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Evaluation of the analgesic activity of extracts of Miconia rubiginosa (Melastomataceae).

Summary

The analgesic effects of the hexane, methylene chloride and ethanol extracts of Miconia rubiginosa were evaluated in mice and rats using the acetic acid-induced writhing and hot plate tests. The extracts (100, 200 and 300 mg/kg body wt.) and indomethacin (5 mg/kg body wt.) produced a significant (p < 0.05 and p < 0.01) inhibition of acetic acid-induced abdominal writhing. These same extracts (200 mg/kg body wt.) showed a significant (p < 0.05) antinociceptive effect, lower than that produced by morphine (4 mg/kg body wt.). The fractionation of the methylene chloride extract yielded ursolic and oleanoic acids as the major compounds. Using only gas chromatography, it was possible to identify the following triterpenes in the hexane extract: [alpha]-amyrin, [beta]-amyrin, lupeol and [beta]-sitosterol.

Key words: Miconia rubiginosa, Melastomataceae, analgesic activity, writhing test, hot plate test

* Introduction

Miconia is a genus of approximately 1000 species (Martins et al. 1996) occurring in tropical America (Renner, 1993; Judd et al. 1991). The genus belongs to the family Melastomataceae, which includes species of medicinal value. Miconia extracts and isolated compounds have demonstrated biological activities such as antibiotic, antitumoral, analgesic and antimalarial effects (Lima et al. 1970; Hasrat, 1997; Cunha et al. 2001). Previous studies on Miconia species have shown the presence of triterpenes, coumarins and benzoquinones (Lowry, 1968; Macari et al. 1990; Chan et al. 1992; Gunatilaka et al. 2001). The aim of the present study was to investigate the analgesic properties of the extracts of Miconia rubiginosa, using the writhing and hot plate tests. This is the first investigation of this species for analgesic activity.

* Materials and Methods

Plant material

Miconia rubiginosa (Melastomataceae) was collected along the Franca-Claraval highway, Sao Paulo State, Brazil, and was identified by Dr. Angela Borges Martins, Instituto de Biologia, UNICAMP, Brazil. A voucher specimen has been deposited in the Herbarium of the same Institute (UEC 10830).

Extraction and isolation

The aerial parts were air dried at 40 [degrees]C, and the dried material (1.0 kg) was pulverized and extracted by maceration with hexane, methylene chloride and ethanol, affording 4.4 g, 12.0 g and 15.4 g of crude extract, respectively. The methylene chloride extract was chromatographed over silica gel 60 (70-230 mesh) eluted with hexane, a mixture of hexane:ethyl acetate in increasing polarity and ethanol, resulting in six fractions (F1-F6). Fractions F3 and F4, containing a mixture of ursolic acid and oleanoic acid (2.0 g), were combined. The chemical structures of both compounds were determined using spectrometric methods (IR, MS, [sup.1]H and [sup.13]C NMR) and comparison with reported retention times for pure compounds as determined by HPLC analysis (Mahato and Kundu, 1994, Furuya et al. 1987). The hexane extract was analyzed via CG (HP-1 and HP-50 capillary columns, hydrogen flame ionization detector) by comparison of the relative retention times of triterpene standards, using cholesterol as internal standard. This analysis allowed the identification of [alpha]-amirin, [beta]-amirin, lupeol and [beta]-sitosterol as major compounds in this extract.

Animals

Male Swiss albino mice (25-30 g each) and male Wistar rats (160-170 g each) were used for the writhing test and hot plate tests, respectively. The groups of animals (n = 5) were housed in standard cages at room temperature (25 [+ or -] 4 [degrees]C) with food and water ad libitum. Twenty-four hours before the experiments, they were transferred to the laboratory, where they had access only to water ad libitum.

Acetic acid-induced writhing test

This test was carried out by using the method described by Koster et al. (1959). Writhing was induced by intraperitoneal injection of 0.6% acetic acid (v/v) (80 mg/kg body wt.) into a group of 5 mice. Control animals received a similar volume of vehicle (10% Tween solution in saline). The number of muscular contractions was counted for 20 min following acetic acid injection. Data represent the average of the total number of writhes observed at 5-min intervals and are expressed as percentage inhibition. The extracts were administered orally 20 min before all experiments, at doses of 100, 200 and 300 mg/kg body wt. Indomethacin was used as positive control and administered orally. Data were analyzed statistically using Dunnett's Multiple Comparison test, with the level of significance set at * p < 0.05 and ** p < 0.01.

Hot plate test

The test was performed using the method described previously (Woolfe and McDonald, 1944) with little modification for the rats. The parameter evaluated was the latency time for paw licking and jumping responses after exposure to the hot plate surface. The hot plate temperature was maintained at 51 [+ or -] 1 [degrees]C. The animal was kept on the hot plate until it lifted one of its hind paws. The response was determined over 60 min after the sample injection and the data represent the mean reaction time for the animals. Latency time was recorded and the results are expressed as the hot plate analgesic index (Yaksh et al. 1976). The extracts were administered orally at a dose of 200 mg/kg body wt. The standard (morphine sulfate, 4 mg/kg body wt.) was administered subcutaneously.

* Results and Discussion

Deraedt et at. (1980) described the quantification of prostaglandins in the peritoneal exudates of rats by radioimmunoassay, obtained after intraperitoneal injection of acetic acid. They found high levels of prostaglandins PG[E.sub.2[alpha]] e PG[F.sub.2[alpha]] during the first 30 min after acetic acid injection. Nevertheless, it was found that intraperitoneal administration of acetic acid induces not only the liberation of prostaglandins, but also the liberation of sympathetic nervous system mediators (Duarte et al. 1988; Borsato et al. 2000) Therefore, we may state that anti-inflammatory substances may also be involved in peripheral analgesic activity.

The extracts of M. rubiginosa inhibited acetic acid-induced writhing in mice. It can therefore be suggested that the analgesic effect of the extract is peripherally mediated. M. rubiginosa extract produced a reduction in the number of writhes at all three doses used, but the most significant effect was obtained at the dose of 200 mg/kg body wt. (Fig. 1).

[FIGURE 1 OMITTED]

The other algesimetric test used was hot plate test that revels the central analgesic response. The extracts showed a significant increase in the pain threshold, corresponding to a significant increase in the percentage of protection, as compared to the control group and the morphine standard (Fig. 2).

[FIGURE 2 OMITTED]

Therefore, the analgesic effect of the extracts was significant by both measures, a fact that can be explained by the presence of a mixture of substances in the extract, bearing both peripheral and central analgesic properties. The triterpenes and sterols may be the active principles responsible for the observed analgesic effect. The literature contains numerous reports on the analgesic and anti-inflammatory properties of these classes of compounds (Geetha, 2001; Diaz et al. 2000; Gaertner et al. 1999; Santos et al. 1995).

Overall, it can be concluded that the extract of M. rubiginosa posseses possible central and peripheral analgesic activity, probably mediated by the inhibition of prostaglandin synthesis, as well as by central inhibitory mechanisms.

Acknowledgments

This study was supported by FAPESP. We are very grateful to Alba R. B. Araujo and Maria Ines J. Garcia (Universidade de Franca) for helping with plant collection and Dr. Jairo Kennup Bastos from FCFRP-USP for helpful discussions.

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* Address

W. R. Cunha, Nucleo de Pesquisa em Ciencias Exatas e Tecnologicas da Universidade de Franca--Av. Dr. Armando Salles de Oliveira, 201--Pq. Universitario, CEP: 14404-600, Franca--SP, Brazil

Tel.: ++55-16-3711-8871; Fax: ++55-16-3711-8876; e-mail: wrcunha@unifran.br

M. A. Spessoto, D. S. Ferreira, A. E. M. Crotti, M. L. A. Silva, and W. R. Cunha

Nucleo de Pesquisa em Ciencias Exatas e Tecnologicas da Universidade de Franca, Pq. Universitario, Franca--SP, Brazil
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Title Annotation:Short Communication
Author:Spessoto, M.A.; Ferreira, D.S.; Crotti, A.E.M.; Silva, M.L.A.; Cunha, W.R.
Publication:Phytomedicine: International Journal of Phytotherapy & Phytopharmacology
Geographic Code:3BRAZ
Date:Jul 1, 2003
Words:1829
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