therefore , special attention was paid to the acidic fraction of 
 the gum resin extracts derived from different boswellia spp . the acidic fractions ( containing lipophilic acidic ingredients ) 
 of gum resins derived from different boswellia spp . 
 were separated from the neutral components ( i.e. , the essential oil 
 and mucilage fraction ) ; see the supporting information . first , aliquots of the neutral and acidic fractions were analyzed 
 for inhibition of mpges-1 activity in a cell - free assay using microsomes 
 of il-1-stimulated a549 cells as enzyme source and 20 m 
 pgh2 as mpges-1 substrate ; mk-886 ( 10 m ; ic50 = 2.4 m ) was used as reference compound . response 
 analysis revealed ic50 values of 1.9 , 2.8 , 1.6 , and 0.4 
 g / ml for the acidic fraction of gum resins from b. 
 serrata , b. sacra , b. carterii , and b. papyrifera , respectively ( figure 1b ) . in contrast , the neutral fraction ( 10 g / ml ) 
 did not significantly inhibit mpges-1 activity , regardless from which 
 species it originated ( not shown ) . in particular , the acidic fraction 
 of b. papyrifera gum potently suppressed mpges-1 
 activity with a maximal inhibition of 92% at 30 g / ml , which 
 was superior to the control inhibitor mk-886 ( 10 m = 0.49 g / ml , 
 79% inhibition ) under the same assay conditions . therefore , the remarkable 
 potency of the acidic fraction of b. papyrifera gums suggested the presence of highly 
 active constituents . it should be noted that the nature of the ingredients 
 and their contents do not substantially differ between lipophilic 
 extracts of gum resins from these four boswellia spp . , indicating that defined mixtures or compositions 
 of the bioactive components may result in efficient mpges-1 inhibition . microsomal 
 preparations of il-1-stimulated a549 cells were 
 preincubated with the indicated concentrations of acidic fractions 
 derived from gums of b. papyrifera , b. serrata , b. sacra , and b. carterii , or 
 vehicle ( dmso ) , for 15 min at 4 c . the reaction was started 
 by addition of 20 m pgh2 , and after 60 s at 4 c , 
 the reaction was terminated and pge2 was analyzed . besides the four boswellic acids 14 that were recently shown to inhibit mpges-1 , the acidic fractions of frankincense gum resins 
 may contain 
 additional triterpene acids that could interfere with mpges-1 activity 
 as well . we isolated 17 known triterpene acids ( table 1 ) from various gum resins of different boswellia spp . ( see supporting information ) , that 
 is , the four boswellic acids 14 , 
 the six tirucallic acids 510 , the 
 three roburic acids 1113 , the nyctanthic 
 acid 14 , and the three lupeolic acids 1517 , by preparative hplc . the chemical structures 
 of the isolated compounds were analyzed by ms and nmr and compared 
 to literature data ( see supporting information ) . the 13 remaining triterpene acids 517 were tested at a fixed concentration 
 of 10 m ( table 1 ) . the roburic acids 11 and 12 as well as the nyctanthic acid 14 failed to significantly inhibit mpges-1 activity , whereas 
 all tirucallic acids ( 510 ) , the 
 roburic acid 13 , and the three lupeolic acids 1517 markedly suppressed pge2 production 
 ( table 1 ) . just like mk-886 ( at 10 m ) , 
 all these active compounds exerted a maximal inhibition of about 70% 
 to 80% , except 5 , which was able to suppress pge2 formation by 96% ( table 1 ) . those 
 triterpene acids that caused more than 60% inhibition at 10 m were subjected to concentration  response analysis . as depicted 
 from table 1 , the six tirucallic 
 acids 510 , the roburic acid 13 , and the lupeolic acid 17 potently suppressed 
 mpges-1 activity with ic50 values of 0.4 to 3 m . 
 in comparison to the boswellic acids 14 as inhibitors of mpges-1 ( ic50 = 330 m ) the two acetylated tirucallic acids 6 and 10 with ic50 = 0.4 m , 
 each , were most potent , whereas the corresponding deacetylated analogues 5 and 9 were less efficient ( ic50 = 
 1.1 and 3.0 m , respectively ) ( figure 2 ) . concentration microsomal preparations of il-1-stimulated 
 a549 cells were preincubated with ( a ) 3--hydroxy-8,24-dienetirucallic 
 acid ( 5 ) , ( b ) 3-acetoxy-8,24-dienetirucallic acid 
 ( 6 ) , ( c ) 3--hydroxy-7,24-dienetirucallic acid 
 ( 9 ) , and ( d ) 3-acetoxy-7,24-dienetirucallic acid 
 ( 10 ) at the indicated concentrations for 15 min at 4 
 c . the reaction was started by addition of 20 m pgh2 , and after 60 s at 4 c the reaction was terminated 
 and pge2 was analyzed data are given as mean + sem , n = 47 . from this structure  activity - relationship ( sar ) study , 
 we 
 conclude that the acetylation of the free hydroxyl moiety at the 3 
 position of 6 and 10 is seemingly beneficial . 
 on the other hand , replacement of the 3-hydroxy group of 5 ( ic50 = 1.1 m ) by a keto group yielding 8 exhibited no significantly improved potency ( ic50 = 0.9 m ) . moreover , 3-configured 7 showed 
 almost the same inhibitory potency ( ic50 = 1.2 m ) 
 as its 3-isomer 5 , indicating that the steric 
 positioning of the 3-hydroxy group has negligible impact on the bioactivity . 
 among the roburic acids , the derivative 13 , carrying 
 a keto moiety , potently inhibited mpges-1 activity ( ic50 = 1.0 m ) , whereas 11 and 12 ( without 
 the keto group ) were not active up to 10 m . along these lines , 
 the nyctanthic acid 14 , also lacking a keto moiety , which 
 is structurally related to the roburic acid 12 , was also 
 inactive . inhibition of mpges-1 by lupeolic acids was evident for 
 all tested derivatives 1517 , but 
 only the acetylated derivative 17 reached an ic50 ( i.e. , 0.9 m ) lower than 10 m , suggesting that the 
 additional hydroxyl moiety at position c(28 ) is responsible for this 
 molecule s bioactivity . in contrast to boswellic acids , 
 few studies have addressed the 
 bioactivities and pharmacological properties of tirucallic acids and 
 lupeolic acids . thus , tirucallic acids were found to modulate 5-lipoxygenase 
 product synthesis and to inhibit the 
 serine / threonine kinase akt in prostate cancer cells in association 
 with apoptotic cell death . cancer chemopreventive 
 and cytotoxic activities in neuroblastoma cell lines were reported 
 for the lupeolic acids 15 and 16 , albeit 
 at high concentrations ( ic50 = 4.186.7 m ) , and both compounds ( applied topically ) inhibited 
 phorbol ester - induced ear inflammation in mice . for roburic acids , only one study on bioactivity has been 
 published that describes moderate inhibitory effects on isolated cox 
 enzymes ( ic50 > 10 m ) . therefore , the identification of mpges-1 as a target for the tirucallic 
 acids 510 , the roburic acid 13 , and the luepolic acid 17 is a substantial 
 insight into the pharmacology of these triterpene acids and underlines 
 their anti - inflammatory and anticancer potential , which was proposed 
 for other mpges-1 inhibitors . the 
 fact that several different structural classes of triterpene acids 
 inhibit mpges-1 , while other derivatives failed , led us to deduce 
 sars in order to speculate about a common binding pattern of active 
 compounds . in silico docking studies were performed 
 to surmise binding modes of triterpene acids in mpges-1 . the tirucallic 
 acids 5 and 10 , the lupeolic acid 17 , and the boswellic acid 1 were analyzed for 
 common chemical features relevant to potent mpges-1 inhibition and 
 to compare the binding mode of these triterpene acids with less active 
 derivatives . the tirucallic acid 10 , one of the most 
 potent derivatives ( ic50 = 0.4 m ) , exhibited various 
 molecular interactions , which were subjected to subtle modifications 
 during the mmff94-based minimization within ligandscout ( figure 3a ) . for instance , the arg73 of subunit a , which 
 we refer to as arg73a , extended as a solvent - exposed residue into 
 the central pore of the homotrimeric mpges-1 . during the minimization , 
 arg73a shifted toward the acidic moiety of 10 , making 
 an ionic interaction feasible . additionally , the acidic group of 10 formed an ionic interaction with arg70a , a residue near 
 the cofactor glutathione ( gsh ) . we additionally 
 observed various hydrophobic interactions between 10 and 
 residues embedded in the central pore ( e.g. , leu69a , val65b , leu69b , 
 and val65c ) . for comparison , the tirucallic acid 5 ( ic50 = 1.1 m ) ( figure 3b ) and the 
 lupeolic acid 17 ( ic50 = 0.9 m ) ( figure 3c ) formed an ionic interaction or a hydrogen bond 
 to arg73c or arg73a , respectively . additionally , in the case of 5 , a hydrogen bond was formed between the hydroxyl group of 
 the triterpene acid and gsh , while , in the case of 17 , a hydrogen bond was formed between the acetoxy group and the backbone 
 amide of his53a . predicted binding modes for representative triterpene 
 acids using 
 molecular docking . predicted binding modes are shown for ( a ) 3-acetoxy-7,24-dienetirucallic 
 acid ( 10 ) ( b ) , 3--hydroxy-8,24-dienetirucallic 
 acid ( 5 ) , ( c ) 3-o - acetyl-28-hydroxylupeolic 
 acid ( 17 ) , and ( d ) -boswellic acid ( 1 ) . protein ligand interactions are color - coded : red arrow , 
 hydrogen - bond acceptor ; green arrow , hydrogen - bond donor ; yellow sphere , 
 hydrophobic interaction ; red star , negatively ionizable . interestingly , the sec - propenyl 
 group of 17 protruded into the region adjacent to the 
 cofactor gsh 
 and formed a hydrophobic interaction with leu69b . finally , the boswellic 
 acid 1 , with less pronounced potency toward mpges-1 ( ic50 = 5 m ) , was docked into 
 the 3d structure of mpges-1 ( figure 3d ) . this 
 boswellic acid was predicted to form a hydrogen bond to arg73b , similar 
 to the other triterpene acids . although hydrophobic interactions were 
 formed ( e.g. , with val65b and leu69b ) , similar to other triterpene 
 acids , these molecular interactions were less frequently observed 
 for 1 compared to the other more potent compounds . together , a hydrogen bond or an ionic interaction of the carboxylic 
 group to arg73 , which extends into the central pore of the homotrimeric 
 enzyme , is observed in all docking poses . thus , the acidic group of 510 , 13 , and 17 may essentially contribute to the potent interference of these triterpene 
 acids with mpges-1 . however , additional interactions involving oxygen - containing 
 substituents determine the potency , since 11 , 12 , and 14 , lacking an additional oxygen substituent , 
 failed to inhibit mpges-1 , and also 15 and 16 , lacking the c(28)oh , were less active than 17 . in contrast , all six tirucallic acids ( 510 ) that carry an oxygen ( hydroxyl , keto , or acetoxy group ) 
 distant from the carboxylic acid moiety potently suppressed mpges-1 
 activity with ic50 values of 0.4 to 3 m . also , 13 and 17 substituted with a hydroxyl or keto 
 group potently suppressed mpges-1 activity . therefore , the presence 
 of oxygen distant from the carboxylic moiety is seemingly important 
 for molecular interactions with mpges-1 . moreover , the docking studies 
 imply that hydrophobic interactions , which were frequently formed 
 to residues embedded in the central pore ( e.g. , val65 , leu69 , and 
 met76 ) , seem to contribute to mpges-1 interference . furthermore , among 
 the most potent triterpene acids ( e.g. , 5 and 10 ) , an ionic interaction to a residue near gsh ( e.g. , arg70 ) or a 
 hydrogen bond formed directly with gsh could be observed in the molecular 
 docking . for generation of the proinflammatory pge2 , the release 
 of aa as substrate and its conversion by cox enzymes are essential 
 upstream processes leading to the formation of pgh2 as 
 substrate for various prostanoid synthases . as mpges-1 is considered to be a valuable drug target for selective 
 inhibition of pge2 biosynthesis , a concomitant suppression 
 of cox enzymes would obliterate the selectivity of such compounds . in fact , the boswellic acids 14 , the tirucallic acid 6 , and the roburic acid 11(22 ) were reported to inhibit cox enzymes , though at higher concentrations , 
 in the two - digit micromolar range . to estimate the selectivity of 
 the triterpene acids that potently inhibited mpges-1 activity with 
 ic50 < 3 m ( i.e. , 510 , 13 , and 17 ) , the impact on cox 
 enzymes was analyzed . in cell - free assays using purified ovine cox-1 
 or purified human recombinant cox-2 , the triterpene acids ( 10 m ) 
 elicited only moderate inhibitory effects without statistical significance 
 ( p > 0.05 ; table 2 ) . , 
 we analyzed the major triterpene acids present in 
 gum resins of boswellia spp . for 
 their ability to interfere with mpges-1 , and we identified six tirucallic 
 acids ( 510 ) , one lupeolic acid ( 17 ) , and one roburic acid ( 13 ) as potent inhibitors 
 of this enzyme . of interest , these triterpene acids , except 9 , inhibit mpges-1 activity with improved potency ( ic50 = 0.41.2 m ) as compared to boswellic acids 
 ( ic50 = 330 m ) . molecular docking simulations confirm the improved potencies of 
 tirucallic acids and lupeolic acids , as hydrogen bonds to or ionic 
 interactions with the cofactor gsh , the neighboring arg70 , or arg73 
 mediate tight binding , whereas the boswellic acid 1 forms 
 hydrophobic interactions at a greater distance to the cofactor gsh , 
 implying weaker binding . we suggest that mpges-1 is a major target 
 of several triterpene acids that are contained in gum resins of certain boswellia spp . , and suppression of mpges-1 by these compounds 
 may underlie their beneficial properties in the treatment of painful 
 inflammatory disorders . the structures of the 
 triterpene acids used in this study have been described previously 
 and were isolated from the gum resin of b. serrata , b. papyrifera , b. carterii , and b. socotrana , respectively . the gum resins from b. serrata , b. papyrifera , b. carterii , and b. sacra were purchased from gerhard eggebrecht 
 vegetabilien & harze , sderau , germany , whereas the gum 
 resin of b. socotrana was a gift by the botanical 
 garden and botanical museum ( bgbm ) in berlin - dahlem , germany . fractions obtained by extraction and/or chromatography 
 were stored in a refrigerator at 30 c until they were 
 used . structure elucidation was done by 1d- and 2d - nmr spectroscopy 
 on a bruker av ii 400 and/or a bruker av 500 nmr spectrometer . as 
 1d - nmr spectra , h , c , dept90 , and dept135 
 spectra h - gs - dqf - cosy , hmqc , hmqc - cosy , 
 hmbc , and noesy spectra were recorded . the molecular formula was calculated 
 from hrms spectra obtained with mat95s ( hrms with ci ) from bruker . esi mass spectra were measured with zq4000 from waters ( esi in the 
 negative ion mode ) . for more details , see the supporting information . then , it was finely 
 ground in a laboratory mill and extracted in a soxhlet extractor with 
 distilled dichloromethane for 16 h. a 180 g amount of the finely ground 
 resin was extracted with ca . the solvent was evaporated 
 with the aid of a rotary evaporator at 40 c under vacuum ( ca . the residue ( raw extract ) was dissolved in 200 ml of diethyl 
 ether and extracted with 200 ml of 5% ( m / v ) aqueous koh in a separatory 
 funnel . after separating the phases , the alkaline aqueous phase was 
 extracted three times with 50 ml of diethyl ether each . the combined 
 ethereal phases were washed with brine ( 20 ml ) and dried with mgso4 . after filtration of the drying agent , the diethyl ether 
 was evaporated under vacuum in a rotary evaporator . the alkaline 
 aqueous phase from above was cooled in an ice bath and carefully acidified 
 with ice cold , concentrated aqueous hydrochloric acid . the combined extracts were washed with brine ( 20 ml ) and dried 
 over mgso4 . after filtration of the drying agent , the solvent 
 was evaporated under vacuum . the remaining yellow to orange foam is 
 the acidic fraction of the resin and contains all lipophilic acids 
 of the particular resin . the amounts of the neutral fraction and the 
 acidic fraction depend strongly on the particular resin . the column 
 was packed with a slurry of silica gel ( normal phase 
 np , merck ag , darmstadt , germany ) with particle size from 40 to 63 
 m in the appropriate mobile phase . after covering the silica 
 gel with a small pad of purified sand , the sample , dissolved in a 
 small amount of mobile phase , was applied on top of the column . elution 
 of the compounds was done with 500 ml portions of mobile phase ( usually 
 pentane / diethyl ether + 1% ( v / v ) of acetic acid ) with stepwise increasing 
 polarity ( starting with pentane / diethyl ether , 8:1 , then 7:1 , then 
 6:1 , and so on up to pentane / diethyl ether , 1:2 ) . ml each ) were collected in test tubes with the aid of a fraction 
 collector . analysis of the fractions was done with tlc ( merck glass 
 plates ) with a suitable solvent . fractions with similar composition 
 were combined , and the solvent was evaporated to dryness . usually , 
 a bright yellow to orange foam is obtained . these combined fractions 
 were analyzed by analytical hplc and further separated by preparative 
 hplc . preparative hplc was done with a preparative hplc pump 
 model sykam s1521 ( sykam , frstenfeldbruck , germany ) , a rheodyne 
 injection valve type 7725i , a diode array detector model sykam s3210 
 ( sykam , frstenfeldbruck , germany ) , and chromstar 6.0 software 
 ( scpa , weyhe - leeste , germany ) . as a column , a semipreparative nucleodur 
 100 - 5 c18 ec , 5 m , 250  20 , from machery & 
 nagel , dren , germany , was used . the purity of the isolated 
 compounds used for biological evaluation was at least 95% . g / l ) 
 medium , penicillin , streptomycin , trypsin / edta solution , paa ( coelbe , 
 germany ) , pgh2 ( larodan , malm , sweden ) , 11-pge2 and mk-886 ( biotrend chemicals gmbh , cologne , germany ) , arachidonic 
 acid , and fetal calf serum were used , and all other chemicals were 
 obtained from sigma - aldrich ( deisenhofen , germany ) unless stated otherwise . induction 
 of mpges-1 expression in a549 cells and isolation of microsomes 
 were performed as described . in brief , 
 cells were incubated for 16 h at 37 c and 5% co2 , 
 and after changing the medium , mpges-1 expression was induced by il-1 
 ( 1 ng / ml ) . after 72 h , cells were frozen in liquid nitrogen , ice - cold 
 homogenization buffer ( 0.1 m potassium phosphate buffer ph 7.4 , 1 
 mm phenylmethanesulfonyl fluoride , 60 g / ml soybean trypsin 
 inhibitor , 1 g / ml leupeptin , 2.5 mm gsh , and 250 mm sucrose ) 
 was added , and after 15 min cells were resuspended and sonicated on 
 ice ( 3  20 s ) . the homogenate was then subjected to differential 
 centrifugation at 10000 g for 10 min and at 174000 g for 1 h at 4 c . the pellet ( microsomal fraction ) 
 was finally resuspended in 1 ml of homogenization buffer , and the 
 protein concentration was determined by the coomassie protein assay . microsomal membranes of a549 cells were diluted in potassium phosphate 
 buffer ( 0.1 m , ph 7.4 ) containing 2.5 mm gsh ( 100 l total volume ) , 
 and pge2 formation was initiated by addition of pgh2 ( 20 m , final concentration ) . after 1 min at 4 c , 
 the reaction was terminated with 100 l of stop solution ( 40 
 mm fecl2 , 80 mm citric acid , and 10 m 11-pge2 ) , and pge2 was separated by solid - phase extraction 
 and analyzed by rp - hplc as described . briefly , purified cox-1 ( ovine , 50 units ) or 
 cox-2 ( human recombinant , 20 units ) was diluted in 1 ml of reaction 
 mixture containing 100 mm tris buffer ph 8 , 5 mm glutathione , 5 m 
 hemoglobin , and 100 m edta at 4 c and preincubated with 
 the test compounds for 5 min . samples were prewarmed for 60 s at 37 
 c , and aa ( 5 m for cox-1 , 2 m for cox-2 ) was added 
 to start the reaction . after 5 min at 37 c , 12(s)-hydroxy-5z,8e,10e - heptadecatrienoic acid was extracted and then analyzed by hplc . the binding modes of the investigated 
 triterpene acids were analyzed using the quantum mechanics - polarized 
 ligand docking ( qpld ) workflow , which is available 
 in the maestro suite version 9.2.112 . for this purpose , the x - ray crystal structure of mpges-1 with bound 
 cofactor gsh was used , which is deposited in the protein data bank 
 ( pdb , http://rcsb.org/pdb/ ) , entry 3dww . for the docking procedure , 2d 
 structures of the triterpene acids were converted into 3d coordinates 
 employing maestro s module ligprep . this included the ionization 
 of acidic moieties of the ligands with the module ligprep ( epik / opls-2005 ) . the 
 hydrogen atoms were added , and atom and bond types were assigned , 
 which was followed by exploration of the hydrogen bond assignment 
 in  extensive  mode . within this procedure , furthermore , the protein was refined by 
 a minimization as a final step of the protein preparation within the 
 respective assistant ( opls - aa 2005/rmsd threshold : 0.3  ) . the 
 molecular docking was performed with glide in extra precision ( xp ) 
 mode and a scaling of the receptor van der waals radius by a factor 
 of 0.9 . in the qpld workflow , the proposed orientation of the ligands 
 within the binding site of the macromolecule target is used to calculate 
 atomic ( partial ) charges of the ligands employing the quantum mechanical / molecular 
 mechanical ( qm / mm ) approach performed with the module qsite ( semiempirical 
 method / mulliken charges ) . the initial docking poses were submitted 
 to a second docking procedure with glide in xp mode , involving atomic 
 ( partial ) charges of ligands from the qm / mm approach . the final docking 
 poses were ranked according to the calculations from the glidescore 
 scoring function . the analysis of the docking poses , which were retrieved 
 from the qpld workflow , was performed within ligandscout version 3.1 following an mmff94-based minimization of the investigated triterpene 
 acid and of the binding site residue side chains within ligandscout , 
 which was also used for visualization purposes . , san diego , ca , usa ) 
 was used for statistical comparisons of the data by one - way anovas 
 for independent or correlated samples followed by tukey hsd post hoc tests . where appropriate , student s t test for paired and correlated samples was applied ic50 values of compounds are approximations determined by graphical 
 analysis ( linear interpolation between the points between 50% activity ) .