Cardiovascular effects of the aqueous extract of Gynostemma pentaphyllum Makino.

Abstract

In the present study, the cardiovascular activity of the aqueous extract of Gynostemma pentaphyllum Makino leaves was investigated in the anaestetized guinea-pigs and has been compared with two of its isolated gypenosides (III, VIII) and with verapamil, a well-known Ca-antagonistic drug.

The results obtained showed that the intravenous administration of the decoction of G. pentaphyllum (2.5, 5 and 10 mg/kg) produced a protective effect against pitressin-induced coronaryspasm, arrhythmias and pressor response. Extract also increased the dose of ouabain required to cause ventricular tachyarrhythmias and lethality.

Further extract reversed ouabain-induced persistent ventricular tachycardia and restored sinus rhythm in a dose-dependent manner.

The results obtained have also shown that gypenosides III and VIII caused similar protective effects in both experimental models used; however, the duration of the action is lower than that of the extract containing corresponding quantities of gypenosides III and VIII.

[c] 2005 Elsevier GmbH. All rights reserved.

Keywords: Gynostemma pentaphyllum Makino; Gypenosides; Cardiovascular activity; Leaves extract; Coronaryspasm; Arrhythmias

Introduction

Gynostemma pentaphyllum Makino (Cucurbitaceae) is a perennial liana grows wild in Southern China, Japan, India and Korea. In China it is called Jiaogulan and has been used in traditional medicine to treat bronchitis and asthma. The Japanese name is Amachazuru. Phytochemical studies of this plant have identified about 90 dammarane-type glycosides, mainly named gypenosides (Gy), closely related to the ginseng saponins. Indeed, gypenosides III, IV, VIII, XII and malonyl gypenosides III and VIII are identical to ginsenosides Rb1, Rb3, Rd, F2, and malonyl ginsenosides Rb1 and Rd (Takemoto et al., 1983; Kuwahara et al., 1989; Piacente and Pizza, 1995).

Because of this similarity to the expensive ginseng root, G. pentaphyllum has attracted much interest as a potential new medicinal plant.

Pharmacological studies of G. pentaphyllum and/or the isolated saponins have shown a variety of interesting activities, such as antitumor, cholesterol-lowering, immuno-potentiating, antiulcer, antioxidant, antihypertensive, antithrombotic, etc. (Cour et al., 1995; Lin et al., 2000).

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Materials and methods

Plant material

Authentic leaves of G. pentaphyllum Makino were obtained from the Chinese Academy of Agriculture Sciences (Beijing, China). A voucher specimen is deposited in the Pharmaco-Biological Department, University of Messina, Italy.

The pure Gy III (Rb1) and Gy VIII (Rd) were obtained from the Laboratoires Labservice Analytica s.r.l., Anzola Emilia (BO).

Preparation of extract

The extract was prepared by boiling 100 g powdered dried leaves with 1000 ml distilled water for 30 min. After filtration (whatman No. 4 filter paper) extract was lyophylized. The yield obtained was 15%.

For administration, the residue was dissolved in normal saline, immediately before use, to the desired concentrations.

The gypenosides III and VIII content of this drug, determined by TLC and HPLC, was in terms of dry weight 0.9% of Gy III and 0.4% of Gy VIII (Kuwahara et al., 1989).

Experiments

Male guinea-pigs with body weight ranging from 350 to 400 g were anaesthetized with intraperitoneal injection of urethane (1.2 g/kg). The trachea was intubated and the right external jugular vein was cannulated with a polyethylene catheter for the administration of drugs. The left common carotid artery was cannulated and connected to a pressure transducer (Gould P 23 ID) and the blood pressure was recorded continuously on a Gemini Recorder (Ugo Basile, model 7070). The electrocardiogram (Battaglia-Rangoni app., Model simplex a-r) of limb lead II was recorded continuously by subcutaneous needle electrodes. The aqueous extract of the G. pentaphyllum, at doses of 2.5, 5 and 10 mg/kg, the verapamil (1 mg/kg) and the pure gypenosides (Gy III 0.7 mg/kg; Gy VIII 0.3 mg/kg) were injected into the jugular vein 3 min before starting the infusion of pitressin or ouabain.

In the ouabain-induced stable ventricular tachycardia, the drugs were administered after ouabain.

Control animals were treated in a similar manner but received vehicle only (saline). Each experimental group consisted of five animals.

Pitressin-induced coronaryspasm, arrhythmias and pressor response

Coronaryspasm, arrhythmias and pressor response were induced by rapidly administering 2IU/kg of pitressin (vasopressin--Parke Davis) into the jugular vein (10 s), as previously described (Occhiuto et al., 1991).

The following parameters were monitored before, during and after pitressin infusion (30 min): ST-segment elevation (height, mm) and duration (s); alteration of T-wave; duration (s) of the P-Q and Q-T intervals; and percentage incidence of the arrhythmias.

Ouabain-induced arrhythmias

The first series of experiments was carried out to investigate the abilities of decoction of G. pentaphyllum leaves and of the isolated gypenosides III and VIII in protecting guinea-pigs from ouabain-induced arrhythmias and lethality. After recording a control lead II ECG, a continuous infusion of ouabain in aqueous solution was started at a rate of 10 [micro]g/kg/min into the jugular vein by means of an infusion pump. The cardiac rhythm disturbances were graded in: onset of arrhythmia (OA), premature ventricular beats (PVB), ventricular tachyarrhythmias (VT; denoted by ventricular tachycardia or ventricular fibrillation). The total doses of ouabain in [micro]g/kg required to produce appearance of the various types of arrhythmia were determined by the infusion time. The second series of experiments was performed using the method described by Somani and Lum (1966) and carried out to investigate the abilities of aqueous extract of G. pentaphyllum and of the pure gypenosides in reversing ouabain-induced stable ventricular tachycardia. After recording a control ECG, ventricular tachycardia was produced by the intravenous administration of initial loading dose of ouabain, 50 [micro]g/kg, followed by 20 [micro]g/kg 20 min later, and 10 [micro]g/kg every 10 min thereafter until a persistent ventricular tachycardia developed 10 min after forming stable ventricular tachycardia. The decoction and pure gypenosides were introduced into the jugular vein. The antiarrhythmic activity was estimated from dose required to re-establish normal sinus rhythm.

Statistical analysis

Data were analysed for significance with Student's t-test for unpaired and paired data. Differences between groups were regarded as significant at a level of p<0.05.

Results

Effects on blood pressure and ECG

Intravenous administration of the aqueous extract (2.5, 5 and 10 mg/kg) and of the pure gypenosides (Gy III 0, 7 mg/kg and Gy VIII 0.3 mg/kg) produced a slight and no significant decrease in blood pressure (8-10%) and heart rate (6-7%) in guinea-pigs. No significant electrocardiographic alterations were observed.

Effects on pitressin-induced coronaryspasm, arrhythmias and pressor response

In all control guinea-pigs, intravenous administration of pitressin resulted in the typical electrocardiographic signs of coronary arterial spasm (ST-Segment elevation, increase voltage or inversion of the T-wave, prolongation of the P-Q and Q-T intervals), in an elevation of arterial blood pressure of 40.5[+ or -]3.0 mmHg and in the disturbances of cardiac rhythm characterized by ventricular extrasystoles followed by paroxysmal ventricular tachycardia of short duration, intervalated by bradicardic sinusal rhythm with atrioventricular blocks. The onset of coronaryspasm of the ST-segment was generally 15 s post-infusion of pitressin and persisted for 44.0[+ or -]6.54s with a maximum increase in ST-segment of 5.2[+ or -]0.41 mm.

However, bradicardia changes of T-wave and prolongation of the P-Q and Q-T intervals continued until 5 min after pytressine infusion. Intravenous administration of aqueous extract at all dosages assayed (2.5, 5 and 10 mg/kg) significantly decreased the electrocardiographic signs of coronaryspasm, the pressor response and the incidence of cardiac arrhythmias induced by pitressin. In particular, at 10 mg/kg dosage showed an inhibitory effect on height and duration of ST-segment elevation the intensity of which was similar to that shown by 0.7 mg/kg of Gy III and by 0.3 mg/kg of Gy VIII (Table 1, Fig. 1).

Effects on ouabain-induced arrhythmias

In the first series of experiments, intravenous ouabain continuous infusion resulted in disturbances of cardiac rhythm progressing from sinus arrhythmia, premature ventricular and supraventricular polytopic extrasystoles to ventricular tachycardia, ventricular fibrillation or cardiac arrest, in all control guinea-pigs. At 2.5 mg/kg, aqueous extract caused a significant (p<0.05) increase in the dose of ouabain required to cause lethality (L) and VT, but there was no significant difference in the case of PVB and in the OA; at dose of 5 and 10 mg/kg, produced a significant (p<0.05) increase in the amount of ouabain required to cause VEB, VT and L. Gypenosides at doses corresponded with those contained in 10 mg/kg of G. pentaphyllum extract were able to exert the same antiarrhythmic effect as the extract, but the dose of ouabain necessary to produce L was lower (Table 2).

In the second series of experiments, ouabain at a cumulative dose of 108.6[+ or -]4.8 [micro]g/kg, i.v. produced persistent multifocal ventricular tachycardia in the animals. Intravenous administration of aqueous extract (2.5, 5 and 10 mg/kg) reverted the persistent ventricular tachycardia back to normal and stable sinus rhythm in a dose-dependent manner between dose and duration of the re-established sinus rhythm. The duration of the sinus rhythm re-established after 10 mg/kg of extract was of 23.0[+ or -]3.0 min. In the same experiment, gypenosides III and VIII showed a lower effect to that of extract. Verapamil at the dose of 1 mg/kg i.v. produced a resolution of the arrhythmia in 100% of the animals treated; the duration of this antiarrhythmic action was longer (28.0[+ or -]2.4 min) (Table 3, Fig. 2).

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Discussion

The present results show that the intravenous administration of the aqueous extract from G. pentaphyllum leaves and of the pure gypenosides III and VIII produced a protective effect against vasopressin-induced coronary spasm, arrhythmias and pressor response and a protective and suppressive effect on ouabain-induced arrhythmias in the anaesthetized guinea-pigs. These effects indicate that G. pentaphyllum extract possesses significant cardiovascular properties similar to those exhibited by verapamil and suggested that gypenoside III and gypenoside VIII are two of its active components responsible of these properties.

It is well known that calcium antagonists are effective drugs in the experimental models used in this study (Haas and Bush, 1968; Ribeiro et al., 1981). In fact, the cardiovascular action of vasopressin is related to both direct and indirect effects. The direct effect, calcium-mediated, is the first component of vasopressin action. In addition it is also well known that the electrophysiological mechanism of the arrhythmogenic action of ouabain is related to oscillatory after potentials (delayed after depolarizations). Calcium overload seems to be a underlying mechanism for the digitalis intoxication (Allen et al., 1984; Vassalle and Lin, 1979; Lee and Klaus, 1971). Although the mechanism of cardiovascular action of G. pentaphyllum extract and of the gypenosides have not been investigated, it is likely that the extract and gypenosides action might be mediated through their ability to inhibit calcium overload.

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References

Allen, D.G., Einer, D.A., Orchard, C.H., 1984. Factors influencing free intra-cellular calcium concentrations in quiescent ferret ventricular muscle. J. Physiol. 350, 615-630.

Cour, B., Molgaard, P., Yi, Z., La-Cour, B., 1995. Traditional Chinese medicine in treatment of hyperlipidaemia. J. Ethnopharmacol. 46 (2), 125-129.

Haas, H., Bush, E., 1968. Antiarrhythmische workungen von verapamil und seinen derivativen in vergleich zu propranolol, pronethalol, chinidin, procainamid und ajmalin. Arzneim. Forsch. 18, 401-408.

Kuwahara, M., Kawanishi, F., Komiya, T., Oshio, H., 1989. Dammarane saponins of Gynostemma pentaphyllum Makino and isolation of malonylginsenosides- Rb1, -Rd, and malonylgypenoside. V. Chem. Pharm. Bull. 37, 135-139.

Lee, K.S., Klaus, W., 1971. The subcellular basis for the mechanism of inotropic action of cardiac glycosides. Pharmacol. Rev. 23, 193-210.

Lin, C.C., Huang, P.C., Lin, J.M., 2000. Antioxidant and hepatoprotective effects of Anoectochillus formosanus and Gynostemma pentaphyllum. Am. Chin. Med. 28, 87-96.

Occhiuto, F., Busa, G., Ragusa, S., De Pasquale, A., 1991. Comparative antiarrhythmic and anti-ischaemic activity of some flavones in the guinea-pig and rat. Phytother. Res. 5, 9-14.

Piacente, S., Pizza, C., 1995. New Dammarane-type glucoside Gynostemma pentaphyllum. J. Nat. Prod. 58, 512-519.

Ribeiro, G.T.L., Brandon, G.T.A., Debauche, T.L., 1981. Anthiarrhythmic and haemodynamic effects of calcium channels blocking agents during coronary artery reperfusion. Am. J. Cardiol. 48, 69-75.

Somani, P., Lum, B.K.B., 1966. Blockade of epinephrine- and ouabain-induced cardiac arrhythmias in the dog heart-lung preparation. J. Pharmacol. Exp. Ther. 152, 235-242.

Takemoto, T., Arihara, S., Nakajiama, T., Okuhira, M., 1983. Studies on the constituents of Gynostemma pentaphyllum, I. Structures of gypenoside I-XIV. Yakugaku Zasshi 103, 173-185.

Vassalle, M., Lin, C.I., 1979. Effect of calcium on strophanthidin-induced electrical and mechanical toxicity in cardiac purkinje fibers. Am. J. Physiol. 256, H689-H697.

C. Circosta*, R. De Pasquale, F. Occhiuto

Pharmaco-Biological Department, School of Pharmacy, University of Messina, Messina, Italy

Received 9 February 2004; accepted 15 June 2004

*Corresponding author. Fax: [phone].

E-mail address: [e-mail] (C. Circosta).

Table 1. Effects of the three dosages (2.5, 5 and 10 mg/kg, i.v.) of
aqueous extract from G. pentaphyllum leaves and of the pure gypenosides
(Gy) on the pitressin--induced electrocardiographic and pressor
alterations in the anaesthetized guinea-pigs

                         % animals with  ST (max height, mm)
Treatment  Dose (mg/kg)  coronary spasm  (means [+ or -] s.e.m)

Saline     --            100             5.2 [+ or -] 0.41
Extract     2.5           60             2.1 [+ or -] 0.30*
Extract     5             40             2.0 [+ or -] 0.50*
Extract    10             40             1.8 [+ or -] 0.10*
Gy III      0.7           20             1.8 [+ or -] 0.20*
Gy VIII     0.3           40             1.9 [+ or -] 0.15*
Verapamil   1             20             1.5 [+ or -] 0.20*

           ST (duration, s)
           (means [+ or -]                           % animals with
Treatment  s.e.m)               PA (a) (%)           arrhythmias

Saline     44.0 [+ or -] 6.54   40.5 [+ or -] 3.0**  100
Extract    15.0 [+ or -] 5.25   20.0 [+ or -] 2.0**   40
Extract    14.0 [+ or -] 3.30*  18.0 [+ or -] 2.0     20
Extract    14.5 [+ or -] 2.00*  16.0 [+ or -] 3.0     20
Gy III     16.0 [+ or -] 4.00*  14.0 [+ or -] 4.0     20
Gy VIII    20.5 [+ or -] 3.20*  15.0 [+ or -] 3.0     40
Verapamil  17.0 [+ or -] 2.50*  10.0 [+ or -] 2.0 0

*p<0.05 respected to control (saline, t-test for unpaired data);
** p<0.01 respected to basal values (t-test for paired data).
(a) Mean percentage increase of the arterial blood pressure respect to
basal values.

Table 2. Effects of the aqueous extract from G. pentaphyllum leaves and
of the pure gypenosides on ouabain-infusion induced arrhythmias and
lethality in guinea-pigs

Treatment  Dose (mg/kg)  OA                   PVB

Saline     --             59.0 [+ or -] 6.0    94.0 [+ or -] 7.2
Extract     2.5           70.5 [+ or -] 4.5   117.0 [+ or -] 8.5
Extract     5             80.5 [+ or -] 6.5*  130.8 [+ or -] 5.0*
Extract    10            120.9 [+ or -] 9.0*  135.0 [+ or -] 8.5*
Gy III      0.7          118.0 [+ or -] 5.0*  132.0 [+ or -] 7.5*
Gy VIII     0.3          115.5 [+ or -] 6.0*  127.5 [+ or -] 5.0*
Verapamil   1            123.7 [+ or -] 7.6*  143.1 [+ or -] 8.4*

Treatment  VT                   L

Saline     131.0 [+ or -] 8.3   210.0 [+ or -] 5.3
Extract    160.5 [+ or -] 8.4*  290.0 [+ or -] 7.5*
Extract    175.0 [+ or -] 8.0*  350.5 [+ or -] 7.0*
Extract    238.0 [+ or -] 7.4*  422.4 [+ or -] 6.0*
Gy III     240.5 [+ or -] 9.0*  340.0 [+ or -] 9.5*
Gy VIII    235.0 [+ or -] 7.0*  355.5 [+ or -] 7.0*
Verapamil  266.9 [+ or -] 9.3*  459.3 [+ or -] 8.5*

*p<0.05 compared to saline group (t-test for unpaired data). Values are
expressed as mean [+ or -] s.e.m. of the total doses of ouabain
([micro]g/kg, b.w.) required to cause: onset of arrhythmia (OA),
premature ventricular beats (PVB), ventricular tachyarrhyth-mias (VT)
and lethality (L).

Table 3. Suppressive effects of G. pentaphyllum extract and of the pure
gypenosides on ouabain-induced persistent ventricular tachycardia, at
cumulative dose of 108.6 [micro]g/kg i.v., in guinea-pigs

                         % of animals with         Time (s) resolution
Treatment  Dose (mg/kg)  resolution of arrhythmia  after treatment

Saline     --              0                       --
Extract     2.5           20                       30.0 [+ or -] 4.0
Extract     5             80                       26.0 [+ or -] 2.0
Extract    10             80                       28.0 [+ or -] 6.0
Gy III      0.7           60                       30.0 [+ or -] 3.0
Gy VIII     0.3           60                       32.0 [+ or -] 5.0
Verapamil   1            100                       16.0 [+ or -] 3.0

           Duration (min) of the sinus
           rhythm re-established after
Treatment  treatment

Saline      0
Extract     5.0 [+ or -] 2.5*
Extract    14.5 [+ or -] 2.0*
Extract    23.0 [+ or -] 3.0*
Gy III     13.0 [+ or -] 2.7*
Gy VIII    12.5 [+ or -] 3.0*
Verapamil  28.0 [+ or -] 2.4*

*p < 0.01 compared with saline group.