Juliana Marques Senedese

In vivo protective effect of Copaifera langsdorffii hydroalcoholic extract on micronuclei induction by doxorubicin

Copaifera lansdorffii Desf. is known as ‘copaíba’, ‘copaiva’ or ‘paú‐de‐óleo’, and is found in part of Brazil. The present study was undertaken to evaluate the genotoxic potential of C. langsdorffii leaf hydroalcoholic extract (CLE) and its influence on the genotoxicity induced by the chemotherapeutic agent doxorubicin (DXR) using the Swiss mouse peripheral blood micronucleus test. HPLC analysis of CLE using two monolithic columns linked in series allowed quantification of two major flavonoid heterosides, quercitrin and afzelin. Animals were treated with CLE by gavage at doses of 10, 20, 40 and 80 mg kg−1 body weight per day, each for 20 days. Peripheral blood samples were collected at 24 and 48 h, and 7, 15 and 21 days after the beginning of the treatment. For the antigenotoxicity evaluation, the animals treated with different concentrations of CLE received DXR (15 mg kg−1 body weight, intraperitoneal) at day 20. The peripheral blood samples were collected 24 and 48 h after the treatment with DXR. The results demonstrated that CLE itself was not genotoxic in the mouse micronucleus assay. In animals treated with CLE and DXR, the number of micronucleus was significantly decreased compared with animals receiving DXR alone. The putative antioxidant activity of one or more of the active compounds of CLE may explain the effect of this plant on DXR genotoxicity. Copyright

Assessment of the Mutagenic Activity of Extracts of Brazilian Propolis in Topical Pharmaceutical Formulations on Mammalian Cells In Vitro and In Vivo

Propolis possesses various biological activities such as antibacterial, antifungal, anti-inflammatory, anesthetic and antioxidant properties. A topically applied product based on Brazilian green propolis was developed for the treatment of burns. For such substance to be used more safely in future clinical applications, the present study evaluated the mutagenic potential of topical formulations supplemented with green propolis extract (1.2, 2.4 and 3.6%) based on the analysis of chromosomal aberrations and of micronuclei. In the in vitro studies, 3-h pulse (G1 phase of the cell cycle) and continuous (20 h) treatments were performed. In the in vivo assessment, the animals were injured on the back and then submitted to acute (24 h), subacute (7 days) and subchronic (30 days) treatments consisting of daily dermal applications of gels containing different concentrations of propolis. Similar frequencies of chromosomal aberrations were observed for cultures submitted to 3-h pulse and continuous treatment with gels containing different propolis concentrations and cultures not submitted to any treatment. However, in the continuous treatment cultures treated with the 3.6% propolis gel presented significantly lower mitotic indices than the negative control. No statistically significant differences in the frequencies of micronuclei were observed between animals treated with gels containing different concentrations of propolis and the negative control for the three treatment times. Under the present conditions, topical formulations containing different concentrations of green propolis used for the treatment of burns showed no mutagenic effect in either test system, but 3.6% propolis gel was found to be cytotoxic in the in vitro test.

Chemopreventive effect of Copaifera langsdorffii leaves hydroalcoholic extract on 1,2-dimethylhydrazine-induced DNA damage and preneoplastic lesions in rat colon

BackgroundNatural antioxidants present in common foods and beverages have drawn great attention to cancer prevention due to its health benefits, remarkable lack of toxicity and side effects. Copaifera langsdorffii, known as “copaiba”, “capaiva”, or “pau-de-óleo“, belongs to the Leguminosae family and occurs in fields and grasslands in the northern and northeastern parts of Brazil. Biological studies of Copaifera corroborate its widespread use by the population. This paper describes the effects of C. langsdorffii leaves hydroalcoholic extract on the 1,2-dimethylhydrazine (DMH)-induced DNA damage and aberrant crypt foci (ACF) in the colon of male Wistar rats.MethodsThe hydroalcoholic extract of C. langsdorffii was administered to rats by gavage at daily doses of 20, 40 and 80 mg/kg body weight. To evaluate DNA damage by the comet assay, animals received the C. langsdorffii extract for seven days and a single subcutaneous injection (sc) of 1,2-dimethylhydrazine (DMH) at a dose of 40 mg/kg on day 7. Animals were sacrificed 4 h after injection of DMH, to assess DNA damage. For the ACF assay, animals were acclimatized for one week (week 1) and then treated with the C. langsdorffii extract five times a week for four weeks (weeks 2 to 5). The rats received sc injections of DMH (40 mg/kg) on days 2 and 5 of weeks 2 and 3, to induce ACF. Animals were euthanized at week 5; i.e., four weeks after the first DMH treatment.ResultsAnimals treated with different doses of the C. langsdorffii extract combined with DMH had significantly lower frequency of DNA damage as compared with the positive control (animals treated with DMH only). The percentage of reduction in the frequency of DNA damage ranged from 14.30% to 38.8%. The groups treated with 40 and 80 mg/kg C. langsdorffii extract during and after DMH treatment presented significantly lower numbers of ACF and aberrant crypts compared with the control.ConclusionThe C. langsdorffii extract significantly reduced the extent of DNA damage and ACF induced by DMH, suggesting that the extract has a protective effect against colon carcinogenesis.

Copaifera langsdorffii oleoresin and its isolated compounds: antibacterial effect and antiproliferative activity in cancer cell lines.

BackgroundNatural products display numerous therapeutic properties (e.g., antibacterial activity), providing the population with countless benefits. Therefore, the search for novel biologically active, naturally occurring compounds is extremely important. The present paper describes the antibacterial action of the Copaifera langsdorffii oleoresin and ten compounds isolated from this oleoresin against multiresistant bacteria; it also reports the antiproliferative activity of the Copaifera langsdorffii oleoresin and (-)-copalic acid.MethodsMICs and MBCs were used to determine the antibacterial activity. Time-kill curve assays provided the time that was necessary for the bacteria to die. The Minimum Inhbitory Concentration of Biofilm (CIMB50) of the compounds that displayed the best results was calculated. Cytotoxicity was measured by using the XTT assay.ResultsThe diterpene (-)-copalic acid was the most active antibacterial and afforded promising Minimum Inhibitory Concentration (MIC) values for most of the tested strains. Determination of the bactericidal kinetics against some bacteria revealed that the bactericidal effect emerged within six hours of incubation for Streptococcus pneumoniae. Concerning the antibiofilm action of this diterpene, its MICB50 was twofold larger than its CBM against S. capitis and S. pneumoniae. The XTT assay helped to evaluate the cytotoxic effect; results are expressed as IC50. The most pronounced antiproliferative effect arose in tumor cell lines treated with (-)-copalic acid; the lowest IC50 value was found for the human glioblastoma cell line.ConclusionsThe diterpene (-)-copalic acid is a potential lead for the development of new selective antimicrobial agents to treat infections caused by Gram-positive multiresistant microorganisms, in both the sessile and planktonic mode. This diterpene is also a good candidate to develop anticancer drugs.

Copaifera multijuga oleoresin and its constituent diterpene (−)-copalic acid: Genotoxicity and chemoprevention study

Copaiba oleoresins are used in alternative medicine as anti-inflammatory, antitumoral, and antimicrobial treatments. (-)-Copalic acid (CA) is the major diterpene found in exudates from Copaifera species. We have examined the genotoxicity and the chemopreventive potential of Copaifera multijuga oleoresin (CM) and CA. Genotoxicity assessment was examined with the peripheral blood micronucleus test and the comet assay (male Swiss mouse hepatocytes). In the chemoprevention study, we evaluated the effects of CM and CA on the formation of 1,2-dimethylhydrazine (DMH)-induced aberrant crypt foci (ACF) in male Wistar rat colon. Neither agent caused a significant increase in micronucleus frequency relative to controls, but the highest CM dose tested (400mg/kg b.w.) caused DNA damage in the comet assay. Both agents significantly reduced the frequency of DMH-induced ACF. Both CM and CA suppressed ACF formation and may have a protective effect against colon carcinogenesis.

Assessment of the antibacterial, cytotoxic and mutagenic potential of the phenolic-rich hydroalcoholic extract from Copaifera trapezifolia Hayne leaves.

Copaifera trapezifolia Hayne occurs in the Atlantic Rainforest, which is considered one of the most important and endangered tropical forests on the planet. Although literature works have described many Copaifera spp., their biological activities remain little known. In the present study, we aimed to evaluate (1) the potential of the hydroalcoholic extract from C. trapezifolia leaves (CTE) to act against the causative agents of tooth decay and apical periodontitis and (2) the cytotoxicity and mutagenicity of CTE to ensure that it is safe for subsequent application. Concerning the tested bacteria, the MIC and the minimum bactericidal concentration of CTE varied between 100 and 400 µg ml-1. The time-kill assay conducted at a CTE concentration of 100 µg ml-1 evidenced bactericidal activity against Porphyromonas gingivalis (ATCC 33277) and Peptostreptococcus micros (clinical isolate) within 72 h. CTE at 200 µg ml-1 inhibited Porphyromonas gingivalis and Peptostreptococcus micros biofilm formation by at least 50 %. A combination of CTE with chlorhexidine dichlorohydrate did not prompt any synergistic effects. The colony-forming assay conducted on V79 cells showed that CTE was cytotoxic at concentrations above 156 µg ml-1. CTE exerted mutagenic effect on V79 cells, but the micronucleus test conducted on Swiss mice and the Ames test did not reveal any mutagenicity. Therefore, the use of standardized and safe extracts could be an important strategy to develop novel oral care products with antibacterial action. These extracts could also serve as a source of compounds for the discovery of new promising biomolecules.

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

ABSTRACT 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.

Assessment of genotoxic activity of oleoresins and leaves extracts of six Copaifera species for prediction of potential human risks

ETHNOPHARMACOLOGICAL RELEVANCE Copaifera species are used in folk medicine for a wide variety of pharmacological properties. This paper reports the cytotoxic and genotoxic analyses of oleoresins and leaves extracts of Copaifera species: C. duckei, C. multijuga, C. paupera, C. pubiflora, C. reticulata and C. trapezifolia. MATERIALS AND METHODS In vitro assays were performed using Chinese hamster lung fibroblasts (V79 cells). The clonogenic efficiency and cytokinesis-block micronucleus assays were employed for the cytotoxicity and genotoxicity assessment, respectively. The mouse bone marrow micronucleus test was used for in vivo studies. RESULTS The cytotoxicity results using the clonogenic efficiency assay showed IC50 values ranging from 9.8 to 99.2 µg/mL for oleoresins and 66.4-721.5 for leaves extracts. However, no cytotoxic effect was observed in the in vivo studies. Additionally, the treatments with oleoresins and leaves extracts did not significantly increase the frequency of micronuclei in both in vitro and in vivo mammalian cells. The UPLC-MS/MS and CG/MS analyses of Copaifera oleoresins allowed the identification of 10 acid diterpenes and 11 major volatile sesquiterpenes. Leaves are rich in phenolic compounds including two flavonoid heterosides and 16 galloylquinic acid derivatives. CONCLUSIONS The oleoresins and leaves extracts of studied Copaifera species were not cytotoxic in vivo, as well as not genotoxic in both in vitro and vivo assays, under the experimental conditions used. Therefore, the obtained results should be sufficient to demonstrate the absence of significant genotoxic risk of these Copaifera products for human use in the evaluated concentrations range.