Acessibilidade / Reportar erro

Anti-inflammatory, antinociceptive, and antipyretic effects of methanol extract of Cariniana rubra stem bark in animal models

Abstracts

Cariniana rubra Miers (Lecythidaceae), popularly known as "jequitibá-vermelho'', is a large Brazilian tree whose bark is used in infusion and decoction for the treatment of inflammatory conditions. This study aims to assess the anti-inflammatory, antinociceptive, and antipyretic effects of Cariniana rubra methanolic stem bark extract (EM Cr) using experimental animals. Anti-inflammatory activity of EM Cr was tested on carrageenan and dextran-induced rat paw edema, carrageenan-induced pleurisy in rats and acetic acid-increase vascular permeability in mice. Antinociceptive and antipyretic activities were evaluated using acetic acid-induced writhing, formalin and hot-plate tests in mice, as well as brewer's yeast-induced pyrexia in rats. The extract inhibitied carrageenan and dextran-induced edema, reduced exudate volume and leukocyte migration on the carrageenan-induced pleurisy and on the vascular permeability increase induced by acetic acid. The EM Cr inhibited nociception on the acetic acid-induced writhing and in the second phase of formalin test, and decreased rectal temperature. It was, however, inactive against thermal nociception.Phytochemical analysis with EM Cr showed the occurrence of saponins, triterpenes, sterols and phenolic compounds. Phytosterols (β-sitosterol, stigmasterol), pentacyclic triterpenes (α- and β-amyrin as a mixture), arjunolic acid, a phytosterol glycoside (sitosterol 3-O-β-D-glucopyranoside), and triterpenoid saponins (28-β-glucopyranosyl-23-O-acetyl arjunolic acid; 3-O-β-glucopyranosyl arjunolic acid and 28-O-[α-L-Rhamnopyranosyl-(1→2)-β-glucopyranosyl]-23- O-acetyl arjunolic acid) were the main identified compounds. It can be presumed that EM Cr caused their effects by inhibiting the liberation and/or action of different inflammatory mediators. These findings support the traditional use of Cariniana rubra preparations to treat inflammation.

anti-inflammatory; antinociceptive; antipyretic; Cariniana rubra; Lecythidaceae; triterpenoidal saponins


Cariniana rubra Miers (Lecythidaceae), popularmente conhecido como "jequitibá-vermelho'', é uma árvore brasileira de grande porte, cuja casca é utilizada nas formas de infusão e decocção para o tratamento de condições inflamatórias. Os efeitos antiinflamatório, antinociceptivo e antipirético do extrato metanólico da casca do caule de Cariniana rubra (EM Cr) foram avaliados em animais experimentais. A atividade antiinflamatória do EM Cr foi testada nos modelos de edema depata induzido por carragenina e dextrana em ratos, pleurisia induzida por carragenina em ratos e permeabilidade vascular aumentada por ácido acético em ratos. As atividades antinociceptiva e antipirética foram avaliadas utilizando os modelos de nocicepções induzidos por ácido acético e formalina, placa quente em camundongos e de pirexia, pela injeção de levedura de cerveja em ratos. O extrato inibiu o edema induzido porcarragenina e dextrana, reduziu o volume de exsudato e a migração de leucócitos na pleurisia induzida por carragenina eo aumento da permeabilidade vascular induzida por ácidoacético. O EM Cr inibiu a nocicepção nas contorções induzidas por ácido acético e na segunda fase do teste de formalina e diminuiu a temperatura retal. No entanto, foi inefetivo no teste da placa quente. A análise química por via úmida deu resultados positivos para saponinas, triterpenos, esteroides e compostos fenólicos. Fitosteróis e triterpenóides pentacíclicos (β-sitosterol, estigmasterol, α and β-amirinas em mistura e ácido arjunólico) e as saponinas triterpenoidais: 3-O-β-D-glucopiranosideo de sitosterol; ácido arjunólico 28-β-glucopiranosila-23-O-acetila; ácido arjunólico 3-O-β-glucopiranosila e ácido arjunólico 28-O-[α-L-rhamnopiranosil-(1→2)-β-D-glucopiranosila]-23-O-acetila. Pode-se presumir que os efeitos do EM Cr foram causados pela inibição da liberação e/ou ação de diversos mediadores inflamatórios. Estes resultados validam o uso tradicional das preparações caseiras de Cariniana rubra para tratar a inflamação.

antiinflamatório; antinociceptivo; antipirético; Cariniana rubra; Lecythidaceae; saponinas triterpênicas


Anti-inflammatory, antinociceptive, and antipyretic effects of methanol extract of Cariniana rubra stem bark in animal models

Edson N. SantosI; Joaquim C.S. LimaI; Vânia F. NoldinII; Valdir Cechinel-FilhoII; Vietla S. N. RaoIII; Evangelista F. LimaIV; Guillermo Schmeda-HirschmannV; Paulo T. Sousa Jr.IV; Domingos T.O. MartinsI

IDepartamento de Ciências Básicas em Saúde, Faculdade de Medicina, Universidade Federal de Mato Grosso, Av. Fernando Correa da Costa, 2367, Boa Esperança, Campus Universitário, 78060-900 Cuiabá, MT, Brasil

IICentro de Pesquisa Químico-Farmacêutica/NIQFAR, Universidade do Vale do Itajaí, Rua Uruguai, 458, 88302-202 Itajaí, SC, Brasil

IIIDepartamento de Fisiologia e Farmacologia, Faculdade de Medicina, Universidade Federal de Ceará, Rua Cel Nunes de Melo 1127, Porangabussu, 60430-270 Fortaleza, CE, Brasil

IVLaboratório de Pesquisa em Química de Produtos Naturais/ICET, Universidade Federal de Mato Grosso, Av. Fernando Correa da Costa, 2367, Boa Esperança, Campus Universitário, 78060-900 Cuiabá, MT, Brasil

VInstituto de Química de Recursos Naturales (IQRN), Universidad de Talca, Casilla: 747, 2 Norte 685, Talca, Chile

Correspondence to Correspondence to: Domingos Tabajara de Oliveira Martins E-mail: taba@terra.com.br

ABSTRACT

Cariniana rubra Miers (Lecythidaceae), popularly known as "jequitibá-vermelho'', is a large Brazilian tree whose bark is used in infusion and decoction for the treatment of inflammatory conditions. This study aims to assess the anti-inflammatory, antinociceptive, and antipyretic effects of Cariniana rubra methanolic stem bark extract (EM Cr) using experimental animals. Anti-inflammatory activity of EM Cr was tested on carrageenan and dextran-induced rat paw edema, carrageenan-induced pleurisy in rats and acetic acid-increase vascular permeability in mice. Antinociceptive and antipyretic activities were evaluated using acetic acid-induced writhing, formalin and hot-plate tests in mice, as well as brewer's yeast-induced pyrexia in rats. The extract inhibitied carrageenan and dextran-induced edema, reduced exudate volume and leukocyte migration on the carrageenan-induced pleurisy and on the vascular permeability increase induced by acetic acid. The EM Cr inhibited nociception on the acetic acid-induced writhing and in the second phase of formalin test, and decreased rectal temperature. It was, however, inactive against thermal nociception.Phytochemical analysis with EM Cr showed the occurrence of saponins, triterpenes, sterols and phenolic compounds. Phytosterols (β-sitosterol, stigmasterol), pentacyclic triterpenes (α- and β-amyrin as a mixture), arjunolic acid, a phytosterol glycoside (sitosterol 3-O-β-D-glucopyranoside), and triterpenoid saponins (28-β-glucopyranosyl-23-O-acetyl arjunolic acid; 3-O-β-glucopyranosyl arjunolic acid and 28-O-[α-L-Rhamnopyranosyl-(1→2)-β-glucopyranosyl]-23- O-acetyl arjunolic acid) were the main identified compounds. It can be presumed that EM Cr caused their effects by inhibiting the liberation and/or action of different inflammatory mediators. These findings support the traditional use of Cariniana rubra preparations to treat inflammation.

Key words: anti-inflammatory, antinociceptive, antipyretic, Cariniana rubra, Lecythidaceae, triterpenoidal saponins.

RESUMO

Cariniana rubra Miers (Lecythidaceae), popularmente conhecido como "jequitibá-vermelho'', é uma árvore brasileira de grande porte, cuja casca é utilizada nas formas de infusão e decocção para o tratamento de condições inflamatórias. Os efeitos antiinflamatório, antinociceptivo e antipirético do extrato metanólico da casca do caule de Cariniana rubra (EM Cr) foram avaliados em animais experimentais. A atividade antiinflamatória do EM Cr foi testada nos modelos de edema depata induzido por carragenina e dextrana em ratos, pleurisia induzida por carragenina em ratos e permeabilidade vascular aumentada por ácido acético em ratos. As atividades antinociceptiva e antipirética foram avaliadas utilizando os modelos de nocicepções induzidos por ácido acético e formalina, placa quente em camundongos e de pirexia, pela injeção de levedura de cerveja em ratos. O extrato inibiu o edema induzido porcarragenina e dextrana, reduziu o volume de exsudato e a migração de leucócitos na pleurisia induzida por carragenina eo aumento da permeabilidade vascular induzida por ácidoacético. O EM Cr inibiu a nocicepção nas contorções induzidas por ácido acético e na segunda fase do teste de formalina e diminuiu a temperatura retal. No entanto, foi inefetivo no teste da placa quente. A análise química por via úmida deu resultados positivos para saponinas, triterpenos, esteroides e compostos fenólicos. Fitosteróis e triterpenóides pentacíclicos (β-sitosterol, estigmasterol, α and β-amirinas em mistura e ácido arjunólico) e as saponinas triterpenoidais: 3-O-β-D-glucopiranosideo de sitosterol; ácido arjunólico 28-β-glucopiranosila-23-O-acetila; ácido arjunólico 3-O-β-glucopiranosila e ácido arjunólico 28-O-[α-L-rhamnopiranosil-(1→2)-β-D-glucopiranosila]-23-O-acetila. Pode-se presumir que os efeitos do EM Cr foram causados pela inibição da liberação e/ou ação de diversos mediadores inflamatórios. Estes resultados validam o uso tradicional das preparações caseiras de Cariniana rubra para tratar a inflamação.

Palavras-chave: antiinflamatório, antinociceptivo, antipirético, Cariniana rubra, Lecythidaceae, saponinas triterpênicas.

INTRODUCTION

Cariniana rubra Miers (Lecythidaceae), popularly known as "jequitibá-vermelho'', is a large tree thatgrows abundantly in the hills and forests of the Brazilian states of Mato Grosso, Goiás, Tocantins and Minas Gerais States of Brazil. Its stem bark is a reputed traditional remedy in the form of decoctions and infusions for the treatment of inflammatory conditions, especially sore throat, oophoritis and venereal diseases(De La Cruz 2008).

No scientific reports in support of the traditional use of this plant are available, although phytochemical studies indicated the presence of phenolic compoundsin C. rubra, such as β-sitosterol, stigmasterol, α- and β-amyrins, arjunolic acid, sitosterol 3-O-β-D-glucopyranoside, the triterpenoidal saponins 28-β-glucopyranosyl-23-O-acetyl arjunolic acid; 3-O-β-glucopyranosyl arjunolic acid and 28-O-[α-L-Rhamnopyranosyl-(1→2)-β-glucopyranosyl]-23-O-acetyl arjunolic acid (Lima et al. 2002).

The traditional knowledge on the medicinal use of plants should be assessed under laboratory conditions using appropriate biological assays to disclose if the traditional claims are evidence-supported. In this context, the ethnopharmacological approach provides specific targets for pharmacological and chemical investigation, looking for a possible development of newplant-derived drugs. With this objective, the present study aims to verify the possible anti-inflammatory, antinociceptive, and antipyretic effects of methanol extract from C. rubra stem bark in selected animal models. In addition, the plant extract was also subjetcted to phytochemical analysis.

MATERIALS AND METHODS

PLANT MATERIAL AND EXTRACTION

The stem bark of Cariniana rubra was collected in November 1997 by Liberio Amorim Neto, at Serra de São Vicente, Cuiabá Municipality, Mato Grosso State, Brazil. It was identified by Prof. Dr Germano Guarim Neto, Department of Botany and Ecology, Federal University of Mato Grosso, Brazil.

A voucher specimen (# 18337) was deposited in the Herbarium of the University. The collection was authorized by the Brazilian Institute of Environment and Renewable Natural Resources.

The finely powdered bark of C. rubra (1 kg) was packed into a Sohxlet extractor and extracted for 48 h, with absolute methanol. The solvent was removed under reduced pressure using a rotavapor to obtain 333 g (33%) of a dark brown solid. The dry extract (EM Cr) was suspended in distilled water for the pharmacological studies.

PHYTOCHEMICAL ANALYSIS

The air-dried plant material (150 g) was extracted with methanol: water (80:20 v/v, 500 mL) in a Soxhletapparatus to obtain the hydrophilic extract. The lipophilic extract was obtained as above, using diethyl ether (500 mL) as solvent. The chemical analysis was performed on the hydrophilic and the lipophilic extracts, according to the literature (Matos 1988).

Chromatographic fractionation was carried outusing silica gel chromatography and permeation on Sephadex LH-20, as well as by high speed countercurrent chromatography (HSCCC) and medium pressure chromatography. The structure determination of the main compounds was carried out using spectroscopic techniques as previously reported (Harbone 1998, Matos 1988, Lima et al. 2002, E.F. Lima, unpublished data).

ANIMALS

Male Wistar rats (170-250 g) and male Swiss mice(25-30 g) were used. Experimental groups consisted of 8 animals per group. They were housed at 22 ± 1°C under a 12 h light/12 h dark cycle, and had free access to standard pellet diet (Purina® chow) and tap water. The animals were deprived of food for 15-24 h before experimentation, but had free access to drinking water.

The Animal Care and Use Committee of the Universidade Federal do Mato Grosso approved the experimental protocols in accordance with the ethical guidelines for the investigation of experimental pain in conscious animals (Zimmermann 1983).

ANTI-INFLAMMATORY ASSAYS

Hind paw-edema induced by carrageenan and dextran

Paw edema was induced in the left hind paw of rats (180-200 g) by sub-plantar injection of 0.1 mL of 1% l-carrageenan or 1.5% of dextran (w/v) in 0.9% of saline. The paw volume was measured before (0) and 3 h after carrageenan injection or 2 h after dextran (Parratand West 1957) using a plethysmometer (Ugo Basile). The edema was expressed as an increase in paw volume. Different groups of animals were treated by gastric gavage with EM Cr (250, 750 and 2,000 mg/kg), indomethacin (5 mg/kg), cyproheptadine (5 mg/kg) orvehicle (10 mL/kg) in a volume of 10 mL/kg, 1 h before carrageenan or dextran injection (Winter et al. 1962).

Carrageenan-induced pleurisy in rats

Pleurisy was induced in rats (200-220 g) by intrapleural injection of 2% l-carrageenan suspension (0.1 mL/rat)in 0.9% of saline (Vinegar et al. 1978).The EM Cr(250, 750, 1,500, and 2,000 mg/kg), dexamethasone(0.5 mg/kg) or vehicle (10 mL/kg) were given by gastric gavage 1 h before the injection of irritant agent. Six hours after the injection of carrageenan, the rats were killed by excess of ether. The pleural cavity was opened and rinsed with 1 mL of saline solution containing 0.1% of EDTA. The exudate and washing solution were collected by aspiration and the total volume measured (mL). The mobilized leukocyte number in the exudate was quantified using improved Neubauer counting chambers.

Acetic acid-induced increase in vascular permeability

Groups of mice (25-30 g) were treated by gastric gavage with vehicle (10 mL/kg), EM Cr (50, 250, and 750 mg/kg) or dexamethasone (0.5 mg/kg) in a volume of 10 mL/kg. One hour after these treatments, each animal received an intravenous injection of 2% Evan'sblue solution (w/v) in 0.9% of saline. Ten minutes later, each mouse received intraperitoneally 0.4 mL of 0.5% acetic acid solution. Twenty minutes after acetic acid injection, the animals were killed, the peritoneal fluidwas collected and the concentration of Evan's bluewas measured by absorbance at 590 nm in a spectrophotometer.

The dye extravasation was quantified from a standard curve and expressed in mg (Whittle 1964).

ANTINOCICEPTIVE ASSAYS

Writhing test

Abdominal constriction induced by intraperitoneal injection of acid acetic was carried out according toa method described earlier (Koster et al. 1959). In thistest, groups of mice were treated with the vehicle,EM Cr (50, 250 and 750 mg/kg) or indomethacin (5 mg/ kg), 1 h before the administration of acetic acid (0.6%, 10 mL/kg, i.p.). The number of writhings was counted for each animal, starting 10 min after acetic acid injection over a period of 20 min.

Formalin test

In formalin test, groups of mice were treated by gastric gavage with vehicle or EM Cr (50, 250, and 750 mg/kg) and, one hour later, each mouse was given 25mL of 2.5% formalin (in 0.9% of saline, subplantar) into the left hind paw (Hunskaar and Hole 1987). The duration of paw licking (s) as an index of painful response was determined at 0-5 min (early phase, neurogenic) and 20-25 min (late phase, inflammatory) after formalin injection. Meperidine (25 mg/kg, s.c., 30 min before the test) pre-treated animals were included in the study asa positive control.

Hot-plate test

In thermal nociceptive test, the reaction time (timein seconds elapsed between placement and the animal starting to lick its hind paw or jumping as an index of painful response) in a hotplate maintained at 56 ± 1°C was measured before and after 15, 30, 60, 120, and 180 min of drug administration (Eddy and Leimbach1953). Mice with baseline latencies of more than 15 s were eliminated from the study. Animal groups were treated with the vehicle, EM Cr (50, 250, and 750 mg/ kg) or meperidine (25 mg/kg, s.c.) 60 or 30 min (in caseof meperidine) before the hot-plate test. The cut-offtime was set at 45 s.

ANTIPYRETIC ASSAY

Brewer's yeast induced pyrexia

Pyrexia was induced in rats (200-220 g) by subcutaneous injection of 10 mL/kg of 20% brewer's yeastsuspension (w/v) in 0.9% of saline (Al-Ghamdi 2001).Twenty-one hours later, the animals were treated bygastric gavage with vehicle, EM Cr (250, 750, and 2,000 mg/kg) or phenacetin (175 mg/kg). The rectal temperature of the rats was determined with a digital thermometer. It was inserted 3 cm into the rectum anus and the temperature measured before and 1, 2, and 3 hafter the treatments. In another group, the animals received a subcutaneous injection from 0.9% of normal saline (1 mL/100g), and 21 h later received the vehicleby gastric gavage. The rectal temperature was determined and included in the study as a negative control.

Statistical analysis

All parametric values are given as Mean ± S.E.M and were analyzed by One-way ANOVA followed by Student Newman-Keuls test for significance at p<0.05.

RESULTS

PHYTOCHEMICAL ANALYSIS

The chemical analysis of the methanolic stem bark extract of C. rubra (EM Cr) showed the presence of saponins, tannins, free steroids, flavonols and flavones. Several constituents were isolated and identified by spectroscopic means, including phytosterols and pentacyclic triterpenoids (β-sitosterol, stigmasterol, α and β-amyrins, arjunolic acid), phenolic compounds, thesteroid glycoside sitosterol 3-O-β-D-glucopyranoside, and triterpenoidal saponins (28-β-glucopyranosyl-23-O-acetyl arjunolic acid; 3-O-β-glucopyranosyl arjunolic acid and 28-O-[β-L-rhamnopyranosyl-(1→2)-β-glucopyranosyl]-23- O-acetyl arjunolic acid). The structures are in agreement with literature data and co-chromatography with authentic samples (Lima et al. 2002, E.F. Lima, unpublished data).

ANTI-INFLAMMATORY EVALUATION

EM Cr was evaluated in the model of rat paw edema induced by carrageenan or dextran. Table I shows that the EM Cr (750, and 2,000 mg/kg) and indomethacin (5 mg/kg) significantly reduced the carrageenan-induced rat hind paw edema by 29 (p<0.01), 38 (6p<0.001) and 55% (p<0.001), respectively, compared to control values. However, in the dextran-induced paw edema, EM Cr was active only at 2,000 mg/kg (60 - p<0.001). Under the same experimental conditions, cyproheptadine (5 mg/kg) reduced inflammation by 54% (p<0.001).

The results on the effect of plant extract on carrageenan pleurisy test are presented in Figure 1. In the present investigation, we compared the effect of the EM Cr at 250, 750, 1,500, and 2,000 mg/kg and dexamethasone on the cell migration. The EM Cr at 1,500 and 2,000 mg/kg markedly inhibited the carrageenan-induced pleuritic exudate by 46 (p<0.01) and 80% (p<0.001), and the leukocyte migration by 37 (p<0.01) and 80% (p<0.001), respectively. Dexamethasone, the reference drug used in the study, produced 83 (p<0.001) and 86% (p<0.001) inhibitions, respectively. The increased vascular permeability caused by intraperitoneal acetic acid was found to be significantly reduced in mice pretreated with 50, 250, and 750 mg/kg of the plant extract in a dose-dependent manner (Fig. 2).



ANTINOCICEPTIVE EVALUATION

Figure 3 shows that the mean number of writhes following intraperitoneal administration of 0.6% aceticacid in vehicle-treated control mice was 44 ± 1. Animal groups that received the plant extract at 250, and750 mg/kg or indomethacin (5 mg/kg) demonstrated significantly diminished number of writhes (12, 57 - P<0.01 and 70% - P<0.01, respectively).


In the formalin test, vehicle-treated animals showed the mean licking times (s) of 81 ± 4 in the first phase, and 173 ± 12 in the second phase (Fig. 4). Pretreatment with the EM Cr at 750 mg/kg did not show significant effect on the first phase response, but significantly diminished the second phase (inflammatorypain) response by 28% (p<0.01). However, meperidine (25 mg/kg, s.c), a known analgesic, produced marked inhibition at both phases (84 - p<0.001 and 99% - p<0.001, respectively).


The present study indicates that, unlike meperidine (25 mg/kg, s.c.), the EM Cr (50, 250, and 750 mg/kg) failed to show any significant analgesia in the hot-plate test (data not shown).

ANTIPYRETIC EVALUATION

The subcutaneous injection of brewer's yeast suspension 20% increased considerably the rectal temperature of the rats 21 h after administration (38.3 ± 0.13°C vs. 37.1 ± 0.16°C, p<0.001). The EM Cr treatment, with 750 and 2,000 mg/kg, significantly reduced the rectal temperature of the animals in the second and third hafter administration, reaching the peak of antipyretic effect with the highest dose in the second h (37.5 ± 0.08°C, p<0.001), in relation to control (38.5 ± 0.01°C). The phenacetin treatment (30 mg/kg) caused significant antipyretic effect at all time periods, reaching the peak in the second h (37.0 ± 0.09°C, p<0.001), in relation to control (38.3 ± 0.08°C) (Fig. 5).


DISCUSSION

Natural products of plant origin are used in folk medicine all over the word. They exhibit a wide range of pharmacological activities and may provide relief of symptoms often comparable to that obtained from allopathic medicines. The results reached in this study suggest that EM Cr possesses anti-inflammatory, antinociceptive and antipyretic properties and support the traditional use of this plant to treat inflammatory conditions. In rats and mice, oral administration of EM Cr reduced the carrageenan and dextran-induced edema. Ithas been referred that, after the carrageenan injection on the rat's paw, several mediators are released sequentially like histamine, serotonin and bradykinin in theinitial phase (0-1h), and an increase in the production of prostaglandins through the activation of cyclo oxygenase-2 and release of nitric oxide in the later phase(1-6 h) are reported (Silva et al. 2005). It is likely thatthe extract might have suppressed the edema formation by the inhibition of the inflammatory mediator substances. In addition, the extract effectively reduced the vascular permeability in intraperitoneal acetic acid test wherein many components of inflammatory cascade participate involving resident cells, macrophages and mast cells (Whittle 1964, Ribeiro et al. 2000). The increase in vascular permeability induced by acetic acid is known to correspond to the initial exudative inflammation and, conversely, its inhibition may contribute to the reduction of edema formation and to decrease the migration of neutrophils. Histamine and serotonin are the principal mediators involved in the dextran-induced paw edema, and their release is a result of mast cell degranulation (Lo et al. 1982). EM Cr effectively suppressed the dextran edema, and its effect at 750 mg/kg was similar to that of 0.5 mg/kg dexamethasone.

The injection of carrageenan into the pleural cavity of rodents provokes an acute inflammatory response characterized by the accumulation of fluids in the pleural cavity with a large number of polymorphonuclear leukocytes. This eventually leads to the increased levels of prostaglandin E2, reactive oxygen intermediates, lipid peroxidation, and cytokines such as TNF-α and IL-1β (Di Paola et al.2004). Cell migration occurs as a result of different processes including adhesion and cell mobility (Meade et al. 1986). The extract treated animals displayed less neutrophils in the pleural cavity fluidsthan in the controls, suggesting the inhibition of neutrophil influx.

Besides anti-inflammatory activity, the EM Cr demonstrated antinociceptive activity in the experimental models of chemical nociception induced by acetic acid and in the second phase of subplantar formalin. However, it was inactive in the first phase of formalin andin hot-plate tests, suggesting that the effect of EM Cr is related to the anti-inflammatory action.

The acetic acid-induced abdominal writhing testis a visceral pain model commonly used to assess thein flammatory pain for its high sensitivity, despite itslow specificity. Acetic acid unleashes the release of several mediators such as bradykinin, substance P and prostaglandins, as well as cytokines such as IL-1β, TNFα and IL-8 (Sekiya 1982). The antinociceptive activity demonstrated by EM Cr in this model implies that it is likely to inhibit these mediators and, thus, the activation of chemosensitive nociceptors that contribute to the development of inflammatory pain.

The formalin test is more specific and simulate clinical pain. Several reports suggest that formalin-induced licking response is mediated by the release of the excitatory amino acid glutamate at the first phase, and by sensory neuropeptides like substance P released from sensory neurons at the spinal cord at the second phase (Otuki et al. 2001). Since EM Cr failed to show antinociception in the first phase of formalin test, possibly EM Cr has only peripheral antinociceptive activity.

Most of non-steroidal anti-inflammatory drugs (NSAIDs) exert antipyretic effects inhibiting prostaglandins production (Dinarello 1989), thereby suppressing inflammation-associated hyperthermia (Kaufmannet al. 1997). The injection of brewer's yeast causes the liberation of proinflammatory cytokines and stimulates the synthesis of prostaglandin E2 in the surroundings of the hypothalamic thermoregulator centers (Chan and Fiscus 2004). Since the extract manifests a significant antipyretic effect in yeast-induced pyrexia, this may berelated to its anti-inflammatory effect.

The phytochemical analysis of the extract confirmed the presence of several constituents previously isolated and identified from this crude drug. Arjunolic acid has been reported as an active compound reducing diabetic injury and other pathological conditions related to oxidative stress (Manna et al. 2009, Ghosh et al. 2010a, b). Furthermore, sitosterol and its glycosides have been shown to display anti-inflammatory andanalgesic effect (Bouic et al. 1996, Yuk et al. 2007, Aragão et al. 2007, Messias et al. 2008). Other still unidentified constituents of the extract, including tannins, also contribute to the total activity of the crudedrug. Tannins have been recognized as active and widespread compounds in several medicinal and food plants (Okuda 2005, Yoshida et al. 2000, 2005).The data presented here indicate that C. rubra extract possesses anti-inflammatory, antinociceptive and antipyretic effects and support to the traditional indication of C. rubra stem bark in inflammatory conditions.

ACKNOWLEDGMENTS

The authors are grateful to Superintendência do Desenvolvimento da Amazônia (SUDAM), Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq), Fundação de Amparo à Pesquisa do Estado de Mato Grosso (FAPEMAT), Instituto Nacional de Ciência eTecnologia em Áreas Úmidas (INAU) and Centro de Pesquisas do Pantanal (CPP) for the financial support.

Manuscript received on October 26, 2009

accepted for publication on August 27, 2010

  • AL-GHAMDI MS. 2001.The anti-inflammatory, analgesicand antipyretic activity of Nigella sativa J Ethnopharmacol 76: 45-48.
  • ARAGÃO GF, CUNHA PINHEIRO MC, NOGUEIRA BANDEIRA P, GOMES LEMOS TL AND DE BARROS VIANA GS. 2007. Analgesic and anti-inflammatory activities of the isomeric mixture of alpha- and beta-amyrin from Protium heptaphyllum (Aubl.) march. J Herb Pharmacother 7(2): 31-47.
  • BOUIC PJD, ETSEBETH S, LIEBENBERG RW, ALBRECHT CF, PEGEL K AND VAN JAARSVELD PP. 1996.Beta-sitosterol and beta-sitosterol glucoside stimulate human peripheral blood lymphocyte proliferation: Implications for their use as an immunomodulatory vitamin combination. Int J Immunopharmacol 18: 693-700.
  • CHAN GH AND FISCUS RR. 2004. Exaggerated productionof nitric oxide (NO) and increases in inducible NO-synthase mRNA leves induced by the pro-inflammatory cytokine interleukin-beta in vascular smooth muscle cells of elderly rats. Exp Gerontol 39(3): 384-394.
  • DE LA CRUZ MG. 2008. Plantas Medicinais de Mato Grosso: a Farmacopéia Popular dos Raizeiros, Cuiabá: Carlini & Caniato Eds, 224 p.
  • DI PAOLA R, DI MARCO R, MAZZON E, GENOVESE T, BENDTZEN K, MACRI B, NICOLETTI F AND CUZZOCREA S. 2004. Prevention of carrageenan-induced pleurisy in mice by anti-CD30 ligand monoclonal antibody. Clin Immunol 113: 64-73.
  • DINARELLO CA. 1989.The endogenous pyrogens in host-defense interactions. Hosp Pract 24: 111-128.
  • EDDY NB AND LEIMBACH D. 1953. Synthetic analgesics II. Dithienylbutenyl and dithienylbutyl-amines. J Pharmacol Exp Ther 107: 385-393.
  • GHOSH J, DAS J, MANNA P AND SIL PC. 2010a. Arjunolic acid, a triterpenoid saponin, prevents acetaminophen (APAP)-induced liver and hepatocyte injury via the inhibition of APAP bioactivation and JNK-mediated mitochondrial protection. Free Radical Bio Med 48: 535-553.
  • GHOSH J, DAS J, MANNA P AND SIL PC. 2010b.Acetaminophen induced renal injury via oxidative stress and TNF-a production: Therapeutic potential of arjunolicacid. Toxicology 268: 8-18.
  • HARBORNE JB. 1998.Phytochemical Methods (A Guideto Modern Techniques of Plant Analysis), London: Chapman and Hall Ltd, 302 p.
  • HUNSKAAR S AND HOLE K. 1987.The formalin test inmice: dissociation between inflammatory and non-inflammatory pain. Pain 30: 103-114.
  • KAUFMANN WE, ANDREASSON KL, ISAKSON PC AND WORLEY PF. 1997. Cyclooxygenases and the centralnervous system. Prostaglandins 54: 601-624.
  • KOSTER R, ANDERSON M AND DE BEER EJ. 1959. Acetic acid for analgesic screening. Fed Proc 18: 412-414.
  • LIMA EF, SOUSA-FILHO PT, BASTIDA J AND SCHMEDA-HIRSCHMAN G. 2002. Saponins from Cariniana rubra (Lecythidaceae). Bol Soc Chil Quim 47: 441-447.
  • LO TN, ALMEIDA AP AND BEAVEN MA. 1982.Dextran and carrageenan evoke different inflammatory responses in rat with respect to composition of infiltrates and effect of indomethacin. J Pharmacol Exp Ther 221: 261-267.
  • MANNA P, SINHA M AND SIL PC. 2009. Prophylactic role of arjunolic acid in response to streptozotocin mediated diabetic renal injury: Activation of polyol pathway and oxidative stress responsive signaling cascades. Chem-Biol Interact 181: 297-308.
  • MATOS FJA. 1988. Introdução à Fitoquímica Experimental, Fortaleza: Edições UFC, 128 p.
  • MEADE CJ, TURNER GA AND BATEMAN PE. 1986.The role of polyphosphoinositides and their breakdown products in A23187-induced release of arachidonic acid from rabbit polymorphonuclear leucocytes. Biochem J23: 425-436.
  • MESSIAS KLS, CAMPOS-BUZZI F, FISCHER LGO, MALHEIROS A, MONACHE FD AND CECHINEL-FILHO V. 2008. Chemical composition and analgesic activity ofthe leaves and branches of Marlierea tomentosa Camb.Quim Nova 31(7): 1747-1749.
  • OKUDA T. 2005. Systematics and health effects of chemically distinct tannins in medicinal plants. Phytochemistry 66: 2012-2031.
  • OTUKI MF, LIMA FV, MALHEIROS A, CECHINEL-FILHO V, MONACHE FD, YUNES RA AND CALIXTO JB. 2001. Evaluation of the antinociceptive action caused by ether fraction and a triterpene isolated from resin of Protium kleinii Life Scie 69(19): 2225-2236.
  • PARRAT JR AND WEST GB. 1957. Inhibition by various substances of edema formation in the hind paw of the rat induced by 5-hydroxytryptamine, histamine, dextran, egg white and compound 48/80. Brit J Pharm Chemot 13: 65-70.
  • RIBEIRO AR, VALE ML, THOMAZZI SM, PASCHOALATO ABP, POOLE S, FERREIRA SH AND CUNHA FQ. 2000. Involvement of resident macrophages and mast cells inthe writhing nociceptive response induced by zymosanand acetic acid in mice. Eur J Pharmacol 387: 111-118.
  • SEKIYA K, OKUDA H AND ARICHI S. 1982. Selective inhibition of platalet lipoxygenase by esculetin. Biochim Biophys Acta 713: 68-72.
  • SILVA MG, OLIVEIRA FS, QUINTANS-JÚNIOR LJ, THENIO OML AND DINIZ MFM. 2005. Investigação de efeito analgésico central e antiinflamatório de Conocliniopsis prasiifolia (DC) RM King and H Robinson em roedores. Acta Farm Bom 24(4): 533-537.
  • VINEGAR R, TRUAX JF, SELPH JL, LEA A AND JOHNSTON PR. 1978. Quantitative "in vivo'' studies of the acute actions of anti-inflammatory drugs in the rat. Eur J Rheumatol Infl 1: 204-211.
  • WHITTLE BA. 1964. The use of changes in capillary permeability in mice to distinguish between narcotic and nonnarcotic analgesics. Brit J Pharmacol 22: 246-253.
  • WINTER CA, Risley EA and Nuss GW. 1962.Carrageenin-induced edema in hind paw of rat as an assayfor anti-inflammatory drugs. P Soc Exp Biol Med 3:544-547.
  • YOSHIDA T, HATANO T AND ITO H. 2005. High molecular weight plant poplyphenols (tannins): Prospective functions. Recent Adv Phytochem 39: 163-190.
  • YOSHIDA T, HATANO T, ITO H AND OKUDA T. 2000. Chemical and biological perspectives of ellagitannin oligomers from medicinal plants. Stud Nat Prod Chem 23: 395-453.
  • YUK JE, WOO JS, YUN C-Y, LEE J-S, KIM J-H, SONG G-Y, YANG EJ, HUR IK AND KIM IS. 2007. Effectsof lactose-β-sitosterol and β-sitosterol on ovalbumin-induced lung inflammation in actively sensitized mice. Int Immunopharmacol 7: 1517-1527.
  • ZIMMERMANN M. 1983. Ethical guidelines for investigation of experimental pain in conscious animals. Pain 16(2): 109-110.
  • Correspondence to:

    Domingos Tabajara de Oliveira Martins
    E-mail:
  • Publication Dates

    • Publication in this collection
      27 May 2011
    • Date of issue
      June 2011

    History

    • Accepted
      27 Aug 2010
    • Received
      26 Oct 2009
    Academia Brasileira de Ciências Rua Anfilófio de Carvalho, 29, 3º andar, 20030-060 Rio de Janeiro RJ Brasil, Tel: +55 21 3907-8100 - Rio de Janeiro - RJ - Brazil
    E-mail: aabc@abc.org.br