Journal of Ethnopharmacology 260 (2020) 112841

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Journal of Ethnopharmacology
journal homepage: www.elsevier.com/locate/jethpharm

Oral treatments with a flavonoid-enriched fraction from Cecropia hololeuca T

and with rutin reduce articular pain and inflammation in murine zymosan-
induced arthritis
Felipe Marques Teixeiraa,1, Mariana Neubarth Coelhob,1,
Fernanda do Nascimento José-Chagasb,c,2, David do Carmo Malvara, Alexandre Kanashirod,
Fernando Queiroz Cunhae, Marcelo Dias Machado Vianna-Filhof,3, Angelo da Cunha Pintoc,4,
Frederico Argollo Vanderlindea,∗∗, Sônia Soares Costab,∗
a
Laboratory of Pharmacology, Department of Physiological Sciences, Institute of Biological and Health Sciences, Federal Rural University of Rio de Janeiro (UFRRJ), BR
465, Km 07, 23890-000, Seropédica, RJ, Brazil
b
Laboratory of Bioactive Natural Products Chemistry, Natural Products Research Institute (IPPN), Federal University of Rio de Janeiro (UFRJ), Av. Carlos Chagas Filho,
373, 21941-902, Rio de Janeiro, RJ, Brazil
c
Institute of Chemistry, Federal University of Rio de Janeiro (UFRJ), Av. Athos da Silveira Ramos, 149, 21941-909, Rio de Janeiro, RJ, Brazil
d
Department of Neurosciences and Behavior, Ribeirão Preto Medical School, University of São Paulo, Av. Bandeirantes 3900, 14049-900, Ribeirão Preto, SP, Brazil
e
Department of Pharmacology, Ribeirão Preto Medical School, University of São Paulo, Av. Bandeirantes 3900, 14049-900, Ribeirão Preto, SP, Brazil
f
Program of Graduate Studies in Plant Biology, Rio de Janeiro State University, Rua São Francisco Xavier, 524, 20550-013, Maracanã, Rio de Janeiro, RJ, Brazil

A R T I C LE I N FO A B S T R A C T

Keywords: Ethnopharmacological relevance: Cecropia Loefl. species (Urticaceae) are widely spread across the rainforest in
Cecropia hololeuca tropical and subtropical regions of Central and South America. Inhabitants of different regions of Brazil employ
Arthritis leaves, fruits and sprouts of Cecropia hololeuca Miq. mainly as anti-inflammatory, anti-asthmatic, expectorant,
Antinociceptive effect fever suppressant, and against cough.
Anti-inflammatory effect
Aim of the study: To evaluate the antinociceptive and anti-inflammatory activities of an aqueous leaf extract of C.
Rutin
Phenolic compounds
hololeuca in a murine model of zymosan-induced arthritis (ZIA) and characterize compounds contributing to
these effects.
Chemical Compounds Studied In This Article: Materials and methods: The crude aqueous extract of C. hololeuca (CAE) was obtained by infusion, screened for
isoorientin antinociceptive and anti-inflammatory activities, and fractionated (solvent partition; RP-2 and Sephadex G-25
2-(3,4-dihydroxyphenyl)-5,7-dihydroxy-6- column chromatography), yielding fractions that were chemically and pharmacologically investigated. TLC,
[(2S,3R,4R,5S,6R)-3,4,5-trihydroxy-6- HPLC-DAD, HPLC-DAD-ESI-MS/MS and NMR analyses were peformed. The antinociceptive activity was assessed
(hydroxymethyl)oxan-2-yl]chromen-4-one by means of acetic acid-induced writhing, hot-plate and rota-rod tests. ZIA was used to evaluate the anti-arthritic
isoquercitrin activity of oral treatment with CAE, butanolic (BF) and aqueous fraction (AF), as well as the fractions obtained
2-(3,4-dihydroxyphenyl)-5,7-dihydroxy-3- from BF (F2, F2-A and F2–B). Rutin, a flavonoid found in C. hololeuca, was also tested. Mechanical hyperno-
[(2S,3R,4S,5S,6R)-3,4,5-trihydroxy-6-
ciception, joint edema, local neutrophil recruitment and articular TNF-α quantification were performed to
(hydroxymethyl)oxan-2-yl]oxychromen-4-one
isovitexin
measure the severity of arthritis and identify the anti-inflammatory potential of C. hololeuca.
5,7-dihydroxy-2-(4-hydroxyphenyl)-6- Results: CAE (0.03–1 g/kg, p.o.) showed a dose-related inhibitory effect on acetic acid-induced writhing test, but
[(2S,3R,4R,5S,6R)-3,4,5-trihydroxy-6- did not change the pain latency in the hotplate test, nor the first fall time on the rota-rod test. In addition, CAE

∗
Corresponding author. Natural Products Research Institute (IPPN), Federal University of Rio de Janeiro (UFRJ), Av. Carlos Chagas Filho, 373, 21941-902, Rio de
Janeiro, RJ, Brazil.
∗∗
Corresponding author. Department of Physiological Sciences, Institute of Biological and Health Sciences, Federal Rural University of Rio de Janeiro (UFRRJ), BR
465, Km 07, 23890-000, Seropédica, RJ, Brazil.
E-mail addresses: felipemarquesvet@gmail.com (F.M. Teixeira), mari.neubarth@gmail.com (M.N. Coelho), fernandajosechagas@gmail.com (F.d.N. José-Chagas),
dcmalvar@gmail.com (D.d.C. Malvar), alex_bioquimica@yahoo.com.br (A. Kanashiro), fdqcunha@fmrp.usp.br (F.Q. Cunha),
marceloviannafilho@gmail.com (M.D. Machado Vianna-Filho), favanderlinde@gmail.com, vanderlinde@ufrrj.br (F.A. Vanderlinde), sscostabh@gmail.com,
sscosta@ippn.ufrj.br (S.S. Costa).
1
Both authors contributed equally to this study.
2
Present address: National Institute of Industrial Property (INPI), Rio de Janeiro, RJ, Brazil.
3
Present address: Institute of Biology Roberto Alcantara Gomes, Rio de Janeiro State University (UERJ), Rio de Janeiro, RJ, Brazil.
4
in memoriam.

https://doi.org/10.1016/j.jep.2020.112841
Received 4 November 2019; Received in revised form 25 March 2020; Accepted 1 April 2020
Available online 05 April 2020
0378-8741/ © 2020 Elsevier B.V. All rights reserved.
F.M. Teixeira, et al. Journal of Ethnopharmacology 260 (2020) 112841

Abbreviations HPLC-DAD-ESI-MS/MS high-performance liquid chromatography

with diode-array detection-electrospray ioniza-
AF residual aqueous fraction tion–tandem mass spectrometry
BF butanolic fraction HSQC heteronuclear single-quantum coherence
CAE crude aqueous extract i.a. intra-articular
bDMARDs biological disease-modifying anti-rheumatic drugs Indo indomethacin
csDMARDs conventional synthetic disease modifying anti-rheu- i.p. intraperitoneal
matic drugs NMR nuclear magnetic resonance
Dexa dexamethasone NSAIDs non-steroidal anti-inflammatory drugs
EDTA ethylenediaminetetraacetic acid p.o. per os (oral administration)
EtOH ethanol s.c. subcutaneous
F1–F6 fractions 1–6 obtained from BF TLC thin layer chromatography
F2-A and F2-B fractions A and B obtained from F2 TNF-α tumor necrosis factor α
HPLC-DAD high-performance liquid chromatography with diode- UV ultraviolet
array detection ZIA zymosan-induced arthritis

(hydroxymethyl)oxan-2-yl]chromen-4-one
rutin
2-(3,4-dihydroxyphenyl)-5,7-dihydroxy-3-
[(2S,3R,4S,5S,6R)-3,4,5-trihydroxy-6-
[[(2R,3R,4R,5R,6S)-3,4,5-trihydroxy-6-
methyloxan-2-yl]oxymethyl]oxan-2-yl]
oxychromen-4-one
(1 g/kg, p.o.) inhibited by 65% the mechanical hypernociception, 46% the joint edema, 54% the neutrophil
recruitment and 53% the articular TNF-α concentration levels in ZIA. BF (0.4 g/kg, p.o.), AF (0.6 g/kg), F2
(0.1 g/kg) and F2-A (0.045 g/kg), but not F2-B (0.055 g/kg), inhibited the mechanical hypernociception, joint
edema and neutrophil recruitment in ZIA. Rutin (0.001–0.03 g/kg, p.o.) produced dose-related inhibitory effects
in the mechanical hypernociception, joint edema and neutrophil recruitment, and at 0.03 g/kg also inhibited
articular TNF-α synthesis after intra-articular zymosan injection. Isoorientin, isovitexin, rutin and isoquercitrin
were identified in the most active fraction (F2-A), along with luteolin and apigenin derivatives, tentatively
identified as isoorientin-2″-O-glucoside and isovitexin-2″-O-glucoside.
Conclusion: This study corroborates the popular use by oral route of aqueous preparations of C. hololeuca against
joint inflammatory disorders, such as rheumatoid arthritis. Our results demonstrated for the first time that oral
administration of rutin shows antinociceptive and anti-inflammatory effects in ZIA, indicating that this flavonoid
is one of the immunomodulatory compounds involved in the anti-arthritic activity of C. hololeuca.

1. Introduction for novel compounds to control inflammation in arthritis with fewer

Chronic pain is one of the most common disabling factors con-
tributing to cognitive impairments, morbidity, and mortality in mul-
tiple clinical disorders, mainly rheumatoid arthritis (Boyden et al.,
2016; Hewlett et al., 2012; Matcham et al., 2014). Rheumatoid ar-
thritis, a chronic auto-immune disorder characterized by deleterious
articular inflammation, hyperplasia of synovial tissues and damage of
the joint cartilage and bone, affects approximately 0.5–1% of the po-
pulation (Firestein, 2003). The articular inflammation is characterized
by the production of cytokines, mainly tumor necrosis factor alpha
(TNF-α), and excessive infiltration of neutrophils into the synovial
cavity, joint pain, edema, stiffness and subsequent loss of functionality.
Neutrophils are the first type of leukocytes that migrate to infectious
site eliminating microorganisms by the production of cytotoxic pro-
ducts such as reactive oxygen species and proteases (Firestein, 2003;
Sweeney and Firestein, 2004). However, neutrophils do not distinguish
between microbial and host cells causing tissue damage (Mantovani
et al., 2011; Wright et al., 2014). Large amounts of neutrophils are
found in the synovial fluid of both clinical and experimental arthritic
joint inflammation, especially in the early phases, but they can also
activate adaptive immunity (Cascão et al., 2010; Firestein, 2003; Jaillon
et al., 2013; Kolaczkowska and Kubes, 2013; Mohr et al., 1981;
Sweeney and Firestein, 2004; Wright et al., 2014). Therefore, neu-
trophils and their products are an interesting target for the treatment of
arthritis and other inflammatory disorders (Cecchi et al., 2018).
The best current treatments for arthritis are based on glucocorti-
coids, non-steroidal anti-inflammatory drugs (NSAIDs), conventional
synthetic disease-modifying anti-rheumatic drugs (csDMARDs) and
biological DMARDs (bDMARDs), which reduce inflammation and pre-
vent neutrophil activation (Ferro et al., 2017; Upchurch and Kay,
2012). However, glucocorticoids, NSAIDs and csDMARDs produce
serious side effects, while bDMARDs are very expensive and can cause
immunosuppression. Thus, efforts have been highlighted on the search
side effects.
People in various parts of the world routinely use plants and herbal
products to alleviate inflammatory processes and pain (Apu et al., 2012;
Ghasemian et al., 2016; Oguntibeju, 2018; Shilpi et al., 2012). Ex-
tensive literature has demonstrated that plants belonging to several
botanical families showed potential use in the treatment of rheumatoid
arthritis due to their inhibitory properties in the cellular and humoral
immune responses (Falcão et al., 2019; Pinto de Oliveira et al., 2018;
Tanwar et al., 2017).
Inhabitants of Brazilian rural communities use aqueous extracts of
leaves from Cecropia Loefl. (Urticaceae) species, known locally as
“embaúba”, mainly for the treatment of inflammatory, respiratory and
cardiovascular disorders as well as for diabetes and gynecological dis-
eases (Agra et al., 2008; Bieski et al., 2012; Silva et al., 2015). Some
Cecropia species have been extensively studied such as C. glaziovii
Snethlage and C. pachystachya Trécul (de Oliveira Aragão et al., 2013;
Lacaille-Dubois et al., 2001; Maquiaveli et al., 2014; Müller et al., 2016;
Pacheco et al., 2014), while others such as Cecropia hololeuca Miq. still
lack information about their pharmacological effects as well as the
identification of their active compounds, despite being the only species
of its genus to feature in the first Brazilian Pharmacopoeia (BRASIL,
1926). C. hololeuca is a very common tree in the Atlantic Forest. Leaves,
fruits and sprouts from this plant are popularly used as anti-in-
flammatory, anti-asthmatic, expectorant, fever suppressant, diuretic,
antihypertensive, sedative and against cough, among others (Botsaris,
2007).
The flavonoids orientin (luteolin-8-C-glucoside), isoorientin (lu-
teolin-6-C-glucoside), vitexin (apigenin-8-C-glucoside) and isovitexin
(apigenin-6-C-glucoside) have been proposed as chemical markers for
leaves of plants from Cecropia genus, along with chlorogenic acid, rutin
(quercetin-3-O-rutinoside), some catechins, procyanidins, steroids, and
triterpenoids (Rivera-Mondragón et al., 2017). Phenolic compounds
from Cecropia sp. have been used in the development of new

2
F.M. Teixeira, et al.
pharmaceutical products. Formulations containing the ethyl acetate
extract from leaves of C. pachystachya, rich in isoorientin, orientin and
chlorogenic acid, have been tested in mice for the treatment of diabetes
(carboxymethylcellulose syrup) and for wound healing (Carbopol® gel).
In vitro assays showed that a flavonoid-containing sample obtained from
C. obtusifolia methanolic extract (isovitexin-2″-O-rhamnoside, iso-
vitexin-2″-O-glucoside, isovitexin-2″-O-xyloside, isovitexin-O-xyloside
and isoorientin-2″-O-xyloside) inhibited the agonistic effect of 100 nM
angiotensin II on the AT1 receptor with the same intensity as the se-
lective AT1 receptor antagonist losartan (Caballero-George and Rivera
Mondragon, 2018; Fontes et al., 2015a; 2015b).
The use of C. hololeuca against inflammatory processes in Brazilian
folk medicine, in addition to the limited chemical and pharmacological
studies focusing on this plant species, encouraged us to (i) investigate
the potential antinociceptive, anti-inflammatory and im-
munomodulatory effects of the oral treatment with the crude aqueous
leaf extract of C. hololeuca in murine zymosan-induced arthritis, and (ii)
explore the compounds contributing to these activities.
2. Materials and methods
2.1. Chemicals and materials
Acetonitrile used in HPLC-DAD analyses, ethanol, butanol and
formic acid were from Tedia (Fairfield, Ohio, USA). Water containing
0.1% formic acid used in HPLC-DAD-ESI-MS/MS analyses, analytical
standards (isoorientin, isovitexin, isoquercitrin and rutin) and Zymosan
A were from Sigma Aldrich Brasil (São Paulo, São Paulo, Brazil).
Acetonitrile used in HPLC-DAD-ESI-MS/MS analyses, TLC plates and
RP-2 stationary phase were from Merck (Darmstadt, Hesse, Germany);
Sephadex G-25 stationary phase from Pharmacia Fine Chemicals
(Uppsala, Uppsala, Sweden) and DMSO-d6 from Cambridge Isotope
Laboratories (Andover, Massachusetts, USA). Deionized water used in
HPLC-DAD analyses was produced in a Milli-Q Gradient A10 System
from Millipore (Burlington, Massachusetts, USA).
2.2. Plant material and extraction
Leaves of a flowering male specimen of Cecropia hololeuca were
collected in Rio de Janeiro (Brazil) and a voucher (RB 555135) was
deposited in the Herbarium of the Botanical Garden of Rio de Janeiro.
After drying for 10 days in an air-conditioned environment, these leaves
were ground in a stainless-steel industrial blender (Croydon, 2 L). A
sample of this leaf material (400 g) was extracted with distilled water
by infusion (5% w/v). After filtration and lyophilization, 59.2 g of the
crude aqueous extract of C. hololeuca (CAE) were obtained.
2.3. Fractionation of the plant extract
Part of the crude aqueous extract of C. hololeuca (44.28 g) was
partitioned between butanol and water based on previous studies with
leaf extracts of C. glaziovii (Tanae et al., 2007). This process yielded the
butanolic fraction (BF; 17.1 g) and the residual aqueous fraction (AF;
26.71 g). A sample of the butanolic fraction (11.63 g) was chromato-
graphed by multiple injections in a reversed-phase column (RP-2;
Merck; 70–230 mesh; 74.0 g; 11.0 × 3.8 cm) using a multi-step system
of distilled water and ethanol as mobile phase (0%, 10%, 30%, 50%,
70% and 100% EtOH). Samples obtained with each ethanol grade were
lyophilized separately, yielding fractions F1 to F6 (6.95 g; 1.20 g;
1.93 g; 191 mg; 19 mg and 15 mg, respectively). The separation was
monitored by TLC using silica gel 60 F254 plates eluted with butanol/
acetic acid/water (8:1:1), visualized under UV light (254 and 365 nm)
and stained with a cerium sulfate solution.
Part of the sample eluted with 10% ethanol (F2; 286 mg) was fur-
ther fractionated on a Sephadex G-25 column (Pharmacia Fine
Chemicals; Fine, 22 g; 43 × 1.6 cm) eluted with distilled water. After
3
Journal of Ethnopharmacology 260 (2020) 112841
the elution with water, the column was washed with 50% ethanol to
retrieve some adhered compounds. Samples showing characteristic
flavonoid TLC profiles were pooled together into fraction F2-A
(125.3 mg) and the remainders were combined into F2-B (149.7 mg).
2.4. HPLC-DAD analyses
HPLC-DAD analyses were performed using a Shimadzu HPLC system
equipped with a LC-20AT pump, a DGU-20A5R degasser, a SIL-20AHT
autosampler, a CTO20A column oven and a SPD-M20A diode-array
detector (Laboratório de Cromatografia Prof. Affonso do Prado Seabra,
Central Analítica, IPPN, UFRJ, Brazil). Chromatographic separations
were carried out at 40°C using a Merck LiChroCART/LiChrospher 100
RP-18 column (250 mm × 4 mm, 5 μm) guarded by a Merck
LiChroCART/LiChrospher 100 RP-18 guard column (4 mm × 4 mm,
5 μm). The mobile phase consisted of water containing 0.1% formic
acid (solvent A) and acetonitrile containing 0.1% formic acid (solvent
B). The following gradient was used, at a flow rate of 1 mL/min:
0–15 min (10–23% B), 15–28 min (23–25% B), 28–30 min (25–100%
B), 30–40 min (100% B). Samples from C. hololeuca (CAE: 5 mg/mL; F2,
F2-A and F2-B: 1.5 mg/mL) and flavonoid standards (isoorientin, iso-
vitexin, isoquercitrin and rutin: 0.5 mg/mL) were analyzed separately.
For coinjection experiments, mixtures of these solutions were prepared
and analyzed (200 μL of F2-A + 100 μL of isoorientin or rutin stan-
dards; 280 μL of F2-A + 20 μL of isovitexin or isoquercitrin standards).
The injection volume was 20 μL. Chromatograms were reported at
280 nm. UV absorption spectra were recorded between 200 and
400 nm.
2.5. HPLC-DAD-ESI-MS/MS analyses
For HPLC-DAD-ESI-MS/MS analyses, chromatographic separations
were carried out at 40°C on a Shimadzu HPLC system equipped with a
LC-20AD pump, a DGU-20A3R degasser, a SPD-M20A diode-array de-
tector (semi-micro flow cell) and a Thermo Scientific ODS Hypersil C-18
column (150 mm × 2.1 mm, 3 μm) guarded by an ODS Hypersil Drop-
In Guard Cartridge (10 mm × 2.1 mm; 3 μm). This system was coupled
to a Bruker Daltonics Amazon SL Ion Trap mass spectrometer equipped
with an Electrospray Ionization (ESI) source (CEMBIO, Instituto de
Biofísica Carlos Chagas Filho, UFRJ, Brazil). The mobile phase consisted
of water containing 0.1% formic acid (solvent A) and acetonitrile
containing 0.1% formic acid (solvent B) in the following gradient, at a
flow rate of 0.3 mL/min: 0–4 min (10–15% B), 4–5 min (15–23% B),
5–10 min (23–25% B), 10–11 min (25–100% B), 11–27 min (100% B).
A sample (20 μL) of fraction F2-A (100 μg/mL) was injected manually
with the aid of a Rheodyne 7725i Sample Injector with a 20 μL loop.
Chromatograms were observed at 280 nm. UV absorption spectra were
recorded between 190 and 400 nm. In the mass spectrometer, nitrogen
was used as nebulizer (1.25 bar) and drying gas (8 L/min, 250°C). The
capillary voltage was set at 3 kV and the end plate offset at 500 V. Ions
were monitored in the 300–750 m/z range in Data Dependent
Acquisition (DDA) mode with selection of 1 precursor ion per cycle.
Fragmentation of precursor ions was induced by Collision Induced
Dissociation (CID) using helium as collision gas (30-300 V). MS/MS
spectra were recorded in the 100–750 m/z range. All mass spectra were
recorded in the negative mode as an average of three measurements.
2.6. NMR analyses
NMR spectra of fraction F2-A were obtained at 40°C using a Varian
VNMRS-500 MHz spectrometer (1H: 499.77 MHz; 13C: 125.68 MHz)
(LAMAR, IPPN, UFRJ, Brazil) using DMSO-d6 as solvent. Parameters
were as follows: 1H NMR (32 scans; 1.0 s relaxation delay; water sup-
pression by presaturation); multiplicity-edited HSQC (96 scans; 512
increments; 1.0 relaxation delay). MNova 12 (Mestrelab Research,
Santiago de Compostela, Spain) was used to process the data.
F.M. Teixeira, et al. Journal of Ethnopharmacology 260 (2020) 112841

2.7. Animals

Adult male Swiss mice (30–35 g) were obtained from the breeding
facility of the Federal Rural University of Rio de Janeiro. Animals were
housed in a temperature-controlled (22 ± 1 °C) room and maintained
on a 12-h light/dark cycle with lights on at 7:00 a.m. The mice were
housed in groups of six to ten per cage (40 cm × 33 cm × 18 cm) and
given free access to food and water. After the experiments the mice
were euthanized by cervical displacement under isoflurane anesthesia
(2.5%). All the experiments were conducted following the National
Institute of Health guidelines for the welfare of experimental animals,
after approval by the Institute of Biological and Health Sciences from
Federal Rural University of Rio de Janeiro (CEUA Protocol 012/2014).

2.8. Antinociceptive evaluation

2.8.1. Acetic acid-induced writhing test

Groups of seven mice were treated orally (p.o.) with vehicle (filtered
water, 10 mL/kg), CAE (0.03–1 g/kg) or indomethacin (0.01 g/kg)
60 min before intraperitoneal (i.p.) injection of acetic acid (1.2%, 0.1
mL/10 g), and the number of writhing was counted for the following
30 min (Koster et al., 1959).

2.8.2. Hot-plate test

The latency of heat stimulus, characterized by the withdrawal of the
hind paw, was measured (in seconds) every 30 min, starting 30 min
before and up to 2 h after the treatment of the mice (n = 6) with
vehicle (filtered water, 10 mL/kg, p.o.), CAE (1 g/kg, p.o.) or fentanyl
(50 μg/kg, s.c.) (D'Amour and Smith, 1941).

2.8.3. Rota-rod test

Rota-rod test was employed according to a previously described
procedure (Rosland et al., 1990). The animals (n = 8) were treated
with vehicle (filtered water, 10 mL/kg, p.o.), CAE (1 g/kg, p.o.) or
diazepam (0.003 g/kg, s.c.). One hour later, the animals were placed on
a rotating bar (15 rpm) and the time until the first fall was recorded in
seconds (s).

2.9. Anti-inflammatory evaluation

We adapted the zymosan-induced arthritis model in mice for our

study (Bassi et al., 2016). Briefly, the animals (n = 7–8) were orally
treated with vehicle (filtered water, 10 mL/kg), crude aqueous extract
(CAE; 1 g/kg), butanolic fraction (BF; 0.4 g/kg), aqueous fraction (AF;
0.6 g/kg), fraction F2 (0.1 g/kg), fraction F2-A (0.045 g/kg), fraction
F2-B (0.055 g/kg) or rutin (0.001, 0.01 and 0.03 g/kg). The positive
control group was subcutaneously treated with dexamethasone
(0.002 g/kg) diluted in sterile saline (0.9% NaCl). Thirty minutes after
the treatments, 10 μL of zymosan suspension (15 μg/μL) in sterile saline
(0.9% NaCl) was injected into the right femoral-tibial joint (intra-ar-
ticular; i.a.) of mice under light isoflurane anesthesia (1.5%).
Fig. 1. Antinociceptive effect of crude aqueous extract of Cecropia holo-
leuca (CAE). (A) Mice (n = 7) were orally treated with CAE (0.03–1 g/kg),
vehicle (filtered water; 10 mL/kg) or the positive control indomethacin (Indo;
0.01 g/kg) 60 min before intraperitoneal (i.p.) injection of acetic acid (1.2%;
0.1 mL/10 g). The number of writhing was counted for 30 min. (B) The latency
(in seconds) of heat stimulus was measured every 30 min, starting 30 min be-
fore and up to 2 h after the treatment of the mice (n = 6) with CAE (1 g/kg,
p.o.), vehicle (filtered water; 10 mL/kg, p.o.) or the positive control fentanyl
(50 μg/kg, s.c.). Values represent the mean ± SEM of the data. *p < 0.05
compared to vehicle treated groups. (A) one-way ANOVA followed by Tukey's
post hoc test; (B) Two-way ANOVA followed by Bonferroni's post hoc test.

2.9.2. Joint swelling

2.9.1. Joint hypernociception
The joint hypernociception was evaluated 5 h after i.a. zymosan
injection by a modified mechanical hypernociception model using
electronic Von Frey apparatus as described by Pinto et al. (2010).
Briefly, using a large tip (4.15 mm2) adapted to the probe, an increasing
perpendicular force was applied to the central area of the plantar sur-
face of the hind paw to induce flexion of the femur–tibial joint followed
by paw withdrawal. The Von Frey apparatus automatically recorded the
intensity of the force in grams (g) applied when the paw was with-
drawn. Results were expressed as the mean ± SEM of the difference
between the flexion-elicited mechanical threshold before (basal) and
after zymosan administration (Δ g).
Under light isoflurane anesthesia, the knee joint thicknesses were
measured 6 h after zymosan injection by caliper in millimeters (mm).
Results were expressed as the mean ± SEM of the difference between
the diameter before (basal) and after zymosan administration (Δ mm).
2.9.3. Neutrophil recruitment
After edema formation measurement, mice were euthanized by
cervical displacement under isoflurane anesthesia and the knee joint
was opened and washed with a saline solution containing EDTA
(1 mM). Aliquots of the resulting washes were diluted in Turk solution.
Leukocytes were counted with the aid of a Neubauer chamber and a

4
F.M. Teixeira, et al.
light microscope. Differential cell counts were stained with hematox-
ylin–eosin and counted under a light microscope. The number of dif-
ferentiated cells was calculated by the percentage found in the total
number of cells (100 cells in total). Results were expressed as the
mean ± SEM of the number of leukocytes (x 104) per joint cavity 6 h
after zymosan administration.
2.9.4. Articular TNF-α levels determination
The knee joint levels of TNF-α were determined 1.5 h after injection
of zymosan. In brief, joints were dissected out, frozen with liquid ni-
trogen, crushed in a mortar and pestle, and solubilized in PBS con-
taining anti-proteases. Then, TNF-α concentrations were evaluated
using a commercially available enzyme-linked immunosorbent assay
(ELISA) following the manufacturer's instructions (Duo-Set kits; R&D
Systems; Minneapolis, MN, USA). Results were expressed as the
mean ± SEM of cytokine levels in pg/mg of joint tissues.
2.10. Data analysis and graphs
Data from pharmacological assays were plotted and statistically
analyzed by a one-way ANOVA followed by the Tukey's multiple
comparison post hoc test or a two-way ANOVA followed by the
Bonferroni's multiple comparison post hoc test, with the aid of the
GraphPad Prism 6.0 (GraphPad Software; La Jolla, CA, USA).
Differences were considered statistically significant when p < 0.05.
Chromatograms were plotted using OriginPro 8 (OriginLab;
5
Journal of Ethnopharmacology 260 (2020) 112841
Fig. 2. Crude aqueous extract of Cecropia holo-
leuca (CAE) (A–C) and its aqueous (AF) and bu-
tanolic (BF) fractions (D–F) reduce hypernoci-
ception, edema and neutrophil recruitment in
zymosan-induced arthritis (ZIA). Mice were
treated with vehicle (filtered water; 10 mL/kg, p.o.),
CAE (1 g/kg, p.o.), AF (0.6 g/kg, p.o.), BF (0.4 g/kg,
p.o.) or the positive control dexamethasone (Dexa,
0.002 g/kg, s.c.) 30 min before intra-articular injec-
tion of zymosan (15 μg/μL, 10 μL/joint). Mechanical
hypernociception (A and D) was measured 5 h after
zymosan while edema formation (B and E) and
neutrophil recruitment (C and F) were measured 6 h
after i. a. zymosan injection. Data are expressed as
the mean ± SEM (n = 7–8) per group for each
experiment. *p < 0.05 compared to vehicle/ZIA
group (one-way ANOVA followed by Tukey's post
hoc test).
Northampton, MA, USA). Chemical structures were drawn using
ChemDraw 12 (PerkinElmer Informatics; Waltham, MA, USA) and
schemes were prepared using Microsoft PowerPoint 2016 (Microsoft
Corporation; Redmond, WA, USA).
3. Results and discussion
The infusion of dried leaves from C. hololeuca (5%; w/v) afforded
the crude aqueous extract (CAE) in 14.8% yield, after lyophilization.
The choice of water as the extraction solvent was guided by the
ethnomedicinal use of this plant (Agra et al., 2008). Extracting with
water also avoided the use of large amounts of organic solvents,
therefore contributing to an overall greener extraction process (Anastas
and Eghbali, 2010; Chemat et al., 2012). Water was also chosen in some
other studies involving Cecropia species (Brango-Vanegas et al., 2014;
Gazal et al., 2014; Müller et al., 2016; Tanae et al., 2007).
3.1. Oral treatment with crude aqueous extract of C. hololeuca (CAE)
reduces nociception in the acetic acid-induced writhing, but not in the hot-
plate test
In order to evaluate the antinociceptive activity of the crude aqu-
eous extract (CAE), we first performed the acetic acid-induced writhing
test. Acetic acid administration induces acute chemical peritonitis by
the release of several inflammatory mediators such as bradykinin,
substance P, prostaglandins and cytokines (Ribeiro et al., 2000). In this
F.M. Teixeira, et al.
test, the oral treatment with CAE (0.03–1 g/kg) produced a dose-related
inhibition on acetic acid-induced writhing with maximal effect at a dose
of 1 g/kg in mice as compared to vehicle-treated group (Fig. 1A),
suggesting an antinociceptive activity for CAE. Similarly, the non-
steroidal anti-inflammatory indomethacin also inhibited the acetic acid-
induced writhing (Fig. 1A). Although acetic acid-induced writhing test
has a good sensitivity to investigate compounds with antinociceptive
activity, this test does not have specificity since it is sensitive to other
agents with different pharmacological properties (Le Bars et al., 2001).
In another experimental set, CAE was evaluated on a hot-plate test
to investigate central antinociceptive effects. This test produces acute
non-inflammatory nociception and has selectivity to drugs with supra-
spinal antinociceptive effect, such as opioid-like drugs (Fischer et al.,
2008; Pinto de Oliveira et al., 2018). In this test, CAE (1 g/kg, p.o.) did
not show antinociceptive effects, while the positive control fentanyl (50
μg/kg, s.c.) increased the pain latency of heat stimulus of the mice
(Fig. 1B). These results suggest the non-involvement of central me-
chanisms in the antinociceptive effect of CAE evidenced in the acetic
acid-induced writhing test.
The antinociceptive effect of CAE was evidenced by a change in the
nociceptive behavior of the mice. Therefore, the rota-rod test was used
to evaluate if the treatments could influence the motor activity of the
animals in order to avoid false positives in the nociception tests, which
could impair the assessment of these results (Costa et al., 2013). In this
test, CAE did not change the first fall time (58.1 ± 0.1 s) on the rota-
rod compared to vehicle group (58.0 ± 1.2 s). As expected, the
6
Journal of Ethnopharmacology 260 (2020) 112841
Fig. 3. Fractions F2 (A–C) and F2-A (D–F), but not
F2-B (D–F), obtained from Cecropia hololeuca re-
duce hypernociception, edema and neutrophil
recruitment in zymosan-induced arthritis (ZIA).
Mice were treated with vehicle (filtered water;
10 mL/kg, p.o.), F2 (0.1 g/kg, p.o.), F2-A (0.045 g/
kg, p.o.), F2-B (0.055 g/kg, p.o.) or the positive
control dexamethasone (Dexa, 0.002 g/kg, s.c.)
30 min before intra-articular injection of zymosan
(15 μg/μL, 10 μL/joint). Mechanical hypernocicep-
tion (A and D) was measured 5 h after zymosan while
edema formation (B and E) and neutrophil recruit-
ment (C and F) were measured 6 h after i.a. zymosan
injection. Data are expressed as the mean ± SEM
(n = 7) per group for each experiment. *p < 0.05
compared to vehicle/ZIA group (one-way ANOVA
followed by Tukey's post hoc test).
positive control diazepam reduced by 65.1 ± 9.1% (20.3 ± 5.3 s) the
first fall time of the animals. This result validates the specificity of the
antinociceptive effect of CAE evidenced in the acetic acid-induced
writhing test.
3.2. Oral treatments with crude aqueous extract of C. hololeuca (CAE) and
with its butanolic and aqueous fractions reduce hypernociception, edema,
and neutrophil recruitment in zymosan-induced arthritis
Since pain is considered the most common clinical manifestation of
rheumatoid arthritis, with a significant impact on life quality of patients
(Matcham et al., 2014), we also investigated the potential activity of
CAE in an experimental arthritis model. The anti-inflammatory activity
of CAE was evaluated using zymosan-induced arthritis (ZIA) model.
Zymosan is a polysaccharide derived from Saccharomyces cerevisiae,
whose intra-articular injection promotes severe erosive synovitis. In the
acute phase, there is a local increase in vascular permeability (im-
portant for the development of edema), TNF-α production, intense
neutrophil migration, as well as marked inflammatory hypernocicep-
tion and fever response (Bassi et al., 2016; da Silva and da Rocha, 2006;
Kanashiro et al., 2009). Several inflammatory mediators are involved in
arthritis development, but cytokine TNF-α has been reported as a
central mediator. Its blockade by specific antibodies is the most effec-
tive protocol currently used in the treatment of arthritis (da Silva and
da Rocha, 2006).
Oral treatment with CAE (1 g/kg) reduced the mechanical
F.M. Teixeira, et al.
Scheme 1. Steps for the extraction of Cecropia hololeuca leaves and frac-
tionation of the crude aqueous extract (CAE). Highlighted samples showed
distinct presence of flavonoids and were pharmacologically active.
hypernociception (Fig. 2A), joint edema (Fig. 2B) and neutrophil mi-
gration (Fig. 2C) induced by i.a. zymosan administration. Moreover,
CAE inhibited the early edema and articular TNF-α production in ar-
thritic mice, as discussed in section 3.6 (Fig. 7A–B). As expected, dex-
amethasone (0.002 g/kg; positive control) also reduced the hyperno-
ciception, joint edema, neutrophil migration, and TNF-α production
induced by ZIA (Fig. 2A–C and 7).
Like the original crude extract (CAE), the oral treatment with bu-
tanolic fraction (BF, 0.4 g/kg) or aqueous fraction (AF, 0.6 g/kg) re-
duced the mechanical hypernociception (Fig. 2D), joint edema (Fig. 2E)
7
Journal of Ethnopharmacology 260 (2020) 112841
and local neutrophil recruitment (Fig. 2F) induced by i.a. zymosan in-
jection. Dexamethasone (0.002 g/kg) was also effective to reduce the
inflammation induced by i.a. zymosan injection (Fig. 2D–F).
The doses of the fractions were selected according to the yield of
each fraction from CAE. The results evidenced that the butanolic frac-
tion was more active than the original crude extract or the aqueous
fraction.
Plant extracts are complex mixtures of primary and secondary me-
tabolites, and may contain numerous bioactive substances, which may
act synergistically with respect to a therapeutic target (Atanasov et al.,
2015). Our preliminary studies have evidenced the presence of
chlorogenic acid and different flavonoids in leaves and inflorescences of
C. hololeuca, including isoorientin, isovitexin and isoquercitrin (Chagas,
2013; José-Chagas et al., 2014). Chlorogenic acid and isoorientin had
already been described in the ethanolic extract of leaves of this plant,
along with orientin and other compounds (Lacaille-Dubois et al., 2001).
Isoorientin has been shown to possess anti-inflammatory activity
(Anilkumar et al., 2017). In unpublished experiments from our group,
oral treatment with chlorogenic acid (0.0002–0.002 g/kg) dose-de-
pendently inhibited mechanical hypernociception, edema and neu-
trophil migration in zymosan-induced arthritis in mice. The HPLC-DAD
profile of CAE was compatible with the presence of these types of
compounds, as discussed in section 3.4.
3.3. Chemical fractionation of the butanolic fraction (BF) and the effect of
resulting fractions on hypernociception, edema and neutrophil recruitment in
zymosan-induced arthritis
The butanolic fraction (BF) was chromatographed in an RP-2
column yielding fractions F1 to F6. Fraction F2, eluted with 10%
ethanol, was selected for further pharmacological evaluation due to a
remarkable presence of flavonoids in its composition. The oral treat-
ment with fraction F2 (0.1 g/kg) or dexamethasone (0.002 g/kg) pro-
duced a marked reduction in the mechanical hypernociception
(Fig. 3A), joint edema (Fig. 3B) and neutrophil migration (Fig. 3C) in-
duced by i.a. zymosan administration.
In view of these results, an aliquot of F2 was fractionated for further
chemical and pharmacological evaluations. Sephadex G-25 was used in
Fig. 4. HPLC-DAD chromatograms of the crude
aqueous extract of Cecropia hololeuca (A) and
fractions F2 (B), F2-A (C) and F2-B (D) at 280 nm.
Samples were analyzed using a Merck LiChroCART/
LiChrospher 100 RP-18 column (250 mm × 4 mm,
5 μm) eluted with water containing 0.1% formic acid
(solvent A) and acetonitrile containing 0.1% formic
acid (solvent B). Gradient: 0–15 min (10–23% B),
15–28 min (23–25% B), 28–30 min (25–100% B),
30–40 min (100% B); flow rate: 1 mL/min; injection
volume: 20 μL; temperature: 40°C; sample con-
centrations: 5 mg/mL (extract) and 1.5 mg/mL
(fractions). Retention times: peak marked with (*)
(9.1 min), peak 1 (13.6 min), peak 2 (14.7 min),
peak 3 (15.9 min), peak 4 (17.2 min),
peak 5 (17.5 min), peak 6 (18.1 min),
peak 7 (33.9 min).
F.M. Teixeira, et al. Journal of Ethnopharmacology 260 (2020) 112841

Table 1
HPLC-DAD-ESI-MS/MS data for compounds 1–6 (flavonoid glycosides) in fraction F2-A obtained from Cecropia hololeuca.
Peak Proposed structure Rt (min) λmax (nm) [M-H]- (m/z) MS2[M-H]- (m/z) (%)
O,C-diglycosylflavones E1– 0,2
X0–/0,2X1– Z1– Ag+71 Ag+71-18 Ag+41 Ag+41-18
1 isoorientin-2″-O-glucoside 7.6 270; 349 609 591(4) 489(52) 429(100) 357(14) 339(10) 327(2) 309(21)
3 isovitexin-2″-O-glucoside 8.2 270; 338 593 473(7) 413(100) 323(5) 293(32)

mono-C-glycosylflavones E1– X0– (Ag+71)

0,3 0,2
X0– (Ag+41)
2 isoorientin 7.9 269; 349 447 429(24) 357(78) 327(100)
5 isovitexin 8.7 269; 339 431 413(8) 341 (43) 311(100)

O-glycosylflavonols 0,2
X0– Y0− [Y0-H]−•
4 rutin 8.6 255; 354 609 343(6) 301(100) 300(25)
6 isoquercitrin 9.0 255; 353 463 343(2) 301(100) 300(13)

Fig. 5. Proposed structures for flavonoids 1–6 indicating general MS2[M-H]– fragment ions observed on HPLC-DAD-ESI-MS/MS analysis of fraction F2-A.
Fragment ions are designated using the nomenclature proposed by Domon and Costello (1988).

this fractionation step in order to avoid the irreversible adsorption of
red-brown components onto the RP-2 phase, which had been observed
during the fractionation of the BF. The complete fractionation process is
summarized in Scheme 1.
The major mode of separation of Sephadex gel is associated with
size exclusion. However, additional adsorption mechanisms also take
place in this cross-linked dextran, as reported in earlier investigations.
Somers (1966) used a Sephadex G-25 column eluted with 60% ethanol
in acidified water to separate condensed tannins from anthocyanins in
wine, observing that the tannins eluted first. According to the author,
this mobile phase almost eliminated the adsorptive capacity of the gel
in this case. Woof and Pierce (1967) described the behavior of simple
phenols, phenolic acids and flavonoids on Sephadex G-25 columns with
different aqueous mobile phases (water, sodium chloride, ammonium
hydroxide, acetic acid and sodium molybdate solutions), making use of
the adsorptive properties of the gel. More recent studies continue to
explore the use of Sephadex G-25 in the separation of phenolics
(Amarowicz and Naczk, 2006; Rabah et al., 2004).
In our case, Sephadex G-25 proved to be a more suitable and even
greener alternative than the silanized silica gel RP-2. The elution was
performed essentially with water and the irreversible adsorption of
compounds onto the gel was minor. After the elution of the flavonoids
(compounds 1–6) with water, we switched the mobile phase to 50%
ethanol to release the remaining material (mainly compound 7).
Flavonoids (1–6) were pooled together into fraction F2-A, while re-
maining components were combined into fraction F2-B. The oral
treatment with F2-A (0.045 g/kg), but not F2-B (0.055 g/kg), produced
an enhanced inhibition on mechanical hypernociception (Fig. 3D), knee
joint edema (Fig. 3E) and neutrophil recruitment (Fig. 3F) in ZIA.
Dexamethasone (0.002 g/kg) was also effective on this experimental set
(Fig. 3D–F). These results indicated that the mixture of flavonoids re-
tained in fraction F2-A possesses anti-inflammatory activity. The frac-
tionation strategy of CAE showed to be efficient, having concentrated
the most potent anti-inflammatory substances in fraction F2-A.
The decreasing order of anti-inflammatory effect observed was as
follows: F2-A > F2 > BF > CAE.
3.4. HPLC-DAD profiling of the crude aqueous extract (CAE) from C.
hololeuca and its fractions F2, F2-A and F2-B
The HPLC-DAD chromatogram of CAE (Fig. 4A) indicated the pre-
sence of different types of phenolic compounds in this sample. UV

8
F.M. Teixeira, et al. Journal of Ethnopharmacology 260 (2020) 112841

Fig. 6. Rutin reduces hypernociception, edema and neutrophil recruit-

ment in zymosan-induced arthritis (ZIA). Mice were treated with vehicle
(filtered water; 10 mL/kg, p.o.), rutin (0.001–0.03 g/kg, p.o.) or the positive
control dexamethasone (Dexa, 0.002 g/kg, s.c.) 30 min before intra-articular
injection of zymosan (15 μg/μL, 10 μL/joint). Mechanical hypernociception (A)
was measured 5 h after zymosan while edema formation (B) and neutrophil
recruitment (C) were measured 6 h after i. a. zymosan injection. Data are ex-
pressed as the mean ± SEM (n = 7) per group for each experiment.
*p < 0.05 compared to vehicle/ZIA group (one-way ANOVA followed by
Tukey's post hoc test).

(Mabry et al., 1970). The UV spectrum of peak 7 showed a single ab-

sorption band (λmax = 277 nm), indicating the presence of a limited
extent of electron conjugation. Compounds proposed as chemical
markers for this plant genus, such as catechins and procyanidins, are
compatible with the UV absorption profile of this peak (Mabry et al.,
1970). A peak compatible with the presence of chlorogenic acid (*) was
also identified in the chromatogram of CAE based on the UV absorption
profile (Fig. S2) and on our previous study with the CAE sample
(Chagas, 2013).
HPLC-DAD analyses were used to evaluate the fractionation of CAE
and showed that more polar compounds, such as the ones in the peak
compatible with chlorogenic acid (*), were separated from the flavo-
noids during the purification process. This peak was not observed in the
chromatogram of fraction F2, which consisted mainly in a mixture of
flavonoids (1–6) and the additional phenolic compound (7) (Fig. 4B).
HPLC-DAD chromatograms of fractions F2-A and F2-B showed that
Sephadex G-25 was able to separate flavonoids 1–6 from compound 7
(Fig. 4C–D). Our pharmacological studies demonstrated that only F2-A
was effective in reducing inflammatory signs in zymosan-induced ar-
thritis, relating the anti-inflammatory activity of F2 to the mixture of
flavonoids 1–6.

3.5. Chemical characterization of F2-A, the most anti-inflammatory

fraction of C. hololeuca extract

Coinjection of F2-A with analytical standards by HPLC-DAD (Fig.

S3) allowed the identification of flavonoids in peaks 2, 4, 5 and 6 as
isoorientin, rutin, isovitexin, and isoquercitrin, respectively. Among
these, only isoquercitrin has not been proposed as a chemical marker
for the Cecropia genus in the study of Rivera-Mondragón et al. (2017).
However, this flavonoid has been reported in other species such as C.
glaziovii (Lacaille-Dubois et al., 2001) and C. pachystachya (Gazal et al.,
2014). Flavonoids in peaks 1 and 3 were tentatively identified as iso-
orientin-2″-O-glucoside and isovitexin-2″-O-glucoside based on our
preliminary study (Chagas, 2013), in which we isolated small amounts
of these compounds from an aliquot of fraction F2 and confirmed their
identity by NMR analyses. In the present study, HPLC-DAD-ESI-MS/MS
(Table 1) and NMR data (Table S1) were obtained for fraction F2-A in
order to corroborate or check for any discrepancies regarding the pro-
posed structures.
Fragment ions were designated according to the nomenclature for
glycoconjugates proposed by Domon and Costello (1988). k,lXj, Yj, and
Zj represent ions containing the aglycone, where j is the number of the
interglycosidic bond counted from the aglycone and k and l denote the
cleavage within the carbohydrate rings. E1– ions represent the loss of a
water molecule. Additional ions were denoted as in Ferreres et al.
(2007), where ‘Ag’ stands for aglycone. Proposed structures and general
fragmentation patterns are shown in Fig. 5.
Compounds 2 (luteolin derivative) and 5 (apigenin derivative)
showed [M-H]- ions at m/z 447 and m/z 431 respectively. Their
MS2[M-H]– spectra showed 0,2X0– ions as the base peak ([(M-H)-
spectra of peaks 1-7 (Fig. S1) suggested the presence of luteolin deri-
120]–; Ag+41) along with abundant 0,3X0– ions ([(M-H)-90]–; Ag
vatives in peaks 1 and 2 (λmax = 270/269; 349 nm); apigenin deri-
+71), which represent characteristic intraglycosidic cleavages from C-
vatives in peaks 3 and 5 (λmax = 270; 338 nm) and C-3 substituted-
hexosyl moieties (Ferreres et al., 2008). These data are in agreement
quercetin derivatives in peaks 4 and 6 (λmax = 255; 354/353 nm)
with the identification of 2 as isoorientin and 5 as isovitexin by HPLC-

9
F.M. Teixeira, et al.
Fig. 7. Crude aqueous extract of Cecropia hololeuca (CAE) and rutin inhibit
the early edema and the increase of articular TNF-α concentration pro-
duced by zymosan-induced arthritis (ZIA). Mice were treated with vehicle
(filtered water; 10 mL/kg, p.o.), CAE (1 g/kg, p.o.), rutin (0.03 g/kg, p.o.) or the
positive control dexamethasone (Dexa, 0.002 g/kg, s.c.) 30 min before intra-
articular injection of saline (10 μL/joint) or zymosan (15 μg/μL, 10 μL/joint).
Edema formation (A, B) was measured 6 h after i.a. zymosan injection. TNF-α
concentration (C) were measured 1.5 h after i.a. zymosan injection. TNF-α was
quantified by ELISA. Representative images (A) from each experimental group
of the knee joints of the animals. Data are expressed as the mean ± SEM
(n = 7) per group for each experiment. # and * p < 0.05 compared to vehicle/
saline and vehicle/ZIA groups respectively (one-way ANOVA followed by
Tukey's post hoc test).
10
Journal of Ethnopharmacology 260 (2020) 112841
DAD coinjection.
Compound 4 showed an [M-H]– ion at m/z 609. Its MS2[M-H]–
spectrum showed Y0− ions at m/z 301 ([(M-H)-162-146]–; deproto-
nated quercetin) as the base peak. A radical aglycone ion at m/z 300
([Y0-H]–•) was also observed, formed by the homolytic cleavage of the
acetal bond between the internal glycoside and the aglycone (Hvattum
and Ekeberg, 2003). 0,2X0– ions (Ag+41) were also observed, resulting
from the cleavage of interglycosidic bonds from the internal glycoside
unit. These data are consistent with the identification of 4 as rutin
(Cuyckens et al., 2001), corroborating our HPLC-DAD coinjection re-
sults.
Compound 6 showed an [M-H]– ion at m/z 463. Its MS2[M-H]–
spectrum showed Y0− ions at m/z 301 ([(M-H)-162]–; deprotonated
quercetin) as base peak. As seen in the fragmentation of rutin, 0,2X0–
(Ag+41) ions and the radical aglycone ([Y0-H]–•) were also found.
These data are compatible with the structure of isoquercitrin, which is
also in accordance with our HPLC-DAD coinjection results.
Compounds 1 and 3 showed [M-H]- ions at m/z 609 and m/z 593,
respectively, and a very similar fragmentation pattern. A difference of
16 mass units was observed not only between their [M-H]- ions but also
when comparing their fragment ions. In agreement with their UV ab-
sorption spectra, this difference was due to the presence of luteolin and
apigenin aglycones in their structures, respectively. MS2[M-H]– spectra
of 1 and 3 showed Z1– ions as the base peak, which is consistent with
O,C-diglycosylation and might be associated with the presence of O-
glycosylation at position 2″ of the C-glycosyl moiety (Ferreres et al.,
2007). Also, Z1– m/z values of [(M-H)-180]– indicate that the external
glycosyl units in 1 and 3 are hexoses. Therefore, by mass difference, the
internal units should also be hexoses. In this specific type of glycoside
(2″-O-hexosyl-C-hexoside), fragments corresponding to [(M-H)-120]–
may represent either 0,2X0– or 0,2X1– ions, resulting from intra-glyco-
sidic cleavages across internal and external hexosyl units, respectively
(Ferreres et al., 2007). Ions [(M-H)-162-90]– (Ag+71) and [(M-H)-
162-120]– (Ag+41) represent the global loss of the external glycosyl
moiety along with different fractions of the internal C-glycoside
(Ferreres et al., 2007).
In the case of luteolin or apigenin, O,C-diglycosylation occurs al-
most exclusively at C-6 or C-8. However, differentiation between those
position isomers based on this technique only is not simple (Ferreres
et al., 2007). Flavonoid O,C-diglycosides compatible with the [M-H]-
ion of compound 3 (m/z 593) have been described in the Cecropia
genus, such as apigenin-6-C-galactosyl-6″-O-β-galactopyranoside
(Oliveira et al., 2003) and vitexin-2″-O-glucoside (Silva Mathias and
Rodrigues de Oliveira, 2019). In this study, compounds 1 and 3 were
tentatively characterized as isoorientin-2″-O-glucoside (1) and iso-
vitexin-2″-O-glucoside (3) based on NMR analyses and comparison with
previous data (Chagas, 2013).
The NMR spectra of F2-A showed some degree of overlapping since
this fraction contains compounds 1–6. Still, the presence of character-
istic signals corroborated the structures proposed for these compounds
(Table S1). Chemical shifts were consistent with our previous work
(Chagas, 2013), where compounds 1–6 were isolated in small amounts
from this plant material and their structures elucidated by thorough
NMR analyzes and comparison with literature data (Cheng et al., 2000;
Krafczyk and Glomb, 2008; Lim et al., 2007).
3.6. Effect of rutin, a flavonoid component of CAE and F2-A, on
hypernociception, edema and TNF-α production in zymosan-induced
arthritis
It has been reported that flavonoids show anti-inflammatory effect
in several animal models; however, there are few reports of their anti-
inflammatory properties in experimental arthritis (Hughes et al., 2017;
Kelepouri et al., 2018; Lim et al., 2019; Sung et al., 2019). The anti-
inflammatory effect of subcutaneous treatment with the flavonoid rutin
was previously reported in a mice zymosan air-pouch model, which
F.M. Teixeira, et al. Journal of Ethnopharmacology 260 (2020) 112841

Table 2
Oral treatment effects of the crude aqueous extract of C. hololeuca, its fractions and rutin on hypernociception, edema and neutrophil recruitment in ZIA.
Group treatment Dose, route of administration Joint hypernociception Joint swelling Neutrophil recruitment

Δg Inhibition (%) Δ mm Inhibition (%) Neutrophils (x104/cavity) Inhibition (%)

Vehicle 10 mL/kg, p.o. 5.20–7.59 – 0.97–1.81 – 1.11–1.92 –

Dexa 0.002 g/kg, s.c. 0.86–2.90 58.5–85.4 0.19–0.49 65.8–80.9 0.26–0.51 71.2–80.6
CAE 1 g/kg, p.o. 1.84 64.6 0.53 45.6 0.51 54.4
AF 0.6 g/kg, p.o. 4.99 30.4 0.70 29.0 0.67 65.4
BF 0.4 g/kg, p.o. 3.53 50.8 0.48 51.8 0.36 81.1
F2 0.1 g/kg, p.o. 3.00 60.5 0.96 47.2 0.44 73.0
F2-A 0.045 g/kg, p.o. 3.11 55.4 0.59 58.9 0.29 78.0
F2-B 0.055 g/kg, p.o. 6.41 8.2 1.36 6.0 1.11 16.3
Rutin 0.001g/kg, p.o. 4,91 16.9 1.16 0 1.16 27.0
0.01g/kg, p.o. 2.28 61.4 0.61 44.9 0.69 56.8
0.03 g/kg, p.o. 1.09 81.6 0.63 43.6 0.33 79.3

Vehicle: water; CAE: crude aqueous extract of C. hololeuca; Dexa: dexamethasone; AF: aqueous fraction; BF: butanolic fraction; F2: fraction 2 obtained from BF; F2-A
and F2-B: fractions A and B obtained from F2, respectively.

simulates a synovial joint inflammation (Furtado et al., 2016; Torres-
Rêgo et al., 2016), and in collagen- and adjuvant-arthritis models (Gul
et al., 2018; Sun et al., 2017). However, there are no studies showing its
immunomodulatory effects in the innate immunity, which can be ob-
served in ZIA. Therefore, we chose to evaluate the effect of the oral
treatment with rutin, one of the flavonoids found in CAE and fraction
F2-A, on inflammatory parameters in this ZIA model. Our results
showed, in general, that rutin at doses of 0.001, 0.01 and 0.03 g/kg
produced a dose-related inhibition on mechanical hypernociception
(Fig. 6A), joint edema (Figs. 6B and 7A), and neutrophil recruitment
(Fig. 6C) in ZIA. In addition, rutin (0.03 g/kg, p.o.) reduced the ar-
ticular TNF-α level similarly to dexamethasone administration (Fig. 7B)
as compared with the vehicle-treated arthritic mice.
The effects of oral treatment with the crude aqueous extract of C.
hololeuca, its fractions and rutin on the inflammatory process (hy-
pernociception, edema and neutrophil recruitment) induced by intra-
articular administration of zymosan in mice are summarized in Table 2.
Our study shows an important characteristic: the extract and rutin
were orally administered, mimicking the most used route in popular
and conventional pharmacological therapies. It has been reported that
after oral administration, rutin is hydrolyzed by intestinal and/or bac-
terial enzymes to quercetin and other conjugated metabolites in the
gastrointestinal system and then absorbed into the circulation (Manach
et al., 1997; Yang et al., 2005). In agreement with our results, a pre-
vious study demonstrated that intraperitoneal administration of quer-
cetin reduces the hypernociception, edema, neutrophil recruitment and
inflammatory mediators (TNF-α and IL-1β) in murine zymosan-induced
arthritis (Guazelli et al., 2018).
Biological agents, such as monoclonal antibodies that neutralize
TNF-α (infliximab), are considered to be the most effective drugs in the
treatment of arthritis (Vivar and Van Vollenhoven, 2014). However,
TNF-α inhibitors are still expensive, increase the risk of opportunistic
infections and malignancies since they can induce an im-
munosuppressive state, and cannot be self-administrated (i.v. route)
(Inanc and Direskeneli, 2006; Smitten et al., 2008). Indeed, different
types of polyphenolic compounds such as phenolic acids, stilbenes and
flavonoids are able to inhibit inflammatory processes by modulating
cytokines, such as TNF-α, mitogen-activated protein kinase, inter-
leukin, among others, and for this reason are considered potential
agents for the treatment of rheumatoid arthritis (Anilkumar et al., 2017;
Huang et al., 2005; Sung et al., 2019).
treatment with C. hololeuca aqueous leaf extract is able to reduce ar-
ticular pain and inflammation in zymosan-induced arthritis (ZIA), and
that its flavonoid-enriched fraction shows an even higher activity,
suggesting that its components may be responsible for the im-
munomodulatory activity. In addition, we also demonstrated for the
first time that the oral administration of rutin, present in C. hololeuca,
shows antinociceptive and anti-inflammatory effects in ZIA. Further
studies on the anti-inflammatory activity of flavonoids isolated from C.
hololeuca, individually or combined, will expand the knowledge on
immunomodulatory activity of these plants and extracts on joint in-
flammatory diseases.
Funding sources
This work was supported by Fundação de Amparo à Pesquisa do
Estado de São Paulo (FAPESP) - Brazil, Conselho Nacional de
Desenvolvimento Científico e Tecnológico (CNPq) - Brazil and
Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES)
- Brazil.
Author contribution
Conception and design of the study: S. S. Costa and F. A.
Vanderlinde.
Plant collection and taxonomic identification of the studied sample:
M. D. M. Vianna-Filho.
Acquisition, analysis and/or interpretation of pharmacological data:
F. M. Teixeira, A. Kanashiro, D.C. Malvar,F. A. Vanderlinde.
Acquisition, analysis and/or interpretation of chemical data: M. N.
Coelho, F. N. José-Chagas, A. C. Pinto (deceased on October 7, 2015), S.
S. Costa.
Drafting the manuscript: M. N. Coelho, S. S. Costa, A. Kanashiro, F.
Q. Cunha, D. C. Malvar, F. A. Vanderlinde.
All the authors have read the final manuscript and approved the
submission.
Declaration of competing interest
The authors declare no conflict of interest.
Acknowledgments

4. Conclusion F. N. José-Chagas and F. M. Teixeira are grateful to CAPES (Brazil)

and M. N. Coelho to CNPq (Brazil) for PhD fellowships. We thank Prof.
In summary, the present study corroborated the popular use of C. Alaíde B. de Oliveira (UFMG, Brazil) for kindly providing the isovitexin
hololeuca aqueous preparations in joint inflammatory disorders, such as standard. We thank São Paulo Research Foundation (FAPESP - 2013/
rheumatoid arthritis. We demonstrated for the first time that the oral 08216-2) for the reagents utilized in the pharmacological study.

11
F.M. Teixeira, et al.
Appendix A. Supplementary data
Supplementary data to this article can be found online at https://
doi.org/10.1016/j.jep.2020.112841.
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