Preventive activity of Copaifera langsdorffii Desf. leaves extract and its major compounds, afzelin and quercitrin, on DNA damage in in vitro and in vivo models.

Copaifera langsdorffii Desf. is a plant found in South America, especially in Brazil. Oleoresin and the leaves of this plant is used as a popular medicinal agent. However, few studies on the chemical composition of aerial parts and related biological activities are known. This study aimed to examine the cytotoxic, genotoxic, and antigenotoxic potential of C. langsdorffii aerial parts hydroalcoholic extract (CLE) and two of its major compounds afzelin and quercitrin. The cytotoxic and antigenotoxic potential of CLE was determined as follows: 1) against genotoxicity induced by doxorubicin (DXR) or methyl methanesulfonate (MMS) in V79 cells; 2) by direct and indirect-acting mutagens in Salmonella typhimurium strains; and 3) by MMS in male Swiss mice. The protective effects of afzelin and quercitrin against DXR or MMS were also evaluated in V79 and HepG2 cells. CLE was cytotoxic as evidenced by clonogenic efficiency assay. Further, CLE did not induce a significant change in frequencies of chromosomal aberrations and micronuclei; as well as number of revertants in the Ames test demonstrating absence of genotoxicity. In contrast, CLE was found to be antigenotoxic in mammalian cells. The results also showed that CLE exerted inhibitory effect against indirect-acting mutagens in the Ames test. Afzelin and quercitrin did not reduce genotoxicity induced by DXR or MMS in V79 cells. However, treatments using afzelin and quercitrin decreased MMS-induced genotoxicity in HepG2 cells. The antigenotoxic effect of CLE observed in this study may be partially attributed to the antioxidant activity of the combination of major components afzelin and quercitrin.

Selective anticancer effects of Serjania marginata Casar. extract in gastric cells are mediated by antioxidant response.

Gastric cancer is the fifth most common malignancy worldwide. Serjania marginata Casar. (SM) displays anti-inflammatory properties and has been used to treat gastrointestinal disorders. In the current study, we examined whether the hydroethanolic extract of SM leaves exerted cytotoxic, mutagenic, and protective effects in non-tumor gastric epithelium cells (MNP01) and gastric adenocarcinoma cells (ACP02) in vitro and analyzed whether its action was selective. Initially, cell viability (MTT assay), cell cycle kinetics (flow cytometry), and cell proliferation (total protein content) were analyzed. In addition, genomic instability (cytokinesis-block micronucleus cytome assay), anti/pro-oxidant status (CM-H2 DCFDA probe), and transcriptional expression (RT-qPCR) of genes related to cell cycle, cell death, and antioxidant defense were also evaluated. The SM extract was cytotoxic toward MNP01 and ACP02 cells at concentrations greater than 300 and 100 µg·ml-1 , respectively, and decreased protein content only toward ACP02 cells at 200 µg ml-1 . In ACP02 cells, the SM extract at 100 µg·ml-1 associated with doxorubicin (DXR; 0.2 µg ml-1 ) clearly promoted cell cycle arrest at the G2/M phase. The extract alone was not mutagenic to either cell type and reversed DXR-induced DNA damage and H2 O2 -induced oxidative stress in MNP01 cells. The gene expression experiments showed that SM hydroethanolic extract exerts an antioxidant response via NFE2L2 activation in non-tumor gastric cells, and cell cycle arrest (G2/M) in ACP02 gastric cancer cells via the TP53 pathway. The selective action of SM indicates that it is a promising therapeutic agent to treat gastric diseases and merits further studies.

Activity of silver nanoparticles on prokaryotic cells and Bothrops jararacussu snake venom.

Nanoparticle-conjugated venom-toxins of venomous animals and its therapeutic efficacy against emerging or neglecting diseases is a promising strategy. In this study, silver nanoparticles (AgNPs ∼50 nm, 0.081 mg mL-1) were studied against the neuromuscular blockade, myotoxic effects induced by Bothrops jararacussu venom (60 µg mL-1) and also against prokaryotic cells. The neurotoxicity was evaluated on ex vivo mouse phrenic nerve-diaphragm using traditional myographic technique, able to obtain functional contractile responses and to check the neurotransmission. The myotoxicity on mammalian cells was evaluated in muscles resulting from pharmacological assays using routine histological techniques and light microscopy. The toxicity to prokaryotic cells was evaluated on Salmonella typhimurium TA100 without metabolic activation. The in vitro preincubation model between AgNPs and venom was enough to abolish toxic effects of B. jararacussu venom, but mammalian cells were highly sensitive to AgNPs more than prokaryotic cells, by acting as dose-independently and dose-dependently parameters, respectively. These results allowed us to conclude that AgNPs showed promising activity as antivenom agent but for its safer use, the toxicity should be evaluated on experimental animals.

Pouteria ramiflora (Mart.) Radlk. extract: Flavonoids quantification and chemopreventive effect on HepG2 cells.

Pouteria ramiflora (Mart.) Radlk., popularly known as curriola, is commonly used in Brazil as medicinal plant to treat worm infections, dysentery, pain, inflammation, hyperlipidemia, and obesity. At present the safety of this extract when used therapeutically in human remains to be determined. Thus, the aim of this study was to examine cytotoxicity, antiproliferative, and antimutagenic actions of this extract. The hydroalcoholic extract from P. ramiflora leaves consisted of flavonoids identified and quantified as myricetin-3-O-ß-D-galactopyranoside (13.55 mg/g) and myricetin-3-O-α-L-rhamnopyranoside (9.61 mg/g). The extract exhibited cytotoxicity at concentrations higher than 1.5 µg/ml in human hepatocarcinoma (HepG2)and 2.5 µg/ml in non-tumoral primary gastric (GAS) cells using the MTT assay, and at concentrations higher than 3 µg/ml in HepG2 and 3.5 µg/ml in GAS cells by the neutral red assay. The extract did not show antiproliferative effect as evidenced by the nuclear division index (NDI). However, in the presence of benzo[a]pyrene (BaP) (positive control), an enhanced cytostatic effect in the NDI and flow cytometry was noted. It is of interest that when the extract was co-incubated with BaP a significant decrease in DNA damage was observed indicating an antimutagenic action. This protective effect might be attributed to myricetin and gallic acid found in P. ramiflora extract. The low cytotoxicity action and protective effect observed in the present study encourage further studies regarding other biological effects of P. ramiflora, as well as its potential use as a chemopreventive agent.

Antigenotoxicity properties of Copaifera multijuga oleoresin and its chemical marker, the diterpene (-)-copalic acid.

In view of the biological activities and growing therapeutic interest in oleoresin obtained from Copaifera multijuga, this study aimed to determine the genotoxic and antigenotoxic potential of this oleoresin (CMO) and its chemical marker, diterpene (-)-copalic acid (CA). The micronucleus (MN) assay in V79 cell cultures and the Ames test were used for in vitro analyses, as well as MN and comet assays in Swiss mice for in vivo analyses. The in vitro genotoxicity/mutagenicity results showed that either CMO (30, 60, or 120 µg/ml-MN assay; 0.39-3.12 mg/plate-Ames test) or CA (2.42; 4.84, or 9.7 µg/ml-MN assay; 0.39-3.12 mg/plate-Ames test) did not induce a significant effect on the frequency of MN and number of revertants, demonstrating an absence of genotoxic and mutagenic activities, respectively, in vitro. In contrast, these natural products significantly reduced the frequency of MN induced by methyl methanesulfonate (MMS), and exerted a marked inhibitory effect against indirect-acting mutagens in the Ames test. In the in vivo test system, animals treated with CMO (6.25 mg/kg b.w.) exhibited a significant decrease in rate of MN occurrence compared to those treated only with MMS. An antigenotoxic effect of CA was noted in the MN test (1 and 2 mg/kg b.w.) and the comet assay (0.5 mg/kg b.w.). Data suggest that the chemical marker of the genus Copaifera, CA, may partially be responsible for the observed chemopreventive effect attributed to CMO exposure. ABBREVIATIONS: 2-AA, 2-anthramine; 2-AF, 2-aminofluorene; AFB1, aflatoxin B1; B[a]P, benzo[a]pyrene; BOD, biological oxygen demand; BPDE, benzo[a]pyrene-7,8-diol-9,10-epoxide; CA, (-)-copalic acid; CMO, oleoresin of Copaifera multijuga, DMEM, Dulbecco`s Modified Eagles`s Medium; DMSO, dimethylsulfoxide; EMBRAPA, Brazilian agricultural research corporation; GC-MS, gas chromatography-mass spectrometry; HAM-F10, nutrient mixture F-10 Ham; HPLC, high performance liquid chromatography; LC-MS, liquid chromatography-mass spectrometry; MI, mutagenic index; MMC, mitomycin C; MMS, methyl methanesulfonate; MN, micronucleus; MNPCE, micronucleated polychromatic erythrocyte; NCE, normochromatic erythrocyte; NDI, nuclear division index; NMR, nuclear magnetic resonance; NPD, 4-nitro-o-phenylenediamine; PBS, phosphate-buffered saline; PCE, polychromatic erythrocyte; SA, sodium azide; V79, Chinese hamster lung fibroblast.

In vitro toxicological assessment of Arrabidaea brachypoda (DC.) Bureau: Mutagenicity and estrogenicity studies.

Arrabidaea brachypoda (DC.) Bureau is a shrub native Cerrado, known as "cipó-una", "tintureiro" or "cervejinha do campo" and popularly used in Southeastern and Northeastern Brazil to treatment of kidney stones and painful joints (arthritis). Nevertheless, scientific information regarding this species is scarce, and there are no reports related to its possible estrogenic and mutagenic effects. Thus, the principal objective of this study was to assess the mutagenic and estrogenic activities of the hydroalcoholic extracts of the leaves, stalks, roots, their respective fractions and isolated compounds of A. brachypoda. The mutagenic activity was evaluated by the Ames test on Salmonella typhimurium strains TA98, TA97a, TA100 and TA102, in the absence (-S9) and presence (+S9) of metabolic activation system. In the RYA was used Saccharomyces cerevisiae engineered strain BY4741 (MATaura3Δ0 leu2Δ0 his3Δ1 met15Δ0) which reproduce the natural pathway of genetic control by estrogens in vertebrate cells; it has the advantage of its simplicity and a high throughput. All extracts and aqueous fraction of leaves A. brachypoda were mutagenic. The crude extract is more active than the fraction, suggesting a synergic effect. Only hydroalcoholic extracts of leaves and roots of A. brachypoda showed significant estrogenic activity, with ERα-dependent transcriptional activation activity. The obtained results in this study showed the presence of compounds capable of interacting with the estrogen receptor and to induce damage in the genetic material. Thus, we demonstrated the risk which the population is subjected due to indiscriminate use of extracts without detailed study.

Effects of indirubin and isatin on cell viability, mutagenicity, genotoxicity and BAX/ERCC1 gene expression.

CONTEXT: Indigofera suffruticosa Miller (Fabaceae) and I. truxillensis Kunth produce compounds, such as isatin (ISA) and indirubin (IRN), which possess antitumour properties. Their effects in mammalian cells are still not very well understood. OBJECTIVE: We evaluated the activities of ISA and/or IRN on cell viability and apoptosis in vitro, their genotoxic potentials in vitro and in vivo, and the IRN- and ISA-induced expression of ERCC1 or BAX genes. MATERIALS AND METHODS: HeLa and/or CHO-K1 cell lines were tested (3 or 24 h) in the MTT, Trypan blue exclusion, acridine orange/ethidium bromide, cytokinesis-blocked micronucleus (CBMN) and comet (36, 24 and 72 h) tests after treatment with IRN (0.1 to 200 µM) or ISA (0.5 to 50 µM). Gene expression was measured by RT-qPCR in HeLa cells. Swiss albino mice received IRN (3, 4 or 24 h) by gavage (50, 100 and 150 mg/kg determined from the LD50 - 1 g/kg b.w.) and submitted to comet assay in vivo. RESULTS: IRN reduced the viability of CHO-K1 (24 h; 5 to 200 µM) and HeLa cells (10 to 200 µM), and was antiproliferative in the CBMN test (CHO-K1: 0.5 to 10 µM; HeLa: 5 and 10 µM). The drug did not induce apoptosis, micronucleus neither altered gene expression. IRN and ISA were genotoxic for HeLa cells (3 and 24 h) at all doses tested. IRN (100 and 150 mg/kg) also induced genotoxicity in vivo (4 h). CONCLUSION: IRN and ISA have properties that make them candidates as chemotherapeutics for further pharmacological investigations.

Chemical and biological characterisation of Machaerium hirtum (Vell.) Stellfeld: absence of cytotoxicity and mutagenicity and possible chemopreventive potential.

Machaerium hirtum (Vell.) Stellfeld (M.hirtum) is a plant known as 'jacarandá-bico-de-pato' whose bark is commonly used against diarrhea, cough and cancer. The aim of this study was to phytochemically characterise the hydroethanolic extract of this plant, investigate its antimutagenic activities using the Ames test and evaluate its effects on cell viability, genomic instability, gene expression and cell protection in human hepatocellular carcinoma cells (HepG2). Antimutagenic activity was assessed by simultaneous pre- and post-treatment with direct and indirect mutagens, such as 4-nitro-o-phenylenediamine (NPD), mitomycin C (MMC), benzo[a]pyrene (B[a]P) and aflatoxin B1 (AFB1), using the Ames test, cytokinesis blocking micronucleus and apoptosis assays. Only 3 of the 10 concentrations evaluated in the MTT assay were cytotoxic in HepG2 cells. Micronucleated or apoptotic cells were not observed with any of the tested concentrations, and there were no mutagenic effects in the bacterial system. However, the Nuclear Division Index and flow cytometry data showed a decrease in cell proliferation. The extract showed an inhibitory effect against direct (NPD) and indirect mutagens (B[a]P and AFB1). Furthermore, pre- and post-treated cells showed significant reduction in the number of apoptotic and micronucleated cells. This effect is not likely to be associated with the modulation of antioxidant genes, as shown by the RT-qPCR results. Six known flavonoids were identified in the hydroethanolic extract of Machaerium hirtum leaves, and their structures were elucidated by spectroscopic and spectrophotometric methods. The presence of the antioxidants apigenin and luteolin may explain these protective effects, because these components can inhibit the formation of reactive species and prevent apoptosis and DNA damage. In conclusion, the M.hirtum extract showed chemopreventive potential and was not hazardous at the tested concentrations in the experiments presented here. Moreover, this extract should be investigated further as a chemopreventive agent.

In vitro assessment of mutagenic and genotoxic effects of coumarin derivatives 6,7-dihydroxycoumarin and 4-methylesculetin.

Coumarins are naturally occurring compounds, widely distributed throughout the plant kingdom (Plantae), and possess important pharmacological properties, including inhibition of oxidative stress. In this context, newly synthesized coumarin compounds are being produced due to their potent antioxidant activities. Therefore, the aim of the present study was to determine the in vitro cytotoxic, mutagenic, and genotoxic effects of 6,7-dihydroxycoumarin (6,7-HC) and 4-methylesculetin (4-ME) using the Salmonella/microsome test and in cultured human lymphocytes the comet assay and micronucleus test. The three coumarin derivatives concentrations evaluated in comet and MN assays were 2, 8, and 32 µg/mL, selected through a preliminary trypan blue-staining assay. In the Ames test, the 5 concentrations tested were 62.5, 125, 250, 500, and 750 µg/plate. Positive (methyl methane-sulfonate, MMS) and negative (dimethyl sulfoxide, DMSO) control groups were also included in the analysis. Our results showed that 4-ME induced greater cytotoxicity at high concentrations than 6,7-HC. In addition, both compounds were not mutagenic in the Ames test and not genotoxic or clastogenic/aneugenic in cultured human lymphocytes.

Mutagenicity and chemopreventive activities of Astronium species assessed by Ames test.

In the neotropical savannah, Astronium species are used in popular medicine to treat allergies, inflammation, diarrhea and ulcers. Given that natural products are promising starting points for the discovery of novel potentially therapeutic agents, the aim of the present study was to investigate the mutagenic and antimutagenic activities of hydroalcoholic extracts of Astronium spp. The mutagenicity was determined by the Ames test on Salmonella typhimurium strains TA98, TA97a, TA100 and TA102. The antimutagenicity was tested against the direct-acting and indirect-acting mutagens. The results showed that none of the extracts induce any increase in the number of revertants, demonstrating the absence of mutagenic activity. On the other hand, the results on the antimutagenic potential showed a moderate inhibitory effect against NPD and a strong protective effect against B[a]P and AFB1. This study highlights the importance of screening species of Astronium for new medicinal compounds. The promising results obtained open up new avenues for further study and provide a better understanding the mechanisms by which these species act in protecting DNA from damage. However, further pharmacological and toxicological investigations of crude extracts of Astronium spp., as well as of its secondary metabolites, are necessary to determine the mechanism(s) of action to guarantee their safer and more effective application to human health.

Vellozia flavicans Mart. ex Schult. hydroalcoholic extract inhibits the neuromuscular blockade induced by Bothrops jararacussu venom.

BACKGROUND: Snakebite is a significant public health issue in tropical countries. In Brazil, some of the most common snake envenomations are from Bothrops. Bothrops bites trigger local and systemic effects including edema, pain, erythema, cyanosis, infections, and necrosis. Vellozia flavicans is a plant from the Brazilian "cerrado" (savanna) that is popularly used as an anti-inflammatory medicine. Since inflammation develops quickly after Bothrops bites, which can lead to infection, the aim of the present study was to observe possible anti-snake venom and antimicrobial activities of V. flavicans (Vf). METHODS: The chromatographic profile of the main constituents from the Vf leaf hydroalcoholic extract was obtained by thin-layer chromatography (TLC). The anti-snake venom activity was measured by Vf's ability to neutralize the in vitro neuromuscular blockade caused by Bothrops jararacussu venom (Bjssu) in a mouse phrenic nerve-diaphragm model (PND). After a 20 min incubation, preparations of PND were added to Tyrode's solution (control); Vf (0.2, 0.5, 1, and 2 mg/mL); 40 µg/mL Bjssu; pre-incubation for 30 min with Bjssu and 1 mg/mL Vf; and a Bjssu pretreated preparation (for 10 min) followed by 1 mg/mL Vf. Myographic recording was performed, and the contractile responses were recorded. The antimicrobial activity (minimum inhibitory concentration [MIC] and minimum bactericidal concentration [MBC]) was obtained for Staphylococcus aureus, Pseudomonas aeruginosa, Escherichia coli, and Enterococcus faecalis, using gentamicin and vancomycin as positive controls. RESULTS: TLC analysis yielded several compounds from Vf, such as flavonoids (quercetin) and phenolic acids (chlorogenic acid). Bjssu completely blocked the contractile responses of PND preparations, while Vf preserved 97% (±10%) of the contractile responses when incubated with Bjssu. In the PND pretreated with Bjssu, Vf was able to inhibit the neuromuscular blockade progress. MIC and MBC of Vf ranged from 2.5 to 5.0 mg/mL for P. aeruginosa and S. aureus strains, while no antimicrobial activity was observed for E. coli and E. faecalis. CONCLUSIONS: The hydroalcoholic extract from Vf leaves was able to neutralize and decrease the in vitro neuromuscular blockade caused by Bjssu. However, it did not show significant antimicrobial activity against the tested bacteria.

Mutagenicity and antimutagenicity of six Brazilian Byrsonima species assessed by the Ames test.

BACKGROUND: In various regions of Brazil, several species of the genus Byrsonima (Malpighiaceae) are widely used to treat gastrointestinal complications. This genus has about 150 species of shrubs and trees distributed over the entire Neotropical region. Various biological activities have been identified in these plants, especially antioxidant, antimicrobial and topical and systemic anti-inflammatory activities. The aim of this study was to investigate the mutagenicity and antimutagenicity of hydroalcoholic leaf extracts of six species of Byrsonima: B. verbascifolia, B. correifolia, B. coccolobifolia, B. ligustrifolia, B. fagifolia and B. intermedia by the Salmonella microsome assay (Ames test). METHODS: Mutagenic and antimutagenic activity was assessed by the Ames test, with the Salmonella typhimurium tester strains TA100, TA98, TA97a and TA102, with (+S9) and without (-S9) metabolization, by the preincubation method. RESULTS: Only B. coccolobifolia and B. ligustrifolia showed mutagenic activity. However, the extracts of B. verbascifolia, B. correifolia, B. fagifolia and B. intermedia were found to be strongly antimutagenic against at least one of the mutagens tested. CONCLUSIONS: These results contribute to valuable data on the safe use of medicinal plants and their potential chemopreventive effects. Considering the excellent antimutagenic activities extracted from B. verbascifolia, B. correifolia, B. fagifolia and B. intermedia, these extracts are good candidate sources of chemopreventive agents. However, B. coccolobifolia and B. ligustrifolia showed mutagenic activity, suggesting caution in their use.

Characterization and quantification of the compounds of the ethanolic extract from Caesalpinia ferrea stem bark and evaluation of their mutagenic activity.

Caesalpinia ferrea Martius has traditionally been used in Brazil for many medicinal purposes, such as the treatment of bronchitis, diabetes and wounds. Despite its use as a medicinal plant, there is still no data regarding the genotoxic effect of the stem bark. This present work aims to assess the qualitative and quantitative profiles of the ethanolic extract from the stem bark of C. ferrea and to evaluate its mutagenic activity, using a Salmonella/microsome assay for this species. As a result, a total of twenty compounds were identified by Flow Injection Analysis Electrospray Ionization Ion Trap Mass Spectrometry (FIA-ESI-IT-MS/MSn) in the ethanolic extract from the stem bark of C. ferrea. Hydrolyzable tannins predominated, principally gallic acid derivatives. The HPLC-DAD method was developed for rapid quantification of six gallic acid compounds and ellagic acid derivatives. C. ferrea is widely used in Brazil, and the absence of any mutagenic effect in the Salmonella/microsome assay is important for pharmacological purposes and the safe use of this plant.

In vitro assessment of the cytotoxic, apoptotic, and mutagenic potentials of isatin.

Isatin (1H-indole-2,3-dione) is a chemical found in various medicinal plant species and responsible for a broad spectrum of pharmacological and biological properties that may be beneficial to human health, as an anticonvulsant, antibacterial, antifungal, antiviral, and anticancer agent. The aim of the present study was to determine in vitro the cytotoxic, mutagenic, and apoptotic effects of isatin on CHO-K1 and HeLa cells using the MTT viability assay (3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyl tetrazolium bromide), micronucleus (MN) test, apoptosis index, and nuclear division index (NDI). The 5 isatin concentrations evaluated in the mutagenicity and apoptosis tests were 0.5, 1, 5, 10, and 50 µM, selected through a preliminary MTT assay. Positive (doxorubicin, DXR) and negative (phosphate buffered saline, PBS) control groups were also included in the analysis. Isatin did not exert a mutagenic effect on CHO-K1 after 3 and 24 h of treatment or on HeLa cells after 24 h. However, 10 and 50 µM concentrations inhibited cell proliferation and promoted apoptosis in both CHO-K1 and HeLa cells. Data indicate that the cytotoxic, apoptotic, and antiproliferative effects of isatin were concentration independent and cell line independent.

Mutagenicity and antimutagenicity of (-)-hinokinin a trypanosomicidal compound measured by Salmonella microsome and comet assays.

BACKGROUND: The dibenzylbutyrolactone lignan (-)-hinokinin (HK) was derived by partial synthesis from (-)-cubebin, isolated from the dry seeds of the pepper, Piper cubeba. Considering the good trypanosomicidal activity of HK and recalling that natural products are promising starting points for the discovery of novel potentially therapeutic agents, the aim of the present study was to investigate the (anti) mutagenic∕ genotoxic activities of HK. METHODS: The mutagenic∕ genotoxic activities were evaluated by the Ames test on Salmonella typhimurium strains TA98, TA97a, TA100 and TA102, and the comet assay, so as to assess the safe use of HK in the treatment of Chagas' disease. The antimutagenic ∕antigenotoxic potential of HK were also tested against the mutagenicity of a variety of direct and indirect acting mutagens, such as 4- nitro-o-phenylenediamine (NOPD), sodium azide (SA), mitomycin C (MMC), benzo[a]pyrene (B[a]P), aflatoxin B1 (AFB1), 2-aminoanthracene (2-AA) and 2-aminofluorene (2-AF), by the Ames test, and doxorubicin (DXR) by the comet assay. RESULTS: The mutagenicity∕genotoxicity tests showed that HK did not induce any increase in the number of revertants or extent of DNA damage, demonstrating the absence of mutagenic and genotoxic activities. On the other hand, the results on the antimutagenic potential of HK showed a strong inhibitory effect against some direct and indirect-acting mutagens. CONCLUSIONS: Regarding the use of HK as an antichagasic drug, the absence of mutagenic effects in animal cell and bacterial systems is encouraging. In addition, HK may be a new potential antigenotoxic ∕ antimutagenic agent from natural sources. However, the protective activity of HK is not general and varies with the type of DNA damage-inducing agent used.

In vitro and in vivo safety evaluation of Dipteryx alata Vogel extract.

BACKGROUND: Dipteryx alata Vogel popularly known as "baru" is an important commercial leguminous tree species from the Brazilian Cerrado, which possess medicinal properties, besides its fruits consumption by animals and humans. The use of the "naturally occurring plants" as herbal remedies and foods mainly from leaves, seeds, flowers and roots of plants or extracts require precautions before ensuring these are safe and efficacious. The objective of this study was to evaluate the safety of D. alata barks extract. METHODS: Vegetal drugs of D. alata barks were submitted to quality control assays and further to the safety assays under 1) in vitro parameter by Salmonella (Ames) mutagenicity, and 2) in vivo parameter on the pregnancy of rats. RESULTS: The extract was non-mutagenic to any of the assessed strains TA97a, TA98, TA100 and TA102 even after metabolic activation (+S9). All in vivo parameters (reproductive ability evaluation, physical development of rat offsprings, and neurobehavioral development assays) showed no changes related to control group. CONCLUSION: D. alata barks extract is neither mutagenic by the Ames test nor toxic in the pregnancy of rats, with no physical-neurobehavioral consequences on the rat offsprings development.

Mutagenicity of flavonoids assayed by bacterial reverse mutation (Ames) test.

The mutagenicity of ten flavonoids was assayed by the Ames test, in Salmonella typhimurium strains TA98, TA100 and TA102, with the aim of establishing hydroxylation pattern-mutagenicity relationship profiles. The compounds assessed were: quercetin, kaempferol, luteolin, fisetin, chrysin, galangin, flavone, 3-hydroxyflavone, 5-hydroxyflavone and 7-hydroxyflavone. In the Ames assay, quercetin acted directly and its mutagenicity increased with metabolic activation. In the presence of S9 mix, kaempferol and galangin were mutagenic in the TA98 strain and kaempferol showed signs of mutagenicity in the other strains. The absence of hydroxyl groups, as in flavone, only signs of mutagenicity were shown in strain TA102, after metabolization and, among monohydroxylated flavones (3-hydroxyflavone, 5-hydroxyflavone and 7-hydroxyflavone), the presence of hydroxyl groups only resulted in minor changes. Luteolin and fisetin also showed signs of mutagenicity in strain TA102. Finally, chrysin, which has only two hydroxy groups, at the 5-OH and 7-OH positions, also did not induce mutagenic activity in any of the bacterial strains used, under either activation condition. All the flavonoids were tested at concentrations varying from 2.6 to 30.7 nmol/plate for galangin and 12.1 to 225.0 nmol/plate for other flavonoids. In light of the above, it is necessary to clarify the conditions and the mechanisms that mediate the biological effects of flavonoids before treating them as therapeutical agents, since some compounds can be biotransformed into more genotoxic products; as is the case for galangin, kaempferol and quercetin.

Characterization of flavonoids and naphthopyranones in methanol extracts of Paepalanthus chiquitensis Herzog by HPLC-ESI-IT-MSn and their mutagenic activity.

A HPLC-ESI-IT-MSn method, based on high-performance liquid chromatography coupled to electrospray negative ionization multistage ion trap mass spectrometry, was developed for rapid identification of 24 flavonoid and naphthopyranone compounds. The methanol extracts of the capitulae and scapes of P. chiquitensis exhibited mutagenic activity in the Salmonella/microsome assay, against strain TA97a.

Comparative Studies of the (Anti) Mutagenicity of Baccharis dracunculifolia and Artepillin C by the Bacterial Reverse Mutation Test.

Baccharis dracunculifolia is a plant native from Brazil, commonly known as 'Alecrim-do-campo' and 'Vassoura' and used in alternative medicine for the treatment of inflammation, hepatic disorders and stomach ulcers. Previous studies reported that artepillin C (ArtC, 3-{4-hydroxy-3,5-di(3-methyl-2-butenyl)phenyl}-2(E)-propenoic acid), is the main compound of interest in the leaves. This study was undertaken to assess the mutagenic effect of the ethyl acetate extract of B. dracunculifolia leaves (Bd-EAE: 11.4-182.8 µg/plate) and ArtC (0.69-10.99 µg/plate) by the Ames test using Salmonella typhimurium strains TA98, TA97a, TA100 and TA102, and to compare the protective effects of Bd-EAE and ArtC against the mutagenicity of a variety of direct and indirect acting mutagens such as 4-nitro-O-phenylenediamine, sodium azide, mitomycin C, benzo[a]pyrene, aflatoxin B1, 2-aminoanthracene and 2-aminofluorene.The mutagenicity test showed that Bd-EAE and ArtC did not induce an increase in the number of revertant colonies indicating absence of mutagenic activity. ArtC showed a similar antimutagenic effect to that of Bd-EAE in some strains of S. typhimurium, demonstrating that the antimutagenic activity of Bd-EAE can be partially attributed to ArtC. The present results showed that the protective effect of whole plant extracts is due to the combined and synergistic effects of a complex mixture of phytochemicals, the total activity of which may result in health benefits.

Mutagenicity and genotoxicity of isatin in mammalian cells in vivo.

Isatin (1H-indole-2,3-dione) is a synthetically versatile substrate used for the synthesis of heterocyclic compounds and as a raw material for drug synthesis. Isatin and its derivatives demonstrate anticonvulsant, antibacterial, antifungal, antiviral, and anticancer properties. We evaluated the genotoxic and mutagenic effects of acute (24h) and repeated (14d) exposure to isatin in vivo, using the comet assay and the micronucleus test. Three doses (50, 100, and 150mg/kgb.w.) were administered to mice via gavage. Doses were selected according to the LD(50) of isatin, estimated in a preliminary test to be 1g/kgb.w. To evaluate the results, parametric (ANOVA/Tukey) and non-parametric (Kruskal-Wallis/Dunn's post hoc test) tests were used, according to the nature of the data distribution. At all doses (50, 100 and 150mg/kgb.w.), after acute treatment with isatin, alterations in DNA migration (comet assay) were not observed and mutagenic effects were not seen (micronucleus test on peripheral blood cells). After repeated doses, only the highest dose of isatin (150mg/kgb.w.) induced alterations in the DNA that gave rise to micronuclei in the bone marrow and peripheral blood cells of the mice. Our results show that the mutagenic and genotoxic effects of isatin depend on dose and on period of exposure.