Penicillic acid in fruits: method development, validation by liquid chromatography-tandem mass spectrometry and survey in southern China.

BACKGROUND: Penicillic acid (PA) is produced by Aspergillus spp. and Penicillium spp., which are common postharvest and storage fungi of fruits. PA can be of concern for human health because of its toxicity and high fruit consumption by the population. However, no data on PA occurrence in various fruits have yet been reported. A quick, easy, cheap, effective, rugged and safe (QuEChERS) approach for PA determination in various fruits was developed and applied to explore PA incidence in fruits. RESULTS: The modified QuEChERS procedure with extraction by ethyl acetate and purification by multi-walled carbon nanotubes (MWCNTs), primary secondary amine (PSA) and octadecyl silane (C18) was established to determine PA in various fruits by high-performance liquid chromatography-tandem mass spectrometry (HPLC-MS/MS). The average recoveries were 72.9-102.2% and relative standard deviations (RSDs) were 1.3-7.9%. A total of 161 fruits samples, including kiwi, apple, peach, grape and mandarin/orange, were collected in southern China. The incidence of PA in fruits was 14.9% and the levels of PA contamination were 0.200-0.596 µg kg-1 . Our results suggested that orange/mandarin, grape and kiwi were favorable matrices for Aspergillus spp. and Penicillium spp. to produce PA, rather than peach and apple. CONCLUSION: To the best of our knowledge, this is the first report about PA contamination in various fruits in China. Our study emphasizes the necessity of the current established method, which could be used for continuous monitoring of PA and reducing the health risk to Chinese consumers. © 2020 Society of Chemical Industry.

Human pancreatic cancer cells under nutrient deprivation are vulnerable to redox system inhibition.

Large regions in tumor tissues, particularly pancreatic cancer, are hypoxic and nutrient-deprived because of unregulated cell growth and insufficient vascular supply. Certain cancer cells, such as those inside a tumor, can tolerate these severe conditions and survive for prolonged periods. We hypothesized that small molecular agents, which can preferentially reduce cancer cell survival under nutrient-deprived conditions, could function as anticancer drugs. In this study, we constructed a high-throughput screening system to identify such small molecules and screened chemical libraries and microbial culture extracts. We were able to determine that some small molecular compounds, such as penicillic acid, papyracillic acid, and auranofin, exhibit preferential cytotoxicity to human pancreatic cancer cells under nutrient-deprived compared with nutrient-sufficient conditions. Further analysis revealed that these compounds target to redox systems such as GSH and thioredoxin and induce accumulation of reactive oxygen species in nutrient-deprived cancer cells, potentially contributing to apoptosis under nutrient-deprived conditions. Nutrient-deficient cancer cells are often deficient in GSH; thus, they are susceptible to redox system inhibitors. Targeting redox systems might be an attractive therapeutic strategy under nutrient-deprived conditions of the tumor microenvironment.

Cryptic Secondary Metabolites from the Sponge-Associated Fungus Aspergillus ochraceus.

The fungus Aspergillus ochraceus was isolated from the Mediterranean sponge Agelas oroides. The initial fermentation of the fungus on solid rice medium yielded 16 known compounds (4⁻19). The addition of several inorganic salts to the rice medium mainly influenced the accumulation of these secondary metabolites. Fermentation of the fungus on white bean medium yielded the new waspergillamide B (1) featuring an unusual p-nitrobenzoic acid as partial structure. Moreover, two new compounds, ochraspergillic acids A and B (2 and 3), which are both adducts of dihydropenicillic acid and o- or p-aminobenzoic acid, were isolated from the co-culture of the fungus with Bacillus subtilis. Compound 2 was also detected in axenic fungal cultures following the addition of either anthranilic acid or tryptophan to the rice medium. The structures of the new compounds were established by 1D and 2DNMR experiments as well as from the HRMS data. The absolute configuration of 1 was elucidated following hydrolysis and derivatization of the amino acids using Marfey's reagent. Viomellein (9) and ochratoxin B (18) exhibited strong cytotoxicity against the A2780 human ovarian carcinoma cells with IC50 values of 5.0 and 3.0 µM, respectively.

Cytotoxic activity of fungal strains isolated from the ascidian Eudistoma vannamei.

The cytotoxic activity at 50 µg/ml of extracts obtained from eleven fungal strains associated to Eudistoma vannamei, an endemic ascidian from Northeast Brazil, against two cell lines, i.e., the HCT-8 (colon cancer) and the MDA-MB-435 (melanoma) cell lines, was investigated. The most promising extract (EV10) was obtained from a fungus identified as Aspergillus sp. by molecular analysis and was selected for bioassay-guided isolation of its active principals. Large-scale fermentation of EV10 in potato-dextrose broth followed by chromatographic purification of the active extract from the liquid medium allowed the isolation of the isocoumarins mellein, cis-4-hydroxymellein, and trans-4-hydroxymellein, besides penicillic acid. All isolated compounds were tested for their cytotoxicity against the tumor cell lines MDA-MB-435 and HCT-8 and revealed penicillic acid as the only cytotoxic compound (cell growth inhibitions >95%).

Synthesis and characteristics of a novel artificial hapten using the copper mercaptide of penicillenic acid from penicillin G for immunoassay of heavy metal ions.

In this paper, we describe the synthesis of a novel copper ion hapten using the copper mercaptide of penicillenic acid (CMPA) derived from penicillin. Results from tests with immune rabbits indicate that: (i) A new antigen synthesized with CMPA has good stability and is safe for immunizing animals with no toxic phenomena being found in animal experiments; (ii) the immunogenic antigen (CMPA-BSA) can stimulate the immune system to produce specific antibodies with high titrations, up to 150000; and (iii) antibodies in antisera showed higher affinity to OVA-GSH-CuCl than OVA-GSH, which indicates that the antibodies have specific affinity towards copper ions. These results confirm that the novel hapten and relevant antigen for copper ion have been successfully synthesized, giving progress towards an immunoassay for copper ions in environmental and food samples.

First morphomolecular identification of Penicillium griseofulvum and Penicillium aurantiogriseum toxicogenic isolates associated with blue mold on apple.

Postharvest blue mold decay caused by Penicillium spp. is the most important disease of fresh apple fruit in the world, which extend from the field to the store. Two new Penicillium spp. responsible for apple fruit decay were recovered. The morphological and molecular features of Penicillium griseofulvum and Penicillium aurantiogriseum isolated from apple fruits were characterized morphologically and molecularly. Pathogenicity test exhibited that both P. griseofulvum and P. aurantiogriseum were responsible for blue mold decay in storage apple fruits. Lesion diameter indicated that P. aurantiogriseum was more aggressive than P. griseofulvum. All tested isolates were able to synthesize citrinin in addition to patulin. Not all of the isolates belonging to the same species showed the same profile of secondary metabolites. Microsatellite-primed polymerase chain reaction was able to differentiate these isolates at the species level and divided the analyzed isolates into two genetically different groups. Little intraspecific variability was evident. Microsatellite-primed polymerase chain reaction analysis proved to be an objective, rapid, and reliable tool to identify Penicillium spp. involved in blue mold of apple. This is the first report of occurrence of P. griseofulvum and P. aurantiogriseum on imported apple fruits in Saudi Arabia.

Inactivation of GDP-mannose dehydrogenase from Pseudomonas aeruginosa by penicillic acid identifies a critical active site loop.

The pathogenic bacterium Pseudomonas aeruginosa synthesizes alginate as one of a group of virulence factors that are produced during infections. The enzyme GDP-mannose dehydrogenase catalyzes the committed step in alginate biosynthesis. We show here that penicillic acid is an irreversible inactivator of GDP-mannose dehydrogenase. Inactivation occurs with a rate constant of 0.39+/-0.01 mM(-1) min(-1) at pH 8.0, and does not exhibit saturation behavior. Partial protection from inactivation is afforded by GDP-mannose, but not by the other substrate, NAD+. GMP and NAD+ together provide complete protection against inactivation. Analysis by mass spectrometry confirmed that the enzyme is alkylated at multiple cysteine residues by penicillic acid, including Cys 213, Cys 246, and the active site cysteine, Cys 268. However, the pH dependence of the inactivation rate suggested that alkylation of a single cysteine residue is sufficient to inactivate the enzyme. The C268A mutant protein was also susceptible to inactivation by penicillic acid. The presence of NAD+ and GMP provided partial protection of Cys 246 and Cys 268, and almost complete protection of Cys 213. Cys 213 is located on a helix that forms part of the binding pocket for GDP-mannose, and forms a hydrogen bond with Asn 252. Asn 252 is located on a loop that surrounds GDP-mannose. The C213A mutant enzyme exhibits a Vmax that is 1.8-fold greater than the wild-type enzyme, suggesting that the interaction between Cys 213 and Asn 252 helps to hold the loop in place during catalysis, and that opening the loop to release product is partially rate-limiting. Cys 246 is adjacent to the GDP-mannose binding loop, and its alkylation may also interfere with loop movement.

Identity and effects of quorum-sensing inhibitors produced by Penicillium species.

Quorum sensing (QS) communication systems are thought to afford bacteria with a mechanism to strategically cause disease. One example is Pseudomonas aeruginosa, which infects immunocompromised individuals such as cystic fibrosis patients. The authors have previously documented that blockage of the QS systems not only attenuates Ps. aeruginosa but also renders biofilms highly susceptible to treatment with conventional antibiotics. Filamentous fungi produce a battery of secondary metabolites, some of which are already in clinical use as antimicrobial drugs. Fungi coexist with bacteria but lack active immune systems, so instead rely on chemical defence mechanisms. It was speculated that some of these secondary metabolites could interfere with bacterial QS communication. During a screening of 100 extracts from 50 Penicillium species, 33 were found to produce QS inhibitory (QSI) compounds. In two cases, patulin and penicillic acid were identified as being biologically active QSI compounds. Their effect on QS-controlled gene expression in Ps. aeruginosa was verified by DNA microarray transcriptomics. Similar to previously investigated QSI compounds, patulin was found to enhance biofilm susceptibility to tobramycin treatment. Ps. aeruginosa has developed QS-dependent mechanisms that block development of the oxidative burst in PMN neutrophils. Accordingly, when the bacteria were treated with either patulin or penicillic acid, the neutrophils became activated. In a mouse pulmonary infection model, Ps. aeruginosa was more rapidly cleared from the mice that were treated with patulin compared with the placebo group.

Phytotoxins from the fungus Malbranchea aurantiaca.

Bioassay-directed fractionation of an ethyl acetate extract from cultures of the fungus Malbranchea aurantiaca led to the isolation of two phytotoxic compounds, namely, 1-hydroxy-2-oxoeremophil-1(10),7(11),8(9)-trien-12(8)-olide (1) and penicillic acid (2). The structure of 1 was established by spectroscopic and X-ray crystallographic analyses. Metabolites 1 and 2 caused significant inhibition of radicle growth of Amaranthus hypochondriacus with IC(50) values 6.57 and 3.86 microM, respectively. In addition, 1 inhibited activation of the calmodulin-dependent enzyme cAMP phosphodiesterase (IC(50)=10.2 microM).

Cytotoxic and other metabolites of Aspergillus inhabiting the rhizosphere of Sonoran desert plants.

In a study to discover potential anticancer agents from rhizosphere fungi of Sonoran desert plants cytotoxic EtOAc extracts of four Aspergillus strains have been investigated. Two new metabolites, terrequinone A (1) and terrefuranone (2), along with Na-acetyl aszonalemin (LL-S490beta) (3) were isolated from As. terreus occurring in the rhizosphere of Ambrosia ambrosoides, whereas As. terreus inhabiting the rhizosphere of an unidentified Brickellia sp. afforded dehydrocurvularin (4), 11-methoxycurvularin (5), and 11-hydroxycurvularin (6). As. cervinus isolated from the rhizosphere of Anicasanthus thurberi contained two new compounds, 4R*,5S*-dihydroxy-3-methoxy-5-methylcyclohex-2-enone (7) and 6-methoxy-5(6)-dihydropenicillic acid (8), in addition to penicillic acid (9). Penicillic acid was also isolated from As. wentii occurring in the rhizosphere of Larrea tridentata. The structures of 1-9 were elucidated by spectroscopic methods and chemical derivatizations. Acetylation of 2 afforded 14-acetylterrefuranone (13) and 14-deoxy-13(14)-dehydroterrefuranone (14). Metabolites 1-9, the dienone 14, and 5(6)-dihydropenicillic acid (16) were evaluated for cytotoxicity in a panel of four human cancer cell lines and in normal human primary fibroblast cells. Compounds 4 and 5 displayed considerable cytotoxicity, whereas 1, 6, 9, and 14 were found to be moderately active, with 6 and 9 exhibiting selective cytotoxicity against cancer cell lines compared with the normal fibroblast cells.

Three new chlorine containing antibiotics from a marine-derived fungus Aspergillus ostianus collected in Pohnpei.

A marine bacterium Ruegeria atlantica (designated as strain TUF-D) was isolated from a glass plate submerged in the coastal water. Three new chlorine containing compounds (1 to approximately 3), together with penicillic acid (4) were obtained from a marine-derived fungus Aspergillus ostianus strain TUF 01F313 isolated from a marine sponge at Pohnpei as antibacterial components against R. atlantica. The structures of three new antibiotics were determined based on their spectral data as 8-chloro-9-hydroxy-8,9-deoxyasperlactone (1), 9-chloro-8-hydroxy-8,9-deoxyasperlactone (2), and 9-chloro-8-hydroxy-8,9-deoxyaspyrone (3). Compound 1 inhibited the growth of R. atlantica at 5 microg/disc (inhibition zone: 12.7 mm), while 2 and 3 were active at 25 microg/disc (10.1 and 10.5 mm, respectively).

[Studies on chemical constituents of mycelium of fungus Cephalosporium sp. AL031(II)].

Three compounds were isolated from the mycelium of the fungus Cephalosporium sp. AL031 whose metabolites have been proven to possess antifungal and antibacterial activities. Based on the spectral data and elemental analysis, they were identified as 3-hydroxy-ergosta-7,22-dien-6-one(A), penicillic acid(B), 4-hydroxy-3,6-dimethyl-2-pyrone(C). All of these compounds were obtained from the culture medium of this fungus for the first time.