Possible mechanism of antispasmodic action of Garcinia kola seed extract on isolated guinea pig ileum.

Aims: Garcinia kola is used in West African countries for the treatment of various ailments such as cough, tooth decay, asthma and menstrual cramps. The inhibitory effect of Garcinia kola seed extract (GKE) on drug-induced contractions was studied on iliac smooth muscle preparations of guinea pig to ascertain the validity of the use of Garcinia kola in traditional medicine and to elucidate its possible mechanism of action. Place and Duration of Study: The study was done in Post Graduate Laboratory, Department of Pharmacology, College of Medical Sciences, University of Calabar, Calabar-Nigeria, between November 2013 and April 2014. Methodology: The antispasmodic influence of GKE (0.02 – 1 mg/ml) on acetylcholine, histamine and potassium chloride -induced contractions were carried out. The effect of GKE in a Ca 2+ -free Tyrode medium and in the presence of adrenergic antagonists was also investigated. Results: The results revealed that GKE inhibited or attenuated the spasmogenic effects of Original Research Article Udia et al.; BJMMR, 6(7): 688-698, 2015; Article no.BJMMR.2015.245 689 histamine and potassium chloride in a dose-dependent manner and shifted their log. doseresponse curves to the right, with pA2 values of 2.09±0.06 and 3.25±0.07 respectively. Preadministration of propranolol, prazosin or labetalol had no attenuating influence on the antispasmodic effect of GKE. Iliac smooth muscle responses to cumulative increased [Ca 2+ ] in a depolarizing bathing medium and in a Cafree Tyrode solution were also blocked. Comparative antispasmodic potencies indicated that papaverine and aminophylline were more potent than the extract. Conclusion: These findings suggest that Garcinia kola seed extract acts neither via cholinergic nor adrenergic receptor mediation, but may involve interference with Ca 2+ mobilization, thus sharing with papaverine and/or aminophylline similar mechanism(s) of action.


INTRODUCTION
Garcinia kola Heckle (Fam.: Clusiaceae or Guttiferae) is a tree that grows wild but sometimes cultivated near villages, mostly in moist environments. The tree is widespread throughout West Africa and is found up to an elevation of 900 meters [1]. The root and the seed parts of the tree are used in traditional medical practice. The root of Garcinia kola (G. kola) is a bitter chew stick and is used traditionally for the treatment of cough, tooth decay and gonorrhea. The mode of therapeutic application of the root is usually by oral administration of the cold alcoholic extract obtained by soaking G. kola root chippings in locally brewed gin. The seeds of G. kola are said to prevent or relieve intestinal spasm, act as curative for cold in the chest, relieve cough and hoarseness and to improve the voice [1][2][3]. The seeds are also used in traditional medicine for the treatment of asthma, bronchitis, gastroenteritis, rheumatism, menstrual cramps, cough, hepatitis and diarrhea [4,5].
Scientific investigation have been carried out to ascertain the biochemical, microbiological, pharmacological and physiological basis of therapeutic application of roots, seeds and leaves of G. kola in traditional medical practice [4][5][6][7][8][9][10]. The pharmacological activities of G. kola are many and include anti-oxidant [11], antibacterial [12][13][14], antiviral [3], antifungal [15], hepatoprotective and anti-inflammatory [16]. Naiho and Ugwu [17] reported on the efficacy of Garcinia kola in the reduction of blood pressure, an effect attributable to the presence of vasoactive substances in the plant herb. The activities of lactate dehydrogenase and glucose-6-phosephate dehydrogenase have been reported to be increased by aqueous extract of Garcinia kola [18]. The antioxidant potential of G. kola has been attributed to the presence of high content of ascorbic acid [19]. Oloyede and Afolabi [20] reported on the antioxidant potential and Fe 2+ chelating ability of the methanolic extract of Garcinia kola leaf; they concluded that G. kola leaf extract possess a significant natural antioxidant activity. Phytochemical screening revealed that G. kola contains alkaloids, cardiac glycosides, phenols, tannins, saponins and flavonoids [21]. Biflavonoids, some flavonoid derivatives and other phytochemical components have the potential of inhibiting smooth muscle tone and intestinal peristalsis, enabling G. kola to be used in the treatment of asthma [19], hypertension [17], gastric ulcer [22] and as an antispasmodic agent and smooth muscle relaxants [4,17,23,24].
Intestinal tone is present in normal conditions, a factor which renders stability to normal peristalsis [25]. This tone and peristalsis are regulated by acetylcholine and noradrenaline and their analogues which are elaborated by the autonomic nervous system (ANS) coupled with the availability of calcium ion (Ca 2+ ), which is a basic determiner of smooth muscle contraction [26][27][28][29]. While cholinergic stimulation and availability of Ca 2+ result in increase intestinal activity, adrenergic stimulation inhibits intestinal peristalsis with increased tone of sphincters [29,30]. Certain drugs such as isoprenaline or adrenaline which stimulate adrenergic receptors and papaverine or aminophylline which cause increase in cyclic adenosine 3', 5' monophosphate (cAMP) interfere with Ca 2+ fluxes during excitation-contraction coupling, also cause relaxation of smooth muscles [31,32]. Kolaviron, a biflavonoid complex from Garcinia kola, has been reported to induce vasodilation independent of endothelium in rats [23]. The pharmacological basis of G. kola in the treatment of asthma [19], hypertension [17], gastric ulcer [22] and other ailments have been linked to the presence of active phytochemical components present in the plant herb [4,17,[21][22][23][24]. Since G. kola has been shown to inhibit smooth muscle activity and is used in traditional medical practice to relieve intestinal spasms, cough and diarrhea, the present work was designed to investigate the effect of the methanolic extract of this species of kola on isolated tissue preparations, and the extract's possible mechanism of action.

Preparation of Extract
The extract of G. kola seed (GKE) was prepared by modification of the methods previously described [4,24,33]. Briefly, G. Kola seeds were dried and grinded to a powder form. The dried seed powder was Soxhlet extracted twice with petroleum ether at 45ºC. The petroleum ether treated material was then extracted with methanol for 72 hours. The extract was slowly evaporated to dryness at 50ºC.
Starting material of 300g yielded 31.52g of the extract which was stored at -4ºC. Weighed sample of the methanol extract of Garcinia kola seed (GKE) was then used to prepare the test solutions.

Methods
Ileal smooth muscles were obtained from guinea pigs of both sexes weighing 400-450g and set up in an organ bath of 50ml capacity, following. The physiological salt solution (PSS) was Tyrode's solution (composition in g/ml: NaCl 8.00, KCl 0.20, CaCl 2 0.02, MgCl 2 0.10, NaH 2 PO 4 0.05, NaHCO 3 and glucose 1.00). A high K + -(Ca 2+ free) PSS was prepared by omitting CaCl 2 , increasing KCl concentration by 50% and decreasing NaCl by 50% concentration. A Ca 2+ free Tyrode solution was prepared by omitting CaCl 2 in the preparation while NaCl and KCl concentrations remained unchanged. Each preparation was maintained at 37ºC under a resting tension of 1-1.5g and aerated with atmospheric air via an aerator (Type R.301, USA).
The preparations were allowed to equilibrate for at least 30 minutes before commencing experimentation while the PSS was changed every 15 minutes. Drug induced responses were recorded isotonically on graph papers of a slowmoving physiograph (Universal Oscillograph, Harvard Apparatus, Britain) with a horizontal writing lever which produced 10-12 fold magnifications. The physiograph was connected to an organ bath apparatus fitted with a heater (Double heated tissue bath, Harvard Apparatus, Britain). Drug or extract contact time with tissue was 0.5-2 minutes followed by 3-5 washings. An interval of 5-10 minutes was allowed between successive doses. In some experiments, drugtissue contact time was 5-7 minutes. Under the mentioned conditions for each tissue preparation, the graded or cumulative concentration-response curves were registered. Progressive increased concentrations of the agonists were duplicated subsequently. The experiments were repeated later after addition of 2 x10 -5 , 2x10 -4 and lx10 -3 g/mL of G. kola extract.
Spasmolytic activity of G. kola extract was compared with reference drugs (aminophylline and papaverine) dose-dependently by calculating the percentage inhibition of ileal contraction induced by acetylcholine. Reference drugs and GKE were given three minutes before administration of acetylcholine at a dose of 5 x 10 -7 M; a dose which has been previously found to cause appreciable ileal contraction. In another set of experiments, the preparations were washed with depolarizing solution [high K + -(Ca Concentration -responses were obtained by cumulative addition of CaCl 2. Later, the effects of GKE on Ca 2+ -induced contractions of the tissues were tested.
The effects of pretreatment with adrenergic antagonists -propranolol, prazosin and labetalolon the GKE ileal relaxant activities were also determined. The sequence of drug administration in the presence of adrenergic antagonist was adrenergic antagonist, followed by GKE and lastly acetylcholine. The interval between each drug administration was 2-3 minutes.
The drugs used were acetylcholine, histamine and aminophylline (Sigma, USA.), prazosin and labetalol (Allen and Hanburys LTD., London), papaverine (L.I.R.C.Z.A., Synthelabo SPA Ltd., Italy), propranolol (Macclesfield, Great Britain), adrenaline and KCl (M&B, England). All chemicals were of analytical grade. Drugs were dissolved in the deionized distilled water while the extract was dissolved in 1ml of DMSO or diethylether and the volume made up to10ml stock solution.

Result Analysis
The determination of EC 50 values were from the log dose-response curves [35] and confirmed by means of the logit representation from a plot of log (E A /E max -E A ) against log concentration (M). Where E A is the effect of agonist and E max is maximal effect. The EC 50 being the value on the abscissa when the log (E A /E max -E A ) equals zero [28]. The pA 2 values were determined by means of the Schild representation [36] from a plot of the reciprocal of the dose ratio (EC x Ag+Antagonist/EC x Ag) against antagonist concentration.
Where EC x Ag. is the concentration of agonist that produces x percent (30% or 50%) response on the tissue. The intersection on log antagonist concentration axis corresponds to pA 2 value [28].

pA 2 Determination
Data for the determination of pA 2 value were generated from log dose-response curves for agonist drugs and the presence of three different concentrations of GKE. The pA 2 is defined as the negative log of the molar concentration of antagonist drug which reduces the effect of double dose of agonist to that of a single dose. The Schild equation provides an experimental means of determining the pA 2 value [36].

Statistical Analysis
Results are expressed as mean values ± SEM based on at least four experiments. Significance was determined by student's t test. A probability level of 5% (P=.05) or better was considered significant.

Effects of GKE on Histamine and KCl -Induced Contractions
Graded additions of increasing concentrations of histamine (4x10 -10 -6x10 -7 M) induced concentration-dependent contractions of the ileum. Pre-administration of GKE (2x10 -5 g/mL, 2x10 -4 g/ml and 1x10 -3 g/mL) inhibited these contractions and shifted the graded log doseresponse curves of histamine-induced contractions to the right in a dose-dependent manner (Fig. 1). Potassium chloride (KCl, 4x10 -3 -5x10 -2 M) induced contractions were inhibited dose-dependently by the three doses of the extract (2x10 -5 g/mL, 2x10 -4 g/mL and 1x10 -3 g/mL of GKE). The log. dose-response curves for KCl were shifted to the right in the presence of the extract in a dose-dependent manner (Fig. 2).

Comparative Antispasmodic Potencies of GKE and Reference Drugs
Pre-administration of GKE resulted in antispasmodic effect on acetylcholine (5x10 -7 M) -induced spasm on the guinea pig ileum in a dose-dependent manner. At a dose of 0.3 mg/ml and 0.6 mg/ml, GKE significantly (P<0.01) reduced acetylcholine-induced contractions. Papaverine and aminophylline equally exhibited significant (P<0.01) dose-dependent antispasmodic effects on acetylcholine-induced contractions on the guinea pig ileum when compared with control. There was no significant (P=.05) difference between GKE antispasmodic effect and those of reference drugs at low dose levels but significant (P=.05 and P<0.01) difference occurred at higher dose levels. The effect of GKE at a dose of 0.6 mg/ml was comparable to that of papaverine at a dose of 8.0x10 -3 mg/ml (Table 2).

Effect of Pretreatment with Adrenergic Antagonists on the Actions of GKE
(5x10 -7 M) decreased by 9.0±1.3% in the presence of GKE (0.3 mg/ml) alone, it decreased by 11.6±1.0% and 10.0±1.8% when the tissue was pre incubated with propranolol (1.6x10 -2 mg/mL) and prazosin (4x10 -4 mg/mL) respectively, prior to GKE and ACh sequential administration (Fig. 3). There was no significant (P=.05) difference between the effects in the presence of adrenergic antagonists when compared with the effect of GKE alone.

DISCUSSION
The methanolic extract of Garcinia kola (GKE) exhibited does-dependant antispasmodic effects on spasms induced by acetylcholine, histamine and potassium chlorine (KCl). The doseresponse curves for histamine and KCl were non-parallely shifted to the right in the presence  prazosin, a α-adrenergic antagonist could not block the inhibitory effects of GKE. Labetalol, a drug which blocks both α-and β-adrenoceptors, blocked the effect of adrenaline on the ileum but failed to attenuate the inhibitory effect of GKE. These results are in line with the reports of Naiho and Ugwu [17] and indicate that GKE exert its smooth muscle relaxant effect by actions which are not due to neither cholinergic nor adrenergic receptor mediation.
Braide [4] suggested that it was unlikely that G. kola extracts acted by selectively blocking muscarinic, histaminic or serotoninergic receptors indiscriminately to cause the observed inhibition of the agonistic actions of these agents. The fact that the EC 50 values of histamine and KCl were significantly increased in the presence of the extract and that the E max values were diminished, lead us to believe that some components of the extract behave as nonspecific non-competitive antagonists of the said agonists at the level other than their respective receptors. It is known that agents which act on specific receptors to elicit their effects (agonist or antagonist) do so in very small concentrations [31], which can be ascertained experimentally from the values of pA 2 (-loglC 50 ). A high pA 2 value indicates that low concentration of the antagonist is needed to reduce the agonistic effect by 50%, whereas a low pA 2 value indicates that a high concentration of the antagonist is needed. The pA 2 values for GKE, as calculated from the plot of Schild equation [36] were 2.69±0.06 and 3.25±0.07 in the presence of the agonistic action of histamine and KCl respectively. These low pA 2 values indicate that GKE acts via non-specific, non-competitive inhibition.
KCl induced dose-dependent ileal muscle contractions in normal Tyrode solution were attenuated by pre-administration of GKE. In a Ca 2+ -free Tyrode medium, no response to depolarization by KCl was observed. This result confirms the report that KCl induced contraction is dependent on extracellular Ca 2+ entry [38]. Responses of the guinea pig ileum to cumulative increased Ca 2+ concentration in a depolarizing bathing medium and in Ca 2+ -free Tyrode were also blocked. Given that the contraction of the ileum previously depolarized is proportional to the addition of Ca 2+ , and since Ca 2+ is a basic determiner of muscle contraction, it can be proposed that the antispasmodic activity of the extract of G. kola is either by blocking the release of intracellularly bound Ca 2+ or by preventing the entry of extracellular Ca 2+ into the smooth muscle cell. Adesuyi et al. [21] and Mazi et al. [39] on commenting on the phytochemical, proximate composition and nutritive properties of Garcinia kola concluded that the nutritive and health benefits of this plant drug is attributable to the abundant active phytochemical constituents present in it. The blood pressure lowering effect [17], the anti-asthmatic effect [19] and the antimalarial activity [40] of Garcinia kola were all attributable to the presence of pharmacologically active components present in the plant. In a related study, Townsend et al. [41] indicated that the airway relaxant effect of Ginger and its isolated active components relax airway smooth muscles in part by altering intracellular Ca 2+ regulation, an effect mediated by the plant's active components.
Braide [4] proposed that G. kola alkaloid and flavonoid fractions produced muscle relaxation by interference with various calcium pools responsible for excitation -contraction coupling, such as chelation of extracellular Ca 2+ and inhibition of Ca 2+ influx.
Adaramoye and Medeiros [23] indicated that the vasorelaxant effects of kolaviron, a biflavonoid complex from Garcinia kola, involve extracellular Ca 2+ influx blockade, inhibition of intracellular Ca 2+ release and the opening of K + channels which resulted to membrane hyperpolarization/repolarization. In their report, Ebomoyi and Okojie [19] concluded that the anti-asthmatic mechanisms of Garcinia kola include, among others, "inhibition of Ca 2+ influx by acting as a blocker of both receptoroperated and voltage-dependent Ca 2+ channels". In smooth muscles, increase in cAMP concentration alters cell membrane permeability to ions with resultant muscle relaxation, an effect which could be brought about by β-adrenergic agonist, aminophylline and papaverine [32]. Relaxation of smooth muscle occurs when free (unbound) Ca 2+ in the cytoplasm is reduced as a result of inhibition of Ca 2+ influx, reuptake into cellular stores, active extrusion across cell membrane or a combination of all these processes [25,26]. Papaverine has been proposed to act via reuptake and active extrusion processes whereas aminophylline acts mainly by inhibiting Ca 2+ influx [4,32]. The present study suggests a link with the mechanism of action of aminophylline and GKE via inhibition of Ca 2+ influx, since KCl induced contraction, which depends on extracellular Ca 2+ entry [38] was also blocked by GKE. However, if GKE is acting as a chelator of Ca 2+ as propose to be a possible mode of action of flavonoids on divalent cation sensitive steps in the regulation of cellular secretions and smooth muscle activity [4], then it is possible that GKE interacts with Ca 2+ via more than one process that link Ca 2+ with smooth muscle relaxation.

CONCLUSION
Smooth muscle relaxation may result from inhibition of cholinergic receptors or stimulation of adrenergic receptors. In addition, interference with Ca 2+ mobilization results in inhibition of smooth muscle contraction. We demonstrated that GKE exert its smooth muscle relaxant effect by actions which are not due to neither cholinergic nor adrenergic receptor mediation and the low pA 2 values indicate that GKE acts via non-specific, non-competitive inhibition. The results from the present study also showed that responses of the guinea pig ileum to cumulative increased Ca 2+ concentration in a depolarizing bathing medium and in Ca 2+ -free Tyrode were also blocked. Given that the contraction of the ileum previously depolarized is proportional to the addition of Ca 2+ , it is proposed that the antispasmodic activity of the extract of G. kola is either by blocking the release of intracellularly bound Ca 2+ or by preventing the entry of extracellular Ca 2+ into the smooth muscle cell. In summary, the methanolic extract of Garcinia kola exhibit antispasmodic activity via nonreceptor interaction, but may involve interference with Ca 2+ mobilization with subsequent impairment of excitation-contraction coupling.

CONSENT
Not applicable.

ETHICAL APPROVAL
We declare that "Principles of laboratory animal care" (NIH publication No. 85-23, revised 1985) were followed; the laws governing the handling of laboratory animals in College of Medical Sciences of this University were strictly adhered to. All experiments have been approved by the Departmental and Faculty Boards of the College.