Cardiotonic and antidysrhythmic effects of oleanolic and ursolic acids, methyl maslinate and uvaol.

Summary

The cardiotonic and antidysrhythmic effects of four triterpenoid derivatives, namely oleanolic acid (OA), ursolic acid (UA), and uvaol (UV), isolated from the leaves of African wild olive (Olea europaea, subsp. africana) as well as methyl maslinate (MM) isolated from the leaves of Olea europaea (Cape cultivar) were examined. The derivatives showed low toxicity on brine shrimp test. They displayed significant, dose-response vasodepressor effect and sinus bradicardia, most prominent for OA and MM. The derivatives acted as [beta]-adrenergic antagonists, blocking the effect of adrenaline and isoprenaline. The established positive inotropic and dromotropic effects were most distinctive for OA and MM.

The antidysrhythmic effects were evaluated on Ca[Cl.sub.2]- and adrenaline-induced chemical arrhythmias, and on ischemia-reperfusion arrhythmia. OA and UA displayed antidysrhythmic effects on both types of chemical arrhythmia; OA and UV in dose 40 mg/kg conferred significant antidysrhythmic activity on ischemia and reperfusion arrhythmias. The effect was comparable to that of propranolol and suggestive of [beta]-adrenergic antagonistic activity.

On the basis of the vasodepressor, cardiotonic and antidysrhythmic effects of these compounds, it was concluded that OA and UV isolated from wild African olive leaves, or crude extract containing all components, can provide a cheap and accessible source of additive to conventional treatment of hypertension, complicated by stenocardia and cardiac failure.

Key words: oleanolic acid, ursolic acid, methyl maslinate, uvaol, Ca[Cl.sub.2]-, adrenaline-, ischemia-reperfusion-induced arrhythmias

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Introduction

The hypotensive effect of olive leaf (Olea europaea, subsp. africana) has been well documented. Studies on the hypotensive effect of the active principles of the European olive leaf, the two secoiridoids oleuropein and oleacein, have been conducted for decades (Petkov and Manolov, 1972). It was reported that crude extracts of olive leaves given orally at a dose of 40 ml/kg can produce antihypertensive effect in spontaneously hypertensive rats (Ribeiro et al. 1986). Recently, we reported (Somova et al. 2003) the beneficial effects of the triterpenoids oleanolic acid (OA) and ursolic acid (UA) (Fig. 1) isolated from Olea europaea L (Oleaceae) in preventing the development of salt-sensitive, insulin resistant hypertension in genetic Dahl model of hypertension. The effects were attributed to the hypotensive, diuretic/saluretic, antihyperlipidemic, antioxidant and hypoglycemic effects of the acids. The above findings were surprising, since unlike diterpenes, triterpenoids are not generally considered as hypotensive or antiatherosclerotic natural products (Wang and Ng, 1999; Kolak et al. 2001; Ulubelen et al. 2002), and their cardiovascular effects have not been researched. In the literature, we found only one report about the cardiotonic effect of OA (Chang et al. 1985). The most common effects attributed to oleanolic, ursolic acids, methyl maslinate, and uvaol are: antibacterial (Braca et al. 2000); cytotoxic (Ukiya et al. 2002); antiviral, including anti-HIV effect (Serra et al. 1994; Kashiwada, 2000); hypoglycemic/antidiabetic (Taniguchi et al. 2002); anti-ulcer activity (Farina et al. 1998) and hepatoprotective effect (Liu et al. 1994).

[FIGURE 1 OMITTED]

Triterpenoids are largely distributed in vegetable oils (Amelio et al. 1992) and in more than 120 plant species (Price et al. 1987). Many of them are used as medicinal plants in traditional medicine (Hutchings, 1989; Shibata, 2001). We identified large amounts of OA and UA in the leaves of 18 African medicinal plants, including the wild olive tree (unpublished data).

We hypothesized that if OA and UA of plant origin have, in addition to their hypotensive and antioxidant properties, cardiotonic and antiarrhythmic effects as well, they will provide an accessible and cheap traditional medicine source for treatment of complicated hypertension in developing countries. This prompted our research on the cardiotonic and antidysrrhytmic effects of OA, UA and uvaol (UV) isolated from the leaves of Olea europaea, subspecies africana and methyl maslinate (MM) (Fig. 1) recently isolated from the leaves of Olea europaea (Cape cultivar).

Methodology

The procedures followed were approved by the Ethics Committee of the University of Durban-Westville. The principles of laboratory animal care (WIH publication 85-23, revised in 1985) were observed. Wistar male rats weighing 300-320 g were used. They were housed in the University Biomedical Resource Center, exposed to a 12-h light: 12-h dark cycle and constant humidity. Water and standard food were provided ad libitum. Before the experiments the animals were fasted overnight. The test compounds were dissolved originally in DMSO (stock solution) and in 0.9% saline before intraperitoneal application as a bolus injection, 20 minutes before the experimental procedure. DMSO was proven indifferent to any of the studied parameters.

Plant materials

Air dried wild African olive leaves from a tree grown behind the green house of the Department of Botany, University of Durban-Westville, Natal Province, South Africa were used. The material was collected during the summer period in South Africa (January 2001). A Voucher specimen of the plant material was assigned collector's number F.O. Shode/1 and housed at Ward Herbarium, Department of Botany Herbarium Unit, UDW.

Fresh leaves of Olea europaea (Cape cultivar) were harvested in March, 2001 at Vesuvio Estates, Paarl, South Africa. A Voucher specimen was assigned collector's number F.O. Shode/6 and housed at Ward Herbarium, Department of Botany Herbarium Unit, UDW.

Extractives of Olea europaea, subsp. africana and Olea europaea (Cape cultivar)

The dried African olive leaves (about 1.5 kg) were crushed and successively extracted with hexane, ethyl acetate, methanol, and 80% aqueous methanol to give hexane solubles (OAH), ethyl acetate solubles (13.9 g) (OAE), methanol solubles (OAM), and aqueous methanol solubles (OAW). From preliminary experiments we knew that the OAE fraction contained the active material (1:1 mixture of OA and UA), so this fraction was further purified by repeated silica gel column chromatography with gradient elution (100% hexane to 60% hexane/EtOAc) to give UV, OA and UA as powdery substances. UV recristallised from methanol/C[H.sub.2]C[I.sub.2] as colourless crystals, m.p. 221-223 [degrees]C. Its spectral data were identical with literature values (Siddiqui et al. 1986). OA recrystalised from methanol as colourless crystals, m.p. 307-308 [degrees]C. Its spectral data were identical with literature data Maillard et al. (1992). UA recrystallised from methanol as colourless crystals, m.p. 286 [degrees]C. Its spectral data were identical with literature values (Seo et al. 1975a). Methyl mascinate was isolated from O. europaea (Cape cultivar) as follows. Dried ground leaves (518 g) were exhaustively extracted at room temperature with petroleum ether (60-80 [degrees]C), dichloromethane, ethyl acetate, and methanol to give OEP (2.6 g), OED (14.5 g), OEE (25.6 g), and OEM (44.3 g), respectively. A portion of OEE (2.0 g) was subjected to silica gel column chromatography with gradient elution (80%-60% hexane/EtOAc). Eluate numbers 26-55 (25 ml each) afforded pure powder of oleanolic acid (0.79 g) while eluate numbers 95-137 afforded white powder of pure methyl maslinate (80 mg). This sample was used without recrystallisation. Its spectral properties were identical with literature values (Seo et al. 1975b).

Test for toxicity

Acute toxicity of the extracts was evaluated using brine shrimp (Artemia salina) bioassay (Meyer et al. 1982). L[C.sub.50] and 95% confidence intervals were determined from the 24 h counts of the survived naupii by intersection.

Hemodynamic screening in rats

To detect the effect of the purified test compounds on blood pressure and heart rate of anesthetized animals, sixf rats per each compound were used. The rats were anesthetized by i.p. injection of sodium thiopentone, 40 mg/kg body weight (Rhone-Pulenc, SA). The trachea was cannulated and mechanical ventilation was achieved with a positive-pressure rodent respirator (Phipps and Bird Inc, USA), using atmospheric air at a tidal volume of about 5 ml at a rate of 25 breaths/min. The electrocardiogram (ECG) was monitored throughout the experiments (ECG-C1, ESAOTE Biomedica, Italy, and PowerLab System ML410/W, Australia). Changes in ECG were recorded only from II standard lead at 25 mm/sec chart velocity. The evaluation of inotropic and dromotropic effects and lengthening of the action potential duration was based on the computer analyses of ECG. The right jugular vein was cannulated for drug administration, which was standardized to injections of 0.2 ml per 100 g body weight over a period of 1 min. For continuous monitoring of hemodynamic parameters (mean arterial pressures, heart rate), the left common carotid artery was cannulated and the catheter was connected to a pressure transducer (Statham MLT 0380, Ad instruments), compatible with the PowerLab System ML410/W.

After obtaining control values for 15 min, isoproterenol (1 [micro]g/kg, Sigma) was administered intravenously, followed by the test substance. To check for beta-receptor blocking activity, isoproterenol administration was repeated 20 min. after injection of the test compound. In some experiments, beta-blocker (propranolol, 2 mg/kg, i.v.) was used as positive control.

Antidysrhythmic effects

The effects were evaluated on chemical and ischemia-reperfusion models of arrhythmia.

CaC[I.sub.2]-induced arrhythmia was used for evaluation of possible antiarrhythmic calcium antagonistic effect. The arrhythmia was produced by i.v. injection of a 2.5% Ca[Cl.sub.2] solution (140 mg/kg) (Papp et al. 1966).

A model of adrenaline-induced arrhytmia was used by i.v. injection of adrenaline (10 [micro]g/kg) for evaluation of [beta]-blocking activity (Barrett and Cullum, 1968).

Regional myocardial ischemia and reperfusion are both powerful arrhythmogenic stimuli with different underlying mechanisms of arrhythmogenesis (Pogwizd and Corr, 1986). We used both models in the experiment. In anesthetized and artificially ventilated rats the chest was opened using a left thoracotomy, followed by sectioning of ribs 4 and 5, approximately 2 mm to the left sternum. After incising the pericardium the heart was exposed out of the chest. A 6.0 silk non-traumatic suture was passed through the epicardial layer around the major branch of the left coronary artery, about 2 mm of its origin. A small plastic button (diameter about 5 mm) was threaded through the ligature and placed in contact with the heart. The end was passed through a small vinyl tube and exteriorized. The heart was replaced in the chest and the chest was closed after removing the residual air to avoid pneumothorax. Any animal in which the above surgery produced arrhythmia or a sustained fall in blood pressure to less than 70 mm Hg was discarded from the experiment. The animals were left to recover for 15 minutes. The occlusion of the artery was produced by applying tension to the ligature, and reperfusion was achieved by releasing the tension (Kane et al. 1984). The rat heart was used for ischemia-reperfusion arrhytmias because its lack of functioning collaterals (Maxwell et al. 1987) leads to reproducible zones of severe ischemia upon ligation of a coronary artery, and because rat atria receive their blood supply almost exclusively from extracoronary vessels (Halpern, 1957). Therefore, the resulting arrhythmias are of ventricular type.

The main experimental protocol comprised of 30 min. occlusion (ischemia), followed by 20-min. reperfusion. In some experiments drug pre-treatment was replaced by ischemic preconditioning, produced by three 3-min. occlusion periods, interspaced with 3-min. reperfusion. This was followed by 30-min. occlusion and 20-min. reperfusion. The 20-min reperfusion period was chosen for antiarrhythmic evaluation, since in the preliminary studies we found that the untreated reperfusion dysrhythmias occurred mainly during the first 10-20 min.

To confirm that reperfusion was occurring, 0.1 ml/100 g of a 10% solution of fluorescein (Sigma) was administered i. v., immediately before sacrificing the animals. The heart was rapidly removed from the chest and placed in ice cold 10% KCI solution for 1 min to stop it beating. A gross examination of the heart was made under u.v. light to determine whether areas of non-perfused myocardium were present. These appeared dark blue under u.v. light whereas perfused areas were stained green. The control non-ischemic and 30-min ischemic, non-reperfused hearts were treated in the same way.

The Gambeth Convensions' guidelines were used for classification, quantification, and analysis of the arrhythmias in ischemia, infarction and reperfusion (Walker et al. 1988).

Antidysrhythmic positive controls

The following antidysrhythmic reference drugs were used, according to the Vaughan Williams' classification (1991), further developed by Weirich and Antoni (1991):

Class I (sodium fast channel blocker), quinidine chloride (Sigma); Class II ([beta]-adrenergic antagonist), propranolol hydrochloride (inderal) (MacClesfield, Great Britain; Class III (prolonged action potential by blocking potassium channels), amiodarone hydrochloride (Sigma); Class IV (slow calcium channel blocker), verapamil (isoptin) (Knoll AG, Germany). The optimal doses for rats were worked out in the preliminary experiments and are presented in the results.

Statistical analysis

All results were evaluated originally as means [+ or -] SEM, and after statistical analysis were presented, when indicated, as % of the baseline value. For all analyses, INSTAT V2.04 program was used, including one-way and two-way ANOVA, t-test and Chi-Square test.

A p values less than 0.05 were considered to be statistically significant.

Results

OA, UA and UV, showed very low toxicity. L[C.sub.50] was 0.50, 0.95 and 1.10 mg/ml, respectively. We were not able to evaluate the toxicity of MM on brine shrimp test, because it was unsolvable in methanol. The hemodynamic results of OA, UA, MM and UV are presented in Table 1. All four derivatives showed significant, dose-dependent vasodepressor effect and sinus bradycardia, lasting more than 60 minutes with a maximum at 20 min after application. The most potent hymodynamic effects were expressed by OA and MM. The pretreatment with triterpenoids decreased the vasopressor and to less extent the tachicardic effects of isoproterenol. The effect of 10 mg/kg OA was similar to the effect of propranolol at dose 2 mg/kg (Table 2). The positive inotropic and dromotropic effects are presented in Table 3, with OA and UA having the highest activity at the medium dose of 40 mg/kg. OA and UA displayed antidysrhythmic effects on Ca[Cl.sub.2]-induced arrhythmia comparable to that of amiodarone (Table 4), and on adrenaline-induced dysrhythmia, a beneficial effect comparable to that of propranolol (Table 5). Methyl maslinate and uvaol did not show antidysrhytmic effects on the above two types of chemical arrhythmia. The most interesting were the results on ischemiareperfusion dysrhythmia. Only OA and uvaol conferred antiarrhythmic effect, and the effect was comparable to that of the [beta]-blocker propranolol (Table 6), but less pronounced than that of ischemic preconditioning, quinidine or amiodarone.

Discussion

As mentioned in the Introduction, the total olive leave extract displayed a significant hypotensive effect in experimental hypertension (Ribeiro et al. 1986). This effect was ascribed originally to the two secoroids oleuropein and aleacfein, isolated from the olive leaves. In more recent studies (Somova et al. 2003) it was reported, that in addition to the effects of oleuropein and oleacein, triterpenoid derivatives might contribute to the potent overall antihypertensive effect of olive leave extract.

In the present study, for the first time a cardiotonic and antidysrhythmic effect of four triterpenoid derivatives was presented. The experiments of the hemodynamic effect of isoproterenol after pretreatment with the triterpenoids, pointed out that the vasodepressor and bradycardic effects of the test compounds may be due to [beta]-blocking activity, similar but less potent to that of propranolol. The positive inotropic effect can not be explained by this mechanism and requires further investigation.

The antidysrhythmic effect on adrenaline-induced arrhythmia is also suggestive of [beta]-adrenergic antagonistic effect.

According to most authors (Curtis et al. 1989), the ischemia-induced ventricular fibrillation (VF) and reperfusion-induced VF are unlikely to be initiated by a common electrophysiological mechanism. The mechanism of maintenance of early ischemia-induced arrhythmias has been attributed to re-entry (Janse et al. 1980). The arrhythmias are inhibited by sodium conductance inhibitors, some of which have been shown to slow conduction causing bi-directional block, and by potassium conductance inhibitors, some of which have been shown to prolong the refractory period (Curtis et al. 1989). Theoretically, both classes of drug may effectively increase the re-entry wavelength and terminate VF and ventricular tachicardia (VT). It has been suggested (Janse, 1999), but not universally accepted that re-entry in ischemia is often initiated by the flow of injury current between the ischemic and non-ischemic tissue. The release of catecholamines (Schomig et al. 1987) plays an important role as shown by the finding that VF and FT are reduced by surgical or pharmacological denervation, by block of the carrier mechanism and so preventing the release of catecholamines, or acting postsinaptically and blocking [alpha]- and [beta]-receptors (Carmeliet, 1999). Inhibitors of the [Na.sup.+]/[H.sup.+] exchanger exert antiarrhythmic activity (Aye et al. 1997), and this effect can be explained by a reduction of catecholamine release: less [Na.sup.+]/[H.sup.+] exchange reduces the [Na.sup.+] load and avoids reversal of the carrier responsible reuptake of noradrenaline in the nerve terminals. It is also possible, however, that the beneficial effect of these inhibitors occurs at the myocyte level, by reducing [Na.sup.+] and [Ca.sup.2+] load (Carmeliet, 1999). The present results suggested that the beneficial antidysrhythmic efffect of the triterpenoids on ischemia-induced arrhythmia could be attributed to either [beta]-blocker activity or inhibition of [Na.sup.+]/[H.sup.+] exchanger, or both.

The mechanisms of initiation and maintenance of reperfusion-induced arrhythmias are even less well-established. The arhythmias start by an automatic stimulus in the reperfused zone and change afterward in a reentry multiple wavelet type of VT and VF. The underlying factors in the genesis of reentry are extremely short action potential and refractory period and slow conduction (Carmeliet, 1999). It was suggested that oxygen radicals could play an important role, and scavengers of radicals act as antiarrhytmics (Bernier and Hearse, 1988). The beneficial antidysrhythmic effect of the triterpenoids in this type of arrhythmia can be attributed to [beta]-blocking activity and recently reported antioxidant activity (Somova et al. 2003). Additional studies are required to elucidate the mechanism(s) of antidysrhythmic effects of triterpenoids.

In conclusion, on the basis of the vasodepressor, cardiotonic and antidysrhythmic activity, crude olive extract or isolate, standardized on oleanolic acid and uvaol, can be recommended for an additional and rational therapy of hypertension, as a cheap and accessible source of treatment of hypertension, complicated with stenocardia and cardiac failure.

Table 1. Changes in heart rate and mean arterial pressure after
intraperitoneal application of different doses of oleanolic acid,
ursolic acid and methyl maslinate and uvad.

          Baseline                           10 minutes
          HR                MAP              HR

OA
20 mg/kg  495 [+ or -] 8    96 [+ or -] 6    [down arrow] 6%
40 mg/kg  480 [+ or -] 10   92 [+ or -] 4    [down arrow] 6%
60 mg/kg  450 [+ or -] 8   104 [+ or -] 6    [down arrow] 6%

UA
20 mg/kg  480 [+ or -] 6    84 [+ or -] 4    [down arrow] 6%
40 mg/kg  465 [+ or -] 6    96 [+ or -] 4    [down arrow] 6%
60 mg/kg  480 [+ or -] 8    94 [+ or -] 6    [down arrow] 8%

UU
20 mg/kg  420 [+ or -] 8   110 [+ or -] 8    [down arrow] 4%
40 mg/kg  400 [+ or -] 8   100 [+ or -] 10   [down arrow] 6%
60 mg/kg  400 [+ or -] 6   115 [+ or -] 8    [down arrow] 10%

UV
20 mg/kg  435 [+ or -] 7    98 [+ or -] 4    [down arrow] 3%
40 mg/kg  430 [+ or -] 10  100 [+ or -] 6    [down arrow] 7%
60 mg/kg  420 [+ or -] 8    96 [+ or -] 6    --

          10 minutes        20 minutes
          MAP               HR                MAP
OA
20 mg/kg  [down arrow] 10%  [down arrow] 6%   [down arrow] 18%
40 mg/kg  [down arrow] 22%  --                [down arrow] 40%
60 mg/kg  [down arrow] 38%  [down arrow] 8%   [down arrow] 60%

UA
20 mg/kg  --                [down arrow] 6%   [down arrow] 2%
40 mg/kg  --                [down arrow] 5%   [down arrow] 5%
60 mg/kg  [down arrow] 4%   [down arrow] 10%  [down arrow] 10%

UU
20 mg/kg  [down arrow] 40%  [down arrow] 7%   [down arrow] 40%
40 mg/kg  [down arrow] 50%  [down arrow] 10%  [down arrow] 60%
60 mg/kg  [down arrow] 60%  --                --

UV
20 mg/kg  [down arrow] 10%  [down arrow] 4%   [down arrow] 20%
40 mg/kg  [down arrow] 15%  [down arrow] 9%   [down arrow] 25%
60 mg/kg  --                [down arrow] 10%  [down arrow] 30%

          30 minutes                          40 minutes
          HR                MAP               HR
OA
20 mg/kg  [down arrow] 6%   [down arrow] 20%  [down arrow] 6%
40 mg/kg  [down arrow] 6%   [down arrow] 40%  [down arrow] 6%
60 mg/kg  [down arrow] 10%  [down arrow] 50%  [down arrow] 8%

UA
20 mg/kg  [down arrow] 6%   [down arrow] 14%  [down arrow] 6%
40 mg/kg  [down arrow] 5%   [down arrow] 20%  [down arrow] 6%
60 mg/kg  [down arrow] 6%   [down arrow] 22%  [down arrow] 8%

UU
20 mg/kg  [down arrow] 7%   [down arrow] 54%  [down arrow] 7%
40 mg/kg  --                --                --
60 mg/kg  --                --                --

UV
20 mg/kg  [down arrow] 6%   [down arrow] 25%  --
40 mg/kg  [down arrow] 9%   [down arrow] 30%  [down arrow] 10%
60 mg/kg  [down arrow] 10%  [down arrow] 30%  [down arrow] 12%

          40 minutes        60 minutes
          MAP               HR                MAP
OA
20 mg/kg  [down arrow] 12%  [down arrow] 8%   [down arrow] 8%
40 mg/kg  [down arrow] 20%  [down arrow] 10%  [down arrow] 20%
60 mg/kg  [down arrow] 50%  [down arrow] 10%  [down arrow] 50%

UA
20 mg/kg  [down arrow] 14%  [down arrow] 6%   [down arrow] 20%
40 mg/kg  [down arrow] 30%  [down arrow] 6%   [down arrow] 30%
60 mg/kg  [down arrow] 36%  [down arrow] 6%   [down arrow] 40%

UU
20 mg/kg  [down arrow] 54%  [down arrow] 5%   [down arrow] 54%
40 mg/kg  --                --                --
60 mg/kg  --                --                --

UV
20 mg/kg  [down arrow] 30%  [down arrow] 6%   [down arrow] 20%
40 mg/kg  [down arrow] 40%  [down arrow] 10%  [down arrow] 30%
60 mg/kg  [down arrow] 50%  [down arrow] 12%  [down arrow] 50%

Mean [+ or -] SEM; [down arrow] decrease; All changes are significant
compared to control values. OA -- Oleanolic acid; UA -- Ursolic acid;
MM -- Methyl Maslinate; UV -- Uvaol; HR -- Heart rate (beats/min);
MAP -- Mean arterial pressure (mm Hg).

Table 2. Beta-adrenolitic effects of triterpenoids isolated from Olea
Africana: Effects of isoproterenol (1 [micro]g/kg) after 15 minutes
pretreatment with reference substance (propranolol) and different
isolates.

                   Baseline                            1 minute
TREATMENT          HR                MAP                HR

Propranolol i.v.
  (2 mg/kg)        150 [+ or -] 4.2  450 [+ or -] 8.0   [down arrow] 25%
Oleanolic acid
  i.p. (10 mg/kg)  145 [+ or -] 6.0  462 [+ or -] 8.8   [down arrow] 20%
Ursolic acid i.p.
  (10 mg/kg)       135 [+ or -] 4.0  440 [+ or -] 9.0   [down arrow] 10%
Methyl maslinate
  i.p. (10 mg/kg)  142 [+ or -] 6.6  410 [+ or -] 8.8   [down arrow] 1%
Uvaol i.p.
  (10 mg/kg)       148 [+ or -] 8.0  416 [+ or -] 12.0  [down arrow] 5%

                   1 minute          3 minutes
TREATMENT          MAP               HR                MAP

Propranolol i.v.
  (2 mg/kg)        [down arrow] 26%  [down arrow] 35%  [down arrow] 29%
Oleanolic acid
  i.p. (10 mg/kg)  --                [down arrow] 22%  [down arrow] 8%
Ursolic acid i.p.
  (10 mg/kg)       --                [down arrow] 15%  [down arrow] 9%
Methyl maslinate
  i.p. (10 mg/kg)  --                [down arrow] 5%   --
Uvaol i.p.
  (10 mg/kg)       [down arrow] 4%   [down arrow] 5%   [down arrow] 8%

                   5 minutes                           10 minutes
TREATMENT          HR                MAP               HR

Propranolol i.v.
  (2 mg/kg)        [down arrow] 27%  [down arrow] 24%  [down arrow] 27%
Oleanolic acid
  i.p. (10 mg/kg)  [down arrow] 30%  [down arrow] 6%   [down arrow] 28%
Ursolic acid i.p.
  (10 mg/kg)       [down arrow] 12%  [down arrow] 5%   [down arrow] 12%
Methyl maslinate
  i.p. (10 mg/kg)  [down arrow] 10%  --                [down arrow] 10%
Uvaol i.p.
  (10 mg/kg)       [down arrow] 12%  [down arrow] 4%   [down arrow] 20%

                   10 minutes        15 minutes
TREATMENT          MAP               HR                MAP

Propranolol i.v.
  (2 mg/kg)        [down arrow] 27%  [down arrow] 30%  [down arrow] 23%
Oleanolic acid
  i.p. (10 mg/kg)  [down arrow] 8%   [down arrow] 21%  [down arrow] 8%
Ursolic acid i.p.
  (10 mg/kg)       [down arrow] 10%  [down arrow] 10%  [down arrow] 9%
Methyl maslinate
  i.p. (10 mg/kg)  --                [down arrow] 10%  --
Uvaol i.p.
  (10 mg/kg)       [down arrow] 2%   [down arrow] 15%  [down arrow] 4%

Baseline values: Mean [+ or -] SEM; [down arrow] percentage compared to
the original effect of isoproterenol before treatment.
MAP -- Mean arterial pressure (mm Hg); HR -- Heart rate (beats/min).

Table 3. Evaluation of inotropic (QRS Complex) and dromotropic (PQ
interval) effects of oleanolic acid, ursolic acid, methyl maslinate and
uvaol.

                  Baseline                         3 minute
Treatment         PQ               QRS             PQ
                  (sec)            (mV)            (sec)

Oleanolic         0.020 [+ or -]   0.141 [+ or -]  0.025 [+ or -]
Acid (40 mg/      0.002            0.006           0.002
kg i.p)
Ursolic Acid      0.028 [+ or -]   0.268 [+ or -]  0.027 [+ or -]
(40 mg/kg i.p.)   0.002            0.002           0.002
Methyl            0.018 [+ or -]   0.120 [+ or -]  0.020 [+ or -]
maslinate         0.002            0.007           0.002
(40 mg/kg i.p)
Uvaol             0.015 [+ or -]   0.120 [+ or -]  0.010 [+ or -]
(40 mg/kg i.p.)   0.003            0.007           0.002

                 3 minute          5 minute
Treatment        QRS               PQ                QRS
                 (mV)              (sec)             (mV)

Oleanolic        0.162 [+ or -]    0.040 [+ or -]    0.166 [+ or -]
Acid (40 mg/     0.006*            0.001*            0.006*
kg i.p)          [down arrow] 15%  [down arrow] X 2  [down arrow] 18%
Ursolic Acid     0.283 [+ or -]    0.020 [+ or -]    0.287 [+ or -]
(40 mg/kg i.p.)  0.006             0.001             0.003*
                 [down arrow] 5%                     [down arrow] 7%
Methyl           0.143 [+ or -]    0.015 [+ or -]    0.153 [+ or -]
maslinate        0.006*            0.002             0.006
(40 mg/kg i.p)   [down arrow] 19%                    [down arrow] 28%
Uvaol            0.150 [+ or -]    0.015 [+ or -]    0.246 [+ or -]
(40 mg/kg i.p.)  0.006*            0.002             0.006*
                 [down arrow] 25%                    [down arrow] X 2

                 10 minute                           20 minute
Treatment        PQ                QRS               PQ
                 (sec)             (mV)              (sec)

Oleanolic        0.040 [+ or -]    0.181 [+ or -]    0.040 [+ or -]
Acid (40 mg/     0.001*            0.002*            0.001*
kg i.p)          [down arrow] X 2  [down arrow] 28%  [down arrow] X 2
Ursolic Acid     0.023 [+ or -]    0.294 [+ or -]    0.022 [+ or -]
(40 mg/kg i.p.)  0.002             0.004*            0.002
                                   [down arrow] 10%
Methyl           0.020 [+ or -]    0.180 [+ or -]    0.025 [+ or -]
maslinate        0.001             0.007*            0.003
(40 mg/kg i.p)                     [down arrow] 50%  [down arrow] 38%
Uvaol            0.015 [+ or -]    0.229 [+ or -]    0.015 [+ or -]
(40 mg/kg i.p.)  0.001             0.007*            0.002
                                   [down arrow] X 2

                 20 minute         30 minute
Treatment        QRS               PQ                QRS
                 (mV)              (sec)             (mV)

Oleanolic        0.188 [+ or -]    0.040 [+ or -]    0.207 [+ or -]
Acid (40 mg/     0.005*            0.001*            0.003*
kg i.p)          [down arrow] 33%  [down arrow] X 2  [down arrow] 46%
Ursolic Acid     0.296 [+ or -]    0.025 [+ or -]    0.293 [+ or -]
(40 mg/kg i.p.)  0.003*            0.002             0.003*
                 [down arrow] 10%                    [down arrow] 9%
Methyl           0.170 [+ or -]    0.025 [+ or -]    0.184 [+ or -]
maslinate        0.007*            0.003             0.006*
(40 mg/kg i.p)   [down arrow] 42%  [down arrow] 38%  [down arrow] 53%
Uvaol            0.245 [+ or -]    0.015 [+ or -]    0.182 [+ or -]
(40 mg/kg i.p.)  0.006*            0.002             0.006*
                 [down arrow] X 2                    [down arrow] 52%

                 40 minute                           60 minute
Treatment        PQ                QRS               PQ
                 (sec)             (mV)              (sec)

Oleanolic        0.040 [+ or -]    0.192 [+ or -]    0.040 [+ or -]
Acid (40 mg/     0.001*            0.002*            0.002*
kg i.p)          [down arrow] X 2  [down arrow] 36%  [down arrow] X 2
Ursolic Acid     0.023 [+ or -]    0.298 [+ or -]    0.022 [+ or -]
(40 mg/kg i.p.)  0.003             0.006*            0.002
                                   [down arrow] 11%
Methyl           0.020 [+ or -]    0.180 [+ or -]    0.020 [+ or -]
maslinate        0.001             0.005*            0.002
(40 mg/kg i.p)                     [down arrow] 50%
Uvaol            0.018 [+ or -]    0.180 [+ or -]    0.015 [+ or -]
(40 mg/kg i.p.)  0.002             0.006*            0.003
                                   [down arrow] 50%

                 60 minute
Treatment        QRS
                 (mV)

Oleanolic        0.201 [+ or -]
Acid (40 mg/     0.001*
kg i.p)          [down arrow] 43%
Ursolic Acid     0.296 [+ or -]
(40 mg/kg i.p.)  0.003*
                 [down arrow] 10%
Methyl           0.170 [+ or -]
maslinate        0.006*
(40 mg/kg i.p)   [down arrow] 42%
Uvaol            0.186 [+ or -]
(40 mg/kg i.p.)  0.007*
                 [down arrow] 55%

Mean [+ or -] SEM
* The difference is significant compared to the control value
i.p. -- intraperitoneally

Table 4. Effects of antidysrhythmic drugs and oleanolic and ursolic
acids on Ca[Cl.sub.2]-induced arrhythmia.

                   Control                    Ca[Cl.sub.2] (140 mg/kg)
                                              i.v.
                   Heart rate decrease (%)    Heart rate decrease (%)
Treatment          1 min    10 min   20 min   1 min    3 min

Saline              0        0        0       20       26
Quinidine p.o.
  (5 mg/kg)         5       12        9        8        6
Propranolol i.v.
  (2 mg/kg)        25       30       30       20       20
Amiodarone p.o.
  (10 mg/kg)        4        4        2       14        6
Verapamil i.v.
  (2 mg/kg)        18       30       32       10        8
Oleanolic acid
  i.p. (40 mg/kg)   0        4       12        1        1
Ursolic acid i.p.
  (40 mg/kg)        1       10       18        1        1

                   Ca[Cl.sub.2] (140 mg/kg) i.v.
                                    Restoration of sinus
                                    rhythm (sec)          Mortality (%)
Treatment          VPB (%)  VF (%)

Saline             100      80      40 [+ or -] 5         67
Quinidine p.o.
  (5 mg/kg)        100      80      60 [+ or -] 8         60
Propranolol i.v.
  (2 mg/kg)        100      60      60 [+ or -] 6         60
Amiodarone p.o.
  (10 mg/kg)        80      12      10 [+ or -] 3         10
Verapamil i.v.
  (2 mg/kg)         20       0      20 [+ or -] 2          4
Oleanolic acid
  i.p. (40 mg/kg)   60       8      10 [+ or -] 3         16
Ursolic acid i.p.
  (40 mg/kg)        60       8      10 [+ or -] 4         18

Sinus rhythm restoration of the survived rats: Mean [+ or -] SEM; 6 rats
per group. VPB -- Ventricular premature beats; VF -- Ventricular
fibrillation; p.o. -- Orally; i.v. -- Intravenously;
i.p. -- Intraperitoneally.

Table 5. Effects of antidysrhythmic drugs and oleanolic and ursolic
acids on adrenaline-induced arrhythmia.

Treatment          Control                  Adrenaline (10 [micro]g/kg)
                                            i.v.
                   Heart rate decrease (%)  VPB  VT   VF
                                            (%)  (%)  (%)
                   1 min   10 min  20 min

Saline              0       0       0       100  100  100
Quinidine p.o.
  (5 mg/kg)         4       4       8        80   60   60
Propranolol i.v.
  (2 mg/kg)        20      15      15        20    0    0
Amiodarone p.o.
  (10 mg/kg)        2       9       2       100   80   80
Verapamil i.v.
  (2 mg/kg)        30      25      20        80   80   60
Oleanolic acid
  i.p. (40 mg/kg)   6      21      21        20    2    8
Ursolic acid i.p.
  (40 mg/kg)        3       5       5        40   10    8

Treatment          Adrenaline (10 [micro]g/kg)
                   i.v.
                   Restoration     Mortality
                   of sinus        (%)
                   rhythm (sec)

Saline              0              66
Quinidine p.o.
  (5 mg/kg)        60 [+ or -] 11  60
Propranolol i.v.
  (2 mg/kg)        20 [+ or -] 8    0
Amiodarone p.o.
  (10 mg/kg)        0              60
Verapamil i.v.
  (2 mg/kg)         0              40
Oleanolic acid
  i.p. (40 mg/kg)  10 [+ or -] 6    8
Ursolic acid i.p.
  (40 mg/kg)       14 [+ or -] 7   10

Sinnus rhythm restoration of the survived rats: Mean [+ or -] SEM; 6
rats per group
VPB -- Ventricular premature beats
VT -- Ventricular tachycardia
VF -- Ventricular fibrillation

Table 6. Effects of antidysrhythmic drugs and triterpenoids on ischemia-
reperfusion dysrhythmia in rats.

                                   30 minute myocardial ischemia
Treatment                          VPB (%)  VT (%)  VF (%)  VPB (%)

Saline-treated preconditioned      77       77      22      22
Saline-treated non-preconditioned  89       77      67      44
Quinidine p.o. (5 mg/kg)           83       66      17      33
Propranolol i.v. (2 mg/kg)         66       33      17      33
Amiodarone p.o. (10 mg/kg)         66       66      17      33
Verapamil i.v. (2 mg/kg)           83       66      50      66
Oleanolic acid i.p. (40 mg/kg)     66       33      33      33
Ursolic acid i.p. (40 mg/kg)       83       50      50      66
Methyl maslinate (40 mg/kg)        83       66      66      83
Uvaol i.p. (40 mg/kg)              66       33      33      33

                                   20 minute reperfusion
Treatment                          VT (%)  VF (%)  Total
                                                   Mortality (%)

Saline-treated preconditioned      33       0      22
Saline-treated non-preconditioned  44      44      67
Quinidine p.o. (5 mg/kg)           33       0      17
Propranolol i.v. (2 mg/kg)         33      33      33
Amiodarone p.o. (10 mg/kg)         33       0      17
Verapamil i.v. (2 mg/kg)           50      50      50
Oleanolic acid i.p. (40 mg/kg)     33      33      33
Ursolic acid i.p. (40 mg/kg)       50      50      50
Methyl maslinate (40 mg/kg)        50      66      66
Uvaol i.p. (40 mg/kg)              33      33      33

The two control (saline) groups consisted of 9 rats each, the rest--6
rats per group.
Design: The preconditioning was performed by three 3 minute coronary
occlusion periods interdispersed with 3 minute reperfusion.
Preconditioning was followed (where applicable) by 30 minute coronary
occlusion and 20 minute reperfusion. Except for the first saline-treated
group, all other groups were non-preconditioned.
VPB -- Ventricular premature beats
VT -- Ventricular tachycardia
VF -- Ventricular fibrillation

Acknowledgements

Acknowledgements are due to Ms C. Govender for typing the manuscript and Ms K. Moodley for the computer analysis of ECGs.

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L.I. Somova (1), F.O. Shode (2), and M. Mipando (1)

(1) Department of Human Physiology

(2) Department of Chemistry, University of Durban-Westville, Durban, South Africa

Address

L. I. Somova, Department of Human Physiology, University of Durban-Westville, Private Bag X54001, Durban 4000, South Africa

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