under innate immune conditions , the inflammatory responses mediated by macrophages , mast cells , and neutrophils comprise an important barrier against infectious pathogens , such as viruses , fungi , and bacteria , as well as chemical toxins [ 1 , 2 ] . among the cellular components of innate immunity
, macrophages are regarded as central inflammatory cells , as they identify external pathogens using special surface receptors ( e.g. , toll - like receptors ( tlrs ) ) and are widely distributed in the human body .
the inflammatory responses mediated by macrophages and their role in pathophysiology have been previously studied .
activated macrophages induce various intracellular signaling cascades , including src , syk , phosphatidylinositide 3-kinase ( pi3k ) , akt , inhibitor of b ( ib ) kinase ( ikk )  , and ib [ 46 ] .
the signaling pathway also stimulates the nuclear translocation of nuclear factor- ( nf- ) b and activator protein ap-1 , triggering the expression of inflammatory genes that lead to secretion of inflammatory mediators ( e.g. , nitric oxide ( no ) , reactive oxygen species ( ros ) , prostaglandin e2 ( pge2 ) , chemokines , and cytokines ( e.g. , tumor necrosis factor- ( tnf- )  ) ) [ 79 ] .
recently , ample evidence has suggested that unchecked , prolonged inflammatory responses can cause serious immunological diseases , including diabetes , septic shock , cancer , arthritis , and cardiovascular disease .
the understanding of inflammatory responses and exploration of strategies for suppressing inflammation are thus considered appropriate approaches to reducing disease incidence [ 1013 ] .
the cordyceps genus including cordyceps sinensis , cordyceps militaris , cordyceps pruinosa , and cordyceps bassiana grow in korea , japan , china , and the congo .
the cordyceps genus can be administered through traditional routes and is known to ameliorate various inflammatory diseases , including chronicle bronchitis , asthma , and eczema .
the biological and pharmacological activities of cordyceps genus are antioxidative , antiviral , antifibrotic , anti - inflammatory , antinociceptive , antiangiogenic , antiplatelet aggregation , and antidiabetic [ 14 , 15 ] .
studies have also demonstrated the anti - inflammatory mechanisms of butanol ( bf ) and hexane ( hf ) fractions of cordyceps bassiana . however , the specific chemical compounds responsible for the plant 's anti - inflammatory properties have not yet been elucidated .
recently , we isolated a promising novel compound [ kth-13 : 4-isopropyl-2,6-bis(1-phenylethyl)phenol ] with anticancer activity from cordyceps bassiana . despite the novel chemical structure of this compound ,
we have established a method for its total synthesis and derivatization to develop more effective molecules .
so far , almost 60 compounds were newly synthesized and tested to check their activities by employing no assay and antiproliferative activity . of them , interestingly , kth-13-amine - diastereomer 1 [ 4-isopropyl-2,6-bis(1-phenylethyl)aniline 1 ( kth-13-ad1 ) ] has been reported to have stronger activity than that of the original compound in terms of anticancer activity ( data not shown ) . in this study , therefore , we further aimed to demonstrate the anti - inflammatory potential of kth-13-ad1 , a derivative of kth-13 , and to explore its mechanism of action using activated macrophages .
sodium nitroprusside ( snp ) , 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide ( mtt ) , dihydrorhodamine 123 ( dhr123 ) , fluorescein isothiocyanate- ( fitc- ) dextran , ascorbic acid , and lps ( e. coli 0111:b4 ) were purchased from sigma chemical co. ( st . louis , mo , usa ) . fetal bovine serum and rpmi 1640
the murine macrophage cell line raw264.7 and human embryonic kidney ( hek ) 293 cells were purchased from the american type culture collection ( rockville , md , usa ) .
luciferase constructs containing binding promoters for nf-b and ap-1 were gifts from professors chung , hae young ( pusan national university , pusan , korea ) and rhee , man hee ( kyungpook national university , daegu , korea ) .
phospho- and total protein - specific antibodies to p65 , p50 , c - fos , c - jun , ib , ikk , akt , p85 , src , syk , lamin a / c , and -actin were obtained from cell signaling technology ( beverly , ma , usa ) .
primers ( table 1 ) designed in our laboratory were synthesized by bioneer ( daejeon , korea ) . to synthesize kth-13-ad1 ( figure 1(a ) ) , a solution of 4-isopropylaniline ( 4.00  g , 29.6  mmol ) in xylene ( 14  ml ) was mixed with styrene ( 9.48  g , 91.1  mmol ) and cf3so3h ( 1.0  ml , 11.4  mmol ) .
the reaction mixture was allowed to heat to 160c and stirred for 24  h. at that time , the reaction was allowed to cool to room temperature and the volatiles were removed under vacuo .
the resulting residue was purified by silica gel column chromatography ( hexanes  :  etoac = 9  :  1 ) to afford the desired kth-13-ad1 ( 2.60  g , 7.57  mmol , and 1  :  1 diastereomers ) in 26% yield as a brown oil . infrared ( ir ) spectra of kth-13-ad1 were recorded on a bruker vertex 70 spectrophotometer , max in cm .
bands are characterized as strong ( s ) , medium ( m ) , and weak ( w ) .
h nuclear magnetic resonance ( nmr ) spectra of this compound were recorded on a jeol jnm - al400 ( 400  mhz ) spectrometer .
chemical shifts are reported in ppm from tetramethylsilane with the solvent resonance as the internal standard ( cdcl3 :  7.27  ppm ) .
data are reported as follows : chemical shift , multiplicity ( s = singlet , d = doublet , t = triplet , q = quartet , and m = multiplet ) , coupling constants ( hz ) , and integration .
c nmr spectra were recorded on a jeol jnm - al400 ( 100  mhz ) spectrometer with complete proton decoupling .
chemical shifts are reported in ppm from tetramethylsilane with the solvent resonance as the internal standard ( cdcl3 :  77.16  ppm ) .
low - resolution mass spectrometry was performed on an agilent 6890n gc ( hewlett - packard co. , palo alto , california , usa ) . 

h nmr ( 400  mhz , cdcl3 , diastereomer 1 ) :  7.407.12 ( m , 12h ) , 4.07 ( q , j = 6.9  hz , 2h ) , 3.29 ( br s , 2h ) , 3.00 ( sept , j = 6.9  hz , 1h ) , 1.67 ( d , j = 7.1  hz , 6h ) , 1.37 ( d , j = 7.1  hz , 6h ) ; c nmr ( 100  mhz , cdcl3 ) :  145.8 , 139.4 , 138.9 , 130.2 , 128.7 , 127.5 , 126.3 , 123.5 , 40.2 , 33.7 , 24.4 , 22,2 ; ir ( neat ) : 3471 ( s ) , 3384 ( s ) , 3060 ( m ) , 3024 ( m ) , 2982 ( s ) , 2869 ( s ) , 2293 ( w ) , 1947 ( w ) , 1878 ( w ) , 1803 ( w ) , 1623 ( s ) , 1600 ( s ) , 1471 ( s ) , 1450 ( s ) , 1373 ( m ) , 1318 ( m ) , 1257 ( m ) , 1172 ( m ) , 1027 ( m ) , 881 ( s ) , 761 ( s ) , 700 ( s ) cm ; lr - ms ( esi ) : m / z calculated for c25h30n ( [ m + h ] ) 344.2 , and found 344.2 .
raw264.7 and hek293 cells were cultured with rpmi1640 medium supplemented with 10% heat - inactivated fbs , glutamine , and antibiotics ( penicillin and streptomycin ) at 37c in a 5% co2 atmosphere . in each experiment , cells were detached with a scraper .
examination of cell densities at 2  10 cells / ml revealed that the proportion of dead cells was consistently < 1% according to trypan blue dye exclusion as the criterion for viability .
raw264.7 macrophage cells ( 1  10 cells / ml ) were cultured for 18  h , pretreated with kth-13-ad1 ( 0 to 200  m ) for 30  min , and further incubated with lps ( 1  g / ml ) for 24  h. the inhibitory effect of kth-13-ad1 on lps - induced no production was determined by analyzing no level using griess reagent , as previously described [ 18 , 19 ] .
the od at 550  nm ( od550 ) was measured using a spectramax 250 microplate reader ( molecular devices , sunnyvale , ca , usa ) .
the level of intracellular ros was determined by recording the change in fluorescence resulting from the oxidation of the fluorescent probe dhr123 .
briefly , 5  10 raw264.7 cells were exposed to kth-13-ad1 ( 0 to 150  m ) for 30  min and then incubated with snp ( 0.25  mm ) at 37c for 20  min to induce ros production .
the cells were further incubated with 20  m of the fluorescent probe dhr123 for 30  min at 37c .
the degree of fluorescence , which corresponded to the level of intracellular ros , was determined using a facscan flow cytometer ( becton - dickinson ) , as reported previously . to measure the phagocytic activity of raw264.7 cells , we modified a previously reported method .
raw264.7 cells ( 5  10 ) were pretreated with kth-13-ad1 ( 0 to 150  m ) for 1  h , resuspended in 100  l phosphate buffered saline ( pbs ) containing 1% human ab serum , and then incubated with fitc - dextran ( 1  mg / ml ) at 37c for 20  min .
the reactions were stopped by adding 2  ml ice - cold pbs containing 1% human serum and 0.02% sodium azide .
the cells were then washed three times with cold pbs - azide and analyzed on a facscan flow cytometer ( becton - dickinson , san jose , ca , usa ) , as reported previously .
the level of fitc - dextran or ros in raw264.7 cells was determined through flow cytometric analysis [ 22 , 23 ] .
10 cells / ml ) treated with kth-13-ad1 in the presence or absence of fitc - dextran ( 1  mg / ml ) or dhr123 ( 20  m ) were washed with staining buffer containing 2% rabbit serum and 1% sodium azide in pbs and then incubated with direct - labeled antibodies for an additional 45  min on ice . after washing three times with staining buffer , stained cells were analyzed on a facscan flow cytometer ( becton - dickinson ) .
raw264.7 cells ( 1  10 cells / ml ) were cultured for 18  h , after which kth-13-ad1 ( 0 to 150  m ) was added for the final 24 or 8  h of culture .
the cytotoxic effects of ats - e3 kth-13-ad1 were then evaluated using a conventional mtt assay , as reported previously [ 24 , 25 ] . for the final 3  h of culture
, 10  l mtt solution ( 10  mg / ml in pbs , ph 7.4 ) was added to each well .
the incubation was stopped by the addition of 15% sodium dodecyl sulfate ( sds ) to each well , which solubilized the formazan .
the absorbance at 570  nm ( od570630 ) was measured using a spectramax 250 microplate reader ( biotek , bad friedrichshall , germany ) .
raw264.7 cells ( 1  10 cells / ml ) were cultured for 18  h , pretreated with kth-13-ad1 ( 0 to 150  m ) for 30  min , and further cultured with lps ( 1  g / ml ) for 6  h. the inhibitory effect of kth-13-ad1 on the expression of inos and tnf- was determined using semiquantitative rt - pcr and real - time quantitative reverse transcription - polymerase chain reaction ( qrt - pcr ) , as reported previously [ 18 , 27 ] .
the primers ( bioneer , daejeon , korea ) used in these reactions are listed in table 1 .
hek293 cells ( 1  10 cells / ml ) were transfected with 1  g plasmids to drive the expression of -galactosidase and either nf-b - luc or ap-1-luc in the presence or absence of an inducing molecule ( myd88 , trif , or ha - src ) .
transfections were performed using the pei method in 12-well plates , as previously outlined [ 28 , 29 ] .
luciferase assays were performed using the luciferase assay system ( promega , madison , wi , usa ) , as previously reported .
raw264.7 cells ( 5  10 cells / ml ) were washed three times in cold pbs supplemented with 1  mm sodium orthovanadate , resuspended in lysis buffer ( 20  mm tris - hcl , ph 7.4 , 2  mm edta , 2  mm ethyleneglycotetraacetic acid , 50  mm -glycerophosphate , 1  mm sodium orthovanadate , 1  mm dithiothreitol , 1% triton x-100 , 10% glycerol , 10  g / ml aprotinin , 10  g / ml pepstatin , 1  mm benzamide , and 2  mm pmsf ) , lysed by sonication , and rotated for 30  min at 4c .
the lysates were clarified by centrifugation at 16,000  g for 10  min at 4c and stored at 20c until use .
the soluble fractions of the cell lysates were immunoblotted , and total and phosphoprotein levels of c - fos , c - jun , p50 , p65 , ib , ikk , akt , p85 , src , syk , lamin a / c , and -actin were visualized , as previously reported .
data are expressed as the mean  standard deviation ( sd ) , as calculated from one ( n = 6 ) of two independent experiments .
other data are representative of three different experiments with similar results . for statistical comparisons ,
the results were analyzed using analysis of variance / scheffe 's post hoc test and the kruskal - wallis / mann - whitney test . a p value < 0.05 was considered to be statistically significant .
all statistical tests were conducted using spss ( spss inc . , chicago , il , usa ) .
in previous studies , we and other investigators have reported on the anti - inflammatory , anticancer , and immunomodulatory activities of cordyceps bassiana [ 3235 ] . unlike other cordyceps species , such as cordyceps militaris and cordyceps sinensis ,
recently , we identified a promising new compound called 4-isopropyl-2,6-bis(1-phenylethyl)phenol with anticancer activity against several cancer cell lines , such as c6 glioma and mda - mb-231 .
we further established a method for total synthesis of this compound in order to facilitate its mass production and derivatization . in this study , we tested one ( kth-13-ad1 ( figure 1(a ) ) ) of its derivatives , specifically to see whether this compound is able to suppress macrophage - mediated inflammatory responses .
as shown in figure 1(b ) , left panel , the production of no was reduced by up to 70% in a dose - dependent manner by kth-13-ad1 in lps - stimulated macrophage - like raw264.6 cells at 150  m , though it did not downregulate no released from snp , a drug that directly releases no   in vitro ( figure 1(b ) , right panel ) .
these results indicate that kth-13-ad1 does not act as a chemically directed neutralizing agent but rather as a modulator of no biosynthesis during tlr4 stimulation following lps treatment . in similar fashion ,
the radical scavenging activity of kth-13-ad1 was observed only at 100 and 150  m when applied to snp - treated cells ( figure 1(c ) , left panel ) but not in the dpph assay ( figure 1(c ) , right panel ) . since kth-13-ad1 did not suppress the viability of raw264.7 cells ( figure 1(d ) ) , the results demonstrate that though this compound does not have direct antioxidative activity , it displays anti - inflammatory properties that indirectly suppress the production of inflammatory mediators , such as no , similarly to ginsenosides that have only anti - inflammatory properties [ 37 , 38 ] .
however , unlike these compounds , flavonoids ( e.g. , quercetin , luteolin , and kaempferol ) and anthraquinones ( e.g. , rhodoptilometrin ) , are active components of numerous medicinal plants and have been reported to demonstrate both antioxidative and anti - inflammatory activities [ 3942 ] . to determine the mechanism by which kth-13-ad1 suppresses no production , we investigated tlr4-mediated transcriptional activation levels in lps - treated raw264.7 cells pretreated with kth-13-ad1 .
first , we evaluated mrna levels in proinflammatory genes , including inos and tnf- , using quantitative real - time and semiquantitative pcr .
as shown in figures 2(a ) and 2(b ) , kth-13-ad1 ( 150  m ) significantly decreased the mrna levels of inos and tnf- , suggesting that kth-13-ad1-driven suppression of inflammatory responses occur at the transcriptional level .
since nf-b and ap-1 are representative of transcription factors regulating inflammatory responses [ 43 , 44 ] , we determined the ability of this compound to suppress the activation of nf-b and ap-1 using a luciferase reporter gene assay with promoter sites for nf-b and ap-1 . to determine how kth-13-ad1 regulates promoter activity in inflammatory genes
, we pretreated the kth-13-ad1 for 30  min before activating hek293 cells with pma , an activator of pkc that induces the inflammatory signaling pathway .
as shown in figure 2(c ) , luciferase activity mediated by nf-b and ap-1 was greatly enhanced by pma , up to 500- and 40-fold , respectively .
interestingly , kth-13-ad1 ( 100 and 150  m ) strongly suppressed nf-b - mediated luciferase activity ( figure 2(c ) , left panel ) but not ap-1 ( figure 2(c ) , right panel ) .
it has also been reported that myd88 and trif , adaptor molecules regulating the tlr4-mediated intracellular signaling pathway , are good inducers of luciferase activity in hek293 cells [ 47 , 48 ] .
the results in figure 2(d ) support this conclusion , as cotransfection of these molecules increased both nf-b- and ap-1-mediated luciferase activity up to 4,000- and 250-fold , respectively , as seen in previous studies . comparing these two conditions ,
myd88-induced nf-b activation was strongly suppressed by kth-13-ad1 , with 100  m of the compound inhibiting activation by 45% ( figure 2(d ) ) . in addition
, kth-13-ad1 reduced the nuclear translocation of the nf-b subunits p65 and p50 at 30 and 60  min , although there was no inhibition at 15  min ( figure 2(e ) ) .
these results suggest that the nf-b signaling pathway affecting the activation of nf-b dimerization at 30 to 60  min might be a potential target for kth-13-ad1 .
these inhibitory patterns observed by kth-13-ad1 treatment seem to also imply that nf-b activation signals might be timely regulated by some upstream signaling units . to prove such raised possibility , we subsequently focused on identifying the nf-b regulatory pathways that are recognized by this compound .
first , we determined the phosphorylation level of ib , an essential step in the nuclear translocation of nf-b .
the phosphorylation of ib was blocked by kth-13-ad1 at 15 , 30 , and 60  min , while the total form of ib was greatly increased at 30 and 60  min , compared to lps treatment alone ( figure 3(a ) ) , implying that this compound is able to suppress both phosphorylation and degradation of ib between 30  min and 1  h. in addition , kth-13-ad1 was found to reduce the phosphorylation of ikk/ , upstream of ib , at 5  min ( figure 3(a ) ) .
similarly , the phosphorylation of akt and p85/pi3k , upstream enzymes for ikk activation , was blocked by kth-13-ad1 at 3 to 5  min ( figure 3(b ) ) .
additionally , src phosphorylation occurring at 1 and 3  min was also suppressed by this compound ( figure 3(b ) ) . to confirm our results
, we evaluated the dose - responsive activity of kth-13-ad1 on the phosphorylation levels of putative target proteins , p85/pi3k and src .
as seen in figure 3(c ) , the phosphorylation of p85 and src was continuously diminished by kth-13-ad1 ( 100 and 150  m ) under the same conditions . as these results strongly suggest that the putative target of kth-13-ad1 is src , we next analyzed whether this compound is able to regulate the autophosphorylation of src , using an overexpression strategy , as reported previously [ 50 , 51 ] .
as shown in figure 3(d ) , kth-13-ad1 ( 100 and 150  m ) clearly decreased the level of src phosphorylation , implying that src is directly modulated by this compound .
this is further supported by the fact that the promoter activity of nf-b , as stimulated by src overexpression , was also blocked by kth-13-ad1 ( figure 3(e ) ) . based on these results
, we propose that src serves as a potential target protein in kth-13-ad1-mediated anti - inflammatory actions .
many anti - inflammatory compounds , both naturally occurring and chemically synthesized , have been found to possess direct inhibitory activity against src kinase [ 5052 ] .
additionally , previous studies have reported on src activity under nf-b activation and inflammatory gene expression [ 51 , 53 , 54 ] , suggesting an important role of src in inflammatory responses and in oncogenic activity .
finally , to demonstrate whether kth-13-ad1 modulates macrophage phagocytic uptake , fitc - dextran was applied to raw264.7 cells during exposure to this compound . as shown in figure 4
, there was no inhibition of phagocytosis by kth-13-ad1 , as measured by fitc - derived fluorescence [ 55 , 56 ] ; however , significant enhancement was observed at 100 and 150  m concentrations , implying that the compound stimulates macrophage phagocytosis , an important step in the innate immune response .
this result suggests that innate immune responses might be regulated by kth-13-ad1 via different cellular mechanisms .
in summary , kth-13-ad1 demonstrated impressive suppression of inflammatory mediator production , including ros and no , without inducing cytotoxicity , while this compound strongly enhanced the phagocytotic uptake , suggesting that kth-13-ad1 is able to regulate cellular responses related to innate immunity .
in addition , the transcriptional activation of nf-b and its upstream signaling pathway , composed of src , p85/pi3k , and akt , was blocked by kth-13-ad1 , as summarized in figure 5 .
thus , the present study proposes that kth-13-ad1 , a chemical analog of kth-13 extracted from cordyceps bassiana , has clinical utility as an anti - inflammatory drug .
 the cordyceps species has been a good source of compounds with anticancer and anti - inflammatory activities . 
 recently , we reported a novel compound ( 4-isopropyl-2,6-bis(1-phenylethyl)phenol , kth-13 ) with anticancer activity isolated from cordyceps bassiana and created several derivatives to increase its pharmacological activity . in this study , we tested one of the kth-013 derivatives , 4-isopropyl-2,6-bis(1-phenylethyl)aniline 1 ( kth-13-ad1 ) , with regard to anti - inflammatory activity under macrophage - mediated inflammatory conditions . kth-13-ad1 
 clearly suppressed the production of nitric oxide ( no ) and reactive oxygen species ( ros ) in lipopolysaccharide ( lps ) and sodium nitroprusside- ( snp- ) treated macrophage - like cells ( raw264.7 cells ) . 
 similarly , this compound also reduced mrna expression of inducible no synthase ( inos ) and tumor necrosis factor- ( tnf- ) , as analyzed by rt - pcr and real - time pcr . 
 interestingly , kth-13-ad1 strongly diminished nf-b - mediated luciferase activities and nuclear translocation of nf-b family proteins . in accordance 
 , kth-13-ad1 suppressed the upstream signaling pathway of nf-b activation , including ib , ikk/ , akt , p85/pi3k , and src in a time- and dose - dependent manner . 
 the autophosphorylation of src and nf-b observed during the overexpression of src was also suppressed by kth-13-ad1 . 
 these results strongly suggest that kth-13-ad1 has strong anti - inflammatory features mediated by suppression of the src / nf-b regulatory loop . 