Journal of Ethnopharmacology 192 (2016) 225–235

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Journal of Ethnopharmacology

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Aqueous extract from Ipomoea asarifolia (Convolvulaceae) leaves andits phenolic compounds have anti-inflammatory activity in murinemodels of edema, peritonitis and air-pouch inflammation

Allanny A. Furtado a, Manoela Torres-Rêgo a, Maíra C.J.S. Lima a, Mariana A.O. Bitencourt a,Andréia Bergamo Estrela a, Nayara Souza da Silva a, Emerson Michell da Silva Siqueira b,José Carlos Tomaz c, Norberto Peporine Lopes c, Arnóbio Antônio Silva-Júnior a,Silvana M. Zucolotto b, Matheus F. Fernandes-Pedrosa a,n

a Laboratório de Tecnologia e Biotecnologia Farmacêutica, Departamento de Farmácia, Faculdade de Farmácia do Rio Grande do Norte-UFRN, Natal, RN,Brazilb Laboratório de Farmacognosia, Departamento de Farmácia, Faculdade de Farmácia do Rio Grande do Norte-UFRN, Natal, RN, Brazilc Núcleo de Pesquisa em Produtos Naturais e Sintéticos, Departamento de Física e Química, Faculdade de Ciências Farmacêuticas de Ribeirão Preto, RibeirãoPreto, SP, Brazil

a r t i c l e i n f o

Article history:Received 28 March 2016Received in revised form15 July 2016Accepted 15 July 2016Available online 19 July 2016

Keywords:Anti-inflammatoryIpomoea asarifoliaConvolvulaceaeIn vivo models

x.doi.org/10.1016/j.jep.2016.07.04841/& 2016 Elsevier Ireland Ltd. All rights rese

espondence to: Laboratório de Tecnologia e BCordeiro de Farias, Petrópolis, Natal, RN, Braail address: mpedrosa@ufrnet.br (M.F. Fernan

a b s t r a c t

Ethnopharmacological relevance: Ipomoea asarifolia (Desr.) Roem. and Schult.(Convolvulaceae), popularlyknown as salsa or salsa-brava, is a plant of which the decoction of leaves is used in folk medicine to treatvarious inflammatory disorders such of dermatitis, scabies, symptoms of syphilis, skin ulcers and ex-ternal wounds. However, little is known about possible compounds and mechanisms of action of theplant to support the activities reported by popular use.Aim of the study: The study aimed to identify bioactive molecules present in the crude extract of I.asarifolia leaves and investigate the anti-inflammatory potential of this extract in different experimentalin vivo models to improve the understanding on that activity.Material and methods: Aqueous extract of I. asarifolia leaves was prepared by decoction (1:10 m/v) and itschromatographic profile was obtained by high performance liquid chromatography coupled with diodearray detector (HPLC-DAD) and liquid chromatography diode array detector coupled with mass spec-trometry (LC-DAD-MS). The potential anti-inflammatory activity of the extract was assessed using thefollowing in vivo models: xylene-induced ear edema (20, 30 and 40 mg/kg), evaluating the degree ofedema formation; carrageenan-induced peritonitis (10, 20 and 30 mg/kg), evaluating leukocyte migra-tion and cytokine levels (IL-1β, IL-6, IL-12 and TNF-α) at 4 h; zymosan-induced air pouch inflammation(20, 30 and 40 mg/kg), evaluating the kinetics of leukocyte migration by total and differential counts at 6,24 and 48 h. The same tests were conducted using pure compounds identified in the aqueous extractfrom I. asarifolia leaves in different doses for each experimental model.Results: The compounds identified in the aqueous extract of I. asarifolia leaves by HPLC-DAD and LC-DAD-MS were rutin, chlorogenic acid and caffeic acid. The extract significantly reduced ear edema in-duced by xylene (81%, 85% and 86% for doses of 20, 30 and 40 mg/kg, respectively, po0.001), as well ascell migration in experimental models of peritonitis (70%, 78% and 83% for doses of 10, 20 and 30 mg/kg,respectively, po0.001) and air pouch inflammation (58%, 67% and 53% for doses of 20, 30 and 40 mg/kg,respectively, po0.001). In addition, the extract demonstrated the ability to significantly inhibit theproduction of cytokines IL-1β, IL-6, IL-12 and TNF-α (po0.001). The secondary metabolites tested (rutin,chlorogenic acid and caffeic acid) also showed the ability to significantly (po0.001) decrease theparameters analyzed above.Conclusion: This is the first study to identify and confirm these phenolic compounds in I. asarifolia leavesextract and to suggest that these compounds contribute to the anti-inflammatory activity in vivo, as

rved.

iotecnologia Farmacêutica, Departamento de Farmácia, Faculdade de Farmácia do Rio Grande do Norte-UFRN, Av. Gal.zil.des-Pedrosa).

A.A. Furtado et al. / Journal of Ethnopharmacology 192 (2016) 225–235226

reported by ethnomedicinal use of this plant. Through the different experimental models performed, wecan conclude that the results obtained with the aqueous extract from I. asarifolia leaves support itspopular use for the treatment of inflammatory disorders.

& 2016 Elsevier Ireland Ltd. All rights reserved.

1. Introduction

Inflammation is a primary process in which the immune sys-tem reacts against an infectious agent, irritation or others injuries.The inflammatory process is characterized by events such as red-ness, increased blood flow to the site of injury, increase of thevascular permeability resulting in extravasation of macro-molecules (e.g. plasma proteins) and leukocytes into the inter-stitium (Serhan et al., 2015). In acute-phase inflammatory re-sponse, there is initially an intense influx of leukocytes (commonlyneutrophils) to the area of inflammatory stimulus. These cellsperform phagocytic activity in an attempt to control the in-flammation (Perez et al., 2014). Subsequently, for activation andamplification of the immune response, there is the production ofproinflammatory mediators such as cytokines, which are keyplayers in the inflammatory process (Cassatela, 1995). The maincytokines present at this stage are IL-1, IL-6 IL-12 and TNF-α, whichare responsible for inducing the expression of adhesion moleculesand promote rolling and adhesion of cells (Zhang et al., 2001; Bi-tencourt et al., 2011). These processes are normally self-controlledand the body is able to return to homeostasis, maintaining tissueintegrity. In cases where inflammation is exacerbated or un-controlled, severe tissue injuries can occur, even leading to func-tion loss (Headland and Norling, 2015). To avoid this outcome, it issometimes necessary to use drugs with anti-inflammatory action,commonly nonsteroidal anti-inflammatory drugs (NSAIDs) andsteroidal anti-inflammatory drugs (SAIDs). However, excessive orprolonged use may cause side effects such as gastric and duodenalulcer, elevated blood pressure, brittle bones and thin skin (Perezet al., 2014; Yuan et al., 2006; Srivastava and Mustafa, 1992). Thesefacts, associated to the difficult access to medicines by part of thepopulation, make the use of plants and their derivatives for thetreatment of various diseases an increasingly common practice inmany parts of the world, reaching 80% of the global population(Brasileiro et al., 2008). The indiscriminate use of these plants,nevertheless, can cause health problems because they are oftenused without pharmacological or toxicity information (Akindeleet al., 2015). Thus, more refined analyses are required to identifyand verify their effects, minimizing damage that may result, forexample, in the case of a poisonous plant (Klein et al., 2009).

The plant Ipomoea asarifolia (Desr.) Roem. and Schult., popu-larly known as salsa or salsa-brava, belongs to the Convolvulaceaefamily and occurs in South and Central America, as well as inAfrica and tropical Asia. In the Brazilian territory, it is found incoastal extensions in the north and northeast (Simão-Bianchiniand Ferreira, 2015); specifically in the semiarid, it is present on thebanks of rivers and dams (Araújo et al., 2008). Despite Ipomoeaasarifolia being considered toxic for cattle, sheep and goats (Riet-Correa and Medeiros, 2001; Mello et al., 2010; Chaves, 2009), arecent study by Akindele et al. (2015), showed that the hydro-ethanolic macerate of this plant when administered for in-traperitoneal route, is relatively safe up to a dose of 1000 mg/kg ina study of subchorionic (90-day) toxicity in rats. In popular med-icine, decoction of salsa leaves is used for various purposes, in-cluding the treatment of dermatitis, scabies, syphilis, skin ulcers,external wounds (Albuquerque et al., 2007; Agra et al., 2007). Twostudies have investigated I. asarifolia for its anti-inflammatory

properties, one using methanolic extract of the leaves in carra-geenan-induced paw edema (Lawal et al., 2010), and another usingaqueous maceration of aerial parts in albumin-induced paw ede-ma model (Jegede et al., 2009); both studies presented a pre-liminary phytochemical test and exercised antiedematogenic ac-tivity significantly. Additionally, our research group has identified,for the first time, the possible presence of the compound rutin inan aqueous extract of the leaves by Thin Layer Chromatographytechnique on a study showing the extract’s ability to minimize theinflammatory effect caused by poisoning scorpion Tityus serrulatusin mice. Other activities studied were: acetylcholinesterase in-hibition potential (Feitosa et al., 2011), hepatoprotective (Faridaet al., 2012), antibacterial (Aliyu et al., 2011) and anti-oxidant ef-fects (Atawodi and Onaolapo, 2010).

The present study aimed to evaluate the anti-inflammatorypotential of aqueous extract I. asarifolia leaves in different ex-perimental in vivo models and identify biomolecules possibly in-volved in the activities indicated by popular use of this plant.

2. Materials and methods

2.1. Plant material

Leaves of Ipomoea asarifolia were collected in the city of Mos-soró, Rio Grande do Norte, Brazil (lat.: �5.1875 long.: �37.3442WGS84), in December 2011. A voucher specimen (registrationnumber MOSS 7096) was deposited in the “Dárdano AndradeLima” Herbarium of “Universidade Federal Rural do Semi-Árido –

UFERSA”. The leaves were washed, dried at room temperature, andmilled. The collection of the plant material was conducted underauthorization of Brazilian Authorization and Biodiversity In-formation System (SISBIO) (process number 35017).

2.2. Preparation of the plant extract

After washed, dried in a circulate air oven at a temperaturer45 °C and ground, the leaves were extracted by decoction incontact with boiling water for 15 min in a 1:10 proportion of plant/solvent (g/mL). The extract was filtered through Whatman paperno.1, and freeze-dried (Liobrass, model L 202, Brazil) for posterioruse.

2.3. Phytochemical screening and Thin Layer Chromatography (TLC)

Aqueous extract of I. asarifolia leaves was subjected to quali-tative tests, based on chemical reaction of color change, foam orprecipitate formation (Matos, 2009). Then, TLC was performedusing aluminum sheets coated with silica gel 60 F254 (Macherey-Nagels) as adsorbent, and ethyl acetate: formic acid: acetic acid:H2O (80:10:5:10 v/v/v/v) as mobile phase. The spots were revealedwith specific chromogenic agents according to the chemical clas-ses analyzed: vanillin sulfuric, Natural Product Reagent A 0.1% –

NP-Reagent – and ferric chloride 1% in methanol. The plates weredried and the spots were observed under short- and long-wave UVlight (254 and 365 nm). Further, co-chromatography of aqueousextract was performed using the following standards: chlorogenic

A.A. Furtado et al. / Journal of Ethnopharmacology 192 (2016) 225–235 227

acid (Sigma-Aldrichs 95%), caffeic acid (Sigma-Aldrichs 98%),rutin (Sigma-Aldrichs 94%), quercetin (Sigma-Aldrichs 95%),kaempferol (Sigma-Aldrichs 97%), ellagic acid (Sigma-Aldrichs

95%), isoquercetin (Sigma-Aldrichs 90%) and vitexin (Fluka 98%).The presence of secondary metabolites identified by color andretention factor (Rf) of the TLC spots of aqueous extract was con-firmed by high performance liquid chromatography coupled withdiode array detector and Liquid Chromatography Diode ArrayDetector coupled with Mass Spectrometry.

2.4. Qualitative High-Performance Liquid Chromatography Analysis(HPLC-DAD)

The identification of the main compounds present in aqueousextract of I. asarifolia was performed using High Performance Li-quid Chromatography (HPLC) in a Varian Pro Star system equippedwith a diode array detector (DAD), a quaternary pump and anautosampler. All HPLC data were processed using the Galaxie™Chromatography software . Chromatographic analyses were per-formed using a reversed-phase column (Luna 5 mm C18,250�4.6 mm, Phenomenexs). The elution system consisted ofacetonitrile 100% (solvent A) and H2O with added acetic acid 0.3%(v/v), adjusted to pH 3.0 (solvent B) at the following gradient: 0–5 min, an isocratic elution with A: B (7:93 v/v); 5–17 min, a linearchange from A:B (7:93 v/v) to A:B (10:90 v/v); 17–25 min, an iso-cratic elution; 25–40 min, a linear change from A:B (10:90 v/v) toA:B (23:77 v/v); 40–50 min, an isocratic elution. The flow rate waskept constant at 1.0 mL/min. The UV spectra were monitored at awavelength of 200–400 nm and the chromatogram was recordedat 340 nm. The peaks were characterized by comparison of theirretention times and UV spectra with the reference standards andby co-injection (extractþstandard). The reference standard solu-tions were prepared in methanol: H2O (3:2, v/v) at a concentrationof 50 mg/mL. The aqueous extract from I. asarifolia leaves wasanalyzed at 3 mg/mL. All solutions prepared for HPLC analysiswere filtered through a 0.45 mm nylon membrane (MILEXs) beforeuse. All analyses were done in triplicate.

2.5. Liquid Chromatography Diode Array Detector coupled with MassSpectrometry Analysis (LC-DAD-MS)

High resolution analyses by LC-DAD-MS was performed on aShimadzu LC-20CE apparatus equipped with an auto sampler (SIL-20A, Shimadzu), diode array detector (SPD-M20AV, Shimadzu) andcoupled to a micrOTOFII (Bruker Daltonics) ESI-qTOF mass spec-trometer. Low resolution analyses applied the same HPLC appa-ratus coupled to amaZon (Bruker Daltonics) ESI-ion trap massspectrometer. A reversed-phase column (Luna 5m C18,250�4.6 mm, Phenomenexs), connected in line, was used forchromatographic analyses. The flow rate was 1.0 mL min, injectionvolume of 20 μL. Mobile phase was H2O with added acetic acid0.3% (v/v) (A) and acetonitrile (B) and the elution gradient was: 0–5 min, an isocratic elution with A:B (13:87 v/v); 5–40 min a linearchange from A:B (13:87 v/v) to (18:82 v/v); 40–45 min, a linearchange from A:B (18:82 v/v) to A:B (19:81 v/v); 45–55 min, a linearchange from A:B (19:81 v/v) at (23:77 v/v); 55–57 min, a linearchange from A:B (23:77 v/v) at (13:87 v/v) and 57–58 min anisocratic elution. The column eluent was split at a ratio of 7:3, thelarger flow going to the DAD detector and the lower one to themass spectrometer. The ion chromatograms of eluted substanceswere recorded between m/z 100 and m/z 1300 in both positive andnegative ionization modes. The spectrum was obtained in highresolution, using capillary and end plate with 3.5 kV and 500 V,respectively. Nitrogen was used as nebulizer (5.5 bar), the flow ofdrying gas was 10 L/mim and drying gas temperature of 220 °C.For chromatographic analysis, ultra-pure water (Millipore, MA,

USA), HPLC-grade acetonitrile (J.T. Bakers) and acetic acid (J.T.Bakers) were used.

2.6. Animals

Male and female Swiss or BALB/c mice (25–35 g), from 6 to8 weeks of age, were utilized. The animals came from the AnimalFacility of the Center for Health Sciences at UFRN and weremaintained under controlled temperature (2272 °C), light/darkcycle of 12 h and with free access to water and commercial feed.Each test group was composed of five animals (n¼5). After theexperiments, all animals were euthanized with an overdose ofthiopental (100 mg/kg) associated with lidocaine 2% (10 mg/kg) i.p. followed by cervical dislocation. The experimental protocol ofthe study was approved by the Committee for Ethics in AnimalExperimentation of the UFRN under protocol number 031/2011 inaccordance to the guidelines of the National Council for the Con-trol of Animal Experimentation (CONCEA).

2.7. Xylene-induced ear edema model

Ear edema was induced according to the procedure previouslydescribed by Parveen et al. (2007). BALB/c mice were randomlyallocated in 14 groups (5 animals in each group) and treated with100 μL, as follows: sterile saline (NaCl 0.9 mg/mL i.p.; group 1);dexamethasone 0.5 mg/kg i.p. (group 2); 20, 30 or 40 mg/kg i.p. ofI. asarifolia aqueous extract (groups 3, 4 and 5, respectively); 2.5,5 or 10 mg/kg i.p. of rutin (groups 6, 7 and 8, respectively); 10, 12.5or 15 mg/kg i.p. of chlorogenic acid (groups 9, 10 and 11, respec-tively); 5, 10 or 15 mg/kg i.p. of caffeic acid (groups 12, 13 and 14,respectively). Subsequently, each animal received 40 μL of xyleneon the anterior and posterior surfaces of the right ear lobe (groups1–14); the left ear received 40 μL of saline and was considered ascontrol. Fifteen minutes later, the mice were euthanized and bothears were removed. Circular sections were taken using a corkborer with a diameter of 7.0 mm, and weighted. The edematousresponse was measured as the weight difference between theright and left ears and the inhibition level (%) was calculated ac-cording to the following equation: Inhibition (%)¼[1�Et/Ec]�100, where Et¼average edema in the treated group (2–14), andEc¼average edema in the untreated group (1).

2.8. Carrageenan-induced peritonitis model

The carrageenan-induced peritonitis model was performedaccording to the procedure previously described by Longhi-Balbi-not et al. (2012), with few modifications. BALB/c mice were ran-domly allocated in 15 groups (5 animals in each group) and treatedwith 100 μL, as follows: sterile saline i.v. (Ctrl; group 1) and (Sal;group 2); dexamethasone 0.5 mg/kg i.p. (Dex; group 3); 10, 20 or30 mg/kg i.v. of I. asarifolia aqueous extract (AE; groups 4, 5 and 6,respectively); 2, 2.5 or 5 mg/kg i.v. of rutin (groups 7, 8 and 9,respectively); 2, 2.5 or 5 mg/kg i.v. of chlorogenic acid (groups 10,11 and 12, respectively); 2, 2.5 or 5 mg/kg i.v. of caffeic acid(groups 13, 14 and 15, respectively). Subsequently, the animalswere injected (i.p.) with saline (group 1) or carrageenan (1 mg/mL,groups 2–16). After 4 h, the animals were euthanized and perito-neal exudates were harvested by peritoneal lavage with 2 mL ofcold sterile saline. Exudates were centrifuged at 1500 rpm for10 min, at 4 °C. One milliliter of the supernatant was stored forcytokine determination, as described below, and the remainingwas removed. Cell pellets was resuspended in 1 mL of sterile salineand this suspension was mixed to Turk's solution (1:10 v/v). Thetotal number of leukocytes was determined using a Neubauerchamber with the aid of a Nikon ECLIPSE E200s microscope at40� magnification (Cunha et al., 1989; Magalhães et al., 2011).

A.A. Furtado et al. / Journal of Ethnopharmacology 192 (2016) 225–235228

The results were expressed as the total number of leukocytes permL.

2.9. Determination of cytokine concentration

The supernatants of peritoneal exudates from animals sub-jected to carrageenan-induced peritonitis model were used tomeasure cytokine levels (IL-1β, IL-6, IL-12 and TNF-α) using ELISAkit (e-Biosciences, San Diego, CA, USA) according to the manu-facturer’s instructions. Results were obtained using the ELISA mi-croplate reader (Epoch, BioTeks) at 450 nm and expressed inpg/mL.

2.10. Zymosan-induced air pouch inflammation model

Air pouch model was performed according to Yoon et al. (2005)with some modifications. The pouch was induced by subcutaneous(s.c.) injection of 5 mL of sterile air into the dorsal region of Swissmice, followed by a second injection of 2.5 mL of air 3 days later,for maintenance. Six days after initial pouch induction, mice wererandomly allocated in 15 groups (5 animals in each group) andtreated with 100 μL, as follows: sterile saline i.p. (Ctrl; group 1)and (Sal; group 2); dexamethasone 2 mg/kg i.p. (Dex; group 3); 20,30 or 40 mg/kg i.p. of I. asarifolia aqueous extract (groups 4, 5 and6, respectively); 2.5, 5 or 10 mg/kg i.p. of rutin (groups 7, 8 and 9,respectively); 2.5, 5 or 10 mg/kg i.p. of chlorogenic acid (groups 10,11 and 12, respectively); 5, 10 or 15 mg/kg i.p. of caffeic acid(groups 13, 14 and 15, respectively). Subsequently, the animalswere injected with saline (group 1) or zymosan (zym; 1 mg/mL,groups 2–15) into the air pouch. After 6 h, the animals were eu-thanized and exudates were harvested from each air pouch bywashing with 2 mL of saline. Exudates were centrifuged at1500 rpm for 10 min, at 4 °C, the supernatants were discarded andcell pellets resuspended with 1 mL of sterile saline and diluted inTurk’s solution (1:10 v/v). The total number of leukocytes wasdetermined using a Neubauer chamber (as described above) andthe number of cells in subpopulations (polymorphonuclear ormononuclear) was determined based on the count of 100 cells onslides obtained by cytocentrifugation (Fialho et al., 2011). Theslides were stained with rapid panoptic staining (Laborclin; Brasil)and were examined under light microscopy (Nikon ECLIPSEE200s) at 100� magnification. The same experimental protocol,including the treatment and control groups, was carried at twodifferent sampling time points (24 and 48 h) with a fixed dose ofaqueous extract (30 mg/kg), rutin (2.5 mg/kg), chlorogenic acid(10 mg/kg) and caffeic acid (10 mg/kg) for temporal analysis.

2.11. Statistical analyses

Data are expressed as mean7standard deviation. Statisticalanalyses were performed by ANOVA test followed by Tukey testusing GraphPad Prism version 5.0 (GraphPad software, San Diego,CA, USA). P-values of less than 0.05 (Po0.05) were considered tobe significant.

3. Results

3.1. Phytochemical screening and Thin Layer Chromatography (TLC)profile

Phytochemical screening of I. asarifolia leaves detected thepresence of phenols, tannins, alkaloids, saponins and flavonoids(data not shown). Thin Layer Chromatography (TLC) of aqueousextracts (data not show) was revealed with vanillin sulfuric acid,ferric chloride (essentially to identify phenolic compounds) and

NP-Reagent (essentially to identify flavonoids). After spraying withvanillin sulfuric acid one yellow spot was observed (Rf¼0.7) sug-gesting the presence of phenolic compounds, as well as a purpleband (Rf¼0.4) suggesting the presence of steroids or terpenes.Spraying the chromatogram with ferric chloride revealed grayzones with Rf similar to those described above, confirming thepresence of phenolic compounds in the extract. To investigate thepresence of flavonoids, the chromatogram was sprayed with NP-Reagent revealing orange and green color bands when analyzedunder UV light at 365 nm. According to Wagner and Bladt (1996),together, these results suggest the presence of flavonoids andphenolic acids in the aqueous extract of I. asarifolia. Throughcomparison with commercial standard of rutin and chlorogenicacid (co-TLC using NP-Reagent), bands were observed (orangecolor at Rf¼0.48 and green color at Rf¼0.66) which werematching the ones observed in the aqueous extract.

3.2. Qualitative Liquid Chromatography profile

Analysis by HPLC-DAD (Fig. 1) showed that the aqueous extractof I. asarifolia leaves presented 10 major peaks at 340 nm. Ac-cording to the UV spectra, all peaks are likely related to phenolicacids, except peaks 5 and 6 which presented spectra characteristicof flavonoids. Of these 10 peaks, 3 peaks were identified, peak 2(tR¼8.3 min), peak 4 (tR¼13 min) and peak 5 (tR¼24 min), aschlorogenic acid, caffeic acid and rutin, respectively. To confirmthe presence of compounds, standard solutions were analyzed byHPLC, showing retention times of 8.3, 13 and 24 min, respectively,similar to the peaks found in the chromatogram of the aqueousextract. Moreover, UV spectra of peaks exhibited UVλmax of325 nm (peak 2), 322 nm (peak 4) and 256 and 351 nm (peak 5),similar to UV spectra of chlorogenic acid, caffeic acid and rutin,respectively (Mabry, 1970). As additional evidence, the co-injec-tion of the standards with aqueous extract resulted in an increasedarea under the respective peaks (data not show).

3.3. Liquid chromatography – mass spectrometry profile

LC-DAD-MS analyses were performed to determine the molarmass of the flavonoids and phenolic acids under investigation andalso to confirm the identification conducted by HPLC-DAD. Thenegative and positive ion mode was used in ESI-MS (electrosprayionization) analysis, where the spectra of compounds were com-pared with those in the MassBank database (http://www.massbank.jp). Negative mode results were used to identify the com-pounds. For compound 2 (RT¼20.1 min), the [M-H]� ion wasobserved at m/z 353, which correspond to chlorogenic acid (354u), while fragment ion at m/z 191 was attributed to [M-H-162]�

with the loss of 162 u corresponding to caffeic acid (180 u) withoutone hydroxyl (17 u) generating an ion at m/z 162 after deproto-nation. For compound 4 (RT¼26.5 min), the [M-H]� ion was ob-served at m/z 179, which correspond to the deprotonated caffeicacid (180 u); the ion of m/z 135 was attributed to [M-H-44]� ,corresponding to the loss of 44 u of CO2 elimination from thecarboxylic acid function. For compound 5 (RT¼40.5 min), the [M-H]� ion was observed at m/z 609, which correspond to deproto-nated rutin (610 u); the other ion fragments found in the spectrumdid not allow a conclusion about the structure of rutin, possiblydue to co-elution with other compounds.

3.4. Effect on xylene-induced ear edema

The anti-edematogenic activity of I. asarifolia leaves extract andselected compounds in xylene-induced ear edema model is pre-sented in Table 1. The groups treated (i.p.) with 20, 30 and 40 mg/kg of aqueous extract before xylene was applied showed a

Fig. 1. (A) HPLC-DAD chromatogram of the aqueous extract from Ipomoea asarifolia leaves. Stationary phase: column (Luna 5 mm C18, 250�4.6 mm, Phenomenexs); mobilephase: acetonitrile: acetic acid 0.3%; flow rate 1.0 mL/min; UV detection at 340 nm. Chemical structures are shown for three posteriorly identified peaks, designated peak 2(tR¼8.3 min), peak 4 (tR¼13 min) and peak 5 (tR¼24 min). UV absorption spectra of the compounds are presented, corresponding to chlorogenic acid (B), caffeic acid (C) andrutin (D), respectively.

Table 1Effects of the aqueous extract (AE) from Ipomoea asarifolia leaves and selectedcompounds on xylene-induced ear edema model. Mice were pretreated (i.p.) withAE or compounds or dexamethasone. Edematous response was measured as thedifference between the right and left ears weight. Inhibition was calculated ac-cording to the following equation: Inhibition (%)¼[1�Et/Ec]�100. WhereEt¼average edema in the treated group, and Ec¼average edema in the untreatedgroup (saline).

Groups treatment Dose (mg/kg) (i.p.) Means (mg) Inhibition (%)

Saline – 32.45072.603 –

Dexamethasone 0.5 6.54072.554*** 79.84AE 20 5.94074.241*** 81.69AE 30 4.56074.199*** 85.94AE 40 4.40071.473*** 86.44Rutin 2.5 7.78072.042*** 76.02Rutin 5 5.56072.248*** 82.86Rutin 10 0.740071.234*** 97.72Chlorogenic acid 10 4.56072.776*** 85.94Chlorogenic acid 12.5 5.54072.569*** 82.92Chlorogenic acid 15 6.50073.978*** 79.97Caffeic acid 5 6.36074.005*** 80.40Caffeic acid 10 3.12072.691*** 90.38Caffeic acid 15 5.26072.676*** 83.79

Values are mean7standard deviation (S.D.), n¼5.*** po0.001, tested-group compared to saline-treated group.

A.A. Furtado et al. / Journal of Ethnopharmacology 192 (2016) 225–235 229

reduction of ear edema of 81.69, 85.94% and 86.44%, respectively,compared to the group which received saline as treatment (con-sidered as 100% edema). These results are similar to the inhibitionof edema formation by the reference drug dexamethasone(0.5 mg/kg; 79.84% inhibition). e compounds analyzed in thismodel showed a significant anti-edematogenic activity at all dosestested. The highest inhibition was achieved at 10 mg/kg, withedema reduction of nearly 98% for rutin, 86% for chlorogenic acid,and up to 90% for caffeic acid.

3.5. Effect on carrageenan-induced peritonitis

The anti-inflammatory effect of the crude extract of Ipomoeaasarifolia leaves and selected compounds were evaluated throughthe carrageenan-induced peritonitis model. According to Bitencourt

et al. (2011), carrageenan 1 mg/mL (i.p.) caused significant accu-mulation of leukocytes at the injection site in animals previouslytreated with saline (i.v.), which was also observed in our results(Fig. 2A). Animals treated with different doses of the aqueous ex-tract (10, 20 and 30 mg/kg) before peritonitis induction presentedsignificantly decreased leukocyte migration into the peritonealcavity, 70.4%, 78.48% and 83.54% at the dose of 10, 20 and 30 mg/kg,respectively (Fig. 2A), compared with the group that were treatedwith sterile saline before carrageenan. In addition, the results ofaqueous extract-treated group were very similar to the group re-ceiving dexamethasone (68.35%), with significantly decreased leu-kocyte migration into the peritoneum of these animals.

To further characterize the anti-inflammatory effect of the ex-tract according to the present experimental model, the con-centration of four different inflammatory cytokines were mea-sured. Aqueous extract in all tested doses, as well as dex-amethasone, were able to significantly reduce the levels of IL-1β,IL-6, IL-12 and TNF-α compared to the group that received sterilesaline before carrageenan (Fig. 2B, C, D and E, respectively).

In addition, the leukocyte migration into the peritoneal cavityand concentrations of cytokines were also measured in groupstreated with the compounds rutin, chlorogenic acid and caffeicacid (Figs. 3–5, respectively). Similar to the aqueous extract anddexamethasone group, each of these compounds was capable ofsignificantly reducing the migration of leukocytes into the peri-toneal cavity, when compared to the saline-treated group (salineand carrageenan). Likewise, concentrations of all tested cytokineswere statistically reduced, except in the case of caffeic acid, whereonly IL1-β and IL-12 levels were significantly decreased.

3.6. Effect on zymosan-induced air pouch inflammation

Mice that received zymosan (s.c.) into the air pouch on thedorsal region and were treated with sterile saline (i.p.) showed astrong influx of exudate along with a large amount of leukocytes tothe site of inflammation. In this model, the treatment with aqu-eous extract had the ability to decrease the inflammatory infiltrateinto the air pouch significantly at all doses tested (58%, 67% and53% for doses of 20, 30 and 40 mg/kg, respectively), compared

Fig. 2. Effects of the aqueous extract (AE) from Ipomoea asarifolia leaves on carrageenan-induced peritonitis model. Mice were treated with AE (i.v.) at doses of 10, 20 and30 mg/kg or with dexamethasone (dex; i.p.) at the dose of 0.5 mg/kg, and subsequently were injected 1 mL of carrageenan. After four hours, the peritoneal lavage wasperformed and the total number of cells was determined in a Neubauer chamber (A). The supernatants were collected for the determination of citokynes IL-1β (B), IL-6(C) and IL-12 (D) and TNF-α (E), which was performed using an enzyme-linked immunosorbent assay. Data represent the mean of the values obtained from five animals, andthe vertical lines indicate the standard deviation. ***po0.001 comparing the group carrageenan (Sal) with treated groups. ### po0.001 comparing the group saline (Ctrl)with all the groups that received carrageenan.

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with the group that received only zymosan and sterile saline(Fig. 6A). The results were similar to the group treated with dex-amethasone 2 mg/kg. In addition, the compounds rutin (Fig. 6B),chlorogenic acid (Fig. 6C) and caffeic acid (Fig. 6D) were also testedin the air pouch model setup. Similarly to the aqueous extract,these compounds were capable of significantly reduce the mi-gration of leukocytes into the air pouch formed in the dorsal re-gion of the mice.

Using a fixed dose of I. asarifolia leaves extract (30 mg/kg), thekinetics of cell migration in the air pouch model was eval-uated, and differential leukocyte counts were performed at threetime points (6, 24 and 48 h). The administration of zymosan(s.c.) caused a significantly increased leukocyte migration at alltime-points (Fig. 7). For animals that were pre-treated with the

Fig. 3. Effect of rutin on carrageenan-induced peritonitis model. Mice were treated withkg, and subsequently were injected 1 mL of carrageenan. After four hours, the peritonealchamber (A). The supernatants were collected for the determination of citokynes IL-1β (immunosorbent assay. Data represent the mean of the values obtained from five animalgroup carrageenan (Sal) with treated groups. ###po0.001 comparing the group saline

extract at a dose of 30 mg/kg, the amount of cells migrating to theair pouch was significantly reduced when compared to the zy-mosan group and saline group at all time points evaluated(Fig. 7A).

Analyzing the cellular profile by differential counts, the crudeextract caused a decrease in the number of polymorphonuclearcells over time, and a parallel increase the number of mononuclearcells (Fig. 7B and C, respectively). The same analyzes were per-formed for rutin (Fig. 7D, E and F), chlorogenic acid (Fig. 7G,H and I) and caffeic acid (Fig. 7J, K and L) as treatments, andall compounds were able to significantly reduce leukocyte mi-gration to the pouch in the dorsal region of the animals during thewhole kinetic study (similarly to the group treated withdexamethasone).

rutin (i.v.) at doses of 2, 2.5 or 5 mg/kg or with dexamethasone (dex; i.p.) at 0.5 mg/lavage was performed and the total number of cells was determined in a NeubauerB), IL-6 (C), IL-12 (D), and TNF-α (E), which was performed using an enzyme-linkeds, and the vertical lines indicate the standard deviation. ***po0.001 comparing the(Ctrl) with all the groups that received carrageenan.

Fig. 4. Effects of chlorogenic acid on carrageenan-induced peritonitis model. Mice were treated with chlorogenic acid (i.v.) at doses of 2, 2.5 or 5 mg/kg or with dex-amethasone (dex; i.p.) at 0.5 mg/kg, and subsequently were injected 1 mL of carrageenan. After four hours, the peritoneal lavage was performed and the total number of cellswas determined in a Neubauer chamber (A). The supernatants were collected for the determination of citokynes IL-1β (B), IL-6 (C), IL-12 (D), and TNF-α (E), which wasperformed using an enzyme-linked immunosorbent assay. Data represent the mean of the values obtained from five animals, and the vertical lines indicate the standarddeviation. ***po0.001 comparing the group carrageenan (Sal) with treated groups. ###po0.001 comparing the group saline (Ctrl) with all the groups that receivedcarrageenan.

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4. Discussion

The use of medicinal plants by the population is a highly re-cognized practice since ancient times, and continues until thepresent day. In the current scenery, in many regions of severalcountries, access to medicines is still very limited and therefore,many people resort to the use of plants, seeking relief from paincaused by certain diseases, or even a cure. Inflammation is acommon event taking place in various disease processes andtherefore the research on plant extracts with anti-inflammatorypotential is considered a promising approach in this field (Calixto,2005; Bellik et al., 2013).

Previous studies on the flowers of Ipomoea asarifolia revealedthe presence of anthocyanins (Pale et al., 1998, 2003), a flavonoid

Fig. 5. Effects of caffeic acid on carrageenan-induced peritonitis model. Mice were trdexamethasone (dex; i.p.) at 0.5 mg/kg, and subsequently were injected 1 mL of carrageecells was determined in a Neubauer chamber (A). The supernatants were collected for thperformed using an enzyme-linked immunosorbent assay. Data represent the mean ofdeviation. ***po0.001, **po0.01 and *po0.05 comparing the group carrageenan (Sasaline (Ctrl) with all the groups that received carrageenan.

subclass known for its anti-oxidant and anti-inflammatory prop-erties (Cardoso et al., 2011; Lv et al., 2015). In addition, there aretwo studies indicating a possible anti-inflammatory activity ofmethanolic (100, 200, 400 mg/kg i.p.) and aqueous maceratedextract (37.5, 75, 150 mg/kg i.p.) from I. asarifolia and reporting thepresence of saponins, tannins, alkaloids, phenols and carbohy-drates (Jegede et al., 2009; Lawal et al., 2010). These studies,however, did not perform a deeper analysis of the compoundspresent in the extracts beyond detection of metabolites classes byphytochemical screening, and the pharmacological effects werenot further characterized.

A previous study by our research group (Lima et al., 2014) wasthe first to indicate the presence of rutin in salsa. Our presentanalyses of the aqueous extract from I. asarifolia leaves by

eated with standard the caffeic acid (i.v.) at doses of 2, 2.5 or 5 mg/kg, or withnan. After four hours, the peritoneal lavage was performed and the total number ofe determination of citokynes IL-1β (B), IL-6 (C), IL-12 (D), and TNF-α (E), which wasthe values obtained from five animals, and the vertical lines indicate the standardl) with treated groups; ###po0.001, ##po0.01, #po0.05 comparing the group

Fig. 6. Effects of the aqueous extract (AE) from Ipomoea asarifolia leaves (A), rutin (B), chlorogenic acid (C) or caffeic acid (D) on zymosan-induced air pouch model. Micewere treated (i.p.) with EA (20, 30, 40 mg/kg), rutin, chlolorogenic and caffeic acid (2, 2.5 and 5 mg/kg) or dexamethasone (dex) (2 mg/kg) and subsequently injected 1 mL ofzymosan. After 6 h the lavage was performed and the total number of cells was determined. Each column represents the mean of the values obtained from five animals, andthe vertical lines indicate the standard deviation. ***po0.001, **po0.01 and *po0.05 comparing the group zym with treated groups; ###po0.001, ##po0.01, #po0.05comparing the group saline (Sal) with all the groups that received zymosan.

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phytochemical screening and TLC combined to HPLC-DAD and LC-DAD-MS indicated the presence of phenolic compounds and con-firmed the presence of rutin, in agreement to Lima et al. (2014). Inaddition, our results allowed the original identification of twophenolic acids in I. asarifolia leaves: chlorogenic acid and caffeicacid. Such compounds are reported in the literature for theirpharmacological properties, for example, antioxidant activity, anti-carcinogenic and anti-inflammatory effects (Kim et al., 2014; Yehet al., 2014).

Different models of experimental inflammation were appliedhere to evaluate the anti-inflammatory potential of the aqueousextract from I. asarifolia leaves in order to verify the activity andprovide basis to understand the mechanism of action involved.

The classic model of vascular permeability evaluation was usedfor analysis of anti-edematogenic activity of the extract from I.asarifolia leaves, since edema is a sign of inflammation, and atrigger to cellular inflammatory response (Kou et al., 2005). Thephlogistic agent xylene, used as an irritant, has the ability topromote vasodilation at the site where it is applied. Through re-lease of the neuropeptide called substance P (SP), it provokes anacute and severe inflammation with fluid leakage, thus resulting inincreased weight of the animal’s ear (Kim et al., 2007; Kou et al.,2005). The extract from I. asarifolia significantly reduced theedema caused by xylene in all tested doses; the greatest percen-tage inhibition was observed at the highest dose tested (86% at

30 mg/kg; Table 1). Furthermore, the compounds rutin, chloro-genic acid and caffeic acid were also able to reduce edema sig-nificantly at all doses tested, ranging from 76% to 97% inhibition.

These results allow us to suggest that the anti-edematogeniceffect of the extract may be due, at least in part, to the presence ofthese compounds. Studies show the efficacy of phenolic com-pounds in combating cardiovascular and neurodegenerative dis-orders, precisely because these compounds act to protect thevessel, maintaining its integrity (Silva et al., 2002; Karunaweeraet al., 2015). Thus, one can hypothesize that the extract is exertinga vasoprotective activity, lowering the amount of plasma to thesite where xylene was applied.

Following evaluation of the anti-inflammatory activity at thelevel of edema, the aqueous extract from I. asarifolia leaves wastested using carrageenan-induced peritonitis model, to assess itsactivity on cell migration processes. Carrageenan is a poly-saccharide derived from seaweed (Prajapati et al., 2014) which hasthe ability to increase vascular permeability, promote the initialaction of histamine and serotonin, and later induce acute in-flammation by the action of bradykinin and prostaglandins, withneutrophil migration to the site of injection (Vinegar et al., 1969;Rauf et al., 2014). These cells, in an attempt to curb the aggressoragent, produce pro-inflammatory mediators such as cytokines,reactive oxygen species, nitric oxide, among others (Li et al., 2015).However, if the inflammation persists, major tissue damage can

Fig. 7. Effects of the aqueous extract (AE) from Ipomoea asarifolia leaves (A-C), rutin (D-F), chlorogenic acid (G-I) and caffeic acid (J-L) on zymosan-induced air pouch modelover time, determined by leukocyte counts. Mice were treated (i.p.) with EA (20, 30, 40 mg/kg), rutin, chlolorogenic and caffeic acid (2, 2.5 and 5 mg/kg) or dexamethasone(dex) (2 mg/kg) and subsequently were injected 1 mL of zymosan (zym). After 6, 24 and 48 h the lavage was performed and the total and differential number of cells wasdetermined. Each column represents the mean of the values obtained from five animals, and the vertical lines indicate the standard deviation. ***po0.001, **po0.01 and*po0.05 comparing the group zymosan (Sal) with treated groups; ###po0.001, ##po0.01, #po0.05 comparing the group saline (Ctrl) with all the groups that receivedzymosan.

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occur and may even result in loss of function. The aqueous extractfrom the leaves of I. asarifolia, as well as the compounds identifiedin the extract, showed a prominent anti-inflammatory effect in theexperimental model of peritonitis induced by carrageenan, sig-nificantly reducing the number of leukocytes into the peritonealcavity of mice at all doses tested. This was corroborated by thequantification of inflammatory cytokines in the peritoneal wash ofthe treated animals.

The mechanism of action by which the extract is acting for thisactivity is not yet known, but some hypotheses can be raised.Rutin is a flavonoid, chlorogenic acid and caffeic acid are phenolicacids, which are already reported in the literature as potentialanti-inflammatory agents (Yeh et al., 2014; Bellik et al., 2013; Kimet al., 2014). The influx of polymorphonuclear leukocytes acrossthe endothelium is mediated by the binding of these cells to en-dothelial receptors such as L-selectin, P-selectin and E-selectin;diapedesis occurs by activation of platelet endothelial cell adhe-sion molecule 1 (PECAM-1) (Kutlar and Embury, 2014; Biswaset al., 2005). A study with experimental models in vitro performedby Hebeda et al. (2011) showed that chlorogenic acid has theability to reduce leukocyte migration, modulating selectin trans-location from intracellular to extracellular environment and in-hibiting the expression of PECAM-1. The extract of I. asarifolia maybe acting through one of these mechanisms, which could explainthe effective decrease in the number of leukocytes that transmi-grate to the peritoneum of the animals.

In order to assess the anti-inflammatory action of I. asarifolia inthe context of a different signaling pathway, air pouch in-flammation model was performed using zymosan as an

inflammatory agent. Zymosan is commercialized as an extractobtained from the membrane of the yeast Saccharomyces cerevisiaespecies containing particles of polysaccharides from the micro-organisms wall, including β-glucan, mannan and chitin, which areinternalized by innate immune cells, such as neutrophils (Di Carloand Fiore, 1958; Makni-Maalej et al., 2013). The model simulates ajoint inflammation, for instance arthritis, because the sterile airinjected into the back of the animals leads to the formation of acoat of cells, resembling a synovial membrane; this model iswidely used to evaluate efficacy of anti-inflammatory drugs aswell as their kinetics (Cabrera et al., 2001). I. asarifolia extractshowed the ability to significantly reduce the number of poly-morphonuclear cells into the pouch cavity at all doses tested at6 h; as there was no significant difference between the doses, thedose of 30 mg/kg was used to perform the kinetic study. At dif-ferent times analyzed, the extract significantly decreased thenumber of leukocytes migrating to the air pouch, as compared tothe group that received only zymosan. The differential relativecounting was performed to assess the cellular profile change overtime and it was observed that I. asarifolia extract had the ability toreduce the number of polymorphonuclear cells at the site of in-flammation with time, like the reference drug dexamethasone.

The same test was performed for the three compounds iden-tified in the aqueous extract from I. asarifolia leaves (chlorogenicacid, caffeic acid and rutin). All compounds were able to reducethe number of leukocytes which have migrated to the dorsal cavityat all times analyzed, having also altered the cell profile as ob-served in differential counts performed. The kinetics results in-dicate that the extract and compounds may act in chronic

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inflammation processes, as it was effective in inhibiting cell mi-gration for longer periods (up to 48 h), however, further studiesare necessary to explore this possibility, for instance using ex-perimental models of chronic arthritis. In addition, when con-sidering the potential application of the extract or compounds asdrugs, the fact that their anti-inflammatory activity is sustained forlonger periods indicate that less replacement doses would beneeded, and therefore less would be the adverse effects that mightarise.

Again, some hypotheses can be considered regarding the me-chanism of action responsible for the observed activity. Zymosanparticles, in order to be internalized by phagocytic cells, need tocontact a repertoire of receptors on the cell surface, such as Dec-tin-1 and Toll-like Receptor 2 (TRL-2) (Makni-Maalej et al., 2013;Jiang et al., 2013). This in turn triggers a cascade of events thatculminate in inflammatory process. Another explanation would bethat, zymosan can activate complement alternative pathway andinduce the production of proinflammatory mediators such as C5peptide, which is a potent neutrophil chemoattractant (Kelly et al.,2008). These mechanisms are important to protect the bodyagainst invading agents, but the activation of these pathwayswhen exacerbated can develop into a severe pathophysiologicalprocess (Headland and Norling, 2015). Compounds identified inthe extract of the I. asarifolia leaves, could, for instance, competewith zymosan particles for the binding to these cell surface re-ceptors. According to Kelly et al. (2008), Toll-like receptor coupledadapter protein MyD88 (myeloid differentiation factor 88), whenactivated, will trigger NFκ-B pathway activation, which is the mainpathway for initiating inflammation. Some studies have shownthat caffeic acid, chlorogenic acid and rutin are implicated in theblocking of this pathway, by binding to these receptors and pre-venting the phosphorylation of inhibitor of kappa B (IκB), thuspreventing the synthesis of inflammatory cytokines such as IL-1,IL-6 e TNF-α (Kim et al., 2014; Xu et al., 2010; Yeh et al., 2014).

5. Conclusion

The results show that the aqueous extract from Ipomoea asar-ifolia leaves have an anti-inflammatory effect in different experi-mental models, suggesting it could be used as an alternative toassist the treatment of inflammatory diseases, as reported bypopular use. It is noteworthy that the compounds identified in theextract appear to be responsible for the activity displayed by thisplant, at least in part, given that these compounds were also ef-fective in the same experimental models. Nevertheless, in order todraw clearer conclusions, further studies should be conductedtaking into consideration other unidentified compounds also de-tected in the extract, which may be also contributing to the ob-served effects.

Conflict of interest

The authors declare that there are no conflicts of interest.

Acknowledgment

The authors acknowledge all participants for their valuabletime and commitment to this study. This research was supportedby grants from CNPq (400743/2013-2) and financial support fromFAPERN (PRONEM 003/2011) and CAPES (Toxinology 63/2010).M.F.F.P. and N.P.L. are researchers from CNPq.

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