The Fusarium mycotoxins enniatins and beauvericin cause mitochondrial dysfunction by affecting the mitochondrial volume regulation,oxidative phosphorylation and ion homeostasis

The mechanisms of cell toxicity of mycotoxins of the enniatin family produced by Fusarium sp. enniatin B, a mixture of enniatin homologues (3% A, 20% A₁, 19% B, 54% B1) and beauvericin, were investigated. In isolated rat liver mitochondria, exposure to submicromolar concentrations of the enniatin mycotoxins depleted the mitochondrial transmembrane potential, uncoupled oxidative phosphorylation, induced mitochondrial swelling and decreased calcium retention capacity of the mitochondria. The mitochondrial effects were strongly connected with the potassium (K⁺) ionophoric activity of the enniatins. The observed enniatins induced K⁺ uptake by mitochondria. This shows that the enniatins acted as ionophores highly selective for potassium ions. The effects were observed in potassium containing media whereas less or no effect remained to be observed when K⁺ was partially or totally replaced by isomolar concentrations of Na⁺. The rank order of enniatin induced mitochondrial impairment was beauvericin > enniatin mixture > enniatin B. Exposure to the enniatins depleted the mitochondrial membrane potential also in intact human neural (Paju), murine insulinoma (Min-6) cells as well as boar spermatozoa. Exposure to enniatin B in media with physiological (4 mM) or low (<1 mM) but not in high (60 mM) external concentration of K⁺ induced hyperpolarization of the spermatozoal plasma membrane indicating enniatin that catalysed efflux of the cytosolic K⁺ ions. These results indicate that the cellular toxicity targets of the enniatin mycotoxins are the mitochondrion and the homeostasis of potassium ions.     

Structural studies on minor enniatins from Fusarium sp. VI 03441: Novel N-methyl-threonine containing enniatins

A strain of a yet unidentified Fusarium sp. produced in addition to enniatins A1, B and B1 a number of minor enniatins. The strain formed a well supported clade with strains identified as Fusarium acuminatum (Gibberella acuminata) in phylogenetic analyses using the TEF-1α gene sequences. Two of the minor enniatins were easily recognised as hydroxylated species on the basis of their fragment ion spectra. The hydroxylation could be traced to one of the amino acid moieties using multiple-stage ion trap mass spectrometry. Different approaches for acetylation of the isolated compounds and complete hydrolysis supported the elucidation of the amino acid moiety as 3-hydroxy-2-methylamino-butyric acid, which is equivalent with N-methyl-threonine. The primary structures of the two enniatins were tentatively determined to be cyclo[Hiv-N-Me-Val-Hiv-N-Me-Val-Hiv-N-Me-Thr] and cyclo[Hiv-N-Me-Leu-Hiv-N-Me-Val-Hiv-N-Me-Thr]. The two depsipeptides represent new analogues and were named enniatin P1 and P2, respectively.     

Identification of cytotoxic principles from Fusarium avenaceum using bioassay-guided fractionation

The cytotoxicity of extracts from rice cultures of five Fusarium avenaceum strains against the porcine epithelial kidney cell-line PK-15 was investigated using the Alamar Blue™ assay. After the identification of known fungal metabolites, cytotoxic extracts were fractionated using semi-preparative reversed-phase HPLC and normal phase LC, and the fractions were tested for cytotoxicity. In this way, two different groups of metabolites were identified as the major cytotoxic principles of the extracts. High concentrations of enniatins, especially enniatins B and B1, inhibited the metabolic activity of PK-15 cells. Furthermore, an unidentified metabolite, produced in high amounts by a strain that produced relatively small amounts of enniatins, was also found to be cytotoxic to PK-15 cells. This study shows that enniatins, a group of cyclic depsipeptides, which have been ignored as significant contributors to the toxicity of fungal extracts, may account for most of the observed effect for F. avenaceum.     

Antifungal effects of the bioactive compounds enniatins A, A1, B, B1

To produce enniatin (ENs), Fusarium tricinctum CECT 20150 was grown in a liquid medium of potato (PDB), being mycotoxin purified by high performance liquid chromatography (HPLC) with a reverse phase semipreparative column using a mobile phase of acetonitrile/water using gradient condition. The purity of the ENs fractions was verified by analytical HPLC and LC/MS-MS. The pure fractions of ENs were utilized to study the biological activity on several mycotoxigenic moulds as Fusarium verticilloides, Fusarium sporotrichioides, Fusarium tricinctum, Fusarium poae, Fusarium oxysporum, Fusarium proliferatum, Beauveria bassiana, Trichoderma harzianum, Aspergillus flavus, Aspergillus parasiticus, Aspergillus fumigatus, Aspergillus ochraceus and Penicillium expansum.The results obtained demonstrated that in several antibiograms, ENs induced the inhibition of the grown microorganisms tested.     

Hemostatic properties of Venezuelan Bothrops snake venoms with special reference to Bothrops isabelae venom

In Venezuela, Bothrops snakes are responsible for more than 80% of all recorded snakebites. This study focuses on the biological and hemostatic characteristics of Bothrops isabelae venom along with its comparative characteristics with two other closely related Bothrops venoms, Bothrops atrox and Bothrops colombiensis. Electrophoretic profiles of crude B. isabelae venom showed protein bands between 14 and 100 kDa with the majority in the range of 14-31 kDa. The molecular exclusion chromatographic profile of this venom contains five fractions (F1-F5). Amidolytic activity evaluation evidenced strong thrombin-like followed by kallikrein-like activities in crude venom and in fractions F1 and F2. The fibrinogenolytic activity of B. isabelae venom at a ratio of 100:1 (fibrinogen/venom) induced a degradation of Aα and Bβ chains at 15 min and 2 h, respectively. At a ratio of 100:10, a total degradation of Aα and Bβ chains at 5 min and of γ chains at 24 h was apparent. This current study evidences one of rarely reported for Bothrops venoms, which resembles the physiologic effect of plasmin. B. isabelae venom as well as F2 and F3 fractions, contain fibrinolytic activity on fibrin plate of 36, 23.5 and 9.45 mm²/μg, respectively using 25 μg of protein. Crude venom and F1 fraction showed gelatinolytic activity. Comparative analysis amongst Venezuelan bothropoid venoms, evidenced that the LD₅₀ of B. isabelae (5.9 mg/kg) was similar to B. atrox-Puerto Ayacucho 1 (6.1 mg/kg) and B. colombiensis-Caucagua (5.8 mg/kg). B. isabelae venom showed minor hemorrhagic activity, whereas B. atrox-Parguasa (Bolivar state) was the most hemorrhagic. In this study, a relative high thrombin-like activity was observed in B. colombiensis venoms (502-568 mUA/min/mg), and a relative high factor Xa-like activity was found in B. atrox venoms (126-294 mUA/min/mg). Fibrinolytic activity evaluated with 10 μg protein, showed that B. isabelae venom contained higher specific activity (50 mm²/μg) than B. colombiensis and B. atrox venoms, which should encourage the isolation of these fibrinolytic molecules to improve the quality of immunotherapy.     

Toxicity assessment of crude and partially purified extracts of marine Synechocystis and Synechococcus cyanobacterial strains in marine invertebrates

Among the Cyanoprokaryota, the genera Synechocystis and Synechococcus have rarely been studied with respect to potential toxicity. This is particularly true with marine environments where studies about the toxicity of cyanobacteria are restricted to filamentous forms at the warmer temperate and tropical regions and also to filamentous forms at cold seas such as the Baltic Sea. In this study, we describe the effects of cyanobacterial strains of the Synechocystis and Synechococcus genera isolated from the marine coast of Portugal, on marine invertebrates. Crude and partially purified extracts at a concentration of 100 mg/ml of freeze-dried material of the marine strains were tested for acute toxicity in nauplii of the brine shrimp Artemia salina, in the rotifer Brachionus plicatillis and in embryos of the sea urchin Paracentrotus lividus and the mussel Mytilus galloprovincialis. The cyanobacterial extracts, especially the crude extract, had an impact on A. salina nauplii. No significant toxic effects were registered against the rotifer. A negative impact of all strains was recorded on the embryonic development of the sea urchin, with toxic effects resulting in an inhibition of embryogenesis or development of smaller larvae. To the mussel embryos, the effects of cyanobacterial extracts resulted in a complete inhibition of embryogenesis. The results of all assays indicate that Synechocystis and Synechococcus marine strains contained toxic compounds to marine invertebrates.     

Fusarium mycotoxin enniatin B: Cytotoxic effects and changes in gene expression profile

The mycotoxin enniatin B, a cyclic hexadepsipeptide produced by the plant pathogen Fusarium, is prevalent in grains and grain-based products in different geographical areas. Although enniatins have not been associated with toxic outbreaks, they have caused toxicity in vitro in several cell lines. In this study, the cytotoxic effects of enniatin B were assessed in relation to cellular energy metabolism, cell proliferation, and the induction of apoptosis in Balb 3T3 and HepG2 cells. The mechanism of toxicity was examined by means of whole genome expression profiling of exposed rat primary hepatocytes. Enniatin B altered cellular energy metabolism and reduced cell proliferation in Balb 3T3 and HepG2 cell lines. Furthermore, the proportion of apoptotic cell populations of Balb 3T3 cells slightly increased. On the other hand, enniatin B caused necrotic cell death in primary hepatocytes. Gene expression studies revealed the alteration of energy metabolism due to effects on mitochondrial organization and function and the assembly of complex I of the electron transport chain.     

Toxicity of cylindrospermopsin, and other apparent metabolites from Cylindrospermopsis raciborskii and Aphanizomenon ovalisporum, to the zebrafish (Danio rerio) embryo

Cyanobacteria produce a diverse array of toxic or otherwise bioactive compounds that pose growing threats to human and environmental health. We utilized the zebrafish (Danio rerio) embryo, as a model of vertebrate development, to investigate the inhibition of development pathways (i.e. developmental toxicity) by the cyanobacterial toxin, cylindrospermopsin (CYN), as well as extracts from various isolates of Cylindrospermopsis raciborskii and Aphanizomenon ovalisporum. CYN was toxic only when injected directly into embryos, but not by direct immersion at doses up to 50 μg/ml. Despite the dose dependency of toxicity observed following injection of CYN, no consistent patterns of developmental defects were observed, suggesting that toxic effects of CYN may not target specific developmental pathways. In contrast, direct immersion of embryos in all of the extracts resulted in both increased mortality and reproducible, consistent, developmental dysfunctions. Interestingly, there was no correlation of developmental toxicity observed for these extracts with the presence of CYN or with previously reported toxicity for these strains. These results suggest that CYN is lethal to zebrafish embryos, but apparently inhibits no specific developmental pathways, whereas other apparent metabolites from C. raciborskii and A. ovalisporum seem to reproducibly inhibit development in the zebrafish model. Continued investigation of these apparent, unknown metabolites is needed.     

Sequence Divergence of the Enniatin Synthase Gene in Relation to Production of Beauvericin and Enniatins in Fusarium Species

Beauvericin (BEA) and enniatins (ENNs) are cyclic peptide mycotoxins produced by a wide range of fungal species, including pathogenic Fusaria. Amounts of BEA and ENNs were quantified in individual rice cultures of 58 Fusarium strains belonging to 20 species, originating from different host plant species and different geographical localities. The species identification of all strains was done on the basis of the tef-1α gene sequence. The main aim of this study was to analyze the variability of the esyn1 gene encoding the enniatin synthase, the essential enzyme of this metabolic pathway, among the BEA- and ENNs-producing genotypes. The phylogenetic analysis based on the partial sequence of the esyn1 gene clearly discriminates species producing exclusively BEA from those synthesizing mainly enniatin analogues.     

Global distribution of ciguatera causing dinoflagellates in the genus Gambierdiscus

Dinoflagellates in the genus Gambierdiscus produce toxins that bioaccumulate in tropical and sub-tropical fishes causing ciguatera fish poisoning (CFP). Little is known about the diversity and distribution of Gambierdiscus species, the degree to which individual species vary in toxicity, and the role each plays in causing CFP. This paper presents the first global distribution of Gambierdiscus species. Phylogenetic analyses of the existing isolates indicate that five species are endemic to the Atlantic (including the Caribbean/West Indies and Gulf of Mexico), five are endemic to the tropical Pacific, and that two species, Gambierdiscus carpenteri and Gambierdiscus caribaeus are globally distributed. The differences in Gambierdiscus species composition in the Atlantic and Pacific correlated with structural differences in the ciguatoxins reported from Atlantic and Pacific fish. This correlation supports the hypothesis that Gambierdiscus species in each region produce different toxin suites. A literature survey indicated a >100-fold variation in toxicity among species compared with a 2 to 9-fold within species variation due to changing growth conditions. These observations suggest that CFP events are driven more by inherent differences in species toxicity than by environmental modulation. How variations in species toxicity may affect the development of an early warning system for CFP is discussed.     

Co-exposure of Fusarium mycotoxins: In vitro myelotoxicity assessment on human hematopoietic progenitors

Mycotoxins such as beauvericin (BEA), deoxynivalenol (DON), enniatin B (ENB), fumonisin B1 (FB1), T-2 toxin and zearalenone (ZEA) can co-occur in food commodities.This aim of this study was to assess the myelotoxicity of these mycotoxins in couple using in vitro human granulo-monocytic (Colony Forming Unit-Granulocyte and Macrophage, CFU-GM) hematopoietic progenitors. Clonogenic assays have been performed in the presence of the following couples of fusariotoxins: DON + BEA, DON + FB1, DON + T-2, DON + ZEA, T-2 + ZEA and BEA + ENB.Co-exposure of human CFU-GM to DON + BEA resulted in synergic myelotoxic effects. The combination of DON + T-2 presented additive or synergic myelotoxic effects. The couples DON + ZEA, T-2 + ZEA and BEA + ENB had additive myelotoxic effects, while the combination of DON + FB1 showed antagonist myelotoxic effects.These in vitro results suggested that the simultaneous presence of mycotoxins in food commodities and diet may be more myelotoxic than the presence of one mycotoxin alone. Diminution of hematopoietic progenitors could give rise to a decrease number of mature blood cells, inducing agranulocytosis and/or thrombocytopenia and in severe cases aplastic anemia.     

Toxicities of destruxins against Bemisia tabaci and its natural enemy, Serangium japonicum

The bioactivities of destruxins against whitefly, Bemisia tabaci and its natural enemy, ladybird beetle Serangium japonicum were evaluated. Destruxins A and B (DA and DB) showed insignificant ovicidal, oviposition deterrent and systemic insecticidal activities to B. tabaci; however, DA and DB had certain contact virulence to its nymphs. The LC₅₀ values of DA at 120 h to 2nd, 3rd and 4th instars were 89.8 (95% confidence interval as 85.4-94.4), 199.3 (187.7-211.5) and 270.7 (251.5-291.5) mg/L, while the LC₅₀s of DB at 120 h were 96.5 (92.0-101.2), 216.7 (203.0-231.2), 359.4 (326.6-395.4) mg/L, respectively. In addition, DA exhibited moderate acute contact toxicities towards S. japonicum, the LC₅₀s at 48 h were 165.4 (132.3-229.4) and 192.5 (148.1-289.2) mg/L for 4th instar larvae and adults. Furthermore, the results from experiments of residual toxicities of DA towards mortalities of 4th instar larvae and adults, pupation rate, emergence rate, average number of egg/female and hatching rate suggested that DA had minimal effects to the ladybird beetle. Generally, the toxicity decreased about 50% from 1st to 3rd-5th day of post-treatment. Specially, the residual toxicity at 50 mg/L and the 7th day post-treatment was down to a value not differing significantly from the control.     

Comparative study of the efficacy and safety of two polyvalent, caprylic acid fractionated [IgG and F(ab′)2] antivenoms, in Bothrops asper bites in Colombia

The efficacy and safety of two polyvalent horse-derived antivenoms in Bothrops asper envenomings were tested in a randomized, double-blind, clinical trial performed in Colombia. Both antivenoms were manufactured from the same pool of hyperimmune plasma. Antivenom A was made of F(ab′)₂ fragments, generated by pepsin digestion and caprylic acid precipitation, whereas antivenom B consisted of whole IgG molecules produced by caprylic acid precipitation followed by ion-exchange chromatography. Besides the different nature of the active substance, antivenom B had higher protein concentration, slightly higher turbidity and aggregate content. No significant differences were observed in the efficacy of antivenoms. Both halted local and systemic bleeding (P = 0.40) within 6-12 h of treatment in 100% of the cases, and restored blood coagulation (P = 0.87) within 6-24 h in 84.7% of patients, and within 48 h in all of them, in agreement with restoration of plasma fibrinogen concentration. Venom concentrations in serum dropped significantly (P < 0.001), to very low levels, 1 h after antivenom infusion. Nevertheless, eight patients (11.1%), four for each antivenom, presented recurrence of venom antigenaemia at different times, from 6 to 96 h, with clinical significance (recurrent coagulopathy) only in one group B patient (2.9%). Serum creatine kinase (CK) activity was increased, as a consequence of local myonecrosis. There was no significant difference (P = 0.51) in the incidence of early adverse reactions to antivenom administration (28.9% for patients of group A and 20.6% for patients of group B), most of the reactions being mild, mainly cutaneous. The most frequent complications were cellulitis (16.7%), abscess formation (5.6%), acute renal failure (8.3%), and compartmental syndrome (5.6%). In conclusion, IgG and F(ab′)₂ antivenoms, prepared by caprylic acid fractionation, presented similar efficacy and safety profiles for the treatment of B. asper envenomings in Colombia.     

Comparison of the effect of Crotalus simus and Crotalus durissus ruruima venoms on the equine antibody response towards Bothrops asper venom: Implications for the production of polyspecific snake antivenoms

Antivenoms are preparations of immunoglobulins purified from the plasma of animals immunized with snake venoms. Depending on the number of venoms used during the immunization, antivenoms can be monospecific (if venom from a single species is used) or polyspecific (if venoms from several species are used). In turn, polyspecific antivenoms can be prepared by purifying antibodies from the plasma of animals immunized with a mixture of venoms, or by mixing antibodies purified from the plasma of animals immunized separately with single venom. The suitability of these strategies to produce polyspecific antibothropic-crotalic antivenoms was assessed using as models the venoms of Bothrops asper, Crotalus simus and Crotalus durissus ruruima. It was demonstrated that, when used as co-immunogen, C. simus and C. durissus ruruima venoms exert a deleterious effect on the antibody response towards different components of B. asper venom and in the neutralization of hemorrhagic and coagulant effect of this venom when compared with a monospecific B. asper antivenom. Polyspecific antivenoms produced by purifying immunoglobulins from the plasma of animals immunized with venom mixtures showed higher antibody titers and neutralizing capacity than those produced by mixing antibodies purified from the plasma of animals immunized separately with single venom. Thus, despite the deleterious effect of Crotalus sp venoms on the immune response against B. asper venom, the use of venom mixtures is more effective than the immunization with separate venoms for the preparation of polyspecific bothropic-crotalic antivenoms.     

Coronatin-1 isolated from entomopathogenic fungus Conidiobolus coronatus kills Galleria mellonella hemocytes in vitro and forms potassium channels in planar lipid membrane

Entomopathogenic fungi are important natural regulatory factors of insect populations and have potential as biological control agents of insect pests. The cosmopolitan soil fungus Conidiobolus coronatus (Entomopthorales) easily attacks Galleria mellonella (Lepidoptera) larvae. Prompt death of invaded insects is attributed to the action of toxic metabolites released by the invader. Effect of fungal metabolites on hemocytes, insect blood cells involved in innate defense response, remains underexplored to date.C. coronatus isolate 3491 inducing 100% mortality of G. mellonella last instar larvae exposed to sporulating colonies, was cultivated at 20 °C in minimal medium. Post-incubation filtrates were used as a source of fungal metabolites. A two-step HPLC (1 step: Shodex KW-803 column eluted with 50 mM KH₂PO₄ supplemented with 0.1 M KCl, pH 6.5; 2 step: ProteinPak™ CM 8HR column equilibrated with 5 mM KH₂PO₄, pH 6.5, proteins eluted with a linear gradient of 0.5 M KCl) allowed the isolation of coronatin-1, an insecticidal 36 kDa protein showing both elastolytic and chitinolytic activities.Addition of coronatin-1 into primary in vitro cultures of G. mellonella hemocytes resulted in rapid disintegration of spherulocytes freely floating in culture medium and shrinkage of plasmatocytes adhering to the bottom of culture well. Coronatin-1 stimulated pseudopodia atrophy and, in consequence, disintegration of nets formed by cultured hemocytes.After incorporation of coronatin-1 into planar lipid membrane (PLM) ion channels selective for K⁺ ions in 50/450 mM KCl solutions were observed. Potassium current flows were recorded in nearly 70% of experiments with conductance from 300 pS up to 1 nS. All observed channels were active at both positive and negative membrane potentials. Under experimental conditions incorporated coronatin-1 exhibited a zero current potential (E_rev) of 47.7 mV, which indicates K⁺-selectivity of this protein.The success of the purification of coronatin-1 will allow further characterization of the mode of action of this molecule, including ability of coronatin-1 to form potassium channels in immunocompetent hemocytes.     

Phomenins and fatty acids from Alternaria infectoria

Taxa of the Alternaria infectoria species group are the predominant Alternaria spp. found in cereals in Northern Europe. While several pyrones have been isolated from A. infectoria and described as taxonomical markers for species identification, information about the bioactivity of metabolites from the fungus is missing. Bioassay-guided fractionation of rice culture extracts from several strains of A. infectoria linked the observed toxicity of the extracts in MRC-5 cells to free fatty acids, i.e. linoleic acid and α-linolenic acid. The fungus also produced a cytotoxic pyrone, which upon isolation and NMR spectroscopic analysis was identified as a mixture of phomenins A and B (approximately 10:1), which have not previously been isolated from an Alternaria species.     

Literature review on the safety of Toyocerin,a non-toxigenic and non-pathogenic Bacillus cereus var. toyoi preparation

Bacillus cereus var. toyoi is a naturally occurring, non-toxigenic and non-pathogenic strain of B. cereus. Safety studies were conducted on a B. toyoi preparation (Toyocerin^®), including but not limited to enterotoxicity, eye irritation, genotoxicity, acute, subchronic and chronic toxicity studies and human clinical trials. In rabbits, Toyocerin^® did not exhibit enterotoxicity and was only slightly irritating to the eyes. It was non-mutagenic in an Ames assay at up to 10,000 μg/plate and did not exhibit clastogenic activity in a chromosomal aberration test at up to 450 mg/ml. It was non-toxic in acute and repeated-dose (30 and 60 days and 1 year) toxicity studies in rats and mice at up to 3 × 10¹¹ spores/kg bw/day. In an eight-day human clinical trial, Toyocerin^® did not cause any adverse effects in healthy male and female subjects at 1 × 10⁹ and 1 × 10¹⁰ spores/kg bw/day. In feeding trials, Toyocerin^® did not cause any adverse effects in rabbits, pigs, chickens, turkeys and cattle at doses ranging from 8.5 × 10⁷ to 4 × 10⁹ spores/kg bw/day for durations of 2 weeks to 18 months. Taken together, these studies demonstrate that Toyocerin^® is safe at the doses tested.     

Isolation and purification of enniatins A, A1, B, B1, produced by Fusarium tricinctum in solid culture, and cytotoxicity effects on Caco-2 cells

Enniatins (ENs) are antibiotic compounds of hexadepsipeptidic structure produced by several strains of Fusarium spp. The ENs A, A₁, B, B₁ were purified from extracts of Fusarium tricinctum grown on a solid medium of corn, by a low pressure liquid chromatography (LPLC) on reverse phase of Amberlite XAD-7 followed by semipreparative LC. The purity and the structure of the isolated compounds were confirmed by LC-MS/MS.The technique of the purification of the fungal extract enabled complete separation of the ENs A, A₁, B, B₁ with a mean purity of 97% for all the compounds.The cytoxicity of the ENs was tested in the cell lines of human origin (epithelial colorectal adenocarcinoma cells, Caco-2) by MTT assays. Only EN A₁ and B₁ evoked toxicity at the tested concentrations. The inhibitory concentration (IC₅₀) for EN A₁ on Caco-2 cells was 12.3 μM, whereas the IC₅₀ produced by the EN B₁ was 19.5 μM.This study indicates that ENs, fungal metabolites that are commonly found in corn and in general in product composed by corn, may have a toxic potential for human health.     

Inhibitory effect of cyclic terpenes (limonene, menthol, menthone and thymol) on Fusarium verticillioides MRC 826 growth and fumonisin B1 biosynthesis

The minimum inhibitory concentration (MIC) of cyclic terpenes (limonene, menthol, menthone and thymol) against Fusarium verticillioides MRC 826 was assessed by using the semisolid agar antifungal susceptibility (SAAS) technique. Limonene, menthol, menthone and thymol were evaluated at final concentrations of 25, 50, 75, 150, 200, 250, 500 and 1000 μL/L of culture medium. Limonene and thymol showed the highest inhibitory effects on F. verticillioides development. Thus, the effects of monoterpenes on fumonisin B1 (FB1) biosynthesis were evaluated by using corn grain (Zea mays) as substrate. The monoterpenes were inserted on maize 1 day before inoculation with a conidial suspension of F. verticillioides to give final concentrations of 75 ppm. At this concentration, thymol was the most active inhibitor on FB1 biosynthesis.     

Antimicrobial and cytotoxic constituents from leaves of Sapium baccatum

Six compounds, namely, Lupeol (1), Betulin (2), β-Taraxerol (3), Taraxerone (4), Stigmasterol (5) and β-Sitosterol (6) were isolated from the petroleum ether extract of the leaves of Sapium baccatum based on spectroscopic evidence. Lupeol (1), Betulin (2) and Stigmasterol (5) were isolated for the first time from this plant. The cytotoxic potential of the different solvent extracts (methanol, petroleum ether, carbon-tetrachloride and dichloromethane); six column fractions (F-4, F-7, F-10, F-12, F-18 and F-22) of petroleum ether extract and three pure compounds 1, 4 and 6 were determined by using brine shrimp lethality bioassay. The LC₅₀ of all the tested samples were showed to be lethal to brine shrimp nauplii. However, petroleum ether, carbon-tetrachloride extract, column fractions F-4 and F-18 of petroleum ether extract and pure compound 6 showed quite potent activity in brine shrimp lethality bioassay with LC₅₀ 1.33, 1.35, 1.40, 1.58 and 1.58 μg/ml, respectively. These result suggested that they might be contain antitumor or pesticidal activity. Further, the methanol extract and four column fractions (F-7, F-12, F-18 and F-22) of petroleum ether showed significant activity against the tested microorganisms.