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J Acupunct Meridian Stud 2013;6(4):199e207

- RESEARCH ART ICLE -

Effects of Extracts and Fractions of Gynuraprocumbens on Rat Atrial Contraction

Omar Saad Saleh Abrika 1,*, Mun Fei Yam 1, Mohd. Zaini Asmawi 1,Amirin Sadikun 1, Hamady Dieng 2, Elssanousi Ali Hussain 1

1 School of Pharmaceutical Sciences, Universiti Sains Malaysia, Penang, Malaysia2 School of Biological Sciences, Universiti Sains Malaysia, Penang, Malaysia

Available online Feb 12, 2013

Received: Jun 2, 2012Revised: Oct 24, 2012Accepted: Oct 31, 2012

KEYWORDSGynura procumbens;inotropic activity;rat;atrial contraction;hypertension

* CE

CopypISSNhttp

orresponding author. School of Ph-mail: edri7@yahoo.com

right ª 2013, International Pharm2005-2901 eISSN 2093-8152

://dx.doi.org/10.1016/j.jams.201

AbstractThere is currently a great deal of research interest in utilizing plant compounds against hu-man diseases, including hypertension. The present study investigated the effects of differ-ent extracts and fractions from leaves ofGynura procumbensMerr. on rat atrial contractionin vitro. Isolated left and right atria, mounted in a 20-ml organ bath, were allowed to equi-librate for 15 min before the application of the extracts or fractions. The extracts (petro-leum-ether extract (PE) and methanol extract (ME)) and the fractions (chloroform fraction(CHL), ethyl-acetate fraction (EA), n-butanol fraction (NB) and water fraction (WA) ofthe methanol extract) were tested at three concentrations (0.25, 0.5 and 1.0 mg/ml), witha b-adrenergic agonist (isoprenaline) as a control. All data on contraction responses werelog-transformed and analyzed. When exposed to the different extracts, both atria tendedto exhibit greater contractive responses with the NB whereas cardiac contractions hada tendency to be reduced withmost other extracts. For a given extract, the contraction re-sponses were particularly greater at 0.5 mg/ml for the right atrium and at 1 mg/ml for theleft atrium. Further analysis focusing on the NB fraction revealed that positive inotropismwas greater in left atria exposed to highly-concentrated F2 and F3 sub-fractions. Taken to-gether, our results suggest that NB extracts and fractions from the G. procumbens-leafmethanol extract have positive inotropic activities and, hence, can be considered as an al-ternative/traditional medicine against increased blood pressure in humans or can be usedin strategies aimed at finding antihypertensive biomolecules from an accessible source.

armaceutical Sciences, Universiti Sains Malaysia, 11800 Minden, Penang, Malaysia.

acopuncture Institute

3.01.020

200 O.S.S. Abrika et al.

1. Introduction

Contemporary lifestyle has become a major threat to publichealth. Frequent cases of arteriosclerosis, obesity, diabetesmellitus, and cancers remind us of the continuous threat ofdiseases related to our lifestyles, and as does the rise incirculatory system diseases such as heart disease, pre-hypertension and hypertension in developed countries[1,2]. Hypertension can be caused by many factors,including increase in the volume of body fluid, resistance ofthe blood vessels, and other factors that trigger a rise inblood pressure [3]. Attempts to control increased bloodpressure have been used as strategies to control arterialhypertension [4,5]. Although treatment of arterial hyper-tension has been shown to be quite efficacious in reducingcardiovascular mortality and morbidity [4], hypertensioncontrol at the population level has been generally consid-ered insufficient. In the United Kingdom, for instance, 94%of hypertensive patients still have their blood pressurehigher than the normal [6], whereas in the United States,only 27% had a normal blood pressure following treatment[7]. This intractability of hypertension has not changed,and there is a noticeable increase in hypertension casesrecently [8]; about 26% of adults worldwide (972 million)are known to have hypertension [2]. Current optimismbased on pharmacological therapy, which consists ofdeveloping drug formulations, is severely impeded by druginteractions, dose dependence, and adverse effects such asthe possibility of depression. Clearly, the development ofalternative strategies complementary to existing controlmethods is needed.

From the beginning of recorded history, plants haveoften been used to cure human diseases [9,10]. Today, withthe increasing threat of intractable diseases, e.g., hyper-tension, a substantial body of research is directed towardsfinding bioactive molecules from plants [11,12]. One suchplant is Gynura procumbens, a perennial herbaceousmember of the Asteraceae family [13].

In South-East Asia, this plant is widely distributed andhas often been used to treat diseases. In Indonesia, leavesof the Compositae family are routinely used for treatingkidney diseases, eruptive fevers, rash, hypertension, dia-betes mellitus, and hyperlipidemia [14]. In Thailand, wherethis plant is empirically used against topical inflammation,rheumatism, and viral ailments, Iskander and colleagueshave reported the anti-inflammatory actions of its extracts[15]. Recently, pharmacological investigations in Singaporehave shown that extracts of G. procumbens reduced serumcholesterol and triglyceride levels in streptozotocin-induced diabetic rats [16]. In Malaysia, evidence existsthat G. procumbens has antidiabetic properties [17,18]. Inthis country, the prevalence of hypertension is high, butlevels of awareness, treatment, and control are low [19].

A few experiments have demonstrated the effects ofG. procumbens on hypertension. Kim et al showed theantihypertensive effects of aqueous extracts of G. pro-cumbens, but this investigation used spontaneously hyper-tensive rats and did not focus on the heart tissues [20]. Inanother study, Hoe et al investigated the effects of leafextract from G. procumbens on rat heart, but this inves-tigation involved only the hypotensive effects [21]. In each

of these studies, the approach was not bioassay directed,an approach fundamental to the discovery of novel naturalproducts from their natural sources and/or to increase theirstandardization preparation to formulate into dosage formsfor human usage after clinical trials.

In summary, there is little analysis on the significance ofthe different extracts and fractions of G. procumbens onheart tissues. Therefore, the present study examined theeffects of G. procumbens leaf extracts and fractions of theactive extract on the contraction of left and right atria (LAand RA, respectively) isolated from Sprague Dawley (SD)rats with the aim of exploring the possibilities of its stan-dardization or industrial scaling up for natural productsagainst hypertension.

2. Materials and methods

2.1. Plant material

Fresh green leaves of the plant Gynura procumbens (Lour.)Merr. were collected from Relau Agricultural Center,Penang. The plant was identified by En Adenan Jaafar ofthe School of Biological Sciences, Universiti Sains Malaysia.A voucher specimen (reference no: 10117) was deposited atthe Herbarium, School of Biological Sciences, UniversitiSains Malaysia. The leaves were washed and oven dried at38�C for 4 days in the School of Pharmaceutical Sciences,Universiti Sains Malaysia. The dried leaf substrates weremilled into powder. Using this procedure, we obtained 720 gof dry powdered leaves that was packed into a cellulosethimble covered with cotton wool.

2.2. Successive extractions

The leaf powder was first extracted using petroleum ether(PE) at 60 to 80�C for 48 hours in a Soxhlet apparatus (Model77-815 Spare extractor, Noida, India) and allowed to dry forthe same period of time. The de-fatted dry product wasthen subjected to methanol extraction for a period of72 hours. The PE and methanol (ME) extracts were con-centrated using a rotary evaporator (Model RE 21, Buchi,Switzerland). The resulting concentrated extracts werefreeze-dried using a Hetovac VR-1 (HETO Lab Equipment,Gydevang 17-19, 3450, ALLERØD, Denmark) freeze dryer(yield of ME extract: 13% of the dry plant).

The ME extract is further fractionated according to theschematic diagram shown in Fig. 1. The extract (110 g) isdissolved in water, transferred to a separating funnel andfractionated to chloroform (CHL), ethyl acetate (EA), n-butanol (NB), and aqueous (WA) fractions. The yield is asfollows: CHL, 26.36%; EA, 14.54%; NF, 15.45%; and WA,36.63% of ME.

2.3. Fractionation and thin-layer chromatographyfinger printing analysis

The active NB fraction was selected for further fractionationby the dry flash chromatography method of Harwood et alwith a slight modification. Three subfractions (F1, F2, andF3; yield: 14.44%, 38.33%, and 31.11% of NB, respectively)

Figure 1 Schematic diagram of bio-guided fraction of G. procumbens on rat atrium contraction.

Effect of Gynura procumbens extracts and fractions on rat atrial contraction 201

were obtained and subsequently tested for an arterial con-traction effect [22]. The most active fraction along with theME and NB extracts were run on thin-layer chromatography(TLC) plates using the modified method of Ling et al [23].Ethyl acetate:formic acid:glacial acetic acid:water(100:11:11:27) was used as the mobile phase, and flavonoidrutin hydrate (RH) was used as a reference compound. Ineach case, 3 mL of the samples were added to the develop-ment chamber holding a silica plate. The time allowed forthe development and maximal separation of the active in-gredients present in the samples was 20e30 minutes. Theplates were then rinsed and dried using a hair dryer. Driedplates were checked under 360-nm UV light. In the fractio-nation analysis, the fractions were obtained by evaporationusing the same rotary evaporator as mentioned before. Inthe TLC analysis, the color and the distance of the unknownspots were compared with RH. Rf values were calculatedusing the formula: Rf Z migration distance of sport/migration distance of solvent [24]. Phytochemical tests werealso carried out for various constituents of F3, ME, and NBusing different reagents: natural product polyethylene gly-col (NP/PEG) reagent for flavonoids; antimony trichloride

reagent for terpenoids; and sulfuric acid reagent forcoumarins.

2.4. High performance thin-layer chromatographyprocedure

Chromatography was performed on a preactivated (100�C)silica gel 60F254 TLC plate (20 � 10 cm; 0.25 mm Layerthickness; Merck). The Camag densitometry (Camag Model-3 TLC scanner equipped with Camag CATS 4 software;Mutten, Switzerland) and a reflectance spectrometer(190e700 nm) were employed for the analysis. The slit wasset to 8 mm � 0.4 mm, and data acquisition and processingwere performed using winCATS software. Four-microlitersamples were applied to the layer at 8-mm width bands,positioned 10 mm from the bottom of the plate usinga Camag Linomat IV automated TLC applicator with nitro-gen flow, providing delivery from the string at a speed of10 mL/s that was maintained for all analyses. The high-performance TLC (HPTLC) plates were developed ina Camag twin-trough glass tank presaturated with the

202 O.S.S. Abrika et al.

mobile phase [ethyl acetate:methanol:water (100:13.5:10)]for 2 hours at room temperature (22e24�C). The solventwas allowed to run across the plate to a height of 8 cm.After development, the TLC plate was dried, andthe components were visualized by observing under UVlight at 365- and 254- nm for kaempferol-3-O-rutinoside andastragalin, respectively. The quantitative determinationwas performed by means of the winCATS software programusing the external calibration method. The calibrationcurve was prepared using kaempferol-3-O-rutinoside andastragalin in the range 15.63 mg/mL to 1000 mg/mL inmethanol. The active subfraction F3 of G. procumbens NBfractions (10 mg/mL) in methanol were subjected to HPTLCanalysis [25].

2.5. Animals

Male SD rats weighing 220e250 g were used as the exper-imental animals. The rats were maintained in the animalhouse and fed with gold-coined chow according to standardfeeding procedures at the School of Pharmaceutical Sci-ences, Universiti Sains Malaysia.

2.6. Statement on ethical issues

This study was carried out in accordance with the principlesexpressed in the Declaration of Helsinki. The study wasconducted following guidelines of the Laboratory AnimalCare (NIH publication #85�23, revised in 1985) andapproved by the Animal Ethical Committee of the School ofPharmaceutical Sciences, Universiti Sains Malaysia.

2.7. Tissue isolation and their setting in organ baths

The rats were killed by stunning, followed by exsanguina-tion, and dissected for the RA and LA. To isolate RA froma given rat, a midline incision was made from its neck re-gion over its chest region down to the abdomen in order toexpose the visceral content and abdominal aorta. The heartwas carefully removed and immediately placed in an ice-cold Petri dish containing Krebs physiological salt solutionaerated with a gas mixture of 95% oxygen and 5% carbondioxide. Krebs solution was prepared according to a previ-ously reported method, and the spontaneously beating RAwas then isolated and care was taken to avoid any damagesto the sinoatrial nodes, the RA pacemaker [26]. The beatingisolated RA, with one end tied to a tissue holder, wascarefully placed into a 20-mL organ bath. The organ bathconsisted of a jacketed glass tube flanked with an inflowthat provides a 37�C water bath. In the middle of thejacket, it holds the experimental physiological solution,which can be poured out through a plastic tube-like tap,which is present at the external base of the jacketed tube.Internally, the jacket consists of a tissue holder used to fixthe second end of the test organ aerated as previously. Thesalt solution is supplied through a coil held inside the jacketaround the organ. Following the same procedure used forthe RA, the LA was dissected from the remaining part ofheart. The undamaged isolated LA was electrically stimu-lated. A suspension of an isolated LA in between electrodes

set at 2 Hz, 5 milliseconds, and threshold voltage plus 50%resulted in continuous beating for many hours.

2.8. Experimental protocol

The experiment aimed to examine the CR patterns of theisolated LA and RA. First, the baseline contraction for allexperimental tissues was established. To this end, a newlyisolated tissue was placed within the organ bath filled withKrebs solution and connected to a force displacementtransducer (Model FT03, Grass Instruments Co., Warwick,Rhode Island, USA). The transducer was connected toa polygraph (Grass Polygraph Model 79D, Grass InstrumentsCo., Warwick, Rhode Island, USA) for the isometric recordof the heart muscle’s contractions. Krebs solution wasrenewed every 15 minutes three times. Once the tissueexhibited steady-state equilibrium, it was allowed to do sofor 15 minutes. Increasing concentrations of isoproterenol(Sigma Chemical Co., St. Louis, MO, USA), a b-adrenergicagonist, was added to Krebs solution holding the tissue,starting with 10�9 mg/mL. When the maximal response wasattained, Krebs solution was replaced as previously, anda given concentration of a given test extract was added.Once the tissue re-expressed a baseline contraction pat-tern, it was then treated with isoproterenol as stated ear-lier. In all experiments, tissues were set at a baselinecontraction state prior to exposure to test extracts. Dif-ferent extracts and an agonist (isoproterenol) were used.All the test extracts were titrated with 0.5%polyethyleneglycol-400 (PEG-400). The three test concen-trations of the extract (0.25, 0.5 and 1.0 mg/mL) wereprepared by dissolving in Krebs solution. In each case, thetest solution was replicated three times. For each testextract, the CRs derived from the polygraph record wereexpressed in mm. The CR induced by any test extract andfor each concentration was derived from the graph andconsidered as the distance in millimeters between the levelof the baseline and that of the maximal response. Thesenumbers were transformed into percentages.

2.9. Statistical analysis

The differences in CRs were evaluated by analysis of var-iance (ANOVA) using the statistical software package(Systat v.11, Systat Software, Inc., Richmond, CA, USA)(Systat 2004) [27]. Mean values of CRs were separated byTukey’s multiple comparison tests. In all statistical ana-lyses, p < 0.05 was taken to indicate the statisticalsignificance.

3. Results

3.1. HPTLC and TLC studies

TLC analysis revealed a greater number of spots in ME. Thechromatogram profile from the NB fraction had a number ofspots similar to that of its F3 subfraction (Table 1) (Fig. 2).The Rf of ME varied from 0.1 to 0.4, a range of values withinthat established by Wagner and Hrazdina [28] for flavonoidglycosides. The Rf range of the NB fraction, which was

Table 1 Results of TLC analysis for ME extract, NB fraction, and F3 subfraction of G. procumbens.

Samples Spot number Spot color Range of Rf value

ME 7 3 blue, 2 greenish yellow, and 2 red spots 0.1e0.4NB 4 Greenish yellow, yellow, orange and pale yellow spots 0.43e0.74F3 subfraction 4 Greenish yellow, yellow, orange and pale yellow spots 0.43e0.74

NB Z n-butanol fraction; ME Z methanol extract; TLC Z thin layer chromatography.

Effect of Gynura procumbens extracts and fractions on rat atrial contraction 203

similar to that of F3 subfraction, oscillated between 0.43and 0.74, a range of values established for flavonoids basedon Wagner and Hrazdina’s [28] classification (Table 1). TheHPTLC study revealed that the F3 subfraction contained7.52 � 0.3% kaempferol-3-O-rutinoside and 2.33 � 0.4%astragalin.

3.2. Effects of extracts, fractions, and subfractionsof G. procumbens on right atrium

3.2.1. Effects of extracts and fractions on right atriumcontraction patternsThe different extracts of G. procumbens and their con-centrations had variable effects on the CRs of the RA. Thepattern of contraction differed significantly between ex-tracts when given at a concentration of 0.25 mg/mL and0.5 mg/mL (Fig. 3). At the lowest concentration, thecontraction baselines induced by isoprenaline were lowerthan that of all extracts. This effect was minor with MEand WA (Fig. 3C and E), intermediate with NB (Fig. 3F) andpronounced with PE, CHL, EA (Fig. 3A, C, and D). At0.5 mg/mL, the CRs induced by WA, EA, PE, and CHL weregreater than the contraction baselines induced by iso-prenaline, which in turn, were lower than those generatedby ME and NB (Fig. 2B and F). At the highest concentration,

Figure 2 TLC profile obtained from G. procumbens (A)methanol extract (B) n-butanol fraction (n-B), n-butanol sub-fraction f 3 (n-Bf 3), and standard rutin hydrate (Rut.) usingethyl acetate, formic acid, glacial acetic acid, and water(100:11:11:27) as the mobile phase after being sprayedwith NP/PEG reagent under 365-nm UV light. NP/PEG Z natural product/polyethylene glycol; TLC Z thin-layerchromatography.

all extracts blocked the spontaneous contractions of RA(Fig. 3).

3.2.2. Effects of n-butanol subfractions on right atriumcontraction patternsThe CRs varied between concentrations of subfractions F1,F2, and F3 (Fig. 3GeI). At 0.25 mg/mL, the contractionbaselines due to the isoprenaline were lower than that ofsubfractions F1 and F2, but higher than that of NB sub-fraction F3 (Fig. 3GeI). A similar pattern of contractionvariations was observed at 0.5 mg/mL, but the decreasingeffect of F3 was more pronounced (Fig. 2GeI). At 1 mg/mL,RA did not exhibit any spontaneous contractions followingexposure to each of the three fractions (Fig. 3GeI). sub-fraction F1 and F2 seem to have greater negative inotropiceffects when compared to subfraction F3 (Fig. 3GeI).

3.3. Effects of extracts, fractions, and subfractionsof G. procumbens on left atrium

3.3.1. Effects of extracts and fractions on left atriumcontraction patternsThe different concentrations of the extracts from the planthad varying effects on CRs of the rat LA. The contractionpatterns significantly varied with type of extracts andfractions at 0.25, 0.5, and 1 mg/mL (Fig. 3AeF). At thelowest concentration, the contraction baselines induced byisoprenaline were lower than those of PE, ME, CHL, and WA(Fig. 4A, B, C, and E, respectively) and higher than those ofEA and NB (Fig. 4D and F, respectively). At the two highestconcentrations, these baseline contractions were greaterthan the contraction level induced by extracts, excludingPE, ME, and EA (Fig. 4A, B, and D, respectively). At thehighest concentration, the NB extract induced the highestdepletion of isoprenaline-induced contraction level. Theinitial mean frequency of maximal contraction of88.6 � 2.75 was reduced to 22.3 � 2.2 following LA expo-sure to the NB fraction (Fig. 4F). It seems likely, therefore,that NB fraction has the highest negative inotropic impactfor antihypertensive effects among the test extracts.

3.3.2. Effects of n-butanol subfractions left atriumcontraction patternsThe fractions induced variable effects on LA contractionsaccording to the concentration at which they were applied(Fig. 4GeI). At 0.25 mg/mL, CRs induced by the isoprena-line were consistently lower than that of subfractions F1and F3, but higher than that of subfraction F2 (Fig. 4GeI).At 0.5 mg/mL, isoprenaline-induced CRs were significantlyhigher than those of subfractions F2 and F3 and lower than

Figure 3 Effects of extracts, fractions, and subfractions of G. procumbens on right atrium contraction. (A) PE; (B) ME; (C) CHLfraction; (D) EA fraction; (E) WA fraction; (F) NB fraction; (G) NB subfraction F1; (H) NB subfraction F2; (I) NB subfraction F3.*p < 0.05 compared to control. **p < 0.01 compared to control. ***p < 0.001 compared to control. CHL Z chloroform; EA Z ethylacetate; NB Z n-butanol; ME Z methanol extract; PE Z petroleum ether extract; WA Z water.

204 O.S.S. Abrika et al.

that of subfraction F1. At 1 mg/mL, subfraction F2-inducedand F3-induced CRs were significantly lower than that dueto isoprenaline, which, in turn, did not differ significantlywith that due to subfraction F1 (Fig. 4GeI). Overall, sub-fraction F1 tended to have an increasing effect and theother two subfractions had a decreasing effect on LA con-tractions. This decreasing effect of subfractions F2 and F3was more pronounced at their highest concentrations.

4. Discussion

The most evident and important observation from our studyis that NB extract is a stronger substrate for cardiac

contraction than the others. The CRs were clearly greaterwith NB extract-exposed atria, particularly in those of theleft cardiac muscle submitted to the highest concentra-tions. ME, PE, and CHL extracts and EA and WA fractionstended to reduce cardiac contractions. The decreasing ef-fect of NB extract on the LA was also more pronounced forits F2 and F3 subfractions. Within the experimental jac-keted glass, we observed an increased negative inotropiceffect of both NB fraction and F2 and F3 subfractions onatrium contraction. Related observations were recentlyreported: in rats, hypotension resulting from synthetic hy-potensive agents was similar to that induced by the extractof G. procumbens, probably by consistently reducing the

Figure 4 Effects of extracts, fractions and subfractions of G. procumbens on left atrium contraction. (A) PE extract; (B) MEextract; (C) CHL fraction; (D) EA fraction; (E) WA fraction; (F) NB fraction; (G) NB subfraction F1; (H) NB subfraction F2; (I) NBsubfraction F3. *p < 0.05 compared to control. **p < 0.01 compared to control. ***p < 0.001 compared to control.CHL Z chloroform; EA Z ethyl acetate; NB Z n-butanol; ME Z methanol; PE Z petroleum ether; WA Z water.

Effect of Gynura procumbens extracts and fractions on rat atrial contraction 205

systolic and diastolic blood pressures, as documentedelsewhere [29].

Hoe et al [21] reported a dose-dependent fall in themean arterial pressure of both hypertensive and normo-tensive rats when they administered intravenously orin vitro a partially purified fraction of the leaves ofG. procumbens. Here, they suggested an inhibitory effectof glycoconjugates and peptides on the angiotensin-converting enzyme. In a related work [20], a decrease inblood pressure was observed in hypertensive rats after theadministration of an aqueous extract of G. procumbens.This effect was attributed to lower serum lactate dehy-drogenase and creatine phosphate kinase, and increasednitric oxide. Most of these molecules are known

vasodilators. In particular, the angiotensin-convertingenzyme plays an important role in the cardiovascular sys-tem through homeostatic regulation of fluids and electro-lytes [30].

Although several active molecules have been implicatedin the effects of G. procumbens on blood pressure, there isstill no definitive evidence for the identity of physiologicalprocesses involved. Several mechanisms have been postu-lated to explain the occurrence of a positive inotropism.These include an activation of endothelial-1 receptors [31],increased concentration of inositol 1,4,5-triphosphate [31],and myocardial contractility [32], as well as a phospho-diesterase-based inhibition of cyclic adenine mono-phosphate (cAMP) [33]. One of the most common

206 O.S.S. Abrika et al.

explanations came from Smith and his collaborators[34,35]; this mechanism is based on cyclic Naþ/Ca2þ ex-changes and the subsequent changes in muscle polarity. Infact, the contraction proceeds by a depolarization anda repolarization, and the contraction occurs in between.Depolarization is triggered by the entering of cations intocontractile elements, whereas repolarization is because oftheir release from cells [32]. Ca2þ that enters the cells viathe L-type Ca2þ channel during depolarization triggers therelease of additional free calcium ions into the cytosol froman intracellular compartment, the sarcoplasmic reticulum(SR) [34,35]. According to some authors [36], positive ino-tropic effect can result from the stimulation of selective b1-adrenoceptors agonists, i.e., albuterol and dobutamine.Such drugs activate the G stimulatory protein linked to anadenylyl cyclase, which in turn, dephosphorylates anadenosine triphosphate (ATP) molecules. The resultingcAMP activates a protein kinase that induces the opening ofcalcium channels. This leads to an increase in calcium ionsentering through the plasma membrane and thus anincreased concentration of the cations within the SR. Theactivation of the protein kinase also triggers the accumu-lation of Ca2þ originating from the SR itself. These freecalcium ions interact with troponin C proteins. This acti-vates the cross-bridge interactions between actin filamentsand myosin cross-bridges, thus leading to cardiac musclecell contraction. The global and simultaneous contractionsof several cells lead to heart tissue contraction. Presum-ably, one of these mechanisms has occurred in our currentstudy. Clearly, an increased exposure to NB fraction,especially to its F2 and F3 subfractions, will tend to resultin low accumulation of Ca2þ in cardiac muscle cells. Witha lengthened exposure to F2 and F3 subfractions of the NBfraction, heart contractile elements are less amenable tocontraction and the animal is less exposed to risks of hy-pertension, as this often results from increased contrac-tions. According to our results, two of the three fractions ofNB extract showed great anticontraction effects on the leftatrium by inducing its relaxation. It is clear that the G.procumbens leaf contains active hypotensive compoundssoluble in NB fraction and it is therefore likely that G.procumbens has potential as an effective antihypertensivemedical plant.

Phytochemical investigations on perennial members ofthe Asteraceae family reveal the presence of inulin.Experimental evidence from recent studies has suggestedthat inulin-containing prebiotics are capable of preventinghypertension [37]. Some species contain flavonoids andsesquiterpene lactones, two compounds reported to beeffective in lowering the blood pressure [38]. Akowuah et al[39] noted that flavonoids have been identified in G. pro-cumbens. Some Compositae plants contain pyrrolizidinealkaloids that cause a decrease in the blood pressure [40].Galduroz et al discovered that the extracts of Allium sat-ivum L. and Ginkgo biloba L. affect the blood flow [41].Diterpenoids identified from the two plants were found toreduce the blood viscosity.

In addition to providing some information on the effectsof several extracts from the medicinal plant G. procumbenson the contractility of cardiac tissues from a vertebrate,this study emphasizes the significance of components fromNB fraction in traditional medicine as a potential remedy

against increased blood pressure in humans. Additionalstudies are needed, however, to identify the hypotensiveagents of the NB fraction and their mechanisms of action.

In conclusion, our in vitro study suggests that NB frac-tions and its subfractions from G. procumbens leaves havepositive inotropic activities, and hence can be consideredin alternative/traditional medicine against increased bloodpressure in humans or used in strategies aimed at findingantihypertensive biomolecules from an accessible source.The mechanism of positive inotropic activities of the polarcompound(s) may be attributed to a direct action on the SAnode, which leads to the decrease in conduction or to thedepression of myocardium of the heart, similar to the ef-fect of quinidine, b-adrenergic blocker drugs, or calcium-channel blockers.

Conflict of interest

The authors declare that there are no conflict of interest.

Acknowledgments

We are grateful to the team of the Departments of Phy-tochemistry and Pharmacology of the School of Pharma-ceutical Sciences, University Sains Malaysia.

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