Absence Of Mutagenicity Research Articles

Preclinical Toxicity Assessment of Polyphenols-Based Standardized Extract of Cinnamomum zeylanicum Bark

Background: Cinnamon (Cinnamomum zeylanicum) bark is used as a spice in culinary practices and has been documented for health and medicinal benefits. Polyphenol (PP) is the major bioactive constituent of cinnamon bark. However, acceptable and safe dose levels and toxicity information of oral exposure to PP-based standardized cinnamon bark extract (PP-CZ) are crucial for safe human consumption. Objective: To evaluate PP-CZ for acute oral, subchronic oral toxicities in vivo, mutagenicity in vitro and genotoxic potential in vitro. Method:. The effects of oral treatment with single- and 90-days repeated dose were evaluated in rates as per OECD Test 423 and 408, respectively. Effects on body weight, food and water intake, organ weight, hematology, biochemistry, and histology were recorded . Mutagenicity and genotoxicity were evaluated using Ames (OECD No. 471) and chromosomal aberrations (OECD Test. 473) tests. Results: A single oral dose of PP-CZ did not cause death or treatment-related toxic effects, indicating a “median lethal dose” > 2,000 mg/kg. In addition, a subchronic dosage (500 mg/kg/day, 90 days) was found safe in rats, suggesting “no observed adverse effect level” (NOAEL) of 500 mg/kg and “Human Equivalent Dose” (HED) of 4.8 g/day. Furthermore, the absence of mutagenicity or genotoxicity of PP-CZ was observed during in vitro tests. PP-CZ showed a robust safety profile without mutagenicity or genotoxicity in rats.

Development of Potent and Selective Monoacylglycerol Lipase Inhibitors. SARs, Structural Analysis, and Biological Characterization.

New potent, selective monoacylglycerol lipase (MAGL) inhibitors based on the azetidin-2-one scaffold ((±)-5a-v, (±)-6a-j, and (±)-7a-d) were developed as irreversible ligands, as demonstrated by enzymatic and crystallographic studies for (±)-5d, (±)-5l, and (±)-5r. X-ray analyses combined with extensive computational studies allowed us to clarify the binding mode of the compounds. 5v was identified as selective for MAGL when compared with other serine hydrolases. Solubility, in vitro metabolic stability, cytotoxicity, and absence of mutagenicity were determined for selected analogues. The most promising compounds ((±)-5c, (±)-5d, and (±)-5v) were used for in vivo studies in mice, showing a decrease in MAGL activity and increased 2-arachidonoyl-sn-glycerol levels in forebrain tissue. In particular, 5v is characterized by a high eudysmic ratio and (3R,4S)-5v is one of the most potent irreversible inhibitors of h/mMAGL identified thus far. These results suggest that the new MAGL inhibitors have therapeutic potential for different central and peripheral pathologies.

Computational Analysis of ZINC Analogues for Antidiabetic Phytochemical with Inhibitory Activity against Aldose Reductase Enzyme

The pathognomonic role of sorbitol pathway in the development of microvascular complications cannot go underappreciated; while it may not be a sole independent initiator, it remains a key contributor to the initiation and progression of diabetic microangiopathies, particularly retinopathy, neuropathy, and nephropathy. The sorbitol pathway is a two-step reaction process initiated by the rate-limiting enzyme aldose reductase. This study aimed to screen ZINC databases for chemical compounds similar to Ellagic acid, Kaempferol, and Mangiferin and analyze their pharmacokinetic, toxicological and docking profile using computational methods. These phytochemicals were chosen based on literature review of ethnobotanical studies and validated via SwissTargetPrediction. An in-silico study design was employed using computational algorithms. Molecular structures of analogues were obtained from ZINC database and prepared using Avogadro software. Docking analysis was carried out using AutoDock Vina embedded in Chimera. Visualization of ligand-enzyme interactions was done using Discovery studio. SWISSADME and Protox-II were used to profile pharmacokinetic and toxicity of the analogs. A total of 44 analogs were analyzed. Sulindac and parent phytochemicals were used as comparators. Kaempferol had strongest binding affinity (-8.7) followed by Ellagic acid and Mangiferin tying at -8.4. Kaempferol analogs had highest binding affinity compared to analogues of Ellagic acid and Mangiferin. In terms of Pharmacokinetic profile, analogues of Ellagic acid demonstrated a favorable profile with no Lipinski rule violations, high GI absorption, and inhibition limited to CYP1A2, which plays a minor role in drug metabolism compared to other enzymes. Toxicology predictions indicated that Kaempferol exhibited a higher safety profile compared to Ellagic acid and Mangiferin, with LD50 values of 3919 mg/kg, 2991 mg/kg, and 2 mg/kg, respectively. Ellagic acid analogues demonstrated chemical safety, absence of mutagenicity, hepatotoxicity, cytotoxicity, and activation of various pathways. Among the Kaempferol analogues, a subset was potentially carcinogenic and mutagenic, while all exhibited potential pathway activation. Mangiferin analogues, except for a few compounds, did not activate specific pathways, nor did they demonstrate hepatotoxicity or cytotoxicity. However, some analogues exhibited potential immunogenicity and mutagenicity. Kaempferol and some of its ZINC analogues had strongest binding affinity compared to Ellagic acid, Mangiferin and their analogues. This can be attributed to the simpler structure of Kaempferol, allowing it to fit snugly into the hydrophobic pocket of aldose reductase. Ellagic acid, with its planar and rigid structure, interacted primarily with the outer surface of the hydrophobic pocket. Similarly, due to its complex and large structure, Mangiferin demonstrated a relatively lower affinity. Analogues ZINC000005004393, ZINC000003872446, and ZINC000031156069 for Kaempferol, Ellagic acid, and Mangiferin respectively depicted the best optimal characteristics required for further development. We recommend an in vitro study be conducted to assess and validate the claims arrived at in this study

Characterization, Preclinical Efficacy and Toxicity Evaluations of Flavonoids Glycosides based Standardized Fenugreek Seed Extract (FEFLG)

Introduction: Fenugreek seeds, a natural food chain raw material, is known to have many flavonoid glycosides. Objective: Characterization, preclinical efficacy, and safety evaluation of flavonoid glycosidebased standardized fenugreek seed extract (FEFLG). Methods: FEFLG was characterized for a group of flavonoid glycoside marker compounds by HPLC. The CD38+ enzyme inhibition efficacy was assessed in vitro. In addition, acute oral toxicity (AOT) and subchronic, 90-day repeated-dose oral toxicity (in vivo), mutagenicity (AMES test, in vitro) and chromosome aberration test (in vitro) of FEFLG were evaluated. Results: The FEFLG was found to have 49.85% of total flavonoid glycosides content in FEFLG (25.15% of Group 1: vitexin, isovitexin and vitexin 2-o- rhamnoside and 24.70% of Group 2 (vicenin derivatives, schaftoside, iso-schaftoside, orientin and iso-orientin). FEFLG showed CD38+ enzyme inhibition in vitro (IC50= 0.96 μg/ml) equivalent to the positive control, apigenin. FEFLG did not show any toxicity at an acute oral dose of more than 2000 mg/kg (median lethal dose, LD50) with a limit dose of 5000 mg/kg. The 90-day repeated-dose oral administration of FEFLG did not induce significant toxicological changes till the maximum dose of 1000 mg/kg in male and female rats, indicating no observed adverse effect level, NOAEL ≥ 1000 mg/kg. FEFLG did not show mutagenicity (up to a concentration of 5000 μg/plate) or structural chromosomal aberrations (up to 5000 μg /ml). Conclusion: The CD38+ enzyme inhibitor efficacy in vitro, oral safety in vivo and absence of mutagenicity or genotoxicity of FEFLG indicated its potential for anti-aging applications.

Structure-Activity Relationship (SAR) and in vitro Predictions of Mutagenic and Carcinogenic Activities of Ixodicidal Ethyl-Carbamates.

Ethyl-4-bromophenyl-carbamate (LQM 919) and Ethyl-4-chlorophenyl-carbamate (LQM 996) are compounds that inhibit egg-laying and hatching of tick larvae that are resistant to conventional ixodicides. The structure-activity relationship (SAR) to get the endpoint predictions of mutagenicity and carcinogenicity of the LQM 919 and LQM 996 was performed and the absence of mutagenicity was confirmed by Ames test. SAR analysis show no structural alerts indicating the ability of ethyl-carbamates to bind biomolecules or estrogen receptors. Endpoint of mutagenicity with and without metabolic activation showed that the ethyl-carbamates were negative (p <0.05) for mutagenicity induction in strains TA97, TA98, TA102, TA1535, TA1537 and TA1538 of Salmonella typhimurium. Pre-incubation with different ethyl-carbamate concentrations did not increase the number of spontaneously reverting colonies; moreover, the compounds did not induce a concentration-dependent increase in the number of reverting colonies in any of the strains used. This confirmed the absence of mutagenic activity in this test system. Exogenous metabolic activation did not modify these observations; suggesting that no metabolites with mutagenic activity were present. The endpoint of carcinogenicity in rats were negative for LQM 919 (p <0.05,) and LQM 996 (p <0.001). The results of the present study strongly suggest that ethyl-carbamates do not represent a risk for cancer in mammals.

Sunitinib-Containing Carborane Pharmacophore with the Ability to Inhibit Tyrosine Kinases Receptors FLT3, KIT and PDGFR-β, Exhibits Powerful In Vivo Anti-Glioblastoma Activity.

Simple SummaryGlioblastoma is one of the most aggressive central nervous system tumors. Combinations of therapies, such as tyrosine kinase receptor inhibition and boron neutron capture therapy (BNCT), could offer greater patients benefits over single-therapies. The aim of our study was to assess the potential of sunitinib-carborane hybrid compound 1 as an anti-glioblastoma agent. We confirmed for 1 the ability to inhibit tyrosine kinase receptors, which could promote canonical and non-canonical effects, absence of mutagenicity, ability to cross the blood–brain barrier, and powerful in vivo anti-glioblastoma activity. The overall attractive profile of 1 makes it an interesting compound for a bimodal therapeutic strategy against high grade gliomas.Malignant gliomas are the most common malignant and aggressive primary brain tumors in adults, the prognosis being—especially for glioblastomas—extremely poor. There are no effective treatments yet. However, tyrosine kinase receptor (TKR) inhibitors and boron neutron capture therapy (BNCT), together, have been proposed as future therapeutic strategies. In this sense in our ongoing project of developing new anti-glioblastoma drugs, we identified a sunitinib-carborane hybrid agent, 1, with both in vitro selective cytotoxicity and excellent BNCT-behavior. Consequently, we studied the ability of compound 1 to inhibit TKRs, its promotion of cellular death processes, and its effects on the cell cycle. Moreover, we analyzed some relevant drug-like properties of 1, i.e., mutagenicity and ability to cross the blood–brain barrier. These results encouraged us to perform an in vivo anti-glioblastoma proof of concept assay. It turned out to be a selective FLT3, KIT, and PDGFR-β inhibitor and increased the apoptotic glioma-cell numbers and arrested sub-G1-phase cell cycle. Its in vivo activity in immunosuppressed mice bearing U87 MG human glioblastoma evidenced excellent anti-tumor behavior.

Antibacterial activities and antiproliferative assays over a tumor cells panel of a silver complex with 4-aminobenzoic acid: Studies in vitro of sustained release using bacterial cellulose membranes as support

The aims of this work were to evaluate the antibacterial and antiproliferative potential in vitro of the metal complex with 4-aminobenzoic acid (Ag-pABA) and a drug delivery system based on bacterial cellulose (BC-Ag-pABA). The Ag-pABA complex was characterized by elemental analysis, high resolution mass spectrometry and single-crystal X-ray diffraction techniques, which indicated a 1:2 metal/pABA composition plus a nitrate ion coordinated to silver by the oxygen atom, with the coordination formula [Ag (C7H7NO2)2(NO3)]. The coordination of pABA to the silver ion occurred by the nitrogen atom. The in vitro antibacterial activity of the complex evaluated by minimum inhibitory concentration assays demonstrated the effective growth inhibitory activity against Gram-positive, Gram-negative biofilm producers and acid-alcohol resistant Bacillus. The antiproliferative activities against a panel of eight tumor cells demonstrated the activity of the complex with a significant selectivity index (SI). The DNA interaction capacity and the Ames Test indicated the absence of mutagenicity. The BC-Ag-pABA composite showed an effective capacity of sustained release of Ag-pABA. The observed results validate further studies on its mechanisms of action and the conditions that mediate the in vivo biological effects using animal models to confirm its safety and effectiveness for treatment of skin and soft tissues infected by bacterial pathogens, urinary tract infections and cancer.

Cyto-genotoxic evaluation of novel anti-tubercular copper (II) complexes containing isoniazid-based ligands

Considering the promising previous results of Cu (II) complexes with isoniazid active ligand against Mycobacterium tuberculosis, the main causative agent of tuberculosis, novel biological assays evaluating its toxicogenic potential were performed to ensure the safe use. The genotoxicity/mutagenicity of the complexes CuCl2(INH)2.H2O (I1), Cu(NCS)2(INH)2.5H2O (I2) and Cu(NCO)2(INH)2.4H2O (I3) was evaluated by the Comet, Micronucleus-cytome and Salmonella microsome (Ames test) assays. The cell viability using resazurin assay indicated that I1, I2 e I3 had moderate to low capacity to reduce the viability of colorectal cells (Caco-2), liver cells (HepG2), lung cells (GM 07492-A and A549) and endothelial cells (HU-VE-C). On genotoxicity/mutagenicity, I1 complex did not induce sizable levels of DNA damage in HepG2 cells (Comet assay), and gene (Ames test) and chromosomal (Micronucleus-cytome assay) mutations. Already, I2 and I3 complexes were considered mutagenic in the highest concentrations used. In light of the above, these results contribute to valuable data on the safe use of Cu(II) complexes. Considering the absence of mutagenicity and cytotoxicity of I1, this complex is a potential candidate for the development of a new drug to the treatment tuberculosis, while I2 and I3 require caution in its use.

Genotoxicological safety assessment of puree-only edible films from onion bulb (Allium cepa L.) for use in food packaging-related applications.

The production of films and coatings from onion (Allium cepa L.) to be applied as packaging is attractive, due to its high nutritional and therapeutic value. Also, it can collaborate to minimize environmental impacts caused by the improper disposal of products made from plastics. However, despite it being an innovative and novel proposal, onion films for the development of edible packaging should be evaluated before being considered nontoxic and safe for human consumption. Thus, the objective of the present study was to elucidate the cytotoxic and mutagenic profile of eluates of polymer films of Allium cepa L. obtained by the casting process and to verify their safety for commercial purposes. The analysis of cellular viability demonstrated greater cytotoxicity for unwashed hydrothermally treated pulp (HTP) than for films of washed hydrothermally treated pulp (W-HTP). Regarding the mutagenic activity, the HTP and W-HTP films were not able to statistically increase the frequencies of the biomarkers for chromosome damage (micronucleus test) at the tested concentrations. However, the HTP films showed signs of mutagenicity in the Ames test (gene mutations), suggesting caution in their use. The detection of genotoxicity is highly recommended in order to avoid the risk of genotoxic exposure to mutagens and carcinogens. In conclusion, the absence of mutagenicity and cytotoxicity observed in this study is extremely relevant, because it provides support for toxicogenic properties of the Allium cepa films with promising applicability in the food industry. PRACTICAL APPLICATION: The bioplastics made from onion bulbs are multifunctional materials, which requires safety profile assessment. The results of the mutagenicity and cytotoxicity tests suggests that especially the W-HTP films are harmless, supporting at the first level of evidence, its safety potential to be used in the food industry (food films), biodegradable packaging, and biomaterials (substrates for drug delivery system).

Rare haplotype load as marker for lethal mutagenesis.

RNA viruses replicate with a template-copying fidelity, which lies close to an extinction threshold. Increases of mutation rate by nucleotide analogues can drive viruses towards extinction. This transition is the basis of an antiviral strategy termed lethal mutagenesis. We have introduced a new diversity index, the rare haplotype load (RHL), to describe NS5B (polymerase) mutant spectra of hepatitis C virus (HCV) populations passaged in absence or presence of the mutagenic agents favipiravir or ribavirin. The increase in RHL is more prominent in mutant spectra whose expansions were due to nucleotide analogues than to multiple passages in absence of mutagens. Statistical tests for paired mutagenized versus non-mutagenized samples with 14 diversity indices show that RHL provides consistently the highest standardized effect of mutagenic treatment difference for ribavirin and favipiravir. The results indicate that the enrichment of viral quasispecies in very low frequency minority genomes can serve as a robust marker for lethal mutagenesis. The diagnostic value of RHL from deep sequencing data is relevant to experimental studies on enhanced mutagenesis of viruses, and to pharmacological evaluations of inhibitors suspected to have a mutagenic activity.

Epigallocatechin Gallate, a Green Tea Polyphenol Inhibits Mycobacterium smegmatis: In silico and In vitro Studies

Presence of multidrug resistant strains makes treatment of tuberculosis difficult. Conventional frontline antituberculosis therapy has high incidence of treatment failure due to non-adherence and severe side effects, and therefore warrants alternative approaches using safe, natural compounds. (-) Epigallocatechin gallate, a green tea catechin with significant antimicrobial properties, and its derivatives have been studied for their inhibitory effects on Mycobacterium smegmatis. Enoyl reductase (Inh A, PDB ID: 2NV6), an important drug target inhibited by the antituberculosis drug, isoniazid, was docked with geometrically optimized conformation of epigallocatechin gallate and two of its derivatives using AutoDock software. Perpropionate and permethyl derivatives of epigallocatechin gallate were synthesized and, along with epigallocatechin gallate, evaluated for their inhibitory and cytotoxic properties against M. smegmatis using microplate dilution method and alamarBlue® assay, respectively. Ames test was performed to assess the mutagenic potential of epigallocatechin gallate. It was docked successfully onto the InhA with docking energy of –9.38 kcal mol-1. Analysis of neighboring groups revealed 11 amino acid residues common to both isoniazid and epigallocatechin gallate. The minimum inhibitory concentrations for epigallocatechin gallate, perpropionate and permethyl were found to be 128 (58.2% inhibition), 8 (32.9%) and 4 (12.5%) µg/ml, respectively. Epigallocatechin gallate showed a cytotoxicity of 18.6% at a concentration of 8 µg/ ml, while the two derivatives did not show any toxicity, even at 64 µg/ml. Ames test revealed the absence of mutagenicity for epigallocatechin gallate, in presence and absence of metabolic activation. Non-mutagenic epigallocatechin gallate inhibits the growth of M. smegmatis and warrants further investigation as an adjunct therapy for pathogenic mycobacteria.

Safety evaluation of rebaudioside A produced by fermentation

The safety of rebaudioside A, produced fermentatively by Yarrowia lipolytica encoding the Stevia rebaudiana metabolic pathway (fermentative Reb A), is based on several elements: first, the safety of steviol glycosides has been extensively evaluated and an acceptable daily intake has been defined; second, the use of Y. lipolytica, an avirulent yeast naturally found in foods and used for multiple applications; and third the high purity of fermentative Reb A and its compliance with internationally defined specifications. A bacterial reverse mutation assay and an in vitro micronucleus test conducted with fermentative Reb A provide evidence for its absence of mutagenicity, clastogenicity and aneugenicity. The oral administration of fermentative Reb A to Sprague–Dawley rats for at least 91 days did not lead to any adverse effects at consumption levels up to 2057 mg/kg bw/day for males and 2023 mg/kg bw/day for females, which were concluded to be the No Observed Adverse Effect Levels. The results were consistent with outcomes of previous studies conducted with plant-derived rebaudioside A, suggesting similar safety profiles for fermentative and plant-derived rebaudioside A. The results of the toxicity studies reported here support the safety of rebaudioside A produced fermentatively from Y. lipolytica, as a general purpose sweetener.

In vivo anti-Trypanosoma cruzi activity of hydro-ethanolic extract and isolated active principles from Aristeguietia glutinosa and mechanism of action studies.

The currently available treatments for Chagas disease show limited therapeutic potential and are associated with serious side effects. Attempting to find alternative drugs isolated from Nature as agents against Trypanosoma cruzi has been our goal. Recently, we have demonstrated the in vitro anti-T. cruzi activities of two secondary metabolites isolated from the hydro-ethanolic extract of the aerial parts of Aristeguietia glutinosa (Lam.), (family Asteraceae). These active principles displayed poor hemolytic activity, low toxicity against murine macrophages, and absence of mutagenicity. Herein, proof of concept in vivo studies of the whole hydro-ethanolic extract of the aerial parts of Aristeguietia glutinosa and of the most active component isolated from the hydro-ethanolic extract, i.e., (+)-15-hydroxy-7-labden-17-al, was done in a murine acute model of Chagas disease. Both treatments caused a decrease in the animals’ parasitemia. Metabolomic mechanism of action studies were done by 1H-NMR, both on the extract and on the active compounds, examining the effects of the metabolites both on membrane sterol biosynthesis and mitochondrial dehydrogenases, whereby we found that one of the metabolites inhibited the activity of the parasite mitochondrial dehydrogenases and the other inhibited the biosynthesis of parasite membrane sterols. The results are interesting in the context of popular use of plants for the treatment of Chagas disease.

Bioactive-guided Identification of Labdane Diterpenoids from Aerial Parts of Aristeguietia glutinosa as anti-Trypanosoma cruzi agents

A bioactive-guided investigation of the hydro-ethanolic extract of aerial parts of Aristeguietia glutinosa Lam. resulted in the isolation of two diterpenoids, (+)-15-hydroxy-labd-7-en-17-al (1) and (+)-13,14,15,16-tetranor-labd-7-en-17,12-olide (2), as the anti-Trypanosoma cruzi active principles. The structures of 1 and 2 were determined by spectroscopic analysis. The hydro-ethanolic extract showed anti-T. cruzi activity (IC50 = 19.6 microg/mL) whereas the isolated compounds 1 and 2 were near to seven- and one and a half-fold (IC50 = 3.0 and 15.6 microg/mL), respectively more active than the original extract. Labdene 1, equipotent as the reference compound (Nifurtimox), displayed low hemolytic activity, low toxicity against murine macrophages, and absence of mutagenicity. These results support the vernacular medicinal use of this plant as an anti-T. cruzi agent.

Preliminary Study by Environmental Indicator Measurements of Sediments in a Mangrove Forest in Ilha Grande Bay, Rio de Janeiro, Southeastern Brazil

Mangroves perform essential functions in biological cycles and are protected by law in Brazil. However, they are being degraded above their support limit, leading to a reduction in the areas around the world. The area investigated in the present study is a potentially unpolluted mangrove ecosystem in the south of Rio de Janeiro state, Brazil. The aim of this work was to investigate the presence of metals and polycyclic aromatic hydrocarbons (PAHs) in mangrove sediment and to correlate them with mutagenic and genotoxic activity. Four seasonal samplings undertaken between 2009 and 2010 at five sites were analyzed. Dichloromethane extracts were obtained under sonication and subjected to mutagenicity assays using Salmonella microsuspension. Metal (Cd, Cr, Cu, Ni and Pb) contents were determined by atomic absorption spectrometry, while the quantification of PAHs was performed by gas chromatography-mass spectrometry. Goniopsis cruentata hemocytes were used to assess potential genotoxic damage (Micronucleus assay). The absence of mutagenicity was observed for all samples using Salmonella typhimurium strains TA97, TA98, TA100 and TA102, in the absence and presence of an exogenous mammalian metabolizing system. Among the metals, Cd and Cu were detected in autumn at values exceeding the threshold effect level and below the probable effect level. Benzo[a] pyrene was detected at levels above the threshold at one site, where small effects could be observed in the biota. Significant differences in the micronucleated cells observed suggest DNA damage had been induced by the PAHs identified.

Comparative study of antioxidant and anti-inflammatory activities and genotoxicity of alcoholic and aqueous extracts of four Fabiana species that grow in mountainous area of Argentina

Fabiana species (Solanaceae family) extracts have long been used in Argentinean traditional medicine as anti-inflammatories, antiseptic, bone fractures and others diseases, but there is no scientific evidence which supports their use. The present study was conducted to evaluate the ability of aqueous and ethanolic extracts of four Fabiana species (Fabiana bryoides Phil., Fabiana punensis A.C. Arroyo, Fabiana densa J. Rèmy and Fabiana patagonica Speg.) to inhibit key enzymes in inflammatory processes, free radical scavenging properties and genotoxic effects. HPLC-DAD of aqueous and ethanolic extracts from four Fabiana species was established. All Fabiana extracts were evaluated on their ability to inhibit hyaluronidase and lipoxygenase enzymes to assess their activity against inflammatory mediators. Antioxidant capacity was determined using the 2,2'-azino-bis 3-ethylbenzothiazoline-6-sulfonic acid (ABTS) and the 1,1-diphenyl-2-picrylhydrazyl (DPPH) scavenging assays and β-carotene-linolenic acid assay. Genotoxicity was evaluated by the Ames assay. The results indicated that the chromatographic patterns of four Fabiana species were different in quantity and absorption intensity of peaks. The alcoholic extract of Fabiana punensis was the most active scavenger of DPPH and ABTS(+) radicals (SC(50) values of 3.85 ± 0.24 and 2.56 ± 0.10 μgGAE/mL, respectively). Fabiana patagonica extracts exhibited the highest peroxyl radical scavenging activity compared with the other three taxa (IC(50) values between 1.00 ± 0.04 and 4.46 ± 0.40 μg GAE/mL for all extracts) and anti-lipoxygenase activity with IC(50) values between 12.5 and 15.5 μg GAE/mL. The absence of mutagenicity indicates that the DNA does not seem to be a relevant target for these extracts. Fabiana bryoides ethanolic extract showed an interesting effect: it inhibited spontaneous mutagenesis, which could be considered as an antimutagenic effect in the TA98 (+S9) and TA100 (+S9/-S9) strains. The potency differences found between the species could be consequence of the different phytochemical pattern observed by HPLC. The inhibitory effects on lipoxygenase and hyaluronidase, free radical scavenging activities and lack of genotoxicity of Fabiana extracts may support the folk use of Fabiana punensis, Fabiana patagonica, Fabiana bryoides and Fabiana densa as inhibitor of inflammatory mediators.

Identification of chalcones as in vivo liver monofunctional phase II enzymes inducers

Cancer preventive agents (CPA) are drugs able to suppress the carcinogen metabolic activation or block the formation of ultimate carcinogens. CPA could act through various molecular mechanisms, for example by interfering with the action of procarcinogen. This could be attained by increasing the phase II enzymes levels of quinone reductase (QR) and glutathione S-transferase (GST). New flavonoids, especially chalcones, have been identified as in vivo monofunctional phase II enzymes inducers. Oral administration of chalcone, 4, and both p-methoxy-substituted chalcones, 6 and 14, increased hepatic QR activity with concomitant decrease in CYP1A1 activity, a member of the most important group of phase I enzymes cytochrome P450. Among them, 4 also increased GST activity. While p-bromo-substituted chalcone 8 was the best inducer of QR it decreased hepatic GST expression and cytochrome P450, being the most effective decreasing cytochrome P450-expression. Thienyl-chalcone 20 being the bioisostere of chalcone 4 did not display the same in vivo profile in the phase I level modification. As chalcone 4 its bioisostere, chalcone 20, displayed low DNA strand breakage and absence of mutagenicity. Also, in our preliminary in vivo tumourigenesis/chemopreventive and acute-toxicity studies, chalcones 4, 6 and 8 showed the best behaviours as CPA justifying additional studies that are ongoing.

Absence of Mutagenicity, Genotoxicity, and Subchronic Oral Toxicity of Touchi Extract

Touchi, a traditional Chinese food used mainly for seasoning is obtained by first steaming soybeans followed by fermentation with Aspergillus oryzae (koji). A series of toxicological studies was conducted to evaluate the mutagenic and genotoxic potential and subchronic toxicity of a water extract of Touchi, a known inhibitor of alpha -glucosidase activity. Touchi extract (TE) did not induce reverse mutations in Salmonella typhimurium strains TA98, TA1537, TA100, TA1535, and Escherichia coli WP2uvrA at concentrations up to 5000 microg/plate, in either the absence or presence of exogenous metabolic activation. No deaths occurred and no abnormal clinical signs were observed in any animal in any group in an in vivo micronucleus test, and TE was devoid of clastogenic activity when administered orally to mice at doses up to 2000 mg/kg/day. Thus, TE was evaluated as negative in the bacterial reverse mutation and mouse bone marrow micronucleus tests under the conditions of these assays. To evaluate its subchronic toxicity, SPF rats were administered TE at doses of 0, 250, 1000, and 2500 mg/kg/day via oral gastric intubation. No treatment-related toxic changes were seen in clinical signs, body weight, food consumption, urinalysis, hematology, blood chemistry, necropsy, organ weight, or histopathology. The no observed adverse effect level for TE was thus considered to be more than 2500 mg/kg/day in both males and females. These results are consistent with Touchi's status as a traditional Chinese food derived from fermented soybeans and its purported long history of use. Specifically, these data are consistent with the expected safety of human consumption of TE up to at least 5 g/day.

Evaluation of mutagenicity and antimutagenicity of cashew stem bark methanolic extract in vitro

Plants remain to be a great source of medicines for humanity. Among them is the Anacardium occidentale L . species, popularly known as the cashew, which has several therapeutic indications, such as cicatrizing, antihypertensive, hypoglycemic and antitumoral properties. The objective of the present work was to assess the mutagenicity and antimutagenicity of cashew stem bark methanolic extract (CSBME) on cell cultures of Chinese hamster lung fibroblasts (V79). The cultures were treated with different concentrations of the CSBME (500, 1000 and 2000 μg/mL) or with the extract associated with doxorubicin (DXR, 0.75 μg/mL) during the cell cycle phases G1, S, and G2 and throughout continuous treatment. Apart from these treatments, the cell cultures were also treated with PBS (negative-control) and DXR (positive-control). The data obtained in the chromosome aberrations (CA) test showed a significant reduction in CA frequency in the cultures treated with DXR and extract in comparison with those that received only DXR during the cell cycle phases G1 and S and throughout the entire cycle, as well as the absence of mutagenicity in all the treatments realized. The antimutagenic effect observed in this work reinforces the presence of the previously described therapeutic properties of cashew and indicates the safe use of this extract.

Studies on the non-pathogenicity of Chryseobacterium proteolyticum and on the safety of the enzyme: Protein–glutaminase

Protein–glutaminase (PG) is a protein-deamidating enzyme produced from the microorganism Chryseobacterium proteolyticum strain 9670. Food safety studies were conducted on both the enzyme and the production organism. The strain was evaluated for pathogenicity and toxigenicity by intravenous and oral inoculation studies in Slc:ICR male SPF mice. The results demonstrate that the tested C. proteolyticum strain is of very low pathogenicity comparable to known food source bacterial strains and is very unlikely to demonstrate any pathogenicity in animals or humans. The level of endotoxin is very low and typical of the endotoxin levels in drinking water and teas. A 90-day study of PG, conducted in Sprague–Dawley rats, showed no adverse effects due to the enzyme up to dose levels of 2500 mg/kg-bw/day (NOAEL). Details of the study are presented, including, organ and body weights, histological findings, and blood and urine chemistry. Additionally, bacterial reverse mutation test (Ames test) and chromosomal aberration test using mammalian established cell line were conducted, resulting in the absence of mutagenicity in PG.