Evaluation of antinociceptive and antiinflammatory effects of Croton pullei var. glabrior Lanj. (Euphorbiaceae)

Avaliação dos efeitos antinociceptivo e antiinflamatório de Croton pullei var. glabrior Lanj. (Euphorbiaceae)

ARTIGO

Evaluation of antinociceptive and antiinflammatory effects of Croton pullei var. glabrior Lanj. (Euphorbiaceae)

Avaliação dos efeitos antinociceptivo e antiinflamatório de Croton pullei var. glabrior Lanj. (Euphorbiaceae)

Fábio F. Rocha^I,*; Evandro M. N. Neves^II; Elson A. Costa^III; Lécia G. Matos^III; Adolfo H. Müller^IV; Giselle M. S. P. Guilhon^IV; Wellington S. Cortes^I; Frederico A. Vanderlinde^I

^IDepartamento de Ciências Fisiológicas, Instituto de Biologia, Universidade Federal Rural do Rio de Janeiro, 23890-000 Seropédica-RJ, Brazil

^IICurso de Medicina Veterinária, Centro Universitário de Barra Mansa, 27330-550 Barra Mansa-RJ, Brazil

^IIIDepartamento de Ciências Fisiológicas, Instituto de Ciências Biológicas, Universidade Federal de Goiás,74001-131 Goiânia-GO, Brazil

^IVDepartamento de Química, Centro de Ciências Exatas e Naturais, Universidade Federal do Pará, 66075-110, Belém-PA, Brazil

ABSTRACT

Croton pullei var. glabrior Lanj. (Euphorbiaceae) is a liana, vastly distributed in the Amazonian Forest. In the folk medicine, several plants of the Croton genus have been used with therapeutic purposes in pathologies that involve painful and inflammatory diseases which justify this work. The aim of this study was to investigate the antinociceptive and antiinflammatory activities of the C. pullei leaves methanol extract (MECP). MECP reduced in a dose-dependent manner the number of acetic acid-induced abdominal writhing (1.2%) in mice, suggesting an antinociceptive activity of the plant. On the other hand, MECP did not significantly modify the reactivity to the thermal stimulation in the hot-plate test and the reactivity to the chemical stimulation in the formalin test first phase, indicating a non-opioid mechanism. MECP reduced the formalin-induced nociception in the second phase, inhibited the croton oil-induced ear edema and reduced the leukocytes migration in the test of the carrageenan-induced peritonitis, indicating an antiinflammatory activity. Although the mechanisms that underlie these plant effects are not completely elucidated, these results appear to support the potential medicinal use of Croton pullei var. glabrior Lanj. against painful and inflammatory diseases.

Keywords: Antiinflammatory, antinociceptive, Croton pullei var. glabrior, phytomedicine.

RESUMO

Croton pullei var. glabrior Lanj. (Euphorbiaceae) é uma liana, amplamente distribuída na Floresta Amazônica. Na medicina popular, diversas plantas do gênero Croton têm sido utilizadas com fins terapêuticos em patologias que envolvem dor e inflamação, o que justifica este trabalho. O objetivo deste estudo foi investigar as atividades antinociceptiva e antiinflamatória do extrato metanólico das folhas de C. pullei (MECP). O MECP reduziu, de forma dose-dependente, o número de contorções abdominais (1,2 %) em camundongos, sugerindo uma atividade antinociceptiva da planta. Por outro lado, o MECP não alterou significativamente a reatividade ao estímulo térmico no teste da placa quente e a reatividade à estimulação química na primeira fase do teste da formalina, indicando um mecanismo não-opioidérgico. O MECP reduziu a nocicepção na segunda fase do teste da formalina, inibiu o edema de orelha induzido pelo óleo de croton e reduziu a migração leucocitária no teste da peritonite induzida por carragenina, indicando uma atividade antiinflamatória. Apesar dos mecanismos responsáveis pelos efeitos da planta ainda não estarem completamente esclarecidos, estes resultados parecem justificar o uso medicinal potencial de Croton pullei var. glabrior Lanj. em patologias que envolvam dor e inflamação.

Unitermos: Antiinflamatório, antinociceptivo, Croton pullei var. glabrior, fitomedicina.

INTRODUCTION

Pain is considered one of the most common complaints worldwide for which patients seek treatment. Along the history different treatments have been used to lessen pain. Nowadays, several antiinflammatory agents are used to treat different types of pain associated or not to the inflammatory process. These agents are efficient in most cases, but the collateral effects are common, especially when they are used in chronic treatments. The most common collateral effect is gastrointestinal disturb (Peura & Goldkind, 2005). An approach to solve this problem was the use of selective COX-2 inhibitors with a low risk of gastrointestinal bleeding (Bombardier et al., 2000; Silverstein et al., 2000). Although these drugs are well tolerated, their cardiovascular safety has been questioned a few years ago (Mukherjee et al., 2001), and recently, rofecoxib was withdrawn from the market because of an increased risk of cardiovascular disease, mainly myocardial infarction and stroke (Singh, 2004).

Considering this situation, new drugs with different spectre of action or less adverse side effects have been searched. The research of medicinal plants can propitiate the discovery of new molecules with innovative mechanisms or less adverse side effects (McCurdy & Scully, 2005). Euphorbiaceae family is well known between the medicinal plants with more than 8,000 species vastly distributed in tropical and temperate regions of the world (Wilson et al., 1979; Agra et al., 2007). In this context, a genus with several species used in the folk medicine to treat different disorders is the genus Croton. Some of Croton species have been exhaustively studied with their medicinal and toxicological properties being scientifically proven (Souza et al., 2006; Costa et al., 2007; Perazzo et al., 2007; Salatino et al., 2007; Torrico et al., 2007). Other species of this genus were poorly studied, and their pharmacological properties and collateral effects are still unknown.

Croton pullei (Euphorbiaceae) is a liana that grows above other trees, distributed in tropical areas with vast distribution in the Amazon forest (Gallenmüller et al., 2001). Until this moment we have not found any report about the use of this species in the folk medicine. On the other hand, this plant belongs to a genus that is used commonly with medicinal purposes which justify this study. The aim of this work was to evaluate the effects of the glabrior Lanj. variety of Croton pullei in models of nociception and inflammation.

MATERIAL AND METHODS

Crude methanol extract

Croton pullei var. glabrior Lanj. leaves were collected in the city of Peixe-boi, State of Pará, (Amazon region - Brazil), in December 1997. Samples were authenticated by Dr. Ricardo de Souza Secco, a botanical from Emílio Goeldi Paranse Museum (MPEG), and voucher specimens were deposited at the herbarium of MPEG under MG-0151738 number. The methanol extract was obtained by percolation and then concentrated dry under reduced pressure, below 40 ºC, to yield the methanol crude extract (MECP) (6.1%).

Drugs and reagents

Formalin, acetic acid, acetone (Merck AG, Darmstadt, Germany), croton oil, indomethacin, carrageenan (Sigma Chemical Co., St. Louis, MO, USA), fentanyl (Janssen Pharmaceutical, Belgium), dexamethasone (Prodome, Brazil). MECP and indomethacin were diluted in tap water, whereas fentanyl and dexamethasone were diluted in saline.

Animals

Male adult Swiss mice weighting 25-30 g were used in all experiments. Animals were maintained on a 12-h light -dark cycle (lights on at 7:00 a.m.) at constant room temperature (23 ± 2 ºC) with free access to food and water, except during the experiments. Control animals received tap water as vehicle (10 mL/kg, p.o.) and each animal was used just once. All experiments were carried out in accordance with current guidelines for the care of laboratory animals and ethical guidelines on the use of animals in pain research (Zimmermann, 1986). The experimental protocols were approved by the local Animal Care and Use Committee (005/2006/CEPEB/UFRRJ).

Acetic acid-induced writhing test

Groups from 6 to 9 mice were treated orally (p.o.) with vehicle, MECP (0.1 to 1.0 g/kg) or indomethacin (10 mg/kg) 60 min before intraperitoneal (i.p.) acetic acid injection (1.2%, 0.1 mL/10 g) and the number of writhes was counted for the following 30 min (Koster et al., 1959).

Hot-plate test

The latency (s) of heat stimulus (55 ± 0.5 ºC) was measured every 30 min, starting 30 min before and up to 2 h after pre-treatment of 8 groups of mice with MECP (1.0 g/kg, p.o.), water or subcutaneous (s.c.) application of fentanyl (200 µg/kg) (D'Amour & Smith, 1941).

Formalin-induced nociception

Groups from 7 to 9 mice were orally treated with vehicle, MECP (1 g/kg) or indomethacin (10 mg/kg) 60 min prior to injection of the formalin solution (1.2 %; 20 µL/paw) into the plantar surface of the hind paw (i.p. injection). Another group was treated with fentanyl (200 µg/kg, s.c.) 15 min before these formalin nociceptive stimuli. The time that animals spent licking the injected paw was measured with a chronometer and was considered as an index of nociception. After the formalin injection, the initial nociceptive response peaked at about 5 min, denominated early phase, which was followed by a second peak (late phase) that occurred at 15-30 min post injection (Hunskaar et al., 1985).

Edema induced by croton oil in mouse ear

One hour after oral administration (n = 7) of water, MECP (1.0 g/kg) or indomethacin (10 mg/kg), each animal was treated with 20 µL of freshly prepared croton oil (2.5% in acetone) on the inner surface of the right ear. The left ear was treated with the same volume of acetone (control). Four hours after treatments, mice were killed by cervical dislocation and a plug (6 mm in diameter) was taken from both the treated and untreated ears with a punch. We monitored the inflammatory response (edema) by weighing (mg) both plugs and testing the differences (Zanini Junior et al., 1992).

Carrageenan-induced peritonitis

Groups of 7 animals were treated with vehicle, dexamethasone (1 mg/kg, s.c.) or MECP (0.1-1.0 g/kg, p.o.), followed by 1% carrageenan (0.25 mL, i.p.) 1 h later. Four hours after the intraperitoneal injection, mice were killed and 2 mL of modified PBS (heparin, 10 IU/mL) were injected into the peritoneal cavity. Total cell counts were performed using a Neubaüer chamber as described by Ferrándiz and Alcaraz (1991). The results were expressed as means ± SEM of the numbers of total leukocytes (x10⁷) per mL of peritoneal wash or percentage of inhibition of leukocyte migration compared to control group.

Statistical analysis

Data were statistically analysed by one-way ANOVA followed by the Dunnet multicomparison test. In the hot-plate test, the two-way ANOVA followed by the Bonferroni's test analysis was used. The values are reported as mean ± standard error of the mean (SEM). P values less than 0.05 (p<0.05) were considered to be significant.

RESULTS

Acetic acid-induced writhing test

Figure 1 shows that MECP (0.3 and 1.0 g/kg, p.o.) produced a significant (p<0.05) dose-related inhibition of acetic acid-induced abdominal writhing by 33.6 and 65.5%, respectively, when compared to the control group. The major dose tested produced inhibition of abdominal constriction similar to positive control indomethacin (10 mg/kg), which produced an inhibition by 65.1%.

Hot-plate test

The MECP was inactive in the hot-plate test at the highest dose effective in acid acetic abdominal writhing test (1.0 g/kg, p.o.). Fentanyl (200 µg/kg s.c.), used as a reference drug, produced a significant antinociceptive effect at 30 min after administration of this drug when compared to control values (Figure 2).

Formalin-induced nociception

In the first phase of formalin-induced nociception, the orally pre-treatment with MECP (1.0 g/kg) did not modify significantly the licking time of the animals when compared to control group. However, in the second phase a significant reduction (71.5%) in the licking time could be observed after MECP treatment, with reduction comparable to that of indomethacin (10 mg/kg), which produced a reduction by 68.5% as showed in Table 1.

Croton oil-induced ear edema test

Table 2 shows that MECP (1.0 g/kg, p.o.) caused an inhibition of 72.4% in croton oil-induced ear edema (p<0.05 vs control). Indomethacin (10 mg/kg), used as a reference drug, inhibited the ear edema by 58.1% when compared to control result.

Carrageenan-induced peritonits

The results listed in Table 3 show that orally administration of MECP (0.1, 0.3 and 1.0 g/kg) produced a dose-related reduction of the number of leukocytes that migrated to the peritoneal cavity, with inhibition by 33.1, 60.1 and 76.7%, respectively. The highest dose of MECP inhibited leukocytes migration comparable to that induced by dexamethasone (1 mg/kg, s.c.) which produced 81.2 % of inhibition.

DISCUSSION

The antinociceptive effect of the MECP was tested in three models of nociception: acetic acid-induced abdominal writhing, hot-plate test and formalin-induced nociception. The treatment with MECP promoted dose-related antinociceptive effect in the acetic acid-induced abdominal writhing model. Although this model could be commonly used as a screening method to identify drugs with antinociceptive activity potential, several groups of drugs with different mechanisms of action can inhibit the abdominal writhing (Koster et al., 1959; Hendershot & Forsaith, 1959). Moreover, the involvement of different mediators such as prostaglandins (Deraedt et al., 1980), neurokinin A (Julia & Buéno, 1997) and CGRP (Friese et al., 1997), for example, was described in this experiment which means that is not possible to suggest any mechanism to the antinociceptive effect of MECP, based only on this model. In the hot-plate test, MECP did not induce any antinociceptive effect. Since supraspinal and spinal opioid receptors play an important role in this assay (Schmauss & Yaksh, 1984) it is possible that MECP does not act on central opioid receptors or produce release of endogenous opioid peptides. This conclusion is reinforced by the fact that MECP did not reduce the licking time in the first phase (neurogenic pain) of formalin test, which is highly sensitive to opioid agents (Hunskaar & Hole, 1987). MECP reduced the licking time in formalin test second phase (inflammatory pain). This phase of the test is sensitive to non-steroid antiinflammatory drugs like indomethacin, used as reference drug (Hunskaar & Hole, 1987). This result suggests that the antinociceptive effect of MECP could be related to antiinflammatory mechanisms.

The antiinflammatory hypothesis was tested in two models that evaluate different aspects of inflammatory process, the edema and the leukocyte migration. MECP reduced significantly the croton oil-induced ear edema by 72%, and also reduced in a dose-related manner the total leukocyte migration in the peritoneum after carrageenan stimulus. Production of exudates in these models is related to local release of vasoactive substances (histamine and kinins) and synthesis of prostaglandins (Morrow & Roberts II, 2001). Although migration of leukocytes could not be directly related to cyclooxygenase products, the process could be inhibited by some non-steroidal antiinflammatory compounds, indicating that many mechanisms may be implicated in its control (Mikami & Miyasaka, 1983). Steroidal antiinflammatory effects on the leukocyte migration may also be multifactorial since it could be related to the inhibition of phospholipase A2 (Morrow & Roberts II, 2001) as well as other mechanisms, for example, inhibition of TNFα and IL-1β production which are potent triggers of many of the actions involved in leukocyte migration (Pereira et al., 2006). Since several mechanisms may be involved in the leukocytes migration more studies must be done to elucidate the mechanism of the antiinflammatory effect of MECP.

Previous work with the trunk wood and bark of Croton pullei var. glabrior Lanj. identified steroids such as sitosterol ester and stigmasterol, the triterpene lupeol and the diterpene kaurenoic acid (Abreu et al., 2001). The antiinflammatory and antinociceptive effects of stigmasterol (Santos et al., 1995; Garcia et al. 1999), beta-sitosterol and beta-sitosteryl-beta-D-glucoside (Villaseñor et al., 2002), kaurenoic acid (Block et al., 1998; Paiva et al., 2003) and lupeol, (Fernández et al., 2001a,b; Nikiéma et al., 2001) isolated from different vegetal species have been demonstrated in pharmacological models and may contribute to the effect of MECP.

Although beta-sitosterol, stigmasterol, kaurenoic acid and lupeol are potential compounds to justify the antinociceptive and anti-inflammatory actions of the MECP, these compounds were isolated from the trunk wood and bark of Croton pullei var. glabrior Lanj. (Abreu et al., 2001) and not from the leaves, material used in the present study. A bioassay-guided fractioning of this extract is now in progress to identify the bioactive substance(s) in the methanol extract of C. pullei as well as the mechanisms of action involved in the effects described before.

ACKNOWLEDGMENTS

The authors thank University Centre of Barra Mansa and Brazilian National Research Council (CNPq) for financial support.

References

• Abreu AS, Barbosa PS, Muller AH, Guilhon GMSP 2001. Constituintes químicos do caule e das cascas do caule de Croton pullei var. glabrior Lanj. (Euphorbiaceae). Revista Virtual de Iniciação Acadêmica da UFPA 1: 1- 9. http://www2.ufpa.br/rcientifica/ ed_anteriores/pdf/ed_02_asa.pdf, acessed october 2006.
• Agra MF, França PF, Barbosa-Filho JM 2007. Synopsis of the plants known as medicinal and poisonous in Northeast of Brazil. Rev Bras Farmacogn 17: 114-140.
• Block LC, Santos ARS, Souza MM, Scheidt C, Yunes RA, Santos MA, Delle Monache F, Cechinel Filho V 1998. Chemical and pharmacological examination of antinociceptive constituents of Wedelia paludosa. J Ethnopharmacol 61: 85-89.
• Bombardier C, Laine L, Reicin A, Shapiro D, Burgos-Vargas R, Davis B, Day R, Ferraz MB, Hawkey CJ, Hochberg MC, Kvien TK, Schnitzer TJ 2000. Comparison of upper gastrointestinal toxicity of rofecoxib and naproxen in patients with rheumatoid arthritis. N Engl J Med 343: 1520-1528.
• Costa MP, Magalhães NSS, Gomes FES, MAM 2007. Uma revisão das atividades biológicas da trans-desidrocrotonina, um produto natural obtido de Croton cajucara. Rev Bras Farmacogn 17: 275-286.
• D'Amour FE, Smith DL 1941. A method for determining loss of pain sensation. J Pharmacol Exp Ther 72: 74-79.
• Deraedt R, Jouquey S, Delevallee F, Flahaut M 1980. Release of prostaglandins E and F in an algogenic reaction and its inhibition. Eur J Pharmacol 61: 17-24.
• Fernández A, Álvarez A, García MD, Saénz MT 2001a. Anti-inflammatory effect of Pimenta racemosa var. ozua and isolation of the triterpene lupeol. Il Farmaco 56: 335-338.
• Fernández A, de las Heras B, García MD, Sáenz MT, Villar A 2001b. New insights into the mechanism of action of the anti-inflammatory triterpene lupeol. J Pharm Pharmacol 53: 1533-1539.
• Ferrándiz ML, Alcaraz MJ 1991. Anti-inflammatory activity and inhibition of arachidonic acid metabolism by flavonoids. Agents Actions 32: 283-288.
• Friese N, Diop L, Chevalier E, Angel F, Riviere PJ, Dahl SG 1997. Involvement of prostaglandins and CGRP-dependent sensory afferents in peritoneal irritation-induced visceral pain. Regul Peptides 70: 1-7.
• Gallenmüller F, Müller U, Rowe N, Speck T 2001. The growth form of Croton pullei (Euphorbiaceae) - Functional morphology and biomechanics of a neotropical Liana. Plant Biology 1: 50-61.
• Garcia MD, Saenz MT, Gomez MA, Fernández MA 1999. Topical anti-inflammatory activity of phytosterols isolated from Eryngium foetidum on chronic and acute inflammation models. Phytother Res 13: 78-80.
• Hendershot LC, Forsaith J 1959. Antagonism of the frequency of phenyquinone-induced writhing in the mouse by weak analgesic and nonanalgesics. J Pharmacol Exp Ther 125: 237-240.
• Hunskaar S, Fasmer OB, Hole K 1985. Formalin test in mice, a useful technique for evaluating mild analgesia. J Neurosci Meth 14: 69-76.
• Hunskaar S, Hole K 1987. The formalin test in mice: dissociation between inflammatory and non-inflammatory pain. Pain 30: 103-114.
• Julia V, Buéno L 1997. Tachykininergic mediation of viscerosensitive responses to acute inflammation in rats: evidence for a CGRP-induced release of tachykinins. Am J Physiol 272: 141-146.
• Koster R, Anderson M, De Beer EJ 1959. Acetic acid for analgesic screening. Fed Proc 18: 412-412.
• McCurdy CR, Scully SS 2005. Analgesic substances derived from natural products (natureceuticals). Life Sci 78: 476-484.
• Morrow JD, Roberts II LJ 2001. Lipid-derived autacoids: Eicosanoids and Platelet-Activating Factor. In: Harman, J.G.; Limbird, L.E. (eds) Goodman & Gilman's, The Pharmacological Basis of Therapeutics 10th edn. New York: McGraw Hill, p. 669-683.
• Mikami T, Miyasaka K 1983. Effects of several anti-inflammatory drugs on the various parameters involved in the inflammatory response in rat carrageenin-induced pleurisy. Eur J Pharmacol 95: 1-12.
• Mukherjee D, Nissen SE, Topol EJ 2001. Risk of cardiovascular events associated with selective COX-2 inhibitors. JAMA 286: 954-959.
• Nikiéma JB, Vanhaelen-Fastré R, Vanhaelen M, Fontaine J, De Graef C, Heenen M 2001. Effects of antiinflammatory triterpenes isolated from Leptadenia hastata latex on keratinocyte proliferation. Phytother Res 15: 131-134.
• Paiva LAF, Gurgela LA, Silva RM, Tomé AR, Gramosa NV, Silveira ER, Santos FA, Rao VSN 2003. Anti-inflammatory effect of kaurenoic acid, a diterpene from Copaifera langsdorffii on acetic acid-induced colitis in rats. Vasc Pharmacol 39: 303-307.
• Perazzo FF, Carvalho JCT, Rodrigues M, Morais EKL, Maciel MAM 2007. Comparative anti-inflammatory and antinociceptive effects of terpenoids and an aqueous extract obtained from Croton cajucara Benth. Rev Bras Farmacogn 17: 521-528.
• Pereira R, Medeiros YS, Fröde TS 2006. Antiinflammatory effects of Tacrolimus in a mouse model of pleurisy. Transplant Immunol 16: 105-111.
• Peura DA, Goldkind L 2005. Balancing the gastrointestinal benefits and risks of nonselective NSAIDs. Arthritis Res Ther 7 (Suppl 4): S7-13.
• Salatino A, Salatino MLF, Negri G 2007. Traditional uses, chemistry and pharmacology of Croton species (Euphorbiaceae). J Braz Chem Soc 18: 11-33.
• Santos ARS, Niero R, Cechinel Filho V, Yunes RA, Pizzolatti MG, Delle Monache F, Calixto JB 1995. Antinociceptive properties of steroids isolated from Phyllanthus corcovadensis in mice. Planta Med 61: 329-332.
• Schmauss C, Yaksh TL 1984. In vivo studies on spinal opiate receptor systems mediating antinociception: II. Pharmacological profiles suggesting a differential association of mu, delta and kappa receptors with visceral chemical and cutaneous thermal stimuli in the rat. J Pharmacol Exp Ther 228: 1-12.
• Silverstein FE, Faich G, Goldstein JL, Simon LS, Pincus T, Whelton A, Makuch R, Eisen G, Agrawal NM, Stenson WF, Burr AM, Zhao WW, Kent JD, Lefkowith JB, Verburg KM, Geis GS 2000. Gastrointestinal toxicity with celecoxib vs nonsteroidal anti-inflammatory drugs for osteoarthritis and rheumatoid arthritis: the class study: a randomized controlled trial. Celecoxib Long-term Arthritis Safety Study. JAMA 284: 1247-1255.
• Singh D 2004. Merck withdraws arthritis drug worldwide. BMJ 329: 816.
• Souza MAA, Souza SR, Veiga Jr VF, Cortez JKPC, Leal RS, Dantas TNC, Maciel MAM 2006. Composição química do óleo fixo de Croton cajucara e determinação das suas propriedades fungicidas. Rev Bras Farmacogn 16(Supl.): 599-610.
• Torrico F, Cepeda M, Guerrero G, Melendez F, Blanco Z, Canelón DJ, Diaz B, Compagnone RS, Suárez AI 2007. Hypoglycaemic effect of Croton cuneatus in streptozotocin-induced diabetic rats. Rev Bras Farmacogn 17: 166-169.
• Villaseñor IM, Angelada J, Canlas AP, Echegoyen D 2002. Bioactivity studies on beta-sitosterol and its glucoside. Phytother Res 16: 417-421.
• Wilson SR, Neubert LA, Huffman JC 1979. The chemistry of the Euphorbiaceae. A new diterpene from Croton californicus. J Am Chem Soc 98: 3669-3674.
• Zanini Junior JC, Medeiros YS, Cruz AB, Yunes RA, Calixto JB 1992. Actions of compounds from Manderilla velutina on croton oil-induced ear edema in mice. A comparative study with steroidal and nonsteroidal antiinflammatory drugs. Phytother Res 6: 1-5.
• Zimmermann M 1986. Ethical considerations in relation to pain in animal experimentation. Acta Physiol Scand Suppl 554: 221-233.

Publication Dates

• Publication in this collection
  29 Oct 2008
• Date of issue
  Sept 2008