Biochemometry identifies ostruthin as pluripotent antimicrobial and anthelmintic agent from masterwort

Summary The root extract of Peucedanum ostruthium (PO-E) was identified as a promising antibacterial source from a screening of 158 extracts against Staphylococcus aureus. It has also recently been shown to significantly decrease the survival of the nematode Caenorhabditis elegans. We used the biochemometric approach ELINA to investigate the phytochemical characteristics of the multicomponent mixture PO-E to identify the anti-infective constituent(s) targeting S. aureus and C. elegans.1H NMR spectra of PO-E-derived microfractions were correlated with their respective bioactivity data. Heterocovariance analyses unambiguously identified ostruthin as an anti-staphylococcal constituent, which potently also inhibited Enterococcus spp.. ELINA demonstrated that anthelmintic activity was due to a combinatorial effect of ostruthin and isoimperatorin. A C. elegans-based survival and motility assay confirmed that isoimperatorin, imperatorin, and verapamil modulated the susceptibility of ostruthin. The combinatorial effect of these natural products was shown in larvae studies to be related to the function of the nematodes’ efflux pump.


INTRODUCTION
Antimicrobial resistance (AMR) is a major concern in public health care due to therapeutic limitations encountered concerning bacterial infections. Therefore, the search for new antimicrobial agents is crucial. Recent developments in drug discovery show that the number of bacterial species with antibiotic resistance has surpassed the number of new antibiotics introduced. 1 With the current pace it is predicted that AMR will cause over 10 million deaths per year by 2050 and is the leading threat to human health. 2 In 2017, the World Health Organization (WHO) released a priority list of drug-resistant microorganisms that pose a major health threat and should be the focus of research and development for new antibiotics. This list was largely comprised of the so-called ''ESKAPEE'' pathogens-Enterococcus faecium, Staphylococcus aureus, Klebsiella pneumoniae, Acinetobacter baumannii, Pseudomonas aeruginosa, Enterobacter spp., and Escherichia coli. 3, 4 The highest-ranked gram-positive bacteria included in this group are vancomycin-resistant (VR) E. faecium and methicillin-resistant S. aureus (MRSA). 5,6 Intestinal nematode infections are among the most common parasitic infections worldwide and are caused by various species of parasitic worms e.g., by the roundworm Ascaris lumbricoides or the hookworm Necator americanus. 7,8 According to the WHO, 9 approximately 1.5 billion people (24% of the human population) are infected with one or more helminth species. Although most nematode infections are non-lethal, they often lead to chronic ailments, such as physical disabilities or delayed development of the affected individual. Parasitic nematodes also pose a huge threat to the health of animal livestock and plants, resulting in a serious threat to the global food supply worldwide. Unfortunately, the arsenal of effective anthelmintic agents is limited, and due to the extensive use of anthelmintic compounds in the past, nematode resistance is on the rise. 10,11 Currently, there are only a handful of anthelmintic compound classes available (e.g., the macrocyclic lactone ivermectin or the imidazotiazole levamisole) with nematode resistance reported for each class. One reason for this limited number of effective drugs discovered is the complex life cycle of parasitic nematodes. These worms rely on a host for propagation, making it difficult to identify lead compounds with high throughput. 12 With the increasing prevalence of nematode resistance, however, it is imperative to shorten the time required for the development of effective anthelmintics with less resistance. 13 Nature has proven to be an outstanding source of compounds with manifold therapeutic applications and has been an inspiration for the development of new antibacterial drugs in the past. 19,20 According to the comprehensive review by Newman and Cragg, 21 162 antibacterial drugs have been approved between 1981 and 2019, of which 11 compounds are natural products (NPs), while 78 are NP-derived (e.g., by semi-synthetic modification). In contrast, the number of antiparasitic compounds is comparably small with only 20 new drugs approved in the same time frame. Of those, only two represent NPs and seven are NP-derived. Six compounds are considered totally synthetic and have been identified via random screening or the modification of an existing drug. 21 A growing number of research articles aims to identify new anthelmintics from natural sources. [22][23][24][25][26] Plant extracts are an important source of new chemical scaffolds with valuable applications for drug discovery. [27][28][29][30] However, the search for bioactive compounds from complex mixtures (i.e., crude extracts) remains a Herculean task. [31][32][33] Various approaches for the identification and prioritization of a bioactive extract have been described, e.g., knowledge from the ethnomedicinal use of a species in folk medicine followed by a phenotypic screening with a readout that is related to the original use of the species. Nevertheless, the assignment of bioactivity to specific compound(s) is a substantial challenge in NP-based drug discovery. 34,35 Bioactivity-guided fractionation approaches are considered the gold standard in NP-based drug discovery. Repetitive fractionation procedures alternate with bioactivity screening and thereby reduce the complexity of the multicomponent mixture successively to isolate the compound(s) responsible for the observed biological effect. A decisive disadvantage is, however, that highly abundant (but potentially inactive) compounds overshadow minor (but potentially active) compounds. 35 Since no structural information of the actual active compound(s) is given, the most time-consuming step in the bioactivity-guided fractionation approach remains the isolation and identification of the bioactive compound(s). In this light, novel biochemometric approaches for the targeted identification of bioactive compounds have been developed in recent years. Thereby, bioactivity data are correlated with their respective metabolite profiles (e.g., obtained from 1 H NMR and/or LC-MS measurements) to pinpoint a particular compound to the observed activity. 30 The biochemometric approach ELINA (Eliciting Nature's Activities) that is employed in this study relies on the deconvolution of a complex mixture (i.e., an active extract or fraction) by generating microfractions with a quantitative variance of constituents over several consecutive fractions. 36 Correlating bioactivity data with their respective 1 H NMR data allows for the generation of so-called heterocovariance analysis (HetCA) coefficient plots. Thus, the structural information of compounds contributing to activity can be detected prior to isolation. [37][38][39] By further implementing LC-MS-CAD/ELSD data in the early phytochemical investigation (i.e., after a single fractionation step), ELINA enables early identification of bioactive compounds (e.g., via dereplication and NMR STOCSY analysis) and a target-oriented isolation of the compound(s) of interest. 36,40 We rationalized the identification of bioactive NPs through the utilization of a multidisciplinary approach: ethnopharmacological knowledge guided the selection of the starting material and was followed by a subsequent phenotypic-based screening for the identification of antimicrobial starting material. We investigated a total number of 158 small-scale extracts of natural origin for their ability to inhibit the growth of S. aureus and E. coli. The root extract of Peucedanum ostruthium (L.) Koch (PO-E) was revealed as a promising anti-staphylococcal agent. Concurrently, PO-E was shown to significantly reduce the survival rate of C. elegans. To accelerate the identification of the bioactive NPs responsible for the observed phenotypic effects on S. aureus and C. elegans, the biochemometric tool ELINA was implemented in this study inter alia for the first time in a whole organism model for anthelmintic screening in C. elegans.  (Table S1). The natural materials have been selected based on their reported use in traditional medicine for the treatment of various kinds of infections without in-depth knowledge of their active principle(s). None of the extracts tested at 100 mg/mL showed a significant antibacterial effect on E. coli (i.e., R50% growth inhibition); however promising results were obtained against S. aureus. Three fungal extracts, one extract prepared from the polypore species Ganoderma applanatum (Pers.) Pat. (extract 47) and two extracts prepared from different strains of Piptoporus betulinus (Bull.) P.Karst (extracts 93 and 94), displayed good antibacterial activity with average inhibition rates of 59%, 52%, and 58%, respectively (Table S1). Two herbal extracts prepared from the roots of Sophora flavescens Aiton (extract 124) and Peucedanum ostruthium (L.) Koch (extract 150; PO-E), as well as the three rootbark extracts prepared from Morus alba L. (extract 156-158), showed excellent inhibitory activities against S. aureus, reaching R90% growth inhibition (Table S1). Anti-staphylococcal activities for S. flavescens [41][42][43] and M. alba 44,45 have already been reported, whereas the promising activity of PO-E against S. aureus has not been described before.

ll
P. ostruthium, commonly known as masterwort, has traditionally been used in the Alpine regions of Austria for the preparation of liqueurs, bitters, and teas. The Swiss physician and alchemist Paracelsus (1493-1541 AD) recommended masterwort for the prevention of infections and as a remedy for tuberculosis and worm infections. 46 The dried and cut roots (i.e., Radix Imperatoriae) are valued for alleviating physiological problems including gastrointestinal conditions, and disorders of the respiratory tract and the cardiovascular system. 47,48 In a recent study, we could observe that PO-E significantly decreases the survival rate of the nematode C. elegans when tested at 100 and 25 mg/mL 17 and exerts anti-inflammatory activities in a model of IL-1 stimulated endothelial cells. 49 Based on these results, PO-E was selected for an in-depth investigation focusing on its anti-infective profile against both S. aureus and C. elegans.
Target-oriented identification of the active constituent(s) in masterwort against S. aureus and C. elegans by implementing the 1 H NMR-based biochemometric approach ELINA The anti-inflammatory potential of P. ostruthium was previously investigated using the biochemometric approach ELINA. 40 For this purpose, 1 H NMR spectra and LC-MS-CAD data from 31 PO-E-generated microfractions were correlated with their respective bioactivity data from three cell-based in vitro assays (i.e., one NF-kB reporter-gene assay and two NF-kB target-gene assays addressing the endothelial adhesion molecules VCAM-1 and E-selectin). By applying this method, several compounds were successfully identified as in vitro anti-inflammatory agents prior to isolation. Hence, for the identification of the anti-infective constituent(s) of PO-E, the 31 previously generated microfractions (PO01_01-PO01_31) were probed in the two phenotypic-based screening assays against S. aureus and C. elegans ( Figure 1). Hence, no phytochemical work-up (i.e., microfractionation of PO-E) or analysis (i.e., 1 H NMR and LC-MS-ELSD measurements) had to be performed as all data necessary for the heterocovariance correlation were already available from the preceding study on masterworts' anti-inflammatory activities. 40 The microfractions from PO01_05 to PO01_09 showed the highest percentage of S. aureus growth inhibition (i.e., R80% at 100 mg/mL). Interestingly, these results were similar to the growth inhibition of PO-E (Table S2; Figure 1A), indicating that the active principle of PO-E against S. aureus is concentrated between PO01_05 and PO01_09. MIC values of those microfractions ranged from 6.25 to 100 mg/mL, with the lowest ones achieved by PO01_06 and PO01_07 (i.e., 6.25 mg/mL, Table 1). Strikingly, PO01_05 and PO01_08 (MIC values of 50 and 100 mg/mL, respectively) showed the highest-nematicidal activity in the C. elegans survival assay ( Figure 1B and Table 1). When tested at 50 mg/mL, both microfractions resulted in an unambiguous reduction of the nematode survival rate of 23% for PO01_05 and 16% for PO01_08. In contrast, the most potent microfractions (i.e., PO01_06 and PO01_07) on S. aureus showed no pronounced effect on the survival rate of C. elegans, assuming that different compounds/compound classes are contributing to the antimicrobial and nematicidal effects observed in this study.
For the identification of the anti-infective constituent(s) present within PO-E, packages of consecutive microfractions with increasing/decreasing activities were generated ( Figure 1 iScience Article (consisting of PO01_05-PO01_07), and package III (consisting of PO01_08-PO01_10) were used to identify the nematicidal compounds. By statistically correlating the activity data with their respective 1 H NMR data, so-called heterocovariance (HetCA) plots were generated ( Figure 2) allowing for the identification of structural features from bioactive (but also inactive) constituents within each individual package. 37,38 For instance, in Figure 2A typical signals given by fatty acids between d H 1 and 2 are shown, which are defined as ''cold features'' (blue signals showing downwards), whereas aromatic signals (between d H 6.0 and 8.5) as well as signals given by e.g., methyl groups (between d H 1.5 and 1.8) are displayed as ''hot features'' (red signals showing upwards). Semi-quantitative LC-MS-ELSD analyses were also performed to visualize the increasing/decreasing peak areas under the curve (AUC) and to facilitate the dereplication of the secondary metabolite(s) present within each package ( Figure S1). For instance, the LC-ELSD stack plot of package I indicates the presence of two peaks at an LC retention time (t R ) of 8.35 and 8.85 min, whereas the AUC of the latter one decreases from the most active microfraction PO01_06 to the least active microfraction PO01_08.
A dereplication of the selected peak was performed considering an m/z value of 299.4 in the positive mode and the structural information given by the generated HetCA plot. Thereby, the coumarin ostruthin (6-geranyl-7-hydroxycoumarin; 1) was identified as an anti-staphylococcal agent without preceding isolation efforts. The previously isolated coumarin 1 40 facilitated the comparison of the positively correlated resonances in the HetCA plot of package I with the 1 H NMR spectra of 1 ( Figure 3). In the upfield chemical shift area, signals at d H 1.59, 1.65, and 1.7 were assigned to the methyl groups at C-3 0 and C-7'. The four protons at C-4 0 and C-5 0 are shown as triplet at d H 2.1 and quartet at d H 2.2, respectively. The two multiplets at d H 5.1 and 5.3 were assigned to the protons at C-2 0 and C-6 0 , respectively. The signal given by the proton at C-1 0 is not visible because of an overlay with the solvent signal at d H 3.31. The signals in the downfield chemical shift area assigned to the protons at C-3 and C-4 were observed as two doublets at d H 6.15 and 7.81. Two singlets at d H 6.70 and 6.27 were assigned to the protons at C-8 and C-5, respectively. Thus, the ELINA allowed for the fast and target-oriented identification of the anti-infective constituent(s) by applying two assays with different readouts. Although a similar bioactivity range (i.e., in PO01_05 to PO01_09) has been disclosed in both assays, the biochemometric approach identified different chemical features contributing to the observed effects against S. aureus and C. elegans.
Coumarins are a major compound class in Peucedanum species, many of which carry beneficial health properties, such as anti-mycobacterial, 50,51 anti-inflammatory, 40,49,52,53 and antioxidant activities. 54 Nevertheless, furanocoumarins, a sub-class of coumarins, are known to cause severe phototoxic reactions to humans, such as erythematous rash. 55 Previously, Vogl et al. 48 performed a quantification of the main coumarins present in various batches of commercial and field-collected roots of P. ostruthium. Compound 1 was identified as the main coumarin present in the dichloromethane extracts of masterwort, with 38-41% of the total coumarin content. In comparison, the content of the furanocoumarins isoimperatorin (2) and imperatorin (3) was comparably low with 9-10% and 12-15%, respectively. 48 No studies indicate phototoxic activity of 1, but some cytotoxic activity has been reported on two pancreatic cell lines. 56 Antibacterial effects of ostruthin on S. aureus and other gram-positive strains To further substantiate the biochemometric correlations with antibacterial activity, minimum inhibitory concentrations (MICs) were determined for compounds 1 and 2 against S. aureus ATCC 29213 (Table 2). Compound 1 was confirmed to be the solely active principle of package I with an MIC of 12.5 mM. Additional MICs for 1 were determined against MRSA strain ATCC 43300 and four enterococcal strains. Compound 1 has already been reported to possess antibacterial activity against several species of Mycobacterium, 50  For the identification of the anthelmintic features in PO-E, the biochemometric approach pinpointed to more than one constituent. As can be seen in package II, the signals of 1 are depicted as ''cold features'' whereas the signals of 2 are shown as ''hot features'' ( Figure 2B). This is also supported by the increasing AUC of 1 (at t R 8.85 min) from the most active (i.e., PO01_05) to the least active microfraction (i.e., PO01_07; Figure S1B). In package III, on the other hand, the signals of both compounds are displayed as hot features.
In Figure 4 the structural predictions delivered by a statistical total correlation spectroscopy (STOCSY) plot are compared to the 1 H NMR spectra of the isolated compounds 1 and 2. By applying this method, multiple 1 H NMR signals from the same molecule can be detected based on their multi-collinearity of their signal intensities in a selected set of 1 H NMR spectra. 36, 58 The 1 H NMR signals of 1 match the dark red signals in the STOCSY plot obtained from the aliphatic signal at 1.715 ppm, whereas the 1 H NMR signals of 2 match the orange-colored signals in the STOCSY plot. Therefore, the combinatorial effects of 1 and 2 were presumed from the applied omics approach. To substantiate the assigned nematicidal principle, 1 and 2 were tested at concentrations ranging from 5 to 500 mM in the C. elegans survival assay ( Figure 5A; Table S3). Intriguingly, when 1 and 2 were tested individually, the survival rate of the worms was not affected. A pronounced and dose-dependent nematicidal response was however observed when 1 and 2 were combined as a 1:1 mixture ( Figure 5B), with an EC 50 of 55.70 mM ( Figure 5C).
Driven by the promising nematicidal properties of masterwort and the combinatorial effects of its constituents, we further investigated the impact of these samples on the locomotor activity of C. elegans.
Motility-based screening deciphers ostruthin (1) as main active agent against C. elegans In C. elegans, the survival rate depends on two crucial factors: locomotion and feeding. While locomotion represents a strong stimulus for the nematodes to regulate their food intake by increasing their pharyngeal  iScience Article pumping rate, feeding directs their locomotory pattern from dwelling to roaming, depending on the presence of food. Hence, locomotion and feeding behaviors are well coordinated to ensure feeding efficiency. 59 Loss of motility (i.e., paralysis) is a characteristic phenotypic readout to detect potentially anthelmintic compounds. Among the classes of anthelmintic drugs on the market, macrocyclic lactones, like ivermectin, are the most prevalently used anthelmintics. Macrocyclic lactones act on the glutamategated chloride channels, an invertebrate-specific family of ion channels, which are expressed in the nematodes neuromuscular system. Levamisole is also commonly used against nematode infections, acting on specific nicotinic acetylcholine receptors (nAChRs). Both compound classes ultimately induce paralysis of the body wall. 60,61 To examine the effect of 1, 2, and their 1:1 mixture on nematodes' body wall muscles, we employed an automated infrared motility reader in our study ( Figure 6; Table S4). This fast and straightforward device provides additional insights into the nematicidal activity 62 of a sample in the context of a whole-organism-based screening on 96-well microtiter plates. By measuring the locomotor activity of treated vs. control worms, pronounced effects on C. elegans' motility were detected for PO01_05 (at 50 mg/mL) with a significant reduction of 81.20% (p < 0.01). In comparison, the levamisole-treated worms (at 10 mM) showed a significant reduction of 88.01% (p < 0.01) on day 3. A dose-dependent reduction of worm motility was shown for the 1:1 mixture of 1 and 2 when tested from 500 to 50 mM on day 3, and from 500 to 5 mM on day 5 and 7, respectively. Strikingly, tendencies of decreasing the nematodes' motility could also be observed for 1 on the 3 rd , 5 th , and 7 th day of the experiment. For instance, on the 7 th day of the experiment, a dose-dependent reduction of worm motility was observed for compound 1-treated nematodes with 72.64, 69.79, 34.03, and 4.82% when tested between 500 and 50 mM, respectively. Although the effects were neither as pronounced iScience Article as for PO01_05, nor as effective as for the 1:1 mixture, these results indicate that 1, but not 2, is the main nematicidal principle against C. elegans. The furanocoumarin 2, when tested as a pure compound, showed no effects on the locomotor activity of C. elegans.
The findings from the survival and motility assays indicate a modulatory effect of 2 on the susceptibility of 1. Interestingly, P. ostruthium and its isolated compounds have recently been investigated for their antimycobacterial properties against Mycobacterium smegmatis. Simunovic et al. 51 investigated inter alia 1 and imperatorin (3; a constitutional isomer of 2) regarding their resistance-modulatory effects and efflux pump inhibition in M. smegmatis. The authors identified 1 as major antimicrobial compound, whereas the furanocoumarin 3 was found to cause potent modulatory effects via inhibition of the bacterial efflux pump. Compound 3 was not investigated in this study. The reported combinatorial effects of 1 and 2 on the mycobacterial efflux pump as well as the outcome of our nematicidal findings in C. elegans prompted us to further investigate a potential interplay of 1 and 2 on the efflux pump of C. elegans.
The modulatory effects of isoimperatorin (2), imperatorin (3), and the efflux pump inhibitor verapamil (4) on the susceptibility of C. elegans larvae toward ostruthin (1) Drug resistance in nematodes can be divided into: (i) specific mechanisms, such as modifications of drug receptors, and (ii) non-specific mechanisms, which are mediated by detoxification enzymes or drug efflux pump pathways. The second group involves multi-drug resistance proteins, such as ATP-binding cassette (ABC) transporters, which attain their protective mechanism through enhanced cellular drug efflux. 63 Among the ABC transporters, the xenobiotic efflux pump P-glycoprotein (PGP) plays an important part in the sensitivity of nematodes to anthelmintic drugs. 14,60,63-66 Inhibition of PGPs decreases the efflux of The effects of 1, 2 and the 1:1 mixture of 1 + 2 on the motility of C. elegans were investigated using an IR-based wormtracker. Worms were treated with control, levamisole (at 10 mM), PO01_05 (at 50 mg/mL) and different concentrations (ranging from 500 mM-5 mM) of (A) the pure compound 1, (B) compound 2, and (C) the 1:1 mixture of 1 + 2. The basal activity of the worms was measured on day 0 and data were normalized to the basal activity. After the worms were treated with the respective samples, their movement was measured on the 3 rd , 5 th , and 7 th day of the treatment. Bars represent the mean locomotor activity (in percentage) G SD of three parallel experiments. One-way ANOVA with Dunnett's post-test was used for statistical evaluation (when compared to the control at the given day). p-value <0.05 was considered as statistically significant. *, p value <0.05; **, p value <0.01; ***, p value <0.001; ****, p value <0.0001.

OPEN ACCESS
iScience Article the active drug from the nematode and thus increases the concentration of the drug within the nematode. For instance, the calcium (Ca 2+ ) channel blocker verapamil has previously been reported as a PGP inhibitor able to potentiate the efficacy of the anthelmintic drug ivermectin against blood-sucking Haemonchus contortus larvae 67 and C. elegans larvae. 14,64 Interestingly, Ca 2+ antagonistic effects for the furanocoumarins 3 and 2 have previously been reported. The activity of 2, 3, and verapamil (4) were investigated by measuring the depolarization-induced 45 Ca 2+ uptake in GH 4 C 1 rat pituitary cells, which resulted in IC 50 values of 6.8, 10.8, and 2.0 mg/mL, respectively. 68 In another study, the Ca 2+ antagonistic activity of a Peucedanum palustre (L.) Moench root extract was pinpointed inter alia to the furanocoumarin 2. 69 Consequently, we investigated the effects of the Ca 2+ channel blockers 2, 3, and 4 on the susceptibility of 1, as well as its impact alone in a larval development-inhibition assay ( Figure 7A; Table S5). 1, when tested alone, impaired the larval development only at the highest concentration tested (i.e., 500 mM) with a larval development inhibition by 93.93%. When tested at concentrations ranging from 250 to 5 mM, the development of C. elegans larvae was hardly affected with only 3.2-13.1% deviation to the untreated control. Conversely, all three Ca 2+ antagonists, when added at 50 mM, dose-dependently increased the susceptibility of 1 toward the larval development, with EC 50 rates of 93.3, 98.6, and 72.1 mM for 2, 3, and 4, respectively ( Figure 7B). Hence, the susceptibility of C. elegans larvae toward 1 with an EC 50 of 173.0 mM was decreased by a factor of 1.854, 1.755, and 2.399 when combined with 2, 3, and 4, respectively. In a previous study, Janssen et al. 14 reported an increased ivermectin susceptibility of C. elegans larvae after inhibition of all PGPs with 4. The authors reported a 2.5-fold increase in ivermectin susceptibility when worms were cotreated with 50 mM and 100 mM of 4 compared to no co-treatment, 14 which is in line with our results. EC 50 calculations from the larval development-inhibition assay were very high, especially when compared to the EC 50 values obtained from the 1:1 mixture of 1 and 2 in the survival assay with adult worms (Figures 5B and 5C). For a better comparison of the treatments, the survival rate of adult worms was re-evaluated after co-treatment with 1 and 50 mM 2 or 4, respectively. The modulatory effects of 2 and 4 on the survival rate of adult worms resulted in much lower EC 50 values than on larval development, with EC 50 values of 27.44 and 19.79 mM for 2 and 4, respectively ( Figure 8). The reason for the higher efficacy in adult C. elegans vs. L1 larvae remains unclear. One explanation could be the differential expression of PGPs in C. elegans larvae and adult worms. For instance, Martin et al. 70 reported a significantly increased expression of the transport protein gene pgp-9 in Parascaris univalens larvae exposed to the anthelmintic drugs pyrantel and thiabendazole. On the contrary, drug exposure to adult worms did not significantly increase gene expression. 70 Nevertheless, the Ca 2+ channel blocker 2 may act in a similar manner on PGPs as the well-known PGP inhibitor (and Ca 2+ channel blocker) 4. However, this is only speculative and warrants further investigations, for example in PGP loss-of-function mutants.
Further, a potential combinatorial effect of 1 and 2 in S. aureus was investigated based on previous results reported for imperatorin (3). 71 Compound 2 did not further increase the antibacterial activity of 1 against S. aureus when 500 mM of 2 was tested with sub-MIC of 1 (3.13 mM; Table S6). This was in agreement with the biochemometric data. Strain ATCC 29213 also presents no antibiotic resistance linked to efflux. Madeiro et al. 71 showed synergistic effects of imperatorin (3) when tested in combination with erythromycin and norfloxacin (4-fold MIC reduction) on S. aureus strain, carrying the gene for overexpression of efflux pumps.
A key benefit of using the whole organism C. elegans in the presented ELINA workflow is the nematodes' simplicity that allows for large numbers of samples to be probed, e.g., in a miniaturized survival assay or by means of a semi-automated motility tracker. Only small amounts of microfractions and their constituents are required. Especially in NP drug discovery, the use of whole-organism assays enables the study of interactions in multicomponent mixtures, fractions, and pure NPs with multiple targets in an organism. 18 By also implementing a semi-automated motility tracker to the anthelmintic screening, the nematicidal 1 was detected as well as the modulatory effect of 2 could be disclosed. The involvement of the nematodes' efflux pump was iScience Article investigated and substantiated the modulatory effects of the known efflux pump inhibitors 2, 3, and 4 on the susceptibility of 1 in a larval development-inhibition assay. In sum, the implementation of C. elegans as a holistic multipurpose tool for bioactivity studies together with the target-oriented biochemometric ELINA approach, aids the exploration of pleiotropic molecular mechanisms in vivo. To our knowledge, this is the first time where a dual antibacterial and nematicidal activity is described for the coumarin ostruthin.

Limitations of the study
The study demonstrated the nematicidal properties of PO-E in C. elegans. However, none of the tested PO-derived microfractions showed similarly strong effects toward the nematodes' survival rate as the complex multi-component mixture PO-E. Therefore, it is likely that further NPs with nematicidal properties are present in PO-E potentiating the observed effects of 1 and 2. The results from the non-parasitic C. elegans model are also not completely transferable to any parasitic nematode, and this warrants further investigation in parasitic nematodes species to substantiate the anthelmintic potential of PO-E.

STAR+METHODS
Detailed methods are provided in the online version of this paper and include the following:

DECLARATION OF INTERESTS
The authors declare no competing interests.

Antibacterial activity
Absorbance values measured at 600 nm using the MultiskanGO plate reader (Thermo Fisher Scientific) were used for evaluating the antibacterial effects, by comparing to untreated controls, and expressed as percentage of growth inhibition. MIC values were defined as the lowest compound concentration at which bacterial growth was inhibited by 90% compared to maximum bacterial growth control after 24 h of incubation at 37 C.

C. elegans assays
Raw data of the survival assay was recorded with MS Excel 2019 to keep track of living/dead population per well. Survival curves for each plate were determined based on the percentage of living nematodes per well plotted versus time. At least 3 wells per trial were used for each condition. The survival rate (%) on the 7 th day of the experiment was used. The deviation of lifespan in comparison to the vehicle control is given as increase/decrease in percentage. For better visualization, all results were depicted as bar charts (GraphPad Prism 6) and data values were reported as the mean G SD. To determine whether the differences between control and treated groups were statistically significant, an ANOVA (analysis of variance) with Dunnett's post-test was performed. Significant activity is based on p < 0.05. For the larval-development inhibition assay, logarithm of doses against percentage of developed larvae were plotted and the IC 50 value of each treatment was calculated from the mean of three replicates (GraphPad Prism 6).