Search Results (22)

The International Agency for Research on Cancer has studied and classified 1045 potential substances. It is therefore important to develop rapid screening methods to identify the mutagenicity of compounds and, further on, the intensity and number of individual mutagenic substances in complex sample mixtures. The current in vitro Ames assay in the microtiter plate format (MPF) uses a pH-sensitive detection as endpoint, however, acidic substances in complex mixtures may interfere the mutagenicity result. Hence, it was transferred to the planar assay format to be more selective for complex mixture testing. The co-culture of Salmonella Typhimurium strains TA98 and TA100 with an optical density of 0.4 at 600 nm was applied on a high-performance thin-layer chromatography silica gel 60 chromatogram and on-surface incubated for 5 h, which period was limited due to zone diffusion. Various positive controls were tested, and 4-nitrochinolin-N-oxide with a limit of detection of 100 ng was established as a positive control. However, due to the shorter incubation time, no mutagenic compounds were detectable or differentiable in the tested perfumes, herbal teas, margarines, and hand creams. This does not mean that the samples are mutagen-free, but it suggests that further improvements to the bioassay are urgently needed to increase the sensitivity and selectivity of the response. Compared to conventional sum value assays, a planar Ames assay performed on the separated and adsorbed sample components advances toxicology research because mutagenic compounds are separated from interfering molecules due to the integrated separation. It thus would provide a more selective detection of mutagens in complex mixtures and allow testing of large sample volumes or concentrated samples without matrix interference. Full article

Seven cyclic depsipeptides, including two new cyclic pentadepsipeptides avenamides A (1) and B (2), were isolated from a plant-derived fungus Fusarium avenaceum W8 by using the bioassay-guided fractionation method. The planar structures were elucidated by using comprehensive spectroscopic analyses, including 1D and 2D NMR, as well as MS/MS spectrometry. The absolute configuration of the amino acid and hydroxy acid residues was confirmed by using the advanced Marfey’s method and chiral HPLC analysis, respectively. Compounds 1–7 were evaluated for their cytotoxic activities against A549 and NCI-H1944 human lung adenocarcinoma cell lines and their antimicrobial activities against Staphylococcus aureus and Saccharomyces cerevisiae. As a result, compounds 1–4 showed moderate cytotoxicity, with IC₅₀ values of 6.52~45.20 µM. Compounds 1 and 3 exhibited significant antimicrobial activities against S. aureus and S. cerevisiae, with an MIC80 of 11.1~30.0 µg/mL. Full article

Biological activities of six under-utilized medicinal leafy vegetable plants indigenous to Africa, i.e., Basella alba, Crassocephalum rubens, Gnetum africanum, Launaea taraxacifolia, Solanecio biafrae, and Solanum macrocarpon, were investigated via two independent techniques. The total phenolic content (TPC) was determined, and six microtiter plate assays were applied after extraction and fractionation. Three were antioxidant in vitro assays, i.e., ferric reducing antioxidant power (FRAP), cupric reduction antioxidant capacity (CUPRAC), and 2,2-diphenyl-1-picrylhydrazyl (DPPH) scavenging, and the others were enzyme (acetylcholinesterase, butyrylcholinesterase, and tyrosinase) inhibition assays. The highest TPC and antioxidant activity from all the methods were obtained from polar and medium polar fractions of C. rubens, S. biafrae, and S. macrocarpon. The highest acetyl- and butyrylcholinesterase inhibition was exhibited by polar fractions of S. biafrae, C. rubens, and L. taraxacifolia, the latter comparable to galantamine. The highest tyrosinase inhibition was observed in the n-butanol fraction of C. rubens and ethyl acetate fraction of S. biafrae. In vitro assay results of the different extracts and fractions were mostly in agreement with the bioactivity profiling via high-performance thin-layer chromatography–multi-imaging–effect-directed analysis, exploiting nine different planar assays. Several separated compounds of the plant extracts showed antioxidant, α-glucosidase, α-amylase, acetyl- and butyrylcholinesterase-inhibiting, Gram-positive/-negative antimicrobial, cytotoxic, and genotoxic activities. A prominent apolar bioactive compound zone was tentatively assigned to fatty acids, in particular linolenic acid, via electrospray ionization high-resolution mass spectrometry. The detected antioxidant, antimicrobial, antidiabetic, anticholinesterase, cytotoxic, and genotoxic potentials of these vegetable plants, in particular C. rubens, S. biafrae, and S. macrocarpon, may validate some of their ethnomedicinal uses. Full article

Two new dipyrroloquinones, namely talaroterreusinones A (1) and B (2), together with four known secondary metabolites, terreusinone A (3), penicillixanthone A (4), isorhodoptilometrin (5), and chrysomutanin (6), were isolated from the solid culture of the endophytic fungus Talaromyces sp. by integrating mass spectrometry-based metabolic profiling and a bioassay-guided method. Their planar structures and stereochemistry were elucidated by comprehensive spectroscopic analysis including NMR and MS. The absolute configuration at C-1″ of terreusinone A (1) was established by applying the modified Mosher’s method. Compounds 1–6 were evaluated for anti-inflammatory activity and cytotoxicity. As a result, 1–3 inhibited the LPS-stimulated NO production in macrophage RAW264.7 cells, with IC₅₀ values of 20.3, 30.7, and 20.6 µM, respectively. Penicillixanthone A (4) exhibited potent cytotoxic activity against Hep G2 and A549 cell lines, with IC₅₀ values of 117 nM and 212 nM, respectively, and displayed significant antitumour effects in A549 cells by inhibiting the PI3K-Akt-mTOR signalling pathway. Full article

Five new polyketides, including two chromones (1–2), two phenyl derivatives (4–5), and a tandyukusin derivative (6), along with five known polyketides (3 and 7–10) were isolated from mangrove endophytic fungus Trichoderma lentiforme ML-P8-2. The planar structures of compounds were elucidated via detailed 1D, 2D NMR, and HR-ESI-MS analysis. ECD spectra, optical rotation values calculation, and alkali hydrolysis were applied in the determination of the absolute configuration of the new compounds. In bioassays, 6 and 9 exhibited promising antifungal activities against Penicillium italicum, with an MIC value of 6.25 μM for both compounds. Moreover, 3 displayed moderate AChE inhibitory activity with an IC₅₀ value of 20.6 ± 0.3 μM. Full article

In today’s medicine, the celerity of the bio-assays analysis is crucial for the timely selection of the appropriate therapy and hence its effectiveness, especially in case of diseases characterized by the late onset of symptoms. In this paper, a lens-less fiber optics-based fluorescence sensor designed for the measurement of labeled bio-assays is presented and its potential for the early diagnosis of sepsis via C-reactive protein (CRP) detection is demonstrated. The sensor performance results from the combination of two key elements: a planar antenna that redirects fluorescence the marker emission and an automated fiber-based optical system for multi-spot analysis. First, the working principle of the device is demonstrated with a well-established antibody–antigen format (immunoglobulin IgG/anti-IgG assay), reporting more than one order of magnitude enhanced limit of detection (LOD) and limit of quantification (LOQ) for the planar antenna with respect to a standard glass substrate. The prototype is then tested against a sample mimicking a realistic case, prepared with commercially available human serum, showing a LOD and LOQ in the clinical range of interest (0.0015 μg/mL and 0.005 μg/mL, respectively) for the investigation of the sepsis biomarker CRP. These results validate the developed prototype as a simple and easy-to-operate device, compatible with standardized micro-well arrays, and potentially suitable for POC applications. Full article

A novel compound (1) along with two known compounds (2 and 3) were isolated from the culture broth of Chlorophyllum molybdites, and three known compounds (4–6) were isolated from its fruiting bodies. The planar structure of 1 was determined by the interpretation of spectroscopic data. By comparing the specific rotation of the compound with that of the analog compound, the absolute configuration of 1 was determined to be R. This is the first time that compounds 2–4 were isolated from a mushroom-forming fungus. Compound 2 showed significant inhibition activity against Axl and immune checkpoints (PD-L1, PD-L2). In the bioassay to examine growth inhibitory activity against the phytopathogenic bacteria Peptobacterium carotovorum, Clavibacter michiganensis and Burkholderia glumae, compounds 2 and 3 inhibited the growth of P. carotovorum and C. michiganensis. In the bioassay to examine plant growth regulatory activity, compounds 1–4 showed a significant regulatory activity on lettuce growth. Full article

During the development of novel, standardized peppermint extracts targeting functional applications, it is critical to adequately characterize raw material plant sources to assure quality and consistency of the end-product. This study aimed to characterize existing and proprietary, newly bred varieties of peppermint and their corresponding aqueous extract products. Taxonomy was confirmed through genetic authenticity assessment. Non-target effect-directed profiling was developed using high-performance thin-layer chromatography–multi-imaging–effect-directed assays (HPTLC–UV/Vis/FLD–EDA). Results demonstrated substantial differences in compounds associated with functional attributes, notably antioxidant potential, between the peppermint samples. Further chemical analysis by high-performance liquid chromatography–photodiode array/mass spectrometry detection (HPLC–PDA/MS) and headspace solid-phase microextraction–gas chromatography–flame ionization/MS detection (headspace SPME–GC–FID/MS) confirmed compositional differences. A broad variability in the contents of flavonoids and volatiles was observed. The peppermint samples were further screened for their antioxidant potential using the Caenorhabditis elegans model, and the results indicated concordance with observed content differences of the identified functional compounds. These results documented variability among raw materials of peppermint leaves, which can yield highly variable extract products that may result in differing effects on functional targets in vivo. Hence, product standardization via effect-directed profiles is proposed as an appropriate tool. Full article

Planar chromatography has recently been combined with six different effect-directed assays for three golden root (Rhodiola rosea L.) samples. However, the profiles obtained showed an intense tailing, making zone differentiation impossible. The profiling was therefore improved to allow for the detection of individual bioactive compounds, and the range of samples was extended to 15 commercial golden root products. Further effect-directed assays were studied providing information on 15 different effect mechanisms, i.e., (1) tyrosinase, (2) acetylcholinesterase, (3) butyrylcholinesterase, (4) β-glucuronidase, and (5) α-amylase inhibition, as well as endocrine activity via the triplex planar yeast antagonist-verified (6–8) estrogen or (9–11) androgen screen, (12) genotoxicity via the planar SOS-Umu-C bioassay, antimicrobial activity against (13) Gram-negative Aliivibrio fischeri and (14) Gram-positive Bacillus subtilis bacteria, and (15) antioxidative activity (DPPH• radical scavengers). Most of the golden root profiles obtained were characteristic, but some samples differed substantially. The United States Pharmacopeia reference product showed medium activity in most of the assays. The six most active compound zones were further characterized using high-resolution mass spectrometry, and the mass signals obtained were tentatively assigned to molecular formulae. In addition to confirming the known activities, this study is the first to report that golden root constituents inhibit butyrylcholinesterase (rosin was tentatively assigned), β-glucuronidase (rosavin, rosarin, rosiridin, viridoside, and salidroside were tentatively assigned), and α-amylase (stearic acid and palmitic acid were tentatively assigned) and that they are genotoxic (hydroquinone was tentatively assigned) and are both agonistic and antagonistic endocrine active. Full article

The safety evaluation of food contact materials requires excluding mutagenicity and genotoxicity in migrates. Testing the migrates using in vitro bioassays has been proposed to address this challenge. To be fit for that purpose, bioassays must be capable of detecting very low, safety relevant concentrations of DNA-damaging substances. There is currently no bioassay compatible with such qualifications. High-performance thin-layer chromatography (HPTLC), coupled with the planar SOS Umu-C (p-Umu-C) bioassay, was suggested as a promising rapid test (~6 h) to detect the presence of low levels of mutagens/genotoxins in complex mixtures. The current study aimed at incorporating metabolic activation in this assay and testing it with a set of standard mutagens (4-nitroquinoline-N-oxide, aflatoxin B1, mitomycin C, benzo(a)pyrene, N-ethyl nitrourea, 2-nitrofluorene, 7,12-dimethylbenzanthracene, 2-aminoanthracene and methyl methanesulfonate). An effective bioactivation protocol was developed. All tested mutagens could be detected at low concentrations (0.016 to 230 ng/band, according to substances). The calculated limits of biological detection were found to be up to 1400-fold lower than those obtained with the Ames assay. These limits are lower than the values calculated to ensure a negligeable carcinogenic risk of 10⁻⁵. They are all compatible with the threshold of toxicological concern for chemicals with alerts for mutagenicity (150 ng/person). They cannot be achieved by any other currently available test procedures. The p-Umu-C bioassay may become instrumental in the genotoxicity testing of complex mixtures such as food packaging, foods, and environmental samples. Full article

Algae are unique natural products that can produce various types of biologically active compounds. The 70% ethanol extract of brown algae Sargassum macrocarpum collected from the East Sea of Korea inhibited human monoamine oxidases A and B enzymes (hMAO-A and hMAO-B) at a 50 μg/mL concentration. The bioassay-guided isolation was performed through solid-phase extraction and the Sepbox system followed by serial high-performance liquid chromatography on the reverse phase condition, resulting in the identification of two new monocyclic terpenoid lactones, sargassumins A and B (1 and 2). The planar structures of the compounds were determined by a combination of spectroscopic data. The absolute configurations were determined by the interpretation of circular dichroism data. Compound 1 exhibited mild hMAO-A inhibition (42.18 ± 2.68% at 200 μM) and docked computationally into the active site of hMAO-A (−8.48 kcal/mol). Although compound 2 could not be tested due to insufficient quantity, it docked better into hMAO-A (−9.72 kcal/mol). Therefore, the above results suggest that this type of monocyclic terpenoid lactone could be one of the potential lead compounds for the treatment of psychiatric or neurological diseases. Full article

Endophytic fungi are one of prolific sources of bioactive natural products with potential application in biomedicine and agriculture. In our continuous search for antimicrobial secondary metabolites from Fusarium oxysporum R1 associated with traditional Chinese medicinal plant Rumex madaio Makino using one strain many compounds (OSMAC) strategy, two diastereomeric polyketides neovasifuranones A (3) and B (4) were obtained from its solid rice medium together with N-(2-phenylethyl)acetamide (1), 1-(3-hydroxy-2-methoxyphenyl)-ethanone (2) and 1,2-seco-trypacidin (5). Their planar structures were unambiguously determined using 1D NMR and MS spectroscopy techniques as well as comparison with the literature data. By a combination of the modified Mosher’s reactions and chiroptical methods using time-dependent density functional theory-electronic circular dichroism (TDDFT-ECD) and optical rotatory dispersion (ORD), the absolute configurations of compounds 3 and 4 are firstly confirmed and, respectively, characterized as (4S,7S,8R), (4S,7S,8S). Bioassay results indicate that these metabolites 1–5 exhibit weak inhibitory effect on Helicobacter pylori 159 with MIC values of ≥16 μg/mL. An in-depth discussion for enhancement of fungal metabolite diversity is also proposed in this work. Full article

Eight new cytochalasins 1–8 and ten known analogs 9–18 were isolated from the endophytic fungus Phomopsis sp. xz-18. The planar structures of the cytochalasins were determined by HR-ESI-MS and NMR analysis. Compounds 1, 2, 9 and 10 were 5/6/6/7/5-fused pentacyclic cytochalasins; compounds 3 and 4 had conjugated diene structures in the macrocycle; and compound 6 had a β,γ-unsaturated ketone. The absolute configuration of 6 was confirmed for the first time by the octant rule. The acid-free purification process proved that the pentacyclic system was a natural biosynthetic product and not an acid-mediated intramolecular cyclized artifact. The new compounds did not exhibit activities against human cancer cell lines in cytotoxicity bioassays or antipathogenic fungal activity, but compounds 1, 3 and 4 showed moderate antibacterial activity in disk diffusion assays. Full article

Three new cyanobactins, trikoramides B (1)–D (3), have been isolated from the marine cyanobacterium, Symploca hydnoides, following a preliminary bioassay-guided isolation of the two most active polar fractions based on the brine shrimp toxicity assay. These new cyanobactins are new analogues of the previously reported cytotoxic trikoramide A (4) with differences mainly in the C-prenylated cyclotryptophan unit. Their planar structures were elucidated from their 1D and 2D NMR spectral data in combination with the HRMS/MS data. Marfey’s method, 2D NOESY NMR spectroscopic and ECD spectra analyses were used to determine the absolute stereochemistry of trikoramides B (1)–D (3). Trikoramides B (1) and D (3) exhibited cytotoxicity against MOLT-4 acute lymphoblastic leukemia cell line with IC₅₀ values of 5.2 µM and 4.7 µM, respectively. Compounds 1 and 3 were also evaluated for their quorum-sensing inhibitory assay based on the Pseudomonas aeruginosa PAO1 lasB-gfp and rhlA-gfp bioreporter strains. Although trikoramide B (1) exhibited weak quorum-sensing inhibitory activity, the Br-containing trikoramide D (3) exhibited moderate to significant dose-dependent quorum-sensing inhibitory activities against PAO1 lasB-gpf and rhlA-gfp bioreporter strains with IC₅₀ values of 19.6 µM and 7.3 µM, respectively. Full article

Overexpression of the histone acetyltransferase and the ¹H NMR spectroscopic experiments of the endophytic fungus Monosporascus eutypoides resulted in the isolation of two new compounds, monosporasols A (1) and B (2), and two known compounds, pestaloficin C (3) and arthrinone (4). Their planar structures and absolute configurations were determined by spectroscopic analysis including high resolution electrospray ionization mass spectroscopy (HRESIMS), one-dimensional (1D) and two-dimensional (2D) NMR, and calculated electronic circular dichroism data. Compounds 1–2 were screened in cytotoxic bioassays against HeLa, HCT-8, A549 and MCF-7 cells. Our work highlights the enormous potential of epigenetic manipulation along with the NMR comparison as an effective strategy for unlocking the chemical diversity encoded by fungal genomes. Full article