
 andrographis paniculata nees , family acanthaceae , has been used since time immemorial in ayurvedic medicine , mainly for liver problems and dysentery .
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 .
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 .
previous researches have shown that a. paniculata extract and its labdane diterpenes have a broad range of pharmacological effects such as the ability to inhibit replication of the hiv virus [ 3 , 4 ] , prevent common cold [ 57 ] , antimalarial , prevent diarrhea , antibacterial , anti - inflammatory [ 1113 ] , antihyperglycemic effect [ 14 , 15 ] , suppress various cancer cells [ 1618 ] , and antifertility and pregnancy - terminating effects . in earlier cardiovascular studies , a. paniculata
extracts significantly reduced atherosclerotic artery stenosis and lowered restenosis rates after angioplasty in rabbits   decreases platelet aggregation in vitro   and were reported to be antihypertensive in rats . nevertheless , the scientific basis for the use of a. paniculata in treating 
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 .
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 .
the aerial parts of andrographis paniculata were supplied by the herbarium unit , department of botany , university of malaya , with herbarium series number kl4930 . dried and ground
aerial parts ( 650.0  g ) of the plant were extracted with dichloromethane for 6  h using a soxhlet apparatus at 45c .
the extract was concentrated under reduced pressure to yield 26.5  g of dichloromethane ( dcm ) crude extract .
dcm ( 3  g ) was chromatographed on a silica gel column with a ch2cl2-meoh gradient solvent system to give 80 fractions , which were combined into 4 subfractions ( a  d ) based on tlc ( thin - layer chromatography ) patterns and h nmr spectra .
subfraction b was subjected to column chromatography on silica gel and gave 14-deoxy-11,12-dihydroandrographolide ( 2 ) ( 264.6  mg , ch2cl2/meoh , 98  :  2 ) and 14-deoxyandrographolide ( 3 ) ( 352.8  mg , ch2cl2/meoh , 99  :  1 ) .
subfractions a , c , and d underwent further isolation by column chromatography analyses over silica gel .
sub fraction c gave andrographolide ( 4 ) ( 882.3  mg , ch2cl2/meoh , 96  :  4 ) , sub fraction d gave neoandrographolide ( 5 ) ( 44.1  mg , ch2cl2/meoh , 90  :  10 ) , and sub fraction a gave 14-deoxy-12- hydroxyandrographolide ( 1 ) ( 176.4  mg , ch2cl2/meoh , 97  :  3 ) .
the structure of each compound was confirmed by comparison of its nmr , ir , uv , and ms data with literature values .
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 . 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 ( 37c ) was pumped to flow at a constant rate ( 10  ml / min ) by using a peristaltic pump ( compact drive masterflex ) , and the gas ( 95% o2 and 5% co2 ) was allowed to pass through this solution .
the krebs - henseleit solution contained the following : nacl 118  mm , d - glucose 11.6  mm , nahco3 25  mm , kcl 4.7  mm , kh2po4 1.2  mm , mgso4 1.2  mm , and cacl2 1.23  mm .
sprague dawley rats ( 250300  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 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  l 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 .
p values less than 0.05 were considered to be significant ( * p < 0.05 ; * * p < 0.01 ; * * * p < 0.001 ) .
the dcm extract of a. paniculata   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 .
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 ) , 14-deoxy-11,12-dihydroandrographolide ( 2 ) ( trans isomer ) , 14-deoxyandrographolide ( 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 .
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 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 ) .
many studies have been reported on the cardiovascular activity of a. paniculata [ 2028 ] and on other pharmacological aspects [ 319 ] .
however , no cardiovascular activity study involving a direct effect on the isolated hearts of rats has been published previously . in this current study ,
the dcm crude extract showed potent cardiovascular activity on the isolated perfused rat heart , except that it did not affect cardiac contractility .
fractionation and purification of the dcm extract led to the isolation of five active compounds ( 15 ) , with 14-deoxy-11,12-dihydroandrographolide ( 2 ) and 14-deoxyandrographolide ( 3 ) showing the largest reduction in coronary perfusion pressure ( 18.96  6.19 and 19.5  7.00  mm hg , resp . ) ( p < 0.05 ) .
the results from our study support the previous finding by zhang and tan   that showed 14-deoxy-11,12-dihydroandrographolide ( 2 ) and 14-deoxyandrographolide ( 3 ) caused significant falls in mean arterial pressure ( map ) in the hearts of anaesthetized rats .
this observation suggests that compound / s other than compounds 15 may be responsible for this activity .
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
another mechanism involved in the cardiovascular activity of 14-deoxy-11,12-dihydroandrographolide ( 2 ) and 14-deoxyandrographolide ( 3 ) has been reported [ 2224 , 26 ] .
the ability of a. paniculata extract to dilate coronary vessels has not been reported before , but both 14-deoxy-11,12-dihydroandrographolide ( 2 ) and 14-deoxyandrographolide ( 3 ) have been reported to dilate aortic rings .
both compounds exert their vasorelaxant activity by the release of nitric oxide ( no ) and activation of the guanylate cyclase pathway , as well as the blockade of ca influx through both voltage- and receptor - operated ca channels .
in conclusion , the ability of 14-deoxy-11,12-dihydroandrographolide ( 2 ) and 14-deoxyandrographolide ( 3 ) to dilate coronary vessels and aortic rings   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 .
 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 . 
1. Introduction
2. Materials and Method
3. Results and Discussion
4. Conclusions