inflammation is one of the self - protective abilities against tissue injury , and it has clinical symptoms like pain , heat , redness , and loss of function . if severe inflammation occurs , organs and cells may be destroyed , and changes such as decomposition of proteins and suppression of dna synthesis may be caused by oxidative stress . salviae miltiorrhizae radix is obtained from the dried root rhizome of salvia miltiorrhiza bunge , which belongs to the labiatae family as a perennial herb . usually , it is gathered in spring and autumn and is washed and dried before use . its effects , such dispelling blood - activated stasis , controlling menstrual pain , blood - culturing mind relaxation , and dispelling blood - cooling furunculus , are reported in the literature on korean medicine . the study of salviae miltiorrhizae radix has been advanced with subjects , and it may have an effect on cell inflammation [ 4 - 6 ] , injured cells and muscles [ 7 - 9 ] , and tumor cells . therefore , we surmise that it may have anti - inflammation , antioxidant efficacy , but sufficient evidence supporting this hypothesis does not exist . thus , to investigate the anti - inflammation , antioxidant effect of salviae miltiorrhizae radix hot aqueous extract , we designed an experiment to study its influence on no ( nitric oxide ) and pge2 ( prostaglandin e2 ) production and on dpph ( 1,1-diphenyl-2-picryl hydrazyl ) radical scavenging in lps ( lipopolysaccharide)-induced macrophages . for experimentation , we purchased salviae miltiorrhizae radix from hmax co. , ltd . , of south korea . for preparing the hot aqueous extract , first , we extracted 300 g of salviae miltiorrhizae radix in 2 l of distilled water at 100 for 4 hours . next , the volume of the filtered extract was reduced to 100 ml by using a rotary evaporator ( korprotech , korea ) ; then , the extract was frozen at 80. lastly , the reduced - volume extract was freeze - dried by using a freeze - drying system ( labconco , usa ) for 7 days . the raw 264.7 macrophages that were used in this experiment were parceled out from atcc : the global bioresource center ( manassas , usa ) . afterward , we cultured the cells by using dulbecco s modified eagle s medium ( dmem ) including 10% fetal bovine serum ( fbs ) and 1% antibiotic ( 100-u / ml penicillin and 100-/ml streptomycin : gibco ) . the macrophages were kept in the culture medium at 37 , with 5% co2 sustained . we measured the cell viability by using 3-(4 , 5-dimrthylthiazol-2-yl)-2 , 5-diphenyltetrazolium bromide ( mtt ) assays . first , a 96-well plate that included 110 cells in each well was stabilized in culture medium at 37 , with 5% co2 sustained . we cultured stabilized macrophages by using salviae miltiorrhizae radix hot aqueous extracts with 1- , 5- , 25- , 125- and 625-/ml concentrations for 16 hours , after which we cultivated them with mtt reagent for 2 hours . we measured the absorbance at 570  with formazan dissolved in dmso ( dimethyl sulfoxide ) after having removed the supernatant liquid . a 96-well plate that included 110 cells in each well we cultured stabilized macrophages by using 10- /ml lps and salviae miltiorrhizae radix hot aqueous extracts with 1- , 5- , 25- , 125- and 625-/ml concentrations for 16 hours . then , we measured the absorbance at 540  of a mixture of 100- supernatant liquid and 100-  griess reagent . we made the griess reagent with 0.1% naphthylethylenediamine dihydrochloride ( 50  ) and 1% sulfanilamide ( 50  ) dissolved in 5% h3 po4 . we measured the pge2 concentration by using commercial competitive enzyme immunoassay kits purchased from r&d systems ( minneapolis , usa ) . a 96-well plate that included 110 cells in each well we cultured stabilized macrophages by using 10-/ml lps and salviae miltiorrhizae radix hot aqueous extracts with 1- , 5- , 25- , 125- and 625-/ml concentrations for 18 hours , and we used cultured supernatant liquid for measuring the pge2 concentration . we first loaded cultured liquid ( 100  ) into a 96-well plate ; then , we coated the plate with goat anti - mouse antibody . finally , the plate was kept overnight at 4 after the primary antibody solution ( 50  ) and the pge2 conjugate ( 50  ) had been added . we measured the absorbance at 450  after having added a substrate solution ( 200  ) for 5 - 20 minutes . a 96-well plate that included 110 cells in each well was stabilized in culture medium at 37 , with 5% co2 sustained for 16 hours . at first , we diluted salviae miltiorrhizae radix hot aqueous extracts with 1- , 5- , 25- , 125- and 625-/ml concentrations with methanol mixed with 0.15-mm dpph ( sigma , usa ) and filled a 96-well plate with 80  of the diluted salviae miltiorrhizae radix hot aqueous extract . after the specimen had reacted for 30 minutes with blocking light at room temperature , we measured the absorbance at 520  by using a microplate reader ( tecan , italy ) . the dpph radical - scavenging capability was evaluated by using the following mathematical formula : we used the spss windows program ( ver . when the cell viability of the group not treated with the extract was set at 1.00  0.01 , the cell viabilities for the groups treated with 1- , 5- , 25- , 125- and 625-/ml salviae miltiorrhizae radix hot aqueous extract were 1.00  0.03 , 0.97  0.02 , 0.96  0.01 , 0.89  0.01 and 0.80  0.02 , respectively . because these differences were not statistically significant , we can conclude that salviae miltiorrhizae radix hot aqueous extract showed no significant cell cytotoxicity at any concentration used in this experiment ( fig . 1 ) . the no production of the control group , the group treated solely with 10-/ml lps , was 74.49  4.64 /ml whereas the no productions for the groups treated with 10-/ml lps and 1- , 5- , 25- , 125- , and 625-/ml salviae miltiorrhizae radix hot aqueous extract were 71.86  4.31 , 70.85  2.38 , 68.11  2.80 , 65.79  2.96 and the values of the no production for the groups treated with 125- and 625-/ml salviae miltiorrhizae radix hot aqueous extract were significantly lower than that of the control group ( fig . pge2 production of the control group , the group treated with 10-/ml lps only , was 31.52  7.68 pg / well , but its values for the groups treated with 10-/ml lps and 1- , 5- , 25- , 125- , and 625-/ml salviae miltiorrhizae radix hot aqueous extract were 337.37  7.68 , 321.19  4.04 , 304.24  8.01 , 276.64  12.22 and 188.66  9.58 thus , the pge2 production values for the groups treated with 25- , 125- , and 625-/ml salviae miltiorrhizae radix hot aqueous extract were significantly lower than the value for the control group ( fig . * statistically significant difference from the normal group , as determined by using the student s t - test as p < 0.05 . * statistically significant difference from the control group , as determined by the student s t - test as p < 0.05 . * statistically significant difference from the control group , as determined by using the student s t - test as p < 0.05 . the dpph radical - scavenging capabilities of the groups treated with 1- , 5- , 25- , 125- , and 625-/ml salviae miltiorrhizae radix hot aqueous extract were measured as 5.58  0.83 , 11.71  1.35 , 37.84  1.52 , 50.23  3.11 and 87.59  2.58 percent , respectively . thus , the values for the groups treated with 125- and 625-/ml salviae miltiorrhizae radix hot aqueous extract were higher than 50% ( fig . the nature of salviae miltiorrhizae radix is a little cold and bitter , and it affects the heart and liver channel . in korean medicine literature , salviae miltiorrhizae radix is reported to have the effects of dispelling blood - activated stasis , controlling menstrual pain , blood - culturing mind relaxation , and dispelling blood - cooling furunculus . thus , salviae miltiorrhizae radix has been used to treat menstrual irregularity , dysmenorrhea , amenorrhea , disorders after delivery , cardialgia , myoma uteri , contusions , anxiety , insomnia and ulcers . the study of salviae miltiorrhizae radix has been advanced with subjects , and it may have an effect on inflammations such as hepatitis , pancreatitis and cancer [ 11 - 18 ] . inflammation is an immune response by activated immunocytes . when macrophages are stimulated by using lps of a gram - negative bacterium , virus , microbe , et cetera , an inflammatory factor is activated to protect the host from the bacterium . however , if pro - inflammatory cytokines , such as il-6 , il-1 , tnf- , no and pge2 activated from macrophages by using lps , are overproduced , the inflammation response continues , finally causing arterioles , capillaries and veinlets to dilate , plasma to exude , and leukocytes to move to inflamed sites . when they are unbalanced and oxidative stress occurs , organs and cells may be harmed . oxidative stress radicals are reactive oxygen species ( ros ) and reactive nitrogen species ( rns ) , such as superoxide ( o2 - ) , nitric oxide ( no ) , hydroxyl radical ( ho ) , singlet oxygen ( o2 ) and hydrogen peroxide ( h2 o2 ) . no is a free radical related with inflammation and is a transporter of the cardiovascular system , the nervous system , and the immune system . its maintains homeostasis , transports neurotransmitters , serves as an anticancer substance , controls blood pressure and serves as an antibiotic , but excessive no causes vasodilation , cytotoxicity and tissue damage . dpph , which is used for measuring antioxidant activity , is a very stable free radical and violet compound presenting specific absorption at 517 . the dpph radical is very stable in an organic solvent such as alcohol , and discoloration by a proton - radical scavenger can easily be seen , so we used the dpph free radical to measure antioxidant activity . generally , an unstable dpph free radical changes to a stable dpph - h by gaining an electron or an atom of hydrogen from the antioxidant . in this study we wanted to determine the anti - inflammation effect and the antioxidant activity of salviae miltiorrhizae radix . we subcultured the raw 264.7 macrophage , and in order to evaluate cytotoxicity , we performed mtt assays . also , we measured the no and the pge2 productions and the dpph free - radical scavenging capability . in yoon s study   and kim s study , salviae miltiorrhizae radix extract seems to have an anti - inflammation and pain - control effect . however , few studies have reported on the anti - inflammation effect and the antioxidant activity of salviae miltiorrhizae radix hot aqueous extracts , so in this study , we investigated the effects of salviae miltiorrhizae radix hot aqueous extracts . the cell viability of the group not treated with the extract was 1.00  0.01 whereas the cell viabilities of the groups treated with 1- , 5- , 25- , 125- and 625-/ml salviae miltiorrhizae radix hot aqueous extract were 1.00  0.03 , 0.97  0.02 , 0.96  0.01 , 0.89  0.01 and 0.80  0.02 , respectively . thus , we can conclude that no significant cell cytotoxicity was observed at any of the concentrations used in this study ( fig . 1 ) . no production of the control group , the group treated with 10-/ml lps only , was 74.49  4.64 /ml , but for the groups treated with 10-/ml lps and 1- , 5- , 25- , 125- , and 625-/ml salviae miltiorrhizae radix hot aqueous extract , the values were 71.86  4.31 , 70.85  2.38 , 68.11  2.80 , 65.79  2.96 and 14.93  4.00 /ml , respectively . thus , we can conclude that the values for the groups treated with 125- and 625-/ml salviae miltiorrhizae radix hot aqueous extract were significantly lower than the value for the control group ( fig . pge2 production of the control group , the group treated with 10-/ml lps only , was 331.52  7.68 pg / well , but for the groups treated with 10-/ml lps and 1- , 5- , 25- , 125- , and 625-/ml salviae miltiorrhizae radix hot aqueous extract , the values were measured to be 337.37  7.68 , 321.19  4.04 , 304.24  8.01 , 276.64  12.22 and 188.66  9.58 thus , we can conclude that the values for the groups treated with 25- , 125- , and 625-/ ml salviae miltiorrhizae radix hot aqueous extract were significantly lower than the value for the control group ( fig . the dpph radical - scavenging capabilities of the groups treated with 1- , 5- , 25- , 125- , and 625-/ml salviae miltiorrhizae radix hot aqueous extract were measured to be 5.58  0.83 , 11.71  1.35 , 37.84  1.52 , 50.23  3.11 and 87.59  2.58 percent , respectively . thus , the values for the groups treated with 125- and 625-/ml salviae miltiorrhizae radix hot aqueous extract were higher than 50% ( fig . we conclude that salviae miltiorrhizae radix hot aqueous extract suppresses the no and the pge2 productions and improves the dpph free - radical scavenging capability . thus , salviae miltiorrhizae radix hot aqueous extract seems to have an anti - inflammation effect and an antioxidant activity , and the best concentration of salviae miltiorrhizae radix hot aqueous extract appears to be 125/ml . more studies on cytotoxicity , the best effective concentration , and the method of extract , as well as more clinical trials , are needed . we investigated whether salviae miltiorrhizae radix hot aqueous extract could have an influence on no and pge2 production and on dpph radical - scavenging capability in lps - induced macrophages , and found the following results : in the groups treated with 1 , 5 , 25 , 125 , 625 /ml of salviae miltiorrhizae radix hot aqueous extract , no significant cell cytotoxicity was noted . in the groups treated with 125 and 625 /ml of salviae miltiorrhizae radix hot aqueous extract , no production was significantly decreased compared to that in the control group . in the groups treated with 25 , 125 , and 625 /ml of salviae miltiorrhizae radix hot aqueous extract , pge2 production was significantly decreased compared to that in the control group . in the groups treated with 125 and 625 /ml of salviae miltiorrhizae radix hot aqueous extract , high dpph radical - scavenging capabilities were noted . based on the results , we could conclude that salviae miltiorrhizae radix hot aqueous extract had no significant cell cytotoxicity at any of the concentrations used in this study , that it suppressed no and pge2 production , and that it had dpph radical - scavenging capability . this suggests that salviae miltiorrhizae radix hot aqueous extract may have an anti - inflammation effect and antioxidant activity .
objectives : the objective of this study is to investigate the effects of salviae miltiorrhizae radix hot aqueous extract on nitric oxide ( no ) and prostaglandin e2 ( pge2 ) production and on 1,1-diphenyl-2-picryl hydrazyl ( dpph ) free - radical scavenging in macrophages.methods : 
 salviae miltiorrhizae radix(300 g ) was heated at 100 with distilled water ( 2 l ) for 4 hours . 
 the extract was filtered and concentrated to 100 ml by using a rotary evaporator , was frozen at -80 , and was then freeze - dried by using a freezing - drying system . 
 the raw 264.7 macrophage was subcultured by using 10-/ml lipopolysaccharide ( lps ) . in order to evaluate cytotoxicity , we performed ( 3-(4,5-dimrthylthiazol-2-yl)-2,5-diphenyltetrazolium bromide ) ( mtt ) assays and measured the cell viability 
 . the no production was measured by using griess assays , and the pge2 production was measured by using enzyme immunoassays . 
 the antioxidant activity , the dpph free - radical scavenging capability , was measured by using the dpph method.results:cell viability with the 1- , 5- , 25- , 125- and 625-/ml salviae miltiorrhizae radix hot aqueous extract was not significantly decreased compared to the cell viability without the extract . 
 when 125 and 625 /ml of salviae miltiorrhizae radix hot aqueous extract were used , no production in lps - stimulated raw 264.7 macrophages was significantly inhibited compared to that in the control group . 
 when 25 , 125 , and 625 /ml of salviae miltiorrhizae radix hot aqueous extract were used , pge2 production in lps - stimulated raw 264.7 macrophages was significantly inhibited compared to that in the control group . 
 the 125- and 625-/ml salviae miltiorrhizae radix hot aqueous extracts had high dpph free - radical scavenging capabilities in raw 264.7 macrophages.conclusion:this study indicates that salviae miltiorrhizae radix hot aqueous extract suppresses no and pge2 production and improves dpph free - radical scavenging capability . 
 thus , it seems that salviae miltiorrhizae radix hot aqueous extract may have an anti - inflammation effect and antioxidant activity .