Cardiovascular Activity of Labdane Diterpenes from Andrographis paniculata in Isolated Rat Hearts

The dichloromethane (DCM) extract of Andrographis paniculata Nees was tested for cardiovascular activity. The extract significantly reduced coronary perfusion pressure by up to 24.5 ± 3.0 mm Hg at a 3 mg dose and also reduced heart rate by up to 49.5 ± 11.4 beats/minute at this dose. Five labdane diterpenes, 14-deoxy-12-hydroxyandrographolide (1), 14-deoxy-11,12-didehydroandrographolide (2), 14-deoxyandrographolide (3), andrographolide (4), and neoandrographolide (5), were isolated from the aerial parts of this medicinal plant. Bioassay-guided studies using animal model showed that compounds, (2) and (3) were responsible for the coronary vasodilatation. This study also showed that andrographolide (4), the major labdane diterpene in this plant, has minimal effects on the heart.


Introduction
Andrographis paniculata Nees, family Acanthaceae, has been used since time immemorial in Ayurvedic medicine, mainly for liver problems and dysentery [1]. The plant is also known as "Indian Echinacea" and "King of bitter." Phytochemical screening on this herbal plant showed that it contains a lot of flavonoids and terpenoids while moderate in alkaloids and tannins compounds [2]. This plant has been featured in at least 26 Ayurvedic formulae, whereas in traditional chinese medicine, A. paniculata is an important "cold property". In Malaysia, A. paniculata is more commonly known as "hempedu bumi" and is widely used in traditional medicine, especially for the treatment of cardiovascular disorders.
In earlier cardiovascular studies, A. paniculata extracts significantly reduced atherosclerotic artery stenosis and lowered restenosis rates after angioplasty in rabbits [20] decreases platelet aggregation in vitro [21] and were reported to be antihypertensive in rats [22]. Nevertheless, the scientific basis for the use of A. paniculata in treating "heart problems" is still unclear. To our knowledge, its direct effects on the isolated heart of an animal model are not known. Therefore, in this study, the dichloromethane (DCM) extract of A. paniculata was tested against coronary vessels, cardiac muscle contractility, and heart rate in Langendorff perfused rat hearts. Our preliminary study showed that the dichloromethane extract caused coronary vasodilation, reduced heart rate while not affecting the cardiac contractility of isolated perfused rat hearts. Further isolation work was performed on the dichloromethane extract in order to identify the active compound(s) responsible for the cardiovascular activity observed.

Materials and Method
2.1. General. All spectral data were obtained on various instruments; infrared (IR) spectrum was recorded on the Perkin Elmer FT-IR (fourier transform) spectrometer RX1, ultraviolet (UV) spectrum was taken on a Shimadzu UV-160A UV-visible recording spectrophotometer, nuclear magnetic resonance spectrum (NMR) was obtained from JEOL JNM-LA400 FT-NMR spectrometer (400 MHz), and mass spectrum (MS) was recorded on a Shimadzu GC-MS (gas chromatograph-mass spectrometry) spectrometer (HP 6890 Series Mass Selective Detector and HP 6890 Series GC System). The cardiovascular activity was measured using polygraph model 7D Grass, peristaltic pump model Compact Drive MasterFlex, and pressure transducer model GOULD Statham USA.

Plant
Material. The aerial parts of Andrographis paniculata were supplied by the Herbarium Unit, Department of Botany, University of Malaya, with herbarium series number KL4930.

Cardiovascular Activity on Isolated Rat Hearts.
The extracts and isolated compounds were screened for their cardiovascular effects in isolated perfused rat hearts by using the Langendorff-perfused model and method, with modification [23]. The Polygraph model (Grass Instrument Co. model 7D) was calibrated to measure heart rate, developed tension, and coronary perfusion pressure of the heart. The modified Krebs-Henseleit solution (37 • C) was pumped to flow at a constant rate (10 mL/min) by using a peristaltic pump (Compact Drive Masterflex), and the gas (95% O 2 and 5% CO 2 ) was allowed to pass through this solution. The Krebs-Henseleit solution contained the following: NaCl 118 mM, D-glucose 11.6 mM, NaHCO 3  Sprague Dawley rats (250-300 g) were anaesthetized with sodium pentobarbitone (72 mg/1000 g) and killed by cervical dislocation. The heart was isolated and put in a Petri dish containing cold Krebs-Henseleit solution to stop the heartbeat. After the fat and connective tissues had been removed from the heart, the aorta was cannulated and perfused with the Krebs-Henseleit solution. The coronary vessel tone was indicated by coronary perfusion pressure, which was measured with a pressure transducer (GOULD Statham USA) and monitored with the Grass Model 7D Polygraph.
The apex of the left ventricle was then attached with a hook and connected to the isometric tension transducer with a piece of thread, which passed through a pulley system. The resting heart tension was adjusted to 2 g to the optimum contractile force. The contractility was monitored by an isometric tension transducer and recorded as developed tension. Heart rate was also detected through this transducer.
The perfused isolated rat heart (PIH) was stabilised for about 20 min before any drug samples were injected. All injections were made at the rubber tubing near the cannula into the Krebs-Henseleit buffer. The labdane diterpenes compounds of A. paniculata were prepared as 1 mg/mL solutions. The first compound at 100 μL was injected into the PIH soon after its heart rate, contractility, and coronary perfusion pressure stabilized. The effect of the first compound/dose was allowed to disappear, and then administered with the second compound until the last compound followed by 40% ethanol as a vehicle control and finally 1000 nmoles isoprenaline and sodium nitroprusside as positive controls, all in the same heart. The compounds administred to the next heart were arranged randomly in order to avoid bias.

Statistical
Analyses. Data obtained were expressed as mean standard error (± S.E.M.). The data were analyzed for statistical significance using Student's t-test. P values less than 0.05 were considered to be significant ( * P < 0.05; * * P < 0.01; * * * P < 0.001).

Results and Discussion
The DCM extract of A. paniculata [24] significantly reduced the coronary perfusion pressure by up to 24.5 ± 3.0 (P < 0.05) and 29.4 ± 8.5 mm Hg (P < 0.05) at doses of 3 mg and 1 mg, respectively, and the heart rate by up to 49.5 ± 11.4 beats/minute (P < 0.05) at the 3 mg dosage. Four isolated rat hearts were used to investigate the effect of the DCM. Further isolation work was performed on the dichloromethane extract in order to identify the compound(s) responsible for the cardiovascular activity. Fractionation and purification of the DCM extract led to the isolation and purification of five labdane diterpenes, identified as 14-deoxy-12-hydroxyandrographolide (1), 14deoxy-11,12-dihydroandrographolide (2) (trans isomer), 14deoxyandrographolide (3), andrographolide (4), and neoandrographolide (5) (Figure 1). The structure of each compound was identified by comparison of their NMR, IR, UV, and MS data with literature values [25]. At least seven rat hearts were used to investigate the effect of each diterpenoid  on coronary perfusion pressure (a measure of coronary tone), developed tension (a measure of cardiac contractility), and heart rate. All compounds were prepared as 1 mg/mL solutions in 40% ethanol. The dose used for each compound was 0.1 mg. All of these diterpenoids reduced coronary perfusion pressure (CPP) significantly with compound 3 showing the largest reduction (−19.5 ± 7.0 mm Hg, P < 0.05) (Figure 2). This value was comparable with sodium nitroprusside (positive control), which reduced CPP by 22.0 ± 5.4 mm Hg (P < 0.05). The combination of all five diterpenoids in the same amounts reduced CPP by 10.8 ± 3.7 mm Hg (P < 0.05). None of the five diterpenoids showed any significant effect on cardiac contractility and heart rate as compared with the positive control, isoprenaline (Figures 3 and 4).
The decreased coronary perfusion pressure is a measurement for coronary artery vasorelaxation of an isolated rat heart. Due to the constant perfusion of Krebs-Henseleit into the heart during the experiment, any change in coronary perfusion pressure therefore would reflect change in vascular resistance [28]. Activation of β-adrenoceptors caused vasorelaxation of coronary channel.

Conclusions
In conclusion, the ability of 14-deoxy-11,12-dihydroandrographolide (2) and 14-deoxyandrographolide (3) to dilate coronary vessels and aortic rings [23] supports the traditional use of A. paniculata in the treatment of cardiovascular disorder and also in alleviating hypertension. These results also suggest that both compounds (2) and (3) might be good drug candidates for the treatment of angina and hypertension. Therefore, further in vivo pharmacological, pharmacokinetic, and toxicological studies need to be undertaken. This result also shows that the major compound, andrographolide (4), is not the major contributor to the cardiovascular activity of this plant.