metabolites were isolated from roots and aerial 
 parts of the plant using similar isolation methods . fractionation 
 of extracts of d. searlsiae by silica gel vlc , sephadex 
 lh-20 , and gradient chromatography over silica gel afforded the four 
 new flavanones malheurans a  d ( 14 ) and two known prenylated flavanones , 5 and 
 prostratol f ( 6 ) , from the roots . aerial parts of the 
 plant yielded the known pterocarpans tephrosin ( 7 ) and 
 milletosin ( 8) and the known isoflavonoids griffonianone 
 e ( 9 ) and calopogonium isoflavone a ( 10 ) . the structures of all 10 compounds were determined by nmr and hresims , 
 and the absolute configurations of compounds 15 , 7 , and 8 were determined by electronic 
 circular dichroism ( ecd ) . compounds 2 and 3 were analogous to two known metabolites of d. versicolor(10 ) and d. scandens var . paucifolia(7 ) that differed only 
 in the presence of hydroxy groups at c-5 . the hresims , c nmr ( table 1 ) , and dept data for compound 1 indicated a molecular formula of c25h28o5 . a pattern characteristic of a flavanone core structure 
 was indicated by hsqc correlations between oxymethine ( h 5.70 ; table 1 ) and methylene ( h 2.94 and 2.72 ) proton resonances of the c - ring and carbons 
 at c 76.0 and 44.0 . the c and dept 
 data indicated the presence of 10 nonprotonated carbons , including 
 a ketocarbonyl at c 191.7 and four oxygenated sp carbons between c 156 and 163 . three hydroxy 
 protons and the multiplicities of the five observed aromatic protons 
 permitted the placement of two substituents each on the flavanone 
 a- and b - rings , while the h-5 o - coupled ( 8.6 hz ) 
 doublet indicated a 7,8-disubstituted flavanone a - ring . the molecular 
 formula supported the presence of a geranyl group as the fourth aromatic 
 substituent , and its placement at c-8 was confirmed through hmbc correlations 
 from h2 - 1 to c-7 , c-8 , and c-9 and from h-2 
 to c-8 . the c-2 double bond was assigned an e - configuration , as the c-3 methyl resonated at c 16.4 instead of at c 23 , as would 
 be expected for a z - configuration . the h nmr data indicated a b - ring with an abx spin system , and hmbc correlations 
 from h-6 to c-2 , c-2 , and c-4 and from h-2 
 to c-1 , c-2 , and c-6 verified the substitution 
 pattern of the b - ring and its connectivity to the c - ring . the overall 
 structural connectivity was established by hsqc and hmbc spectroscopic 
 data ( figures s10 and s11 , supporting information ) and by comparison with known compounds . thus , in conjunction with ecd data ( vide infra ) , the structure of 1 was assigned as the new compound ( 2s , e)-8-(3,7-dimethylocta-2,6-dien-1-yl)-7,2,4-trihydroxyflavanone 
 and named malheuran a. assignments 
 may be interchanged . the 
 molecular formula , c25h28o5 , of compound 2 was established via its c nmr and hresims data . 
 as with 1 , hsqc data ( table 2 ) 
 confirmed a flavanone core structure , with oxymethine ( h 5.71 ) and methylene ( h 3.00 and 2.74 ) proton 
 resonances correlating to carbons at c 76.3 ( c-2 ) 
 and 44.0 ( c-3 ) , respectively . three hydroxy protons and the multiplicities 
 of the four aromatic protons indicated di- and trisubstituted a- and 
 b - rings , respectively . similar to 1 , the a - ring of 2 contained o - coupled ( 8.6 hz ) doublets for 
 h-5 and h-6 . the structure and placement of the c-8 prenyl group and 
 the overall substitution pattern of the a - ring were readily deduced 
 from hmbc correlations . the nmr assignments of h2 - 3 were 
 based on coupling constants relative to h-2 . h-3a and h-3b exhibited 
 hmbc correlations to c-2 , c-10 , and c-1 similar to those observed 
 for compound 1 . the b - ring protons of 2 resonated 
 as singlets , assignable to p - oriented h-3 
 ( h 6.49 ) and h-6 ( h 7.44 ) . hmbc correlations from h-6 to c-2 and to the quaternary c-2 
 ( c 40.8 ) verified the regiochemistry of this portion 
 of the b - ring and the nature of the c-5 prenyl group . all 
 three protons characteristic of the vinylic portion of this group 
 ( h 6.29 , 5.01 , and 4.95 ) correlated to c-2 
 in the hmbc spectrum . the c-3 proton correlated to c-5 , 
 establishing the location of the prenyl group at this carbon . the 
 remaining regiochemistry of the b - ring was assigned via 1d- and 2d - nmr 
 methods ( figures s12s15 , supporting information ) . following ecd analysis ( vide infra ) , the structure of the new 
 compound 2 was assigned as ( 2s)-5-(2-methylbut-3-en-2-yl)-8-(3-methylbut-2-en-1-yl)-7,2,4-trihydroxyflavanone 
 and named malheuran b. assignments may be interchanged . the hresims and c nmr 
 data of compound 3 indicated a molecular formula of c26h30o5 . spectroscopic data for 3 ( table 2 ; figures s16s19 , supporting information ) were similar to those 
 of 2 with the additional feature of an o - methyl group ( h 3.79 , c 55.9 ) , 
 consistent with the difference in the molecular formula as compared 
 to 2 . the placement of the o - methyl 
 and two prenyl groups of 3 was verified by hmbc correlations 
 from o - methyl , methylene , and methine protons to 
 neighboring a- and b - ring carbons , respectively . remaining structural 
 assignments were made based on analysis of hsqc and hmbc correlations , 
 comparison to the spectroscopic data of 2 , and ecd analysis 
 ( vide infra ) . the structure of the new compound was therefore assigned 
 as ( 2s)-7,4-dihydroxy-2-methoxy-5-(2-methylbut-3-en-2-yl)-8-(3-methylbut-2-en-1-yl)flavanone 
 and named malheuran c ( 3 ) . the h nmr 
 data for 4 resembled those of 1 , except 
 for a six - proton singlet ( h 1.48 ) and deshielded 
 resonances ( h 6.29 , 5.01 , and 4.95 ) with multiplicities 
 and coupling constants ( table 1 ) characteristic 
 of a vinyl group . the hresims and c nmr data of 4 revealed a molecular formula of c30h36o5 , consistent with the presence of an additional prenyl 
 group as compared to 1 . two aromatic protons resonating 
 as singlets indicated a p - disubstituted b - ring . the 
 remaining structural assignments were made based on hsqc and hmbc 
 data ( figures s22 and s23 , supporting information ) , comparison with 1 , and ecd analysis ( vide infra ) . the structure of the new compound was therefore assigned as ( 2s , e)-8-(3,7-dimethylocta-2,6-dien-1-yl)-5-(2-methylbut-3-en-2-yl)-7,2,4-trihydroxyflavanone 
 and named malheuran d ( 4 ) . spectroscopic data for 
 compound 5 resembled those of 2 with the 
 addition of a c-5 hydrogen - bonded hydroxy proton , as indicated by 
 a h nmr resonance at h 12.52 . comparison 
 of the nmr and ecd data ( figures s24s27 , supporting information ) with those of 2 and confirmation 
 of the molecular formula c25h28o6 by hresims and c nmr data further indicated that 5 is the 5-oxy analogue of compound 2 . the absolute configurations for compounds 15 were assigned using electronic circular 
 dichroism and nmr data . all five compounds produced similar ecd spectra 
 ( figures s1s5 , supporting information ) containing high - amplitude positive cotton effects ( ces ) between 
 320 
 and 360 nm and high - amplitude negative ces between 280 and 320 nm . these ces correspond to the respective n   * and  
   * electronic transitions of the acetophenone chromophore and allowed for the assignment of the 2s - configuration 
 for all five compounds . all compounds also gave high - amplitude positive 
 ces near 240 nm , corresponding to the la electronic 
 transition of the aromatic chromophores , consistent with 2s - configuration . this configuration was supported by nmr 
 data , which indicated that the aryl substituent was in the thermodynamically 
 preferred equatorial position . accordingly , the vicinal jhh coupling constants of 13 and 2.8 hz 
 for h-2 ( h 5.70 ) in each compound indicated 
 that this proton was in the axial position , with axial axial 
 coupling at 13 hz to h-3a ( h 2.93.1 ) 
 and axial  equatorial coupling at 2.8 hz to h-3b ( h 2.672.75 ) . additionally , in the 
 hmbc spectrum ( optimized for jch = 8 hz ) , h-3a showed correlations to c-2 and c-1 
 but not to c-10 , while the converse was true of h-3b , which correlates 
 to c-10 but not to c-2 or c-1. the thermodynamically preferred 
 conformation for the flavanone c - ring leads to p - helicity and a corresponding 2s - configuration based 
 on the modified octant rule applied to aryl ketones as proposed by 
 snatzke in 1965 . the h and c nmr spectra of 6 showed similarities 
 to 1 and 4 , with methyl groups at h 1.55 , 1.60 , and 1.64 and the characteristic vinylic proton 
 resonances at h 5.07 and 5.26 indicative of a geranyl 
 substituent . an aabb spin system at h 7.42 and 6.90 ( j = 8.5 hz ) indicated a p - substituted b - ring . comparison of the ms and nmr data 
 ( figures s29s32 , supporting information ) of compound 6 with those of the known compound prostratrol 
 f confirmed their identical gross structures and relative configuration . the specific rotation of 6 ( [ ]d 40 ) closely matched the reported value 
 ( [ ]d 42 ) of prostratol f , hence establishing the 2s absolute 
 configuration . compound 6 was therefore assigned as the known compound prostratol f , ( 2s , e)-7,4-dihydroxy-8-(3,7-dimethylocta-2,6-dien-1-yl)flavanone . the structures of the known compounds 710 were determined by comparison of their ms , nmr , and ecd 
 data ( figures s6 , s7 , and s33s48 , supporting 
 information ) with reported values . initial screening in simple disk - diffusion 
 assays ( 100 g / disk ) revealed zones of inhibition of 1114 
 mm for the root extract of d. searlsiae against the 
 tested gram - positive bacteria , while extracts of the aerial portions 
 of the plant were inactive . the results of antibacterial broth 
 microdilution assays using the isolated root compounds 16 are shown in table 3 . all compounds had moderately potent minimum inhibitory concentration 
 ( mic ) values of 2.05.3 g / ml against the cariogenic 
 bacterium s. mutans . these results 
 are consistent with those of previous studies on the antibacterial 
 properties of flavonoids of glycyrrhiza(16 ) and suggest potential applications of these 
 metabolites for oral health . standard errors for all mics and mbcs 
 were in the range 01.7 
 except those for mics of oxacillin against orsa ( 5.3 ) and b. cereus ( 21.3 ) . all compounds also had mics of 2.38.0 g / ml against 
 the endemic soil bacterium bacillus cereus.(19 ) this supports an ecological view that d. searlsiae may allocate antibacterial resources toward 
 gram - positive 
 organisms in the root environment . b. cereus is also 
 an opportunistic pathogen , associated with food - borne illness , and 
 is a close relative of b. anthracis , the causative 
 agent of anthrax . compounds 16 exhibited antistaphylococcal mic values 
 of 3.16.8 g / ml , a level of activity ( < 10 g / ml ) 
 that qualifies as  very interesting  as recently defined 
 by cushnie and lamb . mics of 16 ranged from 3.1 to 6.8 g / ml against 
 oxacillin - sensitive staph . aureus ( ossa ) and from 3.4 to 6.5 g / ml against the resistant strain 
 ( orsa ) . in contrast , the positive control , oxacillin , was effective 
 against ossa ( 0.4 g / ml ) and s. mutans ( 0.5 
 g / ml ) , 
 but had the expected low activity against orsa ( 21.3 g / ml ) . the similarity in mics for 16 against 
 both ossa and orsa suggests that the antibacterial mechanism of action 
 for these compounds is different than that of oxacillin and related 
 -lactams . previous structure  activity analyses 
 of similar flavonoids associated increased antibacterial activity 
 with oxygenation at c-5 , although the 
 present data reveal only a slight increase in potency of 5 compared to 14 and 6 . it may be that an increase in activity due to the presence of lipophilic 
 groups ( e.g. , geranyl or prenyl ) , also concluded previously , is an overriding factor for the activity of 
 this suite of compounds . the confirmatory measurements of minimum 
 bactericidal concentrations ( table 3 ) , defined 
 as the lowest concentration that results in 99.9% reduction of bacterial 
 density , were only slightly higher than the mics of all compounds 
 tested , suggesting that these compounds work via a bactericidal mechanism 
 of action . coupled with their differing mechanism of action from -lactam 
 agents , the levels of bactericidal activity of 16 suggest a potential clinical use against resistant strains 
 of staph . these results also support previous reports of flavonoid 
 activities against -lactam - resistant gram - positive bacteria . the presence 
 of rotenoids 7 and 8 prompted our investigation 
 of the potential antiinsectan properties of d. searlsiae using the generalist herbivore spodoptera frugiperda ( fall armyworm ) to evaluate antifeedant and possible larvicidal 
 properties of extracts and isolated flavonoids . prior reports have 
 demonstrated antifeedant activity of flavonoids toward insects , and other natural products have been shown to 
 have such activity specifically toward s. frugiperda . initial testing of crude methanolic extracts 
 of the roots and aerial parts of the plant was done at concentrations 
 constituting 0.8% ( 8 g / mg disk ) and 1.4% ( 14 g / mg disk ) 
 of a pinto bean diet , respectively . these concentrations were estimated 
 from isolated amounts of the compounds from the original plant material 
 and by taking into account the approximate water content of plants that would be encountered by s. frugiperda . after 4 days , the root extract caused a reduction in growth of s. frugiperda larvae of 5075% ( as indicated by comparing 
 weights of survivors ) , while the extract of the aerial portions caused 
 75% reduction in growth . these results demonstrate that antiinsectan 
 compounds may be distributed throughout the plant , with slightly higher 
 concentrations in aerial portions , and support the premise that secondary 
 metabolites defend against insect larvae feeding on both roots and 
 shoots . further assessment of compounds 110 using pinto bean diet disks containing 1% ( 10 g / mg 
 disk ; 10  000 ppm ) of the test compounds was performed , and 
 the reductions in growth and associated mortality relative to controls 
 are shown in table 4 . in all cases , larvae 
 were observed feeding on diet treatments , rather than on agar , as 
 would be seen for antifeedant activity , so effects were likely due 
 primarily to toxicity . as expected , rotenoids 7 and 8 were both highly active , exhibiting greater than 90% reduction 
 in growth , with associated mortality . the flavanone malheuran d ( 4 ) was also active , causing 90% growth reduction , with 37% 
 mortality . notable reductions in growth were observed for all 10 compounds 
 at this test concentration , which was selected for comparisons of 
 relative activity . the three most active compounds were then assayed 
 further at lower concentrations , ranging from 0.0023 to 0.08% ( 23831 
 ppm ) , approximating those that might be typically encountered by herbivorous 
 lepidoptera larvae . growth reduction of > 50% relative to controls 
 remained significant ( p < 0.0001 ) for both tephrosin 7 and rotenone ( table 4 ) , while 4 and 8 were less active at the lower test concentrations . the level of effectiveness observed at these concentrations suggests 
 a potential ecological role in plant defense against insect herbivores . the mechanism of action of rotenone toward insects is via inhibition 
 of cellular respiration , specifically within the mitochondrial complex 
 i electron transport system . the mechanism 
 of action of nonrotenoid phenolic compounds ( e.g. , compound 4 ) toward insects is , for the most part , undetermined . control 
 disks were treated only with acetone , the solvent used for the test 
 materials . experimental diet disks containing 1% compound 
 ( 10  000 ppm , or 10 g / mg disk ) . where mortality occurs , reduction in growth and 
 mortality are typically decreased ( but not measured ) due to cannibalism 
 of dead insects by survivors . test concentrations : 4 = 0.08% ( 831 ppm ) , 7 = 0.016% ( 163 ppm ) , 8 = 0.0023% ( 23 ppm ) , rotenone 
 = 0.016% ( 163 ppm ) . overall , for crude extracts 
 and isolated compounds of d. searlsiae , the apparent 
 larvacidal activities toward s. frugiperda appear 
 to be greater in the aerial portions of the plant , a phenomenon partially 
 attributable to the presence of rotenoids 7 and 8 . conversely , gram - positive antibacterial metabolites appear 
 to be selectively allocated to root portions of the plant , potentially 
 for defense against soil pathogens . melting points were 
 measured on a barnstead international mel - temp apparatus , model 12010 . optical rotations were recorded on a perkinelmer 341 polarimeter ( na 
 lamp , 589 nm ) ; concentrations were reported in g/100 ml . ecd 
 spectra were obtained on a jasco j-815 circular dichroism spectrometer 
 using high - purity meoh as the solvent and a 1 cm path length quartz 
 cuvette . ecd spectra were obtained for each compound at concentrations 
 of 0.01 , 0.02 , 0.04 , 0.06 , 0.08 , and 1.0 mm , and raw data were smoothed 
 using a binomial smoothing algorithm with 10 passes as provided with 
 the jasco spectra manager ver . 1.54 software . all nmr spectra 
 were obtained on a bruker avance 400 mhz system with topspin 1.3 software . h2o using flow injection analysis ( 100 l / min ) with no 
 column . positive or negative esims was used to generate [ m + h ] , [ m + na ] , or [ m  h ] ions . tlc plates ( emd chemicals , inc . ; silica gel 60 , f254 ) were eluted with 9:1 ch2cl2meoh or 
 mixtures of hexanes  etoac and visualized with uv ( 254 nm ) and 
 the spray reagent vanillin / h2so4 ( 1 g/100 ml 
 w / v ) after gentle heating . the plants were grown from seed at the oregon state 
 university , malheur experiment station , ontario , or . plant materials were air - dried for approximately 72 h from 
 the time of collection . roots were washed with water to remove dirt 
 and debris , aerial portions were separated , and material was stored 
 at 20 c until further use . tom cottrell , department of biological sciences , central washington 
 university , and was deposited in the herbarium of the same department . whole d. searlsiae roots ( 44 g ) were shaved into 36 cm slices , soaked for 48 
 h in meoh at room temperature , and blended in a waring blender for 
 23 
 min with 700 ml of meoh . the mixture was filtered and the filtrate 
 evaporated under reduced pressure to afford 3.7 g of crude extract . this material was preadsorbed in meoh solution onto 10 g of 
 silica gel , the solvent removed under vacuum , and the resulting powder 
 subjected to vlc over a prepacked column bed ( 10  6 cm ; i.d . the column 
 was eluted using a stepwise gradient of solvents beginning with hexanes 
 ( 1 l ) and continuing with mixtures ( 500 ml each ) of etoac in hexanes 
 ( 20 , 40 , 60 , 80 , and 100% etoac ) , followed by mixtures of meoh in 
 ch2cl2 ( 2 , 5 , 8 , 10 , and 30% meoh ) . fractions 
 3 and 4 from this column were preadsorbed ( 3 g of silica gel ; 
 meoh ) , dried , and rechromatographed on a smaller scale ( 3.5  
 7 cm ; i.d . h ) , eluting with 300 ml of 100% hexanes , followed 
 by 300 ml each of 10 , 20 , 30 , 35 , 40 , 75 , and 100% etoac in hexanes . fractions 47 of this column were combined with fractions 5 
 and 6 from the first vlc column to afford an enriched flavonoid fraction 
 ( 1.67 g ) . the enriched flavonoid fraction was further purified 
 by sephadex lh-20 ( sigma ) cc ( 2.5  60 cm ) eluting with 1 l of 
 3:1:1 hexanes  toluene  meoh , followed by 1 l of 100% 
 meoh at a flow rate of 0.30.5 ml / min with 8 ml per 
 fraction tube . materials of similar composition as determined by tlc 
 were pooled to give 46 fractions . fractions 27 and 28 were combined 
 ( 76 mg ) and further purified over silica gel ( 2.5  8 cm ; 60100 
 mesh , 20 ml / min ) using a continuous linear gradient of etoac 
 ( 040% ) 
 in hexanes . fractions eluting with 3640% etoac afforded compound 1 ( malheuran a ; 18 mg ) , and fractions eluting with 3034% 
 etoac afforded ( 2s)-5-(2-methylbut-3-en-2-yl)-8-(3-methylbut-2-en-1-yl)-5,7,2,4-tetrahydroxyflavanone 
 ( 5 ; 13 mg ) . fraction 24 ( 245 mg ) from the sephadex lh-20 
 column was further purified in a similar manner to that used for 27 
 and 28 , using a continuous linear gradient of etoac in hexanes ( 050% ; 
 2.5  11 cm column ) . fractions eluting with 3135% etoac 
 yielded compound 2 ( malheuran b ; 129 mg ) . fraction 6 
 ( 36 mg ) from the sephadex lh-20 column was further purified over silica 
 gel in two successive stages using gradients of 030% etoac 
 in hexanes ( 1.5  8 cm column ) , followed by 04% meoh 
 in ch2cl2 ( 1.5  7 cm column ) to afford 
 compound 3 ( malheuran c ; 17 mg ) . fractions 8 and 9 from 
 the sephadex lh-20 column were combined ( 153 mg ) and further purified 
 over silica gel in two successive stages using gradients of 05% 
 meoh in ch2cl2 ( 2.5  10 cm column ) , followed 
 by 025% etoac in hexanes ( 1.5  7 cm column ) to yield 
 prostratrol f ( 6 ; 23 mg ) . fraction 10 ( 96 mg ) from the 
 sephadex lh-20 column was purified over silica gel using 05% 
 meoh in ch2cl2 ( 2.5  10 cm column ) to 
 yield compound 4 ( malheuran d ; 69 mg ) . aerial portions 
 of d. searlsiae ( leaves , stems , and flowers ; 196 
 g ) were blended in a waring blender for 23 min with 1.2 l 
 of meoh . the mixture was filtered , and the filtrate was evaporated 
 under reduced pressure to afford 27.4 g of crude extract . this material 
 was fractionated by vlc over silica gel ( 10  6 cm ; i.d . h ) , using methods and eluting solvents similar to those used for vlc 
 of the crude root extract . material from fractions 3 and 4 , eluted 
 with 4060% etoac in hexanes from this vlc column , were combined 
 ( 1.5 g ) and fractionated by sephadex lh-20 chromatography as described 
 above to yield 37 fractions following combinations based on tlc analyses . fractions 1821 from the sephadex lh-20 column were 
 combined ( 54 mg ) and further purified over silica gel ( 60100 
 mesh ) in successive stages with step gradients of meoh in ch2cl2 ( 01% ; 1.5  13 cm column ) and 100% ch2cl2 ( 1.5  5 cm column ) to yield ( )-milletosin 
 ( 8 ; 8 mg ) . fractions 10 and 11 from the sephadex lh-20 
 column were combined ( 72 mg ) and further purified over silica gel 
 with continuous linear gradient conditions and solvents identical 
 to those used for 1 above , to afford griffonianone e 
 ( 9 ; 18 mg ) and calopogonium isoflavone a ( 10 ; 12 mg ) . off - white , amorphous 
 solid ; [ ]d 66 ( c 0.1 , chcl3 ) ; uv ( meoh ) max ( log  ) 
 203 ( 4.64 ) , 220 ( sh ) ( 4.42 ) , 236 ( sh ) ( 4.22 ) , 286 ( 4.14 ) , 315 ( sh ) 
 ( 3.79 ) nm ; ecd ( c 0.0016 , meoh )  (  ) 
 204 ( 4.70  10 ) , 223 ( 4.15  10 ) , 247 ( 3.77  10 ) , 303 ( 2.54 
  10 ) , 334 ( 1.34  10 ) nm ; ir ( film 
 on kbr ) max 3342 ( br oh ) , 2966 , 2920 , 1655 , 1585 , 
 1520 , 1440 , 1284 , 1167 , 1114 , 974 cm ; h and c nmr data , see table 1 ; hmbc correlations ( acetone - d6 ) h-2 
  c-4 , 1 , 2 , 6 ; h-3a c-2 , 4 , 
 1 ; h-3b  c-4 , 10 ; h-5  c-4 , 7 , 8 * ; h-6  
 c-7 , 10 ; oh-7  c-6 , 7 , 8 ; oh-2  c-1 , 2 , 3 ; h-3 
  c-1 , 2 , 4 , 5 ; oh-4 c-3 , 4 , 5 ; h-5 
  c-1 , 3 ; h-6  c-2 , 2 , 
 3 * , 4 ; h2 - 1  c-7 , 8 , 9 , 
 2 , 3 ; h-2  c-8 , 1 , ch3 - 3 , 4 ; ch3 - 3 c-2 , 3 , 4 ; 
 h2 - 4  c-2 , 3 , ch3 - 3 , 5 ; h2 - 5  
 c-3 , 4 , 6 , 7 ; h-6  
 c-4 , 8 , ch3 - 7 ; 
 ch3 - 7 c-6 , 7 , 
 8 ; h3 - 8  c-6 , 7 , ch3 - 7 ( * indicates weak four - bond correlation ) ; 
 hresims found m / z 407.1863 [ m  
 h ] , calcd for c25h27o5 407.1859 . yellow , 
 amorphous solid ; [ ]d 90 ( c 0.1 , chcl3 ) ; uv ( meoh ) max ( log  ) 203 ( 4.65 ) , 220 ( sh ) ( 4.43 ) , 235 ( sh ) ( 4.24 ) , 287 
 ( 4.16 ) , 314 ( sh ) ( 3.10 ) nm ; ecd ( c 0.0016 , meoh ) 
  (  ) 208 ( 3.66  10 ) , 228 ( 2.01 
  
 10 ) , 250 ( 1.91  10 ) , 305 ( 2.41 
  10 ) , 335 ( 1.19  10 ) nm ; ir ( film 
 on kbr ) max 3405 ( br oh ) , 2967 , 2916 , 1655 , 1586 , 
 1504 , 1437 , 1384 , 1287 , 1213 cm ; h 
 and c nmr data , see table 2 ; hmbc 
 correlations ( acetone - d6 ) h-2  
 c-3 , 4 , 1 , 2 , 6 ; h-3a  c-2 , 4 , 1 ; 
 h-3b  c-4 , 10 ; h-5  c-4 , 7 , 8 * , 9 ; h-6  c-7 , 
 8 , 10 ; h-3  c-2*,1 , 2 , 4 , 
 5 , 2 * ; h-6  c-2 , 1 , 2 , 
 3 * , 4 , 2 ; h2 - 1  
 c-7 , 8 , 9 , 2 , 3 ; h-2 c-8 , 1 , ch3 - 3 , 4 ; ch3 - 3  c-2 , 3 , 4 ; 
 h3 - 4  c-2 , 3 , ch3 - 3 ; h3 - 1  c-5 , 
 2 , ch3 - 2 , 3 ; ch3 - 2  c-5 , 1 , 
 2 , 3 ; h-3  c-5 , 1 , 
 2 , ch3 - 2 ; h2 - 4  c-5 * , 1 * , 2 , ch3 - 2 ( * indicates weak four - bond correlation ) ; 
 hresims found m / z 407.1865 [ m  
 h ] , calcd for c25h27o5 407.1858 . yellow 
 oil ; [ ]d 100 ( c 0.03 , chcl3 ) ; uv ( meoh ) max  ( log 
  ) 
 204 ( 4.66 ) , 222 ( sh ) ( 4.40 ) , 235 ( sh ) ( 4.26 ) , 286 ( 4.14 ) , 316 ( sh ) 
 ( 3.71 ) nm ; ecd ( c 0.0017 , meoh )  (  ) 
 209 ( 4.96  10 ) , 230 ( 1.94  10 ) , 246 ( 2.66  10 ) , 303 ( 2.65 
  10 ) , 334 ( 1.20  10 ) nm ; ir ( film 
 on kbr ) max 3467 ( br oh ) , 2965 , 2925 , 1653 , 1586 , 
 1506 , 1443 , 1383 , 1286 , 1207 cm ; h 
 and c nmr data , see table 2 ; hmbc 
 correlations ( acetone - d6 ) h-2  
 c-3 , 4 , 9 , 1 , 2 , 6 ; h-3  c-2 , 
 4 , 1 ; h-3  c-4 , 10 ; h-5  c-4 , 8 * , 9 ; 
 h-6  c-7 , 8 , 10 ; oh-7  c-6 , 7 , 8 ; 
 och3 - 2  c-2 , 3 * ; 
 h-3  c-2*,1 , 2 , 4 , 5 , 
 2 * ; oh-4  c-3 , 4 , 
 5 ; h-6  c-2 , 1 , 2 , 3 * , 
 4 , 2 ; h2 - 1  c-7 , 8 , 2 , 
 3 , ch3 - 3 * ; h-2 
  c-8 , 1 , 4 , ch3 - 3 ; h3 - 4  c-2 , 3 , ch3 - 3 ; ch3 - 3  c-2 , 3 , 4 ; h-1 
  c-5 , 2 , ch3 - 2 , 
 3 ; ch3 - 2  c-5 , 
 1 , 2 , 3 ( * indicates weak four - bond correlation ) ; 
 hresims found m / z 423.2172 orange 
 oil ; [ ]d 76 ( c 0.1 , chcl3 ) ; uv ( meoh ) max ( log  ) 
 204 ( 4.75 ) , 221 ( sh ) ( 4.50 ) , 236 ( sh ) ( 4.32 ) , 287 ( 4.24 ) , 316 ( sh ) 
 ( 3.88 ) nm ; ecd ( c 0.0019 , meoh )  (  ) 
 207 ( 5.90  10 ) , 227 ( 3.36  10 ) , 248 ( 3.69  10 ) , 304 ( 3.48 
  10 ) , 335 ( 1.65  10 ) nm ; ir ( film 
 on kbr ) max 3394 ( br oh ) , 2967 , 2925 , 1655 , 1586 , 
 1505 , 1437 , 1383 , 1288 , 1213 cm ; h 
 and c nmr data , see table 1 ; hmbc 
 correlations ( acetone - d6 ) h-2  
 c-3 , 4 , 9 , 1 , 2 , 6 ; h-3a  c-2 , 4 , 
 1 ; h-3b  c-4 , 10 ; h-5  c-4 , 6 , 7 , 8 * ; h-6 
  c-7 , 8 , 10 , 1 * ; oh-7  c-8 ; 
 oh-2  c-1 , 2 , 3 ; 
 h-3  c-2 * , 1 , 2 , 4 , 5 , 
 2 * ; oh-4  c-3 , 4 , 
 5 ; h-6  c-2 , 1 , 2 , 3 * , 
 4 , 2 ; h2 - 1  c-7 , 8 , 9 , 
 2 , 3 ; h-2  c-8 , 1 , 4 , 
 5 ; ch3 - 3  c-2 , 
 3 , 4 ; h2 - 4  c-2 , 
 3 , 5 , 6 ; h2 - 5  
 c-3 , 4 , 6 , 7 ; h-6  
 c-7 , ch3 - 7 , 8 ; 
 ch3 - 7  c-6 , 7 , 
 8 ; h3 - 8  c-6 , 7 , ch3 - 7 ; ch3 - 1  
 c-5 , 2 , ch3 - 2 , 
 3 , 4 * ; ch3 - 2  
 c-5 , 1 , 2 , 3 , 4 * ; h-3 
  c-5 , 1 , 2 , ch3 - 2 ; h2 - 4  c-5 * , 
 1 * , 2 , ch3 - 2 * , 
 3 ( * indicates weak four - bond correlation ) ; hresims found m / z 477.2638 [ m + h ] , calcd 
 for c30h37o5 477.2641 . orange oil ; [ ]d 50 ( c 0.1 , chcl3 ) ( lit . [ ]d 79.1 ) ; uv ( meoh ) max ( log  ) 203 ( 4.68 ) , 
 230 ( sh ) ( 4.29 ) , 291 ( 4.21 ) , 338 ( sh ) ( 3.54 ) nm ; ecd ( c 0.0017 , meoh )  (  ) 210 ( 2.83  10 ) , 230 ( 1.11  10 ) , 254 ( 648 ) , 294 ( 2.49 
  10 ) , 335 ( 3.91  10 ) nm ; ir ( film 
 on kbr ) max 3412 ( br oh ) , 2967 , 2927 , 1635 , 1604 , 
 1504 , 1432 , 1384 , 1300 , 1169 , 1076 cm ; h and c nmr data , see table 2 ; hmbc correlations ( acetone - d6 ) h-2 
  c-4 , 1 , 2 , 6 ; h-3a  c-2 , 
 4 , 1 ; h-3b  c-4 , 10 ; oh-5  
 c-5 , 6 , 10 ; h-6  c-4 * , 5 , 7 , 8 , 10 , 1 ; oh-7  c-8 ; oh-2  c-1 , 
 2 , 3 ; h-3  c-2*,1 , 2 , 
 4 , 5 , 2 * ; oh-4  
 c-3 , 4 , 5 ; h-6  c-2 , 1 , 
 2 , 3 * , 4 , 2 ; h2 - 1 
  c-7 , 8 , 9 , 2 , 3 ; h-2  c-1 , ch3 - 3 , 4 ; h3 - 4 
  c-2 , 3 , ch3 - 3 ; 
 ch3 - 3  c-2 , 3 , 
 4 ; h3 - 1  c-5 , 2 , ch3 - 2 , 3 ; ch3 - 2  c-5 , 1 , 2 , 
 3 ; h-3  c-5 , 1 , 2 , ch3 - 2 ; h2 - 4  
 c-2 ; 3 ( * indicates weak four - bond correlation ) ; hresims 
 found m / z 423.1809 [ m  h ] , calcd for c25h27o6 423.1808 . off - white 
 solid ; mp 5564 c ; [ ]d 40 ( c 0.03 , chcl3 ) ( lit . [ ]d 42 ) ; uv 
 ( meoh ) max ( log  ) 202 ( 4.47 ) , 219 ( sh ) ( 4.36 ) , 
 239 ( sh ) ( 4.16 ) , 283 ( 4.04 ) , 315 ( sh ) ( 3.77 ) nm ; ir ( film on kbr ) 
 max 3325 ( br oh ) , 2923 , 2854 , 1655 , 1597 , 1518 , 
 1440 , 1384 , 1335 , 1283 , 1218 cm ; h 
 and c nmr data ( table 1 ) are consistent 
 with reported data ; cosy correlations 
 ( acetone - d6 ) h-2  h-3a , 3b ; h-3a 
  h-2 , 3b ; h-3b  h-2 , 3a ; h-5  h-6 ; h-6  
 h-5 ; h-2/6  h-3/5 ; h-3/5 
  h-2/6 ; h2 - 1  h-2 , 
 ch3 - 3 * , h2 - 4 * ; 
 h-2  h2 - 1 , ch3 - 3 * , h2 - 4 * ; ch3 - 3  h2 - 1 * , h-2 * , 
 h2 - 4 * ; h2 - 4  h2 - 1 * , h-2 * , ch3 - 3 * , 
 h2 - 5 ; h2 - 5  h2 - 4 , h-6 , ch3 - 7 * , 
 h3 - 8 * ; h-6  h2 - 5 , 
 ch3 - 7 * , h3 - 8 * ; 
 ch3 - 7  h2 - 5 * , 
 h-6 * ; h3 - 8  h2 - 5 * , 
 h-6 * ( * indicates long - range correlation ) ; hresims found m / z 393.2060 [ m + h ] , calcd 
 for c25h29o4 393.2066 . yellow , microcrystalline solid ; mp 8496 
 c ; [ ]d 100 ( c 0.1 , chcl3 ) ( lit . [ ]d 100 ) ; uv ( meoh ) max ( log  ) 
 203 ( 4.69 ) , 237 ( 4.47 ) , 251 ( 4.47 ) , 271 ( 4.51 ) , 300 ( 4.17 ) , 319 ( sh ) 
 ( 4.14 ) nm ; ecd ( c 0.0016 , meoh )  (  ) 
 204 ( 2.52  10 ) , 218 ( 1.04  10 ) , 
 236 ( 4.01  10 ) , 275 ( 1.30  10 ) , 329 ( 1.14  10 ) nm ; ir ( film on 
 kbr ) max 3475 ( br oh ) , 2980 , 2930 , 1674 , 1638 , 1598 , 
 1578 , 1511 , 1443 , 1331 , 1272 , 1112 cm ; h and c nmr data ( acetone - d6 , 400 mhz ) were consistent with reported values and further supported with hsqc and hmbc data ( figures 
 35 and 36 , supporting information ) ; hresims 
 found m / z 433.1269 [ m + na ] , calcd for c23h22o7na 433.1263 . off - white , microcrystalline 
 solid ; mp 8190 c ; [ ]d 132 ( c 0.1 , chcl3 ) ; uv ( meoh ) 
 max ( log  ) 202 ( 4.49 ) , 228 ( sh ) ( 4.26 ) , 241 
 ( 4.31 ) , 250 ( 4.30 ) , 271 ( 4.35 ) , 305 ( 4.07 ) nm ; ecd ( c 0.0016 , meoh )  (  ) ; 203 ( 2.33  10 ) , 219 ( 1.64  10 ) , 241 ( 1.94  10 ) , 275 ( 1.37  10 ) , 300 ( 4.08  10 ) , 329 ( 2.40  10 ) nm ; ir ( film on 
 kbr ) max 3446 ( br oh ) , 2975 , 2926 , 1674 , 1635 , 1599 , 
 1578 , 1482 , 1331 , 1273 , 1211 , 1166 cm ; h and c nmr data ( acetone - d6 , 400 mhz ) were consistent with reported values and further supported with hsqc and hmbc data ( figures 
 39 and 40 , supporting information ) ; hresims 
 found m / z 395.1136 [ m + h ] , calcd for c22h19o7 395.1131 . . 130131 c ) ; uv ( meoh ) max ( log  ) 
 205 ( 4.69 ) , 221 ( sh ) ( 4.57 ) , 245 ( 4.53 ) , 252 ( 4.52 ) , 303 ( 4.38 ) nm ; 
 ir ( film on kbr ) max 3447 ( br oh ) , 2918 , 1643 , 1617 , 
 1601 , 1485 , 1425 , 1268 , 1193 cm ; h 
 and c nmr data ( acetone - d6 , 400 mhz ) were consistent with reported values and further supported with hsqc and hmbc data ( figures 
 43 and 44 , supporting information ) ; hresims 
 found m / z 395.1491 [ m + h ] , calcd for c23h23o6 395.1495 . 138141 
 c ) ; uv ( meoh ) max ( log  ) 202 ( 4.57 ) , 216 ( sh ) ( 4.45 ) , 241 ( sh ) ( 4.60 ) , 261 
 ( 4.78 ) , 311 ( 4.03 ) , 321 ( sh ) ( 4.03 ) nm ; ir ( film on kbr ) max 3447 ( br oh ) , 3073 , 2927 , 1629 , 1591 , 1512 , 1440 , 1395 , 
 1246 , 1211 , 1178 , 1113 cm ; h and c nmr data ( acetone - d6 , 400 mhz ) 
 were consistent with reported values and 
 further supported with hsqc and hmbc data ( figures 47 and 48 , supporting information ) ; hresims found m / z 335.1284 [ m + h ] , calcd 
 for c21h19o4 335.1283 . aureus atcc 43300 ( orsa ) , bacillus cereus ( environmental 
 isolate ) , and streptococcus mutans atcc 25175 were 
 used for these studies . stock cultures of all bacteria were maintained 
 at 35 c under aerobic conditions on nutrient broth / agar , and b. cereus and s. mutans were additionally 
 grown on brain - heart infusion broth / agar . b. cereus was isolated from the environment and identified using standard 
 microscopy and staining techniques as a gram - positive rod that exhibited 
 beta - hemolysis on sheep s blood agar . three colonies from agar 
 plates were transferred to broth and grown for 24 h followed by a 
 broth - to - broth transfer using a calibrated loop ( 5 l ) and growing 
 for 24 h. biologically active extracts and pure compounds were 
 initially identified using disk - diffusion bioassay - guided fractionation . paper disks ( 6 mm ) were permeated with test materials in meoh ( 100 
 g / disk ) and placed on mueller - hinton agar plates containing 
 bacterial inoculum . following incubation ( 37 c ; 3 days ) , the 
 diameters ( mm ) of the resulting zones of inhibition were recorded . broth microdilution was performed according to antimicrobial susceptibility 
 testing standards ( nccls ) to determine 
 mics of pure compounds against ossa , orsa , b. cereus , and s. mutans . dilutions of compounds were made 
 at concentrations incrementally decreasing by 1 g / ml in the 
 appropriate broth with 2% dmso . oxacillin was used as a positive control 
 and diluted in broth using a serial 2-fold method . double - strength 
 test samples were added ( 75 l ) to sterile 96-well round - bottomed 
 culture plates . a bacterial cell suspension corresponding to 1  
 10 cfu / ml ( 75 l ) was added to test wells . the final 
 concentration of bacteria in the assay wells after dilution with broth 
 and sample was 5  10 cfu / ml with 1% dmso . culture 
 plates were incubated at 35 c for 24 h for all strains other 
 than s. mutans , which was incubated for 48 h. at 
 the end of the incubation periods mics were determined by identifying 
 the drug concentration at which no bacterial growth was visible in 
 the wells . mbcs were determined by transferring 20 l of broth 
 from wells without visible growth onto either nutrient agar for staph . averages and standard errors were calculated 
 for mics and mbcs using measurements of at least three . laboratory colonies of s. frugiperda were reared on a pinto bean - based diet at 27  1 c , 50 
  10% relative humidity , and a 14:10 h light : dark photoperiod . pinto bean - based insect 
 diet disks were prepared by  casting  slabs of diet 
 1 mm thick , punching out 8 mm diameter disks , and freeze - drying . disks 
 ( 15 mg each ) were stored in a sealed container at 4 c . 
 for the bioassay , test materials were absorbed into freeze - dried diet 
 disks using acetone at a rate of 25 l per disk to yield the 
 target concentrations . petri dishes with tight - fitting lids ( falcon 
 1006 , becton dickinson & co. ) containing 5 ml of 3% agar were 
 used as bioassay containers . a 1 cm diameter teflon disk ( scientific 
 specialties service ) was added to each plate , and the diet disks with 
 incorporated test materials were placed on top of the teflon disks . the diet disks were rehydrated with sterile distilled water ( 25 l ) , 
 and 10 first instar larvae were added to each dish . each disk provided sufficient material for 
 ad libitum feeding by larvae over the duration of the assay period . the dishes were incubated under the same conditions used in insect 
 rearing except plates were kept in the dark . dishes were evaluated 
 for dead larvae daily and frozen at the end of the assay period until 
 all survivors of the bioassay could be weighed . larvae were weighed 
 to the nearest 0.01 mg using an analytical balance ( mettler instrument 
 corp . ; model ae163 ) . individual treatment replicates were statistically 
 analyzed , and data were pooled where no statistical differences at p < 0.05 were noted ( all cases ) . reductions in larval 
 growth were assessed by comparing the average weight of corresponding 
 control larvae to the average weight of larvae on treatment plates , 
 and results are reported as a percentage of the control . mortality 
 was assessed by calculating the percentage of dead larvae compared 
 to the total larvae found . sas version 8.0 was used for analysis of 
 variance for weights ( proc glm ) and for chi square analysis of mortality 
 ( proc freq ) . crude extracts of d. searlsiae root and aerial parts were tested at concentration percentages 1/10 
 that of the percent of crude extracts resulting from the plant material 
 extracted ( e.g. , 27.4 g of crude extract from 196 g aerial parts = 
 14% , tested at 1.4% of diet disks ) in order to account for the wet 
 weight of fresh plant material . initial testing of compounds 110 was performed at concentrations of 1% of disk weight ( 10 g / mg 
 disk ) over 3 days , for relative activity comparisons . compounds 4 , 7 , 8 , and rotenone were tested 
 at lower doses ( see table 4 ) corresponding 
 to their probable concentrations , based on isolated amounts , in fresh 
 plant material that contains 5065% water , using the same methods as described above . average weights 
 and standard errors for these were as follows : control ( acetone only ) 
 = 0.33 mg  0.2 ; compound 4 = 0.31 mg  0.2 ; 
 compound 7 = 0.14 mg  0.1 ; compound 8 = 0.27 mg  0.2 ; rotenone = 0.14 mg  0.2 .
continued interest in the chemistry 
 of dalea spp . led to investigation of dalea 
 searlsiae , a plant native to areas of the western united 
 states . 
 methanol extractions of d. searlsiae roots 
 and subsequent chromatographic fractionation afforded the new prenylated 
 and geranylated flavanones malheurans a  d ( 14 ) and known flavanones ( 5 and 6 ) . 
 known rotenoids ( 7 and 8) and isoflavones 
 ( 9 and 10 ) were isolated from aerial portions . 
 structure determination of pure compounds was accomplished primarily 
 by extensive 1d- and 2d - nmr spectroscopy . 
 the absolute configurations 
 of compounds 15 , 7 , 
 and 8 were assigned using electronic circular dichroism 
 spectroscopy . 
 antimicrobial bioassays revealed significant activity 
 concentrated in the plant roots . 
 compounds 16 exhibited mics of 28 g / ml against streptococcus mutans , bacillus cereus , 
 and oxacillin - sensitive and -resistant staphylococcus aureus . 
 aerial metabolites 710 were inactive 
 against these organisms , but the presence of 7 and 8 prompted investigation of the antiinsectan activity of d. searlsiae metabolites toward the major crop pest spodoptera frugiperda ( fall armyworm ) . while compounds 110 all caused significant reductions 
 in larval growth rates , associated mortality ( 3366% ) was highest 
 with flavanone 4 and rotenoids 7 and 8 . 
 these findings suggest a differential allocation of antimicrobial 
 and antiinsectan plant resources to root and aerial portions of the 
 plant , respectively .