Tingenone, a pentacyclic triterpene, induces peripheral antinociception due to opioidergic activation

Original Papers

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Authors

Clarice de Carvalho Veloso 1, Vanessa Gregório Rodrigues 2, Renata Cristina Mendes Ferreira 1, Lucienir Pains Duarte 2, Andre Klein 1, Igor Dimitri Duarte 1, Thiago Roberto Lima Romero 1, Andrea de Castro Perez 1

Affiliations

1

2

Key words " Maytenus imbricata l " Celastraceae l " tingenone l " pentacyclic triterpene l " opioid receptors l " peripheral antinociception l

Department of Pharmacology, Institute of Biological Sciences, Federal University of Minas Gerais (UFMG), Belo Horizonte, Minas Gerais, Brazil Department of Chemistry, Institute of Exact Sciences, UFMG, Belo Horizonte, Minas Gerais, Brazil

Abstract !

Plants belonging to the genus Maytenus are routinely used in folk medicine for the treatment of pain diseases. Our previous phytochemical study of the roots of Maytenus imbricata resulted in the isolation and characterization of tingenone, a pentacyclic triterpene. Natural triterpenoids are of growing interest because they have several biological activities, including analgesic properties. The present study assessed the involvement of the opiodergic pathway in the tingenone-induced antinociceptive effect against hyperalgesia induced by prostaglandin E2 (2 µg) in the peripheral pathway. We evaluated the effect of several antag-

Introduction !

received revised accepted

May 7, 2014 Sept. 12, 2014 Sept. 14, 2014

Bibliography DOI http://dx.doi.org/ 10.1055/s-0034-1383147 Published online October 22, 2014 Planta Med 2014; 80: 1615–1621 © Georg Thieme Verlag KG Stuttgart · New York · ISSN 0032‑0943 Correspondence Andrea de Castro Perez Department of Pharmacology Institute of Biological Sciences Federal University of Minas Gerais (UFMG) Av Antonio Carlos 6627 31270–100 Belo Horizonte, Minas Gerais Brazil Phone: + 55 3 13 40 92 72 Fax: + 55 31 34 09 26 95 [email protected]

The development of new analgesics for clinical uses without side effects is the main goal of studies regarding pain. While opioids are the most potent and effective drugs in relieving severe pain, their utilization has several effects, such as respiratory depression, nausea, constipation, physical dependence, and tolerance [1]. Natural substances derived from plants play an important role in the development of new analgesic drugs. Some antinociceptive secondary metabolites discovered include alkaloids, terpenoids, and flavonoids [2, 3]. The isolation and identification of triterpenes with antinociceptive effects have been shown in many studies [4–6]. Maytenus is a genus of the Celastraceae family, and its species are used in traditional medicine for the treatment of gastric disorders, inflammatory diseases, and pain as well as other disorders [7–9]. For example, the hexane and ethyl acetate extracts of Maytenus ilicifolia inhibited formaldehyde-induced nociception and paw edema in mice and carrageenan-induced paw edema in rats [10], while the chloroform extract of Maytenus

onists to opioid receptors using the mouse paw pressure test. Tingenone administered into the right hind paw induced a local antinociceptive effect that was antagonized by naloxone, a nonselective antagonist to opioid receptors. Clocinnamox, naltrindole, and nor-binaltorphimine are selective antagonists to µ, δ, and κ receptors, respectively, which reverted the peripheral antinociception induced by tingenone. Bestatine acts as an inhibitor of aminopeptidase, an enzyme that degrades endogenous opioid peptides, and was shown to intensify the antinociceptive effect of tingenone. The results suggest that the opioidergic system participates in the peripheral antinociception induced by tingenone.

senegalensis reduced edema induced by croton oil in mice [8]. The antinociceptive effect of Maytenus rigida [11] and the anti-inflammatory effect of Maytenus heterophylla [12] were also demonstrated. Our previous phytochemical study of Maytenus imbricata roots resulted in the isolation and characterization of pentacyclic triterpenes, including tingenone [13]. The antinociceptive effect of orally administered tingenone in the second phase of the formalin test, which is characterized by inflammatory pain, was demonstrated [14]. Pain can be effectively diminished by various endogenous mechanisms in the central nervous system (CNS) and in the terminals of primary afferent neurons, such as by endogenous opioids in the inflamed peripheral tissue [15]. The aim of this study was, thus, to investigate whether tingenone induces a peripheral antinociceptive effect via the activation of the opioidergic system that is free from major side effects.

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Tingenone, a Pentacyclic Triterpene, Induces Peripheral Antinociception Due to Opioidergic Activation

Original Papers

Fig. 1 The effects of tingenone on prostaglandin E2-induced hyperalgesia in mice. Tingenone (Ting) was injected in the third hour after the local administration of PGE2 (2 µg). The antinociceptive response was measured by the paw pressure test, as described in Material and Methods. Each column represents the mean ± the standard deviation of Δ of the measurement of the nociceptive threshold expressed in grams (g) for four animals. * Indicates the statistical significance p < 0.05 when compared to the PGE2 2 µg + V control group. V is 20 % DMSO + 1% Tween 20 in saline and Et is ethanol. Δ Denotes the difference between the nociceptive threshold obtained in the beginning of the experiment (basal value) before any injection (time zero) and the threshold measured 10 min after the third hour following the injection of PGE2 (at which time the maximum antinociceptive effect of tingenone was observed).

Fig. 2 Exclusion of the antinociceptive effects of tingenone on the outside paw. PGE2 (2 µg) was administered in both the right (R) and left (L) hind paws. Tingenone (200 µg/paw) was administered 3 h after PGE2 to the right hind paw. Its vehicle was administered to the left paw. Each column represents the mean ± the standard deviation of Δ of the measurement of the nociceptive threshold expressed in grams (g) regarding four animals. * Indicates the statistical significance p < 0.05 when compared to the PGE2 2 µg + V (R Paw) control group and #p < 0.05 when compared to the PGE2 2 µg + V (L Paw) group. V is 20 % DMSO + 1 % Tween 20 in saline. Δ Denotes the difference between the nociceptive threshold obtained in the beginning of the experiment (basal value) before any injection (time zero) and the threshold measured 10 min after the third hour following the injection of PGE2 (at which time the maximum antinociceptive effect of tingenone was observed).

Results !

The intraplantar administration of dosages of 50, 100, and 200 µg/paw of tingenone in the third hour after the injection of prostaglandin E2 (PGE2; 2 µg) induced a peripheral antinociceptive effect. Tingenone (25 µg) showed no statistically significant difference when compared to the group treated with PGE2 + ve" Fig. 1). hicle (V; 20 % DMSO + 1 % Tween 20) (l Intraplantar administration of 200 µg tingenone did not alter the nociceptive threshold when injected in the absence of PGE2. The solutions of 2 % ethanol (vehicle for PGE2) and 20 % DMSO + 1 % Tween 20 (vehicle of tingenone) did not induce antinociception " Fig. 1). or hyperalgesia when injected alone (l To exclude any possible systemic effects, PGE2 was administered in the right paw and left paw of the animals, while tingenone (200 µg) was administered only to the right paw and its vehicle was administered to the left paw. The evaluation of the nociceptive threshold showed that this dosage of tingenone induced an antinociceptive effect restricted to the treated paw because it did not alter the hyperalgesia induced by PGE2 in the contralater-

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" Fig. 2). The same al paw, suggesting a peripheral site of action (l dosage was used in subsequent experiments. The intraplantar administration of naloxone (NX; 25, 50, and 100 µg/paw), a nonselective antagonist to opioid receptors, reverted the peripheral antinociception induced by 200 µg/paw " Fig. 3). doses of tingenone in a dosage-dependent manner (l To investigate the selective involvement of the opioid receptor subtypes in the antinociceptive effect observed, clocinnamox, naltrindole, and nor-binaltorphimine, selective antagonists to µ, δ, and κ receptors, respectively, were administered. Clocinnamox (Cloc; 10, 20, and 40 µg/paw) and naltrindole (NTD; 15, 30, 60, and 120 µg/paw) reverted the peripheral antinociception induced by 200 µg/paw doses of tingenone in a dosage-dependent " Figs. 4 and 5, respectively). manner (l The intraplantar administration of nor-binaltorphimine (Norbin; 100 µg/paw) did not antagonize the antinociceptive effect of tingenone, but this effect was partially antagonized by the local " Fig. 6). Bestatine is an inadministration of Nor-bin (200 µg) (l hibitor of aminopeptidase, an enzyme that degrades endogenous opioid peptides, and was shown to intensify the antinociceptive effect of tingenone when administered in a lower dosage (50 µg/

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Fig. 3 Effects of the intraplantar administration of naloxone on the peripheral antinociceptive effect induced by tingenone. NX was administered 30 min before tingenone. Each column represents the mean ± the standard deviation of Δ of the measurement of the nociceptive threshold expressed in grams (g) regarding four animals. * Indicates the statistical significance p < 0.05 when compared to the PGE2 2 µg + V1 + V2 control group and # p < 0.05 when compared to the PGE2 2 µg + V1 + Ting 200 µg group. V1 is saline, V2 is 20 % DMSO + 1% Tween 20 in saline, and Et is ethanol. Δ Denotes the difference between the nociceptive threshold obtained in the beginning of the experiment (basal value) before any injection (time zero) and the threshold measured 10 min after the third hour following the injection of PGE2 (at which time the maximum antinociceptive effect of tingenone was observed).

Fig. 4 Effects of the intraplantar administration of clocinnamox on the peripheral antinociceptive effect induced by tingenone. Cloc was administered 30 min before tingenone. Each column represents the mean ± the standard deviation of Δ of the measurement of the nociceptive threshold expressed in grams (g) regarding four animals. * Indicates the statistical significance p < 0.05 when compared to the PGE2 2 µg + V1 + V2 control group and # p < 0.05 when compared to the PGE2 2 µg + V1 + Ting 200 µg group. V1 is saline, V2 is 20 % DMSO + 1% Tween 20 in saline, and Et is ethanol. Δ Denotes the difference between the nociceptive threshold obtained in the beginning of the experiment (basal value) before any injection (time zero) and the threshold measured 10 min after the third hour following the injection of PGE2 (at which time the maximum antinociceptive effect of tingenone was observed).

Fig. 5 Effects of the intraplantar administration of naltrindole on the peripheral antinociceptive effect induced by tingenone. NTD was administered 30 min before tingenone. Each column represents the mean ± the standard deviation of Δ of the measurement of the nociceptive threshold expressed in grams (g) for four animals. * Indicates the statistical significance p < 0.05 when treated groups were compared to the PGE2 2 µg + V1 + V2 control group and # p < 0.05 when compared to the PGE2 2 µg + V1 + Ting 200 µg group. V1 is saline, V2 is 20% DMSO + 1% Tween 20 in saline, and Et is ethanol. Δ Denotes the difference between the nociceptive threshold obtained in the beginning of the experiment (basal value) before any injection (time zero) and the threshold measured 10 min after the third hour following the injection of PGE2 (at which time the maximum antinociceptive effect of tingenone was observed).

paw). When each of the antagonists (NX, Cloc, NTD) and bestatine were administered alone in the highest dosage, no antinoci-

" Fig. 3, 4, 5, and 7, respecception or hyperalgesia was induced (l tively).

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Original Papers

Original Papers

Fig. 6 Effects of the intraplantar administration of nor-binaltorphimine on the peripheral antinociceptive effect induced by tingenone. Nor-bin was administered 30 min before tingenone. Each column represents the mean ± the standard deviation of Δ of the measurement of the nociceptive threshold expressed in grams (g) for four animals. * Indicates the statistical significance p < 0.05 when treated groups were compared to the PGE2 2 µg + V1 + V2 control group and # p < 0.05 when compared to the PGE2 2 µg + V1 + Ting 200 µg group. V1 is saline, V2 is 20 % DMSO + 1% Tween 20 in saline, and Et is ethanol. Δ Denotes the difference between the nociceptive threshold obtained in the beginning of the experiment (basal value) before any injection (time zero) and the threshold measured 10 min after the third hour following the injection of PGE2 (at which time the maximum antinociceptive effect of tingenone was observed).

Fig. 7 Effects of the intraplantar administration of bestatine on the peripheral antinociceptive effect induced by tingenone. Bestatine (Best) was administered 30 min before tingenone. Each column represents the mean ± the standard deviation of Δ of the measurement of the nociceptive threshold expressed in grams (g) for four animals. * Indicates the statistical significance (p < 0.05) when treated groups were compared to the PGE2 2 µg + V1 + V2 control group and # p < 0.05 indicates significance when the treated groups were compared to the PGE2 2 µg + V1 + Ting 50 µg group. V1 is saline, V2 is 20% DMSO + 1% Tween 20 in saline, and Et is ethanol. Δ Denotes the difference between the nociceptive threshold obtained in the beginning of the experiment (basal value) before any injection (time zero) and the threshold measured 10 min after the third hour following the injection of PGE2 (at which time the maximum antinociceptive effect of tingenone was observed).

Discussion !

Natural products have greatly contributed to the development of important drugs that are used in contemporary medicine. One of the most important analgesic drugs employed in the clinic is the alkaloid morphine, and other substances with antinociceptive effects include flavonoids and terpenes [2]. Tingenone, a pentacyclic triterpene, presents an antinociceptive effect in the second phase of the formalin test for inflammatory pain when administered orally [14]. To study the mechanisms involved in this effect, we chose a model in which mechanic hyperalgesia is induced by the intraplantar injection of exogenous PGE2 into mice. In the present study, the utilization of peripherally administered PGE2 (2 µg/paw) induced hyperalgesia with a maximum intensity in the third hour after its application. These results are in agreement with a previous study [16]. Tingenone (200 µg/paw) induced a peripheral antinociceptive effect because the intraplantar administration of this triterpene in the right paw did not alter the hyperalgesia induced by PGE2 observed in the contralateral paw. Thus, this dose was utilized in subsequent experiments.

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The result showed once again that triterpenes are capable of inducing central [17] and peripheral [4, 18, 19] antinociceptive effects. We further demonstrated the peripheral antinociceptive effect of tingenone as well as its mechanism of action. Pain can be effectively mitigated by several endogenous mechanisms in the CNS and in first-order afferent neuron terminals. For example, endogenous opioids in the inflamed peripheral tissue can induce analgesia [15]. Opioid peptides such as β-endorphin and Met-enkephalin were detected in immune cells in the inflamed subcutaneous tissues of rat paws, indicating that these peptides are synthetized by resident and migratory immune cells such as neutrophils, lymphocytes, monocytes, and macrophages [15, 20, 21]. Opioid peptides are also synthetized by keratinocytes [20] and peripheral sensory neurons [1]. The pretreatment with orally administered tingenone was demonstrated to induce an antinociceptive effect in the second phase of the formalin test for inflammatory pain. However, a lack of anti-inflammatory effects was observed in the paw edema test and in the recruitment of total leukocytes and neutrophils in the pleural cavity test, both induced by carrageenan. The absence of antinociceptive effects in the tail flick and in the first phase of the

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Fig. 8 Structure of tingenone, a natural pentacyclic triterpene isolated from the roots of M. imbricata.

volvement of endogenous opioid peptides in modulating the antinociceptive peripheral effects of tingenone administered in low doses. The intraplantar administration of bestatine intensified the peripheral antinociceptive effect of tingenone, suggesting that endogenous opioid peptides participate in this effect. Therefore, we suggest that the endogenous opioid peptides involved in tingenone peripheral antinociception are derived from the migratory immune cells that contain and secrete opioid peptides because they are found in peripheral inflamed tissues and not in non-inflamed tissues [31]. In conclusion, tingenone presents a peripheral antinociceptive effect, evidencing the involvement of selective endogenous opioid peptides for receptors µ, δ, and κ. Tingenone has the potential to be used as a new analgesic drug with no systemic collateral effects.

Materials and Methods !

Plant material The roots of M. imbricata were carefully collected to prevent damage to the specimen. The collection area was the Ouro Preto municipality, Minas Gerais state, Brazil. The plant material was identified by the botanists Rita M. de Carvalho Okano, Botanic Department of the Federal University of Viçosa, and M. Cristina Teixeira Braga Messias, Botanic Department of the Federal University of Ouro Preto. A voucher specimen (number 27 780) was deposited in the Herbarium collection at the Botanic Department of the Federal University of Viçosa, Brazil. The M. imbricata roots were dried at room temperature and powdered in a mill. The powder (1.5 kg) was submitted to extraction with the organic solvent hexane/ethyl ether (1 : 1) in a Soxhlet apparatus. The solvent was removed in a rotator evaporator. The quantity of the residue obtained was 16.1 g in the hexane/ethyl ether (1 : 1) extract. From this extract, 1.5 g of tingenone " Fig. 8) was isolated and characterized as previously described (l [13].

Animals Male Swiss mice (30–35 g) obtained from the Bioterism Center of Federal University of Minas Gerais (CEBIO/UFMG) were used in the experiments (n = 4 per group). The mice were housed in standard cages and kept at a constant temperature of 23 °C with a 12h light-dark cycle and free access to food and tap water. All testing procedures were conducted in accordance with the ethical guidelines of the International Association for the Study of Pain (IASP) [32] and approved by the Ethics Committee in Animal Experi-

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formalin test was also observed [14]. Thus, the peripheral antinociceptive action of tingenone in the second phase of the formalin test does not suggest an anti-inflammatory action, but instead indicates the activation of other intracellular signaling pathways, such as the opioid receptors present in the peripheral nerve endings. Previous studies have shown that the activation of peripheral and central opioidergic receptors result in a reduction in the licking time during the second stage of the formalin test [22, 23]. Earlier studies have also shown that µ opioid receptors are upregulated in the peripheral sensory nerve endings during the inflammation of the rat paw in addition to an increase in the number of immune cells containing β-endorphin [24], an opioid peptide that is responsible for antinociception in inflammatory pain [25]. Therefore, we suggest that tingenone activates the opioidergic pathway during the second phase of the formalin test rather than during the first phase. Tingenone may not be acting directly on opioid receptors but instead on immune cells, which in turn release endogenous opioid peptides. Considering that immune cells migrate with effects during the second phase (which corresponds to inflammatory pain), it is possible that the antinociceptive effects of tingenone are dependent on the release of opioids from immune cells during the second phase, which may also be linked to the absence of an anti-inflammatory effect. In other words, tingenone does not interfere with cell migration, but those cells are still important in the process of pain modulation. Thus, to evaluate the involvement of the opioidergic pathway in the peripheral antinociceptive effects of tingenone, paws were pretreated with NX, a nonselective antagonist for opioid receptors. NX reverted the antinociceptive effects of the triterpene in a dosage-dependent manner. We chose the following selective antagonists for the opioid receptors: Cloc, a selective antagonist of the µ receptor; NTD, a selective antagonist of the δ receptor; and Nor-bin, a selective antagonist of the κ receptor. Anatomical, molecular, and electrophysiological studies have shown that all three opioid receptors (µ, δ, and κ) are expressed by sensory neurons. These receptors have been found on cell bodies in the dorsal root ganglia (DRG) and on peripheral terminals of primary afferent neurons in both animals and humans [26]. According to our results, it seems that the peripheral antinociceptive mechanism of tingenone involves the µ, δ, and κ receptors. The endogenous opioid peptides β-endorphin and enkephalins play their roles via the µ and δ receptors, while dynorphin acts through the κ opioid receptor. Endorphins 1 and 2 have a high selectivity for the µ opioid receptor [27]. Opioid peptides originating from immune cells may activate the opioidergic receptors present in the peripheral nerve endings of inflamed cells [24, 25]. In a previous study, it was demonstrated that the peripheral administration of µ, δ, and κ opioid receptor agonists produced analgesic effects in inflamed tissues but not in non-inflamed tissues, suggesting that peripheral opioid receptors can be functionally activated during inflammation [28]. The inhibition of peptidases is used as a pharmacological strategy to maximize the effects of the endogenously released opioid peptides. Aminopeptidase N, which is responsible for inactivating endogenous opioid peptides, is a transmembrane peptidase expressed on leukocytes and neurons that contains a large extracellular domain containing the active site [1, 29]. A previous study has shown that leukocytes and peripheral sensory neurons are a major source of aminopeptidase N and that blockage of this enzyme intensifies the action of opioids in peripheral receptors, which locally inhibits inflammatory pain [30]. In this study, we used bestatine to inhibit aminopeptidase N in order to evaluate the in-

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mentation at the Federal University of Minas Gerais (protocol 115/2012).

!

The authors declare that there are no conflicts of interest in relation to this study.

Measurement of hyperalgesia Hyperalgesia was induced by subcutaneous injection of PGE2 (2 µg) into the plantar surface of the hind paw. Hyperalgesia was measured according to the rat paw pressure test [33], which has been adapted to mice [34]. An analgesimeter equipped with a cone-shaped paw-presser with a rounded tip was used (Ugo-Basile) to apply a linearly increasing force to the hind paw. The weight in grams (g) required to elicit the nociceptive response of paw flexion was determined to be the nociceptive threshold. A cutoff value of 160 g was used to reduce the possibility of damage to the paws. The nociceptive threshold (Δ) was calculated as the difference between the nociceptive threshold obtained in the beginning of the experiment (basal value) before any injection (time zero) and the threshold measured 10 min after the third hour following PGE2 injection (at which time the maximum antinociceptive effect of tingenone was observed). A nociceptive threshold value of Δ > 0 indicated hyperalgesia induced by PGE2 injection, whereas decreases in this value indicated the antihyperalgesic effect of the tested drug.

Drugs administration All drugs were administered using an injected volume of 20 µL/ paw. Tingenone (purity > 98 %) was dissolved in 20 % DMSO and 1 % Tween 20 in saline. NX (purity > 98 %; Sigma), Cloc (Tocris), NTD (purity > 99 %; Tocris), Nor-bin (purity > 98%; Tocris), and bestatine (purity > 99 %; Tocris) were dissolved in isotonic saline. PGE2 (Sigma) was dissolved in 2% ethanol in saline. Abbreviations of the solvents are: V is 20% DMSO + 1 % Tween 20 " Fig. 1), V1 is saline, and V2 is 20% DMSO in saline, Et is ethanol (l + 1% Tween 20 in saline as shown in the figure legends.

Experimental protocol Tingenone was administered subcutaneously in the right hind paw 3 h after a local injection of PGE2. To determine whether tingenone was acting outside the injected paw, PGE2 was injected into both hind paws, while tingenone was administered to the right paw and the vehicle was administered to the left paw. After 10 min (corresponding to the peak action of tingenone), the nociceptive threshold was measured in both hind paws. NX, Cloc, NTD, Nor-bin, and bestatine were administered 30 min prior to the administration of tingenone. It should be noted that the protocols concerning the doses and times of administration of each drug used in this study were obtained from the literature data [35] and pilot experiments.

Statistical analysis The obtained results were analyzed using Graph Pad Prism 3.0 and expressed as mean ± SD. Statistical differences between groups were calculated by one-way ANOVA followed by the Bonferroni test. Statistical significance was set at p < 0.05.

Acknowledgements !

This work was supported by CAPES and CNPq (Brazil).

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Conflict of Interest

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References 1 Stein C, Machelska H. Modulation of peripheral sensory neurons by the immune system: implications for pain therapy. Pharmacol Rev 2011; 63: 860–881 2 Calixto JB, Beirith A, Ferreira J, Santos ARS, Filho VC, Yunes RA. Naturally occurring antinociceptive substances from plants. Phytother Res 2000; 14: 401–418 3 Yunes RA, Filho CV, Ferreira J, Calixto JB. The use of natural products as sources of new analgesic drugs. In: Atta-ur-Rahman, editor. Studies in natural products chemistry. Amsterdam: Elsevier; 2005: 191–212 4 Gaertner M, Müller L, Roos JF, Cani G, Santos ARS, Niero R, Calixto JB, Yunes RA, Monache FD, Cechinel-Filho V. Analgesic triterpenes from Sebastiania schottiana roots. Phytomedicine 1999; 1: 41–44 5 Ferreira J, Floriani AEO, Filho VC, Monache FD, Yunes RA, Calixto JB, Santos ARS. Antinociceptive properties of the methanolic extract and two triterpenes isolated from Epidendrum monsenii stems (Orchidaceae). Life Sci 2000; 66: 791–802 6 Bortalanza LB, Ferreira J, Hess SC, Monache FD, Yunes RA, Calixto JB. Anti-allodynic action of the tormentic acid, a triterpene isolated from plant, against neuropathic and inflammatory persistent pain in mice. Eur J Pharmacol 2002; 453: 203–208 7 Baggio CH, Freitas CS, Mayer B, Dos Santos AC, Twardowschy A, Potrich FB, Cipriani TR, de Souza LM, Sassaki GL, Iacomini M, Marques MC, Mesia-Vela S. Muscarinic-dependent inhibition of gastric emptying and intestinal motility by fractions of Maytenus ilicifolia Mart ex. Reissek. J Ethnopharmacol 2009; 123: 385–391 8 Sosa S, Morelli CF, Tubaro A, Cairoli P, Speranza G, Manitto P. Anti-inflammatory activity of Maytenus senegalensis root extracts and of maytenoic acid. Phytomedicine 2007; 14: 109–114 9 Niero R, de Andrade SF, Filho VC. A review of the ethnopharmacology, phytochemistry and pharmacology of plants of the Maytenus genus. Curr Pharm Des 2011; 17: 1851–1871 10 Jorge RM, Leite JPV, Oliveira AB, Tagliati CA. Evaluation of antinociceptive, anti-inflammatory and antiulcerogenic activities of Maytenus ilicifolia. J Ethnopharmacol 2004; 94: 93–100 11 Martins MV, Estevam CS, Santos ALLM, Dias AS, Cupertino-da-Silva YK, Araújo-Júnior JX, Miranda ALP, Barreiro EJ, Pizza C, Piacente S, Montoro P, Quintans-Júnior LJ, Araujo BS, Alexandre-Moreira MS, SantʼAna AEG. Antinociceptive effects of an extract, fraction and an isolated compound of the stem bark of Maytenus rigida. Rev Bras Farmacogn 2012; 22: 598–603 12 Da Silva G, Taniça M, Rocha J, Serrano R, Gomes ET, Sepodes B, Silva O. In vivo anti-inflammatory effect and toxicological screening of Maytenus heterophylla and Maytenus senegalensis extracts. Hum Exp Toxicol 2011; 30: 693–700 13 Rodrigues VG, Duarte LP, Silva GDF, Silva FC, Góes JF, Takahashi JA, Pimenta LPS. Evaluation of antimicrobial activity and toxic potential of extracts and triterpenes isolated from Maytenus imbricata. Quím Nova 2012; 35: 1375–1380 14 Veloso CC, Rodrigues VG, Azevedo AO, Oliveira CO, Gomides LF, Duarte LP, Duarte ID, Klein A, Perez AC. Antinociceptive effects of Maytenus imbricata Mart. ex. Reissek (Celastraceae) root extract and its tingenone constituent. J Med Plants Res 2014; 8: 68–76 15 Stein C. The control of pain in peripheral tissue by opioids. N Engl J Med 1995; 332: 1685–1690 16 Alves DP, Soares AC, Francischi JN, Castro MSA, Perez AC, Duarte IDG. Additive antinociceptive effect of the combination of diazoxide, an activator of ATP-sensitive K+ channels, and sodium nitroprusside and dibutyryl-cGMP. Eur J Pharmacol 2004; 489: 59–65 17 Tapondjou LA, Lontsi D, Sondengam BL, Choi J, Lee KT, Jung HJ, Park HJ. In vivo anti-nociceptive and anti-inflammatory effect of the two triterpenes, ursolic acid and 23-hydroxyursolic acid, from Cussonia bancoenesis. Arch Pharm Res 2003; 26: 143–146 18 Lima FV, Malheiros A, Otuki MF, Calixto JB, Yunes RA, Filho VC, Monache FD. Three triterpenes from the resinous bark of Protium kleinii and their antinociceptive activity. J Braz Chem Soc 2005; 16: 578–582 19 Longhi-Balbinot DT, Martins DF, Lanznaster D, Silva MD, Facundo VA, Santos ARS. Further analyses of mechanisms underlying the antinoci-

Downloaded by: IP-Proxy CONSORTIUM:CAPES (UFMG Universidade Federal de Minas Gerais), Dot. Lib Information. Copyrighted material.

1620

20 21

22

23 24

25

26 27

ceptive effect of the triterpene 3β,6β,16β-trihydroxylup-20(29)-ene in mice. Eur J Pharmacol 2011; 653: 32–40 Stein C, Schäfer M, Machelska H. Attacking pain at its source: new perspectives on opioids. Nat Med 2003; 9: 1003–1008 Rittner HL, Hackel D, Yamdeu RS, Mousa SA, Stein C, Schäfer M, Brack A. Antinociception by neutrophil-derived opioid peptides in noninflamed tissue – role of hypertonicity and the perineurium. Brain Behav Immun 2009; 23: 548–557 Oluyomi AO, Hart SL, Smith TW. Differential antinociceptive effects of morphine and methylmorphine in the formalin test. Pain 1992; 49: 415–418 Hong Y, Abbott FV. Peripheral opioid modulation of pain and inflammation in the formalin test. Eur J Pharmacol 1995; 277: 21–28 Mousa SA, Zhang Q, Sitte N, Ji R, Stein C. β-Endorphin-containing memory-cells and µ-opioid receptors undergo transport to peripheral inflamed tissue. J Neuroimmunol 2001; 115: 71–78 Stein C, Hassan AH, Przewłocki R, Gramsch C, Peter K, Herz A. Opioids from immunocytes interact with receptors on sensory nerves to inhibit nociception in inflammation. Proc Natl Acad Sci U S A 1990; 87: 5935–5939 Stein C, Machelska H, Binder W, Schäfer M. Peripheral opioid analgesia. Curr Opin Pharmacol 2001; 1: 62–65 Millan MJ. Descending control of pain. Prog Neurobiol 2002; 66: 355– 474

28 Stein C, Millan MJ, Shippenberg TI, Peter K, Herz A. Peripheral opioid receptors mediating antinociception in inflammation. Evidence for involvement of µ, δ and κ receptors. J Pharmacol Exper Ther 1989; 248: 1269–1275 29 Luan Y, Xu W. The structure and main functions of aminopeptidase N. Curr Med Chem 2007; 14: 639–647 30 Schreiter A, Gore C, Labuz D, Fournie-Zaluski MC, Roques BP, Stein C, Machelska H. Pain inhibition by blocking leukocytic and neuronal opioid peptidases in peripheral inflamed tissue. FASEB J 2012; 26: 5161–5171 31 Stein C, Pflüger M, Yassouridis A, Hoelzl J, Lehrberger K, Welte C, Hassan AHS. No tolerance to peripheral morphine analgesia in presence of opioid expression in inflamed synovia. J Clin Invest 1996; 98: 793–799 32 Zimmermann M. Ethical guidelines for investigations of experimental pain in conscious animals. Pain 1983; 16: 109–110 33 Randall LO, Selitto JJ. A method for measurement of analgesic activity on inflamed tissues. Arch Int Pharmacodyn Ther 1957; 111: 409–419 34 Kawabata A, Nishimura Y, Takagi H. L-Leucyl-L-arginine, naltrindole and D-arginine block antinociception elicited by L-arginine in mice with carrageenin-induced hyperalgesia. Br J Pharmacol 1992; 107: 1096–1101 35 Guzzo LS, Perez AC, Romero TR, Azevedo AO, Duarte ID. Cafestol, a coffeespecific diterpene, induces peripheral antinociception mediated by endogenous opioid peptides. Clin Exp Pharmacol Physiol 2012; 39: 412– 416

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