Evaluation of Analgesic Effects of Hydroalcoholic Extract of Marrubium parviflorum by Formalin Test in Mice
In this research, hydroalcoholic extract obtained from the aerial parts of Marrubium parviflorum (Lamiaceae) was subjected to evaluation of analgesic effects using formalin test at the doses of 50, 100 and 200 mg kg-1 in mice. Duration of licking and biting time (min) of the injected paw was recorded at 5 min intervals for 40 min after formalin injection as a pain index. Results of study showed that the dose of 100 mg kg-1 of the extract decreased duration of licking and biting time between 15 and 40 min, but this effect was not statistically significant (p>0.05), while the dose of 200 mg kg-1 of extract showed significant analgesic effects (p<0.05) in the chronic phase (15-40 min) of formalin induced pain that this analgesic effect was equal to morphine. Considering that chronic phase of formalin induced pain is a secondary response to formalin induced inflammation, results of study introduce the M. parviflorum as a valuable analgesic herbal medicine that can be used in treatment of inflammatory painful disease. It is possible to assume that phytochemical contents of M. parviflorum reduce inflammatory pain by inhibiting the formation of inflammatory mediators such as prostaglandins followed by inhibiting COX-II enzyme.
Received: November 28, 2011;
Accepted: March 27, 2012;
Published: May 14, 2012
The genus Marrubium from Labiateae family includes about 40 species
all over the world (Rigano et al., 2006). In
traditional medicine some of these species are used to treat respiratory conditions,
painful and inflammatory diseases and also as cholagogues and sedative agents
(Meyre-Silva and Cechinel-Filho, 2010). Various activities
such as antimicrobial (Hayet et al., 2007), antioxidant
(Weel et al., 1999), anti-hypertensive (Bardai
et al., 2001), antidiabetic (Elberry et al.,
2011), antispasmodic (Schlemper et al., 1996),
hepatoprotective (Ahmed et al., 2010) and analgesic
effects (De Souza et al., 1998) have been shown
from this plant genus during pharmacological and biological investigations.
On the other hand, phytochemical studies have confirmed occurrence of flavonoids
(Nawwar et al., 1989), phenylpropanoids (Sahpaz
et al., 2002), di- and triterpenes (Karioti et
al., 2005; Nicholas, 1958) and volatile oils
(Baher Nik et al., 2004) in Marrubium
species. Marrubium parviflorum Fisch. and C. A. Mey. is one of the ten
species from this genus that grows in Iran (Mozaffarian, 2007).
Previous studies have demonstrated presence of high level of phenolic compounds
such as flavonoids and phenolic acids in the leaves of M. parviflorum
and its potent antioxidant activities (Yumrutas and Saygideger,
2010) as well as bicyclogermacrene (26.3%), germacrene D (21.5%) and β-caryophyllene
(15.6%) as major constituents of its essential oil (Khanavi
et al., 2005). It has been shown that M. vulgar L. (white
horehound), another member of this genus, has significant analgesic effects
in various pharmacological studies (De Souza et al.,
1998; Roghani, 2006). Because of close relation
between phytochemical contents of the species belong to same genus and occurrences
of flavonoids with established analgesic effects in this plant (Rylski
et al., 1979; Yumrutas and Saygideger, 2010)
we assumed that M. parviflorum also may act as an analgesic agent. So,
we put this hypothesis to test by evaluating analgesic effects of hydroalcoholic
extract of its aerial parts in mice by using formalin-induced inflammatory pain
MATERIAL AND METHODS
Plant material: Aerial parts of M. parviflorum were collected from Khalkhal region (Ardabil province, Iran) during its flowering stage in July 2009. This plant species has been identified by herbarium of Institute of Medicinal Plants (Academic Centre for Education, Culture and Research (ACECR), Tehran, Iran.
Extraction: Hydroalcoholic extract was obtained by maceration method from air-dried and grinded aerial parts of M. parviflorum (200 g) with 70% mixture of Methanol in water (3x1 L) for 72 h. The obtained extract was concentrated by using rotary evaporator at the maximum temperature of 45°C.
Animals: Thirty six male Swiss albino mice (25-30 g) were purchased from Razi institute (Karaj, Iran). The animals were kept in groups of six in standard polypropylene cages with easily access to water and food. The cages were housed in a room under standard laboratory conditions (24-26°C) with 12:12 h light/dark cycle. All experiments were carried out regarding to guidelines of Tehran University of Medical Sciences Ethics Committee.
Chemicals: Morphine hydrochloride was obtained from Sigma Company (England). Extract and morphine were all dissolved in normal saline, freshly on experimentation days, to prepare appropriate designed concentrations.
Formalin test: Potential analgesic effects of the Hydroalcoholic extract
was evaluated using formalin test. Briefly, drug solutions (Hydroalcoholic extract
of M. parviflorum (50,100 and 200 mg kg-1), morphine (2 mg
kg-1) as positive control and normal saline as negative control)
were administered intraperitoneally (IP), 15 min before injection of formalin.
Formalin (2.5%) was administered subcutaneously (SC) into the dorsal surface
of the right hind paw of the mice, then duration of the licking and biting time
(min) of the injected paw was recorded at 5 min intervals for 40 min after formalin
injection as a pain index. The responses were measured for two distinct phases,
the initial 5 min after formalin injection is known as acute or early phase
of formalin induced pain and the duration between 15 and 40 min as the chronic
or late phase (Hunskaar and Hole, 1987; Hunskaar
et al., 1985; Rosland et al., 1990;
Tjolsen et al., 1992).
Statistics: All data were presented as Mean±SEM. One-way ANOVA
(analysis of variance) was applied to data analysis followed by Tukeys
t-test. Differences between groups at the level of p<0.05 were considered
According to previous reports, intraperitoneal administration of morphine at
the dose of 2 mg kg-1, 15 min before the injection of formalin significantly
inhibited both the acute and chronic phases of formalin-induced inflammatory
pain (p<0.05) (Fig. 1a-d). Results of
study showed that the dose of 200 mg kg-1 from hydroalcoholic extract
exerts significant analgesic effect (p<0.05) characterized by decreasing
duration of licking and biting time in the chronic or second phase (15-40 min)
of formalin induced pain. This analgesic effect was equal to morphine (Fig.
1d). Dose of 100 mg kg-1 of the extract decreased duration of
licking and biting time between 15 and 40 min, but this effect was not statistically
significant (p>0.05) (Fig. 1c). All of administrated doses
of extract have not showed any analgesic effects in the first or acute phase
(initial 5 min) of formalin induced pain compared with morphine (Fig.
Discussion: On the basis of previous pharmacological studies, formalin
test, by providing two distinct phases of pain, is a useful model to determination
of possible mechanisms of analgesic effects (Hunskaar and
Hole, 1987; Hunskaar et al., 1985; Rosland
et al., 1990; Tjolsen et al., 1992).
Drugs such as opioids which induced analgesic effects by affecting central nervous
system inhibit both acute and chronic phases of formalin induced pain (Shibata
et al., 1989). While drugs such as indomethacin and other non-steroidal
anti-inflammatory drugs (NSAIDs) inhibit chronic or late phase of the pain,
only (Zvejniece et al., 2006). In fact, chronic
or late phase of formalin induced pain is a secondary response to produced inflammation
as a result of subcutaneous formalin injection (Hunskaar
and Hole, 1987). According to the results of this study, analgesic effect
of M. parviflorum was appeared at the dose of 200 mg kg-1
in the chronic phase of induced pain. It is possible to assume that phytochemical
contents of M. parviflorum reduce inflammatory pain by inhibiting the
formation of inflammatory mediators such as prostaglandins followed by inhibiting
COX-II enzyme. Probably, Marrubiin, a furane labdane diterpene, which has been
recognized as the main analgesic compound present in M. vulgare, could
be involved in the analgesic effects of this species (Jesus
et al., 2000; Meyre-Silva et al., 2005).
||Effects of hydroalcoholic extract of Marrubium parviflorum
in different doses and morphine on formalin test. (a): Normal saline as
negative control and morphine (2 mg kg-1) as positive control,
(b): M. parviflorum (50 mg kg-1), (c): M. parviflorum
(100 mg kg-1) and (d): M. parviflorum (200 mg kg-1).
Morphine and extracts were administrated IP, 15 min before of the formalin
injection. Data are expressed as Mean±SEM for six mice. The duration
of licking and biting time (min) of the injected paw was recorded at 5 min
intervals for 40 min after formalin injection
Furthermore, flavonoids and phenylethanoids compounds that its anti-inflammatory
and analgesic activities have been confirmed during previous studies can also
be effective in the appearing of this activity (Fu et
al., 2008; Kim et al., 2009; Pelzer
et al., 1998; Rylski et al., 1979).
Results of this study introduce the M. parviflorum as a valuable analgesic herbal medicine that can be used in treatment of inflammatory painful disease such as rheumatoid arthritis, inflammatory bowel disease (IBD), etc. Certainly, more studies on the analgesic effects of fractions and isolated compounds of M. parviflorum and on its toxicity properties can be useful to evaluating of use of M. parviflorum as an analgesic agent.
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