Ileal Relaxation Induced by Mentha longifolia (L.) Leaf Extract in Rat
Mohammad Kazem Gharib Naseri,
Zahra Gharib Naseri,
Marzie Omidi Birgani
The effect of Mentha longifolia (L.) leaf hydroalcoholic
extract (MLE) was examined on rat ileal smooth muscle contractions. Last
portion of ileum from male adult Wistar rat was mounted in an organ bath
containing Tyrode solution. The tissue was contracted by carbachol (CCh,
10 μM), KCl (60 mM) and BaCl2 (4 mM) and then MLE (0.0625-1
mg mL-1) was added to the bath cumulatively. The effect of
MLE on KCl-induced contraction was examined after tissue incubation with
propranolol (1 μM), naloxone (1 μM) and Nω-nitro-L-arginine
methyl ester (L-NAME, 100 μM). The effect of MLE on CaCl2-induced
ileal contraction in Ca2+-free with high potassium Tyrode solution
was also evaluated. The role of potassium channels was examined by ileum
incubation (5 min) with tetraethylammonium (TEA, 1 mM). The results showed
that KCl-, CCh and BaCl2-induced ileal contractions were inhibited
(p<0.001) by cumulative concentrations of MLE with the same potency.
In addition, MLE (0.25-1 mg mL-1) inhibited (p<0.01) ileal
contractions induced by CaCl2 (0.45-2.7 mM) in a concentration-related
manner. The antispasmodic effect of MLE was affected neither by propranolol,
L-NAME nor by naloxone. The MLE concentration-response curve was shifted
to the right (p<0.05) by tissue incubation with TEA. From results it
may be suggested that Mentha longifolia hydroalcoholic leaf extract
induces its spasmolytic activity mainly through disturbance in calcium
mobilization and partly by potassium channels activation. Present results
show that Mentha longifolia leaf extract exerts relaxant effects
on intestinal smooth muscle, consistent with the traditional use of the
plant to treat gastrointestinal disorders such as diarrhea and colic.
The genus Mentha belongs to the family Lamiaceae consisting of about
25-30 species (Shaiq Ali et al., 2002); most of them are found
in temperate regions of Eurasia, Australia and South Africa. The aromatic
Mentha herbs are perennials found in damp or wet places and members of
this genus are the most important sources of essential oil production
in world (Shaiq Ali et al., 2002). In Northern area of Pakistan,
Mentha longifolia (L.) or horsemint (locally called bonooh) is
used for stomach, liver problems and vomiting (Khan and Khatoon, 2008).
In Iranian traditional medicine, M. longifolia, locally called
poneh kohi, is used for gastric disorders. Latif et al. (2006)
has reported that this herb is used traditionally for treating diarrhea
in children and preventing vomiting. The following properties have been
demonstrated in M. longifolia enhancing bactericide effects of
some drugs (Shahverdi et al., 2004), inhibitory activity against
HIV-1 (Amzazi et al., 2003), antimicrobial and scavenging free
radical activity (Mimica-Dukic et al., 2003), antimycotic (Abu-Jawdah
et al., 2002), anthelmintic activities (Kozan et al., 2006),
antiemetic effect in young chickens (Hosseinzadeh et al., 2004).
Piperienone and piperitone oxide and five flavonoids have been isolated
from essential oil of this herb (Ghoulami et al., 2001) and isolation
of β-sitosterol glycoside (longiside-A and B) and flavanone glycoside
(longitin) (Shaiq Ali et al., 2002; Shaiq Ali et al., 2006)
from M. longifolia have been reported. Although, this herb has
been used to treat various gastrointestinal disorders such as abdominal
pain, flatulence and colic, however, its effect on smooth muscle has not
been scientifically evaluated yet. Therefore, the aim of the present study
was to investigate the effects of M. longifolia leaf hydroalcoholic
extract (MLE) on rat ileum and to study the involved mechanism(s).
MATERIALS AND METHODS
Plant material and powder preparation: Mentha longifolia
was collected from Masjed Soleiman Mountains (Northeastern of Khuzestan
province) in October 2007 and authenticated by Dr. Sedighi Dehkordi from
Ahwaz Shahid Chamran University, Department of Horticultural Science and a voucher was deposited at herbarium of the
same department for further references. The leaves were dried under shade
and powdered by an electrical grinder. The powder was extracted by macerating
method using 70% alcohol for 72 h at room temperature and mixed occasionally
daily. The mixture was then filtered (Whatman No. 1), filtrate was concentrated
in rotary evaporator and dried at room temperature to obtain a dark green
powder (yield: 22.7%). The extract was stored at 4°C until being used
and dissolved in bath solution before using in experiments.
Chemicals and reagents: Propranolol, carbachol, Nω-nitro-L-arginine
methyl ester (L-NAME), tetra- ethylammonium (TEA) were purchased from
Sigma (USA) and naloxone was purchased from Tolidaru (Iran). Other chemicals
were purchased from Merck (Germany).
Animals: All rats used in this study were treated in accordance
with principals and guidelines on animals care of Ahwaz Jundishapur University
of Medical Sciences (AJUMS). Male Wistar adult rats (194.4±5.8
g) were obtained from AJUMS Animal House and kept at 12 h light/dark cycle
and at 20-24°C with free access to food and water. Rats were starved
of food but not water for 24 h before experiment.
Ileum preparation: On the day of experiment the rats were sacrificed
by a sharp blow on the head. A piece (2 cm) was prepared from the terminal
ileum (taken within a distance of 2-3 cm from the caecum) and mounted
in an organ bath containing Tyrode solution (10 mL) between two stainless
steel hooks vertically. The lower hook was fixed at the bottom of the
organ bath and upper one was connected to an isotonic transducer (Harvard
transducer, UK) connected to a recorder (Harvard Universal Oscillograph,
UK). The Tyrode solution (pH 7.4 and 37°C) composition was (in mM):
NaCl (136); KCl (5); CaCl2 (2); NaHCO3 (11.9); MgCl2
(0.98) NaH2PO4 (0.36) and glucose (5.55) which continuously
was bubbled with air (Madeira et al., 2002). The initial tension
was 1 g throughout the experiment and equilibrium period was 60 min. After
equilibrium period, the ileum was contracted either by KCl (60 mM), carbachol
(CCh, 10 μM) or BaCl2 (4 mM) and once the plateau was
achieved, the extract was added cumulatively (0.0625-1 mg mL-1)
to the organ bath. The effect of extract was also studied in separate
tissues after either 30 min incubations with 1 μM of propranolol,
30 min with naloxone or 20 min (Izzo et al., 1998) with L-NAME
(100 μM) as non-selective β-adrenoceptors, opioid receptors
antagonists and nitric oxide synthase inhibitor, respectively. To evaluate
the MLE effect on CaCl2-induced ileum contraction, in Ca2+-free
and rich KCl (60 mM) Tyrode solution, the tissue was depolarized and then
CaCl2 was applied cumulatively (0.45-2.7 mM) before and after
tissue incubation (3 min) with extract (0.25-1 mg mL-1).
To evaluate the role of potassium channels, tissue preparation was incubated
(5 min) with tetra- ethylammonium (TEA, 1 mM), then contraction was induced
by CCh (10 μM) and thereafter MLE was added cumulatively (0.0625-1
mg mL-1). Separate ileum preparations were used for each spasmogens
Statistical analysis: The plateau of ileal contraction induced
by KCl or CCh was regarded as 100% and percentage of relaxation was calculated
from changes in the contraction. Results were expressed as mean±SEM
of n experiments (n indicates the number of tissues and coincides with
the number of animals). Comparison between to sets of data was made by
Student`s t-test. For comparison of one control with several experimental
groups, a one-way Analysis of Variance (ANOVA) was used. Analysis of variance
(two-way) was used to compare different cumulative concentration-effect
curves. A p-value <0.05 was considered significant.
Effect of MLE on ileal contractions induced by applied spasmogens:
Mentha longifolia leaf hydroalcoholic extract (MLE) reduced the
ileum contractions induced either by KCl (60 mM), CCh (10 μM) or
BaCl2 (4 mM) significantly (one-way ANOVA, p<0.001) and
in a concentration dependent manner. As Fig. 1 shows
the MLE antispasmodic effects on contractions induced by these spasmogens
are identical. Eight animals were used for each spasmogen.
Effect of the MLE on CaCl2-induced ileal contractions:
In Ca2+-free with high K+ (60 mM) Tyrode solution,
applying cumulative concentrations of calcium chloride (0.45 to 2.7 mM)
induced ileal contractions in a concentration dependent manner (p<0.01)
as control curve shows in Fig. 2. Three minutes incubation
of tissue preparation with MLE (0.25, 0.5 and 1 mg mL-1) reduced
the contractions evoked by CaCl2 in a concentration-related
manner. The CaCl2-induced contractions in ileum before and
after incubation with extract (0.5 mg mL-1) were significantly
different (two-way ANOVA, p<0.01). Seven animals were used for each
||Effect of Mentha longifolia hydroalcoholic extract
(MLE) on rat ileal contractions induced by KCl (60 mM), BaCl2
(4 mM) and carbachol (10 μM). Two-way ANOVA indicated that these
three concentration-related responses are not significantly different.
Each point represents mean±SEM of 8 observations for each extract
||Spasmogenic effect of CaCl2 on rat ileum
before (0.0 mg mL-1) and after 3 min tissue incubation
with different concentrations of Mentha longifolia hydroalcoholic
extract (MLE). The extract antispasmodic effect at 0.5 mg mL-1
in compare to 0.0 mg mL-1 is significantly different (two-way
ANOVA, p<0.01). Each point represents mean±SEM of 7 observations
for each MLE concentration. (0.0 mg mL-1 vs 0.5 mg mL-1,
Student`s t-test, **p<0.01, ***p<0.001, ****p<0.0001)
Effect of MLE after ileum incubation with propranolol, naloxone or
L-NAME: Incubation of tissue preparation either 30 min with propranolol
(1 μM, n = 7), naloxone
||Antispasmodic effect of Mentha longifolia hydroalcoholic
extract (MLE) on KCl (60 mM)-induced ileal contraction before (Control,
n = 8) and after tissue incubation either with propranolol (1 μM,
30 min, n = 7), L-NAME (100 μM, 20 min, n = 9) or naloxone (1
μM, 30 min, n = 6). The extract spasmolytic activity is not affected
by tissue incubation with these antagonist or inhibitor. Each point
represents mean±SEM of number of observations (n) mentioned
above for each protocol
||Spasmolytic effect of Mentha longifolia hydroalcoholic
extract (MLE) on carbachol 10 μM-induced ileal contraction before
(Control) and after tissue incubation with tetraethylammonium (TEA,
1 mM, 5 min) as a non-specific K+ channels blocker. Incubation
of tissue preparation with TEA attenuated (two-way ANOVA, p<0.05)
the MLE antispasmodic activity. Each point represents mean±SEM
of 8 and 7 experiments for control and TEA protocols respectively
(Student`s t-test, *p<0.05, **p<0.001).
(1 μM, n = 6), or 20 min with L-NAME (100 μM, n = 9), as β-adrenoceptor
antagonist, opioid receptor antagonist and nitric oxide synthase inhibitor,
respectively, did not alter the spasmolytic effect of MLE on KCl-induced
ileum contractions (Fig. 3).
Effect of ileum incubation with TEA on the MLE antispasmodic activity:
The antispasmodic effect of MLE (0.0625-1 mg mL-1) on CCh-induced
ileal contraction was shifted to right after 5 min tissue incubation with
1 mM of TEA (two-way ANOVA, p<0.05, n = 7) which its results are shown
in Fig. 4.
The distinctive finding in this study is that extract from Mentha
longifolia leaf hydroalcoholic extract (MLE) has a myorelaxant effects
on isolated preparations of rat intestinal ileum. The contraction of gastrointestinal
smooth muscle depends on the mediation of intracellular Ca2+
and is accomplished by the process of excitation-contraction coupling
(Zhang et al., 2005). A high-K+ medium could depolarize
the cellular membrane of ileum smooth muscle (Bolton, 1979; Zhang et
al., 2005). Moreover, it is well known that KCl-induced contraction
in smooth muscle is due to an increase in Ca2+ influx through
voltage-operated Ca2+ channels (Kaya et al., 2002; Borrelli
et al., 2006) which the L-type of these channels has been shown
to exist in rat ileum (El Bardai et al., 2004). In addition, it
has been suggested that the substance that inhibits high K+
contractions is considered as a blocker of Ca2+ influx (Gilani
et al., 2005). Ghoulami et al. (2001) has reported that
Mentha longifolia has high content piperitenone and piperitenone
oxide and the relaxant activity of piperitenone oxide on guinea pig ileum
has been reported (Sousa et al., 1997).
It is accepted that CCh-induced contractile response following receptor
activation require an increase in intracellular Ca2+ which
is provided by both Ca2+ influx through L-type Ca2+
channels and Ca2+ release from intracellular calcium stores
(Tanovic et al., 2000). On the other hand, BaCl2 may
act directly on the smooth muscle (Ozaki et al., 2006) or induce
smooth muscle contraction by nonspecifically blocking the K+
channels (Liu et al., 2001). Although, the applied spasmogens have
different modes of action but identical concentration-response curves
effect of MLE on these spasmogens contractile activity indicates that
MLE might be acting via a non-specific mechanism and also at Ca2+
entry level as a common step in the contraction mechanism elicited by
the agonists. Furthermore, this suggestion is supported by the MLE spasmolytic
effect on CaCl2-induced contractions since, in the Ca2+-free
and high K+ Tyrode solution, the tissue was depolarized by
high K+ (Fujimoto and Mori, 2004) however, in this study ileal
contractions was occurred only after applying CaCl2 in the
organ bath as reported by Zhang et al. (2005). Therefore, it may
be assumed that MLE has inhibited the Ca2+ influx. The same
suggestion has been made for vasorelaxatory effect of rotundifolone which
has been found in Mentha longifolia (Guedes et al., 2004).
It is very unlikely that the antispasmodic effect of MLE is due to antimuscarinic
action, since the extract also inhibited the contractions induced by BaCl2
and KCl which do not act through a receptor-mediated mechanism.
It has been shown that activation of β-adrenoceptor in ileal smooth
muscle leads to relaxation (Brown and Summers, 2001). In order to assess
if the extract relaxed intestine by binding on β-adrenoceptor, the
relaxing effect of the extract was examined in the presence of propranolol.
We found that propranolol does not attenuate the activity of the extract,
suggesting that MLE does not have any effect on beta adrenergic receptors.
Nitric oxide synthase elevates nitric oxide (NO) production which in turn
relaxes ileum by promoting cGMP synthesis (Kanada et al., 1992).
But, the ineffectiveness of L-NAME (as a nitric oxide synthase inhibitor)
to reduce the extract spasmolytic effect indicates that NO was not involved
in the extract activity. This result is in agreement to a report describing
that rotundifolone found in Mentha longifolia essential oil exhibits
aorta relaxation was not dependent to nitric oxide synthesis (Guedes et
Activation of opioid receptors relaxes ileum (Gray et al., 2005)
but the extract activity was unaffected by naloxone (as a non-selective
opioid antagonist) which indicates that the MLE activity was not mediated
via these receptors. Another possible mechanism was potassium channels
activation by the extract. Present results show that TEA, as a non-specific
potassium channel blocker, attenuated the MLE effect on CCh-induced ileal
contraction which may indicate that MLE induces its effect, at least in
part, through activation of these channels. Considering the demonstration
of ceramides such as longifoamide-A and B in Mentha longifolia
(Shaiq Ali et al., 2006) and the spasmolytic activity of these
agents (Jang et al., 2005) the spasmolytic activity of MLE could
be attributed to the ceramides. Furthermore, it has been reported that
β-sitosterol which exist in Mentha longifolia, exhibits Ca2+
channel-blocking action (Gilani et al., 2008), therefore, the spasmolytic
activity of MLE could be related to activity of this compound as well.
It seems that the Mentha longifolia leaf extract spasmolytic effect
on rat ileum has been occurred mainly through the voltage operated Ca2+
channels. This study showed the relaxant effect of MLE on the ileum contractions
induced by three different spasmogens. Thus, the in vitro antispasmodic
activity of MLE supports a rational suggesting basis for folk and traditional
use of the Mentha longifolia in gastrointestinal cramps, diarrhea
and colic. Present results may suggest the beneficial effect of this herb
for treatment of diarrhea. However, more detailed phytochemical studies
are necessary to identify the active principle(s) and exact mechanism(s)
The authors wish to thank the Physiological Research Center of AJUMS
for supporting this research and also to thank Dr. Sedighi Dehkordi for
authentication of the herb.
1: Abou-Jawdah, Y., H. Sobh and A. Salameh, 2002. Antimycotic activities of selected plant flora, growing wild in Lebanon, against phytopathogenic fungi. J. Agric. Food Chem., 50: 3208-3213.
2: Amzazi, S., S. Ghoulami, Y. Bakri, A. Idrissi, S.W. Fkil-Tetouani and A. Benjouad, 2003. Human immunodeficiency virus type 1 inhibitory activity of Mentha longifolia. Therapie, 58: 531-534.
PubMed | Direct Link |
3: Bolton, T.B., 1979. Mechanism of action of transmitter and other substances on smooth muscles. Physiol. Rev., 59: 606-718.
4: Borrelli, F., F. Capasso, R. Capasso, V. Ascione, G. Aviello, R. Longo and A.A. Izzo, 2006. Effect of Boswellia serrata on intestinal motility in rodents: Inhibition of diarrhoea without constipation. Br. J. Pharmacol., 148: 553-560.
5: Brown, K.J. and R.J. Summers, 2001. β1 and β3-adrenoceptor mediated smooth muscle relaxation in hypothyroid rat ileum. Eur. J. Pharmacol., 415: 257-263.
6: El-Bardai, S., M.C. Hamaide, B. Lyoussi, J. Quetin-Leclercq, N. Morel and M. Wibo, 2004. Marrubenol interacts with the phenylalkylamine binding site of the L-type calcium channel. Eur. J. Pharmacol., 492: 269-272.
7: Fujimoto, S. and M. Mori, 2004. Characterization of capsaicin-induced, capsazepine-insensitive relaxation of ileal smooth muscle of rats. Eur. J. Pharmacol., 487: 175-182.
8: Ghoulami, S.A., I.L. Idrissi and S. Fkih-Tetouani, 2001. Phytochemical study of Mentha longifolia of Morocco. Fitoterapia, 72: 596-598.
CrossRef | PubMed | Direct Link |
9: Gilani, A.H., S. Bashir, K.H. Janbaz and A.J. Shah, 2005. Presence of cholinergic and calcium channel blocking activities explains the traditional use of Hibiscus rosasinensis in constipation and diarrhoea. J. Ethnopharmacol., 102: 289-294.
CrossRef | PubMed | Direct Link |
10: Gilani, A.H., A.U. Khan, M. Raoof, M.N. Ghayur, B.S. Siddiqui, W. Vohra and S. Begum, 2008. Gastrointestinal, selective airways and urinary bladder relaxant effects of Hyoscyamus niger are mediated through dual blockade of muscarinic receptors and Ca2+ channels. Fundam. Clin. Pharmacol., 22: 87-99.
CrossRef | PubMed | Direct Link |
11: Gray, A.C., P.J. White and I.M. Coupar, 2005. Characterisation of opioid receptors involved in modulating circular and longitudinal muscle contraction in the rat ileum. Br. J. Pharmacol., 144: 687-694.
CrossRef | PubMed |
12: Guedes, D.N., D.F. Silva, J.M. Barbosa-Filho and I.A. Medeiros, 2004. Calcium antagonism and the vasorelaxation of rat aorta induced by rotundifolone. Braz. J. Med. Biol. Res., 37: 1881-1887.
13: Izzo, A.A., N. Mascolo and F. Capasso, 1998. Effect of sodium rhein on electrically-evoked and agonist-induced contractions of the guinea-pig isolated ileal circular muscle. Br. J. Pharmacol., 124: 825-831.
14: Jang, G.J., D.S. Ahn, Y.E. Cho, K.G. Morgan and Y.H. Lee, 2005. C2-ceramide induces vasodilation in phenylephrine-induced pre-contracted rat thoracic aorta: Role of RhoA/Rho-kinase and intracellular Ca2+ concentration. Naunyn Schmiedebergs Arch. Pharmacol., 372: 242-250.
15: Kanada, A., F. Hata, N. Suthamnatpong, T. Maehara, T. Ishii, T. Takeuchi and O. Yagasaki, 1992. Key roles of nitric oxide and cyclic GMP in nonadrenergic and noncholinergic inhibition in rat ileum. Eur. J. Pharmacol., 216: 287-292.
16: Kaya, T.T., G. Kokhan, A.S. Soydan, M. Arpacik and B. Karadas, 2002. Effects of nimesulide and pentoxifylline on decreased contractile responses in rat ileum with peritonitis. Eur. J. Pharmacol., 442: 147-153.
17: Khan, S.W. and S. Khatoon, 2008. Ethnobotanical studies on some useful herbs of Haramosh and Bugrote valleys in Gilbit, Northern areas of Pakistan. Pak. J. Bot., 40: 43-58.
Direct Link |
18: Kozan, E., E. Kupeli and E. Yesilada, 2006. Evaluation of some plants used in Turkish folk medicine against parasitic infections for their in vivo anthelmintic activity. J. Ethnopharmacol., 108: 211-216.
19: Latif, A., Z.K. Shinwari, J. Hussain and S. Murtaza, 2006. NTFPS: An alternative to forest logging in Minadam and Sultanar Valley Swat. Lyonia, 11: 15-21.
Direct Link |
20: Liu, S., H.Z. Hu, J. Ren, C. Gao, N. Gao, Z. Lin, Y. Xia and J.D. Wood, 2001. Pre-and postsynaptic inhibition by nociceptin in guinea pig small intestinal myenteric plexus in vitro. Am. J. Physiol. Gastrointest. Liver Physiol., 281: G237-G246.
21: Madeira, S.V.F., F.J.A. Matos and D.C. Leal-Criddle, 2002. Relaxant effects of the essential oil of Ocimum gratissimum on isolated ileum of the guinea pig. J. Ethnopharmacol., 81: 1-4.
CrossRef | PubMed |
22: Mimica-Dukic, N., B. Bozin, M. Sokovic, B. Mihajlovic and M. Matavulj, 2003. Antimicrobial and antioxidant activities of three Mentha species essential oils. Planta Med., 69: 413-419.
PubMed | Direct Link |
23: Ozaki, M., T. Nagatomo, T. Maeda, S. Kishioka and H. Yamamoto, 2006. Pharmacological differences between Liu-Jun-Zi-Tang, a traditional Chinese herbal medicine and domperidone on isolated guinea-pig ileum. Biol. Pharm. Bull., 29: 1349-1354.
24: Shaiq Ali, M., M. Saleem, W. Ahmad, M. Parvez and R. Yamdagni, 2002. A chlorinated monoterpene ketone, acylated β-sitosterol glycosides and a flavanone glycoside from Mentha longifolia (Lamiaceae). Phytochemistry, 59: 889-895.
25: Ali, M.S., W. Ahmed, M. Saleem and T. Khan, 2006. Longifoamide-A and B: Two new ceramides from Mentha longifolia (Lamiaceae). Nat. Prod. Res., 20: 953-960.
26: Shahverdi, A.R., F. Rafii, F. Tavassoli, M. Bagheri, F. Attar and A. Ghahraman, 2004. Piperitone from Mentha longifolia var. chorodictya Rech F. reduces the nitrofurantoin resistance of strains of enterobacteriaceae. Phytother. Res., 18: 911-914.
CrossRef | PubMed | Direct Link |
27: Sousa, P.J., P.J. Magalhaes, C.C. Lima, V.S. Oliveira and J.H. Leal-Cardoso, 1997. Effects of piperitenone oxide on the intestinal smooth muscle of the guinea pig. Braz. J. Med. Biol. Res., 30: 787-791.
28: Tanovic, A., M. Jimenez and E. Fernandez, 2000. Lack of effect of nitric oxide on KCl, acetylcholine and substance P induced contractions in ileal longitudinal muscle of the rat. Life Sci., 67: 531-541.
29: Zhang, W.W., Y. Li, X.Q. Wang, F. Tian, H. Cao, M.W. Wang and Q.S. Sun, 2005. Effects of magnolol and honokiol derived from traditional Chinese herbal remedies on gastrointestinal movement. World J. Gastroenterol., 11: 4414-4418.
PubMed | Direct Link |