PHYTOTHERAPY RESEARCH

Phytother. Res.

17

, 1135–1139 (2003)Published online in Wiley InterScience (www.interscience.wiley.com).

DOI

: 10.1002/ptr.1132

Copyright © 2003 John Wiley & Sons, Ltd.

Received 13 November 2000Accepted 16 July 2001

John Wiley & Sons, Ltd.

Possible Mechanisms of Action of the Neutral Extract from

Bidens pilosa

L. Leaves on the

Cardiovascular System of Anaesthetized Rats

CARDIOVASCULAR EFFECT OF

B. PILOSA

T. Dimo*

1

, T. B. Nguelefack

2

, P. V. Tan

1

, M. P. Yewah

1

, E. Dongo

3

, S. V. Rakotonirina

1

, A. Kamanyi

2

and M. Bopelet

1

1

Department of Animal Biology and Physiology, Faculty of Sciences, PO Box 812, University of Yaounde I, Yaounde, Cameroon

2

Department of Animal Biology, Faculty of Sciences, PO Box 67 Dschang, University of Dschang, Dschang, Cameroon

3

Laboratory of Organic Chemistry, Faculty of Sciences, PO Box 812, University of Yaounde I, Yaounde, Cameroon

The aim of this study was to investigate the hypotensive and cardiac effects of the neutral extract from

Bidenspilosa leaves. Intravenous administration of the extract resulted in a biphasic dose-related hypotensiveactivity. In normotensive rats (NTR), B. pilosa decreased systolic blood pressure by 18.26%, 42.5% and 30%at doses of 10, 20 and 30 mg/kg, respectively. In spontaneously hypertensive rats (SHR), the decrease insystolic blood pressure was 25.77%, 38.96% and 28.64% at the above doses, respectively. These dosesinduced hypotension by 27%, 34.13% and 18.73% respectively in salt-loaded hypertensive rats. In NTR,B. pilosa reduced heart rate by 23.68% and 61.18% at doses of 20 and 30 mg/kg, respectively. The force ofcontraction of the heart was only affected at 30 mg/kg. The initial phase of hypotensive response was partiallyinhibited by atropine while propranolol increased this effect. These results suggest that B. pilosa exhibitedits first hypotensive effects by acting on the cardiac pump efficiency and secondly through vasodilation.Copyright © 2003 John Wiley & Sons, Ltd.

Keywords: Bidens pilosa

; hypotensive effect; vasodilation; rats.

INTRODUCTION

Bidens pilosa

L. (Asteraceae, Heliantheae) is a wide-spread annual herb which grows to a maximum heightof 1.5 m, with yellow flowers of 5–15 mm diameter.The plant is widely used in traditional medicine all overthe world. In Africa, it is used for the treatment ofdiarrhoea, dysentery, otitis, ophthalmia, headache, musclepain, malaria and snake bite (Bouquet, 1969; Adjanohoun

et al.

, 1988; Brandao

et al.

, 1997). The Kallawaya inthe Bolivian Andes take it in combination with thejuice of

Valeriana officinalis

and of peas to lower bloodpressure (Bastien, 1987). They also use B. pilosa as adiuretic and a choleretic, as well as for fever in the caseof rubella and scarlatina (Girault, 1984). Phytochemicalstudies of the leaves revealed the presence of vari-ous constituents, mainly polyacetylenes and flavonoids(Geissberger and Séquin, 1991). Several flavonoids(aglycones and glycosides) have been isolated, amongthem the chalcones okanin and butein, as well asquerietin 3-o-glucoside and a series of correspondingglucosides (Hoffmann and Hölzl, 1988, 1989). In previousreports the antihypertensive effect of lyophilizedaqueous and methanol leaf extracts of B. pilosa usingunanaesthetized rats have been studied. In unanaesthet-ized rats, it has been found that the aqueous (Dimoet al., 1996) and methanol (Dimo et al., 1999) leafextracts exhibit antihypertensive action. It has also been

shown that the leaf aqueous extract of B. pilosa has avasorelaxant activity (Dimo et al., 1998).

In the present study, the effect of the neutral extractobtained from the leaves of B. pilosa was tested byintravenous administration to spontaneously hypertens-ive and salt-loaded hypertensive rats.

MATERIALS AND METHODS

Plant extracts.

Fresh leaves of

B. pilosa

were collectedaround the campus of the University of Yaounde I inNovember. The plant material was identified at theNational Herbarium in Yaounde, where a voucherspecimen no HNC/58742 has been deposited.

Air-dried leaves (1.5 kg) were macerated in a 1:1 (v/v) mixture of methanol and methylene chloride for 48 hat room temperature. The solution obtained after filtra-tion was concentrated under reduced pressure to obtain204 g of a dark solid. The methylene chloride/methanolextract (100 g) was neutralized with NaOH (1 N) andHCl (10.75 N); the resulting filtrate was concentratedunder reduced pressure to give 38 g of neutral extract of

B. pilosa

(NBp). 600 mg of this extract was dissolvedin 0.5 mL of dimethyl sulphoxide (DMSO) and thevolume of solution was made up to 20 mL using distilledwater. Dilution was later made so that all animalsreceived the same volume of solution (0.1 mL/100 gbody weight), independent of the dose. The effects ofsolvent (2.5% DMSO) were tested in order to ascertainthat the results obtained were exclusively due to theextract.

* Correspondence to: Dr T. Dimo, Department of Animal Biology andPhysiology, Faculty of Sciences, PO Box 812, University of Yaounde I,Yaounde, Cameroon.

Copyright © 2003 John Wiley & Sons, Ltd.

Phytother. Res.

17

, 1135–1139 (2003)

1136

T. DIMO

ET AL

.

Animals.

Normotensive rats (NTR), spontaneouslyhypertensive rats (SHR) and salt-loaded hypertensiverats (SLHR) aged 8–12 weeks and weighing 150–200 gwere used. The animals were fed with broilers mash,with access to food and water

ad libitum

. Salt-loadedhypertensive rats were obtained from normotensiveWistar rats as described by Dimo et al. (1999). Normoten-sive rats weighing 80–120 g (4 weeks) were given bygastric intubation once a day, a solution containing9% NaCl at 2 mL/100 g body weight with free access tonormal rat chow and 1.2% NaCl solution as drinkingwater. Systolic blood pressure (SBP) was verified twicea week using the plethysmographic method and ratsshowing a SBP higher than 150 mmHg were consideredas hypertensive.

Effect of neutral extract of B. pilosa on blood pressure.The rats were anaesthetized using an intraperitonealinjection of urethane (1 g/kg). The trachea was exposedand cannulated to facilitate spontaneous respiration.Arterial blood pressure was measured from the leftcarotid artery via an arterial cannula connected to apressure transducer (PANLAP S-5021) coupled witha Universal Harvard polygraph. The animals wereallowed to stabilize for at least 30 min before adminis-tration of any test substances. The plant extract wasinjected via a cannula inserted into the right femoralvein. The effects of the plant extract were comparedwith those of clonidine (0.08 mg/kg) and isoprenaline(1 mg/kg). A dose of 30 mg/kg was examined afteradministration of atropine (1 mg/kg) and propranolol(1 mg/kg). Atropine and propranolol were injectedintravenously, 5 min before administration of the plantextract.

β

-adrenoceptor was established by its specificblocker, propranolol. The effectiveness of blockade wastested by injecting 1 mg/kg of agonist (isoprenaline) inan effective dose. The mean blood pressure was deter-mined from the sum of diastolic pressure (DBP) plusone-third-pulse pressure. Changes in blood pressurewere expressed as a percentage of the control valuesobtained immediately before the administration of thetest substance.

Effect of neutral extract of

B. pilosa

on heart rate andcontractile force.

The animals were anaesthetized andthe trachea and the right femoral vein were cannulatedas above. The left chest was opened between the thirdand fourth ribs. The apex of the heart was pinched withfine nippers connected to a PANLAB isometrictransducer under a tension of 2 g. The transducer wascoupled with a Harvard Universal polygraph to recordheart mechanical activity. The heart was irrigated withan oxygenated physiological salt (McEwen) solutionmaintained at 37

°

C. The heart was allowed to stabilizefor 20 min; then the plant extract (10, 20 and 30 mg/kg)or acetylcholine (1 mg/kg) were injected intravenously.

Drugs. In addition to the plant extract, the followingdrugs were used: clonidine hydrochloride, acetylcholinechloride, atropine sulphate and propranolol (SigmaChemical, St Louis, MO, USA). Isoprenaline wasfrom Prolabo, France, and heparin from Sanofi, France.All drugs were dissolved in distilled water except forthe plant extract that was dissolved in dimethylsulphoxide(DMSO) and the solution adjusted with distilledwater.

Statistics. All values are presented as mean ± SEM.Statistically significant differences between the controland each intervention were determined by the Student’st-test.

RESULTS

Effects of neutral extract of

B. pilosa

on blood

pressure

Effects on normotensive rats (NTR).

After i.v. injectionto anaesthetized NTR, the neutral extract of

B. pilosa

(NBp) produced a rapid decrease of blood pressure.SBP dropped from 121.60

±

6.88 mmHg to 99.40

±

7.87 mmHg at a dose of 10 mg/kg, and from 128.00

±

3.66 mmHg to 73.60

±

6.99 mmHg at the next higherdose (20 mg/kg), representing a drop of 18.26% and42.50%, respectively. Figure 1 shows the time courseof the hypotensive action following i.v. injection at adose of 10–30 mg/kg. As shown, the maximal effect atthe highest dose of 30 mg/kg was obtained 50 minafter NBp injection. There was a transient increasein blood pressure immediately after the administra-tion of clonidine (0.08 mg/kg), followed by an appreci-able fall (43.41%

±

1.76%). Isoprenaline (1 mg/kg)produced an initial fall of 36.30%

±

2.87%, but theblood pressure returned to the initial value in under1 h (Fig. 2). This hypotensive effect of isoprenalinewas reduced to 5.12%

±

1.08% in the presence ofpropranolol.

Effects on the spontaneous hypertensive rats (SHR).

In SHR, NBp induced a more significant biphasichypotensive effect than in NTR. There was no differencebetween the first response induced on SBP by NBp at10 mg/kg and at 30 mg/kg. With the 10 mg/kg dose,the second phase started 15 min after administration,earlier than what was observed in NTR, and the fallin SBP was 22.38%

±

2.64% 40 min after administra-tion, compared with 33.41%

±

0.98% with the 30 mg/kgdose (Fig. 3).

Figure 1. Effect of B. pilosa neutral extract (E) on the systolicblood pressure in normotensive rats. Each point representsthe mean ± SEM of 5 animals. *p < 0.01 and **p < 0.001.Significantly different from initial value.

CARDIOVASCULAR EFFECT OF

B. PILOSA

1137

Copyright © 2003 John Wiley & Sons, Ltd.

Phytother. Res.

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, 1135–1139 (2003)

Effects on salt loading hypertensive rats (SLHR).

The10 mg/kg dose of the NBp induced 27.31% ± 3.02% and38.01% ± 1.92% fall, respectively, in SBP and DBPimmediately after intravenous administration. The secondphase started when the blood pressure had not com-pletely recovered and reached respective peaks of15.61%

±

0.84% and 25.24%

±

1.76% fall of SBP andDBP. The higher dose of 30 mg/kg did not show thebiphasic response. Here, the hypotensive effect wasprogressive and was 25% ± 2.06% after 25 min (Fig. 4).

Effects of pharmacological substances

The effects of two possible antagonists, atropine andpropranolol, on the hypotensive action of NBp (30 mg/kg) were investigated. The plant extract was given5 min after the administration of atropine (1 mg/kg)or propranolol (1 mg/kg). It was found that atropineinhibited by 39% the initial hypotensive effect of NBp

and completely inhibited the late hypotensive responseinduced by the plant extract while propranolol had apotentiating effect on the initial response provoked bythe extract (Fig. 5). The fall in mean arterial blood pres-sure induced by propanolol was from 102.24

±

6.13mmHg to 67.33

±

1.67 mmHg. This was followed 5 minlater by a rapid return of blood pressure to 104.64

±

4.48 mmHg. The injection of the extract resulted in areduction of blood pressure to 37.23

±

2.39 mmHg, rep-resenting a 64% drop.

Effects of NBp on the heart

As shown in Table 1, there was no significant change inthe heart rate and force of contraction after i.v. injec-tion of 10 mg/kg. At a dose of 20 mg/kg, NBp induceda decrease of heart rate from 377

±

14 to 288

±

31 beats/min, but did not affect the force of contraction. Adecrease of the heart rate by 61.18%

±

3.67% and anincrease in the force of contraction by 24.48%

±

2.09%

Figure 2. Effect of B. pilosa neutral extract (E), isoprenaline,and clonidine on the systolic blood pressure in normotensiverats. Each point represents the mean ± SEM of 5 animals.*p < 0.01 and **p < 0.001. Significantly different from initialvalue.

Figure 3. Effect of B. pilosa neutral extract (E) on the systolicblood pressure in spontaneously hypertensive rats. Eachpoint represents the mean ± SEM of 5 animals. *p < 0.01and **p < 0.001. Significantly different from initial value.

Figure 4. Effect of B. pilosa neutral extract (E) on the systolicblood pressure in salt-loaded hypertensive rats. Each point rep-resents the mean ± SEM of 5 animals. *p < 0.01 and **p < 0.001.Significantly different from initial value.

Figure 5. Effect of B. pilosa neutral extract (E) on the meanarterial blood pressure (MABP) of normotensive rats and ofnormotensive rats pre-treated with atropine (Atr), and pro-pranolol (Pro.). Each point represents the mean ± SEM of 5animals. *p < 0.01 and **p < 0.001. Significantly differentcompared to the effect on intact normotensive rats.

Copyright © 2003 John Wiley & Sons, Ltd.

Phytother. Res.

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, 1135–1139 (2003)

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ET AL

.

was observed at the highest concentration of 30 mg/kg.Under the same conditions, acetylcholine (1 mg/kg)decreased the heart rate and force of contraction by40.83%

±

2.58% and 26.26%

±

1.29%, respectively. Pre-treatment with atropine (1 mg/kg) inhibited the chrono-tropic effect of the plant extract and acetylcholine by84% and 81%, respectively, while the inotropic effectinduced by the extract was completely inhibited.

DISCUSSION

This study looked into the potential activity on the ratcardiovascular system of the neutral extract of

B. pilosa

in anaesthetized normotensive and hypertensive rats.The solvent (2.5% DMSO) alone did not have any effecton the resting blood pressure, heart rate or contractileforce. Intravenous injection of the extract induced abiphasic fall of blood pressure in NTR, SHR andSLHR. Administration of the plant extract provokedan immediate fall in the blood pressure, suggesting apossible action on the cardiac pump. The initial fallin blood pressure was not dose-related. However, thesustained hypotensive effects that occurred 2 to 5 minafter extract injection were dose-dependent. Cardiacactivity was not affected by the extract at 10 mg/kg.This could be due to the fact that the experimentalheart was under tension or demonstrates that the firstphase of hypotensive activity of the extract is not onlydue to its effect on cardiac efficiency. Therefore, theeffect of the extract on vascular smooth muscle can besuggested. In fact, Dimo

et al.

(1998), while working onthe leaf aqueous extract showed the vasorelaxanteffect of

B. pilosa

on the thoracic aorta. The principleresponsible for the vasorelaxant activity is likely presentin the neutral extract and would be responsible for thesubsequent fall in blood pressure. Since the secondphase of hypotensive activity of the extract is significantin NTR, it can be argued that the extract is able toreduce the myogenic resting tone of blood vessels. Themyogenic resting tone is independent of regional innerva-tion and circulating hormones. It is abolished by theremoval of external Ca

2+

(Noon

et al.

, 1978; Fitzpatrickand Szentivanyi, 1980; Winquist and Bohr, 1983) and byblockade of L-type voltage-dependent Ca

2+

channels(Asano

et al., 1993). In a previous study, Dimo et al. (1998)observed that B. pilosa extract provoked a relaxation ofthe aorta through the blockade of the influx of extra-cellular calcium. The sustained hypotensive effect of theextract was more pronounced in hypertensive rats. It

has been reported that one of the major factors contri-buting to the development of hypertension such as inSHR (Asano et al., 1986) or in SLHR (Casteels et al., 1985;Ebeigbe, 1987) may be the high permeability to Ca2+

and a higher concentration of intracellular Ca2+ in thevascular smooth muscle which tend to increase vasculartone (Carmeliet, 1986; Asano et al., 1995). Moreover, thehypotensive activity of the extract was more potent onthe diastolic than on the systolic blood pressure. Thehigher DBP implies an elevation of vascular resistancein the arteriolar sector and small arteries, and persist-ence of the distensibility of the aorta and large vessels(Salvador and Amar, 1995). The important reductionin DBP should conversely imply arteriolar vasodilationand, therefore, the reduction of after-load, which mayresult in the reduction of left ventricular hypertrophy.

The plant extract induced a decrease of the heart rateand an increase of the contractile heart force in normo-tensive anaesthetized rats following the i.v. injection,whereas acetylcholine provoked a decrease of both theheart rate and the force of contraction. Both theseeffects of acetylcholine and the extract were, however,inhibited by atropine. Hoffmann and Hölzl (1989) haveshown the presence of glucosides in B. pilosa leaves.Although the effect of the extract is similar to thenegative chronotropic and positive inotropic effects ofcardiac glucosides (Lechat et al., 1990), it cannot beconcluded from the present data that they have thesame mechanism of action or potential use.

Propranolol enhanced the first hypotensive responseof the extract. It is possible that this is due to asynergistic bradycardiac effect. Also, atropine andpropranolol suppressed the second hypotensive responseof the extract. It can be argued that B. pilosa exertsits action through β-adrenergic vasodilation. Moreover,it is likely that a decrease in β-adrenoceptor activitywould result in potentiation of vasoconstriction byunopposed α-adrenoceptor stimulation. In fact, specificenhancement of norepinephrine-induced contractionafter administration of β-adrenoceptor antagonist wasdemonstrated in different veins from normotensive rats(Cohen and Wiley, 1997) and on the femoral arterysmooth muscle in SHR (Asano et al., 1982). If B. pilosaextract is capable of provoking the release of norepine-phrine, then the blockade of the β-adrenoceptor stimula-tion of norepinephrine can provide the simplest andmost likely explanation of the inhibition of the secondhypotensive effect of the extract. Furthermore, cyclic AMPwas found to augment the ventricular action potentialand potentiate twitch tension in β-blocked ventricularstrips (Morad et al., 1979). Since AMPc mediated the

Table 1. Effects of neutral extract of B. pilosa on the heart rate and contractile force in anaesthetized NTR

Heart rate (beats/min) Contractile force (gf)

Time (s) 0 10 300 600 900 18000 10 300 600 900 1800Extract (mg/kg)10 363 ± 14 358 ± 11 352 ± 15 348 ± 14 349 ± 17 351 ± 18 3.2 ± 0.2 3.2 ± 0.2 3.3 ± 0.3 3.5 ± 0.3 3.2 ± 0.2 3.2 ± 0.220 377 ± 8 288 ± 12a 363 ± 7 367 ± 9 358 ± 11 333 ± 12 4.6 ± 0.5 4.5 ± 0.5 4.9 ± 0.7 4.9 ± 0.7 4.5 ± 0.7 4.8 ± 0.730 350 ± 17 136 ± 26a 331 ± 14 331 ± 8 330 ± 80 335 ± 60 3.8 ± 0.2 4.8 ± 0.2a 4.2 ± 0.3 4.0 ± 0.2 4.0 ± 0.2 4.1 ± 0.1Ach. (1 µg/kg) 360 ± 14 213 ± 18a 298 ± 25a 332 ± 17 348 ± 21 321 ± 17 3.3 ± 0.3 2.4 ± 0.3a 3.0 ± 0.2 3.2 ± 0.2 3.1 ± 0.2 3.1 ± 0.2Extract/Atrb 320 ± 9 289 ± 19 329 ± 5 334 ± 7 328 ± 90 324 ± 10 4.5 ± 0.2 4.5 ± 0.2 4.3 ± 0.3 4.1 ± 0.2 3.9 ± 0.3 3.9 ± 0.3

Values represent mean ± SEM (n = 5), ap < 0.01. Significantly different from control.b Effect of atropine (1 mg/kg) on the cardiac effect of the extract (30 mg/kg).

CARDIOVASCULAR EFFECT OF B. PILOSA 1139

Copyright © 2003 John Wiley & Sons, Ltd. Phytother. Res. 17, 1135–1139 (2003)

mechanism of the positive inotropic effect of norepine-phrine and epinephrine (Morad et al., 1979; Brown et al.,1985), the inhibition of the second phase of hypotensiveresponse of the extract can be clearly understood.

In conclusion, the neutral extract from B. pilosaleaves is effective in producing hypotensive responses

in NTR, SHR and SLHR. This hypotensive activityappears in two successive phases. The first is due, atleast partially, to the effect of the extract on the cardiacpump efficiency, while the second phase may be due toboth β-receptor stimulation and muscarinic receptor-mediated vasodilation.

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