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Analgesic potential of marrubiin derivatives, a bioactive diterpene present in Marrubium vulgare (Lamiaceae)
Il Farmaco 60 (2005) 321–326 http://france.elsevier.com/direct/FARMAC/
Analgesic potential of marrubiin derivatives, a bioactive diterpene present in Marrubium vulgare (Lamiaceae) C. Meyre-Silva a, R.A. Yunes b, V. Schlemper a, F. Campos-Buzzi a, V. Cechinel-Filho a,* a
Núcleo de Investigações Químico-Farmacêuticas (NIQFAR)/Curso de Farmácia-CCS, Universidade do Vale do Itajaí, Itajaí-SC, Brazil b Departamento de Química, Universidade Federal de Santa Catarina, Florianópolis-SC, Brazil Received 17 November 2004; received in revised form 20 December 2004; accepted 7 January 2005 Available online 02 March 2005
Abstract Marrubiin, a furane labdane diterpene, is the main analgesic compound present in Marrubium vulgare, a medicinal plant used in Brazil and other countries to treat several ailments. Considering its important pharmacological action, as well as its high yield, some structural modifications were performed in order to obtain more active compounds. Success was obtained in reducing the lactonic function, in the formation of marrubiinic acid and two esterified derivatives, which exhibited significant analgesic effect against the writhing test in mice. Marrubiinic acid showed better activity and excellent yield, and its analgesic effect was confirmed in other experimental models of pain in mice, suggesting its possible use as a model to obtain new and potent analgesic agents. © 2005 Elsevier SAS. All rights reserved. Keywords: Marrubium vulgare; Marrubiin derivatives; Analgesic action
1. Introduction Marrubium vulgare (Lamiaceae), known as ″maromba″ or ″marroio″ in Brazil, or horehound in Europe, is frequently used in folk medicine to cure a variety of diseases. Preliminary evaluations of this plant in our laboratory have demonstrated hypoglycemic, analgesic and antiespasmodic effects [1–3]. Chemically, marrubiin (1), a furane labdane diterpene, is the main component of this plant, and exhibits potent antinociceptive properties and vasorelaxant activity [4,5]. It is formed from the genuine premarrubiin during the work-up procedure [6]. In the present study, we have extended our previous investigations and obtained some marrubiin derivatives with the aim of optimizing its antinociceptive activity initially using the writhing test in mice. The most active compound was studied in other experimental models of pain. The results of some reference drugs, such as aspirin and paracetamol, were also included for the purpose of comparison (Scheme 1).
* Corresponding author. Tel.: +55 47 341 7664; fax: +55 47 341 7601. E-mail address: [email protected] (V. Cechinel-Filho). 0014-827X/$ - see front matter © 2005 Elsevier SAS. All rights reserved. doi:10.1016/j.farmac.2005.01.003
Scheme 1.
2. Experimental 2.1. Chemistry IR spectra (cm–1): Perkin-Elmer FT-16-PC; in KBr. NMR spectra: Bruker AC-200F (200 MHz) for 1H; Bruker AC-200F (50 MHz) for 13C; d in ppm, J in Hz. 1H values and 13C values from spectra in DMSO, CDCl3 or acetone-d6. Standard reference for all spectra was TMS. dTMS = 0 ppm. Tethrahydrofuran (THF) was stored with KOH, then refluxed and distilled prior to use. Other solvents were dried/purified according to the literature procedures. Elemental analysis, Elemental CHN Perkin-Elmer 2400. 2.1.1. Plant material The plant material was collected in Bom Retiro, in the State of Santa Catarina, Brazil, in June 2000 and classified by Prof.
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Leila da Graça Amaral. A voucher specimen was deposited at the FLOR Herbarium (UFSC), Florianópolis-SC, Brazil, under number 4725. 2.1.2. Isolation of marrubiin The air-dried leaves (1.5 kg) of M. vulgare were extracted with methanol for 7 days at room temperature (25 ± 3 °C). The solvent was evaporated to the desired level, using 70 °C to promote the formation of marrubiin from premarrubiin of the plant [7]. The extract was then chromatographed on a silica gel column using hexane/ethyl acetate with increasing polarity. Marrubiin (1) was obtained with a yield of about 1% and identified by IR, 1H and 13C-NMR [4,7].
(CH-14), 126.12 (C-13), 129.08–135.88 (CH, C-aromatic), 139.15 (CH-16), 143.55 (CH-15), 181.64 (COO-19). 2.1.3.4. Marrubiinic acid methyl ester (5). Marrubiinic acid (0.143 mmol), acetone, potassium carbonate (0.429 mmol) and dimethyl sulfate (0.315 mmol) were refluxed for 3 h and acidified to give the compound (5) with a yield of 50.4%. This compound was purified by CC (n-hexane: ethyl acetate 7:3). M.p. 91.1–94.8 °C; anal. calcd. for C21H32O5: C, 69.2; H, 8.85; O, 21.95%. Found: C, 69.06; H, 8.74; O, 21.74%. IR: m = 3420 (OH), 1765 (C=O). 1H NMR (acetone-d6): d = 3.76 (s, 3H, OCH3), 4.31 (s, 1H, CH-6), 6.36 (s, 1H, CH-14), 7.35 (s, 1H, CH-16), 7.44 (s, 1H, CH-15). 2.2. Pharmacology
2.1.3. General procedure for the synthesis of marrubiin derivatives 2.1.3.1. Marrubiinic acid (2). Marrubiin (0.150 mmol) (1) was hydrolysed in refluxing with ethoxyethanol and potassium hydroxide (0.214 mmol), 0.03 g/mL, with a minimum of water during 15 h and acidified to give the compound (2) marrubiinic acid. A white solid was obtained using HCl 1 N with 94% of yield. M.p. 153–154 °C. Anal. calcd. for C20H30O5: C, 68.54; H, 8.63; O, 22.84%. Found: C, 68.36; H, 8.41; O, 22.55%. 1H NMR (DMSO-d6): d = 0.9 (s, 3H, CH3-17), 1.0 (s, 3H, CH3-20), 2.15 (d, 2H, CH2-7), 2.38 (d, 1H, CH-5), 4.24 (sl, 1H, CH-6), 6.36 (s, 1H, CH-14), 7.41 (s, 1H, CH-16), 7.52 (s, 1H, CH-15), 13.9 (bs, 1H, acid). 13C NMR (DMSO-d6): d = 16.23 (CH3-17), 20.97 (CH3-20), 28.36 (CH2-3), 29.51 (CH2-7), 43.16 (C-4), 45.90 (CH-5), 65.01 (CH-6), 75.68 (C-9), 111.07 (CH-14), 125.65 (C-13), 138.44 (CH-16), 142.96 (CH-15), 179.13 (C-19). 2.1.3.2. Marrubenol (3). Marrubiin (0.150 mmol) (1) was reduced using lithium aluminium hydride (0.151 mmol) in dried-THF to obtain marrubenol (3). This compound was purified by CC over silica gel (n-hexane: ethyl acetate 7:3) with 70% of yield. This compound has already been isolated from Marrubium vulgare [5,8]. Anal. calcd. for C27H36O5: C, 71.82; H, 9.04; O, 19.14%. Found: C, 71.66; H, 8.96; O, 19.02%. 2.1.3.3. Marrubiinic acid benzyl ester (4). Marrubiinic acid (0.143 mmol) was submitted to stirring with acetone, potassium hydroxide (0.352 mmol) and benzyl bromide (1.684 mmol) for 3 h. The compound was purified by CC over silica gel (n-hexane: ethyl acetate 7:3) with a yield of 45%. M.p. 81.5–83.9 °C; anal. calcd. for C27H36O5: C, 73.60; H, 8.24; O, 18.16%. Found: C, 73.45; H, 8.18; O, 18.12%. IR: m = 3410 (OH), 3088 (C=C), 1636 (C=O). 1H NMR (CDCl3): d = 4.38 (sl, 1H, CH-6), 5.10 (dd, 2H, CH2-20), 6.26 (S, 1H, CH-14), 7.20 (s, 1H, CH-16), 7.34 (s, 1H, CH-15), 7.36 (s, 5H, aromatic). 13C NMR: d = 16.91 (CH3-17), 19.56 (CH3-18), 22.04 (CH3-20), 30.00 (CH2-3), 31.09 (CH2-7), 44.35 (C-4), 47.01 (CH-5), 68.18 (CH-6), 77.07 (C-9), 111.48
2.2.1. Animals Male Swiss mice (25–35 g) were used, housed at 22 ± 2 °C under a 12 h light/12 h dark cycle and with access to food and water ad libitum. The experiments were performed during the light phase of the cycle. The animals were acclimatized to the laboratory for at least 2 h before testing and were used once throughout the experiments. All the experiments reported in this study were carried out in accordance with the current guidelines for the care of laboratory animals and ethical guidelines for investigation of experimental pain in conscious animals [9]. 2.2.2. Drugs The following substances were used: acetic acid, formalin, L-glutamic acid, capsaicin (Calbiochen, San Diego, CA, USA), and morphine hidrochloride (Merck, Darmstadt, Germany). The marrubiinic acid, as well as the reference drugs, was dissolved in Tween 80 (E. Merck), plus 0.9% of NaCl solution, with the exception of glutamate which was prepared only in phosphate buffered saline (PBS, composition mmol/L: NaCl 137, KCl 2.7 and phosphate buffer 10) and capsaicin which was dissolved in ethanol. The final concentration of Tween 80 and ethanol did not exceed 5% and did not cause any effect ’per se’. 2.2.3. General procedures of pharmacological assay 2.2.3.1. Acetic acid-induced writhing. Abdominal constriction was induced in mice by intraperitoneal injection of acetic acid (0.6%), as described by Collier et al. with minor modifications [10,11]. The animals were pre-treated intraperitoneally (3, 6 and 10 mg kg−1 , 30 min before) and orally (50 mg kg−1, 60 min before) with the studied compounds. The control animals received a similar volume of saline solution (10 ml kg−1). The number of abdominal constrictions (full extension of both hind paws) was cumulatively counted over a period of 20 min. Antinociceptive activity was expressed as the reduction in the number of abdominal constrictions between the control animals and the mice pre-treated with compounds.
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2.2.3.2. Formalin test. The observation chamber was a glass cylinder of 20 cm in diameter, with a mirror at a 45° angle to allow clear observation of the animal’s paws. The mice were treated with 0.9% saline solution (i.p.), or marrubiinic acid (2) (10 mg kg–1 , i.p.) 30 min before formalin injection. Each animal was placed in the chamber for 5 min before treatment in order to allow acclimatization to the new environment. The formalin test was carried out as described by Hunskaar and Hole with minor modifications [11,12]. Twenty microlitres of a 2.5% formalin solution (0.92% formaldehyde) in 0.9% saline solution were injected intraplantarly in the right hind paw. The animal was then returned to the chamber and the amount of time spent licking the injected paw was considered as indicative of pain. Two distinct phases of intensive licking activity were identified: an early acute phase and a late or tonic phase (0–5 and 15–30 min after formalin injection, respectively). 2.2.3.3. Capsaicin-induced nociception. In an attempt to provide more direct evidence concerning its possible antinociceptive effect on neurogenic nociception, marrubiinic acid (2) was investigated in capsaicin-induced licking in the mouse paw. The procedure used was similar to that described previously [13]. After the adaptation period, 20 ml of capsaicin (1.6 µg/paw) was injected intraplantarly in the right hindpaw. The animals were observed individually for 5 min following capsaicin injection. The amount of time spent licking the injected paw was timed with a chronometer and was considered as indicative of nociception. The animals were treated with the marrubiinic acid via i.p. (10 mg/kg) 30 min before capsaicin injection, respectively. The control animals received a similar volume of saline, intraperitoneally. 2.2.3.4. Glutamate-induced nociception. The animals were treated with the marrubiinic acid (2) via i.p. (10 mg/kg) 30 min before capsaicin injection, respectively. A volume of 20 µl of glutamate solution (30 µmol/ paw), made up in phosphate buffered saline (PBS), was injected intraplantarly under the surface of the right hindpaw as described previously [14]. After injection with glutamate, the animals were individually placed into glass cylinders 20 cm in diameter and observed from 0 to 15 min. The time spent licking and biting the injected paw was timed with a chronometer and considered as indicative of pain. 2.2.3.5. Hot-plate test. The hot-plate test was used to measure response latencies according to the method described by Eddy and Leimback [15]. The mice were treated with saline solution, morphine (10 mg kg–1 , s.c.) or marrubiinic acid (10 mg kg.1, i.p.) placed individually on a hot plate maintained at 56 ± 1 °C and the time between placing the animal on the hot plate and the occurrence of either the licking of the hind paws, shaking the paw or jumping off the surface was recorded as response latency. Mice with baseline latencies of more than 20 s were eliminated from the study and the cutoff time for the hot-plate latencies was set at 30 s. The animals were treated 30 min before the assay.
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2.2.4. Statistical analysis The results are presented as mean ± S.E.M., except for the ID values (i.e., the dose of marrubiinic acid reducing the 50 nociceptive response by 50% relative to the control value), which are reported as geometric means, accompanied by their respective 95% confidence limits. The ID value was 50 determined by linear regression from individual experiments using linear regression Graph Pad software (Graph Pad software, San Diego, CA). The statistical significance of the differences between the groups was detected by ANOVA, followed by Dunnett’s multiple comparison test. P-values of less than 0.05 (P < 0.05) were considered as indicative of significance.
3. Results and discussion Considering the high yield (~1.0%) and the promising pharmacological activity of marrubiin demonstrated in previous studies carried out in our laboratories [4], some structural modifications were performed in order to obtain more active compounds. The marrubiin (1) was hydrolysed in refluxing ethoxyethanol with potassium hydroxide containing a little water for 15 h and acidified to give marrubiinic acid (2), which was reduced using lithium aluminium hydride in dried-THF to obtain marrubenol (3) with yields of 70% and 98%, respectively. Because of the high yield, easy obtaining and potent antinociceptive activity of compound 2, the present study focused on the structural modifications with this compound through estherification reactions. Compound (4) was obtained from marrubiinic acid using sodium hydroxide and benzyl bromide in dried acetone at room temperature for 3 h, given a yield of 45%. The marrubiinic acid (3) was submitted to the estherification reaction to give compound (5) with a yield of 50.4%. The compounds shown in Fig. 1 were initially evaluated using the writhing test in mice (10 mg/kg) (Table 1). Marrubiinic acid (2), exhibited the most interesting activity, causing 80% inhibition of the abdominal constrictions, and was then submitted to formalin, capsaicin and hot-plate assays. It is important to mention that it presents an important functional group (COOH), which gives the structure an acidic characteristic that can be important for its antinociceptive activity, since several classes of different drugs possess this characteristic [16]. Pharmacological studies indicated that marrubiinic acid (2) presents a significant (p < 0.05) and dose-dependent antinociceptive effect, against the writhing test, given intraperitoneally, with ID50 value of 12 µmol/kg, being about 11-fold more active than the standard drugs used as reference, but less active than marrubiin [4]. This suggests that the opening of the chain led to a decrease of activity in this model (Table 2). In this pharmacological test, since intraperitoneal injection of acetic acid (AA) directly activates visceral and somatic
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Fig. 2. Effect of marrubiinic acid against acetic acid-induced pain in mice. Each group represents the mean of six experiments. **p < 0.01, compared with the corresponding control value.
Fig. 1. Compounds obtained from marrubiin and marrubiinic acid.
nociceptors innervating the peritoneum, and induces inflammation not only in subdiaphragmatic visceral organs but also in subcutaneous muscle walls, the antinociceptive effect observed in AA conditioning may not be purely visceral in origin [17]. Given orally at 50 mg/kg, marrubiinic acid (2) caused a pronounced analgesic effect, reducing 76 ± 0.9% the number of abdominal constrictions induced by acetic acid, which suggests that it can be well absorbed by the gastrointestinal tract (Fig. 2). It was more active than the reference drugs used here for the purpose of comparison, which present ID50 of 100–200 mg/kg in the same model [18]. This can be explained by the increase in water solubility of the compound (2) due to the hydrogen bond-forming potential of the functional group, Table 1 Antinociceptive effect of marrubiin and derivatives against the writhing test at 10 mg/kg, i.p. Compound Marrubiin (1) Marrubiinic acid (2) Marrubenol (3)a Marrubiinic acid benzyl ester (4) Marrubiinic acid methyl ester (5) Aspirin (6)
Inhibition (%) 92 ± 1 80 ± 2 94 ± 0.8 44.1 ± 2 48 ± 1 35 ± 2
Each group represents the mean ± SEM of 6–8 experiments. a From Ref. [8]. Table 2 Comparison of the antinociceptive effect of marrubiinic acid (2) with marrubin (1) and non-steroidal anti-inflammatory and analgesic drugs given inraperitoneally in mice Compound (10 mg/kg) Marrubiinic acid Marrubiin a Aspirin Paracetamol
Writhing test ID50 (µmol/kg, i.p.) 12 (8.4–16.8) 2.2 (1.1–3.0) 133 (73–243) 125 (104–250)
Each group represents the mean ± SEM of 6–8 experiments. a From Ref. [4].
carboxylic acid, and its possibility of ionization. The solubility of a drug molecule in water greatly affects the routes of administration available and its absorption, distribution and elimination [16]. The formalin test, also revealed an antinociceptive profile, though less pronounced, and (2) was more active in preventing the late than the first phase of the formalin-induced licking. The respective inhibitions were 28.3 ± 3.6% for the first phase and 46.7 ± 8.3% for the second phase. It was more active than aspirin, which is inactive for the first phase and caused inhibition of 39.0 ± 4% for the second phase. However, it was less active than the prototype compound, marrubiin, which inhibited both phases of pain (Table 3) [4]. In the formalin test, there is a distinctive biphasic nociceptive response termed early and late phases [12]. Drugs that act primarily on the central nervous system inhibit both phases equally, while peripherally acting drugs inhibit the late phase. The early phase is probably a direct result of the stimulation of nociceptors in the paw and reflects centrally mediated pain, while the late phase is due to inflammation with a release of serotonin, histamine, bradykinin and prostaglandins and, to a certain degree, the sensitization of the central nociceptive neurons [19]. Thus, our results suggest that like marrubiin, compound (2) exhibits both central and peripheral effects. We also observed that marrubiinic acid (2) exhibits a similar analgesic profile to that of marrubiin (1) [4] in the other experimental models. Compound (2) was not effective in abolishing pain in a non-opioid way, showing the lack of antinociceptive effects in the hot-plate test (Fig. 3) [4]. When evaluated against the capsaicin test, which provided more direct evidence of Table 3 Antinociceptive action of marrubiinic acid (2), marrubiin (1) and aspirin (reference drug) against the first (0–5 min) and second (15–30 min) phases in a formalin test in mice (10 mg/kg, i.p.) Compound Marrubiinic acid Marrubiin a Aspirin
First phase (% inhibition) 28.3 ( ± 3.6)** 78.0 ( ± 5)** Inactive
Second phase (% inhibition) 46.7 ( ± 8.3)** 83.0 ( ± 4)** 39.0 ( ± 4)*
Each group represents the mean ± SEM of 6–8 animals. *P values < 0.05 and **<0.01 compared with respective control values. a From Ref. [4].
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the antinociceptive effect of this compound on neurogenic pain, it caused an inhibition of 37.3 ± 3.8% at 10 mg/kg of capsaicin-induced licking (Fig. 4), suggesting its involvement with the antagonism of vanilloid receptor [20]. Of great interest are the findings which demonstrate that the antinociception caused by marrubiinic acid (2) could involve, at least in part, its ability to interact with excitatory amino acids, as demonstrated by the fact that it caused inhibition of 52.5 ± 6.9% of the hyperalgesia induced by intraplantar injection of glutamate in mice (Fig. 5). According to Beirith et al. [14], the intraplantar injection of glutamate into the mouse hindpaw results in a rapid onset and short duration of nociception, the response of which is greatly mediated by both N-methyl-D-aspartate (NMDA) and non-NMDA (aamino-3-hydroxyl-5-methyl-4-isoxazolepropionate) (AMPA), kainite (KA) and metabotropic) receptors and by the glycine modulatory site [14]. In summary, we have reported that marrubiinic acid (2) significantly reduced the number of writhings induced by the 0.6% acetic acid solution. Although this test is a non-specific model (i.e. anticholinergic and antihistaminic and other agents also show activity in this test), it is widely used for analgesic screening and involves local peritoneal receptors (cholinergic and histamine receptor) and the mediators of acetylcholine and histamine [21]. This compound also elicited a spinal and supraspinal antinociception when assessed against both
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Fig. 5. Effect of marrubiinic acid on licking/biting response induced by intraplantar injection of glutamate in mice. Each group represents the mean of six experiments. ** p < 0.01, compared with the corresponding control value.
formalin- and capsaicin-induced neurogenic pain as well as in glutamate-induced hyperalgesia in mice. The precise mechanism underlying the antinociceptive action of marrubiinic acid has yet to be determined, but it is unlikely to be associated with an interaction with opioid peptides. Although marrubiinic acid (2) showed lesser antinociceptive effects than its prototype marrubiin (1), it was more potent than some clinically used drugs. Another important observation is its action by the oral route. Finally, the results showed that (2) could be used as a model to obtain new and more potent analgesic drugs. Acknowledgements The authors are grateful to CNPq and FUNCITEC-SC for financial support. References [1]
Fig. 3. Effect of marrubiinic acid and morphine against hot-plate-induced antinociception in mice. Each group represents the mean of six experiments. **p < 0.01, compared with the corresponding control value.
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[8] Fig. 4. Effect of marrubiinic acid on licking/biting response induced by intraplantar injection of capsaincin in mice. Each group represents the mean of six experiments. **p < 0.01, compared with corresponding control value.
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Analgesic potential of marrubiin derivatives, a bioactive diterpene present in Marrubium vulgare (Lamiaceae) C. Meyre-Silva a, R.A. Yunes b, V. Schlemper a, F. Campos-Buzzi a, V. Cechinel-Filho a,* a
Núcleo de Investigações Químico-Farmacêuticas (NIQFAR)/Curso de Farmácia-CCS, Universidade do Vale do Itajaí, Itajaí-SC, Brazil b Departamento de Química, Universidade Federal de Santa Catarina, Florianópolis-SC, Brazil Received 17 November 2004; received in revised form 20 December 2004; accepted 7 January 2005 Available online 02 March 2005
Abstract Marrubiin, a furane labdane diterpene, is the main analgesic compound present in Marrubium vulgare, a medicinal plant used in Brazil and other countries to treat several ailments. Considering its important pharmacological action, as well as its high yield, some structural modifications were performed in order to obtain more active compounds. Success was obtained in reducing the lactonic function, in the formation of marrubiinic acid and two esterified derivatives, which exhibited significant analgesic effect against the writhing test in mice. Marrubiinic acid showed better activity and excellent yield, and its analgesic effect was confirmed in other experimental models of pain in mice, suggesting its possible use as a model to obtain new and potent analgesic agents. © 2005 Elsevier SAS. All rights reserved. Keywords: Marrubium vulgare; Marrubiin derivatives; Analgesic action
1. Introduction Marrubium vulgare (Lamiaceae), known as ″maromba″ or ″marroio″ in Brazil, or horehound in Europe, is frequently used in folk medicine to cure a variety of diseases. Preliminary evaluations of this plant in our laboratory have demonstrated hypoglycemic, analgesic and antiespasmodic effects [1–3]. Chemically, marrubiin (1), a furane labdane diterpene, is the main component of this plant, and exhibits potent antinociceptive properties and vasorelaxant activity [4,5]. It is formed from the genuine premarrubiin during the work-up procedure [6]. In the present study, we have extended our previous investigations and obtained some marrubiin derivatives with the aim of optimizing its antinociceptive activity initially using the writhing test in mice. The most active compound was studied in other experimental models of pain. The results of some reference drugs, such as aspirin and paracetamol, were also included for the purpose of comparison (Scheme 1).
* Corresponding author. Tel.: +55 47 341 7664; fax: +55 47 341 7601. E-mail address: [email protected] (V. Cechinel-Filho). 0014-827X/$ - see front matter © 2005 Elsevier SAS. All rights reserved. doi:10.1016/j.farmac.2005.01.003
Scheme 1.
2. Experimental 2.1. Chemistry IR spectra (cm–1): Perkin-Elmer FT-16-PC; in KBr. NMR spectra: Bruker AC-200F (200 MHz) for 1H; Bruker AC-200F (50 MHz) for 13C; d in ppm, J in Hz. 1H values and 13C values from spectra in DMSO, CDCl3 or acetone-d6. Standard reference for all spectra was TMS. dTMS = 0 ppm. Tethrahydrofuran (THF) was stored with KOH, then refluxed and distilled prior to use. Other solvents were dried/purified according to the literature procedures. Elemental analysis, Elemental CHN Perkin-Elmer 2400. 2.1.1. Plant material The plant material was collected in Bom Retiro, in the State of Santa Catarina, Brazil, in June 2000 and classified by Prof.
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Leila da Graça Amaral. A voucher specimen was deposited at the FLOR Herbarium (UFSC), Florianópolis-SC, Brazil, under number 4725. 2.1.2. Isolation of marrubiin The air-dried leaves (1.5 kg) of M. vulgare were extracted with methanol for 7 days at room temperature (25 ± 3 °C). The solvent was evaporated to the desired level, using 70 °C to promote the formation of marrubiin from premarrubiin of the plant [7]. The extract was then chromatographed on a silica gel column using hexane/ethyl acetate with increasing polarity. Marrubiin (1) was obtained with a yield of about 1% and identified by IR, 1H and 13C-NMR [4,7].
(CH-14), 126.12 (C-13), 129.08–135.88 (CH, C-aromatic), 139.15 (CH-16), 143.55 (CH-15), 181.64 (COO-19). 2.1.3.4. Marrubiinic acid methyl ester (5). Marrubiinic acid (0.143 mmol), acetone, potassium carbonate (0.429 mmol) and dimethyl sulfate (0.315 mmol) were refluxed for 3 h and acidified to give the compound (5) with a yield of 50.4%. This compound was purified by CC (n-hexane: ethyl acetate 7:3). M.p. 91.1–94.8 °C; anal. calcd. for C21H32O5: C, 69.2; H, 8.85; O, 21.95%. Found: C, 69.06; H, 8.74; O, 21.74%. IR: m = 3420 (OH), 1765 (C=O). 1H NMR (acetone-d6): d = 3.76 (s, 3H, OCH3), 4.31 (s, 1H, CH-6), 6.36 (s, 1H, CH-14), 7.35 (s, 1H, CH-16), 7.44 (s, 1H, CH-15). 2.2. Pharmacology
2.1.3. General procedure for the synthesis of marrubiin derivatives 2.1.3.1. Marrubiinic acid (2). Marrubiin (0.150 mmol) (1) was hydrolysed in refluxing with ethoxyethanol and potassium hydroxide (0.214 mmol), 0.03 g/mL, with a minimum of water during 15 h and acidified to give the compound (2) marrubiinic acid. A white solid was obtained using HCl 1 N with 94% of yield. M.p. 153–154 °C. Anal. calcd. for C20H30O5: C, 68.54; H, 8.63; O, 22.84%. Found: C, 68.36; H, 8.41; O, 22.55%. 1H NMR (DMSO-d6): d = 0.9 (s, 3H, CH3-17), 1.0 (s, 3H, CH3-20), 2.15 (d, 2H, CH2-7), 2.38 (d, 1H, CH-5), 4.24 (sl, 1H, CH-6), 6.36 (s, 1H, CH-14), 7.41 (s, 1H, CH-16), 7.52 (s, 1H, CH-15), 13.9 (bs, 1H, acid). 13C NMR (DMSO-d6): d = 16.23 (CH3-17), 20.97 (CH3-20), 28.36 (CH2-3), 29.51 (CH2-7), 43.16 (C-4), 45.90 (CH-5), 65.01 (CH-6), 75.68 (C-9), 111.07 (CH-14), 125.65 (C-13), 138.44 (CH-16), 142.96 (CH-15), 179.13 (C-19). 2.1.3.2. Marrubenol (3). Marrubiin (0.150 mmol) (1) was reduced using lithium aluminium hydride (0.151 mmol) in dried-THF to obtain marrubenol (3). This compound was purified by CC over silica gel (n-hexane: ethyl acetate 7:3) with 70% of yield. This compound has already been isolated from Marrubium vulgare [5,8]. Anal. calcd. for C27H36O5: C, 71.82; H, 9.04; O, 19.14%. Found: C, 71.66; H, 8.96; O, 19.02%. 2.1.3.3. Marrubiinic acid benzyl ester (4). Marrubiinic acid (0.143 mmol) was submitted to stirring with acetone, potassium hydroxide (0.352 mmol) and benzyl bromide (1.684 mmol) for 3 h. The compound was purified by CC over silica gel (n-hexane: ethyl acetate 7:3) with a yield of 45%. M.p. 81.5–83.9 °C; anal. calcd. for C27H36O5: C, 73.60; H, 8.24; O, 18.16%. Found: C, 73.45; H, 8.18; O, 18.12%. IR: m = 3410 (OH), 3088 (C=C), 1636 (C=O). 1H NMR (CDCl3): d = 4.38 (sl, 1H, CH-6), 5.10 (dd, 2H, CH2-20), 6.26 (S, 1H, CH-14), 7.20 (s, 1H, CH-16), 7.34 (s, 1H, CH-15), 7.36 (s, 5H, aromatic). 13C NMR: d = 16.91 (CH3-17), 19.56 (CH3-18), 22.04 (CH3-20), 30.00 (CH2-3), 31.09 (CH2-7), 44.35 (C-4), 47.01 (CH-5), 68.18 (CH-6), 77.07 (C-9), 111.48
2.2.1. Animals Male Swiss mice (25–35 g) were used, housed at 22 ± 2 °C under a 12 h light/12 h dark cycle and with access to food and water ad libitum. The experiments were performed during the light phase of the cycle. The animals were acclimatized to the laboratory for at least 2 h before testing and were used once throughout the experiments. All the experiments reported in this study were carried out in accordance with the current guidelines for the care of laboratory animals and ethical guidelines for investigation of experimental pain in conscious animals [9]. 2.2.2. Drugs The following substances were used: acetic acid, formalin, L-glutamic acid, capsaicin (Calbiochen, San Diego, CA, USA), and morphine hidrochloride (Merck, Darmstadt, Germany). The marrubiinic acid, as well as the reference drugs, was dissolved in Tween 80 (E. Merck), plus 0.9% of NaCl solution, with the exception of glutamate which was prepared only in phosphate buffered saline (PBS, composition mmol/L: NaCl 137, KCl 2.7 and phosphate buffer 10) and capsaicin which was dissolved in ethanol. The final concentration of Tween 80 and ethanol did not exceed 5% and did not cause any effect ’per se’. 2.2.3. General procedures of pharmacological assay 2.2.3.1. Acetic acid-induced writhing. Abdominal constriction was induced in mice by intraperitoneal injection of acetic acid (0.6%), as described by Collier et al. with minor modifications [10,11]. The animals were pre-treated intraperitoneally (3, 6 and 10 mg kg−1 , 30 min before) and orally (50 mg kg−1, 60 min before) with the studied compounds. The control animals received a similar volume of saline solution (10 ml kg−1). The number of abdominal constrictions (full extension of both hind paws) was cumulatively counted over a period of 20 min. Antinociceptive activity was expressed as the reduction in the number of abdominal constrictions between the control animals and the mice pre-treated with compounds.
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2.2.3.2. Formalin test. The observation chamber was a glass cylinder of 20 cm in diameter, with a mirror at a 45° angle to allow clear observation of the animal’s paws. The mice were treated with 0.9% saline solution (i.p.), or marrubiinic acid (2) (10 mg kg–1 , i.p.) 30 min before formalin injection. Each animal was placed in the chamber for 5 min before treatment in order to allow acclimatization to the new environment. The formalin test was carried out as described by Hunskaar and Hole with minor modifications [11,12]. Twenty microlitres of a 2.5% formalin solution (0.92% formaldehyde) in 0.9% saline solution were injected intraplantarly in the right hind paw. The animal was then returned to the chamber and the amount of time spent licking the injected paw was considered as indicative of pain. Two distinct phases of intensive licking activity were identified: an early acute phase and a late or tonic phase (0–5 and 15–30 min after formalin injection, respectively). 2.2.3.3. Capsaicin-induced nociception. In an attempt to provide more direct evidence concerning its possible antinociceptive effect on neurogenic nociception, marrubiinic acid (2) was investigated in capsaicin-induced licking in the mouse paw. The procedure used was similar to that described previously [13]. After the adaptation period, 20 ml of capsaicin (1.6 µg/paw) was injected intraplantarly in the right hindpaw. The animals were observed individually for 5 min following capsaicin injection. The amount of time spent licking the injected paw was timed with a chronometer and was considered as indicative of nociception. The animals were treated with the marrubiinic acid via i.p. (10 mg/kg) 30 min before capsaicin injection, respectively. The control animals received a similar volume of saline, intraperitoneally. 2.2.3.4. Glutamate-induced nociception. The animals were treated with the marrubiinic acid (2) via i.p. (10 mg/kg) 30 min before capsaicin injection, respectively. A volume of 20 µl of glutamate solution (30 µmol/ paw), made up in phosphate buffered saline (PBS), was injected intraplantarly under the surface of the right hindpaw as described previously [14]. After injection with glutamate, the animals were individually placed into glass cylinders 20 cm in diameter and observed from 0 to 15 min. The time spent licking and biting the injected paw was timed with a chronometer and considered as indicative of pain. 2.2.3.5. Hot-plate test. The hot-plate test was used to measure response latencies according to the method described by Eddy and Leimback [15]. The mice were treated with saline solution, morphine (10 mg kg–1 , s.c.) or marrubiinic acid (10 mg kg.1, i.p.) placed individually on a hot plate maintained at 56 ± 1 °C and the time between placing the animal on the hot plate and the occurrence of either the licking of the hind paws, shaking the paw or jumping off the surface was recorded as response latency. Mice with baseline latencies of more than 20 s were eliminated from the study and the cutoff time for the hot-plate latencies was set at 30 s. The animals were treated 30 min before the assay.
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2.2.4. Statistical analysis The results are presented as mean ± S.E.M., except for the ID values (i.e., the dose of marrubiinic acid reducing the 50 nociceptive response by 50% relative to the control value), which are reported as geometric means, accompanied by their respective 95% confidence limits. The ID value was 50 determined by linear regression from individual experiments using linear regression Graph Pad software (Graph Pad software, San Diego, CA). The statistical significance of the differences between the groups was detected by ANOVA, followed by Dunnett’s multiple comparison test. P-values of less than 0.05 (P < 0.05) were considered as indicative of significance.
3. Results and discussion Considering the high yield (~1.0%) and the promising pharmacological activity of marrubiin demonstrated in previous studies carried out in our laboratories [4], some structural modifications were performed in order to obtain more active compounds. The marrubiin (1) was hydrolysed in refluxing ethoxyethanol with potassium hydroxide containing a little water for 15 h and acidified to give marrubiinic acid (2), which was reduced using lithium aluminium hydride in dried-THF to obtain marrubenol (3) with yields of 70% and 98%, respectively. Because of the high yield, easy obtaining and potent antinociceptive activity of compound 2, the present study focused on the structural modifications with this compound through estherification reactions. Compound (4) was obtained from marrubiinic acid using sodium hydroxide and benzyl bromide in dried acetone at room temperature for 3 h, given a yield of 45%. The marrubiinic acid (3) was submitted to the estherification reaction to give compound (5) with a yield of 50.4%. The compounds shown in Fig. 1 were initially evaluated using the writhing test in mice (10 mg/kg) (Table 1). Marrubiinic acid (2), exhibited the most interesting activity, causing 80% inhibition of the abdominal constrictions, and was then submitted to formalin, capsaicin and hot-plate assays. It is important to mention that it presents an important functional group (COOH), which gives the structure an acidic characteristic that can be important for its antinociceptive activity, since several classes of different drugs possess this characteristic [16]. Pharmacological studies indicated that marrubiinic acid (2) presents a significant (p < 0.05) and dose-dependent antinociceptive effect, against the writhing test, given intraperitoneally, with ID50 value of 12 µmol/kg, being about 11-fold more active than the standard drugs used as reference, but less active than marrubiin [4]. This suggests that the opening of the chain led to a decrease of activity in this model (Table 2). In this pharmacological test, since intraperitoneal injection of acetic acid (AA) directly activates visceral and somatic
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Fig. 2. Effect of marrubiinic acid against acetic acid-induced pain in mice. Each group represents the mean of six experiments. **p < 0.01, compared with the corresponding control value.
Fig. 1. Compounds obtained from marrubiin and marrubiinic acid.
nociceptors innervating the peritoneum, and induces inflammation not only in subdiaphragmatic visceral organs but also in subcutaneous muscle walls, the antinociceptive effect observed in AA conditioning may not be purely visceral in origin [17]. Given orally at 50 mg/kg, marrubiinic acid (2) caused a pronounced analgesic effect, reducing 76 ± 0.9% the number of abdominal constrictions induced by acetic acid, which suggests that it can be well absorbed by the gastrointestinal tract (Fig. 2). It was more active than the reference drugs used here for the purpose of comparison, which present ID50 of 100–200 mg/kg in the same model [18]. This can be explained by the increase in water solubility of the compound (2) due to the hydrogen bond-forming potential of the functional group, Table 1 Antinociceptive effect of marrubiin and derivatives against the writhing test at 10 mg/kg, i.p. Compound Marrubiin (1) Marrubiinic acid (2) Marrubenol (3)a Marrubiinic acid benzyl ester (4) Marrubiinic acid methyl ester (5) Aspirin (6)
Inhibition (%) 92 ± 1 80 ± 2 94 ± 0.8 44.1 ± 2 48 ± 1 35 ± 2
Each group represents the mean ± SEM of 6–8 experiments. a From Ref. [8]. Table 2 Comparison of the antinociceptive effect of marrubiinic acid (2) with marrubin (1) and non-steroidal anti-inflammatory and analgesic drugs given inraperitoneally in mice Compound (10 mg/kg) Marrubiinic acid Marrubiin a Aspirin Paracetamol
Writhing test ID50 (µmol/kg, i.p.) 12 (8.4–16.8) 2.2 (1.1–3.0) 133 (73–243) 125 (104–250)
Each group represents the mean ± SEM of 6–8 experiments. a From Ref. [4].
carboxylic acid, and its possibility of ionization. The solubility of a drug molecule in water greatly affects the routes of administration available and its absorption, distribution and elimination [16]. The formalin test, also revealed an antinociceptive profile, though less pronounced, and (2) was more active in preventing the late than the first phase of the formalin-induced licking. The respective inhibitions were 28.3 ± 3.6% for the first phase and 46.7 ± 8.3% for the second phase. It was more active than aspirin, which is inactive for the first phase and caused inhibition of 39.0 ± 4% for the second phase. However, it was less active than the prototype compound, marrubiin, which inhibited both phases of pain (Table 3) [4]. In the formalin test, there is a distinctive biphasic nociceptive response termed early and late phases [12]. Drugs that act primarily on the central nervous system inhibit both phases equally, while peripherally acting drugs inhibit the late phase. The early phase is probably a direct result of the stimulation of nociceptors in the paw and reflects centrally mediated pain, while the late phase is due to inflammation with a release of serotonin, histamine, bradykinin and prostaglandins and, to a certain degree, the sensitization of the central nociceptive neurons [19]. Thus, our results suggest that like marrubiin, compound (2) exhibits both central and peripheral effects. We also observed that marrubiinic acid (2) exhibits a similar analgesic profile to that of marrubiin (1) [4] in the other experimental models. Compound (2) was not effective in abolishing pain in a non-opioid way, showing the lack of antinociceptive effects in the hot-plate test (Fig. 3) [4]. When evaluated against the capsaicin test, which provided more direct evidence of Table 3 Antinociceptive action of marrubiinic acid (2), marrubiin (1) and aspirin (reference drug) against the first (0–5 min) and second (15–30 min) phases in a formalin test in mice (10 mg/kg, i.p.) Compound Marrubiinic acid Marrubiin a Aspirin
First phase (% inhibition) 28.3 ( ± 3.6)** 78.0 ( ± 5)** Inactive
Second phase (% inhibition) 46.7 ( ± 8.3)** 83.0 ( ± 4)** 39.0 ( ± 4)*
Each group represents the mean ± SEM of 6–8 animals. *P values < 0.05 and **<0.01 compared with respective control values. a From Ref. [4].
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the antinociceptive effect of this compound on neurogenic pain, it caused an inhibition of 37.3 ± 3.8% at 10 mg/kg of capsaicin-induced licking (Fig. 4), suggesting its involvement with the antagonism of vanilloid receptor [20]. Of great interest are the findings which demonstrate that the antinociception caused by marrubiinic acid (2) could involve, at least in part, its ability to interact with excitatory amino acids, as demonstrated by the fact that it caused inhibition of 52.5 ± 6.9% of the hyperalgesia induced by intraplantar injection of glutamate in mice (Fig. 5). According to Beirith et al. [14], the intraplantar injection of glutamate into the mouse hindpaw results in a rapid onset and short duration of nociception, the response of which is greatly mediated by both N-methyl-D-aspartate (NMDA) and non-NMDA (aamino-3-hydroxyl-5-methyl-4-isoxazolepropionate) (AMPA), kainite (KA) and metabotropic) receptors and by the glycine modulatory site [14]. In summary, we have reported that marrubiinic acid (2) significantly reduced the number of writhings induced by the 0.6% acetic acid solution. Although this test is a non-specific model (i.e. anticholinergic and antihistaminic and other agents also show activity in this test), it is widely used for analgesic screening and involves local peritoneal receptors (cholinergic and histamine receptor) and the mediators of acetylcholine and histamine [21]. This compound also elicited a spinal and supraspinal antinociception when assessed against both
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Fig. 5. Effect of marrubiinic acid on licking/biting response induced by intraplantar injection of glutamate in mice. Each group represents the mean of six experiments. ** p < 0.01, compared with the corresponding control value.
formalin- and capsaicin-induced neurogenic pain as well as in glutamate-induced hyperalgesia in mice. The precise mechanism underlying the antinociceptive action of marrubiinic acid has yet to be determined, but it is unlikely to be associated with an interaction with opioid peptides. Although marrubiinic acid (2) showed lesser antinociceptive effects than its prototype marrubiin (1), it was more potent than some clinically used drugs. Another important observation is its action by the oral route. Finally, the results showed that (2) could be used as a model to obtain new and more potent analgesic drugs. Acknowledgements The authors are grateful to CNPq and FUNCITEC-SC for financial support. References [1]
Fig. 3. Effect of marrubiinic acid and morphine against hot-plate-induced antinociception in mice. Each group represents the mean of six experiments. **p < 0.01, compared with the corresponding control value.
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[8] Fig. 4. Effect of marrubiinic acid on licking/biting response induced by intraplantar injection of capsaincin in mice. Each group represents the mean of six experiments. **p < 0.01, compared with corresponding control value.
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