Antifungal activity of extracts and purified saponins from the rhizomes of Chamaecostus cuspidatus against Candida and Trichophyton species.

AIM: To evaluate the antifungal activity of extracts of Chamaecostus cuspidatus against Candida and Trichophyton species. METHODS AND RESULTS: Crude ethanol extracts of leaves, stems and rhizomes were prepared and evaluated for antimicrobial activity. Only the rhizomes extract (RE) showed antifungal activity but had no inhibitory effect against bacteria (Staphylococcus aureus and Escherichia coli). The RE was then submitted to liquid-liquid partition with hexane (Hex), dichloromethane, chloroform, ethyl acetate and water. The Hex fraction (Hex Fr) from the RE was found to have the best antifungal effect. Three known saponins were isolated from the Hex Fr, of which two (dioscin and aferoside A) showed good antifungal activity. In addition, Hex Fr and the two bioactive compounds had no antibacterial effect, but exhibited fungicidal activity, caused significant changes in the morphology of the fungal cells and showed anti-Candida albicans biofilm activity. Finally, the bioactive plant products presented greater selectivity for fungal cells over normal human cells. CONCLUSIONS: The rhizomes of C. cuspidatus have bioactive saponins that function as effective antifungals against Candida and Trichophyton species, and have antibiofilm activity against C. albicans. SIGNIFICANCE AND IMPACT OF THE STUDY: Chamaecostus cuspidatus REs may have potential clinical application towards the management of superficial mycoses caused by Candida and Trichophyton species.

Monitoring of volatile and non-volatile urban air genotoxins using bacteria, human cells and plants.

Urban air contains many mutagenic pollutants. This research aimed to investigate the presence of mutagens in the air by short-term mutagenicity tests using bacteria, human cells and plants. Inflorescences of Tradescantia were exposed to air in situ for 6h, once a month from January to May, to monitor volatile compounds and micronuclei frequency was computed. On the same days PM10 was collected continuously for 24h. Half of each filter was extracted with organic solvents and studied by means of the Ames test, using Salmonella typhimurium TA98 and TA100 strains, and the comet assay on human leukocytes. A quarter of each filter was extracted with distilled water in which Tradescantia was exposed. PM10 concentration was particularly high in the winter season (> 50 µg/m(3)). In situ exposure of inflorescences to urban air induced a significant increase in micronuclei frequency at all the sites considered, but only in January (p < 0.01). Aqueous extracts collected in January and February induced genotoxic effects in Tradescantia exposed in the laboratory (p < 0.01). Ames test showed that organic extracts of winter urban air were able to induce genetic mutations in S. typhimurium TA98 strain (± S9), but not in TA100 strain, with a revertants/plate number nine times higher than the negative control. Comet assay showed that winter extracts were more toxic and genotoxic than spring extracts. All the mutagenicity tests performed confirmed that urban air in North Italy in winter contains both volatile and non-volatile genotoxic substances able to induce genetic damage in bacteria, human cells and plants.

In vitro activity of hypnophilin from Lentinus strigosus: a potential prototype for Chagas disease and leishmaniasis chemotherapy.

Hypnophilin and panepoxydone, terpenoids isolated from Lentinus strigosus, have significant inhibitory activity on Trypanosoma cruzi trypanothione reductase (TR). Although they have similar TR inhibitory activity at 10 µg/mL (40.3 µM and 47.6 µM for hypnophilin and panepoxydone, respectively; ~100%), hypnophilin has a slightly greater inhibitory activity (~71%) on T. cruzi amastigote (AMA) growth in vitro as well as on in vitro phytohemagglutinin (PHA)-induced peripheral blood mononuclear (PBMC) proliferation (~70%) compared to panepoxydone (69% AMA inhibition and 91% PBMC inhibition). Hypnophilin and panepoxydone at 1.25 µg/mL had 67% inhibitory activity onLeishmania (Leishmania) amazonensis amastigote-like (AMA-like) growth in vitro. The panepoxydone activity was accompanied by a significant inhibitory effect on PHA-induced PBMC proliferation, suggesting a cytotoxic action. Moreover, incubation of human PBMC with panepoxydone reduced the percentage of CD16(+) and CD14(+) cells and down-regulated CD19(+), CD4(+) and CD8(+) cells, while hypnophilin did not alter any of the phenotypes analyzed. These data indicate that hypnophilin may be considered to be a prototype for the design of drugs for the chemotherapy of diseases caused by Trypanosomatidae.

In vitro activity of hypnophilin from Lentinus strigosus: a potential prototype for Chagas disease and leishmaniasis chemotherapy

Hypnophilin and panepoxydone, terpenoids isolated from Lentinus strigosus, have significant inhibitory activity onTrypanosoma cruzi trypanothione reductase (TR). Although they have similar TR inhibitory activity at 10 μg/mL (40.3 μM and 47.6 μM for hypnophilin and panepoxydone, respectively; ~100 percent), hypnophilin has a slightly greater inhibitory activity (~71 percent) on T. cruzi amastigote (AMA) growth in vitro as well as on in vitro phytohemagglutinin (PHA)-induced peripheral blood mononuclear (PBMC) proliferation (~70 percent) compared to panepoxydone (69 percent AMA inhibition and 91 percent PBMC inhibition). Hypnophilin and panepoxydone at 1.25 μg/mL had 67 percent inhibitory activity onLeishmania (Leishmania) amazonensis amastigote-like (AMA-like) growth in vitro. The panepoxydone activity was accompanied by a significant inhibitory effect on PHA-induced PBMC proliferation, suggesting a cytotoxic action. Moreover, incubation of human PBMC with panepoxydone reduced the percentage of CD16+ and CD14+ cells and down-regulated CD19+, CD4+ and CD8+ cells, while hypnophilin did not alter any of the phenotypes analyzed. These data indicate that hypnophilin may be considered to be a prototype for the design of drugs for the chemotherapy of diseases caused by Trypanosomatidae.

Alcohol and coffee drinking and smoking habit among subjects with HCV infection.

BACKGROUND/AIMS: The aims were to estimate among patients with hepatitis C virus (HCV) infection the prevalence of alcohol and coffee intake and smoking habit, the reliability of these self-reported data and the possible change of patients' habit after their first contact with a Viral Hepatitis Service. METHODS: 229 patients were initially interviewed personally at the Viral Hepatitis Service and after 6 months they were re-interviewed by phone in regard to their alcohol, coffee drinking and smoking habits. RESULTS: Alcohol drinkers were 55.5% of males and 35.3% of females. Most subjects drank coffee daily, both men (90.0%) and women (84.9%). The proportion of current smokers was higher in males (43.6%) than females (26.9%). We found a fair to good reliability of self-reported data regarding patients' habits, alcohol and coffee intake, and number of cigarettes smoked daily. We observed a statistically significant decrease in alcohol and coffee intake and cigarettes smoked between baseline and follow-up interviews. CONCLUSION: We found a fairly high proportion of HCV-infected patients who regularly drink alcohol and coffee beverages and smoke cigarettes, especially among males. The reliability of self-reported data on these habits seems satisfactory. More decisive action to modify these habits, especially alcohol intake, is required in these patients.

Antifungal activities of compounds isolated from Piper abutiloides Kunth.

Piperaceae is a family of tropical plants known to have antifungal, antibacterial, tumour-inhibitory, antiviral, antioxidant, molluscicidal and leishmanicidal activities. In this work, extracts and fractions from aerial parts of Piper abutiloides (Piperaceae), a traditional medicinal plant, were evaluated against the fungal species Candida albicans, C. parapsilosis, C. krusei, C. glabrata, C. tropicalis, Cryptococcus neoformans and Sporothrix schenckii. The results have shown that the antifungal activity of this plant can be concentrated in the hexanic fraction after partitioning its hydroalcoholic extract between hexane and 90% aqueous methanol. The chromatographic fractionation of the bioactive part was monitored with a bioautographic assay using C. glabrata, and allowed the isolation of three antifungal compounds: pseudodillapiol, eupomatenoid-6 and conocarpan. These compounds presented different potencies against the fungi tested, with the strongest effect being observed for eupomatenoid-6 against C. glabrata, which presented a minimal inhibitory concentration value of 0.3 microg spot(-1). Conocarpan showed antifungal activity without apparent cytotoxic effect on normal human lymphocytes, as assessed by the proliferation assay with human peripheral blood mononuclear cells stimulated with phytohaemaglutinin. This work reveals for the first time the occurrence of these compounds in P. abutiloides and justifies further studies to clarify their mechanisms of action.

Evaluation of chlorite and chlorate genotoxicity using plant bioassays and in vitro DNA damage tests.

In the last few years chlorine dioxide has been increasingly used for disinfecting drinking water in many countries. Although it does not react with humic substances, chlorine dioxide added to water is reduced primarily to chlorite and chlorate ions, compounds that are under investigation for their potential adverse effects on human health. The aim of this research was to study the genotoxicity of chlorite and chlorate and their mixtures. The end-points included two plant tests (chromosomal aberration test in Allium cepa and micronucleus assay in Tradescantia, carried out at different times of exposure) and two genotoxicity tests in human HepG2 cells (comet assay and cytokinesis-blocked micronucleus test). Preliminary toxicity tests were carried out for both plant and HepG2 assays. The results showed that chlorite and chlorate are able to induce chromosomal damage to plant systems, particularly chromosomal aberrations in A. cepa root tip cells, even at concentrations lower than the limit established by Italian normative law and WHO guidelines. In HepG2 cells increased DNA damage was only observed for chlorate at the lowest concentration. No increase in micronuclei frequency was detected in any of the samples tested in human HepG2 cells.

Allium cepa chromosome aberration and micronucleus tests applied to study genotoxicity of extracts from pesticide-treated vegetables and grapes.

The Allium cepa assay is an efficient test for chemical screening and in situ monitoring for genotoxicity of environmental contaminants. The test has been used widely to study genotoxicity of many pesticides revealing that these compounds can induce chromosomal aberrations in root meristems of A. cepa. Pesticide residues can be present in fruit and vegetables and represent a risk for human health. The mutagenic and carcinogenic action of herbicides, insecticides and fungicides on experimental animals is well known. Several studies have shown that chronic exposure to low levels of pesticides can cause birth defects and that prenatal exposure is associated with carcinogenicity. This study evaluated the potential application of plant genotoxicity tests for monitoring mutagens in edible vegetables. The presence of pesticides and genotoxic compounds extracted from 21 treated vegetables and eight types of grapes sampled from several markets in Campania, a region in Southern Italy, was monitored concurrently. The extracts were analysed for pesticides by gas chromatography and high-performance liquid chromatography, and for genotoxicity using two plant tests: the micronucleus test and the chromosomal aberration test in A. cepa roots. Thirty-three pesticides were detected, some of which are not approved. Genotoxicity was found in some of the vegetables and grapes tested. Allium cepa tests proved to be sensitive in monitoring genotoxicity in food extracts. The micronucleus test in interphase cells gave a much higher mutagenicity than the chromosomal aberration test in anaphase-telophase cells.

Genotoxicity of surface water treated with different disinfectants using in situ plant tests.

Disinfection of surface drinking water, in particular water chlorination, results in many by-products with potential genotoxic and/or carcinogenic activity. In the present study, we evaluated the genotoxicity of surface water after treatment with different disinfectants by means of in situ plant genotoxicity assays (micronucleus and chromosomal aberration tests) which can detect both clastogenic and aneugenic effects. The study was carried out at a pilot plant using lake water after sedimentation and filtration. This water supplied four stainless steel basins: three basins were disinfected with sodium hypochlorite, chlorine dioxide, and peracetic acid and the fourth basin containing untreated lake water was used as a control. Plants were exposed in situ in the basins. The study was carried out using water collected in different seasons over a period of about 1 year in order to assess the treatments in different physical and chemical lake water conditions. The micronucleus test in root cells of Vicia faba (Vicia faba/MCN test) revealed genotoxicity in many samples of disinfected water. The micronucleus test in Tradescantia pollen cells and the chromosome aberration test in root cells of Allium cepa showed genotoxic effects only in some disinfected samples, but also revealed genotoxicity in raw water. The results of the study indicated that the Vicia faba/MCN test was the most sensitive plant assay for disinfected water and that peracetic acid disinfection produced similar or lower genotoxicity than sodium hypochlorite or chlorine dioxide treatment.

Evaluation of the migration of mutagens/carcinogens from PET bottles into mineral water by Tradescantia/micronuclei test, Comet assay on leukocytes and GC/MS.

This study monitored the release of mutagenic/carcinogenic compounds into mineral water (natural and carbonated) from polyethylene terephthalate (PET) bottles, using a plant mutagenicity test which reveals micronuclei formation in Tradescantia pollen cells (Trad/MCN test), a DNA damage assay (Comet assay) on human leukocytes and gas chromatography/mass spectrometry (GC/MS) for the characterisation of migrants. The water samples were collected at a bottling plant and stored in PET bottles for a period ranging from 1 to 12 months. Every month some samples were randomly collected and lyophilised, the residual powders were extracted with organic solvents and then analysed by GC/MS and tested for DNA damage in human leukocytes, or reconstituted with distilled water to obtain concentrates for the exposure of Tradescantia inflorescences. Micronuclei increase in pollen was found only in natural mineral water stored for 2 months. DNA-damaging activity was found in many of the natural and carbonated water samples. Spring water was negative in the plant micronuclei test and the Comet assay, whereas distributed spring water showed DNA-damaging effects, suggesting a possible introduction of genotoxins through the distribution pipelines. GC/MS analysis showed the presence in mineral water of di(2-ethylhexyl)phthalate, a nongenotoxic hepatocarcinogenic plasticizer, after 9 months of storage in PET bottles.

Polygodial, the fungitoxic component from the Brazilian medicinal plant Polygonum punctatum.

Polygonum punctatum (Polygonaceae) is an herb known in some regions of Brazil as "erva-de-bicho" and is used to treat intestinal disorders. The dichloromethane extract of the aerial parts of this plant showed strong activity in a bioautographic assay with the fungus Cladosporium sphaerospermum. The bioassay-guided chemical fractionation of this extract afforded the sesquiterpene dialdehyde polygodial as the active constituent. The presence of this compound with antibiotic, anti-inflammatory and anti-hyperalgesic properties in "erva-de-bicho" may account for the effects attributed by folk medicine to this plant species.

Biological screening of Brazilian medicinal plants.

In this study, we screened sixty medicinal plant species from the Brazilian savanna ("cerrado") that could contain useful compounds for the control of tropical diseases. The plant selection was based on existing ethnobotanic information and interviews with local healers. Plant extracts were screened for: (a) molluscicidal activity against Biomphalaria glabrata, (b) toxicity to brine shrimp (Artemia salina L.), (c) antifungal activity in the bioautographic assay with Cladosporium sphaerospermum and (d) antibacterial activity in the agar diffusion assay against Staphylococcus aureus, Escherichia coli, Bacillus cereus and Pseudomonas aeruginosa. Forty-two species afforded extracts that showed some degree of activity in one or more of these bioassays.

A cytotoxic diterpene from Alomia myriadenia.

An extract of the aerial parts from Alomia myriadenia Schultz-Bip. ex Baker (Asteraceae) showed significant cytotoxicity against a panel of human cancer cell lines in a screening of extracts from Brazilian Atlantic Forest plant species. Employing a bioassay-linked HPLC-electrospray/MS method, followed by semi-preparative HPLC, the active component was isolated and characterized as a mixture of epimers of the labdane diterpene 12S,16-dihydroxy-ent-labda-7,13-dien-15,16-olide.

Enhancement of micronuclei frequency in the Tradescantia/micronuclei test using a long recovery time.

The Tradescantia/micronuclei test (TRAD/MCN) is a well-validated test for monitoring environmental genotoxicants. These pollutants induce at the early meiotic stage of pollen mother cells chromosome fragments which become micronuclei at the tetrad stage. The standard test protocol requires some hours of exposure of the inflorescences and a recovery time of about 24 hours to reach the early tetrad stage. Since the recovery period represents a critical step of the TRAD/MCN, experiments were performed to establish its length in plants of clone #4430 of the hybrid T. hirsutiflora x T. subacaulis which is widely used in environmental monitoring. The aim of the present research was to ascertain the exact duration of recovery time in order to improve the sensitivity of the TRAD/MCN test. First, studies were performed to select the flowers at the beginning of the meiosis, and then anthers were sampled and studied for a period of 48-86 hours. The complete meiosis in the plants examined required about 80 hours. Second, exposure to genotoxic substances followed by different recovery times was carried out to demonstrate that effectiveness of the TRAD/MCN test is closely related to the duration of the recovery time. The test was carried out by exposing inflorescences to known mutagens (sodium azide and maleic hydrazide) for six hours followed by different recovery times (24-72 hours). The results showed that the frequency of micronuclei in the pollen mother cells increased with the length of the recovery time.

A new cyclohexadecane derivative from Trixis vauthieri DC (Asteraceae).

The dichloromethane-methanol extract from the fresh leaves of Trixis vauthieri DC (Asteraceae) afforded trixol, a new cyclohexadecane derivative. The structural elucidation of this new compound, with a novel skeleton, was based on NMR studies of the natural product nd its derivatives.

Screening of Asteraceae (Compositae) plant extracts for larvicidal activity against Aedes fluviatilis (Diptera:Culicidae).

Ethanol extracts of 83 plants species belonging to the Asteraceae (Compositae) family, collected in the State of Minas Gerais, Brazil, were tested for larvicidal activity against the mosquito Aedes fluviatilis--Diptera: Culicidae). The extract from Tagetes minuta was the most active with a LC90 of 1.5 mg/l and LC50 of 1.0 mg/l. This plant has been the object of several studies by other groups and its active components have already been identified as thiophene derivatives, a class of compounds present in many Asteraceae species. The extract of Eclipta paniculata was also significantly active, with a LC90 of 17.2 mg/l and LC50 of 3.3 mg/l and no previous studies on its larvicidal activity or chemical composition could be found in the literature. Extracts of Achryrocline satureoides, Gnaphalium spicatum, Senecio brasiliensis, Trixis vauthieri, Tagetes patula and Vernonia ammophila were less active, killing more than 50% of the larvae only at the higher dose tested (100 mg/l).

Antiplasmodial triterpene from Vernonia brasiliana.

The hexane extract from leaves of Vernonia brasiliana (L.) Druce (Compositae) was active in vitro against Plasmodium falciparum and in vivo in mice infected with Plasmodium berghei. This extract was subjected to a bioassay-guided fractionation protocol based on the in vitro model. Lupeol was identified as a compound responsible for the activity, inhibiting the P.falciparum growth by 45% when tested at 25 micrograms/ml. However, this triterpene was inactive in vivo when 15 mg/kg were administered per os during four consecutive days to mice infected with P.berghei. beta-Amyrin and germanicol, isolated from the same fraction that yielded lupeol, were inactive in the in vitro assay.

A diterpene from Mikania obtusata active on Trypanosoma cruzi.

The diterpene ent-kaur-16-en-19-oic acid (1) was identified as the trypanocidal component of the ethanolic extract from Mikania obtusata D. C. (Asteraceae). This compound presents an IC50 of 0.5 mg/ml (1.66 mM) against the trypomastigote blood form of the Trypanosoma cruzi, the causative agent of Chagas' disease (American trypanosomiasis).

Brine shrimp lethality assay as a prescreening system for anti-Trypanosoma cruzi activity.

With the aim of finding an acceptable method for selecting plant extracts to be assayed against the infective blood form of Trypanosoma cruzi, the causative agent of Chagas' disease (American trypanosomiasis), two different strategies were compared: a) screening only medicinal species and b) pre-screening random collected species in the brine shrimp lethality assay (BSLA). Fifty-two plants belonging to the Asteraceae family, including eighteen medicinal species, were collected and their ethanol extracts assayed against both T. cruzi and Artemia salina (brine shrimp). The proportion of trypanocidal extracts among the medicinal species and among the random collection did not differ significantly. On the other hand, the proportion of trypanocidal extracts among those that presented LC(50) of less than 100 ppm to A. salina was four times higher than among the medicinal species.