Evaluation of the effects of the herbal product Catuama [R] in inflammatory and neuropathic models of nociception in rats.

Introduction

The herbal product Catuama[R] consists of the association of four hydroalcoholic extracts obtained from well-known medicinal plants: Trichila catigua (Meliaceae), Paullinia cupana (Sapindaceae), Ptychope-talum olacoides (Olacaceae) and Zingiber officinalis (Zingiberaceae). This product has been marketed in Brazil for more than 20 years for the management of chronic conditions such as physical and mental stress, neuromuscular asthenia and fatigue.

Previous studies demonstrated that the herbal product Catuama[R] displays marked relaxant actions in different vascular preparations obtained from rats, guinea pigs and rabbits, via mechanisms involving the release of nitric oxide or nitric oxide-related substances from the endothelium (Cabrini and Calixto, 1997). Moreover, Catuama[R] has been shown to present long-lasting antinociceptive actions when administered orally inseveral chemical (acetic acid, formalin and capsaicin) and thermal (tail-flick and hot -plate) models of nociception in mice, by an interaction with the nitric oxide pathway and the opioid system (Vaz et al., 1997). Additional evidence has shown that Catuama* causes significant relaxant effects in corpus cavernosum strips obtained from rabbits (Antunes et al., 2001). More recently, Campos et al. (2004) have provided convincing evidence indicating an antidepressant-like effect for Catuama, suggesting primarily the involvement of dopaminergic pathways and secondarily serotoninergic pathways in these effects. Clinical studies (phase 1) carried out in healthy volunteers have revealed that Catuama out in healthy volunteets have recvealed that Catuama is safe and well-tolerated, as no severe adverse reactions or hematological and biochemical changes have been observed (Oliveira et al., 2005).

In the present study, we have further assessed the effects of the herbal product Catuama[R] in several models of inflammatory and neuropathic nociception in rats. Attempts have also been made to analyze some of the possible mechanisms through which Catuama exerts its actions.

Materials and methods

Animals

Male and female Wistar rats (120-350 g each; n = 4-6 per group) used were housed under conditions of optimum light, temperature and humidity (12/12h light-dark cycle; 22[+ or -]1 [degrees]C temperature, 60-80% humidity), with food and water provided ad libitum. The Ethics Committee of the Federal University of Santa Catarina approved all the experimental procedures (protocol number: 263/CEUA and 23080.035336/05/2003-16/ UFSC).

The fingerprint of Catuama[R] established by gas chromatography-mass spectrometry (GC-MS)

A Hewlett-Packard 6890 gas chromatograph, coupled with a Hewlett-Packard 5973N mass spectrometer was used for the establishment of the fingerprint of Catuama[R]. The separation was performed on HP-5MS column, 0.25mm i.d. x30m, 0.25um coating thickness. The temperature of the column was programmed from 150 to 258 [degrees]C at 6 [degrees]C/min, 258 to 278 [degrees]C at 10[degrees]C/min and 278 to 300 [degrees]C at 40[degrees]C/min. The injector temperature and the detector temperature were 300 [degrees]C. Helium was used as carrier gas with a constant flow rate of 0.8ml/min. The mass spectrometer was operated at 70 eV, scan range 20-450 amu. Some of the compounds were identified from the recorded mass spectra by comparison with the mass spectra from the NIST and Wiley libraries.

In vivo pharmacological studies

Hindpaw withdrawal response induced by von Frey hair

To assess mechanical allodynia, rats were placed individually in clear Plexiglas boxes (22 x 14 x 16cm) on elevated wire mesh platforms, to allow access to the ventral surface of the right hindpaw. The withdrawal response frequency was measured following 10 applications (duration of 1 s each) of 2.0 g von Frey hair (VFH, Stoelting, Chicago, USA) according to a method described previously (Rodrigues-Filho et al., 2003).

Assessment of mechanical hyperalgesia 

The mechanical hyperalgesia was evaluated with an analgesymeter (Ugo Basile, Italy), based on the method described by Randall and Sellitto (1957), which applies a linearly increasing mechanical force to the dorsum of the hindpaw. The nociceptive threshold was defined as the force (g) at which the rats withdrew their paws.

Assessment of thermal hyperalgesia

The rats were placed individually in a clear plastic container (22 x 14 x 16 cm) on an elevated floor of clear, heat-tempered glass (Plantar Test, Ugo Basile, Italy). After 15min of habituation, a radiant heat source (50 W halogen reflector bulb with intensity controlled by a constant voltage source) was focused on the plantar surface of the hindpaw. The cut-off time in the absence of a response was 40 s to avoid tissue damage (Hargreaves et al., 1988; Rodrigues-Filho et al., 2003).

Mechanical allodynia induced by CFA or LPS injection

These experiments were performed as described previously (Safieh-Garabedian et al., 1997; Nagakura et al., 2003). The rats received a 100 [micro]l intraplantar (i.pl.) injection of CFA (1 mg/ml heat-killed and dried Mycobacterium tuberculosis, each milliliter of vehicle containing 0.85ml paraffin oil plus 0.15ml mannide monooleate) in the surface of the right hindpaw. The mechanical allodynia and the thermal hyperalgesia were assessed 24 h later. The animals were treated orally with Catuama[R] or with saline 0.9% and their responses were evaluated at different time points (2-24h). To evaluate the effects of chronic treatment, Catuama[R] or saline 0.9% was administered orally once a day (24x24h) for 6 days. The treatment was interrupted on the 7th day. The evaluation of the allodynic response was performed 6 h following the daily administration.

In other groups, the animals were treated with Catuama[R] or saline solution 0.9% and after lh, they received a 100 -[micro]l i.pl. injection of saline containing LPS (0111:B4) serotype; Escherichia coli; 1.25 [micro]g/paw) into the right hindpaw. The mechanical allodynia was assessed as described above, in the ipsilateral andcontralateral hindpaws, at different intervals of time after LPS injection (3-9 h).

Partial sciatic nerve ligation (PSNL)

The animals were anesthetized using 7% chloral hydrate (0.6 ml/kg; i.p.) and the PSNL was performed by tying 1/3 to 1/2 of the dorsal portion of the sciatic nerve, using the procedure described previously (Seltzer et al., 1990). In the sham-operated control group, the sciatic nerve was exposed without ligation. Following 5 days, the operated animals were treated with Catuama[R] or saline. Some groups of animals were chronically treated with Catuama[R] or saline for 3 days. The mechanical allodynia was evaluated at different intervals of time.

Streptozotocin-induced diabetic polyneuropathy 

Diabetes was induced as described by Campos et al. (2001). The animals received a single injection of streptozotocin (75mg/kg body wt., i.p.) (Sigma, EUA) diluted in 0.05 M citrate buffer ([.sub.p]H 4.5). Age-matched control rats received citrate buffer vehicle alone. The onset of diabetes was confirmed by assessing the glucose levels in blood samples collected from the tail vein, 4 weeks later. Only animals with blood glucose levels [greater than or equal to] mg/dl (about 70%) were used in the experiments. Control and diabetic rats received Catuama[R] or saline 0.9%. In another series of experiments, to verify the effects of repeated treatment, Catuama[R] or saline 0.9% was administered for 2 days and the evaluation of mechanical hyperalgesia was performed 4h after each treatment.

Mechanisms involved in the antinociceptive effects of Catuama[R]

To assess some of the mechanisms responsible for the antinociceptive effects of Catuama[R] on the mechanical allodynia induced by LPS, separate groups of animals were pre-treated with one of the following drugs (i.p.): haloperidol (dopamine [D.sub.2]/[D sub.3]/[D.sub.4] receptor antagonist, methysergide (serotonin [5HT sub.1]/[5HT sub.2] receptor antagonist) or yohimbine ([[alpha].sub.2])-adrenergic receptor antagonist). Control groups received saline 0.9%. All the drugs were given 1 h before the administration of Catuama[R] or saline 0.9%. Following 1 h of treatment with Catuama[R] or saline, the animals received an i.pl. injection of LPS into the right hindpaw and the mechanical allodynia was assessed 3 h after.

Biochemical assays

Effects of Catuama on IL-1[beta] and TNF[alpha] production.

IL-1[beta] (Interleukin 1[beta]) and [TNF.sub.[alpha]] (tumor necrosis factor [alpha]) levels were measured as described by Passos et al. (2004). Animals were pre-treated with Catuama[R] 1 h before i.pl. LPS injection. Control animals received saline. The animals were sacrificed 1 and 3h later to assess the TNF[alpha] IL-1[beta] production, respectively. The hindpaw subcutaneous tissues were placed in PBS containing Tween-20 0.005%, phenylemthylsulphohnyl fluoride (PMSF) 2.1 mM, benzethonium chloride 0.1 mM, EDTA 10 mM and aprotinin A 20 KIU, homogenized, centrifuged at 3000g for 10 min and stored at -70[degrees]C until further analysis. Cytokine levels were evaluated using an ELISA DuoSet kit according to the manufacturer's recommendations (R&D Systems).

Effects of Catuama[R] on [PGE.sub.2] and [LTB.sub.4] production

The animals were pre-treated with Catuama[R] or saline solution 0.9%. After 1 h, the rats received LPS (1.25 [micro]g/paw) or saline (0.1 ml/paw) and were later sacrificed after 4h. The injected paw was perfused according to the methodology described by Ferreira et al. (2004). The dialysate was used for the quantification of [PGE.sub.2] (prostaglandin [E.sub.2]) and [LTB.sub.4] (leukotriene [B.sub.4]) levels by means of an ELISA kit (R&D Systems) according to the manufacturer's instructions.

Drugs and reagents

The following drugs and reagents were used: aprotinin, chloral hydrate (Vetec; Brazil); Catuama[R] (Laboratorio Catarinense, Joinville, SC, Brazil); bovine serum albumin (BSA), complete Freund's adjuvant (CFA), ethylene diaminetetraacetic acid (EDTA), haloperidol, LPS, methysergide, phenylmethylsulfonyl fluoride (PMSF), streptozotocin, tetramethylbenzide (TMB), yohimbine (all from Sigma Chemical Company, St Louis, MO, USA); KCl, [KH.sub.2]PO4, NaCl, [Na.sub.2][HPO.sub.4], Tween 20 (all from Merck, Germany).

Statistical analysis

Results are presented as the mean [+ or -] S.E.M. of 4-6 animals per experimental group. The percentages of inhibition are reported as the mean [+ or -] S.E.M. of inhibitions obtained for each individual experiment. Statistical comparison of the data was performed by two-way analysis of variance (ANOVA) followed by Bonferroni's post-test or one-way ANOVA followed by Newman-Keuls' test. p-Values less than 0.05 (p<0.05) were considered significant.

Results

The fingerprint of Catuama[R] was obtained by GC-MS (Fig. 1). From the chromatogram of the mixture, three main component peaks were detected: caffeine (retention time = 10.843 min), [beta]-Sitosterol (retention time-29.836 min) and Lupeol (retention time = 30.983 min).

The product Catuama[R]1 was administered orally (50-200 mg/kg body wt.), and the mechanical allodynia induced by CFA was evaluated at different time points. As shown in Fig. 2A, all tested doses of Catuma [R] significantly inhibited the mechanical allodynia induced by CFA, although only the dose of 200 mg/kg body wt. was effective in reducing the nociceptive response for up to 24 h. The inhibitions observed at the 2-h time point were 46[+ or -]6%, 42[+ or -]6% and 66 [+ or -] 9% for the doses of 50, 100 and 200 mg/kg body wt., respectively. Catumam [R] (200mg/kg body wt., p.o.) did not significantly affect the thermal hyperalgesia between the [1.sup.th] and [5.sup.th] day after CFA injection into the ipsilateral hindpaw, although a slight inhibition was observed on the 6th day (Fig. 2B). The prolonged treatment with Catuama [R] (200 mg/kg body wt., p.o., every 24h) for 6 days, significantly inhibited the mechanical allodynia induced by CFA in the ipsilateral hindpaw, during the entire period of treatment (65[+ or -]11%) (Fig.2c). As observed in Fig. 2D, repeated treatment with Catuama[R] also significantly diminished the mechanical allodynia induced by CFA in the contralateral hindpaw (inhibition of 47[+ or -]10%).

The oral administration of Catuama[R] (200 mg/kg body wt.) 1 h before strikingly reduced the mechanical allodynia by LPS in both ipsilateral (Fig. 3A) and contralateral (Fig. 3B) hind paws, with inhibitions of 81 [+ or -] 8% and 81[+ or -]7%, respectively. Conversely, the acute oral administration of Catuama[R] (200 mg/kg body wt.) was not able to significantly alter the mechanical allodynia, 4 days after the PSLN injury (results not shown). Moreover, the repeated administration of Catuama[R] (200 mg/kg body wt., p.o) for 3 days also failed to alter the mechanical allodynia induced by PSNL (results not shown). Furthermore, the acute and repeated oral administration of Catuama[R] (200 mg/kg body wt.) both failed to significantly modify the hyperalgaesic responses observed in streptozotocin-diabetic rats (results not shown).

An additional series of experiments was designed to assess some of the mechanisms involved in the effects of Catuama in the LPS model. Fig. 4 indicates that antinociceptive actions of Catuama[R] (200mg/kg body wt., p.o., 1 h) were completely reverted by previous treatment with the dopaminergic antagonist haloperidol (1mg/kg body wt., i.p., 1 h). On the other hand, neither the serotonergic antagonist methysergide nor the [[alpha].sub.2]- adrenergic antagonist yohimbine significantly interfered with the antinociceptibve effects of Catuama [R] (Fig. 4).

Finally, the i.pl. injection of LPS into the rat paw produces a marked increase in the levels of several inflammatory mediators, such as IL-1[beta], [TNF-.sub.[Alpha]] [PGE.sub.2] and [LTB.sub.4] Oral treatment with Catuama[R] (200 mg/kg body wt., p.o.) 1 h before the injection of LPS failed to significantly affect the production of these inflammatory mediators (results not shown).

[FIGURE 1 OMITTED]

[FIGURE 2 OMITTED]

Discussion

Analgaesic therapeutic options have been found to be only partially effective, and their use is commonly associated with severe side-effects that hamper their continuous use (Mendell and Sahenk, 2003). The interest in identifying new alternatives to treat inflammatory and neuropathic persistent pain has increased significantly in the last decade (Calixto, 2000; Calixto et al., 2000, 2001, 2003, 2004). Naturally occurring products seem to represent good choices for this purpose. The present study evaluated the effects of the herbal product Catuama[R] in inflammatory and persistent neuropathic models of noviception in rays. We first analyzed the profile of action of Catuama[R] in the inflammatory nociception induced by CFA. CFA produces long-term inflammatory and noviceptive responses (Chan et al., 2000), which are probably associated with significant changes in neurons of the central nervous system, culminating in the sensitization of the contralateral paw (Sluka, 2002). Furthermore, CFA effects have been also associated to a significant increase of [TNF.sub.[alpha]] IL-1[beta] and nerve growth factor (NGF) production in the inflamed paw, which might contribute to the initialization of the inflammatory hyperalgesia (Woolf et al., 1997). The present results extend those of our previous findings and show a consistent antinociceptive effect for the herbal product Catuama[R] in the CFA model, when administered orally, in both acute and repeated schedules of treatment. The actions of Catuama[R] in this model might be related to the interference in the mechanisms involved in central sensitization. This conclusion is based on the findings showing that Catuama[R] reduced the allodynic responses evoked by CFA in both ipsilateral and contralateral hindpaws. Importantly, the effects of the acute treatment with Catuama[R] had a long-lasting profile of duration (up to 24h), whereas the repeated treatment with Catuama[R] practically abolished the mechanical allodynia induced by CFA. In contrast, Catuama [R], tested under the same conditions, was not capable of affecting the thermal hyperalgesia in the CFA model. These data allow us to suggest that the herbal product Catuama[R] reduces mechanical nociception without interfering with thermal sensitization.

[FIGURE 3 OMITTED]

[FIGURE 4 OMITTED]

The oral treatment with Catuama[R] also produced a marked and long-term antinociceptive effect (up to 9h) in the mechanical allodynia caused by LPS in both

ipsilateral and contralateral hindpaws. LPS is a component of the outer membrane of Gram-negative bacteria that is widely recognized as a potent activator of the immune system, capable of inducing various inflammatory genes in different cellular types (Medzitov and Janeway, 2000). The i.pl. injection of LPS is able to increase the local production of numerous pro-inflammatory mediators (such as IL-1[beta], TNF[alpha], [PGE.sub.2]), known to be involved in the modulation of the peripheral nervous system (Safieh-Garabedian et al., 1997, 2002). These considerations prompted us to analyze whether or not the antinociceptive effects of the product Catuama[R] were associated with its ability to inhibit the synthesis and/or release of these mediators. Under the same conditions in which Catuama[R] caused a marked reduction of LPS-induced mechanical allodynia, it was not able to significantly affect the production of IL-I[beta], TNF[alpha], [PGE.sub.2] or [LTB.sub.4] induced by LPS. Therefore, interference with such inflammatory mediators seems unlikely to be responsible for Catuama[R] antinociceptive actions.

Neuropathic pain is a long-lasting syndrome involving complex and incompletely known mechanisms, originating and being modulated in both the peripheral and central nervous systems. It is greatly refractory to most of the available analgaesic drugs (Mendell and Sahenk, 2003). In the present work, the effects of Catuama[R] were evaluated in two models of persistent neuropathic pain: the partial ligation of the sciatic nerve and the streptozotocin-induced diabetic polineuropathy. Curiously, Catuama[R] did not produce any significant alteration of the nociceptive responses observed in these two neuropathic models. Whether or not the product Catuama[R] would be effective in other animal models of neuropathic pain remains to be investigated in future studies.

We also evaluated some of the mechanisms by which Catuama[R] produces its antinociceptive effects. This was achieved by investigating the relevance of certain neurotransmitters known to be involved in the genesis and maintenance of nociception. Our results suggest that anti-allodynic effects of Catuama[R] depend on the stimulation of receptors and/or transductional pathways related to the dopaminergic system, as the dopamine receptor antagonist haloperidol completely reversed the effects of Catuama[R] in the LPS model. On the other hand, neither the serotonin methysergide nor the adrenergic yohimbine receptor antagonists produced any significant alteration of Catuama[R] effects. Of interest, previous neurochemical evidence has indicated that the product Catuama[R] is able to concentration-dependently inhibit the re-uptake and increase the release of monoamines, mainly dopamine, in synaptosomal membrane preparations from rats (Campos et al., 2004). Likewise, it has been recently shown (Campose et al., 2005) that antidepressaant-like actions in mice of the plant T. catigua, one of the main constituents of the product Catuama[R], are also largely dependent on the dopaminergic system. Additional studies are still necessary to better characterize which dopamine receptor subtypes are involved in these effects.

The present work demonstrates for the first time that Catuama[R], an herbal product popularly used to treat stress-associated disorders, presents marked oral antinociceptive properties in both CFA and LPS models of inflammatory nociception in rats. The mechanisms through which Catuama[R] exerts its antinociceptive actions still remain incompletely understood, and require further studies, but they seem in part to involve interference with the dopaminergic system. Catuama[R] could well constitute a new and attractive strategy for the management of human persistent inflammatory pain, as commonly observed in rheumatoid arthritis and other related chronic inflammatory diseases.

Acknowledgments

This work was supported by grants from the Conselho Nacional de Desenvolvimento Cientifico e Tecnologico (CNPq), the Fundacao de Apoio a Pesquisa Cientifica e Tecnologica do Estudo de Santa Catarina (FAPESC), the Programa de Apoio aos Niicleos de Excelencia (PRONEX), and the Laboratorio Catarinense, Brazil. N. L. M. Q. is a Ph.D. student in Pharmacology supported by a grant from CNPq.

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N.L.M. Quintao (a) , J Ferreira (b) , A. Beiriuth (c), M.M.. Campos (d), J.B. Calixto (a), *

(a) Department of Pharmacology, Universidade Federal de Santa Catarina, Florianopolis, SC, Brazil

(b) Department of Pharmacology, Centre of Biological sciences, Universuidade Federal de Santa Maria, Santa Maria, SC, Brazil

(c) Department of Pharmacology, Centre of Biological Sciences, FURB, Blumenau, SC, Brazil

(d) School of Dentistry, Pontificia Universidade Catolica do Rio Grande do Sul, Porto Alegre, RS, Brazil

* Corresponding author. Tel.: [phone]; fax: [phone].

E-mail addresses: [e-mail], [e-mail] (J.B. Calixto).