Citrisorbicillinol, an undescribed hybrid sorbicillinoid with osteogenic activity from Penicillium citrinum ZY-2.

Citrisorbicillinol (1), along with six other known compounds (2-7), was isolated from an endphyte Penicillium citrinum ZY-2 of Plantago asiatica L. Citrisorbicillinol (1) was characterized as a skeletally unprecedented hybrid sorbicillinoid, and its unique framework is likely formed by intermolecular [4 + 2] cycloaddition between intermediates derived from citrinin and sorbicillinoid biosynthetic gene clusters. Compounds 1 and 2 demonstrated to promote osteoblastic differentiation in MC3T3-E1 cells, and to be osteogenic in the prednisolone induced osteoporotic zebrafish. Compounds 3-7 exhibited moderate cytotoxicity against four human cancer cell lines.

Non-monotonic dose-response of di-(2-ethylhexyl) phthalate isolated from Penicillium citrinum XT6 on adipogenesis and expression of PPARγ and GLUT4 in 3T3-L1 adipocytes.

OBJECTIVES: Adipogenesis is the fat cell formation process regulated by peroxisome proliferator-activated receptors (PPARγ). The insulin-responsive glucose transporter 4 (GLUT4) has a major role in glucose uptake and metabolism in insulin target tissues (i.e., adipose and muscle cells). The interplay between PPARγ and GLUT4 is essential for proper glucose homeostasis. This study aimed to isolate, elucidate, and investigate the effect of an isolated compound from Penicillium citrinum XT6 on adipogenesis, PPARγ, and GLUT4 expression in 3T3-L1 adipocytes. METHODS: The isolated compound was determined by analyzing spectroscopic data (LC-MS, FT-IR, Spectrophotometry UV-Vis, and NMR). The adipogenesis activity of the isolated compound in 3T3-L1 cells was determined by the Oil Red O staining method. RT-PCR was used to analyze the gene expression of PPARγ and GLUT4. RESULTS: Di-(2-ethylhexyl)-phthalate (DEHP) was the isolated compound from P.citrinum XT6. The results revealed adipogenesis stimulation and inhibition, as well as PPARγ and GLUT4 expressions. CONCLUSIONS: DEHP showed a non-monotonic dose-response (NMDR) effect on adipogenesis and PPARγ and GLUT4 expression. It is the first study that reveals DEHP's NMDR effects on lipid and glucose metabolism in adipocytes.

Mass Spectrometry Metabolomics Approach Reveals Anti-Trichomonas vaginalis Scaffolds from Marine Fungi.

Trichomoniasis is the most common non-viral sexually transmitted infection (STI) in the world caused by Trichomonas vaginalis. Failures in the treatment with the 5-nitroimidazole class including parasite resistance to metronidazole elicit new alternatives. Marine natural products are sources of several relevant molecules, presenting a variety of metabolites with numerous biological activities. In this work, we evaluated the anti-T. vaginalis activity of fungi associated with marine invertebrates by mass spectrometry-based metabolomics approaches. After screening of six marine fungi, extract from Penicillium citrinum FMPV 15 has shown to be 100% active against T. vaginalis, and the gel permeation column on Sephadex LH-20® yielded twelve organic fractions which five showed to be active. Metabolomics and statistical analyses were performed with all the samples (extract and fractions), and several compounds were suggested to be related to the activity. These components include citrinin, dicitrinin C, citreoisocoumarin, dihydrocitrinone, decarboxycitrinin, penicitrinone C, and others. The minimum inhibitory concentration (MIC) value of anti-T. vaginalis activity of citrinin was 200 µM. The marine fungi metabolites show potential as new alternatives to overcome drug resistance in T. vaginalis infections.

Expression of citrinin biosynthesis gene in Liupao tea and effect of Penicillium citrinum on tea quality.

Similar to Pu-erh tea, Liupao tea is a post-fermented tea that is produced through natural fermentation by microorganisms. Penicillium citrinum is involved in multiple production processes of Liupao tea that can produce citrinin, a secondary metabolite with renal toxicity; however, the effect of P. citrinum on the quality of Liupao tea has not been investigated yet. Citrinin production is regulated by approximately 16 biosynthesis genes. However, little is known about the genetic background of citrinin in the complex Liupao tea system. In the present study, we cultured P. citrinum on potato dextrose agar and Liupao tea powder media and analyzed the changes of its nutritional components in Liupao tea. We selected six citrinin biosynthesis genes identified in Monascus exhibiting homology and high sequence similarity to those in P. citrinum and further analyzed the expression of citrinin biosynthesis genes in Liupao tea and the changes in citrinin yield. The results showed that the changes in nutritional components of Liupao tea were closely related to the growth and metabolism of P. citrinum and the quality of the tea. Decreases in the contents of soluble sugars (from 10.29% to 9.58%), soluble pectins (from 3.71% to 3.13%), free amino acids (from 3.84% to 3.14%), and tea polyphenols (from 22.84% to 18.78%) were noted. The Spearman's correlation analysis indicated that P. citrinum growth can improve the tea quality to some extent. Quantitative real-time PCR demonstrated that ctnA gene was a positive regulator of citrinin production regardless of the culture medium used. ctnA and orf5 expressions greatly influenced the metabolism of citrinin by P. citrinum in Liupao tea. In conclusion, the citrinin biosynthesis genes, ctnA and orf5, may be the promising targets for developing strategies to control P. citrinum infection and citrinin biosynthesis in Liupao tea.

GKK1032B from endophytic Penicillium citrinum induces the apoptosis of human osteosarcoma MG63 cells through caspase pathway activation.

Chemical investigation of the culture extract of an endophytic Penicillium citrinum from Dendrobium officinale, afforded nine citrinin derivatives (1-9) and one peptide-polyketide hybrid GKK1032B (10). The structures of these compounds were determined by spectroscopic methods. The absolute configurations of 1 and 2 were determined for the first time by calculation of electronic circular dichroism (ECD) data. Among them, GKK1032B (10) showed significant cytotoxicity against human osteosarcoma cell line MG63 with an IC50 value of 3.49 µmol·L-1, and a primary mechanistic study revealed that it induced the apoptosis of MG63 cellsvia caspase pathway activation.

Detoxication of Citrinin with Kojic Acid by the Formation of the Citrinin-Kojic Acid Adduct, and the Enhancement of Kojic Acid Production by Citrinin via Oxidative Stress in Aspergillus parasiticus

Our previous work showed that citrinin (CTN) produced bay Penicillium citrinum inhibited the production of aflatoxin by Aspergillus parasiticus. We also reported that CTN was non-enzymatically converted to a novel CTN-KA adduct with kojic acid (KA) in aqueous condition. We herein observed that unlike CTN, the CTN-KA adduct does not show antimicrobial activity against Escherichia coli or Bacillus subtilis or any cytotoxic effect on HeLa cells, suggesting that CTN was detoxified by KA by the formation of the CTN-KA adduct. To examine the function of KA production by fungi, we isolated A. parasiticus mutants with impaired KA production. When the mutants were incubated in either liquid or agar medium supplemented with CTN, they were more susceptible to CTN than the wild KA-producing strain. The same results were obtained when we used the A. oryzae KA-producing strain RIB40 and KA-non-producing strains. When KA was added to the CTN-containing agar medium, the inhibition of growth by CTN was remarkably mitigated, suggesting that the production of KA protected the fungal growth from CTN's toxicity. We also observed that CTN enhanced the production of KA by A. parasiticus as well as A. oryzae strains. Reverse transcription-PCR showed that CTN enhanced the expression of KA biosynthetic genes (kojA, kojR, and kojT) of A. parasiticus. However, the enhancement of KA production with CTN was repressed by the addition of α-tocopherol or butylated hydroxy anisole, suggesting that KA production is enhanced by oxidative stress via the formation of reactive oxygen species caused by CTN. In contrast, α-tocopherol did not affect inhibition of AF production as well as fungal growth by CTN, suggesting that the regulation of these inhibitions with CTN might be different from that of KA production. We propose a regulation scheme of CTN for each of KA production, AF production, and fungal growth in A. parasiticus.

Cytogenotoxic evaluation of the acetonitrile extract, citrinin and dicitrinin-A from Penicillium citrinum.

Endophytic fungi are promising sources of bioactive substances; however, their secondary metabolites are toxic to plants, animals, and humans. This study aimed toevaluate the toxic, cytotoxic, mutagenic and oxidant/antioxidant activities of acetonitrile extract (AEPc), citrinin (CIT) and dicitrinin-A (DIC-A) of Penicillium citrinum. For this, the test substances at 0.5; 1.0; 1.5 and 2 µg/mLwere exposed for 24 and 48 h in Artemia salina, and 48 h in Allium cepa test systems. The oxidant/antioxidant test was evaluated in pre-, co- and post-treatment with the stressor hydrogen peroxide (H2O2) in Saccharomyces cerevisiae. The results suggest that the AEPc, CIT and DIC-A at 0.5; 1.0; 1.5 and 2 µg/mL showed toxicity in A. saline, with LC50 (24 h) of 2.03 µg/mL, 1.71 µg/mL and 2.29 µg/mL, and LC50 (48 h) of 0.51 µg/mL, 0.54 µg/mL and 0.54 µg/mL, respectively.In A. cepa, the test substances also exerted cytotoxic and mutagenic effects. The AEPc, CIT and DIC-A at lower concentrations modulated the damage induced by H2O2 in the proficient and mutant strains of S. cerevisiae for cytoplasmic and mitochondrial superoxide dismutase. Moreover, the AEPc at 2 µg/mL and CIT at the two highest concentrations did not affect the H2O2-induced DNA damage in the test strains. In conclusion, AEPc, CIT and DIC-A of P. citrinum may exert their toxic, cytotoxic and mutagenic effects in the test systems possibly through oxidative stress induction pathway.

Activated production of silent metabolites from marine-derived fungus Penicillium citrinum.

As an attempt to utilize of rare earth elements as a novel method to activate the silent genes in fungus, the marine-derived fungus Penicillium citrinum was cultured under ordinary laboratory fermentation conditions in the presence of scandium chloride (ScCl3, 50 µM), and chemical investigation led to the isolation and characterization of three new peptide derivatives (1-3), along with four known pyrrolidine alkaloids (4-7). Those structures were elucidated by spectroscopic data interpretation, as well as chemical reactions. Comparative metabolic profiling of the culture extracts (with/without scandium chloride) indicated that compounds 1-3 scarcely detected in the absence of ScCl3. In addition, the antibacterial and cytotoxic activities of all isolated products were evaluated.

Bioactive polyketides and alkaloids from Penicillium citrinum, a fungal endophyte isolated from Ocimum tenuiflorum.

Chemical investigation of the endophytic fungus Penicillium citrinum cultured on white beans or on rice led to the isolation of two new alkaloids (1 and 2), along with fourteen known polyketides (6-12, 14-20) and four known alkaloids (3-5, and 13). The structures of the isolated compounds were determined by extensive analysis of the 1D, 2D NMR, and MS data, and by comparison with the literature. Compound 13, which had been previously obtained only by chemical synthesis, was isolated as a natural product for the first time, while compound 6 was firstly reported as a fungal metabolite. A re-isolation of sclerotinin A (14) revealed it to be a diastereoisomeric mixture (14a and 14b), whose stereochemistry was proposed for the first time based on ROESY experiment. All isolated compounds were evaluated for their cytotoxic and antibacterial activities. Compounds 12 and 17 showed significant cytotoxicity against the murine lymphoma cell line L5178Y with IC50 values of 1.0, and 0.78 µg/ml, respectively, while compounds 5, 11, and 15 were moderately active against Staphylococcus aureus ATCC 29213 (MIC 64 µg/ml).