Abstract

Introduction

Materials & Methods

Results

Discussion & Conclusion

References

Discussion
Board

Preparation of plant extract

The plant was purchased from the local market and identified as Andrographis paniculata (Burm. f.) Nees (Acanthaceae) by Professor Wee Yeow Chin, Department of Botany, National University of Singapore. A dried specimen is deposited in the herbarium (voucher No. 569).

The fresh aerial parts of A. paniculata (1kg) were blended and extracted with distilled water (10 litres). Following repeated filtration and concentration, the solution obtained was centrifuged at 2000g for 10 min. The resulting supernatant was freeze-dried to yield 49.9g of extract.

For the dose-response study in conscious SHR and the experiments in conscious SHR and WKY rats, the plant extract was dissolved in distilled water and placed in osmotic pumps (Alzet 2ML2; ALZA Corporation, Palo Alto, California, USA) as described by Yugarani et al. (1993). These pumps were then implanted into the peritoneal cavity of the rats to deliver a 13-day infusion of the extract.

The crude extract was semi-purified by successive partitioning with ethyl acetate and n-butanol. The fractions obtained, FA (ethyl acetate), FB (butanol) and FC (aqueous), were further tested in an animal bioassay using the anaesthetised Sprague-Dawley (SD) rat. The five diterpenoids were also tested for hypotensive activity in this animal model.

The 5 diterpenoids, DA and DDA, andrographolide, andrographiside and neoandrographolide were supplied by Professor M. Kuroyanagi, School of Pharmaceutical Sciences, University of Shizuoka, Japan. These were first dissolved in dimethyl sulphoxide (DMSO) and then diluted with normal saline to obtain a final concentration of 20 nM. The solution was sonicated for 3 min before it was ready to use. Where DA or DDA was used in experiments using the isolated thoracic aortae, the final bath concentration of DMSO did not exceed 0.1%; this did not affect aortic smooth muscle contraction.

Acetylcholine, norepinephrine and phenylephrine were obtained from Sigma Chemical Company, USA. Caffeine and glibenclamide were obtained from Research Biochemicals International, USA. Except glibenclamide, which was first dissolved in DMSO and diluted with normal saline to make a concentration of 10mM in 5% DMSO, all the other drugs were dissolved in normal saline.

Animals

Male SD rats were obtained from the local animal breeding unit at Sembawang Animal Centre. Male SHR and WKY rats aged 14-15 weeks, weighing 290-310g, were purchased from Animal Resource Center, Perth, Western Australia. 

For the dose response study, twenty one SHR were divided into four treatment groups, viz. I: distilled water (control), n=5; II: extract 2.8 g/kg, n=5; III: extract 1.4 g/kg, n=6; IV: extract 0.7 g/kg, n=5. The optimum hypotensive dose was determined from this pilot study, and given to eight SHR and seven WKY rats in a subsequent experiment to demonstrate the effects of a 13-day intraperitoneal (i.p.) infusion of the extract on the SBP in these rats.

Following surgery to implant osmotic pumps, the SBPs of the SHR and WKY rats given a 13-day i.p. infusion of the crude plant extract (or its vehicle, water) by osmotic pumps were measured every other day by the indirect tail cuff method (Bunag, 1973). The mean of two SBP readings, obtained over a one-week period before the experiment, was used as the initial (Day 0) SBP.

Male SD rats (250-300g) were anaesthetized with a mixture of Inactin^R (50mg/kg) and sodium pentobarbital (30mg/kg) by i.p. injection. A 30cm length of polyethylene (PE50) tubing was inserted into the carotid artery for the measurements of mean arterial blood pressure (MAP) and heart rate (HR) via a pressure transducer (Grass model PT 300) coupled to a Grass polygraph (Model 7D). The jugular vein was also cannulated with PE10 tubing for intravenous (i.v.) injections of extract/drugs while the trachea was cannulated to facilitate spontaneous respiration.

After a 20 minute equilibration period, samples were injected i.v. in doses of 2.5, 5.0, 10, 20 and 30 mg/kg (in saline) for WE (crude water extract), FB and FC; 1.0, 2.0 and 2.4 mg/kg (in 5% DMSO and saline) for FA. Each sample was tested in 6 rats. Each injection was given in a fixed volume of 0.1 ml.

The pharmacological antagonist studies were performed by the method of Shaw et al. (1984), using anaesthetised SD rats prepared as described above. The effects of autonomic ganglion transmission, alpha- and beta-adrenergic, muscarinic cholinergic and histaminergic activities on the change of MAP with test drug were examined by using their specific antagonists and agonists. The respective antagonists were hexamethonium (10mg/kg), phentolamine (2mg/kg), propranolol (2mg/kg), atropine (2mg/kg), pyrilamine (15mg/kg, H-1 receptor blocker) and cimetidine (15mg/kg, H-2 receptor blocker). Methoxamine (50 micro g/kg), isoproterenol (1.2 micro g/kg), acetylcholine (2 micro g/kg) and histamine (2 micro g/kg) acted as alpha- and beta-adrenoceptor, cholinergic and histaminergic receptor agonists, respectively.

The protocol was as follows: a test dose of the test drug was given and the change in MAP served as a control. The effect of an agonist on the MAP of the rat was first assessed by injection of the agonist at an effective dose. The appropriate antagonist was then introduced. When the MAP was stable, injection of the agonist was repeated to ensure the blockade of the antagonist. When the desired blockade was attained, the same dose of test drug was given immediately. Each agonist/antagonist was tested in 6 rats.

As there was no hypotensive activity in FA, and WE and FC had high ED50 values greater than that of FB, subsequent pharmacological antagonist studies were done only with FB.

The possible interaction between the hypotensive action of the test drug and the renin-angiotensin system was studied using the angiotensin-converting-enzyme (ACE) inhibitor, captopril. The responses of the rats to the test drug were recorded first. An effective dose of angiotensin I (40 ng/kg) was injected, followed by captopril infusion (20 micro g/min/kg) for 30 min at a rate of 0.078 ml/min using an infusion pump (Harvard Apparatus). Angiotensin I and the test drug were again given at the end of the captopril infusion. The MAPs were then compared with those before the infusion and the changes were then recorded. 

Preparation of isolated rat thoracic aortae

The thoracic aorta was isolated from male SD rats (250-300g) killed by decapitation. The vessels were cut into rings (each 4-5 mm in length) and each was penetrated with two stainless steel hooks. Each ring was then suspended under a resting tension of 1g in an organ bath containing Krebs’ solution. In experiments using high K⁺, Ca²⁺-free solution, CaCl₂ was omitted and 50 mM of NaCl was replaced by an equimolar amount of KCl. The tissue bath solution was maintained at 37 degrees and oxygenated with 95% O₂ + 5% CO₂. Before the start of each experiment, tissues were equilibrated for 60 min with changes of bath fluid every 15 min. Changes in tension were recorded isometrically via a force-displacement transducer connected to a Grass polygraph, Model 7D.

For some experiments, the endothelial cells were removed by gentle rubbing with a forcep. Removal of functional endothelium was verified by the lack of any relaxation in response to acetylcholine (1 micro M) in rings precontracted with phenylephrine (0.1 micro M). Aortic rings with functional endothelium exhibited at least 90% relaxation under the same conditions.

Appropriate parallel control experiments were always carried out in order to correct for the possible changes in the sensitivity of the preparations.

Intact and endothelium-denuded preparations were initially contracted with phenylephrine (0.1 micro M) or KCl (80 mM) [Kaminyi et al., 1981]. After the tonic response became stable, cumulative relaxant curves were obtained for DA (2.5-120 micro M/L), DDA (2.5 - 40 micro M/L) and verapamil (0.001-10 micro M/L). Relaxation was expressed as a percentage of the maximum contraction induced by phenylephrine or KCl.

To further determine whether the action of DA or DDA on the phenylephrine-induced contraction was mediated through alpha- adrenoceptors, cumulative concentration-response curves were obtained for phenylephrine (0.001-10 micro M/L) in the absence or presence of DA (20-60 micro M/L, for 20min) or DDA (10-30 micro M/L, for 20min). Values are expressed as the percentages of the maximum contractile responses induced by phenylephrine before aortic rings were treated with DA or DDA.

To assess the effects of DA or DDA on CaCl₂-induced contractions, the preparations were exposed for a further 60 min to high K⁺, Ca²⁺-free solution after an initial equilibration period of 60 min. Two successive cumulative concentration-response curves for Ca²⁺ (0.1-10 mM/L) were obtained at 60 min intervals. The first curve was obtained in the absence of DA or DDA. Different concentrations of DA (20-80 micro M/L) or DDA (10-40 micro M/L) were then added to the bath and incubated for 20 min before obtaining a second cumulative concentration-response curve for Ca²⁺ in its presence. The maximal contractile response obtained in the first response was taken as the 100% response and all subsequent contractions were calculated as a percentage of this value. Each preparation was exposed to only one concentration of DA or DDA.

In order to clarify the possible action of DA/DDA on Ca²⁺ release from intracellular stores, the inhibitory effects of DA/DDA on caffeine- and norepinephrine-induced contractions in the absence of extracellular Ca²⁺ were performed.

After equilibration in normal Krebs’ solution for 60 min, the preparations were washed and bathed in Ca²⁺-free solution for 15 min. Caffeine (10 mM/L) or norepinephrine (1 micro M/L) was added to the bath. A transient phasic contraction for caffeine or a phasic contraction followed by a tonic contraction for norepinephrine developed in the absence of DA/DDA. After a 20 min loading period in normal Krebs’ solution to replenish the intracellular Ca²⁺ stores, the caffeine-and norepinephrine-induced contractions in Ca²⁺-free solution were again obtained but this time in the presence of DA (20-120 micro M/L) or DDA (10-40 micro M/L). Inhibitions are expressed as a percentage decrease of the initial contractions.

Preparations with intact endothelium were contracted with phenylephrine (0.1 micro M/L). Two successive concentration-response curves for DA (5-120 micro M/L) or DDA (2.5-40 micro M/L) were obtained in the absence or presence of N^G-nitro-L-arginine methyl ester [L-NAME] (25 micro M/L), methylene blue (10 micro M/L), indomethacin (20 micro M) and glibenclamide (10 micro M), which were added to the bath 20 min beforehand.

Similarly, concentration-response curves for DA or DDA were made in endothelium-intact aortic rings precontracted with KCl (80 mM) in the absence or presence of L-NAME (25 micro M/L) and methylene blue (10 micro M/L).

Male SD rats (250-300g) were killed by decapitation. The hearts were rapidly excised, and spontaneously beating right atria dissected and mounted in a 10 ml organ bath with one end attached to an isometric force displacement transducer coupled to a MacLab/8s A/D converting board (AD Instrument). Data were recorded on a Power Macintosh (7600/132). Experiments were carried out at 32 degrees C in Krebs solution bubbled with 95% O₂ plus 5% CO₂ at a resting tension of 0.4 g. Atria were allowed to equilibrate for 1 hour. DDA (2.5-320 M/L) was administered cumulatively at 4 min intervals. The effect of DDA on the beating rate of the atria was expressed as the percent reduction from basal values. To evaluate the beta-adrenergic blocking activity of DDA, two successive cumulative concentration-response curves for the chronotropic effect of isoproterenol (10^-9- 10^-5M) were established at 60 min intervals. The first curve was obtained in the absence of DDA.

Different concentrations of DDA (80-240 micro M/L) or propranolol (0.3 M/L) were then applied to the bath and incubated for 20 min before obtaining a second cumulative concentration-curve for isoproterenol. Control atria were incubated in DMSO (0.12%). Results were expressed as a percentage of the maximum response to isoproterenol in the first curve.

The significance of differences between data from the dose-response study was evaluated by one-way ANOVA, followed by Dunnett’s test. P values <0.05 were considered to be statistically significant.

The SBPs of extract-treated animals and their vehicle-treated controls over the 13-day period were compared by two-way ANOVA.

In studies on the isolated rat thoracic aortae, ED₅₀ values of DA and DDA (concentrations which produced 50% of maximum relaxation) were determined from curve fits to individual concentration-response curves using a four parameter logistic function of the form: response = constant + maximum / (1 + (concentration / ED₅₀)^slope) with data expressed on a log axis (y= a₀ + a₁ (1 + (x/a₂)^a₃), utilising Slidewrite Plus, with a₀, a₁, a₂ and a₃ representing the minima, maxima, ED₅₀ and slope respectively. Dose response curves with the rat atria were similarly constructed with this formula.

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