toll - like receptors ( tlrs ) play crucial roles in innate immunity and can contribute to the development of appropriate adaptive immune responses [ 1 , 2 ] . to date , 13 members of tlr family have been identified in mammals [ 35 ] , and tlrs received special attention as potent adjuvant receptors . mammalian tlr5 , expressed on epithelial cells and phagocytes such as dendritic cells and macrophages , recognizes flagellins of bacteria and subsequently activates the nuclear factor - kappa b ( nf-b ) pathway of host cells [ 7 , 8 ] . we reported that a vibrio vulnificus flagellin b ( flab ) showed strong mucosal vaccine adjuvant activity   and enhanced tumor - specific cd8 t cell immune responses through tlr5 stimulation in a therapeutic cancer vaccine model . recently , flab combined with tnf and ifn was reported to generate potent dendritic cells which produce functionally active cytotoxic t lymphocytes . flagellin is a highly priced protein adjuvant candidate . to identify ligands that potentiate vaccine adjuvant activity of flagellin , we screened a plant extract library using hek293 t cells transiently cotransfected with phtlr5 and pnf-b - seap plasmids . the 90% etoh extract from croton tiglium l. ( euphorbiaceae ) showed significant nf-b transactivation . croton tiglium is a plant grown in tropical and subtropical zones , and the seed of croton tiglium is well known as ba - dou ( or badou ) in china and korea . ba - dou has been used to treat gastrointestinal disorders , intestinal inflammation , rheumatism , headache , peptic ulcer , and visceral pain [ 1214 ] . the sesquiterpenes and monoterpenes as the main components comprise the great parts of the extracted essential oil from seed . the toxic substances were found mainly in the bark and leaves of croton tiglium and croton oil . in this study , we isolated phorbol 12-myristate 13-acetate ( pma ) as an active component from croton tiglium and investigated the action mechanisms in tlr signaling pathways . hek293 t and caco-2 cells ( atcc , manassas , va ) were cultured in dulbecco modified eagle medium ( dmem , welgene , korea ) supplemented with 10% fetal bovine serum ( fbs , gibco , invitrogen , carlsbad , ca ) at 37c in a 5% co2 incubator . t cell is a human embryonic kidney cell line with sv40 large t - antigen for efficient transfection of plasmids . t cells seeded at 10/well in 96-well plates were transfected with pnf-b - secreted alkaline phosphatase ( pnf-b - seap , invivogen , san diego , california ) , pil-8-luciferase , or phtlr5 plasmids using fugene 6 ( roche , hague road , indianapolis ) . one day after transfection , the cells were replaced with fresh dmem containing different concentrations of test agents for 1 day . nf-b - seap activities in cell culture supernatants were determined using quanti - blue ( invivogen ) according to the manufacturer 's instructions . recombinant tag - free flagellin , flab , was purified as described elsewhere and contaminating lipopolysaccharide ( lps ) was removed by using the affinitypak detoxi gel endotoxin removing gel ( pierce biotechnology , inc . , the seeds of croton tiglium were purchased from chonnam seangyack nongob , hwasun - gun , in april 2011 , republic of korea . plant sample was identified botanically by professor y. h. moon . a voucher specimen ( snu2011 - 04 ) was deposited at the herbarium of seoul national university , seoul , republic of korea . the dried seeds of croton tiglium ( 600  g ) were extracted with 90% etoh ( 2  l  3 times ) at room temperature . the combined 90% etoh extract was then evaporated under reduced pressure using a rotary vacuum evaporator ( eyela , japan ) . the dried crude extract of croton tiglium ( 12  g ) was suspended in water and divided successively with n - hexane ( 3  500  ml ) , chcl3 ( 3  500  ml ) , etoac ( 3  500  ml ) , and n - buoh ( 3  500  ml ) . the chcl3 fraction ( 2.9  g ) , which showed strong enhanced activity on nf-b transcription , was chromatographed over a silica gel open column ( 5  40  cm ; 63200  m particle size , merck , darmstadt , germany ) eluting with gradient n - hexane / acetone ( 20  :  1 , 10  :  1 ,  , 1  :  3 , each 200  ml ) to yield ten fractions ( f1f10 ) based on the tlc profile . fraction f8 ( 1.05  g ) was reapplied to an rp - c18 open column ( 4  30  cm ; 4063  m particle size ) with a stepwise gradient of meoh / h2o ( 1  :  2 to 10  :  1 ) to afford nine subfractions ( f81f89 ) . finally , subfraction f87 ( 30.2  mg ) was purified by hplc [ optimapak c18 column ( 10    250  mm , 10  m particle size , rs tech , korea ) ; mobile phase meoh in h2o containing 0.1% hco2h ( 015  min : 85% meoh , 1540  min : 95% meoh , 4045  min : 100% meoh ) ] to yield compound 1 ( tr = 31.3  min , 5.2  mg ) ( figure 2 ) . caco-2 cells were seeded at 5  10/well in 48-well plates and were treated with ligands for 8 hours without fbs supplementation . il-8 in the supernatant was measured by an elisa kit ( biosource international , inc . , t cells in 8-well glass chamber plate ( nalge nunc international , rochester , ny ) were transfected with phtlr5 using fugene 6 ( roche ) . after fixation for 15 minutes with 3.7% paraformaldehyde , the cells were rendered permeable by incubation in pbs with 0.2% triton x-100 for 10 minutes . nf-b p65 protein was detected by immunostaining using a specific antibody ( santa cruz biotechnology , delaware avenue , santa cruz , ca ) and alexa fluor-488-conjugated anti - rabbit - igg antibody ( molecular probes , invitrogen , eugene , or ) . fluorescence images were acquired using a fluorescence microscope ( dxm1200c , nikon ) . to study the action mechanism , various pharmacological inhibitors were tested on pma - mediated nf-b activation . pharmacological inhibitors were used such as wortmannin , bay 11 - 7082 , genistein , gf109203x , pd98059 , sb203580 , sp600125 , and u-73122 ( cell signaling technology , danvers , ma ) for the inhibition of phosphoinositide 3-kinase ( pi3k ) , ikb- phosphorylation , protein tyrosine kinase ( ptk ) , protein kinase c ( pkc ) , mek1 , sapk2 ( p38 ) , jun n - terminal kinase ( jnk ) , and phospholipase c ( plc ) , respectively . five - week - old female balb / c mice were intranasally immunized three times with 10  l of pbs containing oval albumin ( ova ) as an antigen alone or in combination with pma or flab at 7-day intervals . seven days after the last immunization , feces and serum samples were collected from the immunized mice to assess antigen - specific antibody responses . all animal procedures were conducted in accordance with the guidelines of the animal care and use committee of chonnam national university . ova - specific antibodies were determined by elisa followed by the methods as described elsewhere . absorbance was read by an elisa microplate reader ( power wavex340 , nio - tek - ins truments , inc ) at 450  nm . spleen lymphocytes were prepared from the immunized mice by using lymphoprep according to the manufacturer 's instructions ( axis - shield poc as , norway ) . the lymphocytes were cultured in rpmi 1640 medium containing 10% fbs , 100  u / ml penicillin , and 100  g / ml streptomycin and incubated with ova ( 10  g / ml ) at 37c for 2 days . the levels of interleukin 12 ( il-12 ) were measured by using sandwich elisa kits following the manufacturer 's experimental protocols ( biolegend , usa ) . all values are expressed as means  standard error of the mean ( sem ) . all experiments were repeated three times and the results from a representative experiment were shown . tlr5 recognizes bacterial flagellin and activates a transcription factor nf-b hence inducing proinflammatory cytokine production in mammalian cells . we studied to identify herbal medicines that enhanced nf-b activity of a flagellin , flab . tlr5 and nf-b - seap were overexpressed in hek293 t cells by transfection with the plasmids and a plant extract library was screened . the 90% etoh extract from croton tiglium increased nf-b transcription in hek293 t cells in tlr5-independent pathway and enhanced the flab activity inducing tlr5-dependent nf-b activation ( figure 1(a ) ) . to identify active components of croton tiglium , the 90% etoh crude extract was divided into 5 fractions : n - hexane , chloroform , ethyl acetate , n - butanol , and water layers . the chloroform fraction showed a more significant effect than the other fractions on nf-b activity at concentrations of 30~300  ng / ml ( figure 1(b ) ) . in order to isolate an active component , the chloroform fraction from croton tiglium extract was subjected to a succession of chromatographic procedures including silica gel chromatography , rp - c18 , and hplc ( figure 2(a ) ) . each fraction was tested on nf-b transcription in hek293 t cells and the activities were shown in table 1 . when phorbol 12-myristate-13-actate [ pma ; synonym : 12-o - tetradecanoylphorbol-13-actate ( tpa ) ] from sigma co. ( st . louis , usa ) and an isolated compound ( compound 1 ) were coinjected into hplc , the same retention time ( tr ) at 31.3  min suggested that it was an identical compound ( figure 2(b ) ) . for further confirmation of the chemical structure of isolated compound , h and c nmr ( nuclear magnetic resonance ) spectra of 1 were measured on a varian unity inova 600  mhz spectrometer at the korea basic science institute ( kbsi , gwangju center , korea ) . the h and c nmr spectroscopic data of compound 1 showed the characteristic signals for an  , -unsaturated carbonyl group [ ( h 7.57 , 1h , br s , h-1 ; c 160.7 , c-1 ) ; c 132.8 , c-2 ; c 208.8 , c-3 ] , a trisubstituted double bond [ ( h 5.66 , 1h , d , j = 4.6  hz , h-7 ; c 129.2 , c-7 ) ; c 140.4 , c-6 ] , an oxymethine ( h 5.39 , 1h , d , j = 10.1  hz , h-12 ; c 76.5 , c-12 ) , and an oxymethylene [ h 4.01 and 4.00 , ab 2h , j = 12.8  hz , h2 - 20 ; c 68.0 , c-20 ] of a phorbol ester system [ 15 , 16 ] . as the nmr and ms data [ m / z 616.3980 , micromass qtof2 ( micromass , wythenshawe , uk ) ] are identical with those reported for pma [ 16 , 17 ] , compound 1 was finally determined as pma ( figure 2(c ) ) colorless oil ; h  ( 600  mhz , in cdcl3 ) : h 7.57 ( 1h , br s , h-1 ) , 5.66 ( 1h , d , j = 4.6  hz , h-7 ) , 5.51 ( 1h , br s , oh-9 ) , 5.39 ( 1h , d , j = 10.1  hz , h-12 ) , 4.01 and 4.00 ( 2h , ab peaks , j = 12.8  hz , h2 - 20 ) , 3.23 ( 1h , br s , h-10 ) , 3.21 ( 1h , t , j = 5.5  hz , h-8 ) , 2.52 and 2.46 ( 2h , ab peaks , j = 19.3  hz , h-5 ) , 2.30 ( 2h , m , h-2 ) , 2.12 ( 1h , m , h-11 ) , 2.07 ( 3h , s , acetyl ) , 1.76 ( 3h , dd , j = 2.7 , 1.4  hz , h-19 ) , 1.60 ( 2h , m , h-3 ) , 1.181.31 [ 26 , ( 413 methylene ) and 2  methyl ( h-16 and h-17 ) ] , 1.06 ( 1h , d , j = 5.0  hz , h-14 ) , 0.87 ( 3h , d , j = 6.4  hz , h-18 ) , 0.86 ( 3h , t , j = 6.5  hz , h-14 ) ; c nmr data ( 150  mhz , in cdcl3 ) : c 160.7 ( c-1 ) , 132.8 ( c-2 ) , 208.8 ( c-3 ) , 73.7 ( c-4 ) , 38.6 ( c-5 ) , 140.4 ( c-6 ) , 129.2 ( c-7 ) , 39.1 ( c-8 ) , 78.1 ( c-9 ) , 56.2 ( c-10 ) , 42.9 ( c-11 ) , 76.5 ( c-12 ) , 65.6 ( c-13 ) , 36.2 ( c-14 ) , 25.6 ( c-15 ) , 23.8 ( c-16 ) , 16.8 ( c-17 ) , 14.1 ( c-18 ) , 10.1 ( c-19 ) , 68.0 ( c-20 ) , 173.7 ( c-1 ) , 34.6 ( c-2 ) , 25.2 ( c-3 ) , 29.029.6 ( c-4 to c-11 ) , 31.9 ( c-12 ) , 22.7 ( c-13 ) , 14.1 ( c-14 ) , 173.7 and 21.1 ( acetyl ) ; eims m / z 616.3980 ( calcd for c36h56o8 , 616.3975 ) . pma , an active component of croton tiglium , was evaluated whether it could stimulate flab - mediated nf-b activity and il-8 production . pma at a low concentration of 10  ng / ml increased nf-b activity with or without flab ( 20  ng / ml ) ( figure 3(a ) ) . pma also enhanced significantly flab - mediated il-8 production in caco-2 cells expressing tlr5 constitutively ( figure 3(b ) ) . these results suggest that pma is an active component of croton tiglium in nf-b transactivation . like other members of the nf-b family , cellular activation results in the nuclear translocation of p50  :  p65 for initiating gene transcription . we therefore assessed the nuclear translocation of nf-b p65 subunit after its activation by pma treatment ( 10  ng / ml ) to the hek293 t cells by immunostaining . nf-b p65 was translocated from cytosol to nucleus after pma treatment ( figure 4(a ) ) . to study the action mechanism of pma two hours after inhibitor incubation , the cells were treated with pma at a concentration of 10  ng / ml . the supernatants were collected after 1 day of pma treatment and nf-b - seap levels were determined by quanti - blue . pma - induced nf-b activation was blocked by the treatment of a pkc inhibitor gf109203x and an ib- degradation inhibitor bay11 - 7082 ( figure 4(b ) ) . balb / c mice were intranasally immunized three times with antigen ova alone or in combination with flab or pma under anesthesia . seven days after immunization , feces and serum samples were collected from the immunized mice to assess antigen - specific antibody responses . ova - specific iga and igg antibodies were significantly increased by the administration of ova plus pma or flab ( figure 5(a ) ) . spleen lymphocytes were prepared by using lymphoprep from vaccinated mice and cytokines were analyzed by elisa . il-12 production was significantly increased in spleen lymphocytes isolated from ova - vaccinated mice treated with pma ( figure 5(b ) ) . the present study demonstrates the vaccine adjuvant effect of pma isolated from croton tiglium . to identify herbal medicines potentiating the vaccine adjuvant effect of flagellin , a plant library was screened using hek293 t cells transiently cotransfected with phtlr5 and pnf-b - seap plasmids . because hek293 t cells do not express any tlrs , the cells transfected with phtlr5 plasmid dna were used for tlr5-dependent response in the absence of other tlrs . flagellin , a tlr5 agonist , activated a transcription factor nf-b only in the presence of tlr5 ( figure 1 ) . in contrast , the 90% etoh extract of croton tiglium and its chloroform layer significantly stimulated nf-b transcription both in tlr5 and tlr5 + screening systems ( figure 1 ) . pma was isolated and characterized as an active component from croton tiglium ( figure 2 ) by activity - guided fractionation , column chromatography , hplc , nmr , and ms . pma induced nf-b transactivation in a tlr5-independent manner and increased il-8 production in caco-2 cells constituently expressing some tlrs ( figure 3 ) . the effect of pma was dependent on nf-b translocation and pkc activation ( figure 4(b ) ) . finally , pma or flab enhanced antigen - specific igg and iga antibody responses in intranasally ova - immunized mice ( figure 5(a ) ) . in addition , pma increased il-12 production corresponding to t cell responses ( figure 5(b ) ) . the present study suggests that pma activating pkc of tlr signaling pathway has a possibility for being an efficacious mucosal vaccine adjuvant .
toll - like receptor ( tlr ) ligands are being developed for use as vaccine adjuvants and as immunomodulators because of their ability to stimulate innate and adaptive immune responses . 
 flagellin , a tlr5 ligand , was reported to show potent mucosal vaccine adjuvant activity . to identify ligands that potentiate the adjuvant activity of flagellin 
 , we screened a plant library using hek293 t cells transiently cotransfected with phtlr5 and pnf-b - seap plasmids . 
 the 90% etoh extract from croton tiglium showed significant nf-b transactivation in a tlr5-independent manner along with the increase of a flagellin activity . 
 we have studied to characterize an active component from croton tiglium and to elucidate the action mechanisms . 
 phorbol 12-myristate 13-acetate ( pma ) was isolated as an active component of croton tiglium by activity - guided fractionation , column chromatography , hplc , nmr , and ms . 
 pma at a range of nm induced pkc - dependent nf-b activation and il-8 production in both tlr5 and tlr5 + assay systems . in in vivo mouse vaccination model , pma induced antigen - specific igg and iga antibody responses and increased il-12 production corresponding to t cell responses in spleen lymphocytes . 
 these results suggest that pma would serve as an efficacious mucosal vaccine adjuvant .