Penicilloneines A and B, Quinolone-Citrinin Hybrids from a Starfish-Derived Penicillium sp.

Penicilloneines A (1) and B (2) are the first reported quinolone-citrinin hybrids. They were isolated from the starfish-derived fungus Penicillium sp. GGF16-1-2, and their structures were elucidated using spectroscopic, chemical, computational, and single-crystal X-ray diffraction methods. Penicilloneines A (1) and B (2) share a common 4-hydroxy-1-methyl-2(1H)-quinolone unit; however, they differ in terms of citrinin moieties, and these two units are linked via a methylene bridge. Penicilloneines A (1) and B (2) exhibited antifungal activities against Colletotrichum gloeosporioides, with lethal concentration 50 values of 0.02 and 1.51 µg/mL, respectively. A mechanistic study revealed that 1 could inhibit cell growth and promote cell vacuolization and consequent disruption of the fungal cell walls via upregulating nutrient-related hydrolase genes, including putative hydrolase, acetylcholinesterase, glycosyl hydrolase, leucine aminopeptidase, lipase, and beta-galactosidase, and downregulating their synthase genes 3-carboxymuconate cyclase, pyruvate decarboxylase, phosphoketolase, and oxalate decarboxylase.

Penctrimertone, a bioactive citrinin dimer from the endophytic fungus Penicillium sp. T2-11.

Penctrimertone (1), a novel citrinin dimer bearing a 6/6/6/6 tetracyclic ring scaffold, along with two known compounds xerucitrinic acid A (2) and citrinin (3) were isolated from the endophytic fungus Penicillium sp. T2-11. Their structures were unequivocally established by a comprehensive interpretation of the spectroscopic data, with the stereochemistry for 1 was defined by a combination of TDDFT-ECD calculations and the DP4+ probability analysis based on NMR chemical shift calculations. Bioassays revealed that compound 1 exhibited noticeable antimicrobial activities and moderate cytotoxicity. A plausible biosynthetic pathway of 1 was also proposed.

Recent advances in analytical methods for the determination of citrinin in food matrices.

Citrinin is a toxic small organic molecule produced as a secondary metabolite by fungi types Penicillium, Monascus and Aspergillus and is known to contaminate various food commodities during postharvest stages of food production. During the last 10 years, most reported methods for citrinin analysis employed enzyme-linked immunosorbent assays or high-performance liquid chromatography. Over this same time period, liquid extraction, solid-phase extraction, dispersive liquid-liquid microextraction and QuEChERS were the most cited sample preparation and clean-up methods. In this review the advantages and disadvantages of the various sample preparation, separation and detection methods for citrinin analysis over the last decade are evaluated. Furthermore, current trends, emerging technologies and the future prospects of these methods are discussed.

Antitumor effects of citrinin in an animal model of Sarcoma 180 via cytogenetic mechanisms.

Citrinin (CIT) is a cytotoxic, hepatotoxic, nephrotoxic and cardiotoxic metabolite obtained from Penicillium citrinum, that has been increasingly searched as an anticancer drug candidate. In this study, we assessed the antitumor effects of citrinin, using cytogenetic biomarkers for genotoxicity in Sarcoma 180 (S-180) ascitic fluid cells of mice. Citrinin, extracted from P. citrinum acetonitrile extract, was characterized by LC-MS. Cytotoxic assessment was done through using comet (alkaline version) and micronucleus assays. In S-180 cells, CI50 of CIT was 3.77 µg/mL, while at 12.5 and 100 µg/mL, CIT was as cytotoxic as doxorubicin (2 µg/mL). At 0.5, 1.0 and 2.0 µg/mL, it induced genotoxicity and mutagenicity in S-180 cells, especially at 2 µg/mL, triggering oxidative damage similar to hydrogen peroxide (10 mM). The antitumor effects were evidenced by a marked increase in S-180 cells apoptosis and necrosis due to clastogenic and/or aneugenic cytogenetic effects (micronucleus formation), as well as by induction of nucleoplasm bridges and nuclear buds, culminating in S-180 apoptosis and necrosis. CIT has potential as drug candidate for antitumor purposesbyinvolving cytogenetic mechanisms.

A New Citrinin Derivative from the Indonesian Marine Sponge-Associated Fungus Penicillium citrinum.

Sponge-associated fungi are attractive targets for the isolation of bioactive natural products with different pharmaceutical purposes. In this investigation, 20 fungi were isolated from 10 different sponge specimens. One isolate, the fungus Penicillium citrinum strain WK-P9, showed activity against Bacillus subtilis JH642 when cultivated in malt extract medium. One new and three known citrinin derivatives were isolated from the extract of this fungus. The structures were elucidated by 1D and 2D NMR spectroscopy, as well as LC-HRMS. Their antibacterial activity against a set of common human pathogenic bacteria and fungi was tested. Compound 2 showed moderate activity against Mycobacterium smegmatis ATCC607 with a minimum inhibitory concentration (MIC) of 32 µg/mL. Compound 4 exhibited moderate growth inhibition against Bacillus subtilis JH642, B. megaterium DSM32, and M. smegmatis ATCC607 with MICs of 16, 16, and 32 µg/mL, respectively. Furthermore, weak activities of 64 µg/mL against B. subtilis DSM10 and S. aureus ATCC25923 were observed for compound 4.

New citrinin derivatives from the deep-sea-derived fungus Cladosporium sp. SCSIO z015.

During the course of our search for novel bioactive compounds from marine fungi, four new citrinin derivatives, cladosporins A-D (1-4) were isolated from a culture broth of the deep-sea-derived fungus Cladosporium sp. SCSIO z015. Their complete structural assignments were elucidated by the extensive spectroscopic investigation. The absolute configurations of 1-3 were established by quantum chemical calculations of the electronic circular dichroism (ECD) spectra. Compounds 1-4 showed weak toxicity towards brine shrine naupalii with LC50 values of 72.0, 81.7, 49.9 and 81.4 µM, respectively. And 4 also showed significant antioxidant activity against ɑ,α-diphenyl-picrylhydrazyl (DPPH) radicals with an IC50 value of 16.4 µM.

The absolute configuration of anti-Vibrio citrinin dimeric derivative by VCD, ECD and NMR methods.

Citrinin dimeric derivatives are bioactive polyketides previously reported from Penicillium, Aspergillus and Monascus fungi species. Due to the large distance between the stereogenic centers of the two monomer units, it was difficult to determine the absolute configuration of the whole molecule (1). In previous work, the absolute configuration of 1 was just proposed by biogenetic considerations. To address this problem, the experimental VCD of 1 was compared with the corresponding DFT calculations for two diastereomers (1a and 1b). Also, the experimental ECD and NMR spectra of 1 were combined for analysis with the corresponding theoretical predictions for different diastereomers. Additionally, compound 1 showed promising anti-Vibrio activity against pathogenic Vibrio spp. with MIC values ranging from 0.4 to 0.8 µM.

Chemical Composition, Security and Bioactivity of the Red Pigment from Penicillium purpurogenum Li-3.

The chemical composition, security and bioactivity of pigments from Penicillium purpurogenum Li-3 strain screened by our group were firstly studied in this work. DPPH and the filter disc diffusion method were used to determine the biological activities of the red pigments. The pigment was characterized by UV/VIS, FT-IR, NMR and UPLC-Q-TOF-MS. HPLC/MS was used to detect mycotoxins (citrinin) in fermentation broth. An acute toxicity was detected in the embryos of zebrafish. As a consequence, the crude red pigment from the AcOEt fraction showed better DPPH scavenge capacity and antibacterial activity. Spectroscopic (UV, FT-IR, 13 C-NMR) and UPLC-Q-TOF-MS analysis revealed that the Penicillium purpurogenum Li-3 red pigment (RPs) was monascus-like pigment and its molecular weight was 439.1997. Moreover, the red pigment was shown to be weak cytotoxic against the zebrafish embryos. The yield of the red pigment increased 69 % under optimized culture conditions. These outstanding properties will enlarge the application of RPs for natural food additives, new antioxidant and antibacterial drug development.

Novel monoclonal antibody-based immunochromatographic strip for detecting citrinin in fruit from Zhejiang province, China.

Citrinin (CIT) is a hepato-nephrotoxic fungal metabolite produced by the genera Penicillium, Aspergillus and Monascu. There is an increasing demand for rapid and economical methods for detection CIT residues in fruit. In this study, we developed an immunochromatographic strip (ICS) for detection of citrinin (CIT) residues in fruit for the first time. Anti-CIT monoclonal antibody (McAb) 2B9 was prepared, with a binding affinity of 9.39 × 108 L/moL. Conjugates CIT-BSA and McAb 2B9 were used to develop the ICS which could be completed in 5 min, with the detection limit of 50 ng/mL and no cross reactivity with other mycotoxins. Analysis of CIT in 64 fruit samples revealed that data obtained from the ICS test were in good agreement with indirect competitive enzyme-linked immunosorbent assays (ic-ELISAs) and high performance liquid chromatography (HPLC). This result demonstrated that the ICS test could be used as a rapid, reliable, cost-effective and user-friendly qualitative tool for detection of CIT residues on-site.

Detection of trace amounts of citrinin in dried orange peel by using an optimized extraction method coupled with ultra-performance liquid chromatography-tandem mass spectrometry.

A fast and sensitive method involving ultra-performance liquid chromatography-tandem mass spectrometry (UPLC-MS/MS) was introduced to detect citrinin in dried orange peel. A series of extraction, purification and chromatographic conditions was also systematically examined. With the proposed method, the obtained calibration graph was linear, with an R of 0.9996 within a concentration range of 0.5-10 ng/mL. The estimated limits of detection and quantification were 0.05 and 0.17 ng/mL, respectively. Under the selected conditions, the relative recoveries in different citrus products spiked with 1-10 ng/mL citrinin were 89.4-98.7% with RSDs of <2.5%. Compared with previously reported analytical methods, the newly developed UPLC-MS/MS method showed excellent sensitivity and good precision in detecting citrinin. The results indicated that it is a reliable and effective technique for the detection of trace citrinin in dried orange peel.

A pair of enantiomeric 5-oxabicyclic[4.3.0]lactam derivatives and one new polyketide from the marine-derived fungus Penicillium griseofulvum.

A pair of enantiomeric 4-oxabicyclic[4.3.0]lactam derivatives, (+)- and (-)-penicilactam A (1), and one new polyketide derivative penicitrinone F (2) were isolated from the marine-derived fungus Penicillium griseofulvum GT-10. Their structures and absolute configurations were elucidated through extensive spectroscopic analyses combined with the calculated ECD spectra. Penicitrinone F (2) had moderate inhibitory activity towards Bacillus subtilis with a MIC value of 6.3 µM.

Purified citritin in combination with vancomycin inhibits VRE in vitro and in vivo.

Gram-positive pathogens such as methicillin-resistant Staphylococcus aureus (MRSA) and vancomycin-resistant Enterococcus faecium (VRE) have been frequently associated with bacterial resistance mechanisms. These mechanisms, in turn, restrict a range of therapeutic opportunities for the treatment of infections caused by these micro-organisms. Faced with this problem, the present study aims to isolate and characterize molecules with antimicrobial activity derived from the fungus Penicillium citrinum isolated from Cerrado soil. Furthermore, we also tested possible antibacterial potential alone and in combination with commercial antimicrobial agents. In this context, citrinin was isolated and characterized by nuclear magnetic resonance and electrospray ionization. Functional analyses showed MIC of 128 µg ml-1 against S. aureus ATCC 25923, E. faecalis ATCC 29212 and a clinical isolate of vancomycin-resistant E. faecium (VRE01). However, for a clinical strain of methicillin-resistant S. aureus (MRSA01), the MIC was 256 µg ml-1. In order to avoid such high concentrations and reduce the collateral effects, additive effects were evidenced by combining citrinin with cefoxitin against MRSA01 (FIC index=0.5) and also citrinin with vancomycin toward VRE01 (FIC index=0.5). In vivo studies with BALB/c-tipe mice (MRSA assay) demonstrated a clinical ineffectiveness of cefoxitin associated with citrinin (9.8 mg kg-1 of cefoxitin +0.2 mg kg-1 of citrinin), with this combination being inefficient to increase animal survival. However, the combination used in the treatment of VRE (23.5 mg kg-1 of citrinin +1.5 mg kg-1 of vancomycin) sepsis model was extremely promising, leading to an animal survival rate of 80 percent. In summary, our data show, for the first time, the possible successful use of citrinin associated with vancomycin for pathogenic bacteria control.

Adaptive conditions and safety of the application of Penicillium frequentans as a biocontrol agent on stone fruit.

Penicillium frequentans (Pf909) reduces brown rot caused by Monilinia spp. in stone fruit. The registration of a microbial biocontrol agent in Europe requires information on the risks and safety of a biological product. This study focused on the impact of the physical environment on Pf909 survival and growth, Pf909 mycotoxin production on fruit surface, and the Pf909 resistance to commercial antifungal compounds used in animal and human medicine. The effect of temperature (4 to 37°C), water activity (0.999 to 0.900), pH (3 to 11), light intensity (0, 90 and 180lm) and photoperiod (0/24, 12/12, 16/8, 24/0; light/dark) on mycelial growth and sporulation of Pf909 were monitored for 10days in vitro on culture medium. Antifungal activity of antifungal compounds on mycelial growth of Pf909 was also measured by a quantitative micro spectrophotometric assay after 72h of incubation. The presence or absence of four non-volatile mycotoxins (patulin, penicillic acid, ochratoxin A and citrinin) on nectarine surfaces treated with Pf909 was determined by HPLC. Growth rate was significantly influenced by water activity, temperature and light exposure conditions. Pf909 showed maximum growth and sporulation at 22°C and 25°C, in wet conditions (0.999 water activity), with a pH5.6 to 9, and in darkness or a short light photoperiod. Our results showed all antifungal compounds used reduced significantly Pf909 mycelial growth at tested commercial doses. HPLC data analysis showed that 7days after biofungicide (Pf909) application there were no mycotoxin products on the surface of nectarine. Finally, no phylogenetic relationship has been shown among Pf909 and other species of Penicillium that produce mycotoxins. In conclusion, from an ecological point of view, Pf909 would establish and survive actively over a broad range of climatic conditions. The probability of risks to human and animal health is considered very low.

Two new compounds from a marine-derived Penicillium griseofulvum T21-03.

A new phenolic acid compound, 46-dimethylcurvulinic acid (1) and a new citrinin monomer derivative penicitrinol P (2) were isolated from marine-derived Penicillium griseofulvum T21-03. The structures of 1 and 2 were elucidated on the basis of spectroscopic data.

Structurally diverse secondary metabolites from a deep-sea-derived fungus Penicillium chrysogenum SCSIO 41001 and their biological evaluation.

Five new compounds, including a cytotoxic dimeric isocoumarin, bipenicilisorin (1), a merosesquiterpenoid, yaminterritrem C (2), a citrinin dimer, penicitrinone F (3), a alkaloid, terremide D (4), and a δ-valerolacton, (E)-4-(propen-1-yl)-5,6-dihydro-2H-pyran-2-one (5), along with ten known compounds (6-15) were isolated from a deep-sea-derived fungus Penicillium chrysogenum SCSIO 41001. Their structures and absolute configurations were elucidated by NMR spectra, MS, CD, optical rotation, X-ray crystallography, and compared with literature data. Biological evaluation results revealed that 1 exhibited significant cytotoxic activities against K562, A549, and Huh-7 cell lines with IC50 values of 6.78, 6.94, and 2.59µM, respectively. Compound 3 exhibited moderate inhibitory activity against EV71 with IC50 value of 14.50µM. In addition, 13 and 14 showed specific COX-2 inhibitory activities with IC50 values of 1.09 and 1.97µM, respectively.

Penicisulfuranols A-F, Alkaloids from the Mangrove Endophytic Fungus Penicillium janthinellum HDN13-309.

Six new epipolythiodioxopiperazine (ETP) alkaloids, penicisulfuranols A-F (1-6), were isolated from the mangrove endophytic fungus Penicillium janthinellum HDN13-309. All structures including absolute configurations were elucidated on the basis of comprehensive spectroscopic data and ECD calculations. They belong to the unusual family of ETPs containing sulfur atoms on both α- and ß-positions of amino acid residues and a rare 1,2-oxazadecaline core moiety. In addition, compounds 1-6 also possess a rare spiro-furan ring and 1-3 showed cytotoxicity with IC50 values ranging from 0.1 to 3.9 µM.

Citrifelins A and B, Citrinin Adducts with a Tetracyclic Framework from Cocultures of Marine-Derived Isolates of Penicillium citrinum and Beauveria felina.

Citrifelins A (1) and B (2), two citrinin adducts possessing a unique tetracyclic framework, were characterized from a coculture of marine-derived fungal isolates of Penicillium citrinum and Beauveria felina. Neither fungus produced these compounds when cultured alone under the same conditions. The structures of these adducts were elucidated on the basis of spectroscopic analysis, and the absolute configurations were assigned on the basis of TDDFT-ECD calculations. A hypothesis that adducts 1 and 2 might be derived from a citrinin derivative through a non-pericyclic Michael reaction is proposed. Compounds 1, 2, and 5 showed inhibitory activities against several human and aquatic pathogens.

A novel citrinin derivative from the marine-source fungus Penicillium citrinum.

A novel citrinin derivative, penicitrinol L (1), along with two known analogues, penidicitrinin B (2) and pennicitrinone A (3) were isolated from the marine-source fungus Penicillium citrinum. The structure of the new compound was elucidated by spectroscopic methods including one and two-dimensional NMR as well as high-resolution mass spectrometric analysis. Furthermore, compound 1 showed modest cytotoxic activity against HL-60 cell line and compound 3 showed weak cytotoxic activity against A375 cell line.

Penicitols A-C and penixanacid A from the mangrove-derived Penicillium chrysogenum HDN11-24.

Three new citrinin analogues, penicitols A-C (1-3), and one new xanthone derivative, penixanacid A (4), together with four known biogenetically related compounds (5-8), were discovered from the extract of a mangrove-derived fungus, Penicillium chrysogenum HND11-24. The structures of penicitols A-C and penixanacid A were established through analysis of extensive spectroscopic data. Their cytotoxic activity against HeLa, BEL-7402, HEK-293, HCT-116, and A549 cell lines was evaluated.

Multi-commutated fluorometric optosensor for the determination of citrinin in rice and red yeast rice supplements.

Citrinin is a toxic secondary metabolite first isolated from Penicillium citrinum, although is also produced by other species of Penicillium and Aspergillus. It has highly toxic, mutagenic, teratogenic and carcinogenic properties and is often found in crops, vegetables and fruit. To our knowledge there is no specific legislation on maximum levels permitted for citrinin, so no official analytical method is currently available for its determination. Our laboratory developed a fluorometric flow-through optosensor using Sephadex SPC-25 as solid support. Multi-commutated flow injection analysis was used for the construction of the manifold and for handling solutions. In this way, we minimised waste generation and human intervention, which are critical aspects when dealing with highly toxic compounds such as citrinin. The optimum excitation/emission wavelengths were set at 330/494 nm; the calibration curve was linear in the concentration range 35-900 ng ml⁻¹. A detection limit of 10.5 ng ml⁻¹ and relative standard deviations (RSDs) lower than 3% were obtained. The developed optosensor was applied to the determination of citrinin in rice and dietary supplements containing red yeast rice.