on the basis of experimental data from in - house screening 
 and from 
 the literature , two natural compounds , the neolignan honokiol and 
 the diarylheptanoid katsumadain a ( chart 1 ) , 
 were selected as templates for a similarity search . honokiol is a moderately active 
 inhibitor with an ic50 of 3.01 m against the na 
 of the historic influenza a strain 
 pr/8/34 , as determined in a chemiluminescence ( cl)-based na inhibition 
 assay . interestingly , its activity is more potent against the oseltamivir - resistant 
 seasonal h1n1 strain b/55/08 ( ic50 1.39 m ) . katsumadain a was discovered as an nai with an ic50 of 
 1.05 m ( pr/8/34 ) in an earlier study . with its t - shaped structure ( chart 1 ) , this 
 bulky compound represents an unusual and novel influenza na inhibiting 
 scaffold . molecular dynamics simulations and docking have suggested 
 that katsumadain a is likely to bind to an extended ( i.e. , more widely 
 open ) na binding pocket , a result of the conformational flexibility 
 of the 430- and 245-loops . in a follow - up study , katsumadain a served as a lead structure in finding further 
 highly active and resistance - breaking nais using shape - focused virtual 
 screening . in the present work , 
 honokiol and katsumadain a were used as chemically 
 diverse templates for the identification of plant material with an 
 accumulation of constituents that are likely to be active against 
 influenza na . to find novel resistance - breaking nais from natural sources , the 
 two templates selected were applied to a 3d similarity screening of 
 the tcm database@taiwan using the program 
 rocs . the similarity was quantified using the tanimotocombo score , 
 which is a combination of shape similarity ( shapetanimoto score ) and 
 chemical similarity ( colortanimoto score ) ( http://www.eyesopen.com/docs/rocs/current/html/index.html ) . it is the sum of the shapetanimoto 
 and colortanimoto score , which both range from 0 to 1 and equally 
 contribute to the combined score . the higher the scores , the more 
 similar is the conformation of the database molecule to the conformation 
 of the template . the rank - ordered list of molecules obtained was analyzed 
 regarding their origin , knowledge of anti - influenza / antiviral activity 
 of the tcm plants , and hints from traditional medicine about beneficial 
 effects of natural - based influenza remedies that are known to contain 
 the respective compounds as prominent constituent and accessibility 
 of the plant material . virtual screening indicated that the roots 
 of glycyrrhiza species contain several known constituents 
 ( as reported in the tcm 
 database@taiwan ) with significant structural 
 similarities to template a or b. considering the degree of similarity 
 between all 103 glycyrrhizaeradix database entries and both templates , it was found that they generally 
 show a higher degree of similarity to honokiol than katsumadain a. 
 with a tanimotocombo score of 0.70 or higher , constituents were identified 
 that have been isolated reportedly from this plant material ( i.e. , 
 compounds 1 , 3 , 4 , 6 , and 10 ; chart 2 ) . this prediction 
 prompted the investigation of the roots of g. glabra phytochemically and the evaluation of the in vitro na inhibiting 
 activity of its constituents , with a preference for compounds similar 
 to the template structures . dried and processed roots of g. glabra ( liquiritiae radix ) are commonly 
 referred to as licorice . for instance , in mainland china , the remedy is known under 
 the name gan - cao , which includes the dried roots 
 and stolons of three glycyrrhiza species , i.e. , g. uralensis , g. glabra , and g. 
 inflata . major compound classes 
 responsible for the bioactivity of gan - cao are triterpene 
 saponins , flavonoids ( including isoflavones ) , and coumarins . as recently reviewed , in particular flavonoids show a wide variety 
 of biological properties , such as antiulcer , antioxidant , anti - inflammatory , 
 antimicrobial , antispasmodic , antitumor , and metabolic syndrome preventive 
 effects . strikingly , the roots of glycyrrhiza species are the most frequently used tcm herbs 
 for treating respiratory tract infections . in addition , they are described as antiviral herbal remedies against chronic hepatitis b and c , hiv , cytomegalovirus , 
 and herpes simplex and as ingredients of complex chinese antiviral 
 preparations such as ma - xing - shi - gan - tang , feng - refan - wei , and redu-xi-fei.glycyrrhiza roots are usually 
 applied as honey - roasted licorice , i.e. , zhi - gan - cao . as a starting point 
 for phytochemical investigation , a methanol extract of licorice was 
 prepared to obtain a multicomponent mixture embracing a wide range 
 of secondary metabolites . since glycosidic molecules showed only low 
 similarity to both templates , and in order to mimic the use in traditional 
 preparations , extraction in sulfuric acid was chosen with the intention 
 to break down these glycosides . in order to separate the aglycones 
 from the respective sugars , both the methanol extract and the 
 aglycone - enriched fraction were investigated for their na - inhibiting 
 potential in a cl - based enzyme inhibition assay against h1n1 strain 
 pr/8/34 . as a positive control , the well - established nai oseltamivir 
 ( ic50 0.29 nm ) was used . in this assay , the methanol extract 
 showed an ic50 of 1.70 g / ml , whereas the aglycone - enriched 
 fraction exhibited slightly more potent na inhibitory activity , with 
 an ic50 value of 0.33 g / ml . on the basis of 
 these results , both crude preparations were subjected 
 to phytochemical workup . after several chromatographic separation 
 steps , 12 constituents were isolated ( chart 2 ) , namely , ( e)-1-[2,4-dihydroxy-3-(3-methyl-2-butenyl)phenyl]-3-(8-hydroxy-2,2-dimethyl-2h-1-benzopyran-6-yl)-2-propen-1-one ( 1 ) , 3,4-dihydro-8,8-dimethyl-2h,8h - benzo[1,2-b:3,4-b]dipyran-3-ol ( 2 ) , biochanin b ( 3 ) , glabrol ( 4 ) , glabrone ( 5 ) , hispaglabridin 
 b ( 6 ) , licoflavone b ( 7 ) , licorice glycoside 
 b ( 8) , licorice glycoside e ( 9 ) , liquiritigenin 
 ( 10 ) , liquiritin ( 11 ) , and prunin ( 12 ) . the constituents were identified by using hplc and comparison 
 of their specific rotations and spectroscopic data with published 
 values . compounds 1 to 7 were isolated from 
 the 
 aglycone - enriched fraction , while compounds 8 to 12 were isolated from the methanol extract . the isolated glycyrrhiza constituents can be classified 
 as two benzodipyrans ( 2 , 6 ) and glycosidic 
 ( 8 , 9 , 11 , 12 ) 
 and nonglycosidic ( 1 , 35 , 7 , 10 ) flavonoids . compounds 3 and 11 are known from earlier 
 work to exhibit weak activity against the na of influenza viruses 
 a / pr/8/34 ( h1n1 ) , a / jinan/15/90 ( h3n2 ) , and b / jiangsu/10/2003 in fl - based 
 na inhibition assays . compounds 3 , 5 , 10 , and 11 have 
 been mentioned in chinese or korean patents as ingredients of multicomponent 
 medicinal compositions for the treatment of viral respiratory tract 
 infections . however , the anti - influenza actions or detailed studies 
 of these four compounds have not been reported to date . an overview of compounds identified 
 from g. glabra and their similarities to templates 
 a and b is provided in table 1 . four out of 
 the 12 structures disclosed after their isolation and identification 
 ( 2 , 7 , 8 , and 9 ) have not been covered by shape - focused screening , since they were 
 not present in the virtual screening library . for the 3d alignments 
 performed with all 12 isolates from licorice , seven molecules identified 
 in the plant material ( 1 , 2 , 3 , 4 , 6 , 10 , and 12 ) had a reasonably high degree of similarity with the template molecules 
 ( tanimotocombo score 0.70 ) . the latter is a measure of the 
 geometric fit of pharmacophoric features including hydrogen - bond donors / acceptors 
 and hydrophobic , anionic , cationic , and cyclic moieties . the low colortanimoto 
 scores can be explained by the fact that this global descriptor ( as 
 shapetanimoto ) does not discriminate between different parts of a 
 molecular scaffold , such as parts representing the pharmacophore and 
 areas of the ligand not crucial for bioactivity . this may lead to 
 an underestimation of how well a pharmacophore is represented in a 
 molecule . this frequently occurs , as exemplified in the case of glycosylated 
 molecules such as compound 12 , where the presence of 
 multiple hydrogen - bond donors in the sugar moiety may drastically 
 lower the similarity score measured against an aglycone , even if the 
 ligand parts responsible for bioactivity are closely related . carbon atoms of 
 the template structure 
 blue , of the query structure gray . similarity threshold too low . to evaluate 
 experimentally 
 the inhibitory activity against influenza virus na , tests 
 were conducted with a historic h1n1 strain ( pr/8/34 ) , an oseltamivir - resistant 
 seasonal h1n1 strain ( b/55/08 ) , a virus strain of the pandemic h1n1 
 lineage that emerged in 2009 ( j/8178/09 ) , and a h3n2 virus ( hk/68 ) . the resistance of oseltamivir against n1 of b/55/08 was shown previously . except for compound 11 , which showed an ic50 of 83.1 m , all compounds studied inhibited the oseltamivir - susceptible 
 viral na of influenza virus pr/8/34 at concentrations lower than 10 
 m . in general , the na of the oseltamivir - resistant b/55/08 
 was less susceptible to most of the compounds . however , similar ic50 ranges were determined for 6 and 12 against oseltamivir - susceptible and -resistant h1n1 strains . n = 3 , each concentration 
 in duplicate . to further investigate the 
 anti - influenza potential of the constituents 
 from g. glabra , cytopathic effect ( cpe ) inhibitory 
 assays were performed with the pandemic h1n1 j/8178/09 and the h3n2 
 hk/68 influenza a virus ( table 3 ) . to reduce 
 the risk of nonspecific compound action , cytotoxicity was determined 
 in parallel ( table 3 ) . in contrast , and with 
 the exception of 4 , all compounds obtained from the aglycone - enriched 
 fraction exhibited antiviral activity against at least one of the 
 viruses studied . percentage 
 of maximal inhibition 
 of the cpe at a concentration of 50 m . not active ( up to 50 m ) . recently , concerns 
 were raised about the reliability of na inhibition assays for the 
 testing of natural compounds , in particular for flavonoids in combination 
 with fl - based assays . fl quenching was shown to lead 
 to false - positive results with natural compounds containing a substituted 
 phenyl - benzopyran scaffold . it was investigated as to whether 
 licorice - derived compounds can lead to such unfavorable effects by 
 using an fl - based assay . however , this assay did not work for viral 
 na as tested for compound 3 ( results not shown ) , a compound 
 that inhibited j/8178/09 and hk/68 in the cpe inhibition assay . in 
 contrast this allowed a comparative study of compounds 2 to 7 isolated from the aglycone - enriched fraction with this bacterial 
 na in an fl- as well as cl - based assay ( table 4 ) . the percentage of self - fluorescence was calculated setting the fluorescence 
 of 4-methylumbelliferone ( 4-mu ; cleavage product ) in the test buffer 
 to 100% . to determine possible quenching effects , fluorescence of 
 4-mu with and without the presence of compounds the 
 percentage of quenching was determined by setting the fluorescence 
 of 4-mu to 100% . with the exception of 3 ( 15.41% compared 
 to test buffer without compound ) , self - fluorescence of the test compounds 
 was generally low ( < 4% ) . quenching of fl of about 40% and 30% was 
 determined for compounds 4 and 5 , respectively . all licorice constituents tested inhibited bacterial na in both the 
 cl and fl assay . in comparison to viral na , the ic50 values 
 determined for c. perfringens na were about 10 times 
 lower in the cl assay . high concentrations of the virus - specific nai 
 oseltamivir had to be added to inhibit this bacterial na by 50% as 
 reported previously . optical rotations were 
 measured on a perkin - elmer 341 polarimeter at 25 c . 1d and 2d 
 nmr experiments were performed by using bruker drx300 and txi600 nmr 
 spectrometers , operating at 300 and 600 mhz , respectively . the samples 
 were measured in meod ( calibrated to the residual nondeuterated solvent 
 signals ) . ms analysis was performed on an esquire 3000 plus ion - trap 
 mass spectrometer ( bruker daltonics ) equipped with electrospray ionization 
 ( esi ) in the positive and negative modes : spray voltage , 4.5 kv ; sheath 
 gas , n2 , 30 psi ; dry gas , n2 , 6 l min , 350 c ; scanning range , m / z 501000 . column chromatography was performed using merck 
 silica gel 60 ( 4063 m ) and pharmacia sephadex lh-20 
 ( 20100 m ) . the fractions obtained from all chromatographic 
 steps were analyzed by tlc ( mobile phase , ch2cl2meoh ( 9:1 ) , etoac  etoh nh3 ( 13:5:1.8:0.2 ) , or ch2cl2meoh  hcooh 
 ( 9:1.5:0.5 ) ; stationary phase , merck silica gel 60 pf254 , detected with staining reagents anisaldehyde / h2so4 at vis , uv254 , uv366 ) . hplc was performed 
 on a shimadzu uflc - xr instrument ( kyoto , japan ) with a photodiode 
 array detector . lc parameters : stationary phase , phenomenex luna phenyl - hexyl , 
 150  3.00 mm , 3 m , agilent ; temperature , 40 c ; 
 mobile phase , water ( a ) ; acetonitrile ( b ) ; flow rate , 0.2 ml / min ; 
 uv detection wavelength , 205 , 254 , 280 , 310 , 360 nm ; injection volume , 
 10 l ; gradient , 80% a , 20% b , 7 min ; 68% a , 32% b , 20 min ; 
 67% a , 33% b , 23 min ; 2% a , 98% b , 37 min ; 2% a , 98% b. all chemicals 
 and solvents used were analytical grade . honokiol ( 98% purity ) was 
 purchased from quality phytochemicals llc , east brunswick , nj , usa . the roots of g. glabra l. 
 were obtained from  mag . kottas  heilkruter  , 
 eitnergasse 8 , 1230 vienna , austria ( a924304 - 001 ) . voucher specimens 
 ( jr-20091106-a1 ; jr-20091203-e1 ) are deposited in the herbarium of 
 the institute of pharmacy / pharmacognosy , lfu , innsbruck , austria . the dried ground roots of g. glabra ( 529.6 g ) were macerated with meoh ( at room temperature , 
 three times for 72 h each ) . after removal of the solvent under vacuum , 
 the meoh extract ( 87.0 g ) was obtained . another 232.8 g of dried ground 
 roots of g. glabra was extracted with 1 l of 1 n 
 h2so4 for 2 h at a temperature of 100 c 
 to hydrolyze the glycosides . the extract obtained was partitioned 
 with ch2cl2 in order to separate the aglycones 
 from sugars in the remaining aqueous phase . the etoac fraction 
 ( 7.2 g ) was subjected to silica gel column chromatography ( merck silica 
 gel 60 pf254 , 213 g ; 65 cm  4 cm ) using a gradient 
 system of n - hexane  etoac  meoh to give 
 23 fractions ( a123 ) . fractions a69 ( 374.2 mg ) were 
 combined and further separated using silica gel column chromatography 
 ( merck silica gel 60 pf254 , 213 g ; 65 cm  4 cm ) applying 
 a gradient system of ch2cl2acetone to 
 yield 23 fractions ( b123 ) . fraction b7 ( 3.3 mg ) was purified 
 via sephadex lh-20 column chromatography ( mobile phase : meoh ) to give 
 1.3 mg of compound 10 . fraction a13 ( 278.0 mg ) was subjected 
 to sephadex lh-20 column chromatography ( mobile phase : meoh ) , yielding 
 eight fractions ( d18 ) . from fraction d8 fraction a14 ( 942.5 mg ) was chromatographed 
 over a sephadex lh-20 column eluted with meoh to give six fractions 
 ( e16 ) . fraction e4 ( 330.0 mg ) was subjected to silica gel 
 column chromatography ( merck silica gel 60 pf254 , 213 g ; 
 65 cm  4 cm ) eluting with a gradient solvent system of ch2cl2meoh , yielding seven fractions ( f17 ) . fraction f3 ( 150.3 mg ) was further separated by means of a sephadex 
 lh-20 column ( mobile phase : meoh ) to give four fractions ( g14 ) . fraction g1 ( 67.5 mg ) was purified by a lobar chromatography system 
 ( merck lobar commercial column art . : 10624 ; size : a ( 24010 ) ; 
 lichroprep rp-18 4060 mm ; gradient elution with ch3cn the combined fractions g34 ( 41.9 mg ) were purified by lobar 
 chromatography ( gradient elution with ch3cn fraction e6 
 ( 34.1 mg ) was subjected to lobar chromatography ( gradient elution 
 with ch3cn fraction j4 ( 8.2 mg ) was purified via a sephadex lh-20 
 column ( mobile phase : meoh ) , resulting in 7.1 mg of compound 9 . the aglycone - enriched fraction ( 2.2 g ) was submitted 
 to passage 
 over a sephadex lh-20 column ( mobile phase : ch2cl2acetone ) to yield seven fractions ( a17 ) . fractions 
 a46 ( 1.0 g ) were combined and chromatographed over a silica 
 gel column using a gradient solvent system of hexane fraction b2 ( 8.0 mg ) was separated via 
 a sephadex lh-20 column ( mobile phase : meoh ) , giving three fractions 
 ( c13 ) . fraction b5 ( 31.3 mg ) was further purified via a sephadex lh-20 
 column eluted with meoh to give five fractions ( d15 ) . compound 2 ( 3.1 mg ) and compound 1 ( 1.5 mg ) were obtained 
 from fractions d1 and d5 , respectively . fraction b7 ( 110.4 mg ) was 
 separated on a sephadex lh-20 column eluted with meoh to yield seven 
 fractions , e17 . fraction e3 ( 39.9 mg ) was subjected to lobar 
 chromatography ( gradient elution with meoh  h2o ) , 
 yielding five fractions ( f15 ) . then , 23.5 mg of fraction f4 
 was subjected to sephadex lh-20 column chromatography ( mobile phase : 
 meoh ) , yielding six fractions ( g16 ) . from fraction g3 fractions e4 and 
 e5 were combined ( 22.9 mg ) and subjected to lobar chromatography ( gradient 
 elution with meoh  h2o ) , yielding five fractions 
 ( h15 ) . among them , h3 afforded 2.2 mg of pure compound 5 . fraction b8 ( 351.4 mg ) was separated via a sephadex lh-20 
 column using meoh as the eluent . this separation resulted in five 
 fractions ( i15 ) , with 83.9 mg of fraction i3 further purified 
 via lobar chromatography ( gradient elution with meoh fraction 
 j10 ( 5.7 mg ) was purified via passage over a sephadex lh-20 column 
 ( mobile phase : meoh ) , yielding 2.8 mg of pure compound 7 . next , 48.3 mg of fraction i4 was purified via lobar chromatography 
 ( gradient elution with meoh  h2o ) to give 3.9 mg 
 of pure compound 3 . the physical and spectroscopic 
 data of compounds 1 to 12 agreed with those 
 published previously for ( e)-1-[2,4-dihydroxy-3-(3-methyl-2-butenyl)phenyl]-3-(8-hydroxy-2,2-dimethyl-2h-1-benzopyran-6-yl)-2-propen-1-one ( 1 ) , 3,4-dihydro-8,8-dimethyl-2h,8h - benzo[1,2-b:3,4-b]dipyran-3-ol ( 2 ) , biochanin b ( 3 ) , glabrol ( 4 ) , glabrone ( 5 ) , hispaglabridin 
 b ( 6 ) , licoflavone b ( 7 ) , licorice glycoside 
 b ( 8) , licorice glycoside e ( 9 ) , liquiritigenin 
 ( 10 ) , liquiritin ( 11 ) , and prunin ( 12 ) . their purity was checked using 
 tlc and lc - ms and was revealed to be > 98% in all cases . tcm database@taiwan version 1.0 contains 
 about 40  000 molecules related to traditional chinese medicines . the database was converted into mdl sd file format using schrdinger 
 maestro suite ( version 9.2 ) . conformational 
 models for all compounds were calculated using omega ( version 2.3.2 
 openeye scientific software , santa fe , nm , usa ) , with default settings . in total rocs ( version 2.4.2 openeye 
 scientific software ) was used to rank 
 all molecules by decreasing similarity to the templates a and b. the 
 minimum energy conformation for each template ( calculated with omega , 
 default settings ) was used as rocs query . omega and rocs calculations were run 
 on an apple macbook air with an intel core i7 1.8 ghz cpu and 4 gb 
 of ram under mac os x version 10.7 . h1n1 influenza viruses a / jena/8178/09 
 ( j/8178/09 ; isolated from nasal swabs of an influenza patient during 
 the pandemic of 2009 ) , a / pr/8/34 ( pr/8/34 ; department of virology 
 and antiviral therapy , jena , germany ) , and a / berlin/55/08 ( b/55/08 : 
 oseltamivir - resistant , seasonal h1n1 isolate , published using the 
 sample number 342/09 ; robert - koch - institute , 
 berlin , germany ) as well as the h3n2 strain a / hongkong/68 ( hk/68 ; 
 strain collection of the department of virology and antiviral therapy , 
 jena , germany ) were used in antiviral studies . they were propagated 
 in madin darby canine kidney ( mdck ) cells ( friedrich - loeffler institute , 
 riems , germany ) in serum - free eagle s minimum essential medium 
 supplemented with 100 u / ml penicillin as well as streptomycin , 2 g / ml 
 trypsin , and 1.2 mm bicarbonate . titers 
 of virus stocks were determined according to reed and muench , 1938 
 in mdck cells . inhibition of 
 the na of influenza a viruses and/or c. perfringens ( sigma - aldrich , chemie gmbh , munich , germany ) was analyzed by applying 
 the fl- and/or cl - based na inhibition assays na - fluor influenza neuraminidase 
 assay kit and na - star influenza neuraminidase inhibitor resistance 
 detection kit ( both applied biosystems , darmstadt , germany ) , according 
 to the manufacturer s instructions . for determination of 50% 
 inhibitory concentrations ( ic50s ) , na inhibition was tested 
 with serial log 10 ( fl assay ) or half - log ( cl assay ) nai concentrations , 
 as previously published for oseltamivir and zanamivir ( maximum tested 
 concentration for pure compounds : 100 m ) . oseltamivir 
 carboxylate ( tamiflu ; hoffmann - la roche ag , basel , switzerland ) was 
 used as a positive control . fl and cl were read using a microtiter 
 plate luminometer ( fluostar omega , bmg - labtech gmbh , ortenberg , germany ) . means and standard 
 deviations of ic50 values were calculated by linear interpolation 
 ( pr/8/34 and b/55/08 ) and/or according to recommendations of the centers 
 of disease control and prevention ( cdc ) with the jaspr curve fitting software ( kindly provided by larisa 
 v. gubareva , cdc ) . additionally , self - fluorescence and quenching of 
 compounds were analyzed using 100 m compound solutions . to 
 calculate the percentage of self - fluorescence of the test compounds , 
 the fluorescence of 4-methylumbelliferone ( 4-mu , the fluorescent cleavage 
 product ) in the test buffer was set to 100% . to determine quenching , the fluorescence of 3.1 m of 4-mu without and with 100 m 
 of compounds was compared . to calculate the percentage of quenching , the 50% cytotoxic concentration 
 ( cc50 ) as well as 50% 
 inhibitory concentration ( ic50 , inhibition of virus - induced 
 cpe ) was determined on two - day - old confluent mdck cell monolayers 
 grown in 96-well plates as described previously ( maximum tested concentration : 
 50 m ) . a multiplicity of infection of 0.001 and 0.003 
 tcid50/cell of j/8178/09 ( pandemic h1n1 ) and hk/68 ( h3n2 ) 
 resulted in a complete cpe at this time point . each concentration 
 was tested in triplicate or once for determination of cytotoxicity 
 and cpe inhibition , respectively . the mean cc50 and ic50 values and standard derivations were calculated from at 
 least two ( j/8178/09 ) or three ( hk/68 ) experiments .
neuraminidase ( na ) , a key enzyme 
 in viral replication , is the first - line 
 drug target to combat influenza . on the basis of a shape - focused virtual 
 screening , 
 the roots of glycyrrhiza glabra ( licorice ) 
 were identified as plant species with an accumulation of constituents 
 that show 3d similarities to known influenza na inhibitors ( nais ) . 
 phytochemical investigation revealed 12 constituents identified as 
 ( e)-1-[2,4-dihydroxy-3-(3-methyl-2-butenyl)phenyl]-3-(8-hydroxy-2,2-dimethyl-2h-1-benzopyran-6-yl)-2-propen-1-one ( 1 ) , 3,4-dihydro-8,8-dimethyl-2h,8h - benzo[1,2-b:3,4-b]dipyran-3-ol ( 2 ) , biochanin b ( 3 ) , glabrol ( 4 ) , glabrone ( 5 ) , hispaglabridin 
 b ( 6 ) , licoflavone b ( 7 ) , licorice glycoside 
 b ( 8) , licorice glycoside e ( 9 ) , liquiritigenin 
 ( 10 ) , liquiritin ( 11 ) , and prunin ( 12 ) . 
 eleven of these constituents showed significant influenza 
 virus na inhibition in a chemiluminescence ( cl)-based assay . 
 additional 
 tests , including ( i ) a cell - based cytopathic effect inhibition assay 
 ( general antiviral activity ) , ( ii ) the evaluation of cytotoxicity , 
 ( iii ) the inhibition of the na of clostridium perfringens ( cl- and fluorescence ( fl)-based assay ) , and ( iv ) the determination 
 of self - fluorescence and quenching , provided further perspective on 
 their anti - influenza virus potential , revealing possible assay interference 
 problems and false - positive results . 
 compounds 1 , 3 , 5 , and 6 showed antiviral activity , 
 most likely caused by the inhibition of na . 
 of these , compounds 1 , 3 , and 6 were highly ranked in 
 shape - focused virtual screening .