Volume 2, Issue 4 , Pages 288-293, December 2009
In Vitro Cholinomimetic Effect of Loranthus Ferrugineus in Isolated Guinea Pig Ileum
Article Outline
- Abstract
- 1.. Introduction
- 2.. Materials and Methods
- 3.. Results
- 4.. Discussion
- Acknowledgments
- References
- Copyright
Abstract
This study aimed to elucidate the mechanism(s) of the spasmogenic action of Loranthus ferrugineus in isolated guinea pig ileum. Thus the contractile responses of guinea pig ileum to graded additions of either L. ferrugineus methanol extract or its n-butanol fraction were tested in the presence and absence of various pharmacological interventions. The data showed that L. ferrugineus methanol extract and the n-butanol fraction produced a concentration-dependent spasmogenic effect in isolated guinea pig ileum segments. These effects were significantly inhibited in the presence of 1 μM atropine. In contrast, the response to the lowest concentrations of L. ferrugineus methanol extract (0.25, 0.5 and 1 mg/mL) and n-butanol fraction of L. ferrugineus (0.125, 0.25 and 0.5 mg/mL) were considerably enhanced in the presence of 0.05 μM neostigmine. Neither L. ferrugineus methanol extract nor n-butanol fraction contractile responses were affected upon the incubation of the ileal segments with 100 μM hexamethonium. The results of this study show that the spasmogenic effect of L. ferrugineus is possibly mediated through a direct action on intestinal muscarinic receptors. It is suggested that the bioactive constituents of L. ferrugineus serve as a substrate for acetylcholinesterase.
Key Words: acetylcholine , acetylcholinesterase , cholinergic activity , guinea pig ileum , Loranthus ferrugineus , muscarinic receptors
1. Introduction
Herbs have always been an integral part of society, valued for both their culinary and medicinal properties. With the high prevalence of herbal medicine use in the world, health practitioners should be informed about such health practices when taking clinical histories and remain updated about the beneficial or harmful effects of these treatments. Thus continuing research is necessary to elucidate the pharmacological activities of many biologically potent herbal medicines and stimulate future pharmaceutical development of therapeutically beneficial herbal drugs [1].
Loranthus ferrugineus Roxb.(Loranthaceae) is a bushy parasitic shrub that attaches itself to a host tree by unique roots. It characteristically has reddish green leaves and small yellow flowers and berries. The shrub's habitat is located in many countries such as Malaysia, Indonesia, Australia and New Zealand [2].
In Malaysia, this plant is locally known as dedalu or benalu, while a common name of this parasite and some other species from the same family is mistletoe. Preparations from L. ferrugineus have been widely used worldwide in folk medicine for several therapeutic purposes. In Malaysia, dedalu is used as a decoction for general health and gerontological applications. Moreover, this plant is also used for treatment and man agement of elevated blood pressure, as a purgative, and for treatment of different gastrointestinal (GI) tract disorders [3]. The leaves, fruits and flowers of L. ferrugineus are the most common parts used to treat these disorders. The roots attaching itself to the host plant are meant for other therapeutic purposes, such as ulcer and cancer treatment [4].
Previous phytochemical screening of this plant showed the presence of a wide variety of naturally occurring components, including terpenoids, flavonoids, and condensed tannins [3, 5]. Recent investigations from our laboratory revealed the presence of potentially active vasorelaxant components in the methanol extract of this plant [6]. Moreover, a preliminary study on the GI effects of this plant also revealed the presence of possible spasmogenic components in the same extract from this shrub [3].
Despite the availability of modern medications, the use of traditional medicine is growing throughout the world [7], indicating a need for scientific investigations into the therapeutic effects of medicinal plant and their underlying mechanisms. Many researchers have focused on GI dysfunctions, such as indigestion, constipation, and dyspepsia. While no previous investigation has thoroughly reported the GI effects of such a widely used medicinal herb, this investigation set out to further characterize the spasmogenic activity of L. ferrugineus and address the possible mechanistic pathways responsible for the activity of its components. To this end, the methanol extract of L. ferrugineus was purified using solvent-solvent extraction and the most bio-active fraction was chosen for further pharmacological investigation.
2. Materials and Methods
2.1. Plant material, extraction and fractionation
The plant material was collected from the main campus of Universiti Sains Malaysia (USM) and identified by a botanist in the School of Biological Sciences, USM. A specimen (number 10943) was kept in the herbarium of the same institute. For extract preparations, L. ferrugineus methanol extract (LFME) was obtained using an hot extraction procedure as previously described [3, 6]. Briefly, the fresh cleaned aerial parts of the plant were dried in an oven at 42°C for 5 days. The dried plant material was then pulverized into a powder using a milling machine. The powder was extracted in a Soxhelt apparatus (Schott, Duran, Germany) with methanol to obtain LFME. Subsequently, the extract was dried over a rotary evaporator (Büchi, Switzerland) and freeze dried. LFME was then purified using a solvent-solvent extraction pro cedure (40 g of LFME was dissolved in 350 mL distilled water and poured into 1L separatory funnel). The aqueous solution of the extract was then extracted successively with (300 mL × 4) chloroform, (300 mL × 4) ethyl acetate, and (300 mL × 4) n-butanol. All the combined fractions were then concentrated by rotary evaporation and subsequently freeze dried. The n-butanol fraction of LFME (NBFLFME) was observed to have the strongest spasmogenic activity compared with the other fractions of LFME (data not shown). Therefore, NBF-LFME was chosen for further pharmacological investigation.
2.2. Animals and tissue preparation
In this investigation, male and female guinea pigs weighing 500-600 g were used to study the GI effects of L. ferrugineus extracts. The guinea pigs were obtained from the Animal Care Facility of USM and allowed to acclimatize for 1 week in the animal transit room before any experiments. The animals were fed with guinea pig pellets (Gold Coin Feed Mills Sdn Bhd, Malaysia) supplemented with orange as a source of vitamin C. Animal handling and all procedures on animals were performed in accordance with the guidelines of the Animal Ethics Committee, USM, Penang, Malaysia.
The guinea pigs were fasted overnight but had free access to water. On the subsequent day, the animals were sacrificed by cervical dislocation and exsanguination. The abdomen was dissected to expose the GI tract, and the small intestine was carefully isolated, flushed of their contents and trimmed of mesentery. Subsequently, a cut was made approximately 15 cm from the ileocecal junction toward the upper part of the GI tract to obtain the ileum. The ileum was quickly placed in a Petri dish containing Tyrode's solution of the following composition: 2.68 mM of potassium chloride, 136.9 mM of sodium chloride, 1.05 mM of magnesium chloride, 11.90 mM of sodium carbonate, 0.42 mM of sodium phosphate, 1.8 mM of calcium chloride and 5.55 mM of glucose. Special care was exercised while handling the ileum and the use of forceps was strictly avoided. Tyrode's solution was maintained under constant aeration with carbogen gas (95% O2 + 5% CO2). Two-centimeter length segments of the ileum was cut and suspended vertically in 10 mL tissue baths containing Tyrode's solution. The bath solution was also bubbled with carbogen and maintained at 37°C. The contractile responses of the intestine were recorded using a force-displacement transducer connected to Grass polygraph recorder to measure isometric contraction. The tissues were allowed to equilibrate under 1 g tension for 30 minutes before the addition of any agonist/antagonist or extract. Under these experimental conditions, guinea pig ileum behaves as a quiescent preparation and is considered useful for studying the spasmogenic activity [3, 8].
2.3. Drugs and solutions
Acetylcholine (ACh), atropine, neostigmine and hex amethonium were purchased from Sigma Chemicals Co., St. Louis, MO, USA. Salts for Tyrode's solution were obtained from R & M, UK.
LFME, NBF-LFME, ACh, atropine, neostigmine and hexamethonium were dissolved in Tyrode's solution just before starting the experimental protocol.
2.4. Pharmacological studies
The effect of LFME, NBF-LFME on the intestinal responses of guinea pig ileum was examined. The contractile responses to ACh (a standard cholinergic agonist; 0.01-100 μM), LFME (0.25-16 mg/mL) and NBF-LFME (0.065-4 mg/mL) were recorded both before and 15 minutes following addition of 1 μM atropine (a non-selective muscarinic blocker) to the tissue bath.
The effect on the acetylcholine esterase enzyme (AChE) and ganglionic nicotinic receptors was investigated using three concentrations of either ACh (0.01, 0.02 and 0.03 μM), LFME (0.25, 0.5 and 1 mg/mL) or NBF-LFME (0.125, 0.25 and 0.5 mg/mL). The contractile effects induced by the standard, extract and fraction were recorded before and 15 minutes following addition of either 0.05 μM neostigmine (a cholinesterase inhibitor) or 100 μM hexamethonium (a ganglionic nicotinic blocker) to the tissue bath.
2.5. Statistical analysis
All data are expressed in terms of mean ± standard error mean (S.E.M). The statistical analysis of the data was performed using one-way analysis of variance (ANOVA) followed by Bonferroni/Dunnett (all mean) post hoc test (SuperANOVA, Abacus Concepts, Inc., Berkeley, CA, USA). The differences between the means were considered significant at p=0.05, 0.01 and 0.001. The number of determinations n = 8. The median effective concentration (EC50) was analyzed by GraphPad Prism version 5, USA.
3. Results
Our findings clearly showed that the addition of graded concentrations of either LFME, NBF-LFME to the organ bath produced concentration-dependent contractile responses in the ileal segments of guinea pig which was quite similar in pattern to that obtained using ACh (Figure 1B and 1C). ACh contractile responses were significantly abolished with 1 μM atropine pretreatment of the guinea pig ileum preparations (p < 0.001-0.01; Figure 1, Figure 2vs. 2B). Similarly, the contractile effects of LFME and NBF-LFME were significantly inhibited in tissue segments incubated with 1 μM atropine at organ bath concentrations of 0.5-16 mg/mL and 0.25-4 mg/mL, respectively (p < 0.001-0.01; Figure 1, Figure 2vs. 2F and 2Ivs. 2J, respectively). Similarly, the EC50 values obtained from the concentration-response curves of ACh, LFME and NBF-LFME were 0.584 μM, 2.0 mg/mL and 2.4 mg/mL, respectively.
Figure 1.
Effects of increasing concentrations of (A) acetylcholine (ACh), (B) Loranthus ferrugineus methanol extract (LFME) and (C) n-butanol fraction of Loranthus ferrugineus methanol extract (NBF-LFME) on the contrac-tile response in isolated guinea pig ileum subjected to pretreatment with 1 μM atropine. *p < 0.01 and †p < 0.001.
Figure 2.
Effects of increasing concentrations of acetylcholine (ACh) on the contractile response in isolated guinea pig ileum, from a typical tracer in which each centimeter corresponds to 1 minute and 1 g tension. (A) without pretreatment, (B) with 1 μM atropine pretreatment, (C) with 0.05 μM neostigmine pretreatment and (D) with 100 μM hexamethonium pretreatment. The addition of increasing concentrations of Loranthus ferrugineus methanol extract (LFME) is also shown: (E) without pretreatment, (F) with 1 μM atropine pretreatment, (G) with 0.05 μM neostigmine pretreatment and (H) with 100 μM hexamethonium pretreatment. Increasing concentrations of n-butanol fraction of L. ferrugineus methanol extract (NBF-LFME) also produced a response in guinea pig ileum: (I) without pretreatment, (J) with 1 μM atropine pretreatment, (K) with 0.05 μM neostigmine pretreatment and (L) with 100 μM hexamethonium pretreatment.
Data from typical traces demonstrated that the incubation of the ileal segments with 0.05 μM neostigmine markedly enhanced the contractile responses of the lower concentrations (0.01-0.03 M) of ACh (Figure 2Avs. 2C). LFME (0.25-1 mg/mL; Figure 2E vs. 2G) and NBF-LFME (0.125-0.5 mg/mL; Figure 2Ivs. 2K), evidently showed a similar pattern of potentiation in the contractile responses. However, no observable changes in the contractile responses of ACh (Figure 2Avs. 2D), LFME (Figure 2Evs. 2H) and NBF-LFME (Figure 2Ivs. 2L) were seen upon the incubation of the ileal preparations with 100 μM hexamethonium
4. Discussion
This study examined the effects of LFME and its active fraction NBF-LFME on isolated guinea pig ileum in the absence and presence of various antagonists. Different mechanisms are involved in the contraction of GI tract smooth muscle. These include the agonistic action of the parasympathetic neuro-transmitter ACh [9, 10] and the excitatory activity of the autacoid histamine [10, 11]. Thus, it is well known that cholinergic and histaminergic transmission is involved in the regulation of intestinal motility. Drugs that can influence cholinergic or histaminergic transmission by acting on their receptors or by affecting the release of endogenous ACh or histamine can consequently bring about GI tract smooth muscle contraction. The contractile effect of ACh on guinea pig ileum is mediated via muscarinic receptors, and is competitively and selectively blocked by atropine. Histamine induced contraction of guinea pig ileum is mediated by H1 receptors in which the latter effect is antagonized by mepyramine [12, 13].
Our experiments revealed that the pattern of activity of the crude LFME and its active fraction NBF-LFME on guinea pig ileal segments was similar to that of ACh rather than histamine since both the activity of the extract and its fraction was meaningfully inhibited in the presence of atropine. These results confirmed the presence of cholinergic (ACh-like) components in the LFME and its active fraction. Moreover, it is likely that LFME and NBF-LFME might have exerted their action mostly via M3 receptors since the latter receptor subtype is considered the main muscarinic receptor known to mediate contraction in normal smooth muscle of the GI tract [14, 15].
The cholinergic effect may have been due to indirect rather than direct muscarinic receptor activation, i.e. via the inhibition of the cholinesterase enzyme (AChE). Normally, AChE rapidly metabolizes and terminates the action of ACh at the junctions of various cholinergic nerve endings with their effector organs or postsynaptic sites [3, 16]. Neostigmine is an anti-AChE which acts as an indirect cholinergic drug and thus increases the availability of endogenous ACh by preventing its hydrolysis into inactive choline and acetyl CoA moieties [3, 16]. Data from our experiments showed that the effects of lowest concentrations of LFME and NBF-LFME resembled those of ACh whereby they were considerably enhanced in the presence of neostigmine, revealing the notion that the extract and its fraction could have served as a substrate for AChE. Furthermore, it was unlikely that LFME or NBF-LFME could have exerted their actions via the inhibition of the latter enzyme, since the magnitude of contraction with both the extract and its active fraction was not antagonized in the presence of neostigmine.
Another possible activity for ACh and ACh-like substances on the isolated guinea pig ileum might be through ganglionic nicotinic receptor activation and eventual increase in the tone and motor activity of the bowel [16]. Hexamethonium, a blocker of the autonomic ganglia that competes with ACh at nicotinic receptors in this tissue without affecting action potentials [16, 17], did not appreciably affect the contractile responses to either LFME or NBF-LFME following preincubation with isolated ileal segments. These observations rule out any possibility that LFME or NBF-LFME could have exerted their effect indirectly via nicotinic receptor stimulation.
The initial purification step of LFME using solvent-solvent fractionation was important because it contributed to a significant enhancement in the potency of NBF-LFME. Following fractionation, a 75% reduction in the bath concentration range resulted in a similar magnitude of contraction in the guinea pig ileum and an almost similar EC50 to the one observed prior to fractionation of LFME.
In summary, data derived from these experiments show for the first time the ability of LFME and its active fraction NBF-LFME to induce a concentration-dependent spasmogenic effect in isolated guinea pig ileum. These effects resemble those of ACh as they are likely to be involved in the activation of the intestinal M3 receptor subtype. AChE is possibly involved in the metabolism of the active constituents of LFME and NBF-LFME. While ACh is the main excitatory neurotransmitter released from the myenteric plexus and has a major role in regulating GI motility [18], several drugs aimed at influencing ACh release and/or antagonizing its action are used in the clinical setting to treat GI motility disorders. However, the efficacy of these drugs is often limited and they are frequently associated with undesired side effects that restrict their use. Constipation is a common health problem, and its treatment is often unsatisfactory. In the light of the present results, there is a potential beneficial effect for the active components of L. ferrugineus to decrease the bowel transit time. Thus additional research on the isolation of the active constituents is likely to pharmacologically yield unique compounds which may then be formulated or chemically modified to produce a prokinetic drug of natural origin.
Acknowledgments
We would like to express our deepest gratitude to Mr Roseli Hassan for his kind help during the collection of the plant material. Also, we gratefully acknowledge the Institute of Graduate Studies (IPS), USM for a USM Fellowship Award.
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PII: S2005-2901(09)60070-4
doi:10.1016/S2005-2901(09)60070-4
© 2009 Korean Pharmacopuncture Institute. Published by Elsevier Inc. All rights reserved.
Volume 2, Issue 4 , Pages 288-293, December 2009
