Determination of griseofulvin by time-resolved phosphorimetry

The phosphorescence lifetimes of griseofulvin and dechlorogriseofulvin are shown to be 0.11 sec and 1.16 sec, respectively. This tenfold difference is shown to enable the use of time-resolved phosphorimetry for the determination of griseofulvin in mixtures with dechlorogriseofulvin.     

THE PRODUCTION OF ANTIBIOTICS IN SOIL V. BREAKDOWN OF GRISEOFULVIN IN SOIL

When griseofulvin (I; R = Cl, R '= OCH₃), a chlorine-containing antibiotic produced by Penicillium nigricans , was added to fresh garden loam, after an initial lag it disappeared rapidly. When further griseofulvin was added it was inactivated from the start and at rates which increased with each successive addition, suggesting that it was degraded biologically. The numbers of one organism, a Pseudomonas sp., increased in the soil steadily after adding griseofulvin.
When a little soil was added to a solution (pH 7·0) containing inorganic salts and griseofulvin as the sole carbon source, bioassays showed that the griseofulvin disappeared within 5 days. An organism isolated from the broth was identified as the Pseudomonas sp. thought to break down griseofulvin in soil. Griseofulvin also disappeared from a broth at pH 5·0 inoculated with soil, but at this lower pH value a dematiaceous fungus was responsible for its breakdown.
The Pseudomonas sp. also degraded two derivatives of griseofulvin, dechlorogriseofulvin (I; R = H, R'= OCH₃) and the amine (I; R = Cl, R '= NH₂). Cl^– was detected in the solutions after breakdown of griseofulvin by the Pseudomonas ; the amount present agreed well with that calculated on the assumption that all the chlorine in the griseofulvin supplied was liberated as Cl^–. Spectrophotometric examination of the solutions showed no metabolites with the aromatic ring intact, and confirmed the complete breakdown of griseofulvin suggested by the liberation of Cl-.     

Indanone and mellein derivatives from the Garcinia-derived fungus Xylaria sp. PSU-G12

Two new compounds, one indanone (1) and one mellein (2), along with 3-hydroxy-4-methyl-1-indanone (3), griseofulvin (4), dechlorogriseofulvin (5), cytochalasin D (6) and three mellein derivatives (7–9) were isolated from the broth extract of the Garcinia-derived fungus Xylaria sp. PSU-G12. The structures were elucidated by spectroscopic analysis. This is the first report on the isolation of indanone derivatives from the genus Xylaria. The isolated compounds were evaluated for antioxidant activity in DPPH assay.     

Bioactive secondary metabolites from <Emphasis Type="Italic">Nigrospora</Emphasis> sp. LLGLM003, an endophytic fungus of the medicinal plant <Emphasis Type="Italic">Moringa oleifera</Emphasis> Lam.

An endophytic fungus was isolated from the root of the medicinal plant Moringa oleifera Lam. Based on analyzing the rDNA sequence, the fungus was identified as Nigrospora sp. This is the first report of the isolation of endophytic Nigrospora from M. oleifera. By bioassay-guided fractionation, four antifungal secondary metabolites were isolated from liquid cultures of the fungus Nigrospora sp. LLGLM003, and their chemical structures were determined to be griseofulvin (1), dechlorogriseofulvin (2), 8-dihydroramulosin (3) and mellein (4) on the basis of spectroscopic analyses. Compound 2, 3 and 4 were isolated from Nigrospora sp. for the first time. In vitro antifungal assay showed that griseofulvin displayed clear inhibition of the growth of 8 plant pathogenic fungi. Dechlorogriseofulvin and mellein exhibited only weak antifungal activities, whereas 8-dihydroramulosin displayed no antifungal activities.     

Production and fungitoxic activity of Sch 642305, a secondary metabolite of <Emphasis Type="Italic">Penicillium canescens</Emphasis>

Production of fungitoxic extrolites was evaluated in culture filtrates of several isolates belonging to Penicillium canescens and P. janczewskii that showed some extent of inhibitory activity against the plant pathogenic fungus Rhizoctonia solani. In addition to griseofulvin and dechlorogriseofulvin that are already known in these species, curvulinic acid, previously unreported in Penicillium, was produced by all isolates assayed. Another extrolite recently characterized from a P. verrucosum strain by the name of Sch 642305 was detected in 5 isolates of P. canescens only. The purified compound completely inhibited mycelial growth of isolates of Rhizoctonia solani and other plant pathogenic fungi in␣vitro. The role of this extrolite as a possible biochemical determinant of antagonism toward plant pathogenic fungi, and implications concerning chemotaxonomy are discussed.     

Penicillium persicinum, a new griseofulvin, chrysogine and roquefortine C producing species from Qinghai province, China

A unique Penicillium isolate from Chinese soil with terverticillate penicilli and ellipsoidal to cylindrical smooth-walled conidia, produces, in addition to the common metabolite ergosterol, copious amounts of an unknown peach-red pigment and the following secondary metabolites: griseofulvin, dechlorogriseofulvin, lichexanthone, roquefortine C, roquefortine D, chrysogine, 2-pyrovoylaminobenzamide, 2-acetyl-quinazolin-4(3H)-one. This isolate, CBS 111235, is described as Penicillium persicinum sp. nov., which belongs to subgenus Penicillium section Chrysogena but is morphologically similar to P. italicum. On the basis of the production of secondary metabolites it resembles P. griseofulvum and P. coprophilum. Sequence data using part of the beta-tubulin gene showed that it is phylogenetically related to P. chrysogenum and P. aethiopicum in section Chrysogena with which it shares both secondary metabolites and ability to grow at 37 degrees C.     

Griseofulvin interacts with microtubules both in vivo and in vitro

Immunofluorescence microscopy using monospecific tubulin antibody shows that in vivo griseofulvin interferes with the expression of both cytoplasmic and spindle microtubules in tissue culture cells in a concentration-dependent manner. In mouse 3T3 cells cytoplasmic microtubules are destroyed at a griseofulvin concentration of 5 × 10^?5m. At this concentration no increase of the mitotic index is observed but the cells are arrested in interphase, probably due to the destruction of cytoplasmic microtubules. Lowering the drug concentration to 10^?5m allows 3T3 cells to accumulate in c-mitotic (“colchicin-mitotic”) arrest. In HeLa cells the display of spindle microtubules observed in drug-arrested cells appears similar to that seen in normal metaphase cells only at lower griseofulvin concentrations. Higher drug concentrations induce c-mitotic arrest accompanied by an increasing loss of typical metaphase tubulin structures.In vitro polymerization experiments with brain tubulin using both light-scattering and electron microscopy show that in the presence of griseofulvin tubulin can aggregate rapidly in the cold. This behaviour is not found in the absence of the drug. Thus both in vivo and in vitro experiments show that griseofulvin, like other c-mitotic drugs, acts at the level of tubulin polymerization and that its effects are concentration dependent.     

Diverse secondary metabolites produced by marine-derived fungus Nigrospora sp. MA75 on various culture media

Bioassay-guided isolation of a fungal strain Nigrospora sp. MA75, an endophytic fungus obtained from the marine semi-mangrove plant Pongamia pinnata, which was fermented on three different culture media, resulted in the isolation and identification of seven known compounds, 2, 3, and 5-9, from a medium containing 3.5% NaCl, while a new compound, 2,3-didehydro-19α-hydroxy-14-epicochlioquinone B (10) was obtained from the medium containing 3.5% NaI. In addition, two new griseofulvin derivatives, 6-O-desmethyldechlorogriseofulvin (1) and 6'-hydroxygriseofulvin (4), were isolated and identified from the rice solid medium. Dechlorogriseofulvin (2) and griseofulvin (3) were the major components in fermentation extracts of all these culture media, while compounds 1 and 4, 5 and 6, and 10 were only present in the extract of respective culture medium. The structures of these compounds were elucidated by detailed spectroscopic analysis, and the absolute configuration of 1 was determined by CD measurement. Compounds 9 and 10 exhibited antibacterial activities toward five tested bacterial strains, while compounds 5, 6, and 8 selectively inhibited MRSA, E. coli, and S. epidermidis, and compound 3 showed moderate activity against V. mali and S. solani. Moreover, compound 10 potently inhibited the growth of MCF-7, SW1990, and SMMC7721 tumor cell lines with IC(50) values of 4, 5, and 7 μg/ml, respectively.     

ONYCHOMYCOSIS OF THE FEET—Treatment with Griseofulvin

Griseofulvin, a new orally administered antifungal antibiotic which has proved to be effective for the treatment of a wide variety of superficial fungus infections of man, was used in the treatment of 51 patients with infections of the toenails due to T. rubrum. Thirty-four of the patients were treated with griseofulvin alone and seven were treated with griseofulvin combined with surgical avulsion of all involved toenails. The remaining ten had bilateral infections, and avulsion was done on one foot but not the other before griseofulvin therapy was begun.Of 34 patients who were treated with griseofulvin alone, few had complete cure even after prolonged treatment. Some nails showed improvement for a time, then no further gain; some showed no improvement; some showed resistant wedges of infection which penetrated proximally toward the posterior nail fold.In the instances of surgical avulsion, clinically normal nails regrew during griseofulvin therapy. This simple procedure, with thorough removal of all underlying keratinous debris, apparently did away with foci of possible reinfection.The results of the study indicated that surgical avulsion of the toenails in combination with griseofulvin therapy is an effective and practical method of treating onychomycosis of the toenails due to T. rubrum.     

Induction of apoptosis against cancer cell lines by four ascomycetes (endophytes) from Malaysian rainforest

Endophytic fungi have been shown to be a promising source of biologically active natural products. In the present study, extracts of four endophytic fungi isolated from plants of the National Park, Pahang were evaluated for their cytotoxic activity and the nature of their active compounds determined. Those extracts exhibiting activity with IC(50) values less than 17 μg/ml against HCT116, MCF-7 and K562 cell lines were shown to induce apoptosis in these cell lines. Molecular analysis, based on sequences of the rDNA internal transcribed spacers ITS1 and ITS4, revealed all four endophytic fungi to be ascomycetes: three sordariomycetes and a dothideomycete. Six known compounds, cytochalasin J, dechlorogriseofulvin, demethylharzianic-acid, griseofulvin, harzianic acid and 2-hexylidene-3-methyl-succinic acid were identified from a rapid dereplication technique for fungal metabolites using an in-house UV library. The results from the present study suggest the potential of endophytic fungi as cytotoxic agents, and there is an indication that the isolates contain bioactive compounds that mainly kill cancer cells by apoptosis.     

Chromatographic analysis of griseofulvin and metabolites in biological fluids

A simple and accurate assay for the determination of griseofulvin and its metabolites in biological fluids using high-performance liquid chromatography is described. Using a reversed phase column and a mobile phase solvent of 45% acetonitrile in 0.1 M acetic acid, baseline separation of griseofulvin and several analogues was obtained. The described method allows one to quantitatively determine griseofulvin, 6-demethylgriseofulvin, and griseofulvic acid, a newly identified metabolite in man, in urine and plasma samples. Treatment of plasma samples prior to the analysis is simply made by deproteinizing the samples with an equal volume of acetonitrile. For urine samples, the procedure involves diethyl ether extraction with subsequent evaporation to dryness and reconstitution with the mobile phase solvent.     

Griseofulvin-producing Xylaria endophytes of Pinus strobus and Vaccinium angustifolium: evidence for a conifer-understory species endophyte ecology

During three decades of research on conifer endophytes of the Acadian forest, numerous insights have been gained in conifer-fungal ecology and secondary metabolite production. Recently, we have explored endophyte assemblages of understory plants commonly occurring with pine. Here we report for the first time the production of the potent antifungal compound griseofulvin by a fungal endophyte isolated from eastern white pine (Pinus strobus) needles and lowbush blueberry (Vaccinium angustifolium) stems from the Acadian forest of New Brunswick and Nova Scotia, Canada. Maximum likelihood phylogenetic analysis has placed the endophyte strains as an undescribed Xylaria sp. Our study highlights the complexity of endophyte-host lifecycles and points to the existence of a pine-blueberry ecotype. Aside from griseofulvin, piliformic acid was isolated from one of the pine endophytes. This compound has been reported from Xylaria and related species but not from Penicillium species known to produce griseofulvin.     

The effect of salinity and osmotic pressure of the medium on the growth,sporulation and changes in the total organic acid content of Aspergillus flavus and Penicillium chrysogenum

Production of patulin and griseofulvin by a strain of Penicillium griseofulvum is described. Mycelial dry weight, pH and production of patulin and griseofulvin were assayed in a minimal and a complete medium; patulin or griseofulvin production was assayed in apple juice.     

Detection of foliar endophytes and their metabolites in Picea and Pinus seedling needles

The needles of Picea glauca (white spruce) and Pinus strobus (white pine) trees infected with toxigenic fungal endophytes contain varying concentrations of their secondary metabolites that are toxic to either insect pests or needle pathogens. In the present study, liquid chromatography-mass spectrometric methods to determine needle concentrations of metabolites of four endophyte species were developed. The endophytes considered were a Phialocephala sp. (vermiculine) and Phialocephala scopiformis (rugulosin) from white spruce, as well as a Xylaria sp. (griseofulvin) and Lophodermium nitens (pyrenophorol) from white pine needles. To ensure that needles were infected with the associated fungal endophyte, suitable qPCR-based methods were also developed. There was a high degree of concordance between the qPCR analysis of the fungal mycelium and the LC-MS/MS quantification of the associated metabolites. Concentrations of the antifungal compounds griseofulvin and pyrenophorol were present in amounts that affect conifer needle diseases including white pine blister rust caused by Cronartium ribicola. Similarly, concentrations of the antiinsectan compounds vermiculine and rugulosin were in the range known to reduce the growth of Choristoneura fumiferana and mitigate foliage damage.     

Effect of decreased ferrochelatase activity on iron and porphyrin content in mitochondrai of mice with porphyria induced by griseofulvin

The content of iron and protoporphyrin in liver mitochondria from mice with porphyria induced by griseofulvin was measured. The amount of porphyrin was 0.0076 ± 0.0043, 4.11 ± 0.58 and 22.2 ± 6.8 nmol/mg protein (n = 5) in mitochondria from control animals and animals treated with griseofulvin for 3 days and 4–5 weeks, respectively. The energy coupling of the mitochondia was greatly diminished after 4–5 weeks of treatment, and the ferrochelatase activity was inhibited 80–90%, compared to that of control animals. Mitochondrial preparations isolated by differential centrifugation were contaminated with iron-containing lysosomes which could be removed by Percoll density-gradient centrifugation. In purified mitochondrial preparations no change in the amount of non-heme iron was found after griseofulvin feeding, representing 3.36±0.15, 3.97±0.40 and 3.59±0.23 nmol/mg protein for control animals, 3 days- and 4–5 weeks-treated animals, respectively (n = 4). A mitochondrial iron pool previously identified in rat liver mitochondria and shown to be available for heme synthesis in vitro (Tangerås, A. (1985)) Biochim. Biophys. Acta 843 199–207) was also present in mitochondria from mice. The magnitude of this iron pool, as well as its availability for heme synthesis, was not changed after treatment of the animals with griseofulvin. The fact that porphyrin, but not iron, accumulated in the mitochondria when ferrochelatase was inhibited is discussed with regard to our understanding of the process of heme synthesis and its regulation.     

Restoration of mitotic and differentiation processes in the root apices ofAllium cepa L. treated with cyanein and griseofulvin

The roots ofAllium cepa L. were treated with water solutions of cyanein and griseofulvin for 24 and 48 h respectively, thereafter cultured for 48 h in the medium without the antibiotics, and the reversibility of the inhibition caused by the antibiotics was evaluated. Changes in the mitotic index of meristematic cells were followed in squash preparations of root apices. In addition to the cytologic observations, the differentiation of the primary meristem was followed in longitudinal sections. After the treatment with reversible doses of cyanein and griseofulvin respectively, differences were found in both the effects of the antibiotics during the treatment and the restoration of the inhibited processes after the treatment. In the cells treated with cyanein the restoration of mitotic activity was instantaneous but less intensive and in|complete especially in the case of the 48 h treatment. After the treatment with griseofulvin which temporarily interfered also with the differentiation of the primary meristem, the restoration of the inhibited processes was delayed but intensive after both application times, 48 h after the treatment no symptoms of the preceding inhibition could be observed.     

Studies on production of griseofulvin

Griseofulvin is an antifungal antibiotic and is used for the treatment of mycotic diseases of human, veterinary and plant systems. It is synthesised by many species of Penicillium. The exploitation of fermentation process plays a vital role in the industrial applications. This leads to techno-economic feasibility of the processes. The present review mainly discusses on the production of griseofulvin in soil, in plants and by various micro-organisms. This communication also summarises the various factors which influence the griseofulvin production and strain improvement by mutation for enhanced production.     

13C-NMR studies on griseofulvin biosynthesis and acetate metabolism in Penicillium patulum

Incorporations of singly and doubly-labelled acetate-[¹³C] into griseofulvin by a mutant strain of Penicillium patulum confirm its origin from simple folding of a single heptaketide chain. An acetate ‘starter’ effect is observed in the ¹³C-NMR spectra of griseofulvin enriched from acetate-[¹³C], and analysis of the ¹³C—¹³C spin—spin couplings observed indicate a rapid metabolic turnover of added acetate. Methyl, but not carboxyl, of acetate is efficiently metabolised into the C₁ pool.     

Interference of Griseofulvin with the Segregation of Chromosomes at Mitosis in Diploid Aspergillus nidulans

Low concentrations of the antibiotic griseofulvin were found to cause increased frequencies of somatic segregation due to chromosome nondisjunction in a diploid strain of Aspergillus nidulans.     

Uptake and translocation of griseofulvin by wheat seedlings

Summary 1. A roughly quantitative technique for studying uptake and translocation of the antibiotic griseofulvin by wheat plants has been devised. Wheat plants were grown in nutrient solutions containing griseofulvin and translocation measured by bioassay of the griseofulvin appearing in the guttation drops induced by transfer to a humid atmosphere.2. Griseofulvin was phytotoxic at concentrations of 5 µg/ml and above, the first symptoms observed being stunting and swelling of the roots.3. The concentration of griseofulvin in the guttation drops was directly related to the concentration in the nutrient solution; there was evidence of griseofulvin accumulation in the leaves, the concentration in the guttation drops being frequently higher than that in the nutrient solution.4. Atmospheric conditions favouring transpiration increased uptake and translocation of griseofulvin.5. Uptake and translocation of griseofulvin was inhibited by inclusion of respiratory enzyme inhibitors in the nutrient solution.