Detection of antifungal compounds in Polygonum ferrugineum Wedd. extracts by bioassay-guided fractionation. Some evidences of their mode of action.

ETHNOPHARMACOLOGICAL RELEVANCE: Polygonum ferrugineum Wedd. (Polygonaceae) is used to heal infected wounds and as antiseptic, antibiotic or antifungal in the traditional Argentinean medicine. The present investigation was carried out to evaluate the antifungal properties of different extracts of aerial parts of Polygonum ferrugineum, in order to give support to its ethnopharmacological use and to isolate the compounds responsible for the antifungal properties. The most active compounds were tested for their capacity of producing hyphae malformations, similar to those previously observed for crude extracts. MATERIALS AND METHODS: Agar Dilution Method (ADM) and Agar Overlay Bioautography (AOB) were used for bioassay-guided fractionation of the aerial part extracts against a panel of human opportunistic pathogenic fungi. The Neurospora crassa assay, followed by Optical Microscopy and Scanning Electron Microscopy observation, was used for studies of mechanisms of action. RESULTS: MeOH extract and DCM and Hex sub-extracts, but not Aq, EtOAc or BuOH ones possess antifungal activity. Of the seven isolated compounds, cardamonin 2 showed a selective inhibition of Epidermophyton floccosum with a very low MIC (=6.2 µg/mL) and pashanone 1 possessed moderate antifungal activity (MICs=25-50 µg/mL) but a broader spectrum of action. Chalcone 2, but not 1, induced swelling and shortening of the Neurospora crassa hyphae, similar as those caused by the crude DCM extract. CONCLUSIONS: The bioassay-guided fractionation of Polygonum ferrugineum DCM extract allowed the isolation of five active compounds. Among them, cardamonin 2 showed the highest antifungal activity and selectivity towards Epidermophyton floccosum; in addition, it induced Neurospora crassa malformations that are similar than those produced by the crude DCM extract. These results give additional support to the ethnopharmacological use of Polygonum ferrugineum as antifungal agent.

Antifungal activity of sakurasosaponin from the root extract of Jacquinia flammea.

The methanolic crude extract from the roots of Jacquinia flammea showed moderate antifungal activity against dermatophytes and very strong antifungal activity against Colletotrichum gloeosporioides. The bioassay-guided purification of the extract, using a combination of vacuum-liquid chromatography and high performance liquid chromatography, allowed the identification of sakurasosaponin as the main metabolite responsible for the antifungal activity.

Antifungal, cytotoxic and SAR studies of a series of N-alkyl, N-aryl and N-alkylphenyl-1,4-pyrrolediones and related compounds.

The synthesis, in vitro evaluation and SAR studies of 67 maleimides and derivatives acting as antifungal agents are reported. A detailed SAR study supported by theoretical calculations led us to determine that: an intact maleimido ring appears to be necessary for a strong antifungal activity, dissimilarly affected by the substituents in positions 2 and 3. The best activities were shown by 2,3-nonsubstituted followed by 2,3 dichloro- and 2-methyl-substituted maleimides. They all were fungicide rather than fungistatic enhancing the importance of their antifungal activity. 2,3-Dimethyl and 2,3-diphenyl-maleimides possessed marginal or null activity. The presence of a flexible connecting chain in N-phenylalkyl maleimides appears not to be essential for antifungal activity, although its length shows a correlation with the antifungal behavior, displaying maleimides with alkyl chains of n=3 and n=4 the best antifungal activities in most fungi. Different substituents on the benzene ring did not have a clear influence on the activity. Values of chemical potential properties as well as of energy do not sufficiently discriminate between active and inactive compounds. Nevertheless, it was found that, although logP alone is not strong enough to properly predict the antifungal activity, the comparison of its values for compounds within the same sub-type, showed an enhancement of antifungal activity along with an increment of lipophilicity. In addition, the LUMO's electronic clouds of the highly active compounds showed to be concentrated on the imido ring, indicating that their carbon atoms are potential sites for nucleophilic attack. Same results were obtained from MEPs. Most of the active compounds did not show cytotoxic activity against human cancer cell lines and no one possessed hemolytic activity, indicating that their activity is selective to pathogenic fungi and that they are not toxic at MIC concentrations.

Influence of plant part, season of collection and content of the main active constituent, on the antifungal properties of Polygonum acuminatum Kunth.

ETHNOPHARMACOLOGICAL RELEVANCE: Polygonum acuminatum Kunth. (Polygonaceae) is used to heal infected wounds and as antifungal in the traditional Argentinean medicine. AIM OF THE STUDY: The present investigation was carried out to evaluate the antifungal properties of aerial parts of Polygonum acuminatum, in order to give support to its ethnopharmacological use as antifungal agent and to isolate the compound(s) responsible for the antifungal properties. The influence of the plant part used, the season of the year and a study of the correlation of the antifungal activity with the content of the main active constituent were investigated too, with the aim of contributing to determine the most suitable plant extract and season of the year for achieving the best antifungal properties for Polygonum acuminatum traditional use. MATERIALS AND METHODS: For the antifungal evaluation, the microbroth dilution assay recommended by the Clinical and Laboratory Standards Institute (CLSI, formerly NCCLS) was used against a panel of human opportunistic and pathogenic fungi. Bioassay-guided fractionation allowed us to isolate the compounds responsible for the antifungal activity. GC-MS was used to quantify the main component in the different extracts. For the statistical analysis, ANOVA test for analyses of variance followed by the Tukey test of Multiple Comparisons were used. The correlations between content of the antifungal compound and antifungal activity, were calculated with the Spearman Correlation Coefficient. RESULTS: Aerial parts (A) of Polygonum acuminatum showed to possess antifungal properties against yeasts as well as dermatophytes but not against Aspergillus spp. From the most active extract (ADCM), polygodial, isopolygodial, drimenol and confertifolin were isolated, possessing polygodial a broader spectrum of action and lower MICs than the rest of compounds. Among the different parts, leaves (L), stems (S) and fruits (F), that constitute the aerial parts of Polygonum acuminatum, (L) showed to possess the best activities, compared to (S) and (F). The analysis of the content of polygodial in the LHex, LDCM, LEtOAC, LMeOH extracts collected in Summer, Autumn, Winter and Spring showed that LDCM of all seasons possessed higher percentages of this sesquiterpene than the rest of extracts. Among the LDCM of different seasons, that of Autumn was the most concentrated in polygodial. The correlation between content of polygodial with antifungal behavior of the different extracts, showed that LDCM of Autumn contains the highest content of polygodial and concomitantly the lowest MICs. CONCLUSION: The ethnopharmacological use of Polygonum acuminatum aerial parts in the Argentinean traditional medicine for ailments related to fungal infections is supported by the results obtained in this investigation. From the obtained results, LDCM of Autumn, possessing the highest content of polygodial and the lowest MICs, appeared to be the most suitable extract for being used as antifungal in the traditional medicine. Nevertheless, if some other plant collection of another season different from Autumn is available, a LDCM extract would be the better option, because it contains a higher amount of polygodial compared to LHex, LEtOAc or LMeOH and therefore, a better antifungal activity can be expected.

Relation between lipophilicity of alkyl gallates and antifungal activity against yeasts and filamentous fungi.

The antifungal activity of a complete series of 15 n-alkyl gallates and six analogues acting against a representative panel of opportunistic pathogenic fungi was studied in order to analyze their role in: the importance of the fungi tested, the importance of the hydroxyls, the influence of the chain length and the hydrophobicity of the compounds. It was demonstrated that dermatophytes were the most susceptible species and that hydroxyls appear to be necessary but not sufficient for the activity. When the logP of each gallate was calculated and related to the different values of MIC against Microsporum gypseum it was observed that hexyl, heptyl, octyl and nonyl gallates exhibit a significant positive deviation from the curve corresponding to a polynomial equation obtained for the other gallates. This suggests that these compounds have a further mode of action besides their hydrophobicity, possibly the inhibition of some enzyme involved in ergosterol biosynthesis.

Antifungal activity of some Brazilian Hypericum species.

Crude methanolic extracts and fractions from the aerial parts of seven species of Hypericum (H. caprifoliatum Cham. and Schltdl., H. carinatum Griseb., H. connatum Lam., H. ternum A. St.-Hil., H. myrianthum Cham. and Schltdl., H. piriai Arechav. and H. polyanthemum Klotzsch ex Reichardt) growing in southern Brazil were analyzed for their in vitro antifungal activity against a panel of standardized and clinical opportunistic pathogenic yeasts and filamentous fungi, including dermatophytes, by the agar dilution method. Chloroform and hexane extracts of H. ternum showed the greatest activity among extracts tested.

Antifungal activity of fractions and two pure compounds of flowers from Wedelia paludosa (Acmela brasiliensis) (Asteraceae).

Wedelia paludosa (Acmela brasiliensis) (Asteraceae), a traditionally used native Brazilian medicinal plant, showed antifungal activity against dermatophytes in dilution tests. The hexane, dichloromethane and butanol fractions displayed activity against Epidermophyton floccosum, Trichophyton rubrum and Trichophyton mentagrophytes, with minimal inhibitory concentrations between 250 and 1000 microg/mL. Two pure compounds, identified as kaurenoic acid (1) and luteolin (2), also showed activity against these dermatophytes.

Antimicrobial activity of plants used in traditional medicine of San Juan province, Argentine.

Eighteen extracts from Acaena magellanica, Baccharis grisebachii, Ephedra breana, Oxalis erythrorhiza, Pachylaena atriplicifolia and Satureja parvifolia were assessed for antimicrobial activity against bacteria and fungi with the agar dilution method. The hexane (H) and dichloromethane (DCM) extracts of B. grisebachii and O. erythrorhiza showed the broadest spectrum of action against fungi, inhibiting all of the tested dermatophytes with MICs ranging from < or =25 to < or =1000 microg/ml. Trichophyton rubrum was the most susceptible species and Cryptococcus neoformans was inhibited only by the DCM extract of B. grisebachii with MIC of 600 microg/ml. Regarding the antibacterial activity, H and DCM extracts of B. grisebachii as well as the DCM of O. erythrorhiza, were active on methicillin-resistant and methicillin-sensitive Staphylococcus aureus with MIC from < or =125 to < or =500 microg/ml. The DCM extract of B. grisebacchii was more active against methicillin-resistant than methicillin-sensitive strains.

In vitro antifungal evaluation and structure-activity relationships of a new series of chalcone derivatives and synthetic analogues, with inhibitory properties against polymers of the fungal cell wall.

Here we report the synthesis, in vitro antifungal evaluation and SAR study of 41 chalcones and analogues. In addition, all active structures were tested for their capacity of inhibiting Saccharomyces cerevisiae beta(1,3)-glucan synthase and chitin synthase, enzymes that catalyze the synthesis of the major polymers of the fungal cell wall.

Inhibitory activity of xanthine-oxidase and superoxide scavenger properties of Inga verna subsp. affinis. Its morphological and micrographic characteristics.

Hexane, dichloromethane and ethanolic extracts of Inga verna subsp. affinis were evaluated as inhibitors of xanthine-oxidase and as scavengers of the superoxide produced by the action of the enzyme. Ethanolic but not hexane and dichloromethane extracts showed inhibitory properties of xanthine-oxidase (IC50=27.3 microg/ml) with an additional superoxide scavenging capacity (IC50=12.7 microg/ml). The antioxidant potential was confirmed with the free radical 1,1-diphenyl-2-picryl hydrazyl (DPPH) assay, which showed that the ethanolic extract scavenges 50% DPPH free radicals at 11.6 microg/ml. HPLC study of the phenol content of the active extract, revealed the presence of ellagic and gallic acids as its main constituents. The main morphological and micrographic characteristics of Inga verna subsp. affinis are described in this paper too, in order to aid in its inequivocal identification since Inga spp. are noted for their morphological variation, which makes taxonomic classification very difficult.

Inhibitors of the fungal cell wall. Synthesis of 4-aryl-4-N-arylamine-1-butenes and related compounds with inhibitory activities on beta(1-3) glucan and chitin synthases.

As part of our project devoted to the search for antifungal agents, which act via a selective mode of action, we synthesized a series of new 4-aryl- or 4-alkyl-N-arylamine-1-butenes and transformed some of them into 2-substituted 4-methyl-tetrahydroquinolines and quinolines by using a novel three-step synthesis. Results obtained in agar dilution assays have shown that 4-aryl homoallylamines not possessing halogen in their structures, tetrahydroquinolines and quinolines, display a range of antifungal properties in particular against Epidermophyton floccosum and Microsporum canis. Regarding the mode of action, all active compounds showed in vitro inhibitory activities against beta(1-3) glucan-synthase and mainly against chitin-synthase. These enzymes catalyze the synthesis of beta(1-3) glucan and chitin, respectively, major polymers of the fungal cell wall. Since fungal but not mammalian cells are encased in a cell wall, its inhibition may represent a useful mode of action for these antifungal compounds.

In vitro evaluation of antifungal properties of phenylpropanoids and related compounds acting against dermatophytes.

Thirty-four arylpropanoids and related compounds were evaluated in vitro for antifungal properties. Among them, 22 phenyl-, 4 naphthyl-, and 4 phenanthrylpropanoids; naphthalene; phenanthrene; and 2-chloro-1-hexyl-1-propanone were tested against dermatophytes by the agar dilution method. alpha-Halopropiophenones exhibited a broad spectrum of activities against Microsporum canis, Microsporum gypseum, Trichophyton mentagrophytes, Trichophyton rubrum, and Epidermophyton floccosum, with MIC values between 0.5 and >50 microg/mL. Keto, alcohol, and alpha-haloketo propyl derivatives of naphthalene and phenanthrene also showed very good activity against all dermatophytes tested, clearly showing that in these series, a halogen atom is not necessary for activity. Phenanthryl derivatives were more active (MICs, 3-20 microg/mL) than naphthyl ones (MICs, 3-50 microg/mL). A structure-activity relationship study was carried out and aided in establishing the structural requirements of arylpropanoids for antifungal activities. Because dermatophytes are a group of fungi that characteristically infect the keratinized areas of the body, these new series of antifungal compounds open the possibility of discovering new topical antifungal drugs for the treatment of dermatomycoses, which are frequently very difficult to eradicate.

In vitro antifungal evaluation and studies on the mode of action of xanthoxyline derivatives.

This study describes the fungistatic effect of xanthoxyline (CAS 90-24-4) and its derivatives against a panel of yeasts, filamentous fungi and dermatophytes, by using the agar dilution method. Results indicated that simple structural modifications led to more potent derivatives, especially in relation with dermatophytes. The most active compound tested (10), which is a benzenesulphonyl derivative, was 12-fold more potent than xanthoxyline itself against Trichophyton rubrum. The evaluation of the mode of action with the whole cell Neurospora crassa assay, suggested that some selected compounds may be acting by the inhibition of fungal cell-wall polymers synthesis or assembly.

In vitro studies on mode of action of antifungal 8.O.4'-neolignans occurring in certain species of Virola and related genera of Myristicaceae.

Neutral racemic antifungal alcohols of 8.O.4'-neolignan type, were evaluated for inhibitory activity towards the fungal cell wall, using the whole cell Neurospora crassa hyphal growth inhibition assay. Results strongly suggested that these compounds could act by inhibiting cell wall polymer synthesis or assembly. Active compounds were tested for their inhibitory activities against (1,3)-beta-glucan synthase, an enzyme that catalyzes the synthesis of the major wall polymer (1,3)-beta-glucan. Although these compounds were found to be inhibitors of the enzyme (inhibition ranging between 2 and 72% at 250 micro/ml), comparison of these results with those from agar dilution assays, allow us to infer that these compounds do not act via the inhibition of glucan synthase. In addition, ketones with same pattern of substitution as alcohols, which have no antifungal properties in agar dilution assays, still displayed similar glucan synthase inhibition.

In vitro antifungal evaluation and studies on mode of action of eight selected species from the Argentine flora.

Twenty nine extracts belonging to eight species of the Argentine flora reported as antifungal in folk medicine, were assayed for antifungal properties by using the agar dilution method, against a panel of yeasts, filamentous fungi as well as dermatophytes. Nine extracts belonging to six species, exhibited a broad spectrum of activity against Microsporum cants, Microsporum gypseum, Trichophyton mentagrophytes, Trichophyton rubrum and Epidermophyton floccosum, with MICs ranging from 25 to 900 µg/ml. A dichloromethanis extract of Polygonum ferrugineum was the most active extract with MICs from 25-50 µg/ml. To gain an insight into the mode of action of the active extracts they were evaluated for their inhibitory activities toward the fungal cell wall, using the whole cell Neurospora crassa hyphal growth inhibition agar diffusion assay. A hazy zone around the paper disk strongly suggested that the dichloromethane extracts from aerial parts of Polygonum punctatum, Polygonum ferrugineum and the bark of Luehea divaricata acted by inhibiting polymer synthesis or assembly of the cell wall. The clear zone of inhibition produced by the dichloromethane and methanol antifungal extracts of Xanthium spinosum could be ascribed to the fact that these extracts have another effect on fungal cells in addition to inhibition of cell walls.

In vitro evaluation of antifungal properties of 8.O.4'-neolignans.

Eighteen racemic 8.O.4'-neolignans with six different substitution patterns in rings A and B, in their ketone and in their erythro and threo alcoholic forms, were evaluated for antifungal activity by the agar dilution method. Only the alcohols exhibited a broad spectrum of activities against Microsporum canis, Microsporum gypseum, Tricophyton mentagrophytes, Tricophyton rubrum, and Epidermophyton floccosum. (+/-)-erythro-3,4-(methylenedioxy) -7-hydroxy-1'-allyl-3',5'-dimethoxy-8.O.4'-neolignan (11) was the most active compound in the series, and E. floccosum was the most susceptible species.