Analysis of the antinociceptive properties of marrubiin isolated from Marrubium vulgare

Phytomedicine, Vol. 7(2). pp.111 - 115 © Urban & Fischer Verlag 1999 http://www.urbanfischer.de/journalslphytomed

Analysis of the antinociceptive properties of marrubiin isolated from Marrubium vulgare R. A. P. De Jesus, V. Cechinel-Filho, A. E. Oliveira, and V. Schlemp er N iicleo de Investigacoes Qu irnico-Farrnaceuticas (NIQFAR), Curso de Farrnacia/Centro de Ciencias da Saude (CCS), Univer sidade do Vale do Itaja i (UNIVALl), Itajai, SC, Brazil

Summary We have shown previously that Marrnbium vulgare, a medicinal plant employed frequently in folk medicine to treat a variet y of ailments , exhibits ant ispasmodic and antinociceptive effects in different experimental models. This work describes the antinociceptive profile of marrubiin, the main constituent of this plant, which was anal ysed in some models of nociception in mice. The results showed that marrubiin exhibits potent and dose-related antinociceptive effects, whose calculated 1050 values (pmol/kg, i.p.) wer e the following: 2.2 in the writhing test, 6.6 (first phase) and 6.3 (second phase) in the formalin-induced pain test and 28.8 when evaluated in the capsaicin test. It was more potent than some well-known analgesic drugs. The antinociception produced by the marrubiin was not reversed by nalo xone when analyzed against the writhing test. In the hot-plate test, marrubiin did not increase the latency period of pain induced by the thermal stimuli. Its exact mechani sm of action remains to be determin ed, but the result s suggest that marrubiin, like hydroalcoholic extract of M. vulgare, does not interact with opioid systems. Key words: Marrubium vulgare, folk medicine, marru biin, antinociception, mice.

Introduction Marrubium vulgare is a medicinal plant used in the folk medicine of many countries for the treatment of a variety of diseases, especially gastroe nteric and respiratory disorders (Balme, 1982; Newall et al., 1996; Knoss, 1998) . Previous studi es cond ucted by our research gro up have demon strated that the hydr oalcoholi c extract of this plant exerts a significant and non-specific antispasmodic effect on isolated smoo th muscle (Schlemper et al., 1996 ). We have also show n that th e extract exhibited pot ent and dose-dependent antinociceptive action in severa l models of pain in mice (Souza et aI., 1998) . Phytoch emical studies carried out by our laboratory and other authors have con firmed that mar ru biin (1), a furanic labdane diter pene, is an artifact produced du ring the extraction process, being the main cons tituent present in this plant (He nderson and McCrindle, 196 9; Laonigro et al., 1979; Bruneton, 199 1; Rodrigues et al., 1998). Altho ugh the chemical aspects of this comp ound are well documented, its pharmacological properties remain to be determined.

However, it is reported to stimulate secretions of the bronchi al mucosa and to possess anti-arrhythmic effects (Newall et al., 1996). In th is report, we have extended our previous studie s carrie d out using the hydroalcoholic extract (HE) of M. vulgare and determined the possible antinociceptive effects of marrubiin using some models of pain in mice. Moreover, we have compared its potency with HE and some reference drugs, such as diclofenac sa die and aspinn.

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Material and Methods Isolation of marrubiin

Marrubiin was isolated from aerial parts of M. vulgare at a yield of approximately 0.1 % according to the previously described method (Rodrigues et al., 1998). It was identified on the basis of its spectroscopic data eH and 13C-NMR, UV), which were identical to those reported in the literature (Henderson and McCrindle, 1969; Laonigro et al., 1979; Knoss and Zapp, 1998) and by co-TLC with an authentic sample. Animals

Swiss mice of both sexes (25-35 g) were housed in automatically controlled temperature conditions (23 ± 2 °C and 12 h light-dark cycles). The animals were given access to water and Nuvital chow ad libitum unless otherwise indicated. The animals remained in the appropriate laboratory of UNIVALI until some hours before of the experiments. Pharmacological analysis

• Abdominal constriction response caused by intraperitoneal injection of diluted acetic acid: The abdominal constriction induced by intraperitoneal injection of acetic acid (0.6%), was carried out according to procedures described previously (Collier et al., 1968; Souza et al., 1998) with minor modifications. Animals were pretreated with marrubiin (0.9-30 prnol/kg) or standard drugs intraperitoneally 30 min before the acid acetic injection. Control animals received a similar volume of 0.9% NaCI (10 ml/kg, i.p.). In a separate set of experiments, animals were pretreated either with marrubiin (30.0 prnol/kg, i.p.) or with morphine (13.3 umol/kg, s.c.) 30 min before the acetic acid injection. We also analyzed the effect of naloxone (13.8 pmol/kg, i.p.) injected 10 min. beforehand against the antinociceptive effect caused by both morphine and marrubiin. After the challenge, each mouse was placed in a separate glass funnel and the number of abdominal contractions of the abdominal muscles together with stretching, was counted cumulatively over a period of 20 mins. Antinociceptive activity was expressed as the reduction of the number of abdominal contractions between control animals and mice pretreated with marrubiin and standard drugs. • Formalin-induced pain. The procedure used was essentially similar to that described previously (Hunskaar and Hole, 1987; Hunskaar et al., 1985; 1986; Souza et al., 1998). Animals from the same strain were slightly anesthetized with ether, except when used to analyze the first phase of formalin-induced pain, and 20 ul of 2.5% (0.92% formaldehyde) made up of PBS (phosphate buffered solution containing: NaCI 137 mM;

KC12.7 mM and phosphate buffer 10 mM) was injected under the plantar surface of the left hindpaw. Animals were acclimatized to the laboratory for at least 24 h before the experiments. Two mice (control and treated) were observed simultaneously for 0 to 30 mins following formalin injection. The initial nociceptive scores normally peaked after 5 mins (first phase, representing the neurogenic pain), and after 15-30 mins after formalin injection (second phase, representing the inflammatory pain) (Hunskaar and Hole, 1987). Animals were treated with saline 0.9% (10 ml/kg, i.p.), marrubiin (3 to 90 umol/kg by i.p. route or 90 to 900 pmol/kg by v.o.) or with standard drugs 60 min before formalin injection. After intraplantar irritant application, the animals were placed immediately in a glass cylinder (20 ern diameter). The time spent by animals licking or biting the injected paw was timed with a chronometer and was considered indicative of pain. At the end of the experiments the animals were sacrificed with ether, the paws cut at the tibio-tarsic joint and weighed on an analytical balance to investigate the interference of the marrubiin on formalin-induced inflammatory oedema. • Hot-plate test: The hot-plate was used to stimate the latency of responses according to the method described by Eddy and Leimback (1953) with minor modifications. The temperature of the hot-plate was maintained at 56 ± 3 "C. The animals (n = 10) were placed on glass funnels in the heated surface and the time between placing the animals and the beginning of licking paws or jumping were recorded as latency of response, in non-treated (saline 10 rnl/kg, i.p.) or marrubiin (180 pmol/kg, i.p.) animals. • Capsaicin-induced pain: The procedure used was similar to that described previously (Sakurada et al., 1993; Campos et al., 1997). The animals were placed individually in transparent glass cylinders. Following the adaptation period, 20 pl of capsaicin (1.6 ug/paw) was injected under the skin of the plantar surface of the right hindpaw, using a microsyringe. The animals were observed individually for 5 minutes following capsaicin injection. The amount of time spent licking the injected paw was timed with a chronometer and was considered as indicative of nociception. Animals were treated with the marrubiin (3 to 90 prnol/kg, i.p.) or saline (10 mllkg, i.p.) 1 hour before administration of capsaicin. Control animals received a similar volume of 0.9% NaCI (10 mllkg, i.p.).

Statistical analysis

The results are presented as mean ± s.e.m. and statistical significance between groups was analysed using the t-test or analysis of variance followed by Dunnett's multiple comparison test, when appropriate. P values

Ana lysis of th e antino ciceptive pro pert ies of marrubiin

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less th an 0.05 were conside red signif icant. W hen appropriate d, the ID so values (the dose of compound th at reduced formalin-, acetic acid- or capsaicin-induced pain by 50 % rela tive to control values) are reported as geometric means accompanied by th eir respe ctive 95% confidence limits.

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Results

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a. Effect of marrubiin on acetic acid -induced writhing

T he results shown in Table 1 indicate a significant (p < 0.05) an d do se-dependent antinociceptive effect of marrubiin given by i.p. 30 min befor ehand, this being highl y effective in inhibiting acetic acid-ind uced wri th ing responses in mice. As can be observed, it exhibited high potency, with an ID so value (and 95% of confidence limits) of 2.2 (1.1-3 .0) umol/kg. At 90 urnol/kg, i.p. marrubiin practically abolished th e non- specific pai n of the writhing test. The antinociceptive effect was observed over a long time period, extendi ng its action until 5 h after the algesic stimuli with acetic acid (Figure 1). When th e animals were treated with na loxone (5 mg/kg" , i.p. ), a non- selective morphine recep tor antagonist, 10 min before the noxious stimuli, no change was observed in the an tinociceptive effect of marrubiin as compared to morphine (Figure 2).

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Fig. 1. Time-course of antinociceptive effect of marrubiin (1) (90 umol/kg, i.p.) against acetic acid-induced pain in mice. Each group represents the mean of six experiments and the vertical bars indicate the s.e.m. * p < 0.05; .... p < 0.01, compared with corresponding control value.

rubiin was also effective in antagonizing the formalininduced hindpaw oedema (results not sho wn), suggesting an associated antiinflamma tory effect. In th e hot- p late test, murr ibinn (180 umol/kg) was not capable of increasing the lat ency period of pain induced by heating of the plate. c. Effect of marrubinn on the capsaicin test

b. Effect of marrubiin on the formalin-induced pain test and the hot plate test

In the for ma lin test, marrubiin significantly inhibited (p < 0,05) dose -dependently both the first and second phases by i.p. and oral routes. The calc ulate d IDso's values for first and second phases were 6.6 (4.8 -8.4) and 6.3 (5.4-7.2) umol/kg for the systemic rou te, with maximum inhibitions (MI) of 78 ± 5 and 83 ± 4, respectively. When analysed by the oral route, marr ubiin presented IDso's of 126 (109-135) and 108 (97- 137) umol/kg with maximal inhibit ions of 75 ± 3 and 99 ± 1 % for first and second phases, respectively. Mar-

When analysed in the capsaicin-induced neurogenic nociception, the marrubiin promoted a significant and dose-dependent inhibition of chemical-induced pain. The calcu lated ID so values w as 28 .8 (27.3-29.3) umol/kg wit h MI of 76 :!: 5%.

Discussion We have recen tly reported that the hydroalcoh olic extract obtained from M. vulgare exerts a significant nonspecific antispasmodic effect on different proinflamma-

Table 1. Comparison of the antinociceptive effect of marrubiin (1) with non-steroidal antiinflammatory and analgesic drugs given intraperitoneally in mice. Compound

Marrubiin (1) Aspirin Diclofenac

Writhing test ID so (prnol/kg)

Formalin test First phase! m., (urnol/kg)

Capsaicin test Second phaseID so (umol/kg)

ID so (prnol/kg]

2.2 (1.1- 3.0) 133 (73-243 ) 38 (30- 49)

6.6 (4.8-8.4 ) Inactive > 94

6.3 (5.4-7.2) 123 (77- 209) 34.5 (25-47)

28.8 (27.3- 29.3) NT 47.4 (35-65)

NT - not tested. Each group represents the mean ± s.e.m. of six experiments. 1 0-5 min licking (s); 2 15-30 min licking (s).

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Fig. 2. Effect of naloxone on the antinociceptive profile caused by morphine and marrubiin (1) against acid-induced pain in mice. Each group represents the mean of six experiments and the vertical bars indicate the s.e.m. *,' p < 0.01, compared with corresponding control value.

tory agonists in isolated tissues (Schlemper et aI., 1996), but these effects seem to be unrelated to the presence of marrubiin (data not shown). This paper extends previous findings where we have shown that M. vulgare possesses active constituents which exhibit marked antinociceptive activity (Souza et aI., 1998). The results reported in the present investigation demonstrate for the first time that marrubiin, the main furanic labdane diterpene of M. vulgare, produces potent antinociceptive effects on different animal models in vivo. The experimental procedures used here were very similar to those used previously for the hydroalcoholic extract of M. vulgare (Souza et aI., 1998). The antinociceptive properties of marrubiin were observed using both the systemic and oral routes and its action remained over a long period. The high potencies observed in the writhing test and formalin-induced pain test led us to suggest that marrubiin is acting by some peripheral mechanism. There are some signs in common with inflammatory pain associated with the profile of analgesic effects. Besides non-specificity of the writhing test, we have observed that the antinociceptive effects of morphine, but not those of marrubiin or HE (Souza et aI., 1998), were partially but significantly reversed by naloxone, suggesting that marrubiin, like HE, is effective in abolishing acetic acid-induced pain in a non-opiod way. This was confirmed by the lack of antinociceptive effects in the hot-plate test, a technique that has a selectivity for opioid-derived analgesics (Abbott and Melzack, 1982; Abbott and Franklin, 1986). It is interest-

ing to note that marrubiin, on formalin-induced pain, a test which defines two distinct periods of response, i.e., "early response" and "late response" (Hunskaar et aI., 1985), inhibited both phases of pain, suggesting that other mechanisms could be involved. The effect produced in the first phase may be due to immediate and direct effects on sensory receptors, bradykinin receptors or in a glutamatergic way, whereas for the last phase the antinociceptive effect is related to the inflammatory responses induced by arachidonic acid cascade (Dubuisson and Dennis, 1977; Hunskaar et aI., 1985; Souza et aI., 1998). It is important to mention that marrubiin was also effective in antagonizing the formalin-induced hindpaw oedema (results not shown). Thus, the factor by which this compound inhibits such an inflammatory oedema induced by irritant could be through modulating the liberation or blocking B2 receptor and prostaglandin receptor systems (Correa and Calixto, 1993). Another interesting result consisted of the potent action of marrubiin in the capsaicin test, which provided more direct evidence of the antinociceptive effect of marrubiin on neurogenic pain. It is important to mention that some well-known nonsteroidal antiinflammatory drugs, including aspirin, are ineffective in the first phase of the formalin test and in the capsaicin model, but they significantly inhibit the second phase of the formalin-induced licking (Table 1). When compared with HE (Souza et aI., 1998) or these standard drugs (aspirin and diclofenac), marrubiin was more potent in all models used (Table 1). Since marrubiin is an artifact produced from pre marrubiin, we have investigated if it is present in the herbal preparations. The chromatographic analysis by thin layer chromatography (TLC) confirmed that this compound is produced in tea (results not shown), being therefore one of the active compounds responsible for the antinociceptive effect of M. vulgare employed in folk medicine. Studies conducted by other groups with marrubiin indicated that it is obtained in low yields by cell suspension and callus cultures produced from seedlings of M. vulgare (Knoss et aI., 1993). It seems to be biosynthesized in this plant via a non-mevalonate pathway (Kness et aI., 1997; Knoss and Reuter, 1998). In summary, our results demonstrate for the first time that marrubiin of the main active component of M. vulgare exerts a marked antinociceptive effect, either by the oral or systemic routes, in distinct models of nociception in mice. It was several times more potent than some drugs employed clinically against dolorous processes. The mechanism by which marrubiin exerts antinociceptive activity remains undetermined, but our results suggest that, like the hydroalcoholic extract of M. vulgare, it does not involve the inhibition of cyclooxy-

Anal ysis of the antinociceptive properties of marrubiin genase products deri ved from th e arachidonic acid pathway or the participat ion of th e opioid system. Considering its promising ph armacological act ion and goo d yield in the plant, it might present new therapeutic possibilities. Acknowledgements

Th is wor k was supported by gran ts from PIBIClCNPq and ProPPExfUNIVALI.

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Address V. Schlemper, NIQFARl CCS, Curso de Farmacia, UNI VAll, 883 02-2 02, Itajai-SC-Bra zil Fax: 005547341 760 0; e-mai l: Valfredo@mbox L.univa li.rct-sc.com.br.