it is projected that the circulatory system disease in india would rise by 103% in man and 90% in women during the period 1985 to 2015 .
the treatment of cardiovascular diseases with allopathic drugs causes moderate to severe adverse events which could cause death .
ayurvadic system of medicine is practised in india , but the scientific evidence for treating diseases with herbs and fixed herbal formulations are poorly documented .
the marketed polyherbal formulation triglize claims that it can be used for the treatment of obesity , hypertension , palpitation , myocardial necrosis , coronary atherosclerosis , hypercholesterol , ischemic heart diseases and peripheral vascular diseases .
diuretics and antioxidants play a role in the treatment of hypertension and ischemic heart disease .
so , the present study was planned to determine the free radical scavenging property of triglize , a polyherbal formulation ( phf ) , and its diuretic effect using in vitro and in vivo models , respectively .
triglize ( batch no . : 13335002 , mfg date : jun , 2005 , apex laboratories ltd . , chennai ) is a marketed soft gelatin capsule and it was formulated using aqueous extracts of terminalia arjuna , cissus quadrangularis , boerhaavia diffusa , commiphora mukul , phyllanthus embilica , terminalia bellirica , terminalia chebula , tribulus terrestris , allium sativum and trigonella foenumgraecum .
healthy , adult , male albino rats of wistar strain , weighing 170  200 g were obtained from animal house , j.s.s .
the rats were housed under 22  2 c temperature , 40 - 60 % humidity and 12:12  1 h light dark cycle .
the study was approved by the institute animal ethics committee and all the animal experiments were carried out as per cpcsea guidelines .
the antioxidant activity of test compounds was assessed on the basis of the radical scavenging effects of greiss ilosvay reaction .
the aqueous extract of ployherbal formulation ( phf ) and the known antioxidant ascorbic acid were dissolved in dmso ( dimethyl sulfoxide ) ( sigma - aldrich ) separately and used for the in vitro antioxidant testing by dpph ( 2 , 2-diphenyl-1-picrylhydrazyl ) ( sigma - aldrich ) method .
the assay was carried out in a 96 well microtiter plate . to 200 l of dpph solution ,
10 l of each of the test and standard solutions were added separately in wells of the 96 well microtiter plates .
the final concentration of the test , standard solution used was 15.62 to 1000.00 g/ ml .
the plates were incubated at 37 c for 30 min and the absorbance of each was measured at 490 nm using elisa reader against the corresponding test and standard blanks and the remaining dpph was calculated .
ic50 the concentration of the sample to scavenge 50 % of dpph free radicals was calculated and recorded .
male wistar rat ( 200 g ) peritoneal macrophages cells were induced using 3% w / v sodium thioglycolate ( i.p . ) .
after 3 days , the macrophages cells were collected , washed with phosphate buffer saline and resuspended with rpmi 1640 medium ( fisher chemicals ) , supplemented with nahco 3 ( 2 g / i ) , penicillin ( 100 iu / l ) , streptomycin ( 100 g / ml ) , gentamicin ( 20 g / ml ) and fetal calf serum ( 10% ) and plated in 24 wells microtiter plate .
equal amounts of the macrophages cells ( 2.6  10/well ) were incubated in humidified 5% co2 at 37 c for 24 h to allow macrophages adherence and the plate was washed 3 - 4 times with serum free fresh medium to dislodge the non - adherent cells .
the macrophages were cultured for 3 h and incubated with lps 25 ng / ml along with five different concentrations of phf at the concentration of 3 g - 1000 g .
the aqueous extract of phf ( dissolved in dmso ) was added 2 - 5 min to the cells before incubation of cell with lps 25 ng / ml .
finally free nitrites were estimated using greiss reagents and the cell viability was calculated using the mtp ( microculture tetrazolium ) assay method .
thirty - six male wistar rats ( 170 - 190 g ) were divided into six groups containing six animals and grouped as follows : group i - control ( 0.5% w / v cmc ) group ii - standard drug treatment ( furosemide- 5mg / kg ) group iii - phf- 12.5 mg / kg group iv - phf- 50 mg / kg group v - phf- 200 mg / kg group vi - phf- 400 mg / kg seventeen to twenty - four hours prior to the experiment , food and water were withdrawn and animals were placed individually in a metabolic cage .
the aqueous extract of phf at various concentrations like 12.5 , 50 , 200 , and 400 mg / kg and reference standard furosemide ( 5 mg / kg ) made as suspension with 0.5% w / v carboxymethyl cellulose ( cmc ) and administered orally as a single dose . additionally , 5 ml of 0.9% w / v nacl solution per 100 g body weight was given by oral gauge .
urine was collected 0- 5 and 5 - 24 h of dosing and urine volume , acidity ( ph ) was measured immediately .
the sodium , potassium content of urine was determined by flame photometry and concentration of chloride was determined by argentometric titration .
significant difference between groups was determined using one - way anova followed by tukey 's multiple comparison test . a p value less than 0.05 was considered to be significant .
triglize ( batch no . : 13335002 , mfg date : jun , 2005 , apex laboratories ltd . , chennai ) is a marketed soft gelatin capsule and it was formulated using aqueous extracts of terminalia arjuna , cissus quadrangularis , boerhaavia diffusa , commiphora mukul , phyllanthus embilica , terminalia bellirica , terminalia chebula , tribulus terrestris , allium sativum and trigonella foenumgraecum .
healthy , adult , male albino rats of wistar strain , weighing 170  200 g were obtained from animal house , j.s.s .
the rats were housed under 22  2 c temperature , 40 - 60 % humidity and 12:12  1 h light dark cycle .
the study was approved by the institute animal ethics committee and all the animal experiments were carried out as per cpcsea guidelines .
the antioxidant activity of test compounds was assessed on the basis of the radical scavenging effects of greiss ilosvay reaction .
the aqueous extract of ployherbal formulation ( phf ) and the known antioxidant ascorbic acid were dissolved in dmso ( dimethyl sulfoxide ) ( sigma - aldrich ) separately and used for the in vitro antioxidant testing by dpph ( 2 , 2-diphenyl-1-picrylhydrazyl ) ( sigma - aldrich ) method .
the assay was carried out in a 96 well microtiter plate . to 200 l of dpph solution ,
10 l of each of the test and standard solutions were added separately in wells of the 96 well microtiter plates .
the final concentration of the test , standard solution used was 15.62 to 1000.00 g/ ml .
the plates were incubated at 37 c for 30 min and the absorbance of each was measured at 490 nm using elisa reader against the corresponding test and standard blanks and the remaining dpph was calculated .
ic50 the concentration of the sample to scavenge 50 % of dpph free radicals was calculated and recorded .
male wistar rat ( 200 g ) peritoneal macrophages cells were induced using 3% w / v sodium thioglycolate ( i.p . ) . after 3 days
, the macrophages cells were collected , washed with phosphate buffer saline and resuspended with rpmi 1640 medium ( fisher chemicals ) , supplemented with nahco 3 ( 2 g / i ) , penicillin ( 100 iu / l ) , streptomycin ( 100 g / ml ) , gentamicin ( 20 g / ml ) and fetal calf serum ( 10% ) and plated in 24 wells microtiter plate .
equal amounts of the macrophages cells ( 2.6  10/well ) were incubated in humidified 5% co2 at 37 c for 24 h to allow macrophages adherence and the plate was washed 3 - 4 times with serum free fresh medium to dislodge the non - adherent cells .
the macrophages were cultured for 3 h and incubated with lps 25 ng / ml along with five different concentrations of phf at the concentration of 3 g - 1000 g .
the aqueous extract of phf ( dissolved in dmso ) was added 2 - 5 min to the cells before incubation of cell with lps 25 ng / ml .
finally free nitrites were estimated using greiss reagents and the cell viability was calculated using the mtp ( microculture tetrazolium ) assay method .
thirty - six male wistar rats ( 170 - 190 g ) were divided into six groups containing six animals and grouped as follows : group i - control ( 0.5% w / v cmc ) group ii - standard drug treatment ( furosemide- 5mg / kg ) group iii - phf- 12.5 mg / kg group iv - phf- 50 mg / kg group v - phf- 200 mg / kg group vi - phf- 400 mg / kg seventeen to twenty - four hours prior to the experiment , food and water were withdrawn and animals were placed individually in a metabolic cage .
the aqueous extract of phf at various concentrations like 12.5 , 50 , 200 , and 400 mg / kg and reference standard furosemide ( 5 mg / kg ) made as suspension with 0.5% w / v carboxymethyl cellulose ( cmc ) and administered orally as a single dose . additionally , 5 ml of 0.9% w / v nacl solution per 100 g body weight was given by oral gauge .
urine was collected 0- 5 and 5 - 24 h of dosing and urine volume , acidity ( ph ) was measured immediately .
the sodium , potassium content of urine was determined by flame photometry and concentration of chloride was determined by argentometric titration .
the aqueous extract of phf showed a moderate antioxidant activity by dpph method with an ic50 value of 219.22  1.64 mg / ml when compared to standard ascorbic acid ( ic50 = 64.33  0.91 ) . in vitro results indicated that thioglycolate activated macrophages were hypersensitive to lps and produced no between 55 - 59 m/ 2.610 cells , whereas normal cells produced no between 17 - 22 m/2.610 cells under similar conditions .
no production was significantly inhibited by simultaneous and pre - incubation with phf aqueous extract in a dose dependent manner [ table 1 ] .
effect of phf on lps induced no production and cell viability on thioglycolate induced rat macrophages a summary of the diuretic activity is presented in tables 2 and 3 .
the results showed significant increase in urine volume in 0 - 5 h and 5 - 24 h. the percentage differences in urine volume in treatment group at the dose levels of 50 , 200 and 400 mg / kg b.wt were 115.87% , 129.03% and 151.61% for 0 - 5 h and 106.4% , 108.0% and 146.4% for 5 - 24 h when compared to control .
effect of phf on urine volume , ph and electrolytes ( 0 - 5 hour urine biochemical estimation ) effect of phf on urine volume , ph and electrolytes ( 5 - 24 hour urine biochemical estimation ) excretion of potassium ion in urine significantly increased with furosemide 5 mg / kg and phf 50 , 200 and 400 mg / kg ( p < 0.01 ) .
the percentage changes in excretion of potassium ion with phf 50 , 200 and 400 mg / kg were 52.63% , 47.36% and 57.87% for 0 - 5 h urine sample and 40.90% , 45.45% , and 54.45% for 5 - 24 h urine sample as compared to control .
phf showed 15% significant reduction in chloride ion excretion at dose levels of 50 and 400 mg / kg at 0 - 5 h urine sample ( p < 0.05 ) , but there is no significant reduction of chloride ion concentration in phf treated animals at 5 - 24 h urine sample .
the results of present study suggest that phf has free radical scavenging property and diuretic activity . however , the free radical scavenging property or antioxidant property of phf is moderate when compared to ascorbic acid while the diuretic activity is comparable to frusemide .
free radicals are formed in the cells as a consequence of both oxidative biochemical reaction and have been implicated in the pathogenesis of wide variety of clinical disorders , resulting from deficient natural antioxidant defences .
phf possesses a series of substituted phenolic compounds and variety of flavones which have known antioxidant property .
this is the reason why it significantly inhibited no production in a dose - dependent manner in rat peritoneal macrophage cells in our study .
phf exhibits diuretic activity at the dose levels of 50 , 200 and 400 mg / kg and the effect is comparable to furosemide ( 10 mg / kg ) .
since phf exhibits significant diuretic activity , it may be useful in the management of vascular and cardiac diseases .
though the in vitro results can be extrapolated to in vivo system , in the present study , the free radical scavenging property of phf was studied only for no free radical . in a living system
, the oxidative reaction can occur through many mediators/ toxins and their inducing superoxide and hydroxyl free radicals .
hence phf should be ideally tested in an in vivo system to confirm its free radical scavenging property and what mediators are involved .
as there are no published reports on phf , the results of our study could not be compared .
it has been shown that some of the ingredients in the phf have antioxidant and diuretic properties.[1315 ]
phf exhibits significant antioxidant effect in vitro and diuretic effect in vivo . since it is currently approved for use in humans ( as an ayurvedic medicine ) , it is worthwhile to explore the usefulness of the phf in the treatment of diseases of cardiovascular system such as hypertension and cardiac failure .
 objective : to determine the in vitro free radical scavenging property and in vivo diuretic effect of triglize , a marketed polyherbal formulation in experimental models.materials and methods : the aqueous extract of polyherbal formulation ( phf ) triglize was used for the experiment . 
 the free radical scavenging property and antioxidant effect of phf were studied by lps - induced free radicals in rat macrophages cells and dpph ( 2 , 2-diphenyl-1-picrylhydrazyl ) methods , respectively . 
 the diuretic effect of a phf was studied with lipschitz model using male wistar rats.results:phf significantly inhibited lipopolysaccharide -induced free radicals in rat macrophages and it showed moderate antioxidant potential in dpph model . 
 polyherbal formulation at 50 , 200 and 400 mg/ kg significantly increased potassium excretion in urine at 0 - 5 h and 5 - 24 h. the diuretic effect of phf was as similar as furosemide.conclusion : the phf has significant diuretic effect and free radical scavenging properties . 
INTRODUCTION
MATERIALS AND METHODS
Polyherbal formulation
Animals
None
Effect of PHF on lipopolysaccharide (LPS)-induced free radicals in rat peritoneal macrophage cells
Diuretic effect of PHF
Statistical analysis
RESULTS
DISCUSSION
CONCLUSION