Studies on the anti-allergic activity of Mikania glomerata

Journal of Ethnopharmacology 66 (1999) 19 – 24 Studies on the anti-allergic activity of Mikania glomerata Iolanda M. Fierro *, Ana Cristina Borges da Silva, Carlos da Silva Lopes, Roberto Soares de Moura, Christina Barja-Fidalgo Departamento de Farmacologia e Psicobiologia, Instituto de Biologia, Uni6ersidade do Estado do Rio de Janeiro, A6enida 28 de Setembro 87 fds. 5o andar, Vila Izabel, Rio de Janeiro, CEP 20551 -030, Rio de Janeiro, Brazil Received 5 May 1998; received in revised form 18 June 1998; accepted 17 August 1998 Abstract A fraction (MG1) obtained from the ethanolic extract of Mikania glomerata Sprengel (Compositae), popularly known as ‘guaco’ and used as ‘an’ anti-allergic and anti-inflammatory agent, was evaluated for these properties on ovalbumin-induced allergic pleurisy and in models of local inflammation induced by biogenic amines, carrageenan and PAF. Plasma exudation as well as neutrophil and eosinophil infiltration evoked by the intrapleural injection of the antigen were significantly reduced by the fraction. Likewise, PAF-induced pleural neutrophil migration was inhibited by the treatment with MG1. On the other hand, pre-treatment of the animals with MG1 failed to modify the pleurisy induced by histamine, serotonin or carrageenan. These results suggest that MG1 is effective in inhibiting immunologic inflammation but did not affect acute inflammatory response caused by other agents. © 1999 Published by Elsevier Science Ireland Ltd. All rights reserved. Keywords: Mikania glomerata, pleurisy; Anti-allergic activity; Anti-inflammatory activity; Rats 1. Introduction Mikania glomerata Sprengel (Compositae), popularly known as ‘guaco’, is a medicinal plant used in Brazilian folk medicine for various inflammatory and allergic conditions, particularly of the respiratory system. These folk indications in asthma and bronchitis are probably due to its * Corresponding author. Tel.: + 55 21 5876398; fax: + 55 21 5876530; e-mail: fierro@openlink.com.br bronchodilating properties (Lopes, 1997). Although this plant has been widely used, even as commercial preparations, there are few studies to date on its anti-inflammatory and/or anti-allergic properties. It has been reported from previous phytochemical investigations. The presence of sesquiterpenes (Vilegas et al., 1997) and coumarin (Oliveira et al., 1984; Vilegas et al., 1997), specially in the leaves have been reported in phytochemical studies. In this study, we have evaluated the effects of a fraction (MG1) obtained from the 0378-8741/99/$ - see front matter © 1999 Published by Elsevier Science Ireland Ltd. All rights reserved. PII: S0378-8741(98)00151-2 20 I.M. Fierro et al. / Journal of Ethnopharmacology 66 (1999) 19–24 ethanolic extract prepared from the leaves of Mikania glomerata in the allergic pleurisy model in rats. In addition, we have also assessed the anti-inflammatory properties of MG1 fraction using different stimuli. 2. Methodology 2.1. Identification and extraction of plant material Leaves of Mikania glomerata were collected in Petrópolis, Rio de Janeiro, Brazil. A voucher specimen (No. 83.002) has been deposited at Herbarium Bradeanum, Universidade do Estado do Rio de Janeiro, Rio de Janeiro, Brazil. Fresh leaves (0.7 kg) were exhaustively extracted by maceration at room temperature with EtOH (2.1 l). The final concentration of the ethanolic extract was 10% w/v, considering the yield of the dried residue as 30%. Further fractionating of the extract was then performed by liquid– liquid extraction using dichloromethane as organic solvent and two fractions were obtained. The organic phase, called MG1, represents 15.5% w/w of the initial extract. The fraction was further chromatographed on a column of silica gel 60 (53 ×4 cm) and eluted with dichloromethane (1.5 l), ethyl acetate (1.0 l) and MeOH (1.2 l). Three fractions were collected, respectively: MG3, MG4 and MG5. Gas chromatography showed that MG3 was rich in coumarin which represents 90% w/w of total fraction. The analysis by gas chromatography coupled to mass spectrometry showed that the MG1 fraction presented 12.9% of coumarin and, in minor proportions, the following compounds: trans-o-hydrocinnamic acid, dihydrocoumarin, palmitic acid, ethyl-hexadecanoate, phytol, ethyl linoleate and traces of compounds presenting the kaurenoic acid structure. Identifications were carried out by comparison of literature data with the aid of computer matching against the Wiley 138L library (Hewlett Packard). The presence of coumarin, kaurenoic acid methyl ester isomers and sesquiterpenes has been already described in ‘guaco’ leaves (Vilegas et al., 1997). 2.2. Animals and allergic pleurisy Male Wistar rats (180 – 200 g), provided by the Oswaldo Cruz Foundation Breeding Unit (Rio de Janeiro, Brazil), were actively sensitized by means of a single dorsal subcutaneous (s.c.) injection of a mixture containing 50 mg of ovalbumin (OVA) and 5 mg of Al (OH)3 in a final volume of 200 ml. A total of 14 days after sensitization, the animals received an intrapleural (i.pl.) injection of ovalbumin (12 mg/cavity) dissolved in sterile saline. At different times ranging from 1 to 24 h post-challenge, the animals were killed by an overdose of ether and the thoracic cavity was rinsed with 3 ml of heparinized saline (10 IU/ml). Protein extravasation and leukocyte infiltration were further analyzed. These experiments included sensitized animals injected i.pl. with sterile saline as negative control groups. 2.3. Induction of pleurisy by different stimulus in normal rats Non-sensitized rats were stimulated i.pl. with carrageenan (300 mg/cavity), histamine (200 mg/ cavity), serotonin (100 mg/cavity) or PAF-acether (1 mg/cavity) in a final volume of 100 ml. The animals were killed 1 h after administration of the biogenic amines, 4 h after carrageenan and 6h Table 1 Effect of MG1 on protein extravasation and leukocyte migration induced by carrageenan (300 mg/cavity) in normal rats Treatment Protein (mg/cavity) Number of total leukocytes (106/cavity) None MG1 (10 mg/ kg) MG1 (30 mg/ kg) MG1 (100 mg/ kg) 49.70 94.0 41.31 95.2 38.17 9 4.2 33.67 9 6.1 44.10 96.4 41.92 9 6.2 42.10 92.3 37.46 9 1.8 MG1 was administered s.c. and the analysis was made 4 h after carrageenan injection. The values for control animals were 6.409 0.7 mg protein/cavity and 8.819 0.6×106 leukocytes/cavity. Values represent means9 S.E.M. from at least five animals. I.M. Fierro et al. / Journal of Ethnopharmacology 66 (1999) 19–24 21 2.6. Treatments The MG1 (10– 100 mg/kg), MG3 (10 mg/kg) and Coumarin (10 mg/kg) were dissolved in propylene glycol. The drugs were then administered s.c. 12 h and 1 h before the agonist or antigen stimulation in a final volume of 0.5 ml. In control groups, the drugs were replaced by the vehicle which was also injected in a 0.5 ml volume. 2.7. Drugs Fig. 1. Effect of MG1 on plasma protein leakage triggered by ovalbumin (12 mg/cavity) in immunized rats. The analysis was made 4 h after challenge. Each column represents the mean 9 S.E.M. from at least five animals. + , Statistically significant when compared to control (SAL) group. *, Statistically significant when compared to the immunized (OVA) group. The MG1 and MG3 were obtained as previously described. Ovalbumin (grade V) was purchased from Biochemica Fluka (Switzerland). Histamine, 5-hydroxytryptamine, carrageenan, propylene glycol, and coumarin, were obtained from Sigma (St. Louis, MO). PAF-acether (1O-hexadecyl-2-acetyl-sn-glyceryl-3-phosphorylcholine) was from Novabiochem (Switzerland). All solutions were freshly prepared immediately before use. after PAF-acether administration. Protein extravasation and leukocyte influx were evaluated as described below. 2.4. Cell analysis Total leukocyte and mast cell counts were performed in Neubauer chamber by means of an optical microscope after dilution of the pleural fluid with Türk solution (2% acetic acid) and toluidine blue dye (Mota, 1966), respectively. Differential analysis was made in cytospin preparations stained with May-Grünwald-Giemsa dye under an oil immersion objective. 2.5. Protein quantification The fluid recovered from the pleural cavity was centrifuged (1200× g) for 10 min and the total protein content of the supernatant quantified spectrophotometrically (Shimadzu) using the Biuret technique. Fig. 2. Effect of MG1 (100 mg/kg) on intact mast cell count decrease evoked by allergen in the pleural cavity of immunized rats. The number of intact mast cells was evaluated as described in Section 2 and expressed as the mean 9 S.E.M. from at least five animals. + , Statistically significant when compared to control group. *, Statistically significant when compared to the immunized group. 22 I.M. Fierro et al. / Journal of Ethnopharmacology 66 (1999) 19–24 Fig. 3. Effect of MG1 (10– 100 mg/kg) on neutrophils (left panel) and eosinophils (right panel) accumulation triggered by ovalbumin (12 mg/cavity) in immunized rats. The analysis was made 4 and 24 h after antigen stimulation, respectively. Each column represents the mean 9 S.E.M. from at least five animals. + , Statistically significant when compared to control group. *, Statistically significant when compared to the immunized group. 2.8. Statistical analysis Data are reported as means9S.E.M. and were analyzed by analysis of variance followed by the Bonferroni’s t-test. In case of comparison between only two groups, the difference in means was analyzed by unpaired Student’s t-test. P-values of 0.05 or less were considered significant. 3. Results extravasation observed 4 h after antigen, but this reduction was significant only at the highest dose tested. The allergen challenge led to an intense mast cell degranulation reducing the number of intact mast cells in the control pleural effluent and, as shown in Fig. 2, this massive mast cell degranulation was partially impaired by the treatment of the animals with MG1 at 100 mg/kg. Furthermore, increasing concentrations of MG1 (10– 100 mg/kg) dose-dependently inhibit the neutrophil infiltration within 4 h and the late eosinophil accumulation noted 24 h-post challenge (Fig. 3). 3.1. Effect of MG1 on the allergic pleurisy Normal rats injected i.pl. with carrageenan developed a marked inflammatory response, which was not affected by MG1, even at the highest dose used (Table 1). The i.pl. injection of OVA into sensitized rats causes an acute exudation followed by leukocyte infiltration, mainly neutrophils and eosinophils, at 4 and 24 h after stimulation, respectively (Lima et al., 1991). As illustrated in Fig. 1, MG1 reduced the protein 3.2. Effect of MG1 on PAF-triggered pleurisy PAF-acether induces an early exudation followed by a marked cellular influx, which is only apparent 6 h after stimulation (Martins et al., 1989). Pre-treatment with MG1, at 100 mg/kg 12 and 1 h before the i.pl. injection of PAF, failed to modify pleural exudation (SAL: 2.83 90.2; PAF: 11.30 90.8; PAF +MG1: 10.909 0.42 mg/cav- I.M. Fierro et al. / Journal of Ethnopharmacology 66 (1999) 19–24 23 Table 3 Effect of Coumarin (10 mg/kg) and MG3 (10 mg/kg) on protein extravasation and leukocyte migration induced by ovalbumin in sensitized rats Treatment Protein (mg/cavity) Number of total leukocytes (106/cavity) None Coumarin MG3 33.92 9 3.6 39.02 9 2.9 33.48 9 2.1 38.11 9 1.7 38.5 9 3.5 34.58 9 2.7 Coumarin and MG3 were administered s.c. and the analysis was made 4 h after antigen stimulation. The values for control animals were 3.18 9 0.4 mg protein/cavity and 6.19 9 0.3×106 leukocytes/cavity. Values represent means 9 S.E.M. from at least five animals. failed to inhibit exudation induced by vasoactive amines. Exudation was analyzed 1 h after the i.pl. injection of the agonists. Fig. 4. Effect of MG1 (100 mg/kg) on neutrophil infiltration induced by PAF (1mg/cavity) in the pleural cavity of normal rats. The analysis was made 6 h after PAF injection. Each column represents the mean 9 S.E.M. from at least five animals. + , Statistically significant when compared to saline group. *, Statistically significant when compared to PAF group. ity), under conditions where neutrophil influx was significantly inhibited (Fig. 4). 3.3. Lack of effect of MG1 on biogenic amines pleurisy As indicated in Table 2, MG1 (100 mg/kg) Table 2 Effect of MG1 (100 mg/kg) on protein extravasation induced by histamine (200 mg/cavity) or 5-HT (100 mg/cavity) in normal rats Stimulus Treatment Protein (mg/cavity) Histamine None MG1 None MG1 40.97 9 3.6 35.98 9 2.3 15.58 9 1.2 12.34 9 0.8 5-HT MG1 was administered s.c. and the analysis was made 1 h after the injection of the stimuli. The protein content of the pleural washing from the saline-injected rats was 6.40 9 0.7 mg/cavity. Values represent means 9 S.E.M. from at least five animals. 3.4. Lack of effect of MG3 and coumarin on allergic pleurisy It was showed by means of gas chromatography that coumarin corresponded to about 10% w/w of the MG1 extract and 90% of the MG3 fraction. In order to evaluate whether the anti-allergic effect of the extract was due to coumarin, the animals were pretreated with coumarin or MG3 at 10 mg/kg. No difference in total protein content or in leukocyte infiltration was found when compared to control animals (Table 3). 4. Discussion and conclusions Active allergic pleurisy in rats is characterized by marked exudation, followed by two consecutive waves of granulocytes influx: neutrophils followed by eosinophils, both of which accumulate in the pleural cavity (Lima et al., 1991). Pre-treatment of the animals with MG1, a fraction obtained from the ethanolic extract of Mikania glomerata, only reduced pleural oedema at the highest dose tested. In contrast, administration of MG1 dose dependently inhibited leukocyte infiltration detected after antigen challenge, being 24 I.M. Fierro et al. / Journal of Ethnopharmacology 66 (1999) 19–24 active against the early (4 h) neutrophilia and also against the late (24 h) eosinophilia. Plasma exudation in allergic pleurisy has an important component induced by the local release of histamine and serotonin (Lima et al., 1991). However, MG1 has no inhibitory activity at all to pleurisy triggered by these two amines suggesting that the anti-allergic properties of MG1 fraction cannot be ascribed to either an anti-histamine or anti-serotonin activity but rather should be due to other mechanisms. The principal target cell of immediate hypersensitivity reactions is the mast cell (Schwartz, 1994) which secretes a variety of inflammatory mediators including leukotrienes and platelet-activating factor (PAF-acether). Since the allergic pleural eosinophilia seen at 24 h was previously shown to be dependent on eicosanoids and PAF (Silva et al., 1992) and it was inhibited by MG1, one possible explanation for the inhibitory effect of this fraction on rat allergic pleurisy is that it antagonizes the effects and/or the release of this lipid. Therefore, the observation that MG1 significantly inhibits mast cell degranulation suggests that it could be affecting not only the action of PAF, but other mediators including leukotrienes. The exudate volume and leukocyte number in the pleural cavity after carrageenan injection were not affected by the pre-treatment with MG1 suggesting that this fraction is not a non-specific anti-inflammatory agent but rather displays an important effect on mast cell degranulation and granulocyte migration. In conclusion, the present series of experiments have shown that, albeit in rather crude form, MG1 displays three main effects: (a) its ability to inhibit granulocyte infiltration following antigen challenge; (b) inhibition of antigen-induced mast cell degranulation; and (c) partial inhibition of PAF-induced granulocyte infiltration, which together lend support to the folk usage of ‘guaco’ in . allergic diseases. In addition, our results certainly warrant further studies to isolate the active principle and to establish its mechanism of action. Acknowledgements This work was supported by grants from CNPq, CEME and SR-2/UERJ, Brazil. The authors thank M.A. Arruda for technical assistance and Dr J. Assreuy for critical correction of the manuscript. References Lima, M.C.R., Martins, M.A., Perez, S.A.C., Silva, P.M.R., Cordeiro, R.S.B., Vargaftig, B.B., 1991. Effect of azelastine on platelet-activating factor and antigen-induced pleurisy in rats. European Journal of Pharmacology 197, 201 – 207. Lopes, C.S., 1997, Efeitos do guaco (Mikania glomerata) na musculatura lisa respiratória. XII Annual Meeting of FESBE, Caxambu, Brazil, pp. 282. Martins, M.A., Silva, P.M.R., Neto, H.C.C.F., Bozza, P.T., Dias, P.M.F.L., Lima, M.C.R., Cordeiro, R.S.B., Vargaftig, B.B., 1989. Pharmacological modulation of PAF-induced rat pleurisy and its role in inflammation by zymosan. British Journal of Pharmacology 96, 363 – 371. Mota, I., 1966. Histamine and antihistaminics. In: Rocha e Silva, M. (Ed.), Handbook of Experimental Pharmacology. Springer, Berlin, pp. 569 – 636. Oliveira, F., Alvarenga, M.A., Akisue, G., Akisue, M.K., 1984. Avaliação quı́mica de espécimes do gênero Compositae. Revista de Farmácia e Bioquı́mica da Universidade de São Paulo (Brazil) 20, 169 – 183. Schwartz, L.B., 1994. Mast cells: function and contents. Current Opinion in Immunology 6, 91 – 97. Silva, P.M.R., Martins, M.A., Lima, M.C.R., Alves, A.C., Diaz, B.L., Cordeiro, R.S.B., 1992. Pharmacological modulation of the late eosinophilia induced by antigen in actively sensitized rats. International Archives of Allergy and Immunology 98, 355 – 362. Vilegas, J.H.Y., de Marchi, E., Lanças, F.M., 1997. Extraction of low-polarity compounds (with emphasis on coumarin and kaurenoic acid) from Mikania glomerata (Guaco) leaves. Phytochemical Analysis 8, 266 – 270.