gastrointestinal nematode infections of ruminants remain a major threat to the economic viability of the livestock industry ( nieuwhof and bishop , 2005 , sackett et  al . , 2006 ) . the control of these parasitic diseases depends on the use of commercial anthelmintic drugs ( molento et  al . , 2011 ) , together with management practices ( kahn and woodgate , 2012 , miller and waller , 2004 , van wyk et  al . , 2006 , however , resistance to anthelmintics is widespread in helminth populations ( fleming et  al . , 2006 ) ; therefore , there is a continuous need for the development of novel anthelmintic drugs . the cost of developing new anthelmintic compounds is high , and very few new compounds have been discovered or synthesized in the last decade ( csermely et  al . , 2013 , this is augmented by the fact that nations that suffer most from helminth infections are countries with limited resources to invest in drug discovery research and , as such , are also financially unattractive to the global pharmaceutical industry ( brooker , 2010 ) . in the last decade , there has been renewed interest in phytomedicine , and many traditional medicinal plants species are being tested for pharmacological activities ( agyare et  al . , 2009 , , 2011 , waterman et  al . , 2010 ) . although plant - based remedies have been used for centuries in traditional medicine for the treatment of various diseases , including worm infections , there has been limited scientific evaluations of their actual activities , properties and toxicities ( eguale et  al . previously , we have shown that traditional medicinal plants are a rich source of anthelmintic drug candidates ( kumarasingha et  al . , 2014 ) . plant extracts were selected based on the knowledge of traditional healers from borneo rainforests , sarawak , malaysia , and screened for anthelmintic compounds in caenorhabditis elegans ( kumarasingha et  al . , 2014 ) . the present study reports the fractionation of the most active plant extract , derived from the whole plant of picria fel - terrae lour . , and identification of a single fraction ( designated fraction 5 ) that is enriched for anthelmintic activity . p.  fel - terrae lour . is a popular medicinal plant in asia , and has been studied for its anti - microbial and anti - inflammatory activities ( ahmed and halaweish , 2014 , huang et  al . , 1998 , huang et  al . , 1999 ; to the best of our knowledge , apart from our previous work , testing of the anthelmintic activity of this plant has not been reported in the literature . plants were dried , ground into a powder , extracted into 1:1 v / v dichloromethane : methanol and then concentrated using a rotary evaporator . before use , the powdered plant extracts were dissolved in absolute ethanol ( merck , australia ) and diluted in m9 buffer ( 85.6  mm nacl , 4.2  mm na2hpo4 , 2.2  mm kh2po4 , 1  mm mgso4 ) to achieve the desired concentration ( brenner , 1974 ) . a solid phase extraction ( spe ) strata c18-e cartridge ( silica - based sorbent ; phenomenex , usa ) was used for sample fractionation . plant extract ( 100  mg ) was dissolved in 1  ml absolute ethanol and diluted with 19  ml milliq water . the column was washed with 20  ml of 100% acetonitrile ( acn ; sigma , australia ) and then 20  ml of milliq water was sent through to condition the column . the dissolved plant extract was loaded on the column and eluted by passing 20  ml of 10% , 25% , 40% , 55% , 70% , 85% and 100% acn dilution series . individual fractions were solubilised in 1  ml of absolute ethanol and 10  l aliquots were added to 990  l of m9 to make working solutions . c.  elegans all the strains were grown on nematode growth medium ( ngm ) in petri dishes containing a lawn of escherichia coli op50 ( brenner , 1974 ) . strains were grown at 20  c and synchronised populations were obtained by a modified alkaline bleaching method ( lenaerts et  al . , 2008 ) . briefly , worm culture plates with eggs and egg - laying adults were washed in m9 and then incubated in a freshly prepared bleaching solution ( 4  ml commercial bleach , 1  ml 1  m naoh and 9  ml h2o ) for 3.5  min , followed by washing 3 times with m9 . eggs in m9 were kept on a rotaty shaker at 20  c overnight to hatch . first stage larvae ( l1s ) were put on to ngm plates the next morning and incubated at 20  c . the 9 fractions collected following spe fractionation were tested on c.  elegans young adults . assays were performed in 96-well microtitre plates using 150  l media per well , with 3 replicates per condition ( 10 worms per well ) as described previously ( kumarasingha et  al . , 2014 ) . the plates were covered and kept in a humid chamber on a shaker at 20  c , and survivors were counted at specific time points for up to 72  h. worms were considered dead if they were immobile , even after provocation with a platinum wire , and if no pharyngeal pumping was detected . two conventional anthelmintic drugs , doramectin ( pfizer , australia ) and levamisole ( sigma , australia ) were used as positive controls , and m9 with 1% ethanol was used as a negative control . each condition was performed in triplicate , and three biological repeats were performed for each experiment . schwarz et  al . , 2013 ) was maintained in experimental sheep as described previously ( preston et  al . , 2015 ) and in accordance with the institutional animal ethics guidelines ( permit no . 1111938 ; the university of melbourne ) . to produce l3s , faecal samples were incubated at 27  c for 710 days before larvae were harvested ( cf . l3 were stored at 10  c for up to three months prior to use . l3s were exsheathed and sterilised by incubation in 0.15% ( v / v ) sodium hypochlorite ( naclo ) at 37  c for 20  min as described by preston et  al . following exsheathment , l3s ( designated xl3s ) were suspended at a density of 300 xl3 in 50  l of luria bertani medium ( lb ) supplemented with 100 iu / ml of penicillin , 100  g / ml of streptomycin and 2.5  g / ml of amphotericin ( fungizone , thermo fisher scientific , australia ; designated lb * ) . to test for activity , the test fractions ( 0.01  mg / ml ) and the solvent control in lb * with 1% ethanol were arrayed in triplicate in 96 well flat - bottomed plates ( corning , 3650 , life sciences , usa ) . six wells were used for the negative control ( lb *  +  1% ethanol ) ; 300 l3s were dispensed ( in 50  l ) into the wells using a multi - channel pipette and a mini - air pump ( air - pump - s100 ; aquatrade , australia ) to keep the parasites suspended , as described by preston et  al . plates were incubated at 38  c and 10% co2 ( v / v ) . following a 48  h incubation period , the motility of the xl3s was assessed as described previously ( preston et  al . , the plates were agitated on an orbital shaker for 30  min  at 38  c prior to recording a 10  s video of each well using an eyepiece camera ( dino - eye , anmo electronic corporation , taiwan ) attached to a stereo dissecting microscope ( olympus , japan ) . each video was then processed for motility by calculating the changes in pixel intensity using a custom macro in the program imagej ( 1.47v , imagei.nih.gov/ij ) , as described in detail by preston et  al . replicate freeze - dried samples ( n  =  5 ) of each fraction ( 50    2  g ) were prepared for derivatization as previously described ( karpe et  al . the stored samples were volatilized by derivatization for application in gas chromatography - mass spectrometry ( gc - ms ) . methoxymine hcl ( 40  l , 2% w / v in pyridine ) was added to the samples , followed by vortexing at 37  c in a thermomixer ( eppendorf , australia ) at 1400  rpm for 45  min . silylation was performed by adding 70  l of n , o - bis(trimethylsilyl)trifluoroacetamide ( bstfa ) in 1% trimethyl - chlorosilane ( tmcs ) to complete the derivatization . the mixture was then centrifuged at 15,700    g for 5  min and the supernatant was transferred to gc - ms vials . pre - derivatized c - sorbitol ( kovats retention index  =  1918.76 , m / z  =  620.00 [ 10  g / ml , hplc grade , sigma - aldrich , australia ] ) was added as the second internal standard at this point in order to verify instrument stability over the run time . the derivatized samples were analysed using an agilent 6890b gas chromatograph oven coupled to a 5973a mass spectrometer detector ( agilent technologies , australia ) , as described previously ( beale et  al . the gc - ms system was fitted with a 30  m db-5ms column , 0.25  mm i d and 0.25  m film thickness . all injections were performed at 1:10 split mode with 1.0  l volume ; the oven was held at an initial temperature of 70  c for 2  min before increasing to 325  c at 7.5  c min ; the final temperature was held for 4.5  min . the transfer line was held at 280  c and the detector voltages at 1054  v. total ion chromatogram ( tic ) mass spectra were acquired at 45550  m / z , at an acquisition frequency of 1.08 spectra s. the ionisation source used was electron ionization ( ei ) and the energy was 70  ev . the solvent delay time of 7.5  min ensured that the source filament was not saturated and damaged with derivatization reagent . qualitative identification of the compounds was performed according to the metabolomics standard initiative ( msi ) chemical analysis workgroup ( sumner et  al . , 2007 ) using standard gc - ms reference metabolite libraries of wiley , nist 11 and nist epa / together with kovats retention indices based on the referenced n - alkane retention times ( c8-c40 alkanes calibration standard , sigma - aldrich , australia ) . for peak integration , a 5 point detection , filtering ( default settings ) was set with a start threshold of 0.2 and a stop threshold of 0.0 for 10 scans per sample . chemometric and statistical analysis was undertaken predominately using simca 13.0.3 , a chemometric software package ( umetrics ag , sweden ) , and post - analysis fold change analysis using metaboanalyst 2.0 ( xia et  al . , data were normalised with respect to the internal standards ( adonitol and c - sorbitol ) . chromatography peaks were considered significant where the signal to noise ( s / n ) ratio was > 50 , the fold change ( fc ) was > 2.0 , and p - values were 0.05 . the calculated p - values were corrected using the benjamini - hochberg ( bh ) adjustment method taking into account a false discovery rate ( fdr ) threshold of 0.25 . the data generated by mass spectral analyses were normalized with respect to internal standards relative standard deviation ( rsd  =  5.14% ) , where a magnitude of 1 fold change ( fc ) referred to the concentration of 10 the normalised data were log transformed and further analysed by principal component analysis ( pca ) ( fig . dmodx or  distance of observation plot of active and control fractions ( fig . an unsupervised statistical approach using pca was undertaken on the combined dataset in order to investigate any sub - data grouping , which was not evident ( see fig . s2 for the pca and dmodx plots respectively ) . to accommodate the outliers and enable differentiation and greater predictability between the groups based on metabolic features , a partial least square - discriminant analysis ( pls - da ) was employed ( karpe et  al . pls - da is a supervised method used to analyse large datasets and has the ability to assess linear / polynomial correlation between variable matrices by lowering the dimensions of the predictive model , enabling easy discrimination between samples and the metabolite features that cause the discrimination ( wold et  al . , 2001 ) . the data obtained from pls - da were subjected to univariate analyses methods of t - test , fold change analysis and analysis of variance ( anova ) ( vranova et  al . orally administered drugs can potentially disrupt the gut microbiota , therefore it was important to test for antimicrobial activity . the most effective fraction was tested against gram positive ( bacillus cereus atcc 11778 and staphylococcus aureus atcc 12600 ) , gram negative ( pseudomonas aeruginosa atcc 10145 and salmonella entrica serovar typhimurium atcc 13311 ) bacteria for their antimicrobial activity . overnight cultures were prepared by inoculating approximately 2  ml nutrient broth ( nb ) ( oxoid , australia ) with a loopful of microorganism from the slant cultures . all bacterial cultures were incubated at 37  c overnight under aerobic conditions . for the disk diffusion test , brain heart infusion agar ( oxoid , australia ) was used as the growth medium . the disc diffusion technique involved the following procedure ; a sterile cotton - tipped swab was dipped into each bacterial broth and used to inoculate bhi agar plates . the plates were dried for 23  min before pre - sterilised discs ( 6  mm diameter ) were placed on the agar surface and 5  l of the selected fraction were added individually onto the discs . the seeded plates were incubated at 37  c for 24  h. tetracycline ( 30  g ) disks were used as positive controls . understanding the cellular toxicity is important in drug discovery and eukaryotic cell cultures are accepted as the model system of choice to get a first approximation of toxicity ( atterwill and steele , 2009 ) . mammary epithelial mcf10a cells were seeded in black walled , flat bottom 384 well black walled plates ( corning ) at 700  cells / well using a biotek 406 automated liquid handling dispenser ( biotek , vermont , usa ) in a total volume of 40 l / well . cells were cultured in dmem - f12 containing 5% horse serum ( life technologies , australia ) , 20  ng / ml human epidermal growth factor ( egf , life technologies , australia ) , 100  ng / ml cholera toxin ( sigma , australia ) , 0.5  g / ml hydrocortisone ( sigma , australia ) and 10  g / ml insulin ( human ; novo nordisk pharmaceuticals pty ltd ) . after 24  h incubation at 37  c , 5% co2 , the growth medium was aspirated and the cells were treated with fraction 5 and controls ( media    ethanol ( 1% ) and the chemotherapeutic compound doxorubicin ) over an 11 point dose curve and incubated for a further 48  h. on day 4 , cell viability was measured using the celltiter - glo luminescent cell viability assay ( promega , australia ) . briefly , 20  l of celltiter - glo was added to each well and mixed by shaking for 2  min . plates were incubated for 10  min at room temperature and luminescence was read on a cytation3 plate reader ( biotek ) . doxorubicin , starting at 3  m concentration was used as the positive control for cell toxicity ( data not shown ) . ph can influence the stability , wettability , solubility , physical form and particle size of drug compounds . drug degradation often occurs by hydrolysis that can be dependent on the ph of the medium and therefore ph can significantly alter the stability of a drug formulation ( sigfridsson et  al . , 2009 ) . the ph of fraction 5 was changed to ph 1 by the addition of hcl ( sigma , australia ) . after 1  h , the hcl was neutralized by adding 5  m naoh ( sigma , australia ) and the ph was adjusted to 7 ( sigma , australia ) . the ph - adjusted 40% acn fraction was then diluted and tested on adult c.  elegans using the same screening method as described in section 2.4 . plant extract using a c-18 spe catridge and all fractions showed some capacity to kill the worms , but four consecutive fractions ( eluted with 25% , 40% , 55% and 70% acn ) showed a significant effect on c.  elegans young adult survival ( fig .  1 ) . of the four active fractions , the 40% acn fraction ( hereafter mentioned as fraction 5 ) demonstrated the greatest efficacy , killing 85100% of c.  elegans adults . it is possible that the active compound(s ) from fraction 5 may have been present in the other fractions from slight carry over , however the compound / s with the greatest anthelmintic activity have primarily eluted in fraction 5 . the anthelmintic activity of fraction 5 was evaluated on h.  contortus ex - sheathed l3 motility using the motility index assay . we performed three technical replicates and three biological repeats using the same concentration as with c.  elegans ( 0.01  mg / ml ) . fraction 5 decreased the motility of h.  contortus xl3 by almost 50% compared to the negative control ( fig .  2 ) . this suggests the presence of concentrated active compound / s with anthelmintic activity in fraction 5 . untargeted gc - ms analysis of fraction 5 ( active fraction ) and fraction 2 were tested for the identification of any putative anti - nematodal components . fraction 2 , the comparatively inactive fraction of the two , was used as the negative control for comparison against fraction 5 . application of pls - da increased the linearity and the overall model predictability ( fig . the analysis was able to improve the metabolite behavioural distribution , as evident from a distinct metabolite distribution ( fig . overall , of 158 metabolites identified by gc - ms , 132 statistically significant metabolites were common to both fractions ( fig . 4 ) the metabolites present in fraction 5 were differentiated from fraction 2 by the distribution of their fold change ( fc ) values ( table  1 ) . medium chained fatty acids ( mcfas ) were the highest represented species identified in fraction 5 , and included heptadecanoic acid ( fc  =  283.2 ) , 2 , 3-bis [ tms]oxy ) butanedioic acid ( fc  =  66.1 ) and docosanoic acid ( fc  =  45.9 ) . benzoic acid , 4-[(trimethylsilyl)oxy]- , trimethylsilyl ester or 4-hydroxy benzoic acid was also highly represented in this fraction with a fc value of 51.131 , equivalent to a concentration of 511.3  mg / l . this compound has been identified from various higher plants as a secondary metabolic constituent ( faizi et  al . hydroxycinnamic acid ( trimethylsilyl 3,4-bis(trimethylsiloxy)cinnamate ) was also observed at considerable levels in fraction 5 ( fc  =  35.136 or 351.4  mg we expect there are other biologically active metabolites in this fraction that were not identified as major constituents in the gc - ms analysis due to detection limits of the system and current mass spectral databases . purified compounds previously identified from p.  fel - terrae plant extracts have shown antimicrobial activity ( huang et  al . , 1999 , zou et  al . , 2006 ) therefore , we tested fraction 5 against two gram positive ( b.  cereus and s.  aureus ) and two gram negative ( p.  aeruginosa and s.  enterica ) bacteria using the disk diffusion test on brain heart infusion agar . interestingly , fraction 5 did not inhibit growth of any of the tested bacterial strains while as expected tetracycline inhibited the growth of all four bacteria ( data not shown ) , confirming the validity of the test ( galappathie et  al . , 2014 ) . this indicates that the anthelmintic activity of the compounds in fraction 5 is highly specific and is not exhibiting general toxicity . we used the immortalized human mammary epithelial cell line , mcf10a , to examine the potential toxicity of fraction 5 . fraction 5 demonstrated minimal and statistically insignificant cytotoxicity at concentrations less than 0.015  mg / ml compared to the negative control ( non - treated ) ( fig . levamisole did not show statistically significant cytotoxic activity below 0.5  mg / ml . since fraction 5 was used at 0.01  mg / ml for anthelmintic activity testing against both c.  elegans and h.  contortus , it could be concluded that there was minimal toxicity at this concentration . we evaluated the ph stability of fraction 5  at seven different ph levels ( from 1 to 7 ) using the c.  elegans adult viability assays described above . after 72  h , all treated fractions demonstrated statistically similar efficacy compared to the untreated fraction 5 ( fig . plant extracts has significant impacts on mortality and motility of c.  elegans and h.  contortus , respectively . moreover , fraction 5 contains biologically active constituents that were ph stable and showed mimimal cytotoxicity in mammalian cells at the therapeutic concentration used . although we report the 50 most abundant compounds identified , we recognise the possibility that there may be other potent compounds with good anthelmintic activities present at lower concentrations but undetectable in our system . while the biologically active compounds were eluted in fraction 5 , there were other potential bioactive compound / s present in fractions 4 , 6 and 7 , although they showed considerably less anthelmintic activity . of the 158 metabolites identified in fraction 5 , 132 were common to the inactive fraction 2 , leaving 26 specific and highly concentrated molecules that could relate to the effects observed in fraction 5 . further chemical dissection of this fraction will help determine whether a single compound or combination of compounds associate with the anthelmintic activity identified here . additionally , a limitation of single quadrupole gc - ms based metabolomics is the need to derivatise samples prior to analysis and the difficulty in identifying and characterizing some plant based secondary and tertiary metabolites . as such , any future work analysing compounds that are associated with the anthelmintic activity of p.  fel - terrae lour . plant extracts would need to consider a suite of complementary techniques ( i.e. , liquid chromatography ( lc ) , e.g. , lc - tofms ( time of light mass spectrometry ) , lc    lc - tofms and lc - hrt ( high resolution time of flight mass spectrometry ) and more advanced gc techniques ( e.g. , gc - tofms , gc    gc - tofms and gc - hrt ) . this will likely lead to the discovery and identification of key biomarkers within isolated fractions through the comprehensive metabolomics mapping of p.  fel - terrae lour . mcfas were abundant in fraction 5 , and this class of compounds ( such as hexanoic , and decanoic acids at 5  mg / l concentrations ) have been found to inhibit growth and resultant fermentation ( edwards et  al . recently , it has been shown that high concentrations of mcfas , such as hexadecanoate ( palmitic acid ) and dodecanoate , caused growth inhibition of aspergillus niger ( karpe et  al . , 2015b ) . furthermore , some mcfas such as palmitic , oleic , myristic and capric acids have been effective in inhibiting egg hatching by up to 84% , and causing 50% juvenile mortality in root knot nematodes , including meloidogyne incognita ( see zhang et  al . , 2012 ) and m.  halpa ( see sharma et  al . , 2014 ) . the metabolite 4-hydroxybenzoic acid is either related to or is a member of the ecathechins family and has been reported to show biological activity ( faizi et  al . , 2011 ) ; 4-hydroxybenzozic acid identified from the floral parts of tagetes patula l has been shown in an in vitro toxicity bioassay to possess anti - nematodal activity ( 40% mortality at 5  ng / ml ) against the free living larvae of the plant cyst nematode heterodera zeae ( see faizi et  al . , 2011 ) . likewise , nematicidal and paralysis activities of 4-hydroxybenzoic acid analogues , isolated from chinaberry ( melia azedarach l. ) , have been reported against free living larvae of the plant root - knot nematode meloidogyne incognita ( ec50/48h 871  g / ml ) ( aoudia et  al . , 2012 ) . hydroxycinnamic acids are secondary metabolites and antioxidants ( olthof et  al . , 2001 ) , and are an intermediate of lignin biosynthesis in higher plants ( boerjan et  al . , 2003 ) . hydroxycinnamic acids have been shown to possess anti - nematodal effects against free living larvae m.  incognita when isolated from chinaberry ( melia azedarach ) ( ec50  >  1000  g / ml ) ( see aoudia et  al . , 2012 ) and the plant pathogenic nematode pratylenchus scribneri when isolated from lolium arundinaceum ( see bacetty et  al . , 2009 ) . plant - derived compounds have been shown to have clear efficacy and safety and have been used to develop semi - synthetic or synthetic drugs ( tagboto and townson , 2001 ) . this study demonstrates that fraction 5 possesses anthelmintic activity against both free living c.  elegans and parasitic h.  contortus . further analyses should be conducted to understand the mechanism of anthelmintic activity of the active compound / s within fraction 5 .
anthelmintic resistance is widespread in gastrointestinal nematode populations , such that there is a consistent need to search for new anthelmintics . 
 however , the cost of screening for new compounds is high and has a very low success rate . using the knowledge of traditional healers from borneo rainforests ( sarawak , malaysia ) 
 , we have previously shown that some traditional medicinal plants are a rich source of potential new anthelmintic drug candidates . in this study , picria fel - terrae lour . 
 plant extract , which has previously shown promising anthelmintic activities , was fractionated via the use of a solid phase extraction cartridge and each isolated fraction was then tested on free - living nematode caenorhabditis elegans and the parasitic nematode haemonchus contortus . 
 we found that a single fraction was enriched for nematocidal activity , killing 90% of c.  elegans adults and inhibiting the motility of exsheathed l3 of h.  contortus , while having minimal cytotoxic activity in mammalian cell culture . 
 metabolic profiling and chemometric analysis of the effective fraction indicated medium chained fatty acids and phenolic acids were highly represented .