Evaluation of the antinociceptive, anti-inflammatory and gastric antiulcer activities of the essential oil from Piper aleyreanum C.DC in rodents

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Evaluation of the antinociceptive, anti-inflammatory and gastric antiulceractivities of the essential oil from Piper aleyreanum C.DC in rodents

Daniella K.S. Lima a,1, Laudir J. Ballico a,1, Fernanda Rocha Lapa b,d, Hilda P. Gonc-alves a,Lauro Mera de Souza c, Marcello Iacomini c, Maria Fernanda de Paula Werner b, Cristiane Hatsuko Baggio b,Isabela Tiemy Pereira b, Luisa Mota da Silva c, Valdir A. Facundo a, Adair Roberto Soares Santos d,n

a Department of Medicine, Federal University of Rondonia, Porto Velho, RO 78900-500, Brazilb Department of Pharmacology, Sector of Biological Sciences, Federal University of Parana, Curitiba, PR 81531-990, Brazilc Department of Biochemistry and Molecular Biology, Sector of Biological Sciences, Federal University of Parana, Curitiba, PR 81531-990, Brazild Laboratory of Neurobiology of Pain and Inflammation, Department of Physiological Sciences, Center of Biological Sciences, Federal University of Santa Catarina,

Florianopolis, SC 88040-900, Brazil

a r t i c l e i n f o

Available online 12 May 2012

Keywords:

Piper aleyreanum

Essential oil

Antinociception

Anti-inflammation

Gastroprotection

a b s t r a c t

Ethnopharmacological relevance: Piper aleyreanum is a small tree that is widely distributed in tropical

and subtropical regions, mostly in North and South America, and is used as an immunomodulator,

analgesic and antidepressant in folk medicine.

Aim of the study: This study was designed to investigate the antinociceptive, anti-inflammatory and

gastric antiulcer activities of the essential oils from the aerial parts of Piper aleyreanum (EOPa) in rodents.

Materials and methods: The antinociceptive and anti-inflammatory effects of orally administered EOPa were

evaluated in mice subjected to the formalin and pleurisy models, respectively. We also pretreated the rats

with EOPa before acute ethanol-induced gastric lesions and measured gastric lesion extension and mucus and

glutathione (GSH) levels in the gastric mucosa. Finally, we performed a phytochemical analysis of EOPa.

Results: The chemical composition of EOPa was analyzed by gas chromatography and mass spectrometry

(GC/MS), which identified 35 compounds, representing 81.7% of total oil compounds. Caryophyllene oxide

(11.5%), b-pinene (9%), spathulenol (6.7%), camphene (5.2%), b-elemene (4.7%), myrtenal (4.2%), verbenone

(3.3%) and pinocarvone (3.1%) were the major oil constituents. The oral administration of EOPa

(10–1000 mg/kg) significantly inhibited the neurogenic and inflammatory phases of formalin-induced licking,

with ID50 values of 281.2 and 70.5 mg/kg, respectively. The antinociception caused by EOPa (100 mg/kg,

p.o.) was not reversed by naloxone (1 or 5 mg/kg, i.p.) in the formalin test. EOPa (100–300 mg/kg, p.o.) did

not affect animal motor coordination in an open-field model. In carrageenan-induced pleurisy, EOPa

(1–100 mg/kg, p.o.) significantly decreased the total cell count, neutrophils and mononuclear cells with

mean ID50 values of 53.6, 21.7 and 43.5 mg/kg, respectively. In addition, EOPa (1–30 mg/kg, p.o.) protected

the rats against ethanol-induced gastric lesions with an ID50 value of 1.7 mg/kg and increased the mucus and

GSH levels of the gastric mucosa to levels similar to those of the non-lesioned group.

Conclusions: These data show for the first time that EOPa has significant antinociceptive and anti-

inflammatory actions, which do not appear to be related to the opioid system. EOPa also has interesting

gastroprotective effects related to the maintenance of protective factors, such as mucus production and GSH.

These results support the widespread use of Piper aleyreanum in popular medicine and demonstrate that this

plant has therapeutic potential for the development of phytomedicines with antinociceptive, anti-inflam-

matory and gastroprotective properties.

& 2012 Elsevier Ireland Ltd. All rights reserved.

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journal homepage: www.elsevier.com/locate/jep

Journal of Ethnopharmacology

0378-8741/$ - see front matter & 2012 Elsevier Ireland Ltd. All rights reserved.

http://dx.doi.org/10.1016/j.jep.2012.05.016

Abbreviations: List: EOPa, Essential oils from the aerial parts of Piper aleyreanum; GSH, glutathione; GC/MS, gas chromatography and mass spectrometry; ASA,

acetylsalicylic acid; DEX, dexamethasone; IND, indomethacin; SEM, standard error of mean; ANOVA, analysis of variance; NSAIDs, nonsteroidal anti-inflammatory drugs;

COX-1, cyclooxygenase-1; COX-2, cyclooxygenase-2; PGE2, prostaglandin E2; NP-SH, non-protein sulfhydryl groups; ICAM-1, intercellular adhesion molecule-1; VCAM-1,

vascular cell adhesion molecule-1; CRP, C-reactive protein; DMSO, dimethyl sulfoxiden Correspondence to: Departamento de Ciencias Fisiologicas, Universidade Federal de Santa Catarina, Campus Universitario – Trindade, 88040-900, Florianopolis, SC, Brazil.

Tel.: þ55 48 3721 9444x206; fax: þ55 48 3721 9672.

E-mail address: [email protected] (A.R.S. Santos).1 Equally contributed to this work.

Journal of Ethnopharmacology 142 (2012) 274–282


1. Introduction

Inflammation is a basic reaction to infection, irritation or otherinjury and is recognized as a type of nonspecific immune responsethat protects the host against injury and initiates specific immunityresponses. The cardinal signs of inflammation can be clinicallycharacterized as redness, warmth, swelling, loss of functionand pain (Sherwood and Toliver-Kinsky, 2004). Redness andwarmth result from increased blood flow, swelling is associatedwith increased vascular permeability, and pain is the conse-quence of the activation and sensitization of primary afferentnerve fibers (Lawrence et al., 2002; Sherwood and Toliver-Kinsky,2004). Therefore, due to their implication in virtually all humanand animal diseases, inflammation and pain have become thefocus of global scientific research.

The drugs most often used for inflammation and pain relief inhumans are nonsteroidal anti-inflammatory drugs (NSAIDs) andopioids, despite its well-known adverse effects (Shu, 1998;Steinmeyer, 2005; Wallace, 2008). Safer, more effective anti-inflammatory and analgesic drugs are urgently needed. The currenttrend of research is the investigation of medicines of plant originbecause of their affordability and accessibility with minimal sideeffects (Calixto et al., 2001, 2003, 2004; Calixto, 2005).

The Piperaceae family consists of 14 genera and over 1950species that are highly commercially, economically and medicinallyimportant. Piper is the largest genus in the family with 1000 species(Mabberley, 1997); it is widely distributed in tropical and subtropi-cal regions, and there are 260 species found in Brazil (Guimar~aesand Giordano, 2004). Chemical studies performed on some of thespecies have demonstrated that they contain diverse secondarymetabolites, including lignans, neolignans, terpenes, chalcones,flavones, alkaloids, amides and propenyl phenoles (Parmar et al.,1997; Navickiene et al., 2000; Facundo and Morais, 2003; Bezerraet al., 2008; Bokesch et al., 2011; Xie et al., 2011).

Some species of Piper have been shown to possess severalbiological actions, such as immunomodulatory, anti-inflammatory,antinociceptive, antipyretic, anti-platelet, antifungic, cytotoxic, anti-tumor, gastroprotective, anxiolytic and antidepressive properties(Majdalawieh and Carr, 2010; Sunila and Kuttan, 2004; RodriguesSilva et al., 2008; Xie et al., 2011; Yao et al., 2009; Rodrigues et al.,2009; Sireeratawong et al., 2010; Koroishi et al., 2008; Quılez et al.,2010; Morikawa et al., 2004; Zakaria et al., 2010; Cıcero et al., 2007;Bezerra et al., 2006; for review see Sarris et al., 2011).

Piper alyreanum C.DC, a member of the Piperaceae family, is asmall tree that is widely distributed in tropical and subtropicalregions, mostly in North and South America. Here in Brazil, it isfound in the North, mainly in the Amazon forest, and is popularlyknown as ‘‘Jo~ao brandinho’’, ‘‘pimenta longa,’’ ‘‘pimenta longa damata,’’ ‘‘pimenta de cobra’’ and ‘‘pani-nixpu’’. Moreover, this planthas been used as an immunomodulator, analgesic and antide-pressant in folk medicine. Recently, Facundo and Morais, (2003)described the isolation and characterization of b-sitosterol,2-methoxy-4,5-methylenedioxypropi-ophenone and galanginfrom the leaves from Piper alyreanum.

In addition, Facundo et al. (2007) demonstrated the presenceof b-pinene (14.4%), isocaryophyllene (17.5%) and b-caryophyl-lene (18.6%) in the essential oils obtained from the leaves of P.

aleyreanum (EOPa). Taking into account the popular uses andbiological activities of the Piper genus, it is surprising that nopharmacological study has been performed concerning the anti-nociceptive, anti-inflammatory and gastroprotective actions of P.

aleyreanum. Here, we examined: (i) the chemical composition ofEOPa; (ii) the possible antinociceptive, anti-inflammatory andgastroprotective effects of EOPa in standard rodent models ofpain, inflammation, and acute gastric lesions; and (iii) thepossible involvement of opioidergic systems and cytoprotective

factors, such as gastric mucus and GSH levels, respectively, in theantinociceptive and gastroprotective effects of EOPa.

2. Materials and methods

2.1. Plant material

Piper aleyreanum C.DC was collected in September 2008 in thesoutheastern Amazonian Forest, Porto Velho (Rondonia State,Brazil) and was classified by Dr. J. Gomes from the INPA herbar-ium (Instituto Nacional de Pesquisa da Amazonia). A voucherspecimen (no. 223303) was deposited in the INPA herbarium.

2.2. Essential oil extraction and analysis

Essential oil was extracted with vapor dragging and waterdistillation. The fresh aerial (leaves and stems) parts of Piper

aleyreanum (5.0 kg) were placed in a container, and a water vaporcurrent was passed through it under pressure. The volatileproducts present in the aerial parts were dragged by the watervapor, and the mixture was transported to a condenser where thevapors returned to the liquid state and were collected in aseparator flask. EOPa was dried over anhydrous sodium sulfate,and its percentage content was calculated based on the plantfresh weight, resulting in an oil yield of 1%. The oil analysis wasperformed on a gas chromatography with mass spectrometrydetection (GC–MS, model 3800) and ion trap detector (MS, model4000) (Varian, CA), with electron ionization-mass spectrometry(EI-MS �70 eV). The analysis was developed in a VF1-MS capil-lary column (Varian, CA), 30 m long, 0.25 mm thickness and0.25 mm i.d., with helium at 1 ml/min as carrier gas Temperaturesetup: injector at 250 1C, column-oven from 60 to 240 1C at 3 1C/min (Santos et al., 2001), over 70 min. The quantitative analysiswas obtained by peak-area integration, using a Trace GC (Thermo-Scientific), with flame ionization detector (FID) operated undersimilar conditions to those in GC–MS.

The retention index (RI) was calculated for all the volatileconstituents using an n-alkane homologous series, ranging from C8to C30, using a linear temperature programmed equation (Van denDool and Kratz, 1963). Individual components were identified bycomparing the mass spectra and CG-retention data with those ofauthentic compounds previously analyzed and stored in the databasefrom the National Institute of Standards and Technology (NIST). Theinterpretation of RI values was assisted by the Terpenoids Library List(http://massfinder.com/wiki/Terpenoids_Library_List).

2.3. Animals

The experiments were conducted using adult male Swiss mice(25–35 g) and male Wistar rats (200–250 g) that were housed at2272 1C under a 12-h light/dark cycle (lights on at 06:00) with freeaccess to food and water. The animals were habituated to thelaboratory conditions for at least two hours before testing. Theexperiments were performed between 09:00 and 16:00. Each animalwas used only once during the study and immediately after the testthey were sacrificed by CO2 asphyxiation. The experiments wereconducted in accordance with the ‘‘Principles of Laboratory AnimalCare’’ (NIH Publication 85-23, revised 1985) and approved by theEthics Committee for Animal Research of both the Federal Universityof Santa Catarina (protocol number PP00467) and the Federal Uni-versity of Parana (protocol number 446). The number of animals usedand the intensity of the noxious stimuli were the minimum necessaryto obtain reliable data.

D.K.S. Lima et al. / Journal of Ethnopharmacology 142 (2012) 274–282 275


2.4. Nociception induced by formalin

The procedure used was similar to a previously describedprotocol (Santos and Calixto, 1997; Santos et al., 1999). The micereceived 20 ml of a 2.5% formalin solution (0.92% formaldehyde) insaline via an intraplantar injection in the ventral surface of theright hindpaw. The mice received EOPa (30–1000 mg/kg, p.o.) oracetylsalicylic acid (400 mg/kg, p.o., used as positive control)60 min before the formalin injection (Saragusti et al., 2012). Thecontrol mice were treated with vehicle (10 ml/kg, p.o.). Followingthe intraplantar injection of formalin, the mice were immediatelyplaced in a glass cylinder (20 cm diameter), and the time spentlicking the injected paw was recorded with a chronometer forboth the early neurogenic phase (0–5 min) and late inflammatoryphase (15–30 min) of this model. These values were consideredmeasures of nociception.

2.5. Involvement of the opioid system

To investigate the possible involvement of the opioid systemin the antinociceptive effect of EOPa, the mice were pretreatedwith the nonselective opioid receptor naloxone (1 and 5 mg/kg,i.p.) (Santos et al., 1999). After 20 min, the animals received aninjection of EOPa (100 mg/kg, p.o.), morphine (2.5 mg/kg, s.c.) orvehicle (10 ml/kg, p.o.). The other groups were pretreated withvehicle and received morphine, EOPa, or vehicle after 20 min.After 60 and 30 min, they received a formalin (2.5%) injection, andthe time spent licking the injected paw was recorded for both theearly and late phases of the model.

2.6. Evaluation of locomotor activity

The open-field test was used to exclude the possibility that theantinociceptive action of EOPa could be related to non-specificlocomotor activity disturbances. Ambulatory behavior was assessedin an open-field test, as described previously (Rodrigues et al., 2002).The apparatus consisted of a wooden box measuring 40�60�50 cm. The arena floor was divided into 12 equal squares, and thenumber of squares crossed with all paws was counted in a 6-minsession. The mice were treated with EOPa (100 or 300 mg/kg, p.o.) orvehicle (10 ml/kg, p.o.) 60 min before the test.

2.7. Carrageenan-induced pleurisy

Pleurisy was induced in anaesthetized animals by an intrapleuralinjection of carrageenan (1%) or a sterile saline solution (0.9% NaCl)into the right pleural space through the chest skin (final volume0.1 ml) (Henriques et al., 1992; Saleh et al., 1996, 1997). On the dayof the experiments, the animals were challenged with Evans bluedye solution (25 mg/kg, 0.2 ml, i.v.) to evaluate the degree ofexudation into the pleural space (Henriques et al., 1992; Salehet al., 1996). After 1 h, the animals received an injection of EOPa (1–100 mg/kg, p.o.) or vehicle (10 ml/kg, p.o.). The positive controlgroup was pre-treated with indomethacin (5 mg/kg, i.p.) or dex-amethasone (0.5 mg/kg, s.c.) 30 min before carrageenan injection(Saleh et al., 1997). The animals were killed 4 h after the carragee-nan injection, the pleural cavity was washed with 1 ml of sterilesaline solution plus heparin (20 UI/ml), and the fluid in the pleuralspace was collected with automatic pipettes. Total leukocyte countswere performed using pleural fluid diluted with Turk solution (1:20)placed in a Neubauer chamber and assessed with an opticalmicroscope. Cellular smears were prepared with another aliquot ofpleural washing and stained with May–Grunwald–Giemsa for thedifferential analysis, which was performed under an immersionobjective. A sample of fluid (300 ml) collected from the pleural spacewas stored in the freezer (�20 1C) and later used to determine the

concentration of Evans blue dye. This was performed by obtainingthe absorbance values at 600 nm with a spectrophotometer andcomparing them to the standard curve of Evans blue dye in therange of 1–500 mg/ml.

2.8. Induction of acute gastric lesions

The possible gastroprotective effects of EOPa were investigatedon acute ethanol-induced lesions in rats (Robert et al., 1979). Therats were fasted overnight (18 h) prior to the experiment butwere allowed free access to water. Animals were treated withvehicle (C: water or saline plus 0.5% Tween 80, 10 ml/kg, p.o. ori.p.), EOPa (10, 30 or 100 mg/kg, p.o.; or 10 mg/kg, i.p.) oromeprazole (40 mg/kg, p.o. or i.p., used as positive control),60 min (p.o.) or 30 min (i.p.) before the oral administration ofethanol (0.5 ml/200 g)(Baggio et al., 2007). One hour later, theanimals were sacrificed, the stomachs were removed, and thelesioned gastric area (mm2) was measured using the programImage Tool 3.0s, as previously described (Potrich et al., 2010).Gastric lesion extension was measured as the total injured area(length in mm�width in mm), as previously described (Baggioet al., 2007). Finally, the gastric tissues were weighed and used todetermine the mucus and glutathione (GSH) content.

2.9. Determination of gastric mucus

The gastric tissues were immediately transferred to 0.1% alcianblue solution prepared in 0.16 mM sucrose and 50 mM sodiumacetate (pH 5.0) and stained for 2 h at room temperature. Next,the gastric mucosa was rinsed twice with 250 mM sucrosesolution for 15 and 45 min, and the dye complexed with thegastric mucus was extracted with 500 mM magnesium chloridesolution, which was intermittently shaken for 1 min every 30 minfor 2 h. The extract was then mixed with an equal volume ofdiethyl ether and centrifuged at 1450g for 10 min. Absorbancewas read at 598 nm. The amount of mucus was calculated usingstandard curves of alcian blue (6.25–100.0 mg), and the resultswere expressed in mg of Alcian Blue/g tissue (Corne et al., 1974).

2.10. Determination of glutathione (GSH) content

Ulcerated stomach tissue samples were homogenized in200 mM potassium phosphate buffer (pH 6.5, 4 1C) to determinethe GSH levels, according to the method of Sedlak and Lindsay(1968). Aliquots of the tissue homogenate were mixed with 12.5%trichloroacetic acid, vortexed for 10 min and centrifuged for15 min at 9000g. The supernatant was reserved, and Tris buffer(0.4 M, pH 8.9) and 5,50-dithiobis-2-nitrobenzoic acid (DTNB,0.01 M) were added to it. The absorbance of the supernatantwas measured by spectrophotometry at 415 nm. The individualvalues were compared to a standard curve of GSH, and the resultswere expressed as mg of GSH/g of tissue.

2.11. Drugs

The following substances were used: formalin, morphinehydrochloride and Tween 80 (Merck, Darmstadt, Germany);alcian blue, dexamethasone, indomethacin, acetylsalicylic acid,omeprazole and naloxone hydrochloride (Sigma Chemical Co., St.Louis, USA). All the drugs were dissolved in saline solution (0.9%NaCl) with the exception of essential oil and acetylsalicylic acid,which were dissolved in saline plus Tween 80, and indomethacin,which was dissolved in saline with 5% DMSO. The final concen-tration of Tween 80 did not exceed 5% and did not cause anyeffect per se. All the control animals received the vehicle used todissolve the essential oil.

D.K.S. Lima et al. / Journal of Ethnopharmacology 142 (2012) 274–282276


2.12. Statistical analysis

The results are presented as the mean7the standard error ofmean (S.E.M.), except for the ID50 values (i.e., the dose of EOPanecessary to reduce the nociceptive response by 50% relative tothe control value), which are reported as geometric meansaccompanied by their respective 95% confidence limits. TheID50 values were determined by nonlinear regression fromindividual experiments using linear regression with GraphPadsoftware (GraphPad software, San Diego, CA, USA). Statisticalsignificance was determined with an analysis of variance(ANOVA) followed by Newman–Keuls tests. P-values less than0.05 (Po0.05) were considered significant.

3. Results

3.1. Essential oil extraction and analysis

The chemical analysis of the EOPa sample used in the presentinvestigations identified 35 compounds, representing 81.7% of thetotal oil content (Table 1). Caryophyllene oxide (11.5%), b-pinene(9%), spathulenol (6.7%), camphene (5.2%), b-elemene (4.7%),myrtenal (4.2%), verbenone (3.3%) and pinocarvone (3.1%) werefound to be major constituents (Table 1).

3.2. Formalin-induced nociception

The results depicted in Fig. 1A and B show that EOPa (30–1000 mg/kg, p.o.) significantly inhibited both the neurogenic (0–5 min) and inflammatory (15–30 min) phases of formalin-inducedlicking. However, its antinociceptive effects were significantlymore pronounced against the second phase of this pain model.The calculated mean ID50 value (and its respective 95% con-fidence limits) for these effects were: 281.2 (196.3–402.7) and70.5 (53.9–92.0) mg/kg, and the inhibitions observed were7573% and 9971% at a dose of 1000 mg/kg, for the first andsecond phases, respectively. In contrast, the NSAID acetylsalicylicacid (400 mg/kg, p.o.), given 60 min before the assay, onlysignificantly reduced the inflammatory (6677%) phase of for-malin-induced pain (Fig. 1A and B).

3.3. Opioid system involvement

Pre-treatment with the non-selective opioid receptor antago-nist naloxone (1 or 5 mg/kg, i.p.) 20 min beforehand, did notreverse the antinociception caused by EOPa (100 mg/kg, p.o.), butit completely reversed the antinociception caused by morphine(2.5 mg/kg, s.c.) during formalin-induced pain (Fig. 2A and B).

3.4. Locomotor activity evaluation

EOPa treatment (100 or 300 mg/kg, p.o.) did not alter mouseambulation in the open-field test. The crossing numbers afteradministration were 80.173.0, 89.0710.7 and 52.0715.4 forthe control group and the groups receiving 100 or 300 mg/kg ofEOPa, respectively.

3.5. Carrageenan-induced pleural acute inflammation

The intrapleural injection of carrageenan produced acute inflam-mation characterized by plasma leakage and considerable leukocytemigration, represented by neutrophils and mononuclear cells(Fig. 3A–D). Treatment with EOPa (1–100 mg/kg, p.o.) given 1 hprior to carrageenan significantly decreased the total cell count andthe number of neutrophils and mononuclear cells with mean ID50

values of 53.6 (19.9–143.9), 21.7 (10.2–46.0) and 43.5 (16.0–118.0) mg/kg and inhibitions of 54713%, 66710% and 6078%,respectively (Fig. 3A, C, and D). The same doses of EOPa also reducedexudation (4579% at dose of 3 mg/kg, Fig. 3B). Pre-treatment withdexamethasone (0.5 mg/kg, s.c.) and indomethacin (5 mg/kg, i.p.)30 min before the carrageenan injection reduced the total leukocytecount by 9575% and 9272%, the differential neutrophil count by9773% and 9872%, and pleural exudation by 8973% and 9773%,respectively (Fig. 3A–D).

3.6. Effects on gastric injury

EOPa treatment (1–30 mg/kg, p.o.) caused a dose-dependentreduction in ethanol-induced gastric lesions, decreasing the ulcerarea mainly at doses of 10 and 30 mg/kg (Fig. 4A), with a mean

Table 1Chemical composicion of the Piper aleyreanum essential oil.

Peak Compound a% bRt cRIcalc dRIlit Identification

(eEI-MS/fNIST, RI)

1 Tyranton 2.0 3.286 901.0 – EI-MS, NIST

2 Allylcyclohexane 0.3 4.243 926.4 – EI-MS, NIST

3 Camphene 5.2 5.309 954.7 950 EI-MS, NIST, RI

4 n.i. 0.6 6.208 978.5 978 EI-MS, NIST, RI

5 b-pinene 9.0 6.331 981.8 978 EI-MS, NIST, RI

6 o-cymene 1.6 7.55 1014.7 1013 EI-MS, NIST, RI

7 Limonene 0.8 7.822 1022.3 1025 EI-MS, NIST, RI

8 Linalool 0.8 10.102 1085.3 1086 EI-MS, NIST, RI

9 n.i. 1.4 10.324 1091.4 – –

10 a-campholenal 0.8 10.779 1104.0 1105 EI-MS, NIST, RI

11 Norinone 0.7 10.96 1109.0 – EI-MS, NIST

12 (E)-Sabinol 0.4 11.534 1124.9 1120 EI-MS, NIST, RI

13 (Z)-Verbenol 1.0 11.793 1132.0 1132 EI-MS, NIST, RI

14 Pinocarvone 3.1 12.088 1140.2 1137 EI-MS, NIST, RI

15 Myrtenal 4.2 13.303 1173.8 1172 EI-MS, NIST, RI

16 Verbenone 3.3 13.808 1187.8 1183 EI-MS, NIST, RI

17 Linalyl acetate 2.1 16.147 1246.9 1239 EI-MS, NIST, RI

18 d-elemene 0.3 19.719 1335.4 1340 EI-MS, NIST, RI

19 a-cubebene 1.8 20.255 1348.7 1355 EI-MS, NIST, RI

20 n.i. 0.5 20.993 1366.9 – –

21 a-copaene 1.6 21.295 1374.4 1379 EI-MS, NIST, RI

22 b-bourbonene 1.2 21.578 1381.4 1386 EI-MS, NIST, RI

23 b-cubebene 1.1 21.778 1386.4 1390 EI-MS, NIST, RI

24 b-elemene 4.7 21.83 1387.6 1389 EI-MS, NIST, RI

25 (E)-b-Caryophyllene 0.9 22.898 1415.0 1421 EI-MS, NIST, RI

26 n.i. 0.8 23.289 1425.3 – –

27 n.i. 0.5 23.508 1431.1 – –

28 g-muurolene 1.4 25.077 1472.4 1474 EI-MS, NIST, RI

29 b-selinene 0.5 25.418 1481.4 1486 EI-MS, NIST, RI

30 d-selinene 1.4 25.779 1490.9 1496 EI-MS, NIST, RI

31 a-muurolene 0.8 26.021 1497.3 1496 EI-MS, NIST, RI

32 g-cadinene 2.4 26.471 1509.2 1507 EI-MS, NIST, RI

33 Elemol 1.5 27.676 1540.9 1541 EI-MS, NIST, RI

34 (E)-nerolidol 1.2 28.23 1555.5 1553 EI-MS, NIST, RI

35 Spathulenol 6.7 28.694 1567.8 1572 EI-MS, NIST, RI

36 Caryophyllene oxide 11.5 28.801 1570.6 1578 EI-MS, NIST, RI

37 Viridiflorol 2.6 29.285 1583.3 1592 EI-MS, NIST, RI

38 n.i. 2.6 29.719 1594.8 – –

39 6-epi-cubenol 1.1 30.11 1605.6 1602 EI-MS, NIST, RI

40 Isospathulenol 1.2 30.563 1618.8 1625 EI-MS, NIST, RI

41 a-cadinol 2.5 31.438 1644.2 1643 EI-MS, NIST, RI

42 n.i. 1.5 32.716 1681.4 – –

43 n.i. 1.7 34.936 1745.9 – –

44 n.i. 4.1 35.147 1752.0 – –

45 n.i. 4.6 37.068 1808.7 – –

n.i.—not identified.a %—relative abundances from the peak area integration.b Rt—retention time (min) from a linear temperature program.c RIcalc—retention index calculated for each compound.d RIlit—retention index obtained from literature (http://massfinder.com/wiki/

Terpenoids_Library_List).e EI-MS—electron ionization mass spectrometry.f NIST—data bank from National Institute of Standards and Technology.



ID50 value of 1.7 (0.9–3.1) mg/kg (injured control groupvalue¼26.472.9 mm2) and an inhibition of 8774% at 10 mg/kg. The positive control omeprazole (40 mg/kg, p.o.) reducedethanol-induced gastric lesions by 9272%.

Ethanol administration decreased gastric mucus and GSH levelsby 64.973.9% and 35.573.0%, respectively, when compared to thenon-lesioned group (N: 61.574.1 mg alcian blue/g of tissueand 267.7710.9 mg of GSH/g of tissue). EOPa administration

Fig. 1. Effects of Piper aleyreanum essential oil (30–1000 mg/kg, p.o.) or acetylsalicylic acid (ASA, 400 mg/kg, p.o.) on the first (panel A) and second phase (panel B) of

formalin-induced licking in mice. Each column represents the mean7S.E.M. (n¼6–10). Control values (C) indicate vehicle (saline and Tween 80, 10 ml/kg) administration,

and the asterisks denote significance levels when compared with the control group; *Po0.05, ***Po0.001.

Fig. 2. Effect of pre-treatment of animals with naloxone (1 or 5 mg/kg, i.p.) on the antinociceptive profiles of Piper aleyreanum essential oil (100 mg/kg, p.o.) or morphine

(2.5 mg/kg, s.c.) on the first (panel A) and second phase (panel B) of formalin-induced licking in mice. Each column represents the mean7S.E.M. (n¼6–10). Asterisks

denote significance levels compared with control groups (vehicle groups), ##Po0.01. *Po0.05 and **Po0.01 compared with agonists (Piper aleyreanum or morphine

plus vehicle).

Fig. 3. Effects of Piper aleyreanum essential oil (1–100 mg/kg, p.o.), dexamethasone (DEX, 0.5 mg/kg, s.c.) or indomethacin (IND, 5 mg/kg, i.p.) on total leukocyte count

(panel A), pleural leakage (panel B), neutrophils (panel C) and mononuclear cell count (panel D) in carrageenan-induced pleurisy in mice. Each column represents the

mean7S.E.M. (n¼4–8). N represents the group injected with 0.9% saline; (C) indicates the group treated with carrageenan and the vehicle used to dilute the essential oil.

The difference between the groups was determined by an ANOVA followed by Newman–Keuls multiple comparison test. *Po0.05, **Po0.01, ***Po0.001 when compared

with carrageenan-treated group; #Po0.05, ##Po0.001 when compared to saline group (S).



(1–30 mg/kg, p.o.) increased the amount of mucus from 46.274.2to 50.375.6 mg alcian blue/g of tissue and GSH from 255.8714.6to 297.7724.4 mg of GSH/g of tissue when compared to the injuredcontrol (C: 21.472.4 mg alcian blue/g of tissue and 172.778.0 mgof GSH/g of tissue) (Fig. 4B and C). Furthermore, omeprazole(40 mg/kg, p.o.) significantly increased the gastric mucus andGSH levels relative to the injured control (Fig. 4B and C).

Similarly, the i.p. administration of EOPa (10 mg/kg) andomeprazole (40 mg/kg, i.p.) reduced (by 44.8712.8% and78.074.2%, respectively) ethanol-induced gastric lesions andincreased the gastric wall mucus to 34.271.5 and 30.472.7 mgalcian blue/g of tissue, respectively, when compared to lesionedgroup (C: 18.470.34 mg alcian blue/g of tissue) (Fig. 4a and c).Moreover, treatment with EOPa (10 mg/kg, i.p.) and omeprazole(40 mg/kg, i.p.) also increased the GSH levels to 215.3717.8 and213.8720.3 mg of GSH/g of tissue, respectively, compared to theinjured control (138.5715.5 mg of GSH/g of tissue) (Fig. 4b).

4. Discussion

Previous chemical reports of the essential oil obtained fromPiper aleyreanum leaves collected from the southern Amazonforest in Porto Velho (Rondonia State), Brazil, revealed that themajor components were sesquiterpenes b-caryophyllene (18.6%)and isocaryophyllene (17.5%) and monoterpene b-pinene (14.4%)(Facundo et al., 2007). Here, we used GC/MS to identify 35compounds in EOPa, which accounts for 81.7% of the total oilcontent. The main components were sesquiterpenes caryophyl-lene oxide (11.5%), spathulenol (6.7%), b-elemene (4.7%), mono-terpenes b-pinene (9%), camphene (5.2%), myrtenal (4.2%),verbenone (3.3%) and pinocarvone (3.1%). Interestingly, the diver-gent data regarding the previous component characterization andour results might be related to the age of the plant; the partcollected and environmental factors, resulting in a differentchemical composition of EOPa (Maciel et al., 2000). However,further studies are needed to investigate this hypothesis.

As mentioned earlier, the plants of the genus Piper have agreat diversity of secondary metabolites and are known for theirimmunomodulatory, anti-inflammatory, antinociceptive, anti-pyretic, anti-platelet, antifungic, cytotoxic, antitumor, gastropro-tective, anxiolytic and antidepressive properties (Majdalawiehand Carr, 2010; Sunila and Kuttan, 2004; Ganguly et al., 2007;Chiou et al., 2003; Rodrigues Silva et al., 2008; Xie et al.,2011; Yao et al., 2009; Rodrigues et al., 2009; Sireeratawong et al.,2010; Koroishi et al., 2008; Marques et al., 2007; Quılez et al., 2010;Morikawa et al., 2004; Zakaria et al., 2010; Cıcero et al., 2007;Navickiene et al., 2000; Bezerra et al., 2006; for review see Sarriset al., 2011). In addition, the literature suggests that the essential oilobtained from some species of Piper also have various biologicalactivities, such as anti-parasitic, insecticidal, larvicidal, antimicro-bial, antifungal and antioxidant properties (Marques et al., 2010;Misni et al., 2011; Salleh et al., 2011; da Silva et al., 2010, 2011;Monzote et al., 2010; Magalh~aes et al., 2012).

Furthermore, no reports were found in the literature regardingthe biological actions of Piper aleyreanum, and although somepharmacological actions of the essential oil of Piper species havebeen reported, no specific antinociceptive, anti-inflammatory orgastroprotective actions of EOPa have been reported in theliterature. The present results show for the first time that oralEOPa exerts potent antinociceptive and anti-inflammatory effectsagainst nociception, vascular leakage and leukocyte migration intwo different pain and inflammation models. Interestingly, bothadministration routes (oral and intraperitoneal) of EOPa increasedthe protective factors, such as mucus and GSH, and protected thegastric mucosa against ethanol-induced lesions.

In the present study, we demonstrated that EOPa has a signifi-cant antinociceptive effect on formalin-induced pain in mice, aclassical chemical model of nociception. The results reported hereindicate that the oral administration of essential oil producedmarked and dose-related antinociception against both neurogenic(first phase) and inflammatory (late phase) pain responses causedby formalin injection in mice. Also, the antinociception caused byEOPa was unlikely to be secondary to its non-specific musclerelaxant, specific and/or non-specific depressant central effects as

Fig. 4. Effects of Piper aleyreanum essential oil (1–30 mg/kg, p.o., panel A, B, and C or 10 mg/kg, i.p., panels a–c) on ethanol-induced gastric lesions (panel A and a) and

glutathione (panel B and b) and gastric mucus (panel C and c) levels in rats. Each column represents the mean7S.E.M. (n¼6–10). Control values (C) indicate the

administration of vehicle (saline and Tween 80, 10 ml/kg) plus ethanol. N represents the group injected with vehicle; O indicates the group treated with omeprazole

(40 mg/kg). Differences between the groups were determined by an ANOVA followed by Newman–Keuls multiple comparison test. *Po0.05, **Po0.01, ***Po0.001 when

compared with the ethanol-treated group; #Po0.05, ##Po0.001 when compared to the vehicle group (N).



revealed by the lack of important motor dysfunction or detectableside effects in the open-field test.

The formalin test is a satisfactory and comprehensive modelfor evaluating the antinociceptive activity of drugs. The intra-plantar injection of formalin activates nociceptive nerve terminalsand produces neurogenic pain, whereas inflammatory pain ismediated by a combination of peripheral input and spinal cordsensitization (Hunskaar and Hole, 1987; Tjolsen et al., 1992).It has been demonstrated that the intraplantar injection of formalinin rodents increases spinal levels of excitatory amino acids, PGE2,nitric oxide, tachykinin, kinins, among other peptides (Tjolsenet al., 1992; Malmberg and Yaksh, 1992; Santos and Calixto, 1997;Santos et al., 1998). Experimental data indicate that formalinpredominantly evokes activity in C-fibers (Tjolsen et al., 1992),although Ad-fibers are thought to be responsible for fast nocicep-tive transmission in the first phase of the pain response (Juliusand Basbaum, 2001).

It is notable that the nociception produced by formalin (firstphase) is quite resistant to the majority of NSAIDs, such asacetylsalicylic acid (results presented here), indomethacin, para-cetamol, and diclofenac. However, these drugs can dose-depen-dently attenuate the second phase of formalin-induced licking(Hunskaar and Hole, 1987; Malmberg and Yaksh, 1992; Santoset al., 1998). Moreover, it has also been reported that morphine,some tachykinin receptor antagonists, non-selective excitatoryamino acid antagonists and both B1 and B2 bradykinin receptorantagonists are able to inhibit both phases of the formalin test (DeCampos et al., 1996; Santos and Calixto, 1997). The present studyshows that the opioid system is unlikely to be involved in theantinociceptive action of EOPa. This is inferred by the fact that thepre-treatment of animals with naloxone, a nonselective opioidreceptor antagonist, completely inhibited the antinociceptiveeffect of morphine but not the action of EOPa in the formalinmodel. Moreover, our data showed that EOPa inhibited bothphases of formalin test but was more effective against theinflammatory (second phase) pain; this data led us to investigateits anti-inflammatory effect.

Treatment with EOPa resulted in a pronounced anti-inflammatoryeffect against acute carrageenan-induced pleurisy. It has been shownthat the carrageenan-induced mouse pleural inflammatory responseelicits the release of chemical mediators such as histamine, bradyki-nin, substance P, and prostaglandins, which is followed by exudationand leukocyte infiltration into the inflammatory site; this peaks at 4 hafter pleurisy induction (Menegazzi et al., 2008; Tomlinson et al.,1994; Saleh et al., 1996, 1997). This acute inflammatory response isusually inhibited by NSAIDs, such as indomethacin or corticoids suchas dexamethasone, and these effects have been attributed to theinhibition of mediator release and the tissue expression of induciblecyclooxygenase (COX-2) (Nantel et al., 1999).

Here, we confirm these observations and demonstrate thatEOPa treatment reduced the main features of acute inflammation,including exudation and leukocyte number, characterized mainlyby a reduction in neutrophil differential cell count in the pleuralcavity. These findings suggest that EOPa has a critical role incontrolling acute inflammatory events. Singh et al. (2008)reported that the chloroform extract of Piper longum inhibitedneutrophil adherence to the endothelial monolayer by inhibitingthe TNF-a-induced expression of intercellular adhesion molecule-1 (ICAM-1), vascular cell adhesion molecule-1 (VCAM-1) andE-selectin, and this effect is probably mediated by NFkB inhibitionin endothelial cells. Recently, Amran et al. (2011) demonstratedthat the aqueous extract of Piper sarmentosum significantlyreduced the levels of VCAM-1, ICAM-1 and C-reactive protein(CRP) in experimental rabbits fed a 1% cholesterol diet, suggestingthat this plant may have a beneficial effect in preventing athero-sclerosis. Future studies are needed to investigate the possible

effect of EOPa on these inflammatory markers that enhance bothacute and chronic inflammation.

NSAIDs (i.e., indomethacin or acetylsalicylic acid) are knownto induce gastric damage due to nonspecific inhibition ofcyclooxygenase-1 (COX-1) and COX-2, and this dual inhibitionmay lead to gastrointestinal ulceration and bleeding (Wallaceet al., 2000; Wallace, 2008). We evaluated whether EOPa treat-ment could protect the gastric mucosa. Notably, EOPa exertsgastroprotective activity and prevents the formation of acutehemorrhagic erosion caused by oral ethanol administration.Ethanol is a well-known necrotizing agent that destroys themucus barrier, increases vascular permeability, and decreasesthe number of non-protein sulfhydryl groups (NP-SH) of thegastric mucosa (Repetto and Llesuy, 2002; Siegmund, 2003),which leads to hemorrhagic gastric erosion. Gastric mucus isone of the main defensive elements against aggressive agents. It iscontinuously secreted by epithelial cells and serves as a physicalbarrier over the mucosa (Bi and Kaunitz, 2003). Our results clearlyshow that compared to the NSAIDs indomethacin and acetylsa-licylic acid, which are broadly used in the clinical management ofinflammatory diseases, EOPa does not have adverse gastrointest-inal effects. EOPa treatment exerts an anti-ulcer effect on ethanollesions. The cytoprotective mechanism appears to be related toincreased levels of mucus and anti-oxidant factor such as GSH.

In this study, the extract was given by two different routes toevaluate whether the observed effect was due to an adherentproperty of the extract on the gastric mucosa, resulting in theformation of a protective barrier against the necrotizing effects ofethanol. EOPa given by the intraperitoneal route showed the sameeffects on GSH levels and mucus production, suggesting that theobserved results were not due to EOPa’s adherence to thegastric wall.

EOPa is chemically composed of terpenes, such as sesquiter-penes and monoterpenes, and their biological activity could beattributed to the high concentrations of caryophyllene oxide(11.5%) and b-pinene (9%). However, we cannot confirm thatthe antinociceptive, anti-inflammatory and gastric antiulcer activ-ities of EOPa are attributable to the single or synergistic action ofthese main components or even other minor constituents presentin the oil.

In conclusion, the present study demonstrates that EOPaexerts dose-dependent antinociceptive action against formalin-induced nociception, without affecting locomotor activity. Inaddition, the antinociceptive action of EOPa is not dependent onthe opioid system. EOPa treatment exerted an important anti-inflammatory action and interesting gastroprotective effectsrelated to the maintenance of protective factors, such as mucusproduction and GSH. This study provides a pharmacological basisfor EOPa use in folk medicine and shows that this plant haspotential for the development of safe phytomedicines with anti-nociceptive and anti-inflammatory effects and gastroprotectiveproperties.

Acknowledgements

This work was supported by grants from Conselho Nacional deDesenvolvimento Cientıfico e Tecnologico (CNPq), Fundac- ~ao deApoio a Pesquisa Cientıfica e Tecnologica do Estado de SantaCatarina (FAPESC), and Coordenac- ~ao de Aperfeic-oamento dePessoal de Nıvel Superior (CAPES), Brazil.

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Evaluation of the antinociceptive, anti-inflammatory and gastric antiulcer activities of the essential oil from Piper aleyreanum C.DC in rodents

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