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Comprehensive approach for the detection of antifungal compounds using a susceptible strain of Candida albicans and confirmation of in vivo activity with the Galleria mellonella model

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Comprehensive approach for the detection of antifungal compounds using a susceptible strain of Candida albicans and confirmation of in vivo activity with the Galleria mellonella model

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... In the frame of our continuous search for antifungal and acetylcholinesterase inhibitors from natural origin (Favre-Godal et al., 2014;Queiroz et al., 2014), the ethanolic extract of the stem bark of C. heliotropiifolius demonstrated significant activities in both assays. To our knowledge, no thorough phytochemical investigation has been performed on this plant and this study presents the isolation and structure elucidation of the bioactive compounds. ...
... The ethanolic extract of the stem bark of C. heliotropiifolius presented antifungal activity in a TLC bioautographic assay against C. albicans with a minimum amount of 10 mg deposited (Favre-Godal et al., 2014). The same extract also showed significant in vitro AChE inhibition in a dilution assay (58% at 40 mg/mL) (Attaur-Rahman et al., 2005;Di Giovanni et al., 2008). ...
... The extract and the isolated compounds were tested at various concentrations against two strains of C. albicans: the mutant strain (DSY2621) and the wild strain (CAF21) ( Table 1). The mutant strain was genetically modified to improve the detection of minor antifungal compounds present in crude extracts (Favre-Godal et al., 2014), while the wild strain is the common pathogen. Spruceanol (8) was the most active compound demonstrating an interesting antifungal activity with a minimal inhibitory quantity (MIQ) of 2 mg against the mutant strain DSY2621, and 10 mg against the wild strain (Table 2). ...
Article
The ethanolic extract of the stem bark of Croton heliotropiifolius Kunth (Euphorbiaceae) showed significant in vitro inhibition of acetylcholinesterase using a dilution spectrophotometric assay and antifungal activity against Candida albicans with a thin layer chromatography (TLC) bioautographic assay. In order to isolate the active compounds, bioassay-guided fractionation was undertaken using HPLC to localize the active compounds. Different zones of the HPLC-UV chromatogram were linked to acetylcholinesterase inhibition or to antifungal activities. In parallel to this HPLC-based activity profiling, HPLC-PDA-ESI-MS and HPLC-TOF-HRMS were used for the early identification of some of the compounds present. The targeted isolation of the active compounds was performed by medium pressure liquid chromatography (MPLC-UV) and further semi-preparative HPLC. Using this approach, nine compounds were isolated, one of them being a new indole alkaloid derivative. The structures of the isolated compounds were elucidated by spectroscopic methods including UV, NMR, MS and HRMS.
... In the present study, from the active dichloromethane extracts of the aerial parts, five flavonol derivatives (1, 3−6) and six 4-quinolone alkaloids (2, 7−11) were isolated and characterized. Among these, five compounds (2,7,8,10,11) have not yet been described in the literature and 9 is reported for the first time in W. indica. The antifungal activity of these compounds, and of 10 quinoline alkaloids (12−21) previously isolated from the roots, was determined against Candida spp. ...
... In a preliminary screening experiment, a TLC bioautography assay 8 of the dichloromethane extract of the aerial parts from W. indica, at 200 μg/spot, showed significant inhibition of a hypersusceptible strain of C. albicans (DSY2621). This strain allows the detection of a small quantity of bioactive compounds from complex matrices. ...
... Article (2,7,8,10,11) and the (R)-enantiomer of vanessine (9). In addition, four flavonoids were isolated (3−6) from the active zone and a fifth one (1), although outside of the antifungal zone, was also isolated because it was described previously as active against C. albicans. ...
Article
Chemical investigation of a dichloromethane extract of the aerial parts of Waltheria indica led to the isolation and characterization of five polyhydroxymethoxyflavonoids, namely, oxyanin A (1), vitexicarpin (3), chrysosplenol E (4), flindulatin (5), 5-hydroxy-3,7,4'-trimethoxyflavone (6), and six quinolone alkaloids, waltheriones M-Q (2, 7, 8, 10, 11) and 5(R)-vanessine (9). Among these, compounds 2, 7, 8, 10, and 11 have not yet been described in the literature. Their chemical structures were established by means of spectroscopic data interpretation including (1)H and (13)C, HSQC, HMBC, COSY, and NOESY NMR experiments and UV, IR, and HRESIMS. The absolute configurations of the compounds were established by ECD. The isolated constituents and 10 additional quinoline alkaloids previously isolated from the roots of the plant were evaluated for their in vitro antifungal activity against the human fungal pathogen Candida albicans, and 10 compounds (7, 9, 11-16, 18, 21) showed growth inhibitory activity on both planktonic cells and biofilms (MIC ≤ 32 μg/mL). Their spectrum of activity against other pathogenic Candida species and their cytotoxicity against human HeLa cells were also determined. In addition, the cytological effect of the antifungal isolated compounds on the ultrastructure of C. albicans was evaluated by transmission electron microscopy.
... In the frame of our continuous search for antifungal and acetylcholinesterase inhibitors from natural origin (Favre-Godal et al., 2014;Queiroz et al., 2014), the ethanolic extract of the stem bark of C. heliotropiifolius demonstrated significant activities in both assays. To our knowledge, no thorough phytochemical investigation has been performed on this plant and this study presents the isolation and structure elucidation of the bioactive compounds. ...
... The ethanolic extract of the stem bark of C. heliotropiifolius presented antifungal activity in a TLC bioautographic assay against C. albicans with a minimum amount of 10 mg deposited (Favre-Godal et al., 2014). The same extract also showed significant in vitro AChE inhibition in a dilution assay (58% at 40 mg/mL) (Attaur-Rahman et al., 2005;Di Giovanni et al., 2008). ...
... The extract and the isolated compounds were tested at various concentrations against two strains of C. albicans: the mutant strain (DSY2621) and the wild strain (CAF21) ( Table 1). The mutant strain was genetically modified to improve the detection of minor antifungal compounds present in crude extracts (Favre-Godal et al., 2014), while the wild strain is the common pathogen. Spruceanol (8) was the most active compound demonstrating an interesting antifungal activity with a minimal inhibitory quantity (MIQ) of 2 mg against the mutant strain DSY2621, and 10 mg against the wild strain (Table 2). ...
Conference Paper
Full-text available
The ethanolic extract of the stem barks of Croton heliotropiifolius Kunth (Euphorbiaceae) showed significant inhibition of acetylcholinesterase and antifungal activity against Candida albicans on thin layer chromatography bioautographic assays [1,2]. In order to target the isolation of the active compounds at a large scale, HPLC-activity-based-microfractionation in 96 well plates was used in a first step to localize the active compounds. Different regions of the HPLC-UV chromatogram were linked to the acetylcholinesterase inhibition and antifungal activities. Some active compounds were dereplicated by HPLC-PDA-ESI-MS and UHPLC-TOF-HRMS. The target isolation of the active compounds was performed by medium pressure liquid chromatography (MPLC-UV) and semi-preparative HPLC. Using this approach, nine compounds were isolated: one new indole alkaloid derivative, 6-hydroxy-1-(R)-methyl-2-dimethyl-3,4-tetrahydro-β-carboline (1) and several known compounds, (α)-magnoflorine (2), (+)-menisperine (3), taspine (4), moschamine (5), n-propylparaben (6), velamolone acetate (7), spruceanol (8), velamone (9). The isolated compounds presented in the activity region were tested at various concentrations against two strains of C. albicans; the mutant strain (DSY2621) and the wild strain (CAF21). Spruceanol (8) was the most active compound demonstrating an interesting antifungal activity with a minimal inhibitory quantity (MIQ) of 2 µg against the mutant strain, and 10 µg against the wild strain. The AChE inhibitory activity of the isolated compounds was measured and the IC50 values calculated. Compounds 4 and 5 showed the highest AChE inhibitory activity with IC50 values below 10µM. Compounds 1-3 exhibited moderate activity compared to the positive control galanthamine. References: [1] Marston A, Kissling J, Hostettmann K. A rapid TLC bioautographic method for the detection of acetylcholinesterase and butyrylcholinesterase inhibitors in plants. Phytochem Anal 2002; 13: 51 – 54. [2] Favre-Godal Q, Queiroz EF, Wolfender JL. Latest developments in assessing antifungal activity using TLC bioautography. JOAC International 2013; 96: 1175 – 1188.
... This strain has been reported to be highly susceptible and suitable for a better detection sensitivity. 18 The same crude extract did not show activity against a C. albicans wild-type strain, thus indicating the probable presence of the active constituents only in low concentration levels. HPLC-PDA-evaporative light scattering detection (ELSD) analysis revealed a very complex mixture of metabolites. ...
... In order to localize the compounds responsible for the antifungal activity, the crude extract of S. simplex roots was subjected to semipreparative high-pressure liquid chromatography (HPLC) microfractionation on a small size using a highresolution C 18 column (250 × 10 mm i.d.). 18 The amount injected (25 mg) was chosen to allow a satisfactory detection by bioautography after microfractionation, and this yielded 50 fractions of 10 mL each. The gradient used for this separation was transferred geometrically from the analytical HPLC conditions of the metabolite profiling ( Figure 1A) by applying a gradient transfer method to ensure similar selectivity. ...
... The crude CH 2 Cl 2 extract (5.5 g) was first fractionated using reversed-phase MPLC with a 460 × 70 mm i.d. column (Buchi, Flawil, Switzerland), loaded with Zeoprep C 18 15−25 μm as the stationary phase (Zeochem, Uetikon am See, Switzerland), with a linear gradient of MeOH and H 2 O from 5% to 100% MeOH with 0.1% formic acid, and yielded 118 fractions. The separation conditions were optimized at the analytical HPLC level with the same stationary phase chemistry and geometrically transferred by gradient transfer. ...
Article
A dichloromethane extract of the roots from the Panamanian plant Swartzia simplex exhibited a strong antifungal activity in a bioautography assay against a genetically modified hypersusceptible strain of Candida albicans. At-line HPLC activity based profiling of the crude extract enabled a precise localization of the antifungal compounds, and dereplication by UHPLC-HRESIMS indicated the presence of potentially new metabolites. Transposition of the HPLC reversed-phase analytical conditions to medium-pressure liquid chromatography (MPLC) allowed an efficient isolation of the major constituents. Minor compounds of interest were isolated from the MPLC fractions using semipreparative HPLC. Using this strategy, 14 diterpenes (1-14) were isolated, with seven (5-10, 14) being new antifungal natural products. The new structures were elucidated using NMR spectroscopy and HRESIMS analysis. The absolute configurations of some of the compounds were elucidated by electronic circular dichroism spectroscopy. The antifungal properties of these compounds were evaluated as their minimum inhibitory concentrations in a dilution assay against both hypersusceptible and wild-type strains of C. albicans and by assessment of their antibiofilm activities. The potential cytological effects on the ultrastructure of C. albicans of the antifungal compounds isolated were evaluated on thin sections by transmission electron microscopy.
... Previous studies described the isolation of pungent sesquiterpenes lactones from this plant [11]. In a preliminary biological screening, this extract demonstrated antifungal activity against a hypersensitive strain of Candida albicans (DSY2621) in a bioautography assay [12]. In the present study, a comprehensive strategy for the preparative flash MS-guided purification of antifungal com-Abstract ! ...
... The crude ethanolic extract of the aerial parts of the liverwort C. polyanthos presented an interesting antifungal activity (50 µg/ spot) in a TLC bioautographic assay against the hypersensitive strain C. albicans (DSY2621) (l " Table 1) [12]. This mutant yeast (DSY2621) is used to detect minor antifungal compounds present in complex matrices that would not have been detected using classical methodologies [12]. ...
... The crude ethanolic extract of the aerial parts of the liverwort C. polyanthos presented an interesting antifungal activity (50 µg/ spot) in a TLC bioautographic assay against the hypersensitive strain C. albicans (DSY2621) (l " Table 1) [12]. This mutant yeast (DSY2621) is used to detect minor antifungal compounds present in complex matrices that would not have been detected using classical methodologies [12]. The active ethanolic extract was partitioned with dichloromethane (DCM) and a methanol (MeOH)-H 2 O (7 : 3) mixture. ...
Article
The targeted purification of bioactive molecules from complex extracts is of prime importance in the field of drug discovery from natural sources. In this regard, Mass Spectrometry (MS) detection is a key tool enabling the monitoring of specific features for precise fractionation. To improve the isolation process efficiency of antifungal natural products (NPs), an LC-MS-based purification strategy was developed. First, the chromatographic separation of the crude extract was optimized by application of a linear gradient at the analytical scale in HPLC-UV-MS. At-line microfractionation, followed by agar overlay bioautography using a Candida albicans hypersusceptible strain, was performed to identify the bioactive fractions [1]. The gradient was then geometrically transferred from the analytical to the preparative scale using gradient transfer rules based on the calibration of both chromatographic systems [2]. Finally, an MS-triggered isolation of the localized bioactive molecules was realized with a Flash chromatographic system coupled, via splitter, to a single quadrupole mass spectrometer (PuriFlash® – MS). This isolation strategy was applied for the MS-direct purification of the antifungal compound Diplophyllolide A from the Chinese liverwort Chiloscyphus Polyanthos (L.) Cord. The bioactive molecule was directly isolated in large amount, without issued related to MS saturation, thanks to the optimization of the MS splitting geometry. This rational LC-MS-based methodology has high potential for the rapid isolation of compounds of interest identified by MS-based metabolomics and for the efficient purification of bioactive natural products that lack of UV chromophore. Keywords: MS-direct purification, antifungal compounds, Flash Chromatography, Chiloscyphus Polyanthos. References: [1] Q. Favre-Godal, S. Dorsaz, E.F. Queiroz, C. Conan et al., Phytochemistry, Accepted. [2] D. Guillarme, D.T.T. Nguyen, S. Rudaz, J.-L. Veuthey, Eur. J. Pharma. Biopharma. 2008, 68, 430.
... Previous studies described the isolation of pungent sesquiterpenes lactones from this plant [11]. In a preliminary biological screening, this extract demonstrated antifungal activity against a hypersensitive strain of Candida albicans (DSY2621) in a bioautography assay [12]. In the present study, Abstract ! ...
... The crude ethanolic extract of the aerial parts of the liverwort C. polyanthos presented an interesting antifungal activity (50 µg/ spot) in a TLC bioautographic assay against the hypersensitive strain C. albicans (DSY2621) (l " Table 1) [12]. This mutant yeast (DSY2621) is used to detect minor antifungal compounds present in complex matrices that would not have been detected using classical methodologies [12]. ...
... The crude ethanolic extract of the aerial parts of the liverwort C. polyanthos presented an interesting antifungal activity (50 µg/ spot) in a TLC bioautographic assay against the hypersensitive strain C. albicans (DSY2621) (l " Table 1) [12]. This mutant yeast (DSY2621) is used to detect minor antifungal compounds present in complex matrices that would not have been detected using classical methodologies [12]. The active ethanolic extract was partitioned with dichloromethane (DCM) and a methanol (MeOH)-H 2 O (7 : 3) mixture. ...
Article
Full-text available
In natural product research, the efficient purification of molecules from large amounts of complex extracts is a key element. In this regard, an integrative strategy for efficient MS-guided isolation of antifungal compounds has been developed. First, off-line HPLC antifungal activity-based profiling and HPLC-PDA-MS profiling were used to localize the compounds of interest on the analytical scale. Then, the analytical gradient was geometrically transferred to the flash chromatographic level. Finally, an MS-triggered isolation of the localized bioactive molecules was realized using high-resolution flash chromatographic columns (15 µm spherical particles) coupled to a single quadrupole mass spectrometer via a splitter system. This isolation strategy was applied for the MS-targeted purification of antifungal principles from the liverwort Chiloscyphus polyanthos. This rational methodology has high potential for the targeted large-scale purification of bioactive compounds, avoiding the need to repeat a given bioassay at each isolation step. Seven sesquiterpene lactones were isolated, of which five were found to be bioactive and one was reported as a new compound. The absolute configuration of some compounds was established for the first time by electronic circular dichroism spectroscopy. Georg Thieme Verlag KG Stuttgart · New York.
... Tables 2 and 3 were used to organize the information gained from each study [11][12][13][14][15][16][17][18][19]. Table 2 displays data from combined in vivo/in vitro studies whilst Table 3 contains information from in vitro studies only . ...
... Finally, a total of 131 articles met the inclusion criteria and were considered suitable for this systematic review. In total, there were 186 natural products involved in this systematic review; please see Tables 2 and 3 for further details on each study, [11][12][13][14][15][16][17][18][19] . Plants identified in the review originated in 42 countries, with the largest percentages in Brazil (20%), India (9%) and Iran (7%) (Fig. 2). ...
... When investigating the in vivo effects of herbal extracts on C. albicans, 9 articles that matched the search criteria with a total of 11 plants were examined ( Table 2). Most studies used rats as the host organism by infecting them with C. albicans in which, only Vicia faba and Morinda tomentosa show no antifungal activity against C. albicans [12,14]. The most active plants were C. sinensis with MIC values of 40 µg/mL [13]. ...
Article
Background In the era of antimicrobial resistance, fungal pathogens are not an exception. Several strategies, including antimicrobial stewardship programs and high throughput screening of new drugs are being implemented. Several recent studies have demonstrated effectiveness of plant compounds with antifungal activity. Objective In this systematic review we examine the use of natural compounds as a possible avenue to fight fungal infections produced by Candida albicans, the most common human fungal pathogen. Method Electronic literature searches were conducted through PubMed/MEDLINE, Cochrane, and Science Direct limited to the 5 years. A total of 131 articles were included with 186 plants extracts evaluated. Results Although the majority of the natural extracts exhibited antifungal activities against C. albicans (both in vivo and in vitro), the strongest antifungal activity was obtained from Lawsonia inermis, Pelargonium graveolens, Camellia sinensis, Mentha piperita, and Citrus latifolia. Conclusion The main components with proven antifungal activities were phenolic compounds such as gallic acid, thymol, and flavonoids (specially catechin), polyphenols such as tannins, terpenoids and saponins. The incorporation of nanotechnology enhances greatly the antifungal properties of these natural compounds. Further research is needed to fully characterize the composition of all herbal extracts with antifungal activity as well as the mechanisms of action of the active compounds.
... During a preliminary antifungal screening procedure, an ethanolic extract from the stem bark of C. fontanesianus exhibited weak activity at 600 μg/spot against a hypersensitive mutant strain of C. albicans (DSY2621) in a bioautography assay. 10 Bioautography with the hypersusceptible strain was used to detect the presence of minor antifungal compounds with high sensitivity. To identify the active compounds, a crude ethanol extract of the stem bark was partitioned with CH 2 Cl 2 and a MeOH−water (7:3) mixture. ...
... Each separation yielded 60 fractions. Fractions obtained were evaluated in an antifungal bioautography assay according to the methodology described by Favre-Godal et al. 10 Using this procedure, it was possible to correlate the antifungal activity to two chromatographic peaks at the retention time 39.5 min (17) and 41.8 min (18). ...
... Bioautography. Inhibitory activity of compounds 17 and 18 against Candida albicans was evaluated using the method described by Fabre-Godal et al. 10 The C. albicans strains were cultivated in Sabouraud broth medium overnight at 36°C. An inoculum of 10 5 cells/mL (an optical density (OD) equal to 1 at 630 nm, corresponding to approximately l0 7 cells/mL) was prepared in malt agar (malt extract, 30.0 g/L; peptone from soymeal, 3.0 g/L; agar− agar, 15.0 g/L; Merck). ...
Article
Full-text available
A dichloromethane-soluble fraction of the stem bark of Conchocarpus fontanesianus showed antifungal activity against Candida albicans in a bioautography assay. Off-line high-pressure liquid chromatography activity-based profiling of this extract enabled a precise localization of the compounds responsible for the antifungal activity that were isolated and identified as the known compounds flindersine (17) and 8-methoxyflindersine (18). As well as the identification of the bioactive principles, the ultra-high-pressure liquid chromatography-high-resolution mass spectrometry metabolite profiling of the dichloromethane stem bark fraction allowed the detection of more than 1000 components. Some of these could be assigned putatively to secondary metabolites previously isolated from the family Rutaceae. Generation of a molecular network based on MS(2) spectra indicated the presence of indolopyridoquinazoline alkaloids and related scaffolds. Efficient targeted isolation of these compounds was performed by geometric transfer of the analytical high-pressure liquid chromatography profiling conditions to preparative medium-pressure liquid chromatography. This yielded six new indolopyridoquinazoline alkaloids (5, 16, 19-22) that were assigned structurally. The medium-pressure liquid chromatography separations afforded additionally 16 other compounds. This work has demonstrated the usefulness of molecular networks to target the isolation of new natural products and the value of this approach for dereplication. A detailed analysis of the constituents of the stem bark of C. fontanesianus was conducted.
... The plasmatocytes and granulocytes participate in phagocytosis, nodule formation, encapsulation and defence against microbial pathogens [20,21]. G. mellonella has also been used to test the efficacy and toxicity of commercial and new antifungals [22][23][24]. ...
... Furthermore, the survival curve profiles for both species were similar in this model, as previously described [13]. In most other studies, antifungal drugs were administered within the first 4 h post-infection [16,18,22,28] and we choose to administer AmB and itraconazole at 1 h post-infection. Following administration of AmB at 0.5, 1 or 2 mg/kg, a dose-dependent survival curve was produced. ...
Article
Paracoccidioides brasiliensis and P. lutzii belong to a group of thermodimorphic fungi and cause paracoccidioidomycosis (PCM), which is a human systemic mycosis endemic in South and Central America. Patients with this mycosis are commonly treated with amphotericin B (AmB) and azoles. The study of fungal virulence and the efficacy and toxicity of antifungal drugs has been successfully performed in a Galleria mellonella infection model. In this work, G. mellonella larvae were infected with two Paracoccidioides spp. and the efficacy and toxicity of AmB and itraconazole were evaluated in this model for the first time. AmB and itraconazole treatments were effective in increasing larval survival and reducing the fungal burden. The fungicidal and fungistatic effects of AmB and itraconazole, respectively, were observed in the model. Furthermore, these effects were independent of changes in haemocyte number. G. mellonella can serve as a rapid model for the screening of new antifungal compounds against Paracoccidioides and can contribute to a reduction in experimental animal numbers in the study of PCM.
... In a precedent study, we reported on a strategy to identify antifungal NPs from plant crude extracts (12). This strategy relied on the use of a C. albicans isolate highly susceptible to growth inhibitors and in which traces of inhibitory NPs could be detected. ...
... Among these compounds, 53% were previously isolated from crude plant extracts that presented antifungal activity. The bioguided process of isolation of these compounds was performed by bioautography using wild-type and genetically modified strains of C. albicans (12). In parallel, NPs with (i) structures closely related to those of NPs possessing antifungal activity from published sources and (ii) unknown antifungal activity were selected from commercial catalogues and acquired (the compounds are listed in Table S1 in the supplemental material). ...
Article
Candida albicans is a major cause of fungal diseases in humans and its resistance to available drugs is of concern. In an attempt to identify novel antifungal agents, we initiated a small scale screening of a 199 natural plant compounds (NPs) library. In vitro susceptibility profiling experiments identified 33 NPs with activity against C. albicans (MIC 50 ≤ 32 μg/ml). Among the selected NPs, the sterol alkaloid tomatidine was further investigated. Tomatidine originates from Solanum lycopersicum (tomato) and exhibited high fungistatic activity against Candida species (MIC 50 ≤ 1 μg/ml) but no cytotoxicity against mammalian cells. Genome-wide transcriptional analysis of C. albicans tomatidine-treated cells revealed a major alteration (upregulation) of ergosterol genes suggesting that the ergosterol pathway was targeted by this NP. Consistent with this transcriptional response, sterol content analysis of tomatidine-treated cells showed not only inhibition of Erg6 (C-24 sterol methyltransferase) but also of Erg4 (C-24 sterol reductase) activity. A forward genetic approach in Saccharomyces cerevisiae coupled with whole genome sequencing identified 2 non-synonymous mutations in ERG6 (amino acids: D249G and G132D) responsible for tomatidine resistance. Our results therefore identified unambiguously Erg6, a sterol C-24 methyltransferase absent in mammals, as the main direct target of tomatidine. We tested the in vivo efficacy of tomatidine in a mouse model of C. albicans systemic infection. Treatment with a nano-crystal pharmacological formulation successfully decreased the fungal burden in infected kidneys as compared to placebo and thus confirmed the potential of tomatidine as a therapeutic agent.
... The genus Candida has been extensively studied in the G. mellonella model especially for the evaluation of virulence and antifungal efficacy [10,71,72]. G. mellonella was used to test the efficacy of different antifungal compounds against Candida yeasts including conventional antifungal drugs [13][14][15][16][17][18][19][20][21][22][23], new drugs [25][26][27][30][31][32]34] or non-antifungal compounds in a repurposing perspective [24,28,29,33]. Antifungal combinations against Candida spp. ...
... Several drugs, such as atorvastatin, a cholesterol-lowering agent [24], miltefosine, an anti-leishmanial drug [33], miramistin, an antiseptic [29], and pilocarpine, a muscarinic agonist [28] were evaluated in a repurposing perspective. In other instances, new synthetic compounds or natural products were tested in vivo in the model [25][26][27][30][31][32]34]. It must be noticed that in all cases, larvae were infected with C. albicans. ...
Article
Full-text available
The treatment of invasive fungal infections remains challenging and the emergence of new fungal pathogens as well as the development of resistance to the main antifungal drugs highlight the need for novel therapeutic strategies. Although in vitro antifungal susceptibility testing has come of age, the proper evaluation of therapeutic efficacy of current or new antifungals is dependent on the use of animal models. Mammalian models, particularly using rodents, are the cornerstone for evaluation of antifungal efficacy, but are limited by increased costs and ethical considerations. To circumvent these limitations, alternative invertebrate models, such as Galleria mellonella, have been developed. Larvae of G. mellonella have been widely used for testing virulence of fungi and more recently have proven useful for evaluation of antifungal efficacy. This model is suitable for infection by different fungal pathogens including yeasts (Candida, Cryptococcus, Trichosporon) and filamentous fungi (Aspergillus, Mucorales). Antifungal efficacy may be easily estimated by fungal burden or mortality rate in infected and treated larvae. The aim of the present review is to summarize the actual data about the use of G. mellonella for testing the in vivo efficacy of licensed antifungal drugs, new drugs, and combination therapies.
... The virulence of different fungi, including Paracoccidioides and Cryptococcus species (Mylonakis et al., 2005;Thomaz et al., 2013;Scorzoni et al., 2015;de Lacorte Singulani et al., 2016;Sangalli-Leite et al., 2016;Palanco et al., 2017) has been successfully studied in G. mellonella. In addition, the effect of azoles and amphotericin B, as well as natural products, have been evaluated in this model Favre-Godal et al., 2014;de Lacorte Singulani et al., 2016). ...
Article
Full-text available
Invasive fungal infections, such as cryptococcosis and paracoccidioidomycosis are associated with significant rates of morbidity and mortality. Cryptococcosis, caused by Cryptococcus neoformans, is distributed worldwide and has received much attention as a common complication in patients with HIV. Invasive fungal infections are usually treated with a combination of amphotericin B and azoles. In addition, 5-fluorocytosine (5-FC) is applied in cryptococcosis, specifically to treat central nervous system infection. However, host toxicity, high cost, emerging number of resistant strains, and difficulty in developing new selective antifungals pose challenges. The need for new antifungals has therefore prompted a screen for inhibitory peptides, which have multiple mechanisms of action. The honeycomb moth Galleria mellonella has been widely used as a model system for evaluating efficacy of antifungal agents. In this study, a peptide analog from the mastoparan class of wasps (MK58911) was tested against Cryptococcus spp. and Paracoccidioides spp. In addition, peptide toxicity tests on lung fibroblasts (MRC5) and glioblastoma cells (U87) were performed. Subsequent tests related to drug interaction and mechanism of action were also performed, and efficacy and toxicity of the peptide were evaluated in vivo using the G. mellonella model. Our results reveal promising activity of the peptide, with an MIC in the range of 7.8–31.2 μg/mL, and low toxicity in MRC and U87 cells (IC50 > 500 μg/mL). Taken together, these results demonstrate that MK58911 is highly toxic in fungal cells, but not mammalian cells (SI > 16). The mechanism of toxicity involved disruption of the plasma membrane, leading to death of the fungus mainly by necrosis. In addition, no interaction with the drugs amphotericin B and fluconazole was found either in vitro or in vivo. Finally, the peptide showed no toxic effects on G. mellonella, and significantly enhanced survival rates of larvae infected with C. neoformans. Although not statistically significant, treatment of larvae with all doses of MK58911 showed a similar trend in decreasing the fungal burden of larvae. These effects were independent of any immunomodulatory activity. Overall, these results present a peptide with potential for use as a new antifungal drug to treat systemic mycoses.
... PP alone exhibited in vitro antifungal effect against both planktonic and biofilms of E. dermatitidis. Further, we observed the in vivo antifungal activity of PP alone and combined with azoles against E. dermatitidis infection using G. mellonella model, which showed similar immune response to mammals (Vilcinskas, 2011;Favre-Godal et al., 2014). PP alone exhibited comparable antifungal effect as azoles alone in vivo. ...
Article
Full-text available
Infections of Exophiala dermatitidis are often chronic and recalcitrant. Combination therapies with novel compounds and azoles could be an effective solution. Previously, we have demonstrated that pyrvinium pamoate exerted antifungal activity alone and favorable synergy with azoles against planktonic E. dermatitidis. Herein, the underlying antifungal mode of action were investigated. Pyrvinium alone showed sessile MIC50 (SMIC50) of 8->16 μg/ml against E. dermatitidis biofilms. However, synergism of PP with itraconazole, voriconazole, and posaconazole were observed against 16 (88.9%), 9 (50%), and 13 (72.2%) strains of E. dermatitidis biofilms. In accordance with in vitro susceptibilities, pyrvinium alone at concentration of 2 μg/ml resulted in significant growth restriction of planktonic E. dermatitidis. Pyrvinium alone resulted in reduction of biofilm formation. Higher concentration of pyrvinium was associate with more progressive reduction of biofilm mass. The in vivo activity of pyrvinium alone and combined with azoles was evaluated using Galleria mellonella model. Pyrvinium alone significantly improved the survival rate of larvae (P < 0.0001). The combination of pyrvinium and voriconazole or posaconazole acted synergistically in vivo (P < 0.05). Fungal burden determination revealed significant reduction of numbers of colony forming unit (CFU) in larvae treated with pyrvinium-itraconazole and pyrvinium-posaconazole compared to itraconazole or posaconazole alone group, respectively. The effect of pyrvinium on apoptosis, expression of TOR and HSP90, and drug efflux reversal were evaluated by PI/Annexin V staining, Real-Time Quantitative PCR and Rhodamine 6G assay, respectively. Pyrvinium alone or combined with azoles significantly (P < 0.05) increased late apoptosis or necrosis of E. dermatitidis cells. Pyrvinium combined with posaconazole significantly decreased the expression of TOR and Hsp90 compared to posaconazole alone group (P < 0.05). Pyrvinium resulted in significant (P < 0.05) decrease of the efflux of Rhodamine 6G. These findings suggested pyrvinium could be a promising synergist with azoles. The underlying mechanisms could be explained by inducing apoptosis/necrosis, inhibition of drug efflux pumps, and signaling pathways related with stress response and growth control.
... Furthermore, the fungal virulence and antifungal drug activity has recently been investigated in G. mellonella model on account of a similar immune response as mammals and other advantages including lack of ethical concerns, low cost, and easy manipulation [28,31,32]. As illustrated in our study, the survival rate of larvae in voriconazole-amorol ne combination group was signi cantly improved compared to the monotherapy groups (including voriconazole or amorol ne-treated group) (P < 0.05). ...
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Background: Fusarium species are environmentally ubiquitous fungi capable of causing diverse superficial, locally invasive, or disseminated infections, making them important pathogens. Compared with antibacterial drugs, the types of antifungal drugs are still limited, and the adverse reactions are significant. Therefore, novel treatments against Fusarium spp. are an urgent need. Results: Here we investigated the interaction of amorolfine combined with voriconazole on Fusarium spp. Our study demonstrated that amorolfine in combination with voriconazole could inhibited the Fusarium spp. significantly. Galleria mellonella was also used as a model to show the interaction of the two-drug combination in vivo against Fusarium spp.; larval survival rates were significantly higher after treatment with the amorolfine-voriconazole combination compared to the monotherapy group. Conclusions: This study is the first to demonstrate that voriconazole combined with amorolfine has a synergistic effect against Fusarium spp. infection and may be an effective method for antifungal therapy.
... a synergistic interaction in vitro, the in vivo efficacy of LF-D1 alone and in combination with AMB against C. albicans SC5314 or C. neoformans H99 was tested in Galleria mellonella (wax moth) larvae. The G. mellonella infection model is an established model host system that has been used to study a number of clinically important fungi, including Candida and Cryptococcus species, and the results obtained with this model show a strong correlation with the results obtained using mammalian models (25)(26)(27). A range of drug concentrations for LF (64 to 1,024 g/ml) and AMB (8 to 128 g/ml) was initially tested, with the full data provided in Data Set S3. ...
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Lactoferrin (LF) is a multifunctional milk protein with antimicrobial activity against a range of pathogens. While numerous studies report that LF is active against fungi, there are considerable differences in the level of antifungal activity and the capacity of LF to interact with other drugs. Here we undertake a comprehensive evaluation of the antifungal spectrum of activity of three defined sources of LF across 22 yeast and 24 mould species, and assess interactions with six widely used antifungal drugs. LF was broadly and consistently active across all yeast species (MIC 8-64 μg/ml), with the extent of activity strongly affected by iron saturation. LF was synergistic with amphotericin B (AMB) in 19 out of 22 yeast species tested and synergy was unaffected by iron saturation but affected by the extent of LF digestion. LF+AMB combination therapy significantly prolonged the survival of Galleria mellonella infected with Candida albicans or Cryptococcus neoformans and decreased fungal burden 12-25 fold. Evidence that LF directly interacts with the fungal cell surface was seen via SEM, which showed pore formation, hyphal thinning and major cell collapse in response to LF+AMB synergy. Important virulence mechanisms were disrupted by LF+AMB treatment, which significantly prevented biofilms in C. albicans and C. glabrata, inhibited hyphal development in C. albicans , and reduced cell and capsule size and phenotypic diversity in Cryptococcus. Our results demonstrate the potential of LF+AMB as an antifungal treatment that is broadly synergistic against important yeast pathogens, with synergy attributed to the presence of one or more LF peptides.
... Galleria mellonella models have become invaluable to study cellular and humoral immune responses against pathogens [31][32][33], epigenetic influences on the evolution of virulence [32,34,35], and for development and testing antimicrobials [36][37][38][39]. It is relevant to add that the complete genomes of P. destructans and G. mellonella have become available recently [40,41]. ...
Article
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Laboratory investigations of the pathogenesis of Pseudogymnoascus destructans, the fungal causal agent of bat White Nose Syndrome (WNS), presents unique challenges due to its growth requirements (4°-15°C) and a lack of infectivity in the current disease models. Pseudogymnoascus pannorum is the nearest fungal relative of P. destructans with wider psychrophilic – physiological growth range, and ability to cause rare skin infections in humans. Our broad objectives are to create the molecular toolkit for comparative study of P. destructans and P. pannorum pathogenesis. Towards these goals, we report the successful development of an invertebrate model in the greater wax moth Galleria mellonella. Both P. destructans and P. pannorum caused fatal disease in G. mellonella and elicited immune responses and histopathological changes consistent with the experimental disease.
... The antibacterial activity was evaluated using a modification of the method reported by Favre-Godal et al. (2014). B. subtilis grown on potato sucrose medium was used for the assay. ...
Article
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Two new C11-terpenes with an octahydrobenzofuran skeleton, designated ficusnotadiol (1) and ficusnotanone (2), have been isolated from the plant Ficus nota. Their structures were elucidated on the basis of spectroscopic data. The absolute structure of 1 was determined by X-ray crystallographic analysis. The isolated compounds were evaluated for their antibacterial activity against Bacillus subtilis.
... Ethylene (ET), jasmonate (JA), and salicylic acid (SA) are probably the major defenserelated phytohormones, with SA mainly involved in response to biotrophic microbes, and JA and ethylene in response to necrotrophic microbes (Berens et al. 2017;Xu et al. 2018;Li et al. 2019). The production of various antimicrobial agents called phytoalexins (phenols, flavonoids, coumarins, quinones, saponins, xanthones, alkaloids, lectins and polypeptides, terpenoids…) also inhibits the development of pathogens (Arif et al. 2009;Favre-Godal et al. 2014. Expectedly, some antimicrobial compounds were isolated from orchids. ...
Article
Orchids are associated with diverse fungal taxa, including nonmycorrhizal endophytic fungi as well as mycorrhizal fungi. Theorchid mycorrhizal (OM) symbiosis is an excellent model for investigating the biological interactions between plants and fungidue to their high dependency on these symbionts for growth and survival. To capture the complexity of OM interactions,significant genomic, numerous transcriptomic, and proteomic studies have been performed, unraveling partly the role of eachpartner. On the other hand, several papers studied the bioactive metabolites from each partner but rarely interpreted theirsignificance in this symbiotic relationship. In this review, we focus from a biochemical viewpoint on the OM dynamics andits molecular interactions. The ecological functions of OM in plant development and stress resistance are described first,summarizing recent literature. Secondly, because only few studies have specifically looked on OM molecular interactions, thesignaling pathways and compounds allowing the establishment/maintenance of mycorrhizal association involved in arbuscularmycorrhiza (AM) are discussed in parallel with OM. Based on mechanistic similarities between OM and AM, and recent findingson orchids’endophytes, a putative model representing the different molecular strategies that OM fungi might employ to establishthis association is proposed. It is hypothesized here that (i) orchids would excrete plant molecule signals such as strigolactonesand flavonoids but also other secondary metabolites; (ii) in response, OM fungi would secrete mycorrhizal factors (Myc factors)or similar compounds to activate the common symbiosis genes (CSGs); (iii) overcome the defense mechanism by evasion of thepathogen-associated molecular patterns (PAMPs)-triggered immunity and by secretion of effectors such as small inhibitorproteins; and (iv) finally, secrete phytohormones to help the colonization or disrupt the crosstalk of plant defense phytohormones.To challenge this putative model, targeted and untargeted metabolomics studies with special attention to each partner’scontri-bution are finally encouraged and some technical approaches are proposed.
... The choice of hosts (murine and insect models) was motivated by the idea that minihost models such as G. mellonella could replace mammalian models in some experimental infections with C. albicans due to cost and ethical reasons. Several laboratories have investigated fungal virulence and antifungal drug activity with G. mellonella (62)(63)(64)(65)(66)(67). The results obtained from this insect model were consistent with those obtained from the mouse systemic model of infection (62, 64; reviewed in reference 40) and with the pathogenicity of C. albicans strains in human patients (68). ...
Article
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Importance: Understanding the mechanisms utilized by pathogens to infect and cause disease in their hosts is crucial for rational drug development. Transcriptomic studies may help investigations of these mechanisms by determining which genes are expressed specifically during infection. This task has been difficult so far, since the proportion of microbial biomass in infected tissues is often extremely low, thus limiting the depth of sequencing and comprehensive transcriptome analysis. Here, we adapted a technology to capture and enrich C. albicans RNA, which was next used for deep RNA sequencing directly from infected tissues from two different host organisms. The high-resolution transcriptome revealed a large number of genes that were so far unknown to participate in infection, which will likely constitute a focus of study in the future. More importantly, this method may be adapted to perform transcript profiling of any other microbes during host infection or colonization.
... Many researchers are now using G. mellonella to study the impact of particular genes on microbial virulence, to compare the virulence of different clinical isolates, and to assess the efficacy of potential treatments. [11][12][13][14][15][16][17][18][19][20][21][22][23][24][25][26][27][28][29][30] In issue 6(2) of Virulence, Ciesielczuk and colleagues have used G. mellonella to study the pathogenicity of 40 well-characterized clinical isolates of extra-intestinal pathogenic E. coli (ExPEC). 28 This is a task for which G. mellonella is well suited as it can become prohibitive to carry out such experiments using more traditional surrogate hosts, such as mice. ...
... Galleria mellonella larvae have recently been identified as an ideal in vivo model for the preclinical analysis of the antifungal activity of novel drugs, as these larvae exhibit immune responses similar to those of mammals without any of the concomitant ethical concerns, and they also offer advantages such as being inexpensive and easy to manipulate [30,37,38]. To further verify the interaction between AMO and VOR detected in our study, we tested the in vivo effect of this combination against one of the isolates of F. solani complex with G. mellonella model. ...
Article
Full-text available
Fusarium species represent a range of fungal pathogens capable of causing diverse mycotic diseases. Relative to antibacterial drugs, few effective antifungal agents have been developed to date, and all are subject to significant limitations. As such, there is an urgent need to design novel antifungal treatments for infections caused by Fusarium spp. Herein, 15 clinical isolates, including 5 Fusarium oxysporum and 10 Fusarium solani strains, were analyzed to explore the relative inhibitory effects of different combinations of amorolfine (AMO) and voriconazole (VOR) on the growth of these fungal pathogens. These analyses were conducted by measuring minimal inhibitory concentration (MIC) values for these antifungal agents in a broth microdilution assay and by using an in vivo model of Fusarium-infected Galleria mellonella. These experiments revealed that in isolation, AMO and VOR exhibited MIC values ranging from 4 to 16 μg/mL and 2 to 8 μg/mL, respectively. However, these effective MIC values fell to 1–2 μg/mL and 0.5–2 μg/mL, respectively, when AMO and VOR were administered in combination with one another, exhibiting synergistic activity against 73.3% of analyzed Fusarium strains. Subsequent in vivo analyses conducted using the G. mellonella model further confirmed that combination VOR + AMO treatment was associated with significantly improved larval survival following Fusarium spp. infection. Together, these results serve as the first published evidence demonstrating that VOR and AMO exhibit synergistic activity against infections caused by Fusarium spp., indicating that they may represent an effective approach to antifungal disease treatment.
... To facilitate in vivo experimentation, a number of insect models have recently been developed for use with fungal pathogens, such as the larvae of the insect species Galleria mellonella (Cotter et al., 2000). These larvae have been increasingly used to study fungal virulence and antifungal drug activity (Brennan et al., 2002;Mylonakis et al., 2005;Coleman et al., 2011;Thomaz et al., 2013;Favre-Godal et al., 2014). The results obtained from this insect model were consistent with, first, those obtained from the mouse systemic model of infection but for a small number of mutants (Brennan et al., 2002;reviewed in Coste and Amorim-Vaz, 2015) and, second, with data on the pathogenicity of C. albicans strains in human patients (Cotter et al., 2000). ...
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The aim of the present study was to identify Candida albicans transcription factors (TFs) involved in virulence. Although mice are considered the gold-standard model to study fungal virulence, mini-host infection models have been increasingly used. Here, barcoded TF mutants were first screened in mice by pools of strains and fungal burdens (FBs) quantified in kidneys. Mutants of unannotated genes which generated a kidney FB significantly different from that of wild-type were selected and individually examined in Galleria mellonella. In addition, mutants that could not be detected in mice were also tested in G. mellonella. Only 25% of these mutants displayed matching phenotypes in both hosts, highlighting a significant discrepancy between the two models. To address the basis of this difference (pool or host effects), a set of 19 mutants tested in G. mellonella were also injected individually into mice. Matching FB phenotypes were observed in 50% of the cases, highlighting the bias due to host effects. In contrast, 33.4% concordance was observed between pool and single strain infections in mice, thereby highlighting the bias introduced by the "pool effect." After filtering the results obtained from the two infection models, mutants for MBF1 and ZCF6 were selected. Independent marker-free mutants were subsequently tested in both hosts to validate previous results. The MBF1 mutant showed impaired infection in both models, while the ZCF6 mutant was only significant in mice infections. The two mutants showed no obvious in vitro phenotypes compared with the wild-type, indicating that these genes might be specifically involved in in vivo adapt.
... In addition, G. mellonella model recently has been used to study fungal virulence and antifungal drug activity, since it has a similar immune response to mammals (Vilcinskas, 2011;Favre-Godal et al., 2014), and the correlation between virulence of C. albicans mutants in mice and G. mellonella model has been confirmed (Brennan et al., 2002). It is worth mentioning that, with these advantages and the low cost, lack of ethical concerns and easy manipulation, G. mellonella model has gained wide acknowledgment. ...
Article
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Candida albicans (C. albicans) is one of the important opportunistic fungal pathogens that is closely associated with disseminated or chronic infections. The objective of this study is to evaluate the synergistic antifungal effect of licofelone, which is dual microsomal prostaglandin E2 synthase/lipoxygenase (mPGES-1/LOX) inhibitor in combination with fluconazole against C. albicans. Here our results showed that licofelone (16 μg/mL) can synergistically work with fluconazole (1 μg/mL) against planktonic cells of fluconazole-resistant C. albicans. The two-drug combination inhibited the C. albicans biofilm formation over 12 h, and reduced the expression of extracellular phospholipase genes, biofilm-specific genes and RAS/cAMP/PKA pathway related genes. In addition, the two-drug combination inhibited the transition from yeast to hyphal growth form, and decreased the secreted aspartyl proteinase activity, while not affecting the drug efflux pumps activity. Galleria mellonella model was also used to confirm the antifungal activity of the drug combination in vivo. This study first indicates that the combination of fluconazole and licofelone has synergistic effect against resistant C. albicans and could be a promising therapeutic strategy for the antifungal treatment.
... In the present case, based on the resolution achieved in HPLC, the injected quantity was much higher to favour purification yields at a large scale (up to 100 mg of pure products were collected), leading to distorted and broadened peaks, which, however, did not compromise the isolation efficiency. The second example deals with the fractionation of the methanolic extract of Morinda tomentosa L., Rubiaceae, which was used as a model since it contains series of closely related anthraquinones isomers [14]. An efficient separation was performed by HPLC-UV using a generic linear MeOH : H 2 O gradient as the mobile phase (l " Table 1, Fig. 3 A). ...
Article
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In natural product research, the isolation of biomarkers or bioactive compounds from complex natural extracts represents an essential step for de novo identification and bioactivity assessment. When pure natural products have to be obtained in milligram quantities, the chromatographic steps are generally labourious and time-consuming. In this respect, an efficient method has been developed for the reversed-phase gradient transfer from high-performance liquid chromatography to medium-performance liquid chromatography for the isolation of pure natural products at the level of tens of milligrams from complex crude natural extracts. The proposed method provides a rational way to predict retention behaviour and resolution at the analytical scale prior to medium-performance liquid chromatography, and guarantees similar performances at both analytical and preparative scales. The optimisation of the high-performance liquid chromatography separation and system characterisation allows for the prediction of the gradient at the medium-performance liquid chromatography scale by using identical stationary phase chemistries. The samples were introduced in medium-performance liquid chromatography using a pressure-resistant aluminium dry load cell especially designed for this study to allow high sample loading while maintaining a maximum achievable flow rate for the separation. The method has been validated with a mixture of eight natural product standards. Ultraviolet and evaporative light scattering detections were used in parallel for a comprehensive monitoring. In addition, post-chromatographic mass spectrometry detection was provided by high-throughput ultrahigh-performance liquid chromatography time-of-flight mass spectrometry analyses of all fractions. The processing of all liquid chromatography-mass spectrometry data in the form of an medium-performance liquid chromatography x ultra high-performance liquid chromatography time-of-flight mass spectrometry matrix enabled an efficient localisation of the compounds of interest in the generated fractions. The methodology was successfully applied for the separation of three different plant extracts that contain many diverse secondary metabolites. The advantages and limitations of this approach and the theoretical chromatographic background that rules such as liquid chromatography gradient transfer are presented from a practical viewpoint. Georg Thieme Verlag KG Stuttgart · New York.
... Moreover, both the human host and fungi are eukaryotic organisms, limiting the cellular targets for these drugs. These factors, associated with the agents' toxicity, resistance of microorganisms to such agents (Singh et al., 2014;Torabzadeh and Panahi, 2013) and the emergence of new virulence factors (Guirao-Abad et al., 2013), lead to the search for compounds with novel mechanisms of action or actions which are synergistic with the mechanisms already assessed (Favre-Godal et al., 2014). ...
Article
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Infections caused by microorganisms of the Candida genus represent a major cause of morbidity and mortality among the population. Therefore, this study aimed at evaluating the antifungal activity and toxicity of resveratrol-analog Schiff bases. Faced with Candida albicans ATCC 10,231, the broth microdilution method was used, along with amphotericin B as the reference drug. The toxicity was evaluated in human keratinocyte cells and against Artemia salina. All analogs were active against the microorganism and caused structural changes in the yeast. The minimum inhibitory concentration was lower for analog A (156.3 μg mL⁻¹), followed by B (312.5 μg mL⁻¹), C (312.5 μg mL⁻¹) and D (625 μg mL⁻¹); while the minimum fungicidal concentration was lower for A (1,250 μg mL⁻¹), B (1,250 μg mL⁻¹) and D (1,250 μg mL⁻¹), followed by C (2,500 μg mL⁻¹). In keratinocytes, the compounds presented cell viability between 50.9 and 93%. Against A. salina, the compounds presented moderate cytotoxic activity. The compound with the nitro group, an electron withdrawing group, in the para position presented better antifungal activity. Resveratrol analogs can be promising in the development of new antifungal agents.
... Ethylene (ET), jasmonate (JA), and salicylic acid (SA) are probably the major defenserelated phytohormones, with SA mainly involved in response to biotrophic microbes, and JA and ethylene in response to necrotrophic microbes (Berens et al. 2017;Xu et al. 2018;Li et al. 2019). The production of various antimicrobial agents called phytoalexins (phenols, flavonoids, coumarins, quinones, saponins, xanthones, alkaloids, lectins and polypeptides, terpenoids…) also inhibits the development of pathogens (Arif et al. 2009;Favre-Godal et al. 2014. Expectedly, some antimicrobial compounds were isolated from orchids. ...
Article
Full-text available
Orchids are associated with diverse fungal taxa, including nonmycorrhizal endophytic fungi as well as mycorrhizal fungi. The orchid mycorrhizal (OM) symbiosis is an excellent model for investigating the biological interactions between plants and fungi due to their high dependency on these symbionts for growth and survival. To capture the complexity of OM interactions, significant genomic, numerous transcriptomic, and proteomic studies have been performed, unraveling partly the role of each partner. On the other hand, several papers studied the bioactive metabolites from each partner but rarely interpreted their significance in this symbiotic relationship. In this review, we focus from a biochemical viewpoint on the OM dynamics and its molecular interactions. The ecological functions of OM in plant development and stress resistance are described first, summarizing recent literature. Secondly, because only few studies have specifically looked on OM molecular interactions, the signaling pathways and compounds allowing the establishment/maintenance of mycorrhizal association involved in arbuscular mycorrhiza (AM) are discussed in parallel with OM. Based on mechanistic similarities between OM and AM, and recent findings on orchids’ endophytes, a putative model representing the different molecular strategies that OM fungi might employ to establish this association is proposed. It is hypothesized here that (i) orchids would excrete plant molecule signals such as strigolactones and flavonoids but also other secondary metabolites; (ii) in response, OM fungi would secrete mycorrhizal factors (Myc factors) or similar compounds to activate the common symbiosis genes (CSGs); (iii) overcome the defense mechanism by evasion of the pathogen-associated molecular patterns (PAMPs)-triggered immunity and by secretion of effectors such as small inhibitor proteins; and (iv) finally, secrete phytohormones to help the colonization or disrupt the crosstalk of plant defense phytohormones. To challenge this putative model, targeted and untargeted metabolomics studies with special attention to each partner’s contribution are finally encouraged and some technical approaches are proposed.
... This could allow the testing of compounds in larvae at an earlier stage and on a larger scale than would be possible if mammals were used. This approach has already been used to identify novel antimicrobials [53][54][55][56][57][58][59][60][61][62][63][64][65][66][67][68][69][70]. ...
Article
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In the past decade, Galleria mellonella (wax moth) larvae have become widely used as a non-mammalian infection model. However, the full potential of this infection model has yet to be realised, limited by the variable quality of larvae used and the lack of standardised procedures. Here, we review larvae suitable for research, protocols for dosing larvae, and methods for scoring illness in larvae infected with fungal pathogens. The development of standardised protocols for carrying out our experimental work will allow high throughput screens to be developed, changing the way in which we evaluate panels of mutants and strains. It will also enable the in vivo screening of potential antimicrobials at an earlier stage in the research and development cycle.
... Concerning G. mellonella, the efficacy of antifungal drugs show a good relation between in vitro test and in vivo treatment of the larvae infected with C. albicans, C. krusei and C. tropicalis Scorzoni et al., 2013). The efficacy of anthraquinones from a Rubiaceae plant, Morinda tomentosa, against C. albicans was proven in the G. mellonella model, however these compounds did not show significant antifungal activity in the model (Favre-Godal et al., 2014). ...
Article
In the last decades, the increased number of immunocompromised patients has led to the emergence of many forms of fungal infections. Furthermore, there are a restricted arsenal of antifungal available and an increase in the development of resistance to antifungal drugs. Because of these disadvantages, the search for new antifungal agents in natural sources has increased. The development of these new antifungal drugs involves various steps and methodologies. The evaluation of the in vitro antifungal activity and cytotoxicity are the first steps in the screening. There is also the possibility of antifungal combinations to improve the therapy and reduce toxicity. Despite that, the application of the new antifungal candidate could be used in association with photodynamic therapy or using nanotechnology as an ally. In vivo tests can be performed to evaluate the efficacy and toxicity using conventional and alternative animal models. In this work, we review the methods available for the evaluation of the antifungal activity and safety of natural products, as well as the recent advances of new technology in the application of natural products for antifungal therapy.
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Invasive fungal infections have dramatically increased over the last 20 years. They became a major cause of nosocomial infections in developed countries making urgent the need of new antifungal drugs [1]. In this context, NPs have a high potential to become interesting leads for drug discovery. In our search for new antifungal compounds, four South American plants extracts were selected based on their in vitro antifungal activity. These extracts presented interesting activity against Candida albicans in a bioautography assay using hypersusceptible engineered strain [2]. This mutant strain was used in order to isolate the minor active constituent which would not have been detected otherwise. Bioassay-guided microfractionation was undertaken using HPLC to localize the active compounds. Different zones of the HPLC-UV chromatogram were linked to antifungal activities. In parallel to this HPLC-based activity profiling, UHPLC-TOF-HRMS was used for the early identification of some of the compounds present. The targeted isolation of the active compounds was performed by MPLC-UV and further semi-preparative HPLC steps. Structures of the isolated compounds were elucidated by spectroscopic methods including UV, NMR, MS and HRMS. Their absolute configuration was elucidated by ECD. Some of them were new NPs. Belong the fourteen isolated compounds, three saponins, two terpenes and two anthraquinones were specifically active against Candida spp with MIC below 32 µg/mL. The antifungal properties were also evaluated against biofilms of C. albicans and in vivo activity was assessed using the Galleria mellonella model. References: [1] Ostrosky-Zeichner L, Casadevall A, Galgiani J. N, Odds F. C, Rex J. H. An insight into the antifungal pipeline: selected new molecules and beyond. Nat Rev Drug Discov 2010; 9, 719 – 727.
Article
A new anthraquinone, morinquinone, together with 18 known anthraquinones were isolated from the stems of Morinda elliptica Ridl. Their structures were elucidated on the basis of spectroscopic data. They each showed weak inhibitory activity against a susceptible strain of Staphylococcus aureus and a methicillin-resistant S. aureus. Damnacanthal was effective against Microsporum gypseum (MIC 1 μg/mL). Lucidin was active against Entamoeba histolytica (MIC 31.25 μg/mL) and Giardia intestinalis (MIC 7.8 μg/mL).
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Aim: Mouse-rat models have been used for many years in studying the pathogenesis of infectious diseases. Using Galleria mellonella larvae was accepted in the literature as a model host for many microorganisms that are important for medical reseach. In this study, the secretory acid proteinase (SAP) enzyme activity of Candida albicans was investigated on the G. mellonella larvae as a model host. Material and Method: Two clinical C. albicans strains were used for the infection model, which had positive and negative SAP enzyme activity determined by bovine serum albumin agar method. Healthy G. mellonella larvae of 2-3 cm length and 250- 300 mg weight in the last larval stage, were divided into four groups; control, sham (phosphate buffer), SAP positive and SAP negative (105 CFU/ml) C. albicans-infected group. Larvae were left at 37oC. Their health conditions were scored after 96 hours and they were sacrificed. Fungal burden was studied. Results: A significant difference was found between the groups when evaluated in terms of activity, survival, melanization and fungal load of larvae. The control and sham groups showed no death and melanization, no pathogen growth was detected at the end of 96 hours. Fungal burden, melanization and total health score showed significant difference between the SAP positive and SAP negative groups (p<0.05). Conclusion: In our study, the role of the SAP enzyme activity of C. albicans on the virulence was demonstrated in G. mellonella larvae in vivo model. G. mellonella larvae model can be used as a reliable, easily applicable and an inexpensive method in investigating the effects of fungal infections and virulence factors such as SAP on host cells. Widespread use of the larvae model as an alternative to mammalian models will also contribute to reducing ethical concerns.
Article
Twenty-three monoketone derivatives of curcumin were synthesized to investigate the synergy with fluconazole against fluconazole-resistant Candida spp. The minimal inhibitory concentration (MIC80) and the fractional inhibitory concentration index (FICI) of the antifungal synergist fluconazole were measured against fluconazole-resistant C. albicans, C. tropicalis and C. krusei in vitro. Most of these compounds showed good synergistic activities against C. tropicalis. Among them, compound 9 exhibited significant synergistic activities against Candida spp. SARs were also discussed. In particular, a cell growth test exhibited that a combination of 1 μg ml−1 fluconazole and 64 μg ml−1 or 128 μg ml−1 compound 9 showed the most potent fungicidal effect against C. tropicalis. The synergistic effect may be associated with the changes of the intracellular ATP content and cell membrane permeability. Our results provided a basis for future evaluation and development of these compounds as leads for therapeutics for fluconazole-resistant candidiasis.
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Two new prenylated ortho-dihydroxycoumarins, designated fipsomin (1) and fipsotwin (2), were isolated from the fruits of Ficus nipponica together with a known prenylated coumarin, apigravin (3). Their structures were established by spectroscopic data and X-ray crystallographic analysis. Compound 1 exhibited antibacterial activity against Bacillus subtilis with an MIC value of 61 μM, while 2 and 3 showed very weak activity. This article is protected by copyright. All rights reserved.
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A new bibenzyl derivative (4), together with two glycosylated flavonoids (1 and 2), batatasin III (3) and the phenanthrene isohircinol (5) were isolated from the aerial parts of Gavilea lutea. Their structures were elucidated on the basis of spectroscopic studies including 1D and 2D NMR, UV, IR and HRESIMS. All isolated compounds were evaluated for their antifungal activity towards Candida albicans. The new compound 4 showed inhibitory activity with a MIQ of 50 μg. In addition, compound 4 exhibited a selective activity (IC50 = 2.3 μg/mL) against Leishmania donovani.
Article
Aim: This work aimed to evaluate the activity of 3'-hydroxychalcone against Cryptococcus gattii in planktonic and biofilm forms and their toxicity using alternative animal models. Materials & methods: Minimum inhibitory concentration and minimum fungicide concentration were determined. Biofilm formation and the susceptibility tests were performed by the 2,3-bis-(2-methoxy-4-nitro-5-sulfophenyl)-5-[carbonyl(phenylamino)]-2H-tetrazolium hydroxide assay. Toxicity and efficacy were checked in Danio rerio and Galleria mellonella models. Results: The compound 3'-hydroxychalcone showed fungicidal activity against C. gattii in both planktonic and biofilm forms. The toxicity in zebrafish embryos revealed a low lethal concentration. In G. mellonella, the compound did not show antifungal activity and larvae toxicity. Conclusion: Because of the activity of 3'-hydroxychalcone against C. gattii in vitro, molecular modifications should be made to improve efficacy and to reduce toxicity in vivo.
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Significance and impact of the study: The failure to respond to antifungal therapy is complex and is associated with microbiological resistance and increased expression of virulence in fungal pathogens. Thus, this review offers an overview of current challenges in the treatment of fungal infections associated with increased antifungal drug resistance and the formation of biofilms in these opportunistic pathogens. Furthermore, the most recent and potential strategies to combat fungal pathogens are explored here, focusing on new agents as well as innovative approaches, such as combination therapy between antifungal drugs or with natural compounds.
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Unlike superficial fungal infections of the skin and nails, which are the most common fungal diseases in humans, invasive fungal infections carry high morbidity and mortality, particularly those associated with biofilm formation on indwelling medical devices. Therapeutic management of these complex diseases is often complicated by the rise in resistance to the commonly used antifungal agents. Therefore, the availability of accurate susceptibility testing methods for determining antifungal resistance, as well as discovery of novel antifungal and antibiofilm agents, are key priorities in medical mycology research. To direct advancements in this field, here we present an overview of the methods currently available for determining (i) the susceptibility or resistance of fungal isolates or biofilms to antifungal or antibiofilm compounds and compound combinations; (ii) the in vivo efficacy of antifungal and antibiofilm compounds and compound combinations; and (iii) the in vitro and in vivo performance of anti-infective coatings and materials to prevent fungal biofilm-based infections.
Book
Climate change affects the distribution of fungal communities, provoking outbreaks in locations where these mycoses were absent or in low frequencies. Moreover, the reports of clinical cases related to new fungal pathogens have increased due to the description of new fungal species or due to the ability of some species to shift to new hosts. This book, is a contribution to the knowledge of a global environmental phenomenon and its relation to these diseases, and it serves as a guide for health professionals to dive deep into the repercussions of climate change and how they can implement measures for the prevention and control of fungal infections.
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This paper reviews the use of TLC-bioautography in the search for antifungal compounds from natural sources. The main methods used for antifungal screening are presented, with special emphasis on bioautography. Different aspects of the technique, including the latest chromatographic developments such as HPTLC and HPLC microfractionation are presented. The present status and recent advances made in antifungal bioautography are discussed, and a comprehensive review of the applications over the last 6 years is presented. Various strategies applied in the search for antifungal compounds from natural sources are discussed, with a highlight on the challenges faced when screening complex crude mixtures. The activities of approximately 100 antifungal compounds of natural origin are presented with their minimum inhibitory quantity. The most active natural source compounds against Candida, Cladosporium, Colletotrichum, and Fusarium species are highlighted, and the compound activities discussed. In addition, perspectives concerning future improvements in bioautography sensitivity and reproducibility are noted.
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As a result of the strong structural and functional similarities between the innate immune system of mammals and the insect immune response, insects have been exploited for evaluating the virulence of fungal pathogens of humans and for assessing the efficacy of anti-fungal agents. There is a strong correlation between the results obtained using insects and mammals and insects have the added advantage of being cheap to purchase, give results in 24–48h and are without the ethical and legal restrictions associated with the use of mammals. Larvae of Galleria mellonella are excellent in vivo models and have been used with a variety of fungi and anti-fungal agents. Factors affecting the use of G. mellonella larvae are described and examples of where these larvae have been utilized are discussed.
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Pentas micrantha is used in the East African indigenous medicine to treat malaria. In the first investigation of this plant, the crude methanol root extract showed moderate antiplasmodial activity against the W2-(3.37 μg/mL) and D6-strains (4.00 μg/mL) of Plasmodium falciparum and low cytotoxicity (>450 μg/mL, MCF-7 cell line). Chromatographic separation of the extract yielded nine anthraquinones, of which 5,6-dihydroxylucidin-11-O-methyl ether is new. Isolation of a munjistin derivative from the genus Pentas is reported here for the first time. The isolated constituents were identified by NMR and mass spectrometric techniques and showed low antiplasmodial activities.
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Bioactivity-guided fractionation led to the successful isolation of antiosteoporotic components, i.e. physicion (1), rubiadin-1-methyl ether (2), 2-hydroxy-1-methoxy- anthraquinone (3), 1,2-dihydroxy-3-methylanthraquinone (4), 1,3,8-trihydroxy-2-methoxy- anthraquinone (5), 2-hydroxymethyl-3-hydroxyanthraquinone (6), 2-methoxyanthraquinone (7) and scopoletin (8) from an ethanolic extract of the roots of Morinda officinalis. Compounds 4-8 are isolated for the first time from M. officinalis. Among them, compounds 2 and 3 promoted osteoblast proliferation, while compounds 4, 5 increased osteoblast ALP activity. All of the isolated compounds inhibited osteoclast TRAP activity and bone resorption, and the inhibitory effects on osteoclastic bone resorption of compounds 1 and 5 were stronger than that of other compounds. Taken together, antiosteoporotic activity of M. officinalis and its anthraquinones suggest therapeutic potential against osteoporosis.
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Candida albicans is a dimorphic human pathogen in which the yeast to hyphal switch may be an important factor in virulence in mammals. This pathogen has recently been shown to also kill insects such as the Greater Wax Moth Galleria mellonella when injected into the haemocoel of the insect larvae. We have investigated the effect of previously characterised C. albicans mutations that influence the yeast to hyphal transition on virulence in G. mellonella larvae. There is a good correlation between the virulence of these mutants in the insect host and the virulence measured through systemic infection of mice. Although the predominant cellular species detected in G. mellonella infections is the yeast form of C. albicans, mutations that influence the hyphal transition also reduce pathogenicity in the insect. The correlation with virulence measured in the mouse infection system suggests that Galleria may provide a convenient and inexpensive model for the in vivo screening of mutants of C. albicans.
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Phytochemical investigation of the CH 2 Cl 2 extract of Fagara zanthoxyloides Lam. (Rutaceae) led to the isolation of eleven compounds. One phenylethanoid derivative is a new natural product. The isolation of the antifungal and the antioxidant compounds was monitored by direct TLC bioautographic assays. The structures of the isolated compounds were elucidated by classical spectroscopic methods including UV, NMR, MS and HR-MS.
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In the course of a study of medicinal plants from Mali, the root bark of Zanthoxylum zanthoxyloides Lam. (Rutaceae) was investigated. The root bark of this plant is used as a toothbrush by West African people. Phytochemical investigation of the MeOH extract of Zanthoxylum zanthoxyloides Lam. (Rutaceae) led to the isolation of different biologically active compounds. The separation of acetylcholinesterase inhibitors was performed by centrifugal partition chromatography (CPC). The fractions were monitored by direct TLC bioautographic assays. The bio-guided isolation led to the isolation of a new peroxide derivative. In addition, LC/UV/MS analysis performed on the crude MeOH extract allowed on-line identification of some known compounds. The structures of the isolated compounds were elucidated by classical spectroscopic methods including UV, NMR, MS and HR-MS.
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Voriconazole (VRC) is a broad-spectrum antifungal triazole with nonlinear pharmacokinetics. The utility of measurement of voriconazole blood levels for optimizing therapy is a matter of debate. Available high-performance liquid chromatography (HPLC) and bioassay methods are technically complex, time-consuming, or have a narrow analytical range. Objectives of the present study were to develop new, simple analytical methods and to assess variability of voriconazole blood levels in patients with invasive mycoses. Acetonitrile precipitation, reverse-phase separation, and UV detection were used for HPLC. A voriconazole-hypersusceptible Candida albicans mutant lacking multidrug efflux transporters (cdr1Delta/cdr1Delta, cdr2Delta/cdr2Delta, flu1Delta/flu1Delta, and mdr1Delta/mdr1Delta) and calcineurin subunit A (cnaDelta/cnaDelta) was used for bioassay. Mean intra-/interrun accuracies over the VRC concentration range from 0.25 to 16 mg/liter were 93.7% +/- 5.0%/96.5% +/- 2.4% (HPLC) and 94.9% +/- 6.1%/94.7% +/- 3.3% (bioassay). Mean intra-/interrun coefficients of variation were 5.2% +/- 1.5%/5.4% +/- 0.9% and 6.5% +/- 2.5%/4.0% +/- 1.6% for HPLC and bioassay, respectively. The coefficient of concordance between HPLC and bioassay was 0.96. Sequential measurements in 10 patients with invasive mycoses showed important inter- and intraindividual variations of estimated voriconazole area under the concentration-time curve (AUC): median, 43.9 mg x h/liter (range, 12.9 to 71.1) on the first and 27.4 mg x h/liter (range, 2.9 to 93.1) on the last day of therapy. During therapy, AUC decreased in five patients, increased in three, and remained unchanged in two. A toxic encephalopathy probably related to the increase of the VRC AUC (from 71.1 to 93.1 mg x h/liter) was observed. The VRC AUC decreased (from 12.9 to 2.9 mg x h/liter) in a patient with persistent signs of invasive aspergillosis. These preliminary observations suggest that voriconazole over- or underexposure resulting from variability of blood levels might have clinical implications. Simple HPLC and bioassay methods offer new tools for monitoring voriconazole therapy.
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The insecticidal activity of the endemic species Galium melanantherum was evaluated against Crematogaster scutellaris ants and Kalotermes flavicollis termites. Iridoid glucosides 1-7 were isolated for the first time as metabolites of the investigated plant, along with the coumarin scopolin. The main components of the extract were found to be the non-acetylated iridoids: geniposidic acid (1), 10-hydroxyloganin (2), deacetyldaphylloside (3), monotropein (4), deacetylasperulosidic acid (5) and scandoside (6), while asperulosidic acid (7) was present only in minute quantities. All isolated metabolites were identified on the basis of their spectral data. Laboratory bioassays revealed significant levels of toxicity for 1-4 against Kalotermes flavicollis termites and Crematogaster scutellaris ants.
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From Radix Rubiae (dried roots of Rubia cordifolia), mollugin, 1-hydroxy-2-methyl-9, 10-anthraquinone, alizarin, 1, 3-dihydroxy-2-ethoxymethyl-9, 10-anthraquinone, lucidin primeveroside, ruberythric acid and three new anthraquinones, 2-methyl-1, 3, 6-trihydroxy-9, 10-anthraquinone, 2-methyl-1, 3, 6-trihydroxy-9, 10-anthraquinone 3-O-(6'-O-acetyl)-α-rhamnosyl-(1→2)-β-glucoside and 2-methyl-1, 3, 6-trihydroxy-9, 10-anthraquinone 3-O-α-rhamnosyl (1→2)-β-glucoside, were isolated. The new anthraquinone glycosides were also obtained from the roots of R. akane, as the main chemical constituents of anthraquinone glycosides. The structures were established by various chemical and spectroscopic methods.
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A new anthraquinone glycoside was isolated from the hydrophilic fraction of the Chinese medicinal plant Rhynchotechum vestitum. Its structure was determined as damnacanthol-11-O-β-glucoside by spectroscopic evidence. The occurrence of rubiadin-l-methylether-3-O-β-primeveroside, lucidine-3-O-β-primeveroside and rubiadin-3-O-β-primeveroside in the plant is reported for the first time.
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Four naphthohydroquinones and their glycosides, and 11 anthraquinones and their glycosides were isolated from the dried roots of Rubia cordifolia var. pratensis. Six of them, dihydromollugin, 2-carbomethoxy-3-(3′- hydroxy)isopentyl-1,4-naphthohydroquinone 4-O-β-glucoside, 2-methyl-1,3,6-trihydroxy-9,10-anthraquinone 3-O-β-glucoside, 2-methyl-1,3,6-trihydroxy-9,10-anthraquinone 3-O-(3′-O-acetyl)-α-rhamnosyl (1→2)-β-glucoside, 2-methyl- 1,3,6-trihydroxy-9,10-anthraquinone 3-O-(3′,6′-O-diacetyl)-α-rhamnosyl(1→2)-β-glucoside and 2-methyl-1,3,6-tri- hydroxy-9,10-anthraquinone 3-O-(4′,6′-O-diacetyl)-α-rhamnosyl(1→2)-β-glucoside, were isolated for the first time from a natural source. The structures were established by various chemical and spectroscopic methods. The chemotaxonomic significance of these findings is discussed briefly.
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Antifungal testing of filamentous fungi generally suffers from incomparability of results. In order to illustrate this problem, the polyacetylene falcarindiol and the naphthoquinone juglone, two known antifungal natural products, were assayed in various dilution and diffusion bioassays against three selected microfungi, Botrytis cinerea, Cladosporium herbarum and Fusarium avenaceum. Broth microdilution, based on the 96-well microtitre plate format, can be scored by various direct and biochemical methods. As examples, direct observation with image analysis and the fluorescein diacetate scoring method are compared. Of the other methodologies used, results obtained by thin-layer bioautography and the radial growth rate method clearly deviated. Disk diffusion results, however, matched microdilution. In conclusion, microdilution offers the greatest potential of all bioassays to become the future general standard methodology. Copyright © 2000 John Wiley & Sons, Ltd.
Article
TEST PROCEDURES Test procedures are similar to those published in the document entitled “Method for the determination of minimum inhibitory concentration (MIC) by broth dilution of fermentative yeasts” [¹⁰]. The medium recommended is RPMI 1640 supplemented with glucose to a final concentration of 2%. The preparation of stock and working solutions of antifungal agents and the preparation and storage of microdilution plates is identical to that described in the method for fermentative yeasts [¹⁰]. However, inoculum preparation is performed by counting spores in a haemocytometer chamber instead of adjusting the optical density of the culture using a spectrophotometer as this would require separate standardization for each species to compensate for differences in the size and colour of the spores [¹¹, ¹² and ¹³]. In addition, the endpoints are read visually by recording the degree of growth for each well using a viewing mirror. Two different endpoints are obtained, the MIC and the minimum effective concentration (MEC). The MIC is recorded for polyenes, azoles and terbinafine whereas the MEC is reserved for the echinocandins - caspofungin, micafungin and anidulafungin. The MIC is defined as the lowest concentration of drug that yields no growth whereas the MEC is the lowest concentration of drug that results in macroscopic changes in filamentous growth to microcolonies or granular growth when compared with growth control wells. Reading the MEC requires a degree of expertise which can be acquired by examining under the microscope a small volume removed from each of the wells of the microdilution plate.
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Seven known iridoid glycosides (1∼7), together with one known diterpenoid (8) were isolated from the aerial parts of Borreria latifolia K. Schum. (Rubiaceae) collected in Indonesia, and their structures were identified by spectroscopic methods.
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The first echinocandin-type antimycotic (echinocandin B) was discovered in the 1970s. It was followed by the isolation of more than 20 natural echinocandins. These cyclic lipo-hexapeptides are biosynthesized on non-ribosomal peptide synthase complexes by different ascomycota fungi. They have a unique mechanism of action; as non-competitive inhibitors of β-1,3-glucan synthase complex they target the fungal cell wall. Results of the structure-activity relationship experiments let us develop semisynthetic derivatives with improved properties. Three cyclic lipohiexapeptides (caspofungin, micafungin and anidulafungin) are currently approved for use in clinics. As they show good fungicidal (Candida spp.) or fungistatic (Aspergillus spp.) activity against the most important human pathogenic fungi including azole-resistant strains, they are an important addition to the antifungal armamentarium. Some evidence of acquired resistance against echinocandins has been detected among Candida glabrata strains in recent years, which enhanced the importance of data collected on the mechanism of acquired resistance developing against the echinocandins. In this review, we show the structural diversity of natural echinocandins, and we summarize the emerging data on their mode of action, biosynthesis and industrial production. Their clinical significance as well as the mechanism of natural and acquired resistance is also discussed.
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Covering: up to 2012Since the advent of high-throughput screening (HTS) in the early 1990s, a wealth of innovative technologies have been proposed and implemented for the effective localization and characterization of bioactive constituents in complex matrices. The latest developments in this field are reviewed under the perspective of their applicability to natural product-based drug discovery. The approaches discussed here include TLC-based bioautography, HPLC-based assays with on-line, at-line and off-line detection, as well as affinity-based methods, such as frontal affinity chromatography, pulsed ultrafiltration mass spectrometry, imprinted polymers, and affinity capillary electrophoresis. Selected practical examples are given to illustrate the strengths and limitations of these approaches in contemporary natural product lead discovery. In addition, compatibility issues of natural product extracts and HTS are addressed, and selected protocols for the generation of high quality libraries are presented.
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Background: Nature has been a source of medicinal products for millennia, with many useful drugs developed from plant sources. Following discovery of the penicillins, drug discovery from microbial sources occurred and diving techniques in the 1970s opened the seas. Combinatorial chemistry (late 1980s), shifted the focus of drug discovery efforts from Nature to the laboratory bench. Scope of review: This review traces natural products drug discovery, outlining important drugs from natural sources that revolutionized treatment of serious diseases. It is clear Nature will continue to be a major source of new structural leads, and effective drug development depends on multidisciplinary collaborations. Major conclusions: The explosion of genetic information led not only to novel screens, but the genetic techniques permitted the implementation of combinatorial biosynthetic technology and genome mining. The knowledge gained has allowed unknown molecules to be identified. These novel bioactive structures can be optimized by using combinatorial chemistry generating new drug candidates for many diseases. General significance: The advent of genetic techniques that permitted the isolation / expression of biosynthetic cassettes from microbes may well be the new frontier for natural products lead discovery. It is now apparent that biodiversity may be much greater in those organisms. The numbers of potential species involved in the microbial world are many orders of magnitude greater than those of plants and multi-celled animals. Coupling these numbers to the number of currently unexpressed biosynthetic clusters now identified (>10 per species) the potential of microbial diversity remains essentially untapped.
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Abstract Bioactivity guided fractionation of a dichloromethane extract of Morinda lucida Benth. (Rubiaceae) roots has led to the isolation often anthraquinones. Four of these were active against Cladosporium cucumerinum and Candida albicans in bioautographic (TLC) assays. In-depth investigation of activity against three human pathogenic fungi showed that alizarin-1-methyl ether displayed the highest activity.
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In spite of their decreased polarity with respect to previously studied electron-rich analogues, monooxygenated dienes also react regiospecifically with halo quinones. The corresponding adducts can easily be aromatized on silica gel to isomeric polysubstituted naphthoquinones of unambiguous structure and therefore provide ready access to substrates for subsequent regiospecific annulations. The scope of this approach is illustrated by advantageous syntheses of several natural products: chimaphilin, 6-methylalizarin, 6-methylxanthopurpurin, and barleriaquinone. The adducts can also give rise to a series of products in which the oxygen function of the dienes is preserved as a hydroxyl group in the quinone. To this end adducts derived from 1-oxygenated dienes and halo quinones were oxidized effectively with Jones' reagent while those obtained from the 2-oxygenated isomers responded better to manganese dioxide. Relative positions of substituents in the adducts were readily confirmed by comparison of some of the hydroxylated oxidation products with known compounds of unambiguous structure. The method is again illustrated by the ready synthesis of a number of natural products including plumbagin, soranjidiol, isochrysophanol and its 8-methyl ether, and isozyganein and its 5-methyl ether.
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A new `quinone methide' diterpene with a cassane skeleton was isolated from the root bark of Bobgunnia madagascariensis (Desv.) J.H. Kirkbr. et Wiersema (Leguminosae). Its structure was established as (4bS,8aS)-4b,5,6,7,8,8a-hexahydro-4-hydroxy-2-(2-hydroxyethyl)-1,4b,8,8-tetramethylphenanthrene-3,9-dione by spectroscopic methods including single-crystal X-ray analysis. This compound showed strong antifungal properties towards human pathogenic fungi, in particular the yeast Candida albicans.
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The development of medicinal plant research over the last 30 years is reviewed with reference to the search for new active principles. Difficulties inherent to activity guided isolation and the specific requirements of bioassays are discussed. An overview is given on currently used systems for various bioactivities, with emphasis on simple bioassays for phytochemical laboratories. The progress in medicinal plant research is illustrated by selected examples of plant derived compounds of importance as drugs or pharmacological tools.
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This review is focused on planar chromatography and especially on its most important subcategory high-performance thin-layer chromatography (HPTLC). The image-giving format of the open, planar stationary phase and the post-chromatographic evaporation of the mobile phase ease the performance of various kinds of hyphenations and even super-hyphenations. Examples in the field of natural product search, food and lipid analysis are demonstrated, which point out the hyphenation with effect-directed analysis (EDA) and mass spectrometry and illustrate the efficiency gain. Depending on the task at hand, hyphenations can readily be selected as required to reach the relevant information about the sample, and at the same time, information is obtained for many samples in parallel. The flexibility and the unrivalled features through the planar format valuably assist separation scientists.
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Two new benzoquinones, heliotropinones A and B, have been isolated from the aerial parts of Heliotropium ovalifolium. Their structures were elucidated by spectrometric methods including high resolution electrospray ionization (ESI-HR), EI mass spectrometry, 1H, 13C and 2D NMR experiments. The two quinones demonstrated antifungal activities against Cladosporium cucumerinum and Candida albicans as well as antibacterial activity against Bacillus subtilis.