eCommons@AKU

Department of Biological & Biomedical Sciences Medical College, Pakistan

February 2008

Gut modulatory, blood pressure lowering, diureticand sedative activities of cardamomAnwar GilaniAga Khan University

Qaiser JabeenaAga Khan University

Arif-ullah KhanAga Khan University

Abdul Jabbar ShahAga Khan University

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Recommended CitationGilani, A., Jabeena, Q., Khan, A., Shah, A. (2008). Gut modulatory, blood pressure lowering, diuretic and sedative activities ofcardamom. Journal of Ethnopharmacology, 115(3), 463-472.Available at: http://ecommons.aku.edu/pakistan_fhs_mc_bbs/83

Available online at www.sciencedirect.com

Journal of Ethnopharmacology 115 (2008) 463–472

Gut modulatory, blood pressure lowering, diuretic and sedativeactivities of cardamom

Anwarul Hassan Gilani a,∗, Qaiser Jabeen a,b, Arif-ullah Khan a, Abdul Jabbar Shah a

a Natural Product Research Division, Department of Biological and Biomedical Sciences, Aga Khan University, Karachi 74800, Pakistanb Department of Pharmacy and Alternative Medicine, Islamia University, Bahawalpur, Pakistan

Received 6 September 2007; received in revised form 24 September 2007; accepted 14 October 2007Available online 22 October 2007

Abstract

Ethnopharmacological relevance: Cardamom (Elettaria cardamomum) is traditionally used in various gastrointestinal, cardiovascular and neuronaldisorders.Aim of the study: To rationalize cardamom use in constipation, colic, diarrhea, hypertension and as diuretic.Materials and methods: Cardamom crude extract (Ec.Cr) was studied using in vitro and in vivo techniques.Results: Ec.Cr caused atropine-sensitive stimulatory effect in isolated guinea-pig ileum at 3–10 mg/ml. In rabbit jejunum preparations, Ec.Crrelaxed spontaneous and K+ (80 mM)-induced contractions as well as shifted Ca++ curves to right, like verapamil. Ec.Cr (3–100 mg/kg) inducedfall in the arterial blood pressure (BP) of anaesthetized rats, partially blocked in atropinized animals. In endothelium-intact rat aorta, Ec.Crrelaxed phenylephrine (1 �M)-induced contractions, partially antagonized by atropine and also inhibited K+ (80 mM) contractions. In guinea-pigatria, Ec.Cr exhibited a cardio-depressant effect. Ec.Cr (1–10 mg/kg) produced diuresis in rats, accompanied by a saluretic effect. It enhancedpentobarbital-induced sleeping time in mice. Bio-assay directed fractionation revealed the separation of spasmogenic and spasmolytic componentsin the aqueous and organic fractions respectively.Conclusion: These results indicate that cardamom exhibits gut excitatory and inhibitory effects mediated through cholinergic and Ca++ antagonistmechanisms respectively and lowers BP via combination of both pathways. The diuretic and sedative effects may offer added value in its use inhypertension and epilepsy.© 2007 Published by Elsevier Ireland Ltd.

Keywords: Elettaria cardamomum; Cardamom; Gastrointestinal motility; Hypotensive; Diuretic; Sedative

1. Introduction

Elettaria cardamomum Maton (cardamom, family; Scita-minaceae) locally known as “elaichi” is a perennial herb,indigenous to India, Pakistan, Burma and Sri Lanka (Nadkarni,1976). In addition to its wide use for culinary purpose, cardamomhas folkloric repute as carminative, stomachic, diuretic, abortifi-cient, antibacterial, antiviral, antifungal and is considered usefulin treatment of constipation, colic, diarrhea, dyspepsia, vomit-ing, headache, epilepsy and cardiovascular diseases (Khan andRahman, 1992; Duke et al., 2002).

∗ Corresponding author. Tel.: +92 21 4864571;fax: +92 21 493 4294, 493 2095.

E-mail address: anwar.gilani@aku.edu (A.H. Gilani).

Phytochemical studies revealed the presence of multiplechemicals, such as �-terpineol, myrcene, heptane, subinene,limonene, cineol, �-phellandrene, menthone, �-pinene, �-pinene (Shaban et al., 1987), linalol, nerolidol (Okugawa etal., 1988), �-sitostenone, �-sitosterol, phytol, eugenyl acetate(Gopalakrishnan et al., 1990), bisabolene, borneol, citronellol,p-cymene, geraniol, geranyl acetate, stigmasterol and terpinene(Duke, 1992).

Cardamom in combination with some other plants was foundto reverse the liver damage induced by CCl4 (Shirwaikar et al.,1992). The aqueous and methanolic extracts of eight Zingib-eraceae herbs including cardamom were examined in rabbitsfor their effect on gastric secretion and the oral administra-tion of either extract caused a significant decrease in gastricsecretion and pepsin output (Sakai et al., 1989). In anotherstudy, cardamom was found to increase the gastric acid secre-

0378-8741/$ – see front matter © 2007 Published by Elsevier Ireland Ltd.doi:10.1016/j.jep.2007.10.015

464 A.H. Gilani et al. / Journal of Ethnopharmacology 115 (2008) 463–472

tion (Vasudevan et al., 2000). Clinical studies with herbal eyedrop preparation containing cardamom was found useful incases of cataract refractive errors and some infective condi-tions of the eye (Ghosh et al., 1985; Mukherji and Sahai, 1985;Mitra et al., 1986). Two commonly used Ayurvedic medicinesconsisting of Myristica fragrans, Syzygium aromaticum andcardamom exhibited antifertility activity (Sethi et al., 1987).Its volatile oil was found to exhibit analgesic, antiinflamma-tory, antimicrobial and antispasmodic properties (Al-Zuhairet al., 1996; Elgayyar et al., 2001; Daswani and Bohra,2003).

In this study, we report that cardamom exhibits combinationof spasmogenic, spasmolytic, blood pressure (BP)-lowering,vasodilator, cardio-suppressant, diuretic and sedative activities.The activity-directed fractionation of the crude extract resultedin the separation of gut excitatory and relaxant components inthe aqueous and organic fractions respectively.

2. Materials and methods

2.1. Plant material, preparation of crude extract andfractions

Dried fruits of Elettaria cardamomum were purchased froma local market in Karachi and the sample voucher (EC-SE-07-04-54) was submitted to the Department of Biological andBiomedical Sciences herbarium, Aga Khan University, Karachi.After cleaning of adulterant material, the fruits were ground withan electric grinder into a coarse powder. Extraction and frac-tionation was carried out as described previously (Williamsonet al., 1998). About 986 g of ground material was soaked inaqueous-methanol (70%) at room temperature (23–25 ◦C) for 3days with occasional shaking. It was filtered through a muslincloth and then through a Whatman qualitative grade 1 filterpaper. This procedure was repeated twice and the combinedfiltrates were evaporated on rotary evaporator under reducedpressure (−760 mmHg) to a thick, semi-solid pasty mass of darkbrown color; i.e. the crude extract (Ec.Cr), yielding approxi-mately 10.81%. Ec.Cr was completely solubilized both in thedistilled water and saline for use in in vitro and in vivo experi-ments.

Activity-guided fractionation was carried out, using sol-vents of increasing polarity. Ec.Cr was dissolved in 300 ml ofdistilled water. Petroleum spirit was added to it and shakenvigorously in a separating funnel. The petroleum spirit layer(upper) was collected thrice and evaporated on rotary evapo-rator to give the petroleum spirit fraction (Ec.Pt). The lowerlayer was taken in a separating funnel, chloroform was added.The chloroform layer (lower) was collected thrice and evap-orated on rotary evaporator to obtain the chloroform fraction(Ec.Cl). The other layer (upper) was again taken into a sep-arating funnel, ethyl acetate was added into it, separatedand was also evaporated in rotary evaporator to give theethyl acetate fraction (Ec.EtAc). The remaining lower layerwas collected and evaporated to yield the aqueous fraction(Ec.Aq).

2.2. Phytochemical screening

Preliminary screening of the plant extract for various phy-tochemical classes was carried out following the reportedmethods (Harborne, 1984; Akinyemi et al., 2005). Alkaloidswere tested by using Dragendorff’s reagent. Appearance of yel-low color with AlCl3 reagent and green or black with aqueousFeCl3 detects flavonoids and tannins respectively. Plant mate-rial treated with petroleum ether and subsequently extracted withCHCl3 was noted for green to pink or pink to purple color afterreaction with acetic anhydride and HCl in succession to detectsterols and terpenes respectively. Saponins were detected onthe basis of froth upon vigorous shaking. The observation ofyellow florescence under UV light on filter paper impregnatedwith the vapors from boiling extract indicates the presence ofcoumarins. Benzene extract prepared from acidified plant mate-rial was treated with NH4OH for anthraquinones based on theappearance of pink, violet or red color.

2.3. Drugs and animals

Acetylcholine chloride (ACh), atropine sulphate, furosemide,histamine, pentobarbital sodium, phenylephrine hydrochloride(PE), potassium chloride and verapamil hydrochloride werepurchased from Sigma Chemicals Company, St. Louis, MO,USA. Diazepam, pentothal sodium and heparin injectionswere obtained from F. Hoffmann-La Roche, Basel, Switzer-land, Abbot Laboratories, Karachi, Pakistan and Rotex Medica,Trittau, Germany respectively. Chemicals used for makingphysiological salt solutions were: potassium chloride (SigmaChemicals Co.), calcium chloride, glucose, magnesium chloride,magnesium sulfate, potassium dihydorgen phosphate, sodiumbicarbonate, sodium dihydorgen phosphate (Merck, Darmstadt,Germany) and sodium chloride from BDH Laboratory supplies,Poole, England. All chemicals used were of the analytical gradeavailable. Stock solutions of the drugs were made in distillH2O/saline and the subsequent dilutions were prepared freshon the day of experiment.

Animals used in this study such as adult rabbits (1–1.5 kg),guinea-pigs (450–500 g), Sprague–Dawley rats (200–220 g) andSwiss albino mice (20–25 g) of either sex and local breedwere housed at the Animal House of the Aga Khan University,maintained at 23–25 ◦C. Food was withdrawn from rabbits andguinea-pigs 24 h prior to the experiment and rats were deprivedof water during the collection of urine. Guinea-pigs were sacri-ficed by cervical dislocation and rabbits by blow on back of thehead. Experiments performed complied with the rulings of theInstitute of Laboratory Animal Resources, Commission on LifeSciences, National Research Council (NRC, 1996) and approvedby the Ethical Committee of the Aga Khan University.

2.4. Isolated tissue experiments

2.4.1. Guinea-pig ileumThe guinea-pig abdomen was cut, ileum was dissected out and

kept in Tyrode’s solution (Gilani and Aftab, 1992). Segments,each of about 2 cm length, were mounted individually in a 10 ml

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tissue bath, filled with Tyrode’s solution, at 37 ◦C and aeratedwith a mixture of 95% O2 in CO2 (carbogen). The composi-tion of the Tyrode’s solution in mM was: KCl 2.68, NaCl 136.9,MgCl2 1.05, NaHCO3 11.90, NaH2PO4 0.42, CaCl2 1.8 and glu-cose 5.55, with of pH 7.4. A preload of 1 g was applied to eachtissue and isotonic contractions were recorded with a Biosciencetransducer coupled to a Harvard oscillograph. After equilibra-tion period of 30 min, the tissues were repeatedly treated withsub-maximal concentration (0.3 �M) of ACh with 3 min intervaluntil constant responses were recorded. The contractile effect ofthe test material was assessed as percent of the maximum effectproduced by the control drug, ACh.

2.4.2. Rabbit jejunumThe jejunum was dissected out after surgical opening of rabbit

abdomen, kept in Tyrode’s solution and cleaned off mesenter-ies (Gilani et al., 2005a). Each segment of about 2 cm lengthwas suspended in a 10 ml tissue bath containing Tyrode’s solu-tion, maintained at 37 ◦C and aerated with carbogen. Intestinalresponses were recorded isotonically using Bioscience transduc-ers and oscillograph. Each tissue was allowed to equilibrate forat least 30 min before the addition of any drug and then stabi-lized with a sub-maximal concentration of ACh (0.3 �M). Underthese conditions, rabbit jejunum exhibits spontaneous rhythmiccontractions, allowing to test the relaxant (spasmolytic) effectdirectly without use of an agonist.

For the determination of calcium channel blocking (CCB)activity, high K+ (80 mM) was used to depolarize the prepa-rations as described by Farre et al. (1991). K+ was added tothe tissue bath, which produced a sustained contraction. Testmaterials were then added in a cumulative fashion to obtainconcentration-dependent inhibitory responses (Van Rossum,1963).

To confirm the Ca++ antagonist action of the test substance,the tissue was allowed to stabilize in normal Tyrode’s solution,which was then replaced with Ca++-free Tyrode’s solution con-taining EDTA (0.1 mM) for 30 min in order to remove Ca++

from the tissues. This solution was further replaced with K+-richand Ca++-free Tyrode’s solution, having the following compo-sition (mM): KCl 50, NaCl 91.04, MgCl2 1.05, NaHCO3 11.90,NaH2PO4 0.42, glucose 5.55 and EDTA 0.1. Following an incu-bation period of 30 min, control concentration–response curves(CRCs) of Ca++ were obtained. When the control Ca++ CRCswas found super-imposable (usually after two cycles), the tissuewas pretreated with the plant extract for 60 min to test the pos-sible CCB effect. The CRCs of Ca++ were reconstructed in thepresence of different concentrations of the test material.

2.4.3. Rat aortaThoracic aorta from the rats was set up by following the

procedure of Furchgott and Zawadzki (1980) with slight mod-ifications. Care was taken in isolating the tissue to avoid anydamaging of endothelium. It was then carefully cleaned of fatsand then made into rings. Individual rings were mounted in 5 mltissue bath with Kreb’s solution maintained at 37 ◦C and aer-ated with carbogen. Composition of the Kreb’s solution was(mM): NaCl 118.2, NaHCO3 25.0, CaCl2 2.5, KCl 4.7, KH2PO4

1.3, MgSO4 1.2 and glucose 11.7 (pH 7.4). Each tissue wasincubated for 30 min under the resting tension of 1 g. Changesin tension were recorded and analyzed isometrically throughforce transducer (Fort-10, WPI, UK) coupled to a bridge ampli-fier (Transbridge TBM 4M, WPI) and PowerLab ML 845 dataacquisition system (ADInstruments, Sydney, Australia). The tis-sues were than stabilized with PE (1 �M). After stabilization, aninduced contraction was obtained with PE (1 �M). Once plateauwas achieved, ACh (0.3 �M) was than tested on PE-inducedcontraction to observe the endothelium integrity. The tissueswere pre-incubated with atropine (1 �M) for 20 min to explorefor a cholinergic receptor mediated vasodilator effect. The pos-sibility of Ca++ antagonist effect involvement was also ruledout by administering the extract on high K+ (80 mM)-inducedcontractions.

2.4.4. Guinea-pig atriaRight atria from guinea-pigs were dissected out, cleaned of

fatty tissue and mounted individually in 20 ml tissue bath con-taining Kreb’s solution, at 32 ◦C and aerated with carbogen.The tissues were allowed to beat spontaneously (due to thepresence of pacemaker cells) under the resting tension of 1 gand an equilibrium period of 30 min was given (Tanaka et al.,1996). Tension changes in the tissue were recorded via a Grassforce–displacement transducer (model FT-03) coupled with aGrass model 7 Polygraph (Grass instrument company, Quincy,MA, USA).

2.5. In vivo experiments

2.5.1. Measurement of BPThese experiments were performed according to the method

previously described (Gilani et al., 2005b). Rats were anaes-thetized with thiopental sodium (70–90 mg/kg, i.p.) and thearterial BP was recorded through carotid artery cannulation viaa pressure transducer (P23 XL) coupled with a Grass model 7Polygraph. Drugs were injected through a cannula inserted intothe jugular vein. After a 20 min period of equilibrium, the ratswere injected intravenously with 0.1 ml saline (NaCl 0.9%) orwith the same volume of test substance. Arterial BP was allowedto return to the resting level between injections. Changes in BPwere recognized as the difference between the steady-state val-ues before and the lowest readings after injection. Mean arterialblood pressure (MABP) was calculated as the diastolic BP plusone-third pulse width.

2.5.2. Diuretic assayThe protocol of Ratnasooriya et al. (2004) was followed with

some modifications. Rats were divided into five groups of fiveanimals each. The control group received saline (50 ml/kg, i.p.)Another group of animals was given furosemide (10 mg/kg, i.p.)as reference diuretic. The other groups were treated with the dif-ferent doses of extract. Immediately after treatment, each animalwas housed in its metabolic cage. The urine was collected ingraduated vials and volume was measured every hour for 6 h.Total urine excreted out was collected and volume was calcu-lated. The assay was repeated at intervals of 1 week on the same

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group of rats. Treatments for each rat were randomized. Theelectrolyte concentration was measured using Corning flamephotometer 410, UK.

2.5.3. Pentobarbital-induced sleeping timeMice were divided into five groups of five animals each. One

group received normal saline (10 ml/kg, i.p.), another groupwas given diazepam (5 mg/kg, i.p.) while to the rest of thegroups, extract was administered in increasing doses (Gilaniet al., 2000a). The pentobarbital (75 mg/kg, i.p.) was than givenafter 1 h to all the animals. The prolongation of animals sleepingtime was noted.

2.5.4. Acute toxicity testAnimals were divided in groups of five mice each. The test

was performed using increasing doses of the extract, given orallyin 10 ml/kg volume to different groups serving as test groups(Sanmugapriya and Venkataraman, 2006). Another group wasadministered saline (10 ml/kg) as negative control. The micewere allowed food ad libitum and kept under regular observationfor 6 h while the lethality was recorded after 24 h.

2.6. Statistical analysis

Data expressed as mean ± standard error of mean (S.E.M.,n = number of experiments) and the median effective concentra-tions (EC50 values) with 95% confidence intervals (CI). Thestatistical parameter applied is Student’s t-test. p < 0.05 wasconsidered statistically significant. CRCs were analyzed by non-linear regression using GraphPad program (GraphPAD, SanDiego, CA, USA).

3. Results

3.1. Phytochemical screening

Ec.Cr was found to contain alkaloids, flavonoids, saponins,sterols and tannins while tested negative for the rest of classes.

3.2. Effect on ileum

In guinea-pig ileum, Ec.Cr (3–10 mg/ml) caused aconcentration-dependent contractile effect. The efficacy of thestimulant effect was 9.3 ± 2.36, 49.48 ± 5.53 and 70.61 ± 4.4%(n = 5) at the concentrations of 3, 5 and 10 mg/ml respectivelywhen compared to ACh maximum response. Pretreatment ofthe tissue with atropine (1 �M) completely blocked the effectof Ec.Cr, similar to that of ACh, while the histamine responseremained unchanged (Figs. 1 and 2).

3.3. Effect on jejunum

Ec.Cr caused a concentration-dependent inhibition of spon-taneous and K+ (80 mM)-induced contractions of rabbit jejunumwith respective EC50 values of 2.96 (2.61–3.36, 95% CI, n = 5)and 3.37 mg/ml (2.38–4.78, n = 5). Similarly, verapamil relaxedboth types of contractions with EC50 values of 0.46 (0.25–0.55,

Fig. 1. Typical tracing showing the stimulatory effect of Elettaria cardamomumcrude extract (Ec.Cr) in comparison to acetylcholine (ACh) and histamine (Hist)in the absence and presence of atropine in isolated guinea-pig ileum preparations.

n = 4) and 0.04 �M (0.03–0.05, n = 4) respectively (Fig. 3).Ec.Cr(0.3–1.0 mg/ml) shifted the Ca++ CRCs to right (Fig. 4A), likethat caused by verapamil (Fig. 4B).

3.4. Effect on BP

Ec.Cr at the doses of 3, 10, 30 and 100 mg/kg caused arespective fall of 6.81 ± 1.07, 16.49 ± 2.39, 36.78 ± 4.89 and52.6 ± 5.67% (n = 4) in MABP of anaesthetized rats. When thisBP-lowering effect was challenged with atropine (1 mg/kg), par-tial blockade was observed. Fig. 5 shows tracing from a typical

Fig. 2. Concentration–response curves showing the stimulatory effect of Elet-taria cardamomum crude extract (Ec.Cr) and its aqueous (Ec.Aq) fraction inisolated guinea-pig ileum preparations. The responses are expressed as per-cent to that of the acetylcholine maximum (ACh Max.). Values shown aremean ± S.E.M., n = 5.

A.H. Gilani et al. / Journal of Ethnopharmacology 115 (2008) 463–472 467

Fig. 3. Concentration-dependent inhibitory effect of Elettaria cardamomum crude extract (Ec.Cr), its petroleum spirit (Ec.Pt), chloroform (Ec.Cl) and ethylacetate(Ec.EtAc) fractions and verapamil on spontaneous and K+ (80 mM)-induced contractions in isolated rabbit jejunum preparations. Values shown are mean ± S.E.M.,n = 3–5.

experiment, whereas the combined data from different experi-ments are presented in Fig. 6.

3.5. Effect on aorta

When tested on vascular preparations at resting tension, theextract was found devoid of any vasoconstrictor effect up to10 mg/ml. Ec.Cr caused concentration-dependent relaxation ofPE (1 �M)-induced contractions in endothelium intact rat aorticpreparations with EC50 value of 2.94 mg/ml (1.99–4.33, n = 4).In the presence of atropine (1 �M), the inhibitory effect againstPE was partially blocked with significant change at 1 mg/ml(p < 0.05) and 3–5 mg/ml (p < 0.01), shifting the curve to theright, with EC50 value of 7.39 mg/ml (4.63–11.82, n = 4). Ec.Cralso inhibited the K+ (80 mM)-induced contractions with EC50value of 3.66 mg/ml (2.24–5.98, n = 4) as shown in Fig. 7.

3.6. Effect on atria

Ec.Cr caused concentration-dependent suppression of theatrial contractions with EC50 value of 2.94 mg/ml (1.67–5.16,n = 4). The cardio-depressant effect of Ec.Cr was found resistantto atropine (1 �M), while pretreatment of tissue with atropinecompletely blocked the inhibitory response of ACh (Fig. 8).

3.7. Effect on urine output

The urinary output of rats/100 g of the body wt./6 h insaline treated group was 3.4 ± 0.17 ml while with furosemide(10 mg/kg) was 6.93 ± 0.17 ml (p < 0.001 vs. saline). Ec.Cr atthe doses of 1, 3 and 10 mg/kg increased the urinary volume to4.13 ± 0.23, 5.05 ± 0.1 and 5.54 ± 0.26 ml (p < 0.001 vs. saline)respectively indicating diuretic effect (Fig. 9A). In addition tothe increase in urinary volume, Ec.Cr also enhanced the Na+

and K+ excretion. Total Na+ and K+ contents (mmol/100 g bodywt./6 h) in pooled urine samples were 0.63 ± 0.03, 0.69 ± 0.06,0.84 ± 0.08 (p < 0.05) and 0.16 ± 0.01, 0.14 ± 0.01, 0.20 ± 0.01(p < 0.01) respectively at the doses of 1, 3 and 10 mg/kg of Ec.Cr,as compare to the respective Na+ and K+ contents of 0.44 ± 0.13and 0.11 ± 0.03 mmol in saline group. Similarly, furosemide(10 mg/kg) increased the Na+ and K+ excretion to 0.88 ± 0.03(p < 0.01 vs. saline) and 0.19 ± 0.01 mmol (p < 0.05 vs. saline)respectively (Fig. 9B).

3.8. Effect on sleeping time

Ec.Cr (30–300 mg/kg) prolonged the pentobarbital-inducedsleeping time in mice. The sleeping time in the saline treatedgroup was 117.25 ± 2.1 min, while with diazepam (5 mg/kg)

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Fig. 4. Concentration–response curves of Ca++ in the absence and presence ofdifferent concentrations of (A) Elettaria cardamomum crude extract (Ec.Cr)and (B) verapamil in isolated rabbit jejunum preparations. Values shown aremean ± S.E.M., n = 4.

Fig. 5. Typical tracing showing the blood pressure (BP)-lowering effect ofElettaria cardamomum crude extract in comparison to acetylcholine (ACh) inabsence and presence of atropine in an anaesthetized rat. Small triangles showthe times at which the drugs were administered.

Fig. 6. Bar chart representing the dose-dependent effect of Elettaria car-damomum crude extract (Ec.Cr) on mean arterial blood pressure (MABP) inanaesthetized rats. Values shown are mean ± S.E.M., n = 4.

was 348.01 ± 13.27 min (p < 0.001 vs. saline). Ec.Cr at thedoses of 30, 100 and 300 mg/kg prolonged the sleeping time to162.5 ± 2.47 (p < 0.01 vs. saline), 202.75 ± 8.54 (p < 0.001 vs.saline) and 277.4 ± 4.91 min (p < 0.001 vs. saline) respectively(Fig. 10).

3.9. Acute toxicity

Ec.Cr did not cause any mortality up to the dose of 10 g/kg.

3.10. Effect of fractions

The aqueous fraction (Ec.Aq) caused atropine-sensitivemarked stimulatory effect in the guinea-pig ileum. The effi-

Fig. 7. Concentration–response curves showing the effect of (A) Elettaria car-damomum crude extract (Ec.Cr) on phenylephrine (PE)-induced contractionsin the absence (�) and presence of atropine (�) and on K+-induced contrac-tions (�) in isolated endothelium-intact rat aorta preparations. Values shown aremean ± S.E.M., n = 4. *p < 0.05 and **p < 0.01 vs. Ec.Cr on PE in the absenceof atropine curve values, Student’s t-test.

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Fig. 8. Typical tracing showing the cardio-depressant effect of Elettaria car-damomum crude extract and acetylcholine (ACh) in the absence and presenceof atropine in isolated guinea-pig atrial preparations.

cacy of the contractile effect was 34.05 ± 7.9, 52.52 ± 7.16and 83.93 ± 6.12% (n = 5) at 3, 5 and 10 mg/ml respectivelyas compared to ACh maximum response (Fig. 2). In rabbitjejunum, Ec.Aq was found devoid of any spasmolytic activ-

Fig. 9. Bar diagrams showing (A) diuretic effect of Elettaria cardamomum crudeextract (Ec.Cr) and furosemide and (B) effect of Ec.Cr and furosemide on Na+

and K+ excretion in rats. Values shown are mean ± S.E.M., n = 5. *p < 0.05,**p < 0.01 and ***p < 0.001 vs. saline, Student’s t-test.

Fig. 10. Bar diagram showing the effect of Elettaria cardamomum crude extract(Ec.Cr) and diazepam on pentobarbital-induced sleeping time in mice. Valuesshown are mean ± S.E.M., n = 5. **p < 0.01 and ***p < 0.001 vs. saline, Student’st-test.

ity, while the organic fractions, i.e. Ec.Pt, Ec.Cl and Ec.EtAccaused concentration-dependent relaxation of both spontaneousand K+ (80 mM)-induced contractions with respective EC50 val-ues of 0.02 (0.01–0.03, n = 4) and 0.08 (0.06–0.09, n = 5), 0.23(0.15–0.32, n = 4) and 0.79 (0.52–1.21, n = 4), 0.10 (0.06–0.17,n = 3) and 1.02 mg/ml (0.56–1.88, n = 3) as shown in Fig. 3.

4. Discussion

In view of the well-known use in indigestion, the cardamomwas tested for its possible stimulatory effect in guinea-pigileum, a quiescent preparation, considered useful for this pur-pose (Gilani and Aftab, 1992), where it produced excitatoryeffect, like that caused by ACh and histamine. Pretreatmentof the tissues with atropine, a muscarinic receptor antagonist(Arunlakhshana and Schild, 1959) abolished the stimulatoryeffect of extract, similar to that of ACh, without altering theresponse of histamine which indicates that the cardamom causesgut stimulation via cholinergic pathway. ACh is a neurotransmit-ter released by the parasympathetic nervous system, mediates itsaction in the gut by activation of M3 muscarinic receptor sub-types and hence plays an important physiological role to regulatethe peristaltic movements of the gut (Brown and Taylor, 1996).This may explain the medicinal use of cardamom in dyspepsiaand constipation.

The cardamom has also been used in overactive diseases ofthe gut, such as abdominal spasm and diarrhea. To see whetherit also contains some spasmolytic constituent(s), it was tested onrabbit jejunum, a spontaneously contracting hyperactive prepa-ration considered more responsive to spasmolytics (Ghayur andGilani, 2005). Ec.Cr inhibited the spontaneous contractions, thusshowing antispasmodic action. In earlier studies we observedthat the relaxant effect of medicinal plants is usually mediatedthrough blockade of Ca++ channels (Gilani et al., 1994a, 1999,2005b). To investigate whether the spasmolytic effect of car-damom is also mediated via similar mechanisms, the extract wastested on high K+-induced contractions. High K+ (>30 mM) isknown to cause smooth muscle contractions through opening of

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voltage-dependent L-type Ca++ channels, thus allowing influx ofextracellular Ca++ causing a contractile effect (Bolton, 1979) andthe substance causing inhibition of high K+-induced contractionis considered as inhibitor of Ca++ influx (Godfraind et al., 1986).Ec.Cr relaxed the high K+-induced contractions, like that causedby verapamil, a standard Ca++ antagonist (Fleckenstein, 1977),indicating its CCB action. The Ca++ antagonist effect of car-damom was further confirmed when it shifted the Ca++ CRCsto the right, like verapamil. The ability of the extract to inhibitthe high K+-induced contraction, followed by rightward dis-placement of Ca++ CRCs strongly suggest the presence of Ca++

antagonist(s) in cardamom.Both cholinergics and Ca++ antagonists are known to effec-

tively reduce the BP (Ghayur et al., 2005). When injectedintravenously to the normotensive rats under anesthesia, car-damom produced a dose-dependant fall in the arterial BP. Partialblockade of hypotensive responses with atropine indicate thatcardamom lowers BP due to the presence of cholinergic and anadditional (Ca++ antagonist) component(s), as identified in thegut preparations. BP is the product of peripheral resistance andcardiac output (Johansen, 1992), hence the extract was furtherstudied in aorta and heart for the possible vasodilator and cardiacinhibitory effects.

In endothelium-intact rat aorta, cardamom caused relaxationof PE-induced contractions, partially blocked in the presenceof atropine, as expected. Cholinergic receptor (M3) mediatedvasodilator activity in the vasculature is brought about by therelease of nitric oxide from the endothelium (Furchgott andZawadzki, 1980). Ec.Cr also relaxed the contractions induced byhigh K+, due to the presence of Ca++ antagonist component(s).When tested in spontaneously beating guinea-pig atria, the car-damom caused suppression of the atrial contractions, unaffectedby atropine pretreatment. The lack of atropine resistance to thecardamom effect in heart, as opposed to the gut and vessels,indicate the absence of cholinergic component in its effect onthe heart. It is possible that the observed cholinergic compo-nent(s) interacts only with M3 subtype without any effect on M2receptors located in the heart (Eglen and Watson, 1996) and thecardio-depressant effect of the extract is possibly due to CCB.However, further studies are required involving the identifica-tion of active constituents to get a clear answer of this interestingscenario.

The diuretic effect of cardamom was confirmed, when itcaused significant increase in the urine volume (diuresis) inrats, like furosemide, a standard diuretic (Ives, 1998). In par-allel with increase in the volume of urine, Ec.Cr also enhancedthe urinary excretion of Na+ and K+, similar to that caused byfurosemide. Loop diuretics in addition to increase in urinary flowrate are also known to strongly enhance the urinary electrolyteexcretion, considered as saluretic (Jackson, 2001). Diuretics arecommonly prescribed in combinations with antihypertensivedrugs for the control and management of moderate to severehypertension (Shah et al., 2004) and the presence of diuretic con-stituents is likely to complement the BP-lowering effect of thecardamom.

Sedatives are routinely used as adjunct therapy for the man-agement of epilepsy (McNamara, 2006). The extract was tested

for its possible sedative effect as a component which canadd to its overall potential in such condition. Ec.Cr causedprolongation of pentobarbital-induced sleeping time in mice,similar to that observed with diazepam, a standard seda-tive agent (Hobbs et al., 1996). This may also explain themedicinal use of cardamom in epilepsy, though further stud-ies are required to see whether it has direct antiepilepticeffect.

Activity-directed fractionation revealed that the stimulantcomponent (cholinomimetic) was separated in the aqueous frac-tion as it produced atropine-sensitive contractile effect in ileum,but found devoid of any relaxant effect in spontaneously con-tracting rabbit jejunum preparations. The efficacy of the aqueousfraction for its stimulatory effect was more pronounced thanthat of the parent crude extract, apparently due to the shift ofinhibitory component(s) to the organic fractions. All the threeorganic fractions, i.e., petroleum ether, chloroform and ethylacetate were found devoid of any stimulant effect, while causedconcentration-dependent inhibition of both spontaneous andhigh K+-induced contractions of rabbit jejunum. The petroleumspirit fraction was found to be the most potent for its spas-molytic action, being 150, 10 and 5-times more potent thanthe parent extract, chloroform and ethylacetate fraction respec-tively. This pattern of separating gut stimulatory and inhibitoryconstituents respectively in aqueous and organic fractions isin accordance with our previous findings, showing that thecholinergic component is concentrated in the aqueous fraction,while Ca++ antagonist in organic fractions (Gilani et al., 2000b,2006).

The results of phytochemical analysis showed that cardamomcontains alkaloids, flavonoids, saponins, sterols and tannins. Thechemical structures, properties and pharmacological activities ofsaponins and flavonoids are so complex. But the observed choli-nomimetic and CCB effects of the extract may be due to thepresence of saponins and flavonoids, as such phytochemicalshave been reported to exhibit cholinergic and Ca++ antago-nist activities respectively (Gilani et al., 1994b; Revuelta et al.,1997). Among the constituents of the plant, �-pinene possessesspasmogenic, antispasmodic and sedative effects. Geraniol andmyrcene are antispasmodic and hypotensive and citronellolexhibits sedative action (McMahon, 2002).

In acute toxicity testing, the extract was found safe at the doseas high as 10 g/kg, which is in line with the wide therapeutic anddietary use of cardamom.

5. Conclusion

This study showed that cardamom possess gut stimulatoryand inhibitory effects mediated through cholinomimetic andCa++ antagonist mechanisms respectively and lowers BP viacombination of both cholinergic and CCB pathways, thus pro-vide sound mechanistic background for its folkloric use inconstipation, colic, diarrhea and hypertension. The diureticactivity observed is likely to compliment the antihypertensiveuse of the cardamom. Sedative action might be a contribut-ing factor to the use of cardamom in the management ofepilepsy.

A.H. Gilani et al. / Journal of Ethnopharmacology 115 (2008) 463–472 471

Acknowledgement

This study was supported in part by the Higher EducationCommission, Government of Pakistan under the program ofindigenous PhD scholarship awarded to Ms. Qaiser Jabeen.

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