Genotoxicity and toxicological evaluations of Brazilian red propolis oral ingestion in a preclinical rodent model.

ETHNOPHARMACOLOGICAL RELEVANCE: Brazilian red propolis is a natural product known due to its medicinal properties. The efficacy of this natural resin has been proved; however, few studies report the safety of its oral use. Some toxic effects of natural products may not be expressed in traditional use, and preclinical studies are necessary to guarantee their safety. Health regulatory agency currently requires these non-clinical studies to develop drugs and herbal medicines, including genotoxic and oral toxicity tests. AIM OF THE STUDY: Accomplish the preclinical toxicity studies of Brazilian red propolis extract (BRP) in rodents, including genotoxicity, acute and sub-chronic toxicities. MATERIAL AND METHODS: Genotoxicity assays followed the erythrocyte micronucleus test protocol in a range of 500-2000 mg/kg BRP oral treatment on male Swiss mice. After an up-and-down procedure, acute oral toxicity (single dose) was performed on female Wistar Hannover rats, reaching a 2000 mg/kg BRP oral gavage concentration. Animals were monitored periodically until 14 days and euthanized for a macroscopic necropsy analysis. The sub-chronic oral toxicity test (90 days) was achieved with 1000 mg/kg of BRP on Wistar Hannover rats (males/females). Animals were monitored to evaluated behavioral and biometrical changes, then were euthanized to perfomed hematological, biochemical, and histopathological analyses. RESULTS: No genotoxic effect of the BRP was detected. The acute toxicity indicated no toxicity of a single oral dose of 2000 mg/kg of BRP. The long-term oral toxicity performed with 1000 mg/kg of BRP altered water and food intake and the biometrics, hematological and biochemical parameters. Biochemical alterations in hepatic and renal parameters were detected only in the males. Despite the detection of biochemical alterations, no histopathological changes were detected in the organs of any group. CONCLUSIONS: BRP, at a higher dose, showed no signs of immediate toxicity. However, the obtained results suggest that the chemical composition and the intake of higher doses deserve special attention regarding possible toxicity.

Guttiferone E Displays Antineoplastic Activity Against Melanoma Cells.

Guttiferone E (GE) is a benzophenone found in Brazilian red propolis. In the present study, the effect of GE on human (A-375) and murine (B16-F10) melanoma cells was investigated. GE significantly reduced the cellular viability of melanoma cells in a time-dependent manner. In addition, GE demonstrated antiproliferative effect, with IC50 values equivalent to 9.0 and 6.6 µM for A-375 and B16-F10 cells, respectively. The treatment of A-375 cells with GE significantly increased cell populations in G0/G1 phase and decreased those in G2/M phase. Conversely, on B16-F10 cells, GE led to a significant decrease in the populations of cells in G0/G1 phase and concomitantly an increase in the population of cells in phase S. A significantly higher percentage of apoptotic cells was observed in A-375 (43.5%) and B16-F10 (49.9%) cultures after treatment with GE. Treatments with GE caused morphological changes and significant decrease to the melanoma cells' density. GE (10 µM) inhibited the migration of melanoma cells, with a higher rate of inhibition in B16-F10 cells (73.4%) observed. In addition, GE significantly reduced the adhesion of A375 cells, but showed no effect on B16-F10. Treatment with GE did not induce changes in P53 levels in A375 cultures. Molecular docking calculations showed that GE is stable in the active sites of the tubulin dimer with a similar energy to taxol chemotherapy. Taken together, the data suggest that GE has promising antineoplastic potential against melanoma.

In vitro evaluation of the antibacterial and cytotoxic activities of the Euclea natalensis crude extract and fractions against oral infection agents.

OBJECTIVE: This study aimed (i) to evaluate the antibacterial and cytotoxic activities of the crude extract and fractions obtained from Euclea natalensis A.D.C. roots against bacteria that cause periodontal disease and caries and (ii) to identify the isolated compounds. DESIGN: The minimum inhibitory concentration (MIC) and the minimum bactericidal concentration (MBC) of the extract and fractions were determined by the microplate dilution assay. The cytotoxicity of the extract and fractions was evaluated by using the XTT colorimetric assay and normal human fibroblast cells (GM07492A, lung fibroblasts). The compounds present in the most promising fraction were determined by qualitative analysis through liquid chromatography coupled to mass spectrometry (HPLC-MS-ESI). RESULTS: The MIC results ranged from 25 to > 400 µg/mL for the extract and from 1.56 to > 400 µg/mL for the fractions. To evaluate cytotoxicity, the tested concentrations of the extract and fractions ranged from 19.5 to 2500 µg/mL; IC50 values between 625 and 1250 µg/mL were obtained. Analysis of the main bioactive fraction by HPLC-MS-ESI identified phenolic acids, coumarins, naphthoquinones, lignans, and fatty acids. CONCLUSIONS: The E. natalensis root extract and fractions displayed good antibacterial activity against periodontal pathogenic and cariogenic bacteria. The antibacterial activity may be due to compounds present in the extract and fractions, which also showed low cytotoxicity to normal human cells. These data are relevant and encourage further research into this plant species, which may contribute to the discovery of new herbal medicines that will help to mitigate the problems caused by oral pathogenic bacteria.

Ozonated oil is effective at killing Candida species and Streptococcus mutans biofilm-derived cells under aerobic and microaerobic conditions.

This study explores the growth of bacterial, fungal, and interkingdom biofilms under aerobiosis or microaerobic conditions and the effect of ozonated sunflower oil on these biofilms. Candida species and Streptococcus mutans were used to study this interaction due to their importance in oral health and disease as these microorganisms display a synergistic relationship that manifests in the onset of caries and tooth decay. Biofilms were developed in a 96-well microtiter plate at 37ºC for 24 h, under aerobiosis or microaerobic conditions, and treated with ozonated oil for 5 to 120 min. All the microorganisms formed biofilms in both oxygenation conditions. Scanning electron microscopy was used to visualize biofilm morphology. Rodent experiments were performed to verify the oil-related toxicity and its efficacy in oral candidiasis. The growth of all Candida species was increased when co-cultured with S. mutans, whilst the growth of bacterium was greater only when co-cultured with C. krusei and C. orthopsilosis under aerobiosis and microaerobic conditions, respectively. Regardless of the oxygenation condition, ozonated oil significantly reduced the viability of all the tested biofilms and infected mice, showing remarkable microbicidal activity as corroborated with confocal microscopy and minimal toxicity. Thus, ozonated oil therapy can be explored as a strategy to control diseases associated with these biofilms especially in the oral cavity. LAY SUMMARY: We demonstrated that ozonated sunflower oil is effective at killing the biofilms formed by Candida species, by the bacterium Streptococcus mutans, or by both micoorganisms that can interact in the oral cavity, making it a potential therapeutic option for the treatment of these infections.

Toxicological and chemoprevention studies of Dalbergia ecastaphyllum (L.) Taub. stem, the botanical source of Brazilian red propolis.

OBJECTIVES: Dalbergia ecastaphyllum (L.) Taub. is a semi-prostrate species associated with estuaries, mangroves and dunes. This plant species has great ecological and economic importance, especially concerning apiculture pasture and Brazilian red propolis production. In this study, non-clinical toxicological evaluations of the hydroalcoholic extract of D. ecastaphyllum stems (DEHE), the resin production source, were conducted. In addition, the action of DEHE on genomic instability and colon carcinogenesis was investigated. METHODS AND RESULTS: The extract's chemical profile was analysed by HPLC, and medicarpin, vestitol and neovestitol were found as major compounds. DEHE showed an IC50 equivalent to 373.2 µg/ml and LC50 equal 24.4 mg/L, when evaluated using the XTT colorimetric test and the zebrafish acute toxicity assay, respectively. DEHE was neither genotoxic nor cytotoxic at the highest dose, 2000 mg/kg, by peripheral blood micronucleus test. The treatments DEHE (6 and 24 mg/kg) led to the reduction of micronuclei induced by doxorubicin (DXR) in mice. Furthermore, significantly higher serum levels of reduced glutathione were observed in animals treated with DEHE plus DXR, revealing an antioxidant effect. Treatments with DEHE (48 mg/kg) led to a significant reduction in pre-neoplastic lesions induced by the 1,2-dimethylhydrazine (DMH) carcinogen in the rat colon. Immunohistochemical analysis revealed significantly lower levels of expression of COX-2 (86%) and PCNA (83%) in the colon of rats treated with DEHE plus DMH, concerning those treated with the carcinogen. CONCLUSIONS: These results indicate the involvement of anti-inflammatory and antiproliferative pathways in the protective effect of DEHE.

Hypericin, a medicinal compound from St. John's Wort, inhibits genotoxicity induced by mutagenic agents in V79 cells.

This study evaluated the cytotoxic, genotoxic, and the modulatory effects on DNA damage of hypericin in Chinese hamster lung fibroblasts (V79 cells). The hypericin is a natural polycyclic quinone, mainly extracted from St. John's Wort (Hypericum perforatum L.). Along with hyperforin, the hypericins are responsible for the antidepressant activity of St. John's Wort. Cytotoxicity was assessed by the XTT colorimetric assay and the nuclear division index (NDI). The genotoxic activity was studied by the micronucleus test at concentrations of 30, 60, 120, and 240 µg/mL. Mutagenic agents, methyl methanesulfonate (MMS, 44 µg/mL), doxorubicin (DXR, 0.5 µg/mL), and etoposide (VP16, 1 µg/mL) were used in combination with different concentrations of hypericin in order to evaluate the modulatory effect on DNA damage. Results showed that the hypericin was cytotoxic at concentrations above 156.2 µg/mL and genotoxic above 120 µg/mL. The hypericin significantly reduced DNA damage frequency induced by DXR, at concentrations of 30 and 60 µg/mL, and MMS at a concentration of 30 µg/mL, but was unable to reduce damage when combined with VP-16. These results demonstrate the non-photoactivated hypericin toxicological safety limits, its protective effect on DNA damage and provide a basis for future studies that may characterize better its chemopreventive mechanism.

Watermelon Reduces the Toxicity of Cisplatin Treatment in C57BL/6 Mice with Induced Melanoma.

An alternative to reduce the undesirable effects of antineoplastic agents has been the combination of classical treatments with nutritional strategies aimed at reducing systemic toxicity without decreasing the antitumor activity of already used drugs. Within this context, this study evaluated the possible reduction of toxicity when cisplatin treatment is combined with watermelon pulp juice supplementation in C57BL/6 mice with melanoma. Watermelon is a fruit rich in vitamins, minerals, proteins, lycopene, carotene, and xanthophylls, which has shown effectiveness in the treatment of cardiovascular diseases, weight loss, urinary infections, gout, hypertension, and mutagenicity. The following parameters were analyzed: animal survival, bone marrow genotoxicity, serum creatinine and urea, histopathological features of the tumor tissue, tumor weight and volume, and weight of non-tumor tissues (kidney, liver, spleen, heart, and lung). The results showed that watermelon had no antitumor effect but reduced the toxicity of cisplatin, as demonstrated by an increase in the number of bone marrow cells and a decrease in serum creatinine and urea levels. The data suggest that watermelon pulp juice can be an alternative for reducing the side effects of antineoplastic agents.

Chemical study of Adenocalymma axillarum crude leaf extract and isolated compounds

Abstract Adenocalymma axillarum (K.Schum.) L.G. Lohmann is a liana belonging to the family Bignoniaceae. In traditional medicine, the genus Adenocalymma is used to treat fever, skin ailments, and body, joint, and facial muscle pains, and it is also applied as cosmetic. Biological assays conducted with the A. axillarum crude leaf ethanol extract have indicated leishmanicidal activity and absence of cytotoxicity. This study aimed to analyze the A. axillarum leaf ethanol crude extract by high-performance liquid chromatography-high-resolution mass spectrometry- diode array detector (HPLC-HRMS-DAD) and to evaluate the leishmanicidal and cytotoxic activities of this crude extract, its fractions, and isolated compounds. HPLC-HRMS-DAD analysis of this extract revealed that it consisted mainly of flavonoids, with nine major compounds. Extract purification yielded 4-hydroxy-N-methylproline, 6-β-hydroxyipolamiide, quercetin-3-O-robinobioside, hyperin, isorhamnetin-3-O-robinobioside, and 3'-O-methylhyperin, which were identified by Nuclear Magnetic Resonance. The isolated compounds were inactive against Leishmania amazonensis promastigotes and human lung fibroblast cells.

Styrax camporum, a typical species of the Brazilian cerrado, attenuates DNA damage, preneoplastic lesions and oxidative stress in experimental rat colon carcinogenesis.

Styrax camporum Pohl, a typical species from the Brazilian cerrado, commonly known as "benjoeiro", is used to treat gastroduodenal diseases. In previous studies carried out by our research group, hydroalcoholic extract of S. camporum stems (SCHE) exhibited antigenotoxic and antiproliferative effects. For a comparative analysis of the chemopreventive effect of SCHE, the aim of this study was to investigate the influence of SCHE against carcinogen 1,2-dimethylhydrazine (DMH)-induced DNA damage and pre-neoplastic lesions in Wistar rat colon. Animals were treated orally with SCHE at 250, 500 or 1000 mg/kg body weight in conjunction with a subcutaneous injection of DMH. DNA damage was assessed using the comet assay while tpre-neoplastic lesions by aberrant crypt foci (ACF) assay. The following hepatic oxidative stress markers were determined including activities of catalase (CAT) and glutathione S-transferase (GST) as well as levels of reduced glutathione (GSH) and malondialdehyde (MDA). Treatment with SCHE was not genotoxic or carcinogenic at the highest dose tested (1000 mg/kg b.w.). The extract effectively inhibited DNA damage and pre-neoplastic lesions induced by DMH administration at all concentrations tested. Measurement of CAT, and GST activities and levels of GSH showed that SCHE did not reduce oxidative processes. In contrast, treatment with SCHE (1000 mg/kg b.w.) decreased liver MDA levels. Taken together, these findings suggested the chemopreventive effect attributed to SCHE in colon carcinogenesis, may be related to its capacity to inhibit DNA damage as well as an antioxidant action associated with its chemical constituents egonol and homoegonol.

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.

Manool, a diterpene from Salvia officinalis, exerts preventive effects on chromosomal damage and preneoplastic lesions.

The present study aimed to evaluate the effect of the manool diterpene on genomic integrity. For this purpose, we evaluated the influence of manool on genotoxicity induced by mutagens with different mechanisms of action, as well as on colon carcinogenesis. The results showed that manool (0.5 and 1.0 µg/ml) significantly reduced the frequency of micronuclei induced by doxorubicin (DXR) and hydrogen peroxide in V79 cells but did not influence genotoxicity induced by etoposide. Mice receiving manool (1.25 mg/kg) exhibited a significant reduction (79.5%) in DXR-induced chromosomal damage. The higher doses of manool (5.0 and 20 mg/kg) did not influence the genotoxicity induced by DXR. The anticarcinogenic effect of manool (0.3125, 1.25 and 5.0 mg/kg) was also observed against preneoplastic lesions chemically induced in rat colon. A gradual increase in manool doses did not cause a proportional reduction of preneoplastic lesions, thus demonstrating the absence of a dose-response relationship. The analysis of serum biochemical indicators revealed the absence of hepatotoxicity and nephrotoxicity of treatments. To explore the chemopreventive mechanisms of manool via anti-inflammatory pathways, we evaluated its effect on nitric oxide (NO) production and on the expression of the NF-kB gene. At the highest concentration tested (4 µg/ml), manool significantly increased NO production when compared to the negative control. On the other hand, in the prophylactic treatment model, manool (0.5 and 1.0 µg/ml) was able to significantly reduce NO levels produced by macrophages stimulated with lipopolysaccharide. Analysis of NF-kB in hepatic and renal tissues of mice treated with manool and DXR revealed that the mutagen was unable to stimulate expression of the gene. In conclusion, manool possesses antigenotoxic and anticarcinogenic effects and its anti-inflammatory potential might be related, at least in part, to its chemopreventive activity.

Antigenotoxic, antiproliferative and antimetastatic properties of a combination of native medicinal plants from Argentina.

ETHNOPHARMACOLOGICAL RELEVANCE: Jarilla is the common name of an appreciated group of native plants from the semi-arid region in Argentina (Larrea cuneifolia Cav., Larrea divaricata Cav. and Zuccagnia punctata Cav.) that have been historically consumed to heal respiratory, musculoskeletal and skin ailments, as well as recommended for weakness/tiredness, hypertension, diabetes and cancer treatment. It was previously reported that some biological properties could be improved when these plants are used jointly. Infusions of a defined mixture, composed by three Jarilla species, L. cuneifolia: L. divaricata: Z. punctata (0.5:0.25:0.25) (HM2) showed synergistic and additive effect on antioxidant activity even after passing through the gastro-duodenal tract. AIM OF THE STUDY: The main purpose of this work was to evaluate antigenotoxic, antitumor, and anti-metastatic properties of the Jarilla species that grow in the Northwest of Argentina and a herbal combination of them. MATERIAL AND METHODS: Infusions of Jarilla mixture (HM2), and of each single plant species were prepared. Phenolic profiles of infusions were analyzed by HPLC-ESI-MS/MS and two relevant chemical markers were quantified. The antigenotoxic activity was evaluated by using the Ames test and the Cytokinesis-Block Micronucleus (CBMN) assay against direct mutagens. Evaluations of both cytotoxicity and antiproliferative effects were conducted on tumor and non-tumor cell lines. Both in vivo tumoral growth and metastasis inhibition were evaluated by using a carcinoma model on Balb/c mice. RESULTS: HM2 mix could suppress genetic and chromosome mutations induced by 4-nitro-o-phenylendiamine (4-NPD) and doxorubicin. Herbal mixture and single plant infusions showed cytotoxic effect against mammary, uterus, and brain tumoral cells without a selective action vs normal human cell line. HM2 mix was able to reduce mammary tumor mass on the Balb/c mice model and showed a significant reduction in the number of metastatic nodules in the lungs. CONCLUSIONS: Our results suggest that the combinations of three Jarilla species from northwest Argentina would be a promising alternative to treat or slow down the development of chronic diseases, such as cancer.

Evaluation of cytoprotective effects of compounds isolated from Copaifera langsdorffii Desf. against induced cytotoxicity by exposure to methylmercury and lead.

Copaifera langsdorffii L. is one of the most known medicinal species in Brazil. Its leaves are rich in phenolic compounds with potential biological activities as an antioxidant and chelating agent. This paper reports the isolation of four compounds from the hydroalcoholic extract of the leaves of C. langsdorffii and the investigation of their possible cytoprotective effects against heavy metal poisoning. Quercitrin (1), afzelin (2), 3,5-di-O-(3-O-methyl galloyl) quinic acid (3) and 4,5-di-O-(3-O-methyl galloyl) quinic acid (4), were associated with toxic doses of methylmercury and lead and evaluated by Alamar blue cell viability assays in HepG2 and PC12. The compounds displayed significant cytoprotective effect for the HepG2 cell line against both metals. Compounds 1-4 did not protect PC12 cells against methylmercury induced-cytotoxicity, but at lower concentrations, they protected against lead induced-cytotoxicity. The evaluated compounds showed a promising cytoprotection effect against exposure to heavy metals and should be further investigated as protective agents.

Biological properties of volatile oil from Brazilian brown propolis

ABSTRACT Propolis, is a bee product collected from exudates and flower buds of several plants, has strong aroma and several biological applications. This study aimed at evaluating the chemical composition and in vitro antioxidant, antibacterial and cytotoxic properties of volatile oil from Brazilian brown propolis. It was extracted by hydrodistillation and analyzed by gas chromatography-flame ionization detection and gas chromatography-mass spectrometry. Volatile oil from brown propolis exhibited strong antibacterial activity against H. pylori (MIC 3.25 µg/ml), Mycobacterium tuberculosis (MIC 50 µg/ml) and M. avium (MIC 62.5 µg/ml). It was evaluated in vitro for antioxidant activity by DPPH (IC50 25.0 µg/ml) and ABTS (IC50 30.1 µg/ml) methods. Its cytotoxic property was evaluated in normal (human fibroblasts, GM07429A) and tumor (MCF-7-human breast adenocarcinoma; HeLa-human cervical adenocarcinoma and M059J-human glioblastoma) cell lines. IC50 values were 81.32 µg/ml for GM07429A and 85.00, 129.40 and 84.12 µg/ml for MCF-7, HeLa and M059J cells, respectively. Three major dereplicated components of volatile oil from brown propolis were acetophenone (15.2%), nerolidol (13.3%), and spathulenol (11.6%). Our results contribute to a better understanding of the chemical and biological properties of Brazilian brown propolis and provide evidence for its potential medicinal use.

Chemopreventive role of Copaifera reticulata Ducke oleoresin in colon carcinogenesis.

In Brazilian folk medicine, copaiba oleoresin is widely known for its therapeutic activity, especially its wound healing and anti-inflammatory actions. Considering the relationship between inflammatory processes and carcinogenesis, this paper reports on the Copaifera reticulata Ducke oleoresin (CRO) chemopreventive potential in the colon carcinogenesis model in rats. To understand the mechanisms involved in this effect, the anti-inflammatory activity of CRO and its major chemical constituent, the diterpene ent-polyalthic acid (PA), were evaluated on the production of nitric oxide (NO) and prostaglandin E2 (PGE2) in mouse macrophages. For the chemoprevention assessment, the effect of CRO administered by gavage was investigated on DNA damage, pre-neoplastic lesions and mitotic frequencies induced by the 1,2-dimethylhydrazine (DMH; intraperitoneal injection) carcinogen by comet, aberrant crypt focus (ACF) and long-term assays, respectively. CRO reduced DNA damage (average 31.5%) and pre-neoplastic lesions (average 64.5%) induced by DMH, which revealed that CRO has antigenotoxic and anticarcinogenic effects. In the long-term assay, treatment with CRO significantly decreased mitoses in the tumor tissue, which suggested that CRO influenced carcinogenesis progression. PA reduced NO levels induced by lipopolysaccharides in macrophages. However, this diterpene showed no effect on PGE2. Taken together, our results suggest that PA exerts anti-inflammatory action via the NO pathway. The CRO chemopreventive effect may be partly due to the anti-inflammatory property of its major chemical constituent, PA. Our findings indicate that CRO is a promising agent to suppress colon carcinogenesis.

Genotoxic and Chemopreventive Effects of Vochysia divergens Leaves (Pantanal, Brazil).

The medicinal plant Vochysia divergens is a colonizing tree species of the Pantanal, a unique and little explored wetland region in Brazil. This species is used in folk medicine as syrups and teas to treat respiratory infections, digestive disorders, asthma, scarring, and skin diseases. The objectives of this study were to evaluate the antioxidant, cytotoxic, and genotoxic potential of the ethanolic extract of Vochysia divergens leaves (VdE), as well as the influence of VdE and its major component (the flavone 3',5-dimethoxy luteolin-7-O-ß-glucopyranoside; 3'5 DL) on MMS-induced genotoxicity. The extract significantly reduced the viability of V79 cells in the colorimetric XTT assay at concentrations ≥ 39 µg/mL. A significant increase in micronucleus frequencies was observed in V79 cell cultures treated with VdE concentrations of 160 and 320 µg/mL. However, animals treated with the tested doses of VdE (500, 1000, and 2000 mg/kg b.w.) exhibited frequencies that did not differ significantly from those of the negative control group, indicating the absence of genotoxicity. The results also showed that VdE was effective in reducing MMS-induced genotoxicity at concentrations of 20, 40, and 80 µg/mL in the in vitro test system and at a dose of 15 mg/kg b.w. in the in vivo test system. Its major component 3'5 DL exerted no protective effect, suggesting that it is not responsible for the effect of the extract. The results of the 2,2-diphenyl-1-picrylhydrazyl (DPPH) assay showed that VdE was able to scavenge 92.6% of free radicals. In conclusion, the results suggest that the protective effect of VdE may be related, at least in part, to the antioxidant activity of its chemical constituents.

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.

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.

Use of Copaifera multijuga for acute corneal repair after chemical injury: A clinical, histopathological and toxicogenetic study.

Copaiba oil is widely used in medicine, but there are no reports regarding its application in ophthalmology. Therefore, the objective of this study was to evaluate the clinical, histopathological and toxicogenetic effects of eye drops containing 0.1 and 0.5% of Copaifera multijuga Hayne oil on superficial corneal ulcers induced with alkali in the left eye of rats. For histological analysis, the percent reduction in ulcers and thickness of the corneal epithelium and stroma were evaluated 48 and 72 h after ulcer induction. Additionally, neovascularization and polymorphonuclear infiltration were classified in the stroma. The bone marrow micronucleus test was used for toxicogenetic assessment. None of the animals exhibited clinical signs of immediate ocular discomfort after instillation and the eye drops were harmless to the ocular surface. There was a significant difference in percent ulcer reduction and corneal stroma thickness between animals treated with the C. multijuga eye drops and untreated animals with corneal injury and the negative control, respectively, suggesting a healing effect of the oleoresin. Analysis of the thickness of the corneal epithelium at the two time points showed that the eye drops formulated did not significantly reduce the damage caused by alkali. The same was observed for the treatments with the reference drugs. No difference in stromal neovascularization or inflammatory infiltration was observed between the treated groups. The toxicogenetic results revealed the absence of cytotoxicity and genotoxicity of the treatments. In conclusion, the C. multijuga eye drops did not cause damage to the ocular surface under the present experimental conditions and corneal epithelization was similar to the conventional treatments. These results indicate that eye drops containing C. multijuga oleoresin are a promising option for the treatment of superficial keratitis.

Assessment of the antioxidant, cytotoxic, and genotoxic potential of the Annona muricata leaves and their influence on genomic stability.

The popular use of Annona muricata L. is based upon a range of medicinal purposes, and the plant exhibits biological activities including antihyperglycemic, antiparasitic, and antitumor activities. The objectives of this study were to examine the antioxidant, cytotoxic, and genotoxic potential of the hydroalcoholic extract of A. muricata leaves (AMEs), as well as its effects on genotoxicity induced by methyl methanesulfonate (MMS) and hydrogen peroxide (H2O2). The results using 2,2-diphenyl-1-picrylhydrazyl assay showed that AME was able to scavenge 44.71% of free radicals. The extract significantly reduced the viability of V79 cells in the clonogenic assay at concentrations ≥8 µg/ml. No significant differences in micronucleus (MN) frequency were observed between V79 cell cultures treated with different concentrations of the extract (0.125, 0.25, 0.5, and 1 µg/ml) and negative control. When AME concentrations were combined with MMS, data revealed no marked differences from mutagen alone. In contrast, significant reductions in the frequencies of MN were noted in cultures treated with AME combined with H2O2 compared to H2O2 alone. In vivo studies found no significant differences in the frequencies of micronucleated polychromatic erythrocytes (MNPCEs) between animals treated with different AME doses compared to control. Animals treated with AME doses of 125 and 250 mg/kg and MMS exhibited significantly higher frequencies of MNPCE compared to mutagen alone. In conclusion, under current experimental conditions, AME was not genotoxic and exerted a modulatory effect on DNA damage depending upon the experimental conditions. The extract did not influence markedly MMS-induced genotoxicity in in vitro test system. However, the extract increased DNA damage induced by mutagen in mice. In V79 cells, AME reduced the genotoxicity produced by H2O2, and this protective effect was attributed in part to the antioxidant activity of AME.