french marigold ( florets of tagetes patula ) has been used internally for indigestion and externally for sore eyes and rheumatism . the plant , a member of the family asteraceae , is a common cultivated ornamental . in general , these flowers are used as a coloring or flavoring agent in food and have potential medical use against inflammatory diseases of the skin , as well as insecticidal activity [ 1 , 2 ] . previous phytochemical investigations on french marigold have resulted in the isolation of carotenoids [ 3 , 4 ] , flavonoids [ 5 , 6 ] , and monoterpenoids . in our previous studies , we reported the anti - inflammatory action of french marigold [ 8 , 9 ] , and in this paper , we report the inhibitory effects of the flavonol patuletin and its glucoside from methanol extracts of french marigold on acute inflammation models , such as the hind - paw edema induced by -carrageenin and histamine and ear edema induced by arachidonic acid ( aa ) and 12-o - tetradecanoylphorbol-13-acetate ( tpa ) . tagetes patula l. was cultivated at the yamagata prefectural horticultural experiment station in yamagata , japan . a voucher specimen ( er-070 ) of tagetes patula l. ( asteraceae ) was deposited in the herbarium of the yamagata prefectural institute of public health , yamagata - shi , yamagata , japan . raw flower petals of t. patula l. ( 5  kg ) were subjected to extraction twice with meoh ( 5  l ) at room temperature . solvents were evaporated in vacuo to dryness , and an extract was obtained ( yield : 3.9% ) . meoh extract ( 35  g ) was partitioned between etoach2o ( 1  :  1 ) . the etoac extract ( 7.2  g ) was partitioned between n - hexanemeohh2o ( 19  :  19  :  2 ) , which afforded an n - hexane fraction ( 0.94  g ) and a meohh2o fraction ( 6.26  g ) . on the other hand , the h2o solution was partitioned between n - buohh2o ( 1  :  1 ) , yielding an n - buoh extract ( 4.74  g ) and an h2o extract ( 22.04  g ) , respectively . the meohh2o fraction ( 6  g ) the potent active fraction was subjected to same cc using 90% meoh , which yielded two active fractions , fr-3 and fr-7 ( yields : 2.5  g and 1.6  g ) . fr-3 was recrystallized to give compound-1 ( 1.8  g ) from 90% ethanol , and fr-7 was recrystallized to give compound-2 ( 0.9  g ) from ethanol . compound-1 and compound -2 were identified as patuletin and patulitrin by nmr and mass spectrometry [ 10 , 11 ] . ( kanagawa , japan ) ; phenylbutazone , cyproheptadine , and arachidonic acid from sigma chemical co , ( st . louis , mo ) ; tpa from chemicals for cancer research , inc . ( chicago , il ) ; and histamine , methanol , and acetone from wako pure chemical industries , ltd . experiments were performed in accordance with the guidelines of the institutional animal care and use committee of the school of pharmacy , nihon university ( chiba , japan ) . female icr mice ( age , 7 weeks ) and male ddy mice ( 22~26  g ) were purchased from japan slc inc . ( shizuoka , japan ) and were housed in an air - conditioned specific pathogen - free room ( 22 - 23c ) illuminated from 08:0020:00 . the inhibitory activity of flavonoids on carrageenin - induced mouse - paw edema was assessed according to the method of tsurufuji et al . . briefly , mice were treated orally with flavonoids ( each 5 , 15 and 50  mg / kg ) or vehicle ( 10  ml / kg ) . samples were administered orally 30  min before injection of 25  l of a 2% suspension of carrageenin in physiological saline solution into the subcutaneous tissues of the right hind paw . the left hind paw was injected in the same manner with 25  l of physiological saline solution . edema measurements were made using a dial thickness gauge ( ozaki mfg co. , ltd . , tokyo , japan ) at intervals of 1 , 2 , 3 , 4 , 5 , and 6 hours after carrageenin injection . mice in groups of five at each dose level received flavonoids ( each 5 , 15 , and 50  mg / kg ) in water ( 10  ml / kg , p.o . ) . at 30  min after flavonoid administration , mice were injected with histamine ( 5  g ) in physiological saline solution ( 5  l ) into the subplantar tissue of the right hind paw , and with saline solution ( 5  l ) into that of the left hind paw . the difference in foot pad thickness between the right and left paws was then measured with a dial thickness gauge every 6  min . tpa ( 1  g ) or aa ( 1  mg ) dissolved in acetone ( 20  l ) was applied to the right ear only of mice by means of a micropipette . a volume of 10  l was delivered to both the inner and outer surfaces of the ear . flavonoids ( each 0.04 , 0.2 , and 1.0  mg / ear ) or vehicle , meoh - chcl3 ( 1  :  1 , 20  l ) or meoh  chcl3h2o ( 2  :  1  :  1 , 20  l ) were topically applied 30  min before tpa or aa treatment . ear thickness was determined with a pocket thickness gauge ( mitsutoyo co. , ltd . , tokyo , japan ) having a range of 09  mm , graduated at 0.01-mm intervals and modified such that the contact surface area was increased to reduce loading when applied to the tip of the ear . ear thickness was measured before treatment ( a ) and at 6 ( 1 ) h after tpa ( aa ) treatment ( b = tpa ( aa ) plus vehicle ; b = tpa ( aa ) plus sample ) . the following values were then calculated :   ( i)edema a induced by tpa ( aa ) plus vehicle ( b  a ) ;   ( ii ) edema b induced by tpa ( aa ) plus sample ( b  a ) , ( iii)inhibitory  rate  ( % ) = [ ( edema  a  edema  b)/edema  a ]  100 , 
  ( iv)each value was the mean of individual determinations from four mice . edema a induced by tpa ( aa ) plus vehicle ( b  a ) ;    edema b induced by tpa ( aa ) plus sample ( b  a ) , inhibitory  rate  ( % ) = [ ( edema  a  edema  b)/edema  a ]  100 , 
  each value was the mean of individual determinations from four mice . data are expressed as means  standard deviation and were analyzed using prism statistical software . differences between groups ( figures 2 , 3 , 4 , 5 , 6 , and 7 ) were analyzed by one - way anova followed by correction with tukey - kramer test . oral administration of patuletin and patulitrin suppressed carrageenin - induced edema in a dose - dependent manner . at 15  mg / kg and 50  mg / kg patuletin , edematization was significantly suppressed and the inhibition rates were 4152% at 4 - 5  h. at 50  mg / kg patulitrin , hind - paw edema was suppressed at 35  h after administration , and inhibition rates were 4552% . at 15  mg / kg patulitrin , hind - paw edema was suppressed at 4 - 5  h , while 10  mg / kg phenylbutazone suppressed edematization by 4760% at 36  h ( figures 2 and 3 ) . however , these compounds were less effective than phenylbutazone ( 100  mg / kg ) ( figure 1 ) . figures 4 and 5 illustrate the time course of histamine - induced hind - paw edema in mice . at 50  mg / kg patuletin , edematization was significantly suppressed and inhibition rates were 5470% at 1224  min . at 50  mg / kg patulitrin , however , these compounds were less effective than cyproheptadine ( 10  mg / kg ) ( figure 1 ) . patuletin and patulitrin suppressed tpa - induced ear edema in a dose - dependent manner . in comparison with a standard drug , these compounds were less effective than indomethacin ( 0.5  mg / ear ) . patuletin and patulitrin inhibited aa - induced edema in a dose - dependent manner . however , these compounds were less effective than indomethacin ( 0.5  mg / ear ) . carrageenin - induced hind - paw edema is used as an animal model of acute inflammation and is thought to represent the early phase of inflammation . oral administration of patuletin and patulitrin inhibited edematization in a dose - dependent manner , although 50  mg / kg patuletin and patulitrin suppressed hind - paw edema less effectively than 100  mg / kg phenylbutazone , an anti - inflammatory drug . furthermore , the effects of patuletin and patulitrin on edema induced by histamine were investigated . patuletin and patulitrin significantly inhibited histamine - induced hind - paw edema in a dose - dependent manner . the above findings clarified that patuletin and patulitrin are effective in treating acute inflammation by suppressing the chemical mediators of inflammation . these results suggest that the active components in the anti - inflammatory effects of french marigold are patuletin and patulitrin . french marigold has been applied to the human foot to decrease inflammation caused by corns , calluses , hallux valgus , and ulcers and to reduce the associated pain . therefore , we examined the anti - inflammatory effects of patuletin and patulitrin after topical administration . these results suggest that the active components in the anti - inflammatory effects after the topical administration of french marigold are patuletin and patulitrin . they also inhibit the expression of inducible nitric oxide synthase ( inos ) , cyclooxygenase ( cox ) , and lipoxygenase ( lox ) , with subsequent decreases in nitric oxide ( no ) , prostanoids , and leukotrienes , as well as other mediators of the inflammatory process such as cytokines , chemokines , or adhesion molecules . body cells and tissues are continuously threatened by the damage caused by free radicals and reactive oxygen species , which are produced during normal oxygen metabolism or are induced by exogenous factors . on evaluation of the 1,1-diphenyl-2-picrylhydrazyl ( pddf ) , radical - scavenging activity of patuletin and patulitrin exhibited potent radical - scavenging activities with ic50 values of 4.3 and 10.17  g / ml , respectively . in addition , free radicals can attract various inflammatory mediators , contributing to a generalized inflammatory response and tissue damage . indeed , patuletin and patulitrin are powerful in vitro antioxidants , being able to scavenge a wide range of free radical species , as well as to inhibit their formation . this is the first report to find that patuletin and patulitrin inhibit acute inflammation induced by carrageenin , histamine , tpa , and aa in mice .
the major components patuletin and patulitrin were isolated from french marigold ( florets of tagetes patula ) . 
 patuletin and patulitrin were found to inhibit acute inflammation in mice . 
 oral administration of patuletin and patulitrin significantly suppressed hind - paw edema induced by carrageenin and histamine , while topical application of patuletin and patulitrin significantly inhibited ear edema induced by 12-o - tetradecanoylphorbol-13-acetate and arachidonic acid . 
 thus , oral and topical administration of patuletin and patulitrin inhibited acute inflammation in mice . 
 these results suggest the anti - inflammatory efficacy of french marigold .