hypertension is associated with increase of endothelial dysfunction which has been demonstrated in vessels from hypertensive humans and in many experimental models of hypertension ( 1 ) . it serves as a sensor of hemodynamic and humoral variables and a transducer of signals to subjacent vascular smooth muscle cells ( smc ) . subsequently , the alterations of smc growth , migration , differentiation , death , and ecm modifications are responsible for the resulting vascular remodeling ( 2 ) . circulating endothelial cells ( cecs ) are mature cells that are not associated with vessel walls , but are released from vascular wall and circulate within peripheral blood . the presence of cec in the blood has been found to be increased in response to various diseases ( 3 ) . indeed , the level of cecs has been recognized as a useful biomarker for vascular damage . the average lifespan of epcs was recently reported to be shortened by oxidative stress and regulated by anti - oxidative mechanisms ( 8) . the dysfunction of epcs was clearly correlated with vascular injury in the case of various risk factors such as hypertension ( 9 ) . flow cytometry analysis did not show any effects of increasing blood pressure on the number of circulating epcs , which had been measured by several markers ( 4 ) . scurrula atropurpurea ( bl . ) dans . is a parasitic tea plant . in indonesia , especially on the island of java , the stems and leaves of this plant have been empirically used for therapeutic applications . as far we know , there is no study to evaluate the effects of s. atropurpurea ( bl . ) dans . on nitric oxide , therefore , this study aimed to investigate whether methanolic extract of s. atropurpurea ( bl . ) dans . is able to modulate total plasma nitrate / nitrite levels , decrease endothelial damage , and increase endothelial progenitor cells in hypertensive rats . prior to the experiment , the characteristic of botanical determination of the leaves was performed at the indonesian scientific institute ( lipi ) at purwodadi , pasuruan , east java . one hundred milligrams of dry leaf powder was steeped in methanol in a 1000 ml - erlenmeyer flask . the supernatant ; a mixture of methanol and the active constituents , was subjected to evaporation . the methanolic extract of s. atropurpurea ( mesa ) was administered daily by the oral gavage ( 2 ml ) using a catheter for 6 weeks . twenty five male wistar rats , aged 3 - 5 months and weighing 250 - 300 g. the rats were injected subcutaneously with deoxycorticosterone acetate ( doca ) ( sigma aldrich , pte ltd . singapore ) at a dosage of 10 mg / kgbw , 2 times weekly for 6 weeks . the treatment groups consist of the control group , the group of non - mesa hypertensive rats , three groups of hypertensive rats were received mesa at dosages of 50 , 100 , and 200 mg / kgbw ( 6 ) . after 6 weeks of doca with or without mesa treatment , the blood pressure of the rats was determined . total plasma nitrate / nitrite levels were determined indirectly as its metabolic products ( nitrate + nitrite ions ) spectrophotometrically using a test kit ( boeringher , usa ) in which all the nitrate ions in serum were first reduced to nitrite ions by nitrate reductase followed by the reaction between nitrite ions and the greiss reagent ( 0.1% naphthylethylenediamine dihydrochloride in distilled water and 1% sulfanilamide in 5% h3po4 ) to form a blue color solution . the levels of nitric oxide in all groups were determined by extrapolation from absorbance - concentration curve of the sodium nitrate standard solution ( 10100 m ) ( 7 ) . endothelial progenitor cells and circulating endothelial cells were isolated from peripheral blood according to previous studies with modification . ten ml of venous blood with ethylenediaminetetraacetic acid ( edta ) was obtained by peripheral veinpuncture , stored at 4c to 10c , and processed within 6 hr after collection . in order to isolate mononuclear cells , we performed density - gradient centrifugation using ficoll - paque plus ( amercontrol pharmacia biotech , uppsala , sweden ) . after that , the isolated cells were washed twice with pbs and resuspended in 20 ml of pbs supplemented with 0.5% of bovine serum albumin and 2 mm of edta . for endothelial progenitor cells measurement , cd133 + cells in peripheral blood were evaluated by immunostaining with pe - conjugated cd133 monoclonal antibody ( biolegend ) and detected by flow cytometry ( bd facs calibur flow cytometer ) ( 8) . for circulating endothelial cells , cd146 + cells in peripheral blood were evaluated by immunostaining with fitc - conjugated cd146 + monoclonal antibody ( biolegend ) and detected by flow cytometry ( bd facscalibur flow cytometer ) ( 9 ) . the animal handling and details experimental procedures were evaluated and approved by the ethics committee of faculty of medicine , university of brawijaya , malang , east java , indonesia . data are presented as meansd and the differences between groups were analyzed using one - way anova that was performed with spss 16.0 statistical package . post hoc test was used if probability values of p<0.05 , were considered statistically significant . prior to the experiment , the characteristic of botanical determination of the leaves was performed at the indonesian scientific institute ( lipi ) at purwodadi , pasuruan , east java . one hundred milligrams of dry leaf powder was steeped in methanol in a 1000 ml - erlenmeyer flask . the supernatant ; a mixture of methanol and the active constituents , was subjected to evaporation . the methanolic extract of s. atropurpurea ( mesa ) was administered daily by the oral gavage ( 2 ml ) using a catheter for 6 weeks . twenty five male wistar rats , aged 3 - 5 months and weighing 250 - 300 g. the rats were injected subcutaneously with deoxycorticosterone acetate ( doca ) ( sigma aldrich , pte ltd . singapore ) at a dosage of 10 mg / kgbw , 2 times weekly for 6 weeks . the treatment groups consist of the control group , the group of non - mesa hypertensive rats , three groups of hypertensive rats were received mesa at dosages of 50 , 100 , and 200 mg / kgbw ( 6 ) . after 6 weeks of doca with or without mesa treatment , the blood pressure of the rats was determined . total plasma nitrate / nitrite levels were determined indirectly as its metabolic products ( nitrate + nitrite ions ) spectrophotometrically using a test kit ( boeringher , usa ) in which all the nitrate ions in serum were first reduced to nitrite ions by nitrate reductase followed by the reaction between nitrite ions and the greiss reagent ( 0.1% naphthylethylenediamine dihydrochloride in distilled water and 1% sulfanilamide in 5% h3po4 ) to form a blue color solution . the levels of nitric oxide in all groups were determined by extrapolation from absorbance - concentration curve of the sodium nitrate standard solution ( 10100 m ) ( 7 ) . endothelial progenitor cells and circulating endothelial cells were isolated from peripheral blood according to previous studies with modification . ten ml of venous blood with ethylenediaminetetraacetic acid ( edta ) was obtained by peripheral veinpuncture , stored at 4c to 10c , and processed within 6 hr after collection . in order to isolate mononuclear cells , we performed density - gradient centrifugation using ficoll - paque plus ( amercontrol pharmacia biotech , uppsala , sweden ) . after that , the isolated cells were washed twice with pbs and resuspended in 20 ml of pbs supplemented with 0.5% of bovine serum albumin and 2 mm of edta . for endothelial progenitor cells measurement , cd133 + cells in peripheral blood were evaluated by immunostaining with pe - conjugated cd133 monoclonal antibody ( biolegend ) and detected by flow cytometry ( bd facs calibur flow cytometer ) ( 8) . for circulating endothelial cells , cd146 + cells in peripheral blood were evaluated by immunostaining with fitc - conjugated cd146 + monoclonal antibody ( biolegend ) and detected by flow cytometry ( bd facscalibur flow cytometer ) ( 9 ) . the animal handling and details experimental procedures were evaluated and approved by the ethics committee of faculty of medicine , university of brawijaya , malang , east java , indonesia . data are presented as meansd and the differences between groups were analyzed using one - way anova that was performed with spss 16.0 statistical package . post hoc test was used if probability values of p<0.05 , were considered statistically significant . the total plasma nitrate / nitrite levels were significantly ( p<0.05 ) decreased in doca - salt hypertensive rats compared to control group . the administration of mesa100 and mesa200 elevated the total plasma nitrate / nitrite levels but can not reach the level in control group . the administration of doca salt affected the endothelial progenitor cells number , as shown in figure 1 . endothelial progenitor cells level were significantly ( p<0.05 ) decreased in doca - salt hypertensive rats compared to control group . the administration of mesa100 or mesa200 significantly ( p<0.05 ) elevated the endothelial progenitor cell number compared with hypertensive group . the level of endothelial progenitor cells in mesa200 is not significantly different than that of control group ( p > 0.05 ) . the administration of doca salt affected the circulating endothelial cell number , as shown in figure 2 . circulating endothelial cell levels were significantly ( p<0.05 ) increased in hypertensive rats compared to control group . the administration of mesa significantly ( p<0.05 ) decreased the circulating endothelial cells number compared to hypertensive groups . the total plasma nitrate / nitrite levels were significantly ( p<0.05 ) decreased in doca - salt hypertensive rats compared to control group . the administration of mesa100 and mesa200 elevated the total plasma nitrate / nitrite levels but can not reach the level in control group . the administration of doca salt affected the endothelial progenitor cells number , as shown in figure 1 . endothelial progenitor cells level were significantly ( p<0.05 ) decreased in doca - salt hypertensive rats compared to control group . the administration of mesa100 or mesa200 significantly ( p<0.05 ) elevated the endothelial progenitor cell number compared with hypertensive group . the level of endothelial progenitor cells in mesa200 is not significantly different than that of control group ( p > 0.05 ) . the administration of doca salt affected the circulating endothelial cell number , as shown in figure 2 . circulating endothelial cell levels were significantly ( p<0.05 ) increased in hypertensive rats compared to control group . the administration of mesa significantly ( p<0.05 ) decreased the circulating endothelial cells number compared to hypertensive groups . in this study we found that total plasma nitrate / nitrite levels in hypertensive animals was lower than normotensive group . higher production of reactive oxygen species which accompanied by low expression of endothelial no synthase , and/or impaired l - arginine uptake are different mechanisms for decreased no bioavailability in high blood pressure . the administration of mesa100 and mesa200 elevated the total plasma nitrate / nitrite levels but can not reach the level in control group , maybe due to reactive oxygen species - lowering effect . the ability of mesa to modulate total plasma nitrate / nitrite levels is due to its active constituents , among others flavonol glycosides ( quercetin and rutin ) , monoterpene glucosides ( icariside b ) , lignan glycosides ( aviculin ) , flavans ( catechin , epicatechin , epicatechin-3 - 0-gallate , epigallocatechin-3 - 0-gallate , gallocatechin , and epigallocatechin ) . catechin increases enos phosphorylation and no bioavailability by inhibition of nadph oxidase ( 11 ) . in this study , there were significantly ( p<0.05 ) increased circulating endothelial cells levels in hypertensive rats compared to control group . endothelial progenitor cells level were significantly ( p<0.05 ) decreased in doca - salt hypertensive rats compared to control group . all doses administration of mesa significantly ( p<0.05 ) decreased the circulating endothelial cells number compared to hypertensive groups . the administration of mesa100 and mesa200 elevated the endothelial progenitor cell number than that of hypertensive group ( p<0.05 ) . this finding is consistent with previous studies that epcs decreased in hypertensive rats ( 12 ) . the bioactive compound from s. atropurpurea is able to diminish endothelial damage via increasing endothelial progenitor cells . besides , total plasma nitrate / nitrite levels - increasing effect of s. atropurpurea may contribute to endothelial progenitor cells differentiation ( 13 - 15 ) methanolic extract of s. atropurpurea modulate the total plasma nitrate / nitrite levels and diminish endothelial damage via increasing endothelial progenitor cells .
objective(s):to know whether scurrula atropurpurea is able to modulate total plasma nitrate / nitrite levels , decrease endothelial damage , and increase endothelial progenitor cells ( epcs ) in hypertensive rats.materials and methods : the rats were divided in 5 groups : control ( normotensive ) group , desoxy cortico sterone ( doca)-salt hypertensive group , and three doca - salt hypertensive groups . 
 all 5 groups received methanolic extract of s. atropurpurea ( mesa ) at a dosage of 50 ; 100 ; and 200 mg / kgbw . 
 serum nitric oxide ( no ) was assayed by colorimetric . 
 circulating endothelial cells ( cecs ) and epcs were assayed using flow cytometry.results:the administration of mesa100 and mesa200 elevated the total plasma nitrate / nitrite levels but can not reach the level in control group . mesa100 and mesa200 also elevated the epcs number compared with hypertensive group . the administration of mesa significantly ( p < 0.05 ) decreased the cecs number compared to hypertensive groups.conclusion:methanolic extract of s. atropurpurea is able to modulate total plasma nitrate / nitrite levels and diminish endothelial damage via increasing epcs .