Detection of Griseofulvin and Dechlorogriseofulvin by Thin-Layer Chromatography and Gas-Liquid Chromatography

A rapid and accurate method is described for the determination of griseofulvin and dechlorogriseofulvin extracted from Penicillium urticae with chloroform. Thinlayer chromatography was used to tentatively identify griseofulvin or dechlorogriseofulvin, or both. Two gas-liquid chromatographic systems provided additional qualitative information and simultaneous quantitation of the individual compounds.     

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-.     

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.     

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.     

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.     

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.     

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.     

Effect of griseofulvin on lipid composition and membrane integrity inMicrosporum gypseum

The effect of griseofulvin on lipid constituents and membrane permeability ofMicrosporum gypseum has been investigated. Mycelia grown in medium containing griseofulvin (IC₅₀ concentration) possessed a lower content of total lipids, phospholipids and sterols. This inhibitory effect was further supported by decreased incorporation of [¹⁴C] acetate in total lipids, total phospholipids and sterols. Decrease in total phospholipids was also reflected to a varying extent in all individual phospholipids. An increase in the unsaturated to saturated fatty acid ratio was observed in mycelia grown in medium containing griseofulvin. Membrane permeability was affected by griseofulvin as shown by increased K⁺-efflux and greater leakage of intracellular [³²P] labelled components from prelabelled cells. Our results suggest that the antifungal activity of griseofulvin is partially due to its secondary effect on lipid constituents ofMicrosporum gypseum.     

THE PRODUCTION OF ANTIBIOTICS IN SOIL: II. PRODUCTION OF GRISEOFULVIN BY PENICILLIUM NIGRICANS

A study has been made of the conditions affecting production of griseofulvin by Penicilliiim nigricans in two types of soil, an acid, sandy podsol from Wareham Heath and a garden soil. The characteristic morphogenetic response of many fungi to low concentrations of griseofulvin was made the basis of a highly specific bioassay.
The essential prerequisites for production of griseofulvin in either soil were sterilization and enrichment with organic matter; no griseofulvin could be detected in autoclaved soil which had not been supplemented or in normal soil even when organically enriched. Garden soil was a better medium for growth of P. nigricans and production of griseofulvin than Wareham soil although this soil could be improved in this respect by liming.
The yield of griseofulvin was decreased in soil re-infected by other soil organisms, particularly by some which were known to produce antifungal antibiotics, e.g. Penicilliunr expansum, P. frequentons and two strains of Trichoderma viride. The antagonism shown to Penicilliunz nigricuns was not entirely a matter of antibiotic activity, as some fungi believed not to produce antifungal substances had an antagonistic effect. These were mostly fungi with a characteristically rapid growth rate, e.g. Mucor rarnmannianus and one strain of Trichoderma riride. In some cases Penicillium nigricans was itself antagonistic to other fungi irrespective of their ability to produce antibiotics or of their fast-growing habit.
The results were compared with those obtained from a previous study of the soil conditions affecting the production of gliotoxin by Trichoderma viride. A higher level of nutrient was required for the production of griseofulvin, and the effect of antagonism by other soil micro-organisms was more important than in the production of gliotoxin by T. viride in the soil.     

Griseofulvin: Association with tubulin and inhibition of in vitro microtubule assembly

Griseofulvin—shown previously to disrupt the mitotic apparatus $\text{in vivo}$—inhibited the $\text{in vitro}$ microtubule assembly reaction completely at 8 × 10^?4M griseofulvin. In a gel filtration assay, randomly tritiated griseofulvin associated stoichiometrically with purified tubulin, as determined by chromatography on Sephadex G-25. No detectable drug binding was observed when bovine serum albumin was used as a control in an identical column assay. Both gel filtration chromatography and a kinetic analysis of the inhibition of assembly by griseofulvin suggest that the drug interacts directly and stoichimetrically with the tubulin dimer, and that the interaction is both rapid and independent of temperature.     

INVESTIGATIONS ON FUNGICIDES II. ARYLOXY- AND ARYLTHIO-ALKANECARBOXYLIC ACIDS AND THEIR ACTIVITY AS FUNGICIDES AND SYSTEMIC FUNGICIDES

A wide range of aryloxyacetic acids and corresponding acids with alkyl groups in the side chain, their arylthio- analogues and the antibiotic griseofulvin have been assessed in the plate test for fungistatic effect on six fungi, and as systemic fungicides against Botrytis fabae in broad beans and Alternaria solani in tomatoes. The results indicate that in general, arylthio- derivatives are more fungicidal than their aryloxy- analogues. The systemic fungicidal performance of x-(2-chlorophenylthio)propionic acid in the tomato test at 1–100 p. p. m. was found to be of the same order as that shown by griseofulvin at 50–500 p. p. m. Variable results were obtained with griseofulvin in the tornato test and its performance in the bean test was consistently poor. Further evidence is presented which indicates that the protection conferred by certain compounds may not be due to activity per se .     

Simultaneous determination of griseofulvin and 6-desmethylgriseofulvin in plasma by electron-capture gas chromatography

Two methods have been developed for the simultaneous determination of griseofulvin and its major metabolite 6-desmethylgriseofulvin in plasma using electron-capture gas chromatography. The first method was based on the quantitative reversion of the 6-desmethyl metabolite to griseofulvin by diazomethane. Plasma extract was chromatographed both before and after treatment with diazomethane, the former being the measure of griseofulvin and the latter representing the sum of the two compounds. In the second method, plasma extract was treated with diazobutane and griseofulvin and the butylated metabolite were separated by gas chromatography. The sensitivity for griseofulvin was 20 ng/ml by both methods and that for the metabolite was 20 ng/ml and 40 ng/ml by the first and the second method, respectively. The concentrations of the metabolite as well as griseofulvin were determined in dog and human plasma after oral administration of griseofulvin.     

The influence of actinomycin D on the activity of delta-aminolaevulinic acid synthetase and dehydratase in the liver of mice and rats treated by griseofulvin.

The changes of delta-aminolaevulinic acid dehydratase--the second enzyme of porphyrin synthesis--were studied and compared in the liver of mice and rats treated with griseofulvin. The results showed that griseofulvin increased the activity of delta-aminolaevulinic acid dehydratase in the liver of mice and rats treated by griseofulvin. No differences were found between mice and rats as to the effect of griseofulvin on the activity of delta-aminolaevulinic acid dehydratase. The influence of the administration of actinomycin D on the activity of delta-aminolaevulinic acid synthetase and dehydratase in the liver of mice and rats was studied at an early period of experimental porphyria induced by griseofulvin. It was found that the administration of actinomycin D blocked the observed effect of griseofulvin on the activity of both enzymes in the liver of mice and rats.     

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.     

Evaluation of Griseofulvin Binary and Ternary Solid Dispersions with HPMCAS

The stability and dissolution properties of griseofulvin binary and ternary solid dispersions were evaluated. Solid dispersions of griseofulvin and hydroxypropyl methylcellulose acetate succinate (HPMCAS) were prepared using the spray drying method. A third polymer, poly[N-(2-hydroxypropyl)methacrylate] (PHPMA), was incorporated to investigate its effect on the interaction of griseofulvin with HPMCAS. In this case, HPMCAS can form H bonds with griseofulvin directly; the addition of PHPMA to the solid dispersion may enhance the stability of the amorphous griseofulvin due to greater interaction with griseofulvin. The X-ray powder diffraction results showed that griseofulvin (binary and ternary solid dispersions) remained amorphous for more than 19 months stored at 85% RH compared with the spray-dried griseofulvin which crystallized totally within 24 h at ambient conditions. The Fourier transform infrared scan showed that griseofulvin carbonyl group formed hydrogen bonds with the hydroxyl group in the HPMCAS, which could explain the extended stability of the drug. Further broadening in the peak could be seen when PHPMA was added to the solid dispersion, which indicates stronger interaction. The glass transition temperatures increased in the ternary solid dispersions regardless of HPMCAS grade. The dissolution rate of the drug in the solid dispersion (both binary and ternary) has significantly increased when compared with the dissolution profile of the spray-dried griseofulvin. These results reveal significant stability of the amorphous form due to the hydrogen bond formation with the polymer. The addition of the third polymer improved the stability but had a minor impact on dissolution.     

Visual micromethod for assay of fungal growth

A visual micromethod for measuring antifungal effects on germination and growth is described. The antifungal agent griseofulvin and the fungus Trichophyton mentagrophytes were used as materials to compare the micromethod with a standard assay based on dry mycelial weight. The micromethod was more sensitive than the weight method in detecting the minimum inhibitory concentration of griseofulvin (0.18 and 35 microgram/ml, respectively). At higher concentrations of griseofulvin (22.5 microgram/ml), the micromethod measured minimal fungal growth that was undetectable on a weight basis. The micromethod showed that griseofulvin does not change the number of spores forming germ tubes. Progressively severe alterations in fungal morphology occured as the concentration of griseofulvin was increased from 0.09 to 22.5 microgram/ml.     

The Occurrence of Translocated Antibiotics in Expressed, Plant Sap

The details of a convenient laboratory press are presented anda procedure by which plant sap is readily obtained is described.The pressing procedure has been shown to release 53–74per cent. of the total plant water as expressed sap. Preliminary observations on the uptake and translocation ofantibiotics by higher plants obtained by examining expressedsap are described. It is shown that the assay of expressed sapprovides a measure of the griseofulvin and chloramphenicol concentrationin leaves of plants grown in solutions of these antibioticsthat does not differ significantly from the value obtained bythe assay of water extracts. Extraction of leaves from plants grown in griseofulvin solutionswith organic solvents demonstrates a much greater antibioticcontent than is indicated by the assay of expressed sap. Toexplain this difference a ‘free’ antibiotic fraction,obtainable in expressed sap or water extracts, and a ‘bound’fraction recovered from the leaf only by organic solvent extractionare postulated.     

The anti-mitotic drug griseofulvin induces apoptosis of human germ cell tumor cells through a connexin 43-dependent molecular mechanism

Griseofulvin, a widely used antifungal antimitotic drug has been proposed as an anti-tumoral treatment by way of in vitro experiments. Recently, in vivo demonstration of griseofulvin efficacy against multiple myeloma in mice argues for its potential as therapeutics for cancer. Nevertheless, the molecular mechanisms by which griseofulvin disrupts cancerous cell progression are far from being understood. In the present study, we found that griseofulvin inhibits human germ cell tumor cell growth through activation of mitochondrial caspase pathway (caspase 9 and 3) leading to the activation of apoptosis rather than an alteration of cell proliferation. Strikingly, we demonstrated that griseofulvin triggered the expression level of connexin 43 (mRNA and protein), a well described tumor-suppressor gene, known to participate in apoptosis regulation. Consistently, together with microtubule instability, a mechanism classically associated with cell death in response to griseofulvin, we observed a disruption of connexin 43/tubulin association concomitant of an enhanced translocation of connexin 43, or an immunoreactive fragment of the protein, from the cytoplasm to the nucleus. Finally, by using siRNA approaches we demonstrated the requirement of connexin 43 in the apoptotic induction of griseofulvin on our tumor cell model. Altogether, these results described a new molecular mechanism connexin 43-dependent targeted by griseofulvin leading to apoptosis of human germ cell tumor cells.     

Griseofulvin-induced alterations in site of dividing nuclei and structure of septa in a dikaryon ofSchizophyllum commune

Summary Ultrastructural study of a dikaryon of the basidiomyceteSchizophyllum commune showed that treatment with griseofulvin affected the site of the dividing nuclei and the location and structure of the septa. The microtubules were considered to be the primary target of griseofulvin, since they participate in nuclear division and movement in the hyphae, and their assembly is known to be in other organisms than fungi inhibited by griseofulvin. It is pointed out that dikaryotic hyphae with two nuclei and a clamp connection per cell are more sensitive indicators of the effect of griseofulvin than homokaryotic hyphae, whose structure is less complex.