Journal of Ethnopharmacology 280 (2021) 114473 Contents lists available at ScienceDirect Journal of Ethnopharmacology journal homepage: www.elsevier.com/locate/jethpharm Pharmacological validation of Solanum mammosum L. as an anti-infective agent: Role of solamargine Billy Cabanillas a,b, François Chassagne c, Pedro Vásquez-Ocmín c, Ali Tahrioui d, Sylvie Chevalier d, Marieke Vansteelandt c, Asih Triastuti e, Carlos A. Amasifuen Guerra f, Nicolas Fabre c, Mohamed Haddad c,* a Laboratorios de Investigación y Desarrollo, Universidad Peruana Cayetano Heredia (UPCH), Lima, 34, Peru b Instituto de Investigaciones de la Amazonía Peruana, Avenida Abelardo Quiñonez Km. 4.5, Iquitos, Peru c UMR 152 PharmaDev, Université de Toulouse, IRD, UPS, France d Laboratoire de Microbiologie Signaux et Microenvironnement, LMSM EA4312, Normandie Université, Université de Rouen Normandie, Évreux, France e Department of Pharmacy, Universitas Islam Indonesia, Yogyakarta, 55584, Indonesia f Instituto Nacional de Innovación Agraria, Dirección de Recursos Genéticos y Biotecnología, Avenida La Molina 1981, La Molina, Lima, 15024, Peru A R T I C L E I N F O A B S T R A C T Keywords: Ethnopharmacological relevance: Fungal and bacterial infections remain a major problem worldwide, requiring the Solanum mammosum development of effective therapeutic strategies. Solanum mammosum L. (Solanaceae) (“teta de vaca”) is used in Candida albicans traditional medicine in Peru to treat fungal infections and respiratory disorders via topical application. However, Trichophyton mentagrophytes the mechanism of action remains unknown, particularly in light of its chemical composition. Pseudomonas aeruginosa Bioautography Materials and methods: The antifungal activity of TDV was determined against Trichophyton mentagrophytes and Solamargine Candida albicans using bioautography-TLC-HRMS to rapidly identify the active compounds. Then, the minimum LC-HRMS inhibitory concentration (MIC) of the fruit crude extract and the active compound was determined to precisely evaluate the antifungal activity. Additionally, the effects of the most active compound on the formation of Pseudomonas aeruginosa biofilms and pyocyanin production were evaluated. Finally, a LC-HRMS profile and a molecular network of TDV extract were created to characterize the metabolites in the fruits’ ethanolic extract. Results: Bioautography-TLC-HRMS followed by isolation and confirmation of the structure of the active com- pound by 1D and 2D NMR allowed the identification solamargine as the main compound responsible for the anti- Trichophyton mentagrophytes (MIC = 64 μg mL− 1) and anti-Candida albicans (MIC = 64 μg mL− 1) activities. In addition, solamargine led to a significant reduction of about 20% of the Pseudomonas aeruginosa biofilm for- mation. This effect was observed at a very low concentration (1.6 μg mL− 1) and remained fairly consistent regardless of the concentration. In addition, solamargine reduced pyocyanin production by about 20% at con- centrations of 12.5 and 50 μg mL− 1. Furthermore, the LC-HRMS profiling of TDV allowed us to annotate seven known compounds that were analyzed through a molecular network. Conclusions: Solamargine has been shown to be the most active compound against T. mentoagrophytes and C. albicans in vitro. In addition, our data show that this compound affects significantly P. aeruginosa pyocyanin production and biofilm formation in our conditions. Altogether, these results might explain the traditional use of S. mammosum fruits to treat a variety of fungal infections and respiratory disorders. 1. Introduction (TDV). S. mammosum was introduced to the Old World tropics for orna- Solanum mammosum L. (Solanaceae) is an annual or tender perennial mental use as well as medicinal and food purposes (Lim, 2013) and re- plant, reputed to be a tropical American plant, considered native to ported as invasive in Cuba, Philippines, Fiji, Tonga, and Hawaii (Oviedo Central and South America (Acevedo-Rodríguez and Strong, 2008), et al., 2012; Space and Flynn, 2002; Pier, 2014). The fruit, decorative, is named in the traditional medicine of Peruvian Amazon “teta de vaca” a toxic piriform berry, initially light green, to a bright yellow color * Corresponding author. E-mail address: mohamed.haddad@ird.fr (M. Haddad). https://doi.org/10.1016/j.jep.2021.114473 Received 21 May 2021; Received in revised form 26 July 2021; Accepted 27 July 2021 Available online 31 July 2021 0378-8741/© 2021 Elsevier B.V. All rights reserved. B. Cabanillas et al. J o u r n a l o f E t h n o p h a r m a c o l o g y 280 (2021) 114473 when, tending over time to orange-yellow with white spongy mesocarp to dryness under reduced pressure (Rotavapor R-100 (Buchi, Flawil, and numerous semi lenticular seeds. S. mammosum has traditionally Switzerland), the concentrated residual extracts (21.6 g) was stored at - been used to treat athlete’s foot among hunter groups in Peru (Jovel 20 ◦C in a dry airtight container until further use. A stock solution of et al., 1996; Polesna et al., 2011), Belize (Arnason et al., 1980) and S. mammosum in MeOH (10 mg mL− 1) was prepared, aliquoted in Trinidad (Lans et al., 2001), by rubbing leaf juice or cut fruit onto different vials (2 mL), then stored at - 80 ◦C for further analysis. Indi- afflicted areas. In Bolivia, the fruits are mashed and rubbed over the vidual stock solutions of amphotericin B (0.5 mg mL− 1, Sigma) (200 μL affected area to treat skin ulcer, scabies, furunculosis and rashes (Muñoz aliquot, positive control) were prepared in DMSO (analytical grade, et al., 2000; Hajdu and Hohmann, 2012). In Guatemala and in the Sigma) and used as a positive control by spotting onto the plate 1 μL of Philippines, leaves, fruits and seeds are also used in the treatment of the stock solution with disposable micropipettes. respiratory disorders such as asthma, cough, cold and sinusitis (Caceres et al., 1991). Other studies have reported various pharmacological 2.4. Fungal and bacterial strains, media, and growth conditions properties of the plant including antioxidant, anticancer, antimalarial and molluscicidal activities (Burkill, 1966; Grieve, 1971; Muñoz et al., Trichophyton mentagrophytes (18748) and Candida albicans strains 2000; Wiart et al., 2004; DeFilipps et al., 2004; Chaiyasit et al., 2006; (10231, 90028) were purchased from ATCC. The fungi were subcultured Stuart, 2010; Crommett, 2011). Additionally, due to its toxicity, it has and routinely maintained on SDA at 4 ◦C in a cold room until use. A been used in the past as an insecticide, rat poison (Flores, 1984) and for sterile swap was used to inoculate the fungi into the liquid medium fish catching (Levin et al., 2005). Litterature data reports the presence of (MHA). many potentially toxic substances, including alkaloids and saponins that Pseudomonas aeruginosa H103 is a derivative of P. aeruginosa wild- give the plant its pharmacological properties. S. mammosum fruit was type PAO1 strain (Hancock and Carey, 1979). Planktonic cultures found to contain solasodine along with other steroidal glycoalkaloids, were grown aerobically for 24 h at 37 ◦C in LB broth on a rotary shaker including solasonine, solamargine and β-solamarginine (Tarigan, 1980), (180 rpm) from an initial inoculum adjusted to an absorbance at 580 nm as well as diosgenin and phytosterols (Sawariam, 1986). As of 0.08. S. mammosum fruits and/or leaf juices are rubbed onto afflicted areas to treat various health ailments related to the possible presence of 2.5. Inoculum for the bioautography assay T. mentagrophytes (athlete’s foot) and C. albicans, we designed a study to identify the major antifungal compounds in S. mammosum fruit using Inoculum is prepared by picking five distinct colonies of approxi- bioautography-TLC-HRMS, a combination of cutting-edge microbiolog- mately 1 mm from 24 h old culture grown on SDA and incubated at 25 ± ical, chromatographic, and spectrometric tools. Additionally, we eval- 2 ◦C for T. mentagrophytes and 35 ± 2 ◦C for the Candida albicans strains. uated the main active compound of the fruit extract (i.e., solamargine) Colonies were suspended in 5 mL of sterile 0.85% saline solution and the for the first time on P. aeruginosa pyocanin production and biofilm for- turbidity of the resulting suspension was adjusted to yield 1 × 106–5 × mation, since Solanum mammosum is used in the treatment of respiratory 106 cells mL− 1 (i.e. 0.5 McFarland standard). MHA was used as the solid disease due to its antimicrobial activity (Caceres et al., 1991). Finally, a media for the T. mentagrophytes and C. albicans overlays. The molten dereplication and molecular network analysis strategy was used to media were maintained in a water bath at 45 ◦C and inoculated with the explore the chemical composition of S. mammosum fruit through inoculum. The final concentration in the solid medium was approx. 105 UHPLC-ESI-MS/MS. cells mL− 1. The suspension was prepared immediately before carrying out the test. 2. Material and methods 2.6. Thin-layer chromatography (TLC) 2.1. Plant material TLC were carried out on a precoated silica gel 60 F254 (Merck, S. mammosum fruits were collected in the Allpahuayo Mishana Na- Darmstadt, Germany), with an appropriate solvent system in a classical tional Reserve, Iquitos, Maynas Province (Peru) (3◦58′02.3′′S TLC chamber. One μL of the amphotericin B stock solution and 5 μL of 73◦25′03.9′′W; − 3.967295, − 73.417754) by Billy Cabanillas and S. mammosum extract at the concentrations of 10, 20 and 30 mg. Mohamed Haddad. Plant identification was performed by the botanist C. mL− 1were spotted on TLC plates. Standard mobile phases CHCl3/ A. Amasifuen Guerra (Voucher N◦ 26646, Museum of Natural History of MeOH/H2O (65:40:10, v/v/v) and CHCl3/MeOH (50:50, v/v) were used Lima, Peru). Authorization to collect the plant was obtained from rele- to separate components over a wide range of polarities. The standard vant authorities (authorization SERNANP N◦ 010-2017-SERNANP- solutions/extract samples were applied to the TLC plate by using an RNAM/JRR). automatic TLC sampler ATS4 (CAMAG, Muttenz, Switzerland) with the following application conditions: filling speed: 15 μL s− 1, pre-dosage 2.2. Drug and chemicals volume: 200 nL, dosage application speed: 150 nL/s, rinsing cycle 1 with methanol/water (9:1, v/v), rinsing vacuum time: 4 s, filling vac- Methanol, ethanol, chloroform and all solvents used for extraction, uum time: 1 s. The TLC plates were prepared in triplicate: after exami- TLC development and LC/MS grade solvents as well as MHA, glucose nation of all developed chromatograms under ultra-violet light at 254 anhydrous, SDA, and LB were purchased from Fisher Chemicals (Lei- and 366 nm (CAMAG Universal UV lamp TL 600), one plate was sprayed cestershire, UK). MTT and DMSO were obtained from Sigma-Aldrich with vanillin–sulphuric acid reagent, while others were kept for bio- (Saint-Louis, MO, USA). Water was deionized using Milli-Q water puri- autography and TLC-MS, respectively. fication system (Millipore, Bedford, MA, USA). PBS Gibco® was ob- tained from Thermo Fisher Scientific (Waltham, MA, USA). RPMI 1640 2.7. Bioautography: agar-overlay method was purchased from Corning (Corning, NY, USA). Bioautographic agar overlay method was used following the protocol 2.3. Preparation of extracts, sample preparation and stock solutions for described in Rahalison et al. (1991) with slight modifications. Briefly, bioautography TLC plate was placed in a Petri dish and covered with approximately 10 mL of a thin layer of MHA inoculated with T. mentagrophytes or The pulp of 20 fresh fruits (~150 g) was extracted by maceration in C. albicans. After solidification of the medium, TLC plates were incu- MeOH (1 L) at room temperature for 24 h using an orbital shaker from bated overnight at 25 ± 2 ◦C or 35 ± 2 ◦C in polyethylene boxes lined Thermo Scientific (Waltham, MA, USA). After filtration and evaporation with moist chromatography paper. The bioautograms were sprayed with 2 B. Cabanillas et al. J o u r n a l o f E t h n o p h a r m a c o l o g y 280 (2021) 114473 an aqueous solution (2.5 mg mL− 1) of MTT and incubated for 4 h at data were normalized for bacterial cell density (A580 nm). 30 ◦C. Dehydrogenases of living microorganisms convert these salts into colored formazans, and as a result yellow zones of inhibition are 2.12. Quantitative biofilm assay observed on a purple background. Then, the bioautography assay was analyzed by observing inhibition zones (no color) and non-inhibition To assess the propensity of P. aeruginosa H103 strain to form biofilms zone (purple color). Amphotericin B was used as a reference com- in the presence of solamargine, we performed crystal-violet-adhesion pound for the determination of inhibition zone. assays as described by O’Toole (2011). Briefly, overnight cultures were inoculated into a fresh medium and grown for 24 h in a 96-well 2.8. Characterization of bioactive compounds using mass spectrometry microtiter plate. Cell growth was determined from A580 nm. Biofilm was measured by discarding the medium, rinsing the wells with water A TLC-MS Interface with an oval extraction head of 4 × 2 mm and staining any bound cells with crystal violet at 0.1%. The dye was (CAMAG, Muttenz, Switzerland) was connected between an UHPLC dissolved in 30% w/v acetic acid and A595 nm was determined in each (Dionex UltiMate3000, Dionex, USA) and an Orbitrap mass spectrom- experiment, background staining was adjusted by subtracting the crystal eter (LTQ XL, 21880, Thermo Fisher Scientific, USA). The bioactive violet bound to inoculated controls. zones on the TLC plate were marked with a soft pencil based on their RF value and extraction was performed with a mixture of methanol and 2.13. LCMS analytical methodology water (95:5, v/v) at a flow rate of 0.5 mL/min provided by the UHPLC pumps. Mass spectrometric analysis was carried out in negative- and The methanolic extract of S. mammosum was carried out by UHPLC- positive-ion modes. ESI parameters were set as follows: heater temper- DAD-LTQ Orbitrap XL instrument (Ultimate 3000, Thermo Fisher Sci- ature 300.0 ◦C, capillary temperature 350.0 ◦C, capillary voltage 10.0 V, entific), which was equipped with an electrospray ionization probe sheath gas flow rate 10 arbitrary units, aux gas flow rate 5 arbitrary (ESI). Chromatographic separations were performed on an Acquity BEH units, tube lens 80 V, Ion spray voltage 3.50 kV. Data was acquired and C18 column (100 × 2.1 mm i.d., 1.7 μm, Waters, USA). The mobile phase recorded by Thermo Xcalibur Qual Browser software. Different collision comprised acidified solvents (0.1% formic acid), water (A) and aceto- energies were applied for MS/MS analysis in order to obtain more in- nitrile (B) respectively. A stepwise gradient method at constant flow rate formation about fragment ions of the target constituents. of 0.3 mL/min was used to elute the column using the following con- ditions: 0–0.5 min, 95% A; 0.5–12 min, 95–5% A; 12–15 min, 5% A; 2.9. Isolation of the active compound 15–15.5 min, 5–95% A; 15.5–19 min, 95% A. Analyses of the samples (2 μL injected) were performed by a diode array detector (DAD) from 210 The methanolic extract (1 g) of the fruit of S. mammosum was sub- to 400 nm. The column temperature was maintained at 40 ◦C. Mass jected to a flash chromatography instrument (Spot Ultimate, Armen, parameter settings were: negative ESI mode, under the following con- France) on a silica column (Chromabond® Flash RS 25 SiOH 40–63 μm) ditions: capillary voltage at 3.0 kV, capillary temperature at 300 ◦C. Full eluted with the solvent systems CHCl3/MeOH/H2O (80:20:2, v/v/v) to mass spectra were recorded between 100 and 1500 Da. CID mass spectra give six main sub-fractions (B1–B6), including pure solamargine (34.7 were obtained in the data dependent mode for the four most intense ions mg). (top 4) of each MS full scan using the following parameters: 35% normalized collision energy, isolation width 2 Da, activation Q0.250. 2.10. Minimum inhibitory concentrations (MIC) assays External mass calibration was performed before starting the experiment. C. albicans strains (10231, 90028) were purchased from American 2.14. Data processing Type Culture Collection (ATCC). MIC values were determined by the broth microdilution method according to the CLSI (2008). The yeast was Data obtained from high resolution mass spectrometry (.raw) were grown at 35 ◦C on SDA plates for 48 h. The inoculum was prepared by first processed with MZmine 2.52 (Pluskal et al., 2010). Briefly, as a first suspending scraped cell mass in 0.85% NaCl solution, adjusted to 0.5 Mc step, the transformation of chromatograms in a peak list was realized Farland standard with a spectrophotometer at 530 nm, then diluted to following mass detection for the extract. Then, the chromatogram was obtain a final suspension of 5.0 × 102 to 2.5 × 103 cells per mL. RPMI built and deconvolutioned using ADAP chromato-builder and wavelets 1640 medium buffered with MOPS and supplemented with dextrose was (ADAP) algorithms; grouping of isotope patterns (peak grouper algo- used as a growth media. In a 96-well plate, plant extracts (10 mg mL− 1 in rithm) and a unique peaks list aligned was created. The gap filling (peak DMSO) were serial diluted in RPMI 1640 medium so that 8 concentra- finder algorithm) in the list was also performed. Cleaning of the peaks tions in the range of 4–512 μg mL− 1 were obtained. The working culture list was carried out as follows: merge of duplicates in the list, attribution was then added to all wells, and the plates were incubated at 35 ◦C for of scans MS2 to MS1 using group MS2 scans with features algorithm and 48 h. A spectrophotometer was used to determine the MIC by reading finally the peak list was filtered using the peak list rows filter by keeping the 96-well plate at 600 nm. The MIC was defined as the lowest con- only the peaks with MS2 scan. centration of plant extract that completely inhibits growth of C. albicans In the peaks list, the features annotations of known compounds, by in the wells as detected by the unaided eye (CLSI, 2008). Each test was looking for compounds corresponding to the molecular formula derived performed in triplicates. Amphotericin B was used as a positive control. from HRMS data, were realized as follows: 1) identification of fragments Cultures without plant extracts or antifungal were employed as negative and complexes; 2) comparison with an in-house database compiled from control. literature data to gather secondary metabolites isolated from Solanum genus and Solanaceae family (Data not shown) for the annotation of 2.11. Pyocyanin quantification assay known compounds; 3) Pubchem online database was screened for annotating the other compounds. Before the exportation of the peaks list Pyocyanin quantification assay was carried out as described previ- data, these latter were normalized using the linear normalizer parame- ously by Tahrioui et al. (2020). P. aeruginosa H103 cells untreated and ters. Finally, the exportation of generated MS/MS spectra was made in treated with solamargine were grown in a 96-well microtiter plate at MGF format and the list of mass compounds, retention times, row ID and 37 ◦C for 24 h on a rotary shaker (180 r.p.m). One volume of chloroform peak heights was exported in CSV (comma-separated value). Molecular was used to extract free-cell supernatants samples. Then, ½ volume of network (mass spectra similarity) was carried out using the open-source 0.5 M HCl was added to the chloroform layer (blue layer). The absor- software MetGem (Olivon et al., 2018) from final MS/MS data. Values bance of the HCl layer (red-pink layer) was recorded at 520 nm and the used were MS2 m/z tolerance = 0.1 Da, minimum matched peaks = 4 3 B. Cabanillas et al. J o u r n a l o f E t h n o p h a r m a c o l o g y 280 (2021) 114473 and minimal cosine score value = 0.65. Visualization of the network was performed on Cytoscape version 3.8.2 (Shannon et al., 2003). Com- pounds with the same fragmentation pathway were grouped into the same cluster. 3. Results 3.1. Bioautography-TLC-HRMS/MS, isolation and structural determination of the bioactive compound As a preliminary assay, a bioautography-TLC-HRMS/MS of S. mammosum fruit extract was initially performed in order to identify the potential anti-Trichophyton mentagrophytes and anti-Candida albicans metabolites present in S. mammosum, as it relates to its traditional use. One main inhibition zone with Rf=0.72 was identified in both bio- autographies and marked with a pencil on the duplicate TLC plate and extracted for MS analysis, using the TLC-MS interface. The workflow is Fig. 2. Structure of solamargine 1. shown in Fig. 1. Solamargine (1, Fig. 2) was identified as the main compound by TLC-HRMS (m/z = 868.5057 [M+H]+; calcd for C45H73NO15 867.4980). Further confirmation was obtained by the Table 1 isolation of the active spot through Combiflash® on Si60 followed by Antifungal activity (MIC values) of solamargine on two Candida albicans strains structure determination by 1D and 2D NMR and comparison with and one Trichophyton mentagrophytes strain. literature (Burger et al., 2018). MIC (μg.mL− 1) T. C. albicans C. albicans 3.2. Minimum inhibitory concentrations assays mentagrophytes 90028 10231 TDV crude fruit 256 256 256 The results of MIC determination from TDV fruits crude extract and extract solamargine against T. mentagrophytes and two strains of C. albicans are Solamargine 64 64 64 shown in Table 1. TDV exhibited weak anti-Trichophyton and anti- Candida albicans activities (MIC = 256 μg mL− 1) when compared to effect on virulence through the pyocyanin production as well as on Amphotericin B (100% inhibition at 8 μg mL− 1) whereas solamargine biofilm formation were studied. The effect of solamargine against exhibited a moderate activity against the three strains (MIC = 64 μg P. aeruginosa was evaluated at concentrations ranging from 0.8 to 100 mL− 1). μg mL− 1, in a liquid medium using the model bacterium P. aeruginosa H103, a prototroph derivative of PAO1 wild-type strain. Solamargine − 1 3.3. Effect of solamargine on pyocyanin production and biofilm formation reduced pyocyanin production by about 20% at 50 μg mL (Fig. 3a). However, no impact was observed at low concentrations. Solamargine To assess the impact of solamargine on P. aeruginosa physiology, its also led to a significant reduction of about 20% of the biofilm formation Fig. 1. Bioautography-TLC-MS workflow for anti-Trichophyton mentagrophytes and anti-Candida albicans activities. 4 B. Cabanillas et al. J o u r n a l o f E t h n o p h a r m a c o l o g y 280 (2021) 114473 Fig. 3. Effect of solamargine on pyocyanin production and biofilm formation by P. aeruginosa. Statistics were achieved by a two-tailed t-test using Prism GraphPad. The mean with SEM were calculated and plotted. ★, p = 0.01 to 0.05; NS (Not Significant), p ≥ 0.05. (Fig. 3b). This effect was observed for a very low concentration (1.6 μg Vásquez, 1997; Vega, 2001) is commonly used in Peru to treat mycosis mL− 1) and was quite similar for the other solamargine concentrations and scabies (Roumy et al., 2007), to relieve headaches (Luziatelli et al., tested (data not shown). Altogether, our data show that solamargine 2010) and as a poison to kill rats (Ayala Flores, 1984). Among the moderately affects virulence traits of P. aeruginosa at 50 μg mL− 1. available ethnopharmacological data, several authors have reported the traditional use of different botanical parts of S. mammosum against 3.4. Chemical composition of TDV MeOH extract and annotation fungal skin infections (Roumy et al., 2007; Hajdu and Hohmann, 2012; Polesna et al., 2011; Lim, 2013). Particularly, S. mammosum is specif- A list of 143 peaks (Rt-m/z) which corresponded to the retention ically used to treat athlete’s foot infection (Muñoz et al., 2000), a su- times and pseudomolecular ion masses of compounds were obtained perficial inflammatory infection of the feet skin caused by dermatophyte from methanolic extract of S. mammosum, created using the MZmine fungi, especially Trichophyton rubrum, T. mentagrophytes, and Epi- software. Top priority for the annotation of the compounds in the peaks dermophyton floccosum (Hsu and Hsu, 2012; Rinaldi, 2000). In this study, list was made for the Solanum genus in-house database. Thus, seven our result showed that solamargine is the main active ingredients of compounds were annotated (Table 2), including the active compound S. mammosum, exhibiting a moderate activity against T. mentagrophytes solamargine. Then, we crossed the information between compounds and C. albicans (MIC = 64 μg mL − 1 against both strains), which might annotated and bibliographic data (activity on T. mentagrophytes, confirm its traditional use to treat skin fungal infections. In addition, C. albicans and P. aeruginosa). Visualization of chemical class of com- S. mammosum is also traditionally used in the treatment of respiratory pounds annotated from S. mammosum were placed in network according diseases but few studies have been done to better understand the to MS/MS fragmentation similarity (Fig. 4). mechanism of action. Caceres et al. (1991) carried out a screening of 68 plants used in Guatemala for the treatment of respiratory diseases. They 4. Discussion have shown that several plants used for the treatment of respiratory infections have some in vitro activity against pathogenic gram-positive In this study, we investigated the anti-Trichophyton mentagrophytes bacteria, including S. mammosum (moderate activity against Strepto- and anti-Candida albicans activity of TDV MeOH extract through coccus pneumonias). P. aeruginosa is a pathogenic gram-positive bacteria bioautography-TLC-HRMS and isolated the most active compound, responsible of repiratory infections. It is known to produce a range of solamargine. virulence factors that enhance its ability to damage the host tissue. One S. mammosum, named in the traditional medicine of Peruvian of the most important virulence factors is pyocyanin that is highly toxic Amazon chucho de vaca, teta de vaca, tinta uma, cocona venenosa, tintuma, because of its redox-active and zwitterionic properties, contributing to tinctona, resalgal, tintonilla, cocoán and chuf-cha (Pinedo et al., 1997; tissue damage (Lau et al., 2004) and inducing pulmonary pathophysi- ology in mice (Caldwell et al., 2009). Pyocyanin is a blue redox-active secondary metabolite that is readily recovered in large quantities in Table 2 sputum from patients with cystic fibrosis. Pyocyanin can cross the cell Principal molecular ions determined in the methanolic extract of S. mammosum by LCMS and corresponding compounds. membrane and causes oxidative stress by generating reactive oxygen and nitrogen species, which allow P. aeruginosa to kill competitor mi- m/z [M-H]- RT Compound Main fragments crobes inhabiting the same niche, as well as damaging host cells or 851.2234 3.7337 Viarumacid A 689.220; 515.227; 497.196 modulating their immune signaling (Morin et al., 2021). In this study, 207.0658 5.91 Ethyl caffeate 179.018; 135.162; 207.072 our data indicate that Solamargine has a significant effect on 285.0396 4.79 Kaempferol 257.099; 199.109; 217.064 P. aeruginosa biofilm formation and pyocyanin production under our 347.0976 1.5402 Tabaflavone E 161.090; 139.120; 223.017 299.0553 5.1537 Sorbifolin 284.094; 271.135; 267.188 conditions, which brings new insights about the potential of this plant to 609.1449 3.5785 Rutin 447.210; 285.054; 489.332 treat respiratory problems. 867.3099 5.8119 Solamargine 720.4147; 469.243; 549.159 From a chemical point of view, S. mammosum was shown to contain 5 B. Cabanillas et al. J o u r n a l o f E t h n o p h a r m a c o l o g y 280 (2021) 114473 Fig. 4. Molecular networking and visualization of compounds annotated from S. mammosum. The compounds classification was carried out using Classy-Fire (Feunang et al., 2016). metabolites which are both highly toxic to humans but medicinally flavonoids is the scavenging of oxygen-derived free radicals (Nijveldt useful. It is a source of solasodine (Telek, 1977; Roddick and Rijnenberg, et al., 2001). Studies about flavonoids reported that several of these 1986; Hanelt et al., 2001; Lim, 2013), a poisonous, teratogenic, alka- compounds exhibit anti-oxidant, antitumoral, anti-inflammatory and loidal compound that is a precursor to pharmaceutical production of antimicrobial activities including antifungal, antiviral, and antibacterial contraceptive pills and has also been the subject of recent research for its effects (Middleton, 1998; Nijveldt et al., 2001). Kaempferol, which is diuretic, anticancer, antifungal, cardiotonic, antispermatogenetic, anti- one of most representative natural flavonol, was reported to have androgenic, immunomodulatory, and antipyretic effects on the central moderate activity on C. albicans strains with values of MIC between 128 nervous system (Patel et al., 2013). Also, solamargine is a major gly- and 441 μg mL− 1 (449–1547 μM) (Seleem et al., 2017; Shao et al., 2016), coalkaloid in Solanum species and especially in S. mammosum, which has but to be inactive against P. aeruginosa (MIC > 1000 μg mL− 1) been less studied for its antibacterial and antifungal activities and more (Adamczak et al., 2019a, b). Sorbifolin, a flavone previously isolated for its anticancer properties (Kalalinia and Karimi-Sani (2017). Thus, from Astragalus trimestris L. (Fabaceae) displayed mild antibacterial ac- our study provides new data with regards to its antimicrobial properties tivity on Escherichia coli and C. albicans with MIC value of 125 μg mL− 1 and show that solamargine plays a key role in the pharmacological ac- (417 μM) (El-Hawiet et al., 2010). Plants containing rutin (querceti- tion of S. mammosum. Through LC-HRMS and data analyses of com- n-3-O-rutinoside), a flavonol glycoside, are traditionally used as anti- pounds from S. mammosum extract, seven compounds were annotated, microbial, antiarthritic or antiallergic (Sharma et al., 2016). among which four metabolites that matched with flavonoid compounds: Nonetheless, these compounds displayed lack of activities on C. albicans kaempferol (m/z 285.0396), sorbifolin (m/z 299.0553), tobaflavone E (Han, 2009; Tempesti et al., 2011) and P. aeruginosa (Lou et al., 2015) (m/z 347.0976) and rutin (m/z 609.1449). Flavonoids are associated with MIC > 1000 μg mL− 1. Another flavonoid annotated was toba- with a broad spectrum of health-promoting effects and are indispensable flavone E, that expressed activity on virus such as TMV (Tobacco Mosaic components in a variety of nutraceutical, pharmaceutical, medicinal and Virus) with 35.3 ± 3.2% of inhibition (Miao et al., 2015). TMV is one of cosmetic applications (Panche et al., 2016). An important effect of the most damaging plant virus, causing significant yield losses in crop 6 B. Cabanillas et al. J o u r n a l o f E t h n o p h a r m a c o l o g y 280 (2021) 114473 production worldwide (Rybicki, 2015). Moreover, three additional Adamczak, A., Ożarowski, M., Karpiński, T.M., 2019a. Antibacterial activity of some compounds were annotated and placed in the network: ethyl caffeate flavonoids and organic acids widely distributed in plants. J. Clin. Med. 9 (1), 109. https://doi.org/10.3390/jcm9010109. (m/z 207.0658), viarumacid A (m/z 851.2234) and solamargine (m/z Adamczak, A., Ożarowski, M., Karpiński, T.M., 2019b. Antibacterial activity of some 867.3099). First, ethyl caffeate is a compound previously isolated from flavonoids and organic acids widely distributed in plants. J. Clin. Med. 9, 109. Cnestis palala (Lour.) Merr. (Connaraceae), which has been tested on https://doi.org/10.3390/jcm9010109. μ − 1 Arnason, T., Uck, F., Lambert, J., Hebda, R., 1980. Maya medicinal plants of san Jose Staphylococcus aureus and S. epidermidis with MIC value at 500 g mL Succotz, Belize. J. Ethnopharmacol. 2, 345–364. https://doi.org/10.1016/s0378- (2.41 mM) against both microorganisms (Dej-adisai et al., 2015). Sec- 8741(80)81016-6. ondly, viarumacid A, a glucosylated caffeoylquinic acid derivative iso- Ayala Flores, F., 1984. Ethnobotany in the neotropics. In: Advances in economic botany, lated from S. viarum has been showed to display antioxidant activity (Wu 1, pp. 1–8. Burger, T., Mokoka, T., Fouché, G., Steenkamp, P., Steenkamp, V., Cordier, W., 2018. et al., 2012). The diversity of class of compounds visualized in the polar Solamargine, a bioactive steroidal alkaloid isolated from Solanum aculeastrum extract from S. mammosum leaves a door open for further exploration in induces non-selective cytotoxicity and P-glycoprotein inhibition. BMC Compl. the laboratory. Altern. Med. 18 (1), 137. https://doi.org/10.1186/s12906-018-2208-7. Burkill, I.H., 1966. A dictionary of the economic products of the Malay Peninsula. Revised reprint. 2 vols. Ministry of Agriculture and Co-operatives, Kuala Lumpur. 5Conclusion vol. 1 (A–H), pp. 1–1240, vol 2 (I–Z), pp 1241–2444. Caceres, A., Alvarez, A.V., Ovando, A.E., Samayoa, B.E., 1991. Plants used in Guatemala for the treatment of respiratory diseases. 1. Screening of 68 plants against gram- The findings of the present investigation conclude that solamargine positive bacteria. J. Ethnopharmacol. 31 (2), 193–208. https://doi.org/10.1016/ is the main active compound against T. mentagrophytes, C. albicans in 0378-8741(91)90005-x. vitro. In addition, our data indicate that this compound has a significant Caldwell, C.C., Chen, Y., Goetzmann, H.S., Hao, Y., Borchers, M.T., Hassett, D.J., Young, L.R., Mavrodi, D., Thomashow, L., Lau, G.W., 2009. Pseudomonas aeruginosa effect on P. aeruginosa biofilm formation and pyocyanin production exotoxin pyocyanin causes cystic fibrosis airway pathogenesis. Am. J. Pathol. 175 under our conditions. Taken together, these findings may help to explain (6), 2473–2488. https://doi.org/10.2353/ajpath.2009.090166. why S. mammosum fruits have been traditionally used to treat a variety Chaiyasit, D., Choochote, W., Rattanachanpichai, E., Chaithong, U., Chaiwong, P., Jitpakdi, A., Tippawangkosol, P., Riyong, D., Pitasawat, B., 2006. Essential oils as of fungal infections and respiratory disorders. potential adulti- cides against two populations of Aedes aegypti, the laboratory and natural field strains, in Chiang Mai province, northern Thailand. Parasitol. Res. 99 Declaration of competing interests (6), 715–721. https://doi.org/10.1007/s00436-006-0232-x. CLSI, 2008. In: Reference Method for Broth Dilution Antifungal Susceptibility Testing of Filamentous Fungi ; Approved Standard-2nd Edition (MA38–A2). Clinical and The authors declare that they have no known competing financial Laboratory Standards Institute, Wayne, PA. interests or personal relationships that could have appeared to influence Crommett, M.K., 2011. Dominican medicinal plant inventory. http://www.prhdr.org/d the work reported in this paper. ocs/Dominican%20Medicinal%20Plants.pdf. DeFilipps, R.A., Maina, S.L., Crepin, J., 2004. Medicinal Plants of the Guianas (Guyana, Surinam, French Guiana). Department of Botany, National Museum of Natural CRediT authorship contribution statement History. Smithsonian Institution, Washington, DC. Dej-adisai, S., Tinpun, K., Wattanapiromsakul, C., Keawpradub, N., 2015. Bio-activities and phytochemical investigation of cnestis palala (lour.) Merr. African Journal of Billy Cabanillas: Writing – original draft, Formal analysis, collected Traditional. Complement. Alternative Med. 12, 27–37. https://doi.org/10.4314/ the plant, performed the extraction of S. mammosum fruits and analyzed ajtcam.v12i3.3. the data. François Chassagne: Writing – original draft, performed the El-Hawiet, A., Toaima, S.M., Asaad, A., Radwan, M., El-Sebakhy, N., 2010. Chemical constituents from Astragalus annularis Forssk. and A. trimestris L., Fabaceae. Revista microdilution assay against C. albicans. Pedro Vásquez-Ocmín: Brasileira De Farmacognosia 20, 860–865. https://doi.org/10.1590/S0102- Writing – original draft, Formal analysis, performed the LC-HRMS and 695X2010005000047. molecular network experiments. Ali Tahrioui: Formal analysis, per- Feunang, Y.D., Eisner, R., Knox, C., Chepelev, L., Hastings, J., Owen, G., Fahy, E., Steinbeck, C., Subramanian, S., Bolton, E., Greiner, R., Wishart, D.S., 2016. formed the P. aeruginosa pyocyanin production and biofilm formation ClassyFire: automated chemical classification with a comprehensive computable experiments. Sylvie Chevalier: Formal analysis, performed the P. aer- taxonomy. J. Cheminf. 8 https://doi.org/10.1186/s13321-016-0174-y. uginosa pyocyanin production and biofilm formation experiments. Flores, F.A., 1984. Notes on some medicinal and poisonous plants of Amazonian Peru. Marieke Vansteelandt: Formal analysis, performed the LC-HRMS ex- Adv. Econ. Bot. 1, 1–8. Ethnobotany in the Neotropics (18 September 1984). Grieve, M., 1971. A Modern Herbal, 2. Penguin, p. 919. Dover publications, Dover/New periments, All authors read, discussed and agreed on the final manu- York. script. Asih Triastuti: Formal analysis, performed the microdilution Hajdu, Z., Hohmann, J., 2012. An ethnopharmacological survey of the traditional assay against C. albicans. Carlos A. Amasifuen Guerra: identified the medicine utilized in the community of Porvenir, Bajo Paraguá Indian Reservation, Bolivia. J. Ethnopharmacol. 139, 838–857. https://doi.org/10.1016/j. plant. Nicolas Fabre: Formal analysis, performed the LC-HRMS exper- jep.2011.12.029. iments, All authors read, discussed and agreed on the final manuscript. Han, Y., 2009. Rutin has therapeutic effect on septic arthritis caused by Candida albicans. Mohamed Haddad: Writing – original draft, Formal analysis, Int. Immunopharm. 9, 207–211. https://doi.org/10.1016/j.intimp.2008.11.002. Hancock, R., Carey, A.M., 1979. Outer membrane of Pseudomonas aeruginosa: heat- and conceived, designed, performed the experiments (plant collection, 2-mercaptoethanol-modifiable proteins. J. Bacteriol. 140, 902–910. https://doi.org/ bioautography-TLC-HRMS, microdilution assay against T. menta- 10.1128/JB.140.3.902-910.1979. grophytes, isolation and structure elucidation of Solamargine, LC- Hanelt, P., Buttner, R., Mansfeld, R., 2001. Mansfeld’s Encyclopedia of Agricultural and Horticultural Crops (Except Ornamentals). Springer, Berlin, Germany. HRMS). Hsu, A.R., Hsu, J.W., 2012. Topical review: skin infections in the foot and ankle patient. Foot Ankle Int. 33 (7), 612–619. https://doi.org/10.3113/FAI.2012.0612. Acknowledgments Jovel, E.M., Cabanillas, J., Towers, G.H.N., 1996. An ethnobotanical study of the traditional medicine of the Mestizo people of Suni Miraño, Loreto, Peru. J. Ethnopharmacol. 53 (3), 149–156. https://doi.org/10.1016/0378-8741(96) The authors acknowledge the French National Research Institute for 01437-7. Sustainable Development and the Federal University of Toulouse for Kalalinia, F., Karimi-Sani, I., 2017. Anticancer properties of solamargine: a systematic research support, the infrastructure, incentive, and collaboration. Be- review. Phytother Res. 31, 858–870. https://doi.org/10.1002/ptr.5809. Lans, C., Harper, T., Georges, K., Bridgewater, E., 2001. Medicinal and ethnoveterinary sides, the authors acknowledge the Instituto de Investigaciones de la remedies of hunters in Trinidad. BMC Complement. Altern. Med. 1 (10) https://doi. Amazonia Peruana (IIAP) and especially Ms Elsa Rengifo and Dr Kember org/10.1186/1472-6882-1-10. Mejia for the access to the botanical garden in Allpahuayo Mishana. Lau, G.W., Ran, H., Kong, F., Hassett, D.J., Mavrodi, D., 2004. Pseudomonas aeruginosa pyocyanin is critical for lung infection in mice. Infect. Immun. 72 (7), 4275–4278. https://doi.org/10.1128/IAI.72.7.4275-4278.2004. References Levin, R.A., Watson, K., Bohs, L., 2005. A four-gene study of evolutionary relationships in Solanum section Acanthophora. Am. J. Bot. 92, 603–612. https://doi.org/10.3732/ Acevedo-Rodríguez, P., Strong, M.T., 2008. Floristic richness and affinities in the west ajb.92.4.603. indies. Bot. Rev. 74, 5–36. https://doi.org/10.1007/s12229-008-9000-1. Lim, T.K., 2013. Solanum linearifolium. In: Edible Medicinal and Non-medicinal Plants. Springer, Dordrecht. 7 B. Cabanillas et al. J o u r n a l o f E t h n o p h a r m a c o l o g y 280 (2021) 114473 Lou, Z., Tang, Y., Song, X., Wang, H., 2015. Metabolomics-based screening of biofilm- Rinaldi, M.G., 2000. Dermatophytosis: epidemiological and microbial update. J. Am. inhibitory compounds against Pseudomonas aeruginosa from burdock leaf. Acad. Dermatol. 43 (5 Suppl. l), s120–s124. https://doi.org/10.1067/ Molecules 20, 16266–16277. https://doi.org/10.3390/molecules200916266. mjd.2000.110378. Luziatelli, G., Sørensen, M., Theilade, I., Mølgaard, P., 2010. Asháninka medicinal plants: Roddick, J.G., Rijnenberg, A.L., 1986. Effect of steroidal glycoalkaloids of the potato on a case study from the native community of Bajo Quimiriki, Junín, Peru. J. Ethnobiol. the permeability of liposome membranes. Physiol. Plantarum 68, 436–440. https:// Ethnomed. 6, 21. https://doi.org/10.1186/1746-4269-6-21. https://doi.org/10. doi.org/10.1111/j.1399-3054.1986.tb03378.x. 1186/1746-4269-6-21. Roumy, V., Garcia-Pizango, G., Gutierrez-Choquevilca, A.L., Ruiz, L., Jullian, V., Miao, M.M., Li, L., Shen, Q.P., Liu, C.B., Li, Y.K., Zhang, T., Zhang, F.M., He, P., Wang, K. Winterton, P., Fabre, N., Moulis, C., Valentin, A., 2007. Amazonian plants from Peru M., Zhu, R.Z., Chen, Y.K., Yang, G.Y., 2015. Anti-TMV activity flavones from the used by Quechua and Mestizo to treat malaria with evaluation of their activity. leaves of Yunnan local air cured tobacco. Fitoterapia 103, 260–264. https://doi.org/ J. Ethnopharmacol. 112, 482–489. https://doi.org/10.1016/j.jep.2007.04.009. 10.1016/j.fitote.2015.04.014. Rybicki, E.P., 2015. A Top Ten list for economically important plant viruses. Arch. Virol. Middleton Jr., E., 1998. Effect of plant flavonoids on immune and inflammatory cell 160, 17–20. https://doi.org/10.1007/s00705-014-2295-9. function. Adv. Exp. Med. Biol. 439, 175–182. https://doi.org/10.1007/978-1-4615- Sawariam, I., 1986. Studi Kandungan Buah Solanum Mammosum L, Skripsi (BSc Thesis). 5335-9_13. Fakultas Farmasi Universitas Airlangga, Surabaya. Morin, C.D., Déziel, E., Gauthier, J., Levesque, R.C., Lau, G.W., 2021. An organ system- Seleem, D., Pardi, V., Murata, R.M., 2017. Review of flavonoids: a diverse group of based synopsis of Pseudomonas aeruginosa virulence. Virulence 12 (1), 1469–1507. natural compounds with anti-Candida albicans activity in vitro. Arch. Oral Biol. 76, https://doi.org/10.1080/21505594.2021.1926408. 76–83. https://doi.org/10.1016/j.archoralbio.2016.08.030. Muñoz, V., Sauvain, M., Bourdy, G., Callapa, J., Rojas, I., Vargas, L., Tae, A., Deharo, E., Shannon, P., Markiel, A., Ozier, O., Baliga, N.S., Wang, J.T., Ramage, D., Amin, N., 2000. The search for natural bioactive compounds through a multidisciplinary Schwikowski, B., Ideker, T., 2003. Cytoscape: a software environment for integrated approach in Bolivia. Part II. Antimalarial activity of some plants used by Mosetene models of biomolecular interaction networks. Genome Res. 13, 2498–2504. https:// Indians. J. Ethnopharmacol. 69, 139–155. https://doi.org/10.1016/s0378-8741(99) doi.org/10.1101/gr.1239303. 00096-3. Shao, W., Liu, H., Wang, S., Wu, J., Huang, M., Min, H., Liu, X., 2016. Controlled release Nijveldt, R.J., van Nood, E., van Hoorn, D.E., Boelens, P.G., van Norren, K., van and antibacterial activity of tetracycline hydrochloride-loaded bacterial cellulose Leeuwen, P.A., 2001. Flavonoids: a review of probable mechanisms of action and composite membranes. Carbohydr. Polym. 145, 114–120. https://doi.org/10.1016/ potential applications. Am. J. Clin. Nutr. (74), 418–425. https://doi.org/10.1093/ j.carbpol.2016.02.065. ajcn/74.4.418, 2001. Sharma, P., Kalita, M.C., Thakur, D., 2016. Broad spectrum antimicrobial activity of O’Toole, G.A., 2011. Microtiter dish biofilm formation assay. JoVE 47, 2437. https://doi. forest-derived soil actinomycete, Nocardia sp. PB-52. Front. Microbiol. 7 (347) org/10.3791/2437. https://doi.org/10.3389/fmicb.2016.00347. Olivon, F., Elie, N., Grelier, G., Roussi, F., Litaudon, M., Touboul, D., 2018. MetGem Space, J., Flynn, T., 2002. Report to the Government of the Cook Islands on Invasive software for the generation of molecular networks based on the t-SNE algorithm. Plant Species of Environmental Concern. USDA Forest Service, Honolulu, USA. Anal. Chem. 90, 13900–13908. https://doi.org/10.1021/acs.analchem.8b03099. Pacific southwest research station, Institute of Pacific Islands forestry, 146. Oviedo, R., Herrera, P., Caluff, M.G., Regalado, L., Ventosa, I., 2012. Lista nacional de Stuart, G.U., 2010. Philippine alternative medicine. In: Manual of Some Philippine especies de plantas invasoras y potencialmente invasoras en la República de Cuba Medicinal Plants. http://www.stu-artxchange.org/OtherHerbals.html. -2011. Plantas invasoras en Cuba, Bissea. Tahrioui, A., Ortiz, S., Azuama, O.C., Bouffartigues, E., Benalia, N., Tortuel, D., Panche, A.N., Diwan, A.D., Chandra, S.R., 2016. Flavonoids: an overview. J. Nutr. Sci. 5, Maillot, O., Chemat, S., Kritsanida, M., Feuilloley, M., Orange, N., Michel, S., e47. https://doi.org/10.1017/jns.2016.41. Lesouhaitier, O., Cornelis, P., Grougnet, R., Boutefnouchet, S., Chevalier, S., 2020. Patel, K., Singh, R., Patel, D., 2013. Medicinal significance, pharmacological activities, Membrane-interactive compounds from Pistacia lentiscus L. Thwart Pseudomonas and analytical aspects of solasodine: a concise report of current scientific literature. aeruginosa virulence. Front. Microbiol. 11 (1068) https://doi.org/10.3389/ J. Acute Dis. 2, 92–98. https://doi.org/10.1016/S2221-6189(13)60106-7. fmicb.2020.01068. Pier, 2014. Pacific Islands Ecosystems at Risk. HEAR, University of Hawaii, Honolulu, Tarigan, P., 1980. Sapogenin Steroid. Perbit Alumni, Bandung, pp. 96–103. USA. http://www.hear.org/pier/index.html. Telek, L., 1977. Determination of solasodine in fruits of solarium species. J. Pharmaceut. Pinedo, M., Rengifo, E., Cerruti, T., 1997. Plantas medicinales de uso popular en la Sci. 66, 699–702. https://doi.org/10.1002/jps.2600660523. Amazonía Peruana, studio de su uso y cultivo. Instituto de Investigaciones de la Tempesti, T.C., Álvarez, M.G., Araújo, M.F., Júnior, F.E., Carvalho, M.G., Durantini, E., Amazonía Peruana, p. 315. Iquitos, Peru). 2011. Antifungal activity of a novel quercetin derivative bearing a trifluoromethyl Pluskal, T., Castillo, S., Villar-Briones, A., Oresic, M., 2010. MZmine 2: modular group on Candida albicans. Med. Chem. Res. 21, 2217–2222. https://doi.org/ framework for processing, visualizing, and analyzing mass spectrometry-based 10.1007/s00044-011-9750-x. molecular profile data. BMC Bioinf. 11, 395. https://doi.org/10.1186/1471-2105- Vásquez, R., 1997. Flórula de las reservas biológicas de Iquitos. Missouri Botanical 11-395. Garden, Perú, p. 1046p. Iquitos, Peru. Polesna, L., Polesny, Z., Clavo, M.Z., Hansson, A., Kokoska, L., 2011. Vega, M., 2001. Etnobotánica de la amazonía peruana, 1ra. Edicion. Ediciones, Abya- Ethnopharmacological inventory of plants used in coronel Portillo province of Yala, p. 166. Quito, Ecuador. Ucayali department, Peru. Pharm. Biol. 49, 125–136. https://doi.org/10.3109/ Wiart, C., Mogana, S., Khalifah, S., Mahan, M., Ismail, S., Buckle, M., Narayana, A.K., 13880209.2010.504927. Sulaiman, M., 2004. Antimicrobial screening of plants used for traditional medicine Rahalison, L., Hamburger, M., Hostettmann, K., Monod, M., Frenk, E., 1991. in the state of Perak, Peninsular Malaysia. Fitoterapia 75, 68–73. https://doi.org/ A bioautographic agar overlay method for the detection of antifungal compounds 10.1016/j.fitote.2003.07.013. from higher plants. Phytochem. Anal. 2, 199–203. https://doi.org/10.1002/ Wu, S.B., Meyer, R.S., Whitaker, B.D., Litt, A., Kennelly, E.J., 2012. Antioxidant pca.2800020503. glucosylated caffeoylquinic acid derivatives in the invasive tropical soda apple, Solanum viarum. J. Nat. Prod. 75, 2246–2250. https://doi.org/10.1021/np300553t. 8