. The test was performed 20 min after the i.p.
injection of 1 mg/kg, 10 mg/kg and 30 mg/kg of compound 31 or vehicle.
For the competition binding, data were analyzed by nonlinear regression fit to
one site of specific bound vs radioligand concentration. Ki values were calculated
using the Cheng-Prusoff/Chou equation: Ki ¼ IC50/[1 þ (L/Kd)], where Ki refers to the
inhibition constant of the unlabeled ligand, IC50 is the concentration of unlabeled
ligand required to reach half-maximal binding, Kd refers to the equilibrium disso-
ciation constant of the radioactive ligand and L refers to the concentration of
radioactive ligand.
2.5.2.2. Locomotor activity. The spontaneous locomotor activity was automatically
measured as previously described (Fernández et al., 2006) and was expressed as
total light beam counts per 5 min. The test was performed 20 min after the i.p. in-
jection of 1 mg/kg, 10 mg/kg and 30 mg/kg of compound 31 or vehicle.
2.5.2.3. Elevated plus maze test. This test has been described as a simple method for
assessing anxiety responses in rodents. The elevated plus maze set-up consisted of a
maze of two open arms, 25 cm ꢂ 5 cm, crossed by two closed arms of the same
A P value < 0.05 was considered statistically significant. All data were expressed
as mean ꢀ S.E.M. and analyzed with GraphPad Prism 5.00 software.

12
J. Higgs et al. / Neuropharmacology 72 (2013) 9e19
Table 1
Molecular structures and activities on the
m
-opioid receptor binding assay of the investigated compounds. .
R3'
R3'
R2'
R2'
R4'
R4'
R8
R8
O
O
O
O
R7
R6
R7
R6
R3
R3
R5
R5
Flavanone (A)
Flavone (B)
Compound
Moiety
R3
R5
R6
R7
R8
R20
R30
R40
Binding inhibitiona
A) Flavonoid glycosides
b
1
2
3
4
5
6
Hesperidin
Neohesperidin
Naringin
Diosmin
Rutin
Gossypin
A
A
A
B
B
B
H
H
H
H
O-
OH
OH
OH
OH
OH
OH
H
H
H
H
H
H
O-
O-
O-
O-
OH
OH
b
b
b
b
-Rt
H
H
H
H
H
H
H
H
H
H
H
OH
OH
H
OH
OH
OH
OCH3
OCH3
OH
OCH3
OH
ꢃ
-Nh
-Nh
-Rt
ꢃ
ꢃ
þ
þ
þ
b-Rt
OH
O-
b
-Glc
OH
B) Flavonoid aglycones
7
8
9
10
11
12
13
14
15
Flavanone
Hesperetin
Naringenin
Flavone
Diosmetin
Quercetin
Apigenin
A
A
A
B
B
B
B
B
B
H
H
H
H
H
OH
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
OH
H
H
þþ
b
OH
OH
H
OH
OH
OH
OH
H
OH
OH
H
OH
OH
OH
OH
H
OCH3
OH
H
OCH3
OH
OH
H
þþþ
þþþ
þþþ
þþ
H
OH
OH
H
H
H
þþþ
þþþ
þþ
Chrysin
6-Methylflavone
CH3
H
þþþ
C) Synthetic flavonoids
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
6-Nitroflavone
6-Fluorflavone
6-Chloroflavone
B
B
B
B
B
B
B
B
B
B
B
B
B
B
B
A
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
NO2
F
Cl
CH3
CH3
H
H
H
H
H
H
H
H
H
Br
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
NO2
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
NO2
F
ꢃ
þ
ꢃ
6,30-Dimethylflavone
6-Methyl-30-bromoflavone
20-Nitroflavone
CH3
Br
H
NO2
Cl
Br
CH3
H
H
H
NO2
H
H
þ
ꢃ
þ
30-Nitroflavone
ꢃ
30-Chloroflavone
30-Bromoflavone
30-Methylflavone
40-Nitroflavone
þ
þ
þþ
ꢃ
40-Fluoroflavone
ꢃ
40-Bromoflavone
3-Bromo-30-nitroflavone
3,6-Dibromoflavone
3,3-Dibromoflavanone
H
Br
H
H
H
ꢃ
Br
Br
Br2
ꢃ
ꢃ
þþþþ
D) Related compounds
32
33
Chalcone
þþþ
ꢃ
O
Hesperidin methyl chalcone
34
35
Cinnamic acid
ꢃ
ꢃ
Caffeic acid

J. Higgs et al. / Neuropharmacology 72 (2013) 9e19
13
Table 1 (continued )
Compound
Moiety
R3
R5
R6
R7
R8
R20
R30
R40
Binding inhibitiona
36
37
38
Chromone
ꢃ
ꢃ
ꢃ
ꢃ
Ipriflavone
a
-Naphthoflavone
-Naphthoflavone
39
b
a
Capacity of the compounds, at 300
m
M, to inhibit the binding of [3H]-DAMGO to the
m
-opioid receptor indicated as: inhibition > 90% (þþþþ); inhibition 70e90% (þþþ);
-L Rhamnosyl-(1 / 2)-glucose, Nh), rutinose (O-
inhibition 40e70% (þþ); inhibition 20e40% (þ) and inhibition <20% (L). Sugar moieties: glucose (Glc), neohesperidose (O-
a
a
-L Rhamnosyl-(1 / 6)-glucose, Rt).
b
Loscalzo et al., 2011.
3. Results
(unpaired t-test, P ¼ 0.0392), 20 min (P ¼ 0.0002), 30 min
(P ¼ 0.0004) and until 60 min (P ¼ 0.0004), without reaching control
values until 120 min (P ¼ 0.0743).
3.1. Effects of flavonoids on [3H]-DAMGO binding
On the other hand, in the first phase of the formalin test, com-
pound 31 at 3 mg/kg, 10 mg/kg and 30 mg/kg inhibited formalin
induced pain by 29.8%, 60.5% and 45.2% respectively
[F(4,39) ¼ 4.774, P ¼ 0.0035, Fig. 5A]. Mice receiving compound 31 at
10 mg/kg and 30 mg/kg had significantly minor licking time than
control mice (P < 0.01, P < 0.05, respectively, Dunnett’s procedure)
(Fig. 5A). Indomethacin, the positive control drug, showed no effect
in this phase, as expected.
In the second phase the i.p. administration of 3 mg/kg, 10 mg/kg
and 30 mg/kg of compound 31 produced a reduction of the licking
time of 34.1%, 70.4% and 78.4%, respectively [F(4,39) ¼ 5.386,
P ¼ 0.0017, Fig. 5B]. The comparison between the vehicle control
group and experimental groups by the Dunnett’s test indicated that
flavanone 31 at 10 mg/kg and 30 mg/kg (P < 0.01) and indometh-
acin at 10 mg/kg (P < 0.05), significantly reduced the licking time of
mice (Fig. 5B).
The capacity of the compounds, tested at 300
m
M, to inhibit the
binding of [3H]-DAMGO to the
Table 1.
m-opioid receptor is shown in
The results pointed out that compounds 7e15, 25 and 32
exhibited low to medium activity, meanwhile compound 31
showed the highest activity in the binding assay. Competition
binding assays and Ki values (mean ꢀ S.E.M.) of these compounds
are shown in Fig. 2 and Table 2, respectively. In all cases, data ob-
tained were best fitted to one site binding hyperbola.
3.2. Antinociceptive effects of flavanone 31 in the writhing, hot
plate and formalin tests
The effect of compound 31 in the writhing and the hot plate tests
are shown in Figs. 3 and 4, respectively. ANOVA indicated a signifi-
cant effect on the number of writhes [F(3,33) ¼ 4.739, P ¼ 0.0080,
Fig. 3] and on the latency time in the hot plate test [F(3,47) ¼ 5.174,
P ¼ 0.0038, Fig. 4A]. Comparisons between the vehicle control group
and experimental groups (Dunnett’s procedure) indicated that
compound 31 decreased the number of writhes at 10 mg/kg and
30 mg/kg (P < 0.05 and P < 0.01, respectively) (Fig. 3) and increased
the latency time in the hot plate test at 10 mg/kg and 30 mg/kg
(P < 0.05 and P < 0.01, respectively) (Fig. 4A). Alternatively, mice i.p.
Mice i.p. injected with morphine (6 mg/kg) did not lick appre-
ciably their paws in any phase of this assay.
3.3. Behavioral effects of compound 31 in the hole board and the
locomotor activity tests
The effects of compound 31 in the hole board and locomotor
activity tests are shown in Fig. 6. For compound 31 i.p. injected at
1 mg/kg, 10 mg/kg and 30 mg/kg, ANOVA indicated that there is no
significant effect on the number of mouse rearings, the number of
head dips, the time spent head dipping and the locomotor activity
counts; so it failed to show any effect in these assays, at the doses
tested.
injected with morphine (6 mg/kg), a classical
m opioid receptor
ligand used as a reference compound, showed no abdominal cramps
in the writhing test and displayed a high latency time in the hot plate
assay (latency time ¼ 42.6 s ꢀ 4.2 s). The time course of the effect of
compound 31 at 30 mg/kg in the hot plate test is shown in Fig. 4B. A
significant antinociceptive effect could be achieved at 5 min

14
J. Higgs et al. / Neuropharmacology 72 (2013) 9e19
Fig. 2. Competition curves of naltrexone (-) and compounds 8 (
6
), 9 ( ), 10 (þ), 12 (:), 15 (>), 31 (B) and 32 (C) for [3H]-DAMGO binding to Wistar rat forebrain membranes.
;
Each point represents the mean ꢀ S.E.M., expressed as a percentage of [3H]-DAMGO bound, of two to four independent experiments performed in duplicate.
3.4. Effect of compound 31 in the inverted screen test
plate test. The treatment of these antagonists did not evoke any
response by themselves in this test.
Compound 31 at 30 mg/kg, the higher dose used in all the
pharmacological tests performed, did not alter the motor coordi-
nation performance of mice in the inverted screen assay, as the
number of mice that climb to the top of the screen (Chi-
Square ¼ 0.00001; P ¼ 1) (Fig. 7A) and the time mice spent climbing
(unpaired t-test, P ¼ 0.4429) (Fig. 7B) did not have significant dif-
ferences with the control group.
The pre-treatment with naltrexone (a nonselective opioid re-
ceptor antagonist, 5 mg/kg, i.p.) given 20 min before the injection of
compound 31 (30 mg/kg, i.p.) caused an inhibition of the analgesic
effect induced by this flavanone in the hot plate test. In turn, nal-
trindole (a potent and highly selective d opioid receptor antagonist,
5 mg/kg, i.p.), given 20 min before the administration of compound
31 (30 mg/kg, i.p.) and nor-binaltorphimine (a potent and highly
selective
k opioid receptor antagonist, 10 mg/kg, s.c.), given 24 h
before the administration of the flavonoid, did not antagonize the
antinociceptive action in the hot plate test.
3.5. Effects of flavanone 31 in the plus maze test
ANOVA of the results obtained for compound 31 (3 mg/kg,
10 mg/kg and 30 mg/kg) did not yield statistically significant dif-
ferences in the percentage of open arm entries, time spent in open
arms and in the number of total arm entries in the plus maze test
(data not shown).
3.7. Effect of a single administration of morphine or compound 31
on gastrointestinal transit
Compound 31, at 30 mg/kg, did not modify the gastrointestinal
transit of mice compared with vehicle treated mice (mean ꢀ S.E.M.:
62.5% ꢀ 4.5% and 62.1% ꢀ 3.6%, respectively). On the other hand,
3.6. Analysis of the possible mechanism of the antinociceptive
action of compound 31
As shown in Table 3, pre-treatment of the animals with ketan-
serin (0.5 mg/kg, i.p., a selective antagonist for the 5-HT2A/C re-
ceptor), prazosin (0.2 mg/kg, i.p., an a1-adrenoceptor antagonist)
and yohimbine (2 mg/kg, i.p., an a2-adrenoceptor antagonist) given
20 min before the injection of compound 31 (30 mg/kg, i.p.) failed
in to revert the antinociceptive effect of this compound in the hot
Table 2
Binding affinity of selected natural and synthetic flavonoids for the
m
-opioid re-
ceptors present in rat forebrain membranes.
Compound
Ki (m
M)a
8
9
Hesperetin
Naringenin
Flavone
Quercetin
6-Methylflavone
3,3-Dibromoflavanone
Chalcone
39.04 ꢀ 1.32b
98.42 ꢀ 15.59
120.20 ꢀ 16.66
11.32 ꢀ 2.26
10
12
15
31
32
106.50 ꢀ 13.85
0.846 ꢀ 0.263
28.21 ꢀ 9.97
a
Ki ꢀ standard error of the mean values are means of 2e4 independent de-
terminations and estimate the inhibition of [3H]-DAMGO binding to rat forebrain
membranes as described under Materials and methods. Naltrexone, used as a
control, gave a Ki value of 0.21 ꢀ 0.01 nM.
Fig. 3. Effect of the i.p. injection of compound 31 on the acetic acid induced writhing in
mice. Results are expressed as mean ꢀ S.E.M of the number of writhes. The writhing
test was performed 30 min after the i.p. injection of vehicle (VEH) or compound 31.
*P < 0.05, **P < 0.01 significantly different from VEH; Dunnett’s multiple comparison
test after one-way ANOVA. Number of animals per group ranged between 7 and 12.
b
Loscalzo et al., 2011.

J. Higgs et al. / Neuropharmacology 72 (2013) 9e19
15
Fig. 5. Antinociceptive activity of compound 31 on paw licking during (A) first and (B)
second phase of the formalin test. Vehicle (VEH) and flavanone 31 were i.p. adminis-
tered 30 min prior the injection of formalin in the dorsal surface of the right hind paw.
The licking time was measured during a period of 5 min (A) or 15e50 min (B) after the
formalin injection. Indomethacin (10 mg/kg, i.p.) was used as reference drug. Data are
expressed as mean ꢀ S.E.M. *P < 0.05, **P < 0.01 significantly different from VEH;
Dunnett multiple comparison test after ANOVA (n ¼ 6e13 mice/group).
Fig. 4. Effect of flavanone 31 on the hot plate test in mice. A) Mean ꢀ S.E.M. of the
reaction time of mice in the hot plate test, 30 min after an i.p. injection of vehicle (VEH)
or increasing doses of compound 31. *P < 0.05, **P < 0.01 significantly different from
VEH; Dunnett multiple comparison test after ANOVA (n ¼ 8e17 mice/group). B)
Mean ꢀ S.E.M. of the time course of the effect of compound 31 at the dose of 30 mg/kg.
*P < 0.05, ***P < 0.001 significantly different from the corresponding VEH; unpaired t-
test (n ¼ 6e15 mice/group).
receptors present in rat forebrain membranes, with a Ki value of
0.846 ꢀ 0.263
Recently, several flavonoid aglycones from Vitex agnus-castus L.,
were found to bind to both and opioid receptors in a dose-
dependent manner. Casticin, a flavanone, was found to have the
highest affinity for opioid receptors with Ki value of
1.14 ꢀ 0.17 M. Meanwhile, for apigenin a Ki value of 16.2 ꢀ 3.16
was reported (Webster et al., 2011).
mM.
m
d
m
a
morphine
6
mg/kg
decreased
gastrointestinal
transit
m
mM
(mean ꢀ S.E.M.: 24.6% ꢀ 3.2%; ***P < 0.0001, significantly different
from vehicle; Dunnett multiple comparison test after ANOVA
[F(2,17) ¼ 32.25, P < 0.0001], n ¼ 6 mice/group), which means an
inhibition of 60.4%.
An efficient synthesis of compound 31 was developed, the
procedure employed was simply, easy and cheap, and the yield
obtained was appropriate. Then, the in vivo effects of flavanone 31,
after acute treatments, were studied using behavioral and anti-
nociceptive assays. The antinociceptive tests are based on an inhi-
bition of a nocifensive response to a normally painful stimulus, in
which opioid receptors are involved, as they have a fundamental
participation in pain transmission.
4. Discussion
In the present work the capacity of a series of flavonoids to bind
to the
m
opioid receptor was evaluated. We found that flavonoid
opioid receptors. On the
The sequence of the
among human, rat and mouse. Also,
mouse are very similar to those described in the rat (Moskowitz
and Goodmans, 1984). It has been demonstrated that the opioid
receptor ligand DAMGO showed similar specificity in competition
binding studies in whole brain homogenate in rat and mouse. There
was, also, no difference between the mouse and rat in the density
and affinity of DAMGO sites (Yoburn et al., 1991). For practical
reasons, we decided to perform the binding studies using rat
forebrains. Meanwhile, mice were used for the pharmacological
assays.
m
opioid receptors is highly conserved
glycosides seem unlikely to bind to
contrary, several natural aglycones (hesperetin, naringenin,
flavone, quercetin, 6-methylflavone, and chalcone) were able to
displace the binding of the specific
m
m
and site localizations in the
d
m
m
opioid agonist [3H]DAMGO
with Ki values ranging from 10 M to 100 mM. These results point
m
out that the presence of a sugar moiety in the flavonoid glycosides
appears to interfere for the interaction with the receptor. The most
active compound 3,3-dibromoflavanone (31), a synthetic flavonoid
from our library of compounds, presented the highest inhibition of
the binding of the selective tritiated ligand [3H]DAMGO to
m opioid

16
J. Higgs et al. / Neuropharmacology 72 (2013) 9e19
Fig. 7. Effect of the i.p. injection of compound 31 in the inverted screen test in mice.
Results are expressed as mean ꢀ S.E.M. of (A) number of mice which succeed in per-
forming the test and (B) time spent climbing; registered 30 min after an i.p. injection of
vehicle (VEH) or compound 31 (n ¼ 7e10 mice/group).
Fig. 6. Effect of the i.p. injection of compound 31 in the hole board and locomotor
activity tests in mice. Results are expressed as mean ꢀ S.E.M. of (A) the hole board
parameters and (B) spontaneous locomotor activity counts; registered in 5 min ses-
sions, 20 min after an i.p. injection of vehicle (VEH) or compound 31 (n ¼ 7e12 mice/
group).
the misinterpretation of results (Jürgensen et al., 2005). This can be
avoided by complementing the test with other models of noci-
ception and with motor toxicity tests, as the inverted screen test,
where compound 31 showed no motor impairment patterns at the
higher dose it produced complete suppression of the writhing
response.
The formalin induced nociception assay measures the ability of a
substance to attenuate moderate continuous pain generated by
injured tissue. The early and late phases of formalin nociception are
considered to represent neurogenic and inflammatory pain
behavior, respectively. The first phase is acute and represents the
direct chemical stimulation of nociceptors, whereas the second
phase is tonic and appears to be dependent on the combination of
an inflammatory reaction in the peripheral tissue and central
sensitization. Centrally acting drugs inhibit both phases of pain,
while peripheral acting drugs such as acetylsalicylic acid or indo-
methacin, only inhibit the second phase (Bannon and Malmberg,
2007; Tjølsen, 1992). The i.p. administration of 10 mg/kg and
30 mg/kg of compound 31 reduced significantly the licking time in
both phases of the formalin test.
In summary, the present study demonstrates that systemic (i.p.)
administration of 3,3-dibromoflavanone (31) elicits a dose depen-
dent inhibition of the nociceptive behavioral response in mice
submitted to chemical and thermal pain inducing stimuli and
demonstrates the central action of this drug and its effect on
visceral pain.
To further investigate the mechanism of action of compound 31
in vivo blockade experiments of receptors which are involved in the
mechanism of pain were performed. The hot plate test was used as
It is necessary to apply several tests, with differences regarding
stimulus quality, intensity and duration, to obtain a complete pic-
ture of the antinociceptive properties of a drug (Tjølsen, 1992;
Vidyalakshmi et al., 2010). Nociceptive tests use electrical, thermal,
mechanical, or chemical stimuli. They can be of short duration
representing the phasic pain or of long duration stimuli repre-
senting the tonic pain (Le Bars et al., 2001).
In the hot plate test short thermal stimulus is employed and the
behavioral responses measured are considered to be supraspinally
integrated responses, so it is suitable for evaluation of centrally but
not of peripherally acting antinociceptive drugs. Compounds
exhibiting good antinociceptive effect in this method may be
considered as potent analgesics (Bannon and Malmberg, 2007;
Vidyalakshmi et al., 2010). Compound 31 significantly increased the
latency time in the hot plate test at 10 mg/kg and 30 mg/kg. The
antinociceptive effect reached its maximum at 20 min after drug
injection, remained relatively stable until 60 min and slowly
decreased after this time without reaching control values until
120 min.
Different irritating chemical agents can be used as nociceptive
stimuli to assess pain and preclinically evaluate analgesic drugs,
such as acetic acid and formalin (Barrot, 2012). In the writhing test
the irritating agents, usually acetic acid, are administered i.p.
inducing a stereotyped behavior characterized by abdominal con-
tractions evidencing visceral pain (Le Bars et al., 2001). This test has
poor specificity because the abdominal writhing response may be
suppressed by muscle relaxants and other drugs, leaving scope for

J. Higgs et al. / Neuropharmacology 72 (2013) 9e19
17
Table 3
Effect of different antagonists pretreatments on the antinociceptive activity of 3,3-dibromoflavanone in mice measured on the hot plate assay.
Pretreatment
Treatment
Time before
Latency time (s)a
measure (min)
Vehicle
Vehicle
Vehicle
Compound 31 (30 mg/kg)
Vehicle
Compound 31 (30 mg/kg)
Vehicle
Compound 31 (30 mg/kg)
Vehicle
Compound 31 (30 mg/kg)
Vehicle
Compound 31 (30 mg/kg)
Vehicle
Compound 31 (30 mg/kg)
Vehicle (i.p.)
Compound 31 (30 mg/kg, i.p.)
Vehicle (i.p.)
50
50
50
50
50
50
50
50
50
50
50
50
1470
1470
1470
1470
17.9 ꢀ 0.7
25.1 ꢀ 1.1***
19.6 ꢀ 1.4
23.2 ꢀ 2.4*
20.0 ꢀ 2.0
24.4 ꢀ 2.2**
18.0 ꢀ 2.1
25.0 ꢀ 2.0**
20.5 ꢀ 1.2
23.2 ꢀ 0.5*
20.0 ꢀ 1.6
18.3 ꢀ 1.4þþ
17.8 ꢀ 0.6
25.0 ꢀ 1.3***
20.0 ꢀ 1.5
26.7 ꢀ 2.3***
Yohimbine (2 mg/kg)
Yohimbine (2 mg/kg)
Ketanserin (0.5 mg/kg)
Ketanserin (0.5 mg/kg)
Prazosin (0.2 mg/kg)
Prazosin (0.2 mg/kg)
Naltrindole (5 mg/kg)
Naltrindole (5 mg/kg)
Naltrexone (5 mg/kg)
Naltrexone (5 mg/kg)
Vehicle (s.c.)
Vehicle (s.c.)
Nor-binaltorphimine (10 mg/kg, s.c.)
Nor-binaltorphimine (10 mg/kg, s.c.)
Compound 31 (30 mg/kg, i.p.)
Number of animals per group ranged between 6 and 22. The symbols denote significance levels: *P < 0.05, **P < 0.01, ***P < 0.001, significantly different from vehicleevehicle
(two-way ANOVA, Bonferroni post-hoc test); þþP < 0.01 significantly different from vehicle-compound 31 (one-way ANOVA followed by NewmaneKeuls test).
a
Results are expressed as mean ꢀ S.E.M. of the latency time (s). Mice were pretreated with vehicle, yohimbine, ketanserin, prazosin, naltrexone, naltrindole or nor-
binaltorphimine. The hot plate test was performed 30 min after the i.p. injection of compound 31 or vehicle.
it distinguished the strong central antinociceptive actions of
m
selective k and d opioid receptor antagonists, nor-binaltorphimine
opioids and a2-adrenoceptor agonists from the weak, predomi-
nantly peripheral actions of the NSAIDs, which were ineffective in
and naltrindole, respectively, were used to investigate the
possible participation of these receptor subtypes on flavanone 31
action. Only naltrexone could block the antinociceptive action of
this paradigm (Le Bars et al., 2001). Antinociceptive properties of
k
opioid agonists, a1-adrenoceptor agonists and serotonin agonists
could also be detected in this model (Millan, 1994). Moreover, in
advantage to the formalin test it is a faster and easier method to
determine the supraspinal response.
3,3-dibromoflavanone, reinforcing the involvement of the m opioid
receptors in its mechanism of action. In turn, naltrexone completely
abolished the antinociceptive action caused by injection of
morphine (6 mg/kg, i.p.) (Loscalzo et al., 2011).
Adrenergic receptors appear to play an important role in the
modulation of pain and their activation can generate notable
analgesic effects (Carroll et al., 2007). Central a2-adrenoceptors
mediate sedative-hypnotic, analgesic, hypotensive and anxiolytic
responses (Lakhlani et al., 1997). Some data indicate that all three
a2-adrenoceptor subtypes are implicated in the regulation of pain
perception in the mouse (Philipp et al., 2002) and an involvement
of a1-adrenoceptors in nociceptive responses was described
(Tanoue et al., 2002). Prazosin and yohimbine are selective antag-
onists of a1 and a2-adrenoceptors, respectively, but they do not
differ between receptor subtypes (Bourin et al., 2005; Yue et al.,
2007). Both antagonists, prazosin and yohimbine, were ineffective
to reverse the antinociceptive action of flavanone 31 in the hot plate
test at the doses tested, demonstrating that a1 and a2 adreno-
ceptors were not involved in the antinociceptive activity of this
drug.
The serotonergic system is involved in the regulation of many
physiological and behavioral functions, including mood, sleep and
appetite (Schloss and Williams, 1998). The multiple 5-HT receptor
types within the spinal cord appear to fulfill different roles in the
control of nociception (Millan, 1994). Several pieces of evidence
point that 5-HT2 are involved in the modulation of pain perception,
acting at spinal and supraspinal level (Ormazábal et al., 1999). The
subtypes 5-HT2A and 5-HT2C predominate in the CNS. The pre-
treatment of mice with ketanserin, a 5-HT2A/C receptor antago-
nist, did not reverse the antinociceptive action of compound 31 in
the hot plate test at the doses tested, evidencing that 5-HT2A/C re-
ceptors are not implicated in this action.
Efficacy is normally the primary outcome in the evaluation of
analgesics; however, it is equally important to consider that opioid
receptor agonists are not devoid of unpleasant side effects. For
example morphine is considered the “gold standard” analgesic, but
effective analgesic doses can produce sedation, constipation,
dependence and addiction. Previous rodent studies have also
assessed the influence of morphine on locomotion (Neubert et al.,
2007). The effect of compound 31 on general mice behavior was
evaluated using the hole board test, which provides measurements
of the exploratory behavior of mice, and it is sensitive to depressant
effects of a variety of compounds which act by different mechanism
of actions (File and Pellow, 1985). The locomotor activity test was
also performed as it is a commonly used assay to evaluate potential
sedative effects of compounds (Vogel et al., 2002). In both assays,
the i.p. administration of the flavanone 31 did not reduce the
exploration and ambulatory activity of the animals, discarding that
compound 31 presents sedative effects in the CNS at the doses
tested. Constipation is a common morphine undesired side effect
that can appear even after a single dose (Kromer,1988). In this work
we found that, unlike morphine, a single administration of the
maximal dose tested for compound 31 (30 mg/kg) was not able to
produce an inhibition of gastrointestinal transit on the charcoal
meal test.
Patients with chronic pain frequently have comorbid anxiety
disorders. As some flavanone derivatives, such as 6-bromoflavanone
and 5-methoxy-6,8-dibromoflavanone have shown anxiolytic ac-
tivity (Ognibene et al., 2008), we decided to evaluate the possible
anxiolytic activity of compound 31 in the plus maze test. Flavanone
31 did not show anxiolytic activity in mice at the doses tested. It was
suggested that substitution at position 6 of the flavanone nucleus
with a bromine atom is essential for the compounds to manifest
elevated anxiolytic properties (Ognibene et al., 2008).
To study the implication of the opioid receptors in the anti-
nociceptive effect of compound 31, the antagonists naltrexone, nor-
binaltorphimine and naltrindole were used. Naltrexone is consid-
ered a nonselective antagonist of opioid receptors; it has higher
affinity for
m
opioid receptors, then for
k
and even less for
d
opioid
A structure activity relationship (SAR) was developed with the
basic flavone nucleus and many monohydroxy and monomethoxy
receptors (Peng et al., 2007; Takemori and Portoghese, 1984). The

18
J. Higgs et al. / Neuropharmacology 72 (2013) 9e19
Harbone, J.B., Williams, C., 2000. Advances in flavonoid research since 1992.
Phytochemistry 5, 481e505.
Havsteen, B.H., 2002. The biochemistry and medical significance of the flavonoids.
Pharmacol. Ther. 96, 67e202.
Ioja, E., Tourwé, D., Kertész, I., Tóth, G., Borsodi, A., Benyhe, S., 2007. Novel diaste-
reomeric opioid tetrapeptides exhibit differing pharmacological activity pro-
files. Brain Res. Bull. 74, 119e129.
Jäger, A.K., Lasse, S., 2011. Flavonoids and the CNS. Molecules 16, 1471e1485.
Jürgensen, S., Dalbó, S., Angers, P., Santos, A.R., Ribeiro-do-Valle, R.M., 2005.
Involvement of 5-HT2 receptors in the antinociceptive effect of Uncaria
tomentosa. Pharmacol. Biochem. Behav. 81 (3), 466e477.
Katavic, P.L., Navarro, H., Lamb, K., Prisinzano, T.E., 2007. Flavonoids as opioid re-
ceptor ligands: identification and preliminary structureeactivity relationships.
J. Nat. Prod. 70 (8), 1278e1282.
Kaur, R., Singh, D., Chopra, K., 2005. Participation of alpha-2 receptors in the anti-
nociceptive activity of quercetin. J. Med. Food 8 (4), 529e532.
Kieffer, B., Gavériaux-Ruff, C., Befort, K., Massotte, D., Becker, J., 2009. Brain opioid
receptor systems: molecular and functional aspects. Eur. Neuro-
psychopharmacol. 19, S1eS2.
derivatives. It was demonstrated that the flavone nucleus per se has
an inherent analgesic action; the C2eC3 double bond in the flavone
nucleus appears to be essential for the analgesic effect and sub-
stitution of different groups (hydroxyl or methoxyl) at different
positions in the flavone nucleus altered the analgesic potency of
flavone (Thirugnanasambantham et al., 1993). Moreover, the
flavonoid core was studied as a potential new scaffold for the
development of opioid receptor ligands and it was observed that
the stereochemistry of the C2 and C3 positions, and its substitution,
is important for antagonist activity and selectivity (Katavic et al.,
2007). Some dihydroxy flavones (5,30-, 7,30-, 20,30-, 20,40-, 3,30-,
5,6-, 3,7 and 6,30- dihydroxy flavones) with antinociceptive activity
have been already synthesized. These flavones produced dose
related antinociception through mechanisms that involve an
interaction with opioid and GABAergic systems (Vidyalakshmi
et al., 2010). In the present work we demonstrated that 3,3-
dibromoflavanone (31), despite having C2eC3 single bond in its
structure, presents antinociceptive activity.
Koster, R., Anderson, M., Beer, E.J., 1959. Acetic acid for analgesic screening. Fed.
Proc. 18, 412e418.
Kromer, W., 1988. Endogenous and exogenous opioids in the control of gastroin-
testinal motility and secretion. Pharmacol. Rev. 40 (2), 121e182.
Lakhlani, P.P., Macmillan, L.B., Guo, T.Z., McCool, B.A., Lovinger, D., Maze, M.,
Limbird, L.E., 1997. Substitution of a mutant a2a-adrenergic receptor via “hit
and run” gene targeting reveals the role of this subtype in sedative, analgesic,
and anesthetic-sparing responses in vivo. Proc. Natl. Acad. Sci. of U. S. A. 94,
9950e9955.
5. Conclusion
The results of the present study provided convincing evidence
that i.p. administration of 3,3-dibromoflavanone (31), at doses that
do not interfere with the motor performance and the gastrointes-
tinal transit, exerted clear dose dependent antinociception when
assessed in the chemical and thermal models of nociception in mice
Le Bars, D., Gozariu, M., Cadden, S.W., 2001. Animal models of nociception. Phar-
macol. Rev. 53 (4), 597e652.
Lister, R.G., 1987. The use of a plus maze to measure anxiety in the mouse. Psy-
chopharmacology (Berl) 92, 180e185.
Loscalzo, L.M., Wasowski, C., Paladini, A.C., Marder, M., 2008. Opioid receptors
are involved in the sedative and antinociceptive effects of hesperidin as
well as in its potentiation with benzodiazepines. Eur. J. Pharmacol. 580,
306e313.
and it seems that its action is related to the activation of the
opioid system.
m
Loscalzo, L.M., Yow, T.T., Wasowski, C., Chebib, M., Marder, M., 2011. Hesperidin
induces antinociceptive effect in mice and its aglicone, hesperetin, binds to m-
opioid receptor and inhibits GIRK1/2 currents. Pharmacol. Biochem. Behav. 99,
333e341.
It could be suggested that flavanone derivatives can afford
opioid ligands emerging as novel analgesics without motor side
effects.
Manara, L., Bianchi, G., Ferretti, P., Tavani, A., 1986. Inhibition of gastrointestinal
transit by morphine in rats results primarily from direct drug action on gut
opioid sites. J. Pharmacol. Exp. Ther. 237 (3), 945e949.
Marder, M., Paladini, A.C., 2002. GABAA-receptor ligands of flavonoid structure. Curr.
Top. Med. Chem. 2, 853e867.
Acknowledgments
Marder, M., Viola, H., Wasowski, C., Fernández, S.P., Medina, J.H., Paladini, A.C., 2003.
6-Methylapigenin and hesperidin: new valeriana flavonoids with activity on
the CNS. Pharmacol. Biochem. Behav. 75, 537e545.
Martinez-Vazquez, M., Ramirez Apan, T.O., Aguilar, H., Bye, R., 1996. Analgesic and
antipyretic activities of an aqueous extract and of the flavone linarin of Buddleia
cordata. Planta Med. 62, 137e140.
Meotti, F.C., Luiz, A.P., Pizzolatti, M.G., Kassuya, C.A.L., Calixto, J.B., Santos, A.R.S.,
2006. Analysis of the antinociceptive effect of the flavonoid myricitrin: evi-
dence for a role of the L-arginine-nitric oxide and protein kinase C pathways.
J. Pharmacol. Exp. Ther. 316, 789e796.
Milano, J., Rossato, M.F., Oliveira, S.M., Drewes, C., Machado, P., Beck, P., Zanatta, N.,
Martins, M.A., Mello, C.F., Rubin, M.A., Ferreira, J., Bonacorso, H.G., 2008. Anti-
nociceptive action of 4-methyl-5-trifluoromethyl-5-hydroxy-4, 5-dihydro-1H-
pyrazole methylester in models of inflammatory pain in mice. Life Sci. 83 (21e
22), 739e746.
This work has been supported by grants from CONICET (PIP
Number 112-201101-00045) and UBA (UBACyT Number
20020100100415), Argentina.
References
Abbott, F., Melzack, R., Samue, C., 1982. Morphine analgesia in the tail-flick and
formalin pain test is mediated by different neural systems. Exp. Neurol. 75 (3),
644e651.
Bannon, A.W., Malmberg, A.B., 2007. Models of nociception: hot plate, tail flick, and
formalin tests in rodents. Curr. Protoc. Neurosci. S41, 8.9.1e8.9.16.
Barrot, M., 2012. Tests and models of nociception and pain in rodents. Neuroscience
211, 39e50.
Millan, M.J., 1994. Serotonin and pain: evidence that activation of 5HT1A receptors
does not elicit antinociception against noxious thermal, mechanical and
chemical stimuli in mice. Pain 58, 45e61.
Bourin, M., Masse, F., Dailly, E., Hascoët, M., 2005. Anxiolytic-like effect of milna-
cipran in the four-plate test in mice: mechanism of action. Pharmacol. Biochem.
Behav. 81, 645e656.
Bradford, M.M., 1976. A dye binding assay for protein. Anal. Biochem. 72, 248e254.
Carroll, I., Mackey, S., Gaeta, R., 2007. The role of adrenergic receptors and pain:
the good, the bad, and the unknown. Semin. Anesth. Perioper. Med. Pain 26,
17e21.
Carter, R.B., 1991. Differentiating analgesic and non-analgesic drug activities on rat
hot plate: effect of behavioral endpoint. Pain 47, 211e220.
Chou, T.C., Chang, L.P., Li, C.Y., Wong, C.S., Yang, S.P., 2003. The anti-inflammatory
and analgesic effects of baicalin in carrageenan-evoked thermal hyperalgesia.
Anesth. Analg. 97, 1724e1729.
Moskowitz, A.S., Goodmans, R.R., 1984. Light microscopic autoradiographic locali-
zation of
m and d opioid binding sites in the mouse central nervous system.
J. Neurosci. 4, 1331e1342.
Murray, C.W., Porreca, F., Cowan, A., 1988. Methodological refinements to the mouse
paw formalin tests: an animal model of tonic pain. J. Pharmacol. Method 20 (2),
175e186.
Neubert, J.K., Rossi, H.L., Pogar, J., Jenkins, A.C., Caudle, R.M., 2007. Effects of mu- and
kappa-2 opioid receptor agonists on pain and rearing behaviors. Behav. Brain
Funct. 3, 49e59.
Ognibene, E., Bovicelli, P., Adriani, W., Saso, L., Laviola, G., 2008. Behavioral effects of
6-bromoflavanone and 5-methoxy-6,8-dibromoflavanone as anxiolytic com-
pounds. Prog. Neuropsychopharmacol. Biol. Psychiatry 32 (1), 128e134.
Ormazábal, M.J., Goicoechea, C., Alfaro, M.J., Sánchez, E., Martín, M.I., 1999. Study of
mechanisms of calcitonin analgesia in mice. Involvement of 5-HT3 receptors.
Brain Res. 845, 130e138.
Coughenour, L.L., Maclean, J.R., Parker, R.B., 1977. A new device for the rapid mea-
surement of impaired motor function in mice. Pharmacol. Biochem. Behav. 6,
351e353.
De Diesbach, H., Kramer, H., 1945. Sur quelques dérivés de la tétrahydro-quinoléine.
Helv. Chim. Acta 28 (1), 1399e1405.
Fernández, S.P., Wasowski, C., Paladini, A.C., Marder, M., 2006. Central nervous
system depressant action of flavonoid glycosides. Eur. J. Pharmacol. 539,
168e176.
File, S.E., Pellow, S., 1985. The effects of triazolobenzodiazepines in two animal tests
of anxiety and in the hole board. Br. J. Pharmacol. 86, 729e735.
Guzmán-Gutiérrez, S.L., Navarrete, A., 2009. Pharmacological exploration of the
sedative mechanism of hesperidin identified as the active principle of Citrus
sinensis flowers. Planta Med. 75 (4), 295e301.
Peng, X., Knapp, B.I., Bidlack, J.M., Neumeyer, J.L., 2007. Pharmacological prop-
erties of bivalent ligands containing butorphan linked to nalbuphine,
naltrexone, and naloxone at
m, d, and k opioid receptors. J. Med. Chem. 50,
2254e2258.
Philipp, M., Brede, M., Hein, L., 2002. Physiological significance of a2-adrenergic
receptor subtype diversity: one receptor is not enough. Am. J. Physiol. Regul.
Integr. Comp. Physiol. 283, R287eR295.

J. Higgs et al. / Neuropharmacology 72 (2013) 9e19
19
Rathee, P., Chaudhary, H., Rathee, S., Rathee, D., Kumar, V., Kohli, A., 2009. Mech-
anism of action of flavonoids as anti-inflammatory agents: a review. Inflamm.
Allergy Drug Targets 8, 229e235.
Umamaheswari, S., Viswanathan, S., Sathiyasekaran, B.W.C., Parvathavarthini, S.,
Ramaswamy, S., 2006. Antinociceptive activity of certain dihydroxy flavones.
Indian J. Pham. Sci. 68 (6), 749e753.
Schloss, P., Williams, D.C., 1998. The serotonin transporter: a primary target for
antidepressant drugs. J. Psychopharmacol. 12 (2), 115e121.
Schreiber, S., Backer, M.M., Pick, C.G., 1999. The antinociceptive effect of venlafaxine
in mice is mediated through opioid and adrenergic mechanisms. Neurosci. Lett.
273, 85e88.
Vidyalakshmi, K., Kamalakannan, P., Viswanathan, S., Ramaswamy, S., 2010. Anti-
nociceptive effect of certain dihydroxy flavones in mice. Pharmacol. Biochem.
Behav. 96 (1), 1e6.
Viswanathan, S., Sambantham, P.T., Reddy, K., Kameswaran, L., 1984. Gossypin-
induced analgesia in mice. Eur. J. Pharmacol. 98, 289e291.
Schulz, R., Wuster, M., Herz, A., 1979. Centrally and peripherally mediated inhibition
of intestinal motility by opioids. Naunyn-Schmiedeberg’s Arch. Pharmacol. 308,
255e260.
Vogel, H.G., Vogel, W.H., Schölkens, B.A., Sandow, J., Müller, G., Vogel, W.F., 2002. Drug
Discovery and Evaluation: Pharmacological Assays, second ed. Springer, Heidelberg.
Wasowski, C., Marder, M., 2012. Flavonoids as GABAA receptor ligands: the whole
story? J. Exp. Pharmacol. 4, 9e24.
Takemori, A.E., Portoghese, P.S., 1984. Comparative antagonism by naltrexone and
naloxone of
m
,
k
, and
d
agonists. Eur. J. Pharmacol. 104, 101e104.
Webster, D.E., He, Y., Chen, S., Pauli, G.F., Farnsworth, N.R., Wanga, Z.J., 2011.
Opioidergic mechanisms underlying the actions of Vitex agnus-castus L. Bio-
chem. Pharmacol. 81, 170e177.
Tanoue, A., Koshimizu, T., Tsujimoto, G., 2002. Transgenic studies of a1-adrenergic
receptor subtype function. Life Sci. 71, 2207e2215.
Thirugnanasambantham, P., Viswanathan, S., Ramaswamy, S., Krishnamurty, V.,
Mythirayee, C., Kameswaran, L., 1993. Analgesic activity of certain flavone de-
rivatives: a structureeactivity study. Clin. Exp. Pharmacol. Physiol. 20 (1), 59e63.
Tjølsen, A., 1992. The formalin test: an evaluation of the method. Pain 51 (1),
5e17.
Yoburn, B.C., Lutfy, K., Candido, J., 1991. Species differences in
m and d opioid re-
ceptors. Eur. J. Pharmacol. 193 (1), 105e108.
Yue, C.Q., Ye, J., Li, C.L., Li, R.T., Sun, Q., 2007. Antinociceptive effects of the novel
spirocyclopiperazinium salt compound LXM-10 in mice. Pharmacol. Biochem.
Behav. 86, 643e650.