Evaluation of the effect of crude extract of Datura innoxia on the cardiovascular action of detomidine in rabbits

Datura innoxia indigenous shrub in South Asia region belongs to the family Solanaceae which contains medicinal important alkaloids (hyoscine, atropine, hycosamine, etc). Thirty adult rabbits of mixed breed, male and female were pretreated with 0.1 mg/500 mg of crude extract (1% of dimethyl sulfoxide (DMSO) solution) and injected 5 min later with 0.01 mg/500 mg of detomidine (group A, n=15) or Saline (group B, n=15). Mean arterial pressure, measurements and electrocardiography were performed for 65 min. After injecting crude extract of D. innoxia, the heart rate was increased by 45 and 46.34% in groups A and B, respectively. Heart rate remained increased after the injection of detomidine returning to base line values after 15 min. No increase in the mean arterial pressure (MAP) was noted in group B rabbits. Crude extract shortened PR and QT interval in both groups but after detomidine, PR and QT interval were enlarged significantly at the end of the experiment. The second degree atrioventricular was blocked in two rabbits after 40 min only in group B. It was concluded that alkaloid present in D. innoxia prevented detomidine induced Bradycardia and might be useful during combination against the Bradycardia induced by alpha-2 agonist in the rabbits.


INTRODUCTION
Detomidine hydrochloride is an alpha-2 agonist drug that produces profound and long acting sedation by reduction of noradrenalin and dopamine release in the central nervous system (CNS) (Jochle and Hamm, 1986).Both alpha-1 and alpha-2 effects are obtained peripherally producing vasoconstriction and increased blood pressure (Clarke and Taylor, 1986;Sarazan et al., 1989;Short et al., 1986;Wagner et al., 1991).Bradycardia is often accompanied by atrioventricular heart block.Approximately 60 minutes after detomidine administration, the blood pressure decreases as a result of bradycardia and persistent effects on alpha-2 receptors in the CNS (Muir, 1992).Side effects of detomidine administration include transient reduction in cecal, colonic, and jejunal blood flow (Clark et al., 1988).
Anticholinergic agents may be used to prevent bradycardia induced by detomidine in animals (Alitalo et al., 1986).Experiments with medetomidine in dogs (Short, 1991) showed that pretreatment with atropine were effective against medetomidine bradycardic effects.Nevertheless, side effects of anticholinergic use are tachycardia, high myocardial oxygen consumption, and intestinal activity reduction (Short, 1991;Jones, 1993).
Tropane alkaloids present in Datura innoxia's extract are central and peripherally acting anticholinergic are widely used as a spasmolytic in the treatment of abdominal pain in animals.The purpose of this study was to determine the effects of the extract of D. innoxia on the cardiovascular changes induced by detomidine administration in rabbits.

MATERIALS AND METHODS
Thirty adult mixed-breed rabbits, weighing 396 ± 56 g, 20 males and 10 females, were divided in two groups (A and B).Weight range in group A was 373 ± 41 kg (ranging from 300 to 430 g) and in group B was 390 ± 66 g (ranging from 295 to 515 g).The rabbits were held in separate stalls with food and water.Stainless steel needle-electrodes were placed through the skin at the right forelimb (negative), left forelimb (positive), and placed just behind the left elbow over the area of the cardiac apex for recording the electrocardiogram (ECG-1200, China).Both groups received crude extract of D. innoxia (1.0% solution in dimethyl sulfoxide (DMSO)), 0.1 mg/500 g intravenously through a 20 gauge hypodermic needle.Five minutes later, detomidine hydrochloride (0.01 mg/500 g) was injected intravenously into fifteen experimental animals (group A) or IV saline in equal volume into nine control animals (group B).In twelve rabbits (6 animals from group A and 6 from group B), a sterile polypropylene catheter-190 (Medical's Products-SP, Brazil) was inserted aseptically into a facial artery on the ventral surface of the mandible.The catheter was connected to a transducer placed at the cardiac base level for measurement and recording of systolic (SAP) and diastolic (DAP) arterial pressure (Physiograph-DMP-4B Narco Bio-Systems, TA-USA).The mean arterial pressure (MAP) was obtained by calculation of DAP + (SAP -DAP)/3.Heart rate (HR), PR and QT intervals were obtained from measurements of the ECG.Lowering of the head and onset of calm behavior of the rabbits after drug administration was considered to be clinical signs of sedation.The experiments were conducted in the afternoon.
The rabbits were maintained in a restraint stock for 30 min and then the baseline values for SAP, DAP, MAP, ECG (HR, PR and QT) and their behavior were obtained before injection of crude extract.Values were recorded 5 min later, before the injection of detomidine (group A) or saline (group B).The values were recorded again 2 min after detomidine or saline injection.Further recordings were made between 10 and 25 min at 5 min intervals, and then at 10 min intervals for a total of 65 min.
Statistical results are presented as mean ± standard deviation.

RESULTS
All results are summarized in the Table 1.Baseline HR was 40±10 and 41±12 beats/min.MAP was 140±18 and 121±12 mmHg in groups A and B, respectively.The injection of crude extract increased the HR to 58±8 beats/min (group A) and 60±10 beats/min (group B) at the 5 min.Administration of detomidine resulted in typical behavior changes in the experimental rabbits with sedation and dropped heads.Initially, the HR remained elevated, decreasing to baseline values after 15 min, and QT significantly lengthened from 45 min until the end of the observation time.The PR intervals in the group A were stable and the reduction in the group B was not significant.No arrhythmias were noted until 45 min, when atrioventricular heart block was recorded in 2 rabbits.The atrioventricular block occurred regularly at every third to fifth beats.Before administration of detomidine, MAP was 172±24 mmHg.A significant increase in MAP (62%) was measured at 7 min.MAP remained increased until 35 min and progressively decreased to baseline values until the end of the experiment.
The control rabbits were calm throughout the study.The HR was increased until 20 min after the crude extract.Saline injection did not result in further increase in HR and MAP.No arrhythmias were noticed in the control rabbits (Table 2).
Comparison between treatments showed that MAP was higher in group A, from 7 to 35 min.Baseline QT interval was higher in saline group and decreases after crude extract injection.In the detomidine treated group, QT was increased from 35 until 65 min as compared to group B. For groups A and B, PR was not different from time zero, and differences between groups at time 7, 10 and 35 to 65 min were noticed (Figures 1 to 4).

DISCUSSION
The detomidine induced bradycardia and atrioventricular block had been correlated with a decrease in the cardiac output (Wagner et al., 1991).In this study, crude extract increased the HR and prevented the bradycardia and atrioventricular heart block during the initial period in rabbits given detomidine, probably through reduction in vagal activity.
In this study, crude extract increased heart-rate in experimental and control rabbits.The brief period of tachycardia observed in detomidine treated group may be attributed to the alpha-2 agonist mediated increase in vagal tone (Wagner et al., 1991), which counteracts the anticholinergic-induced tachycardia.
It has been stated that prolongation and reduction of PR and QT interval may be associated with changes in heart rate (Tilley, 1991).Indeed, in this study, crude extract induced tachycardia resulted in QT shortening for 15 min in the control group.Additionally, in detomidine treated group, cardiac rate reduction was coincident with significant prolongation of QT interval after 45 min.The observation of second-degree atrioventricular blocks in two rabbits was also coincident with QT enlargement, but no changes were observed at the same period for the PR intervals.These data suggest that reduction of crude   extract action and the late prevalence of central sympatholytic effect of detomidine (Sarazan et al., 1989;Wagner et al., 1991;Savola, 1986) may be responsible for the atrioventricular heart block and increased QT interval.
The cardiac effects of detomidine use in rabbits had been a subject of considerable discussion.Atropine injection was recommended to prevent detomidine induced bradycardia (Alitalo et al., 1986;Jones, 1993).The effect of mixture of alkaloids (crude extract of D. innoxia) for this purpose has not been previously reported.It is well known that the immediate effects of the intravenous injection of this alpha-2 agonist are vasoconstriction and increased blood pressure by stimulation of post-synaptic adenoreceptors (Muir et al., 1992).The mean arterial pressure recorded after extract/detomidine administration was substantially higher than the values reported for detomidine alone (Wagner et al., 1991;Clarke and Taylor, 1986).It may be that hyoscine induced vagal blockade potentiated detomidine hypertensive action (Sarazan et al., 1989;Savola, 1986).In a preliminary report, Young et al. (1994) observed that atropine sulphate also prevented the heart rate drop caused by the alpha-2 agonist romifidine and resulted in marked hypertension.Similarly, Marques et al. (1998) described that hyoscine antagonized the romifidine bradyarrhytmias.Alibhai et al. (1996) also observed prolonged and intense hypertension when anticholinergic was associated with alpha-2 agonists in dogs.The tachycardia and hypertension observed in the aforementioned studies may be due to deleterious by increasing myocardial oxygen demand.Although it is not investigated in the current work, further studies may be conducted to investigate these effects attenuated by combination of bioactive compounds.
Although there is no comparative study available regarding cardiovascular effects of plant extract and recommended drugs, its use for preventing bradycardia may be preferred to prevent or treat bradycardia in rabbits, because of its shorter action and lower reduction in gastrointestinal motility.
Conclusively, pretreatment with anticholinergic agents was effective against the arrhythmoge activity of detomidine, and produced an additional increase in blood pressure.Therefore, its routine use before detomidine must be considered with regard to its hypertensive response.

Figure 1 .Figure 2 .Figure 3 .
Figure 1.Results of the heart rate from cardiovascular activity of Datura innoxia crude extract.

Figure 4 .
Figure 4. Results of the MAP from cardiovascular activity of Datura innoxia crude extract.

Table 1 .
Result of heart rate, PR interval and QT interval from cardiovascular activity of crude extract.

Table 2 .
Result of mean arterial pressure from cardiovascular activity of crude extract.