Analgesic potential of marrubiin derivatives, a bioactive diterpene present in Marrubium vulgare (Lamiaceae)

Article abstract of DOI:10.1016/j.farmac.2005.01.003

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

Full text of DOI:10.1016/j.farmac.2005.01.003

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 modifi-
cations 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. Prelimi-
nary evaluations of this plant in our laboratory have demon-
strated hypoglycemic, analgesic and antiespasmodic effects
[1–3]. Chemically, marrubiin (1), a furane labdane diterpene,
is the main component of this plant, and exhibits potent anti-
nociceptive 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 deriva-
tives with the aim of optimizing its antinociceptive activity
initially using the writhing test in mice. The most active com-
pound was studied in other experimental models of pain. The
results of some reference drugs, such as aspirin and paraceta-
mol, were also included for the purpose of comparison
(Scheme 1).
Scheme 1
.
2. Experimental
2.1. Chemistry
IR spectra (cm–1): Perkin-Elmer FT-16-PC; in KBr. NMR
spectra: BrukerAC-200F (200 MHz) for 1H; BrukerAC-200F
(50 MHz) for 13C; d in ppm, J in Hz. 1H values and 13C val-
ues 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.
* Corresponding author. Tel.: +55 47 341 7664; fax: +55 47 341 7601.
E-mail address: cechinel@univali.br (V. Cechinel-Filho).
0014-827X/$ - see front matter © 2005 Elsevier SAS. All rights reserved.
doi:10.1016/j.farmac.2005.01.003

322
C. Meyre-Silva et al. / Il Farmaco 60 (2005) 321–326
Leila da Graça Amaral. A voucher specimen was deposited
at the FLOR Herbarium (UFSC), Florianópolis-SC, Brazil,
under number 4725.
(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). H 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).
2.1.2. 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 col-
umn using hexane/ethyl acetate with increasing polarity. Mar-
rubiin (1) was obtained with a yield of about 1% and identi-
fied by IR, 1H and 13C-NMR [4,7].
1
2.2. Pharmacology
2.1.3. General procedure for the synthesis of marrubiin
derivatives
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 guide-
lines for investigation of experimental pain in conscious ani-
mals [9].
2.1.3.1. Marrubiinic acid (2). Marrubiin (0.150 mmol) (1)
was hydrolysed in refluxing with ethoxyethanol and potas-
sium 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;
1
H, 8.41; O, 22.55%. H 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).
2.2.2. Drugs
The following substances were used: acetic acid, forma-
lin, L-glutamic acid, capsaicin (Calbiochen, San Diego, CA,
USA), and morphine hidrochloride (Merck, Darmstadt, Ger-
many). 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 pre-
pared 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 concen-
tration of Tween 80 and ethanol did not exceed 5% and did
not cause any effect ’per se’.
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 puri-
fied 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%.
2.2.3. General procedures of pharmacological assay
2.1.3.3. Marrubiinic acid benzyl ester (4). Marrubiinic acid
(0.143 mmol) was submitted to stirring with acetone, potas-
sium 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%.
2.2.3.1. Acetic acid-induced writhing. Abdominal constric-
tion was induced in mice by intraperitoneal injection of acetic
acid (0.6%), as described by Collier et al. with minor modi-
fications [10,11]. The animals were pre-treated intra-
peritoneally (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 solu-
tion (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 ex-
pressed as the reduction in the number of abdominal constric-
tions between the control animals and the mice pre-treated
with compounds.
1
IR: m = 3410 (OH), 3088 (C=C), 1636 (C=O). H 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

C. Meyre-Silva et al. / Il Farmaco 60 (2005) 321–326
323
2.2.3.2. Formalin test. The observation chamber was a glass
2.2.4. Statistical analysis
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 consid-
ered 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 injec-
tion, respectively).
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 deter-
mined by linear regression from individual experiments using
linear regression Graph Pad software (Graph Pad software,
San Diego, CA). The statistical significance of the differ-
ences between the groups was detected by ANOVA, fol-
lowed by Dunnett’s multiple comparison test. P-values of less
than 0.05 (P < 0.05) were considered as indicative of signifi-
cance.
3. Results and discussion
Considering the high yield (~1.0%) and the promising
pharmacological activity of marrubiin demonstrated in pre-
vious studies carried out in our laboratories [4], some struc-
tural modifications were performed in order to obtain more
active compounds.
2.2.3.3. Capsaicin-induced nociception. In an attempt to pro-
vide more direct evidence concerning its possible antinocice-
ptive 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 previ-
ously [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 consid-
ered 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.
The marrubiin (1) was hydrolysed in refluxing ethoxyetha-
nol 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%, respec-
tively. 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). Marru-
biinic acid (2), exhibited the most interesting activity, caus-
ing 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 func-
tional group (COOH), which gives the structure an acidic char-
acteristic that can be important for its antinociceptive activ-
ity, since several classes of different drugs possess this
characteristic [16].
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 indica-
tive of pain.
2.2.3.5. Hot-plate test. The hot-plate test was used to mea-
sure 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 main-
tained 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 cut-
off time for the hot-plate latencies was set at 30 s. The ani-
mals were treated 30 min before the assay.
Pharmacological studies indicated that marrubiinic acid (2)
presents a significant (p < 0.05) and dose-dependent antinoci-
ceptive effect, against the writhing test, given intraperito-
neally, 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 injec-
tion of acetic acid (AA) directly activates visceral and somatic

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C. Meyre-Silva et al. / Il Farmaco 60 (2005) 321–326
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.
carboxylic acid, and its possibility of ionization. The solubil-
ity 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 prevent-
ing the late than the first phase of the formalin-induced lick-
ing. 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. How-
ever, it was less active than the prototype compound, marru-
biin, 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 neu-
rons [19].
Fig. 1. Compounds obtained from marrubiin and marrubiinic acid.
nociceptors innervating the peritoneum, and induces inflam-
mation 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 sug-
gests 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.
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 analge-
sic profile to that of marrubiin (1) [4] in the other experimen-
tal 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
Compound
Inhibition (%)
Marrubiin (1)
Marrubiinic acid (2)
Marrubenol (3)a
Marrubiinic acid benzyl ester (4)
Marrubiinic acid methyl ester (5)
Aspirin (6)
92
80
1
2
94 0.8
44.1
48
2
1
2
35
Each group represents the mean SEM of 6–8 experiments.
a From Ref. [8].
Table 3
Table 2
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.)
Comparison of the antinociceptive effect of marrubiinic acid (2) with mar-
rubin (1) and non-steroidal anti-inflammatory and analgesic drugs given inra-
peritoneally in mice
Compound
First phase
Second phase
(% inhibition)
46.7 ( 8.3)**
83.0 ( 4)**
39.0 ( 4)*
Compound (10 mg/kg)
Marrubiinic acid
Marrubiin a
Writhing test ID50 (µmol/kg, i.p.)
12 (8.4–16.8)
(% inhibition)
28.3 ( 3.6)**
78.0 ( 5)**
Inactive
Marrubiinic acid
Marrubiin a
Aspirin
2.2 (1.1–3.0)
Aspirin
133 (73–243)
Paracetamol
125 (104–250)
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].
Each group represents the mean SEM of 6–8 experiments.
a From Ref. [4].

C. Meyre-Silva et al. / Il Farmaco 60 (2005) 321–326
325
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 involve-
ment 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 inhi-
bition of 52.5 6.9% of the hyperalgesia induced by intra-
plantar 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 (a-
amino-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 (cholin-
ergic and histamine receptor) and the mediators of acetylcho-
line and histamine [21]. This compound also elicited a spinal
and supraspinal antinociception when assessed against both
Fig. 5
.
Effect of marrubiinic acid on licking/biting response induced by intra-
plantar 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 marru-
biinic 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 clini-
cally 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.
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