Phytochemical characterization, isolation, antioxidant and cytogenotoxic activity of leaves of Heliotropium elongatum (Lehm) I.M. Johnst.

Heliotropium elongatum is used to treat inflammation, cough, and flu. This study aimed to characterize the phytochemical profile and determine the total phenolic content (TPC), antioxidant and cytogenotoxic activity of the ethanolic extract (EE), and fractions of H. elongatum leaves. In the phytochemical profile analysis, organic acids, reducing sugars, flavonoids, saponins, anthraquinones, steroids/triterpenes, and depsides/depsidones were detected in the EE and/or fractions (hexanic/FH, chloroformic/FC, ethyl acetate/FAE, and hydromethanolic/FHM). The highest TPC and highest antioxidant activity (DPPH and ABTS) was detected in FHM. In FH, 16 compounds were identified by GC-MS, and ursolic acid was isolated by 1H NMR and 13C NMR. HPLC-DAD from EE, FAE, and FHM demonstrated characteristic wavelengths for flavonoids, flavonols, flavones, and anthraquinones. ESI-IT/MSn analysis of EE, FC, FAE, and FHM revealed alkaloids, steroids, terpenoids, flavonoids, and phenolic acids. In Allium cepa assay there was no significant cytotoxic effect initiated by EE (62.5 to 1,000 µg/ml), FHM (1,000 µg/ml), and FAE (62.5 µg/ml). Genotoxicity was evidenced only with EE at 500 and 1,000 µg/ml, and FHM (62.5 to 1,000 µg/ml) as evidenced by presence of micronuclei (MN) and nuclear buds (NB). Our results identified compounds of medicinal interest with antioxidant activity; however observed cytogenotoxic changes indicated the need for caution when using these compounds for therapeutic purposes.

Antioxidant and in vitro cytogenotoxic properties of Amburana cearensis (Allemão) A.C.Sm. leaf extract.

Amburana cearensis leaves have been used in folk medicine to treat respiratory diseases and inflammations. This study aimed to evaluate the biological potential of A. cearensis leaves by antioxidant and in vitro cytogenotoxic analyses of ethanolic crude extract (EE) and its fractions in healthy human cells. The EE was obtained by percolation, followed by fractionation using dichloromethane, cyclohexane, ethyl acetate (EtOAc), and methanol (MeOH) as organic solvents. Extract and all fractions were evaluated for their antioxidant potential by DPPH and reducing power tests. In vitro cytotoxic activity was determined in human peripheral blood mononuclear cells by MTT assay for the extract, EtOAc and MeOH fractions. In turn, the genotoxic activity was determined in human lymphocytes by the Cytokinesis Block Micronucleus assay only for the EtOAc fraction. Only EtOAc fraction was analyzed via gas chromatography coupled to mass spectrometry due to its higher biological activity. Considering the antioxidant potential, the EtOAc fraction was most effective in DPPH (EC50 43.37 µg/mL) and reducing power (EC50 89.80 µg/mL) assays. GC-MS analysis of the EtOAc fraction led to the identification of guaiacol, 2,3-dihydro-benzofuran, 2-methoxy-4-vinylphenol, isovanillic acid methyl ester, 4-hydroxybenzaldehyde, and 4-(ethoxymethyl)-phenol. The EE (400-1000 µg/mL), EtOAc (≤150 µg/mL) and MeOH (50 and 150-600 µg/mL) fractions were not cytotoxic by MTT test. Additionally, the EtOAc fraction (100-400 µg/mL) did not induce significant genotoxic damage. Concentrations of the EtOAc fraction with antioxidant activity showed no cytotoxicity, nor genotoxicity potential, indicating them as a nontoxic natural antioxidant source.

Identification of bioactive compounds and cytogenotoxicity of the essential oil from the leaves of Croton heliotropiifolius Kunth.

Croton heliotropiifolius Kunth, popularly known as "quince" and "velame," contains a high concentration of volatile oils in the leaves, and widely used in folk medicine as an antiseptic, analgesic, sedative, anti-inflammatory, spasmolytic and local anesthetic. The objectives of this investigation were to (1) identify the phytochemical compounds and (2) assess the cytogenotoxicity of the essential oil extracted from the leaves of C. heliotropiifolius Kunth. The oil was extracted utilizing hydrodistillation and phytochemical profile determined using gas chromatography and mass spectrometry (GCMS). In the toxicogenetics analysis, Allium cepa roots were exposed to 1% dimethylsulfoxide or methylmethanesulfonate (MMS, 10 µg/ml) negative and positive controls, respectively, and to C. heliotropiifolius oil at 6 concentrations (0.32; 1.6; 8; 40; 200 or 1000 µg/ml). The phytochemical profile exhibited 40 chromatographic bands, and 33 compounds identified. α-pinene (16.7%) and 1,8-cineole (13.81%) were identified as the major compounds. Some of these identified secondary metabolites displayed biological and pharmacological activities previously reported including antiseptic, analgesic, sedative, anti-inflammatory as well insecticidal, antiviral, anti-fungal actions. In the A. cepa test, C. heliotropiifolius leaves oil induced cytotoxicity at concentrations of 0.32, 1.6 or 200 µg/ml and genotoxicity at 200 or 1000 µg/ml as evidenced by increased presence of micronuclei and significant chromosomal losses. Based upon our observations data demonstrated that the essential oil of C. heliotropiifolius leaves contain monoterpene hydrocarbons, and oxygenated monoterpenes, sesquiterpenes, and oxygenated sesquiterpenes which are associated with cytotoxic and genotoxic responses noted in on A. cepa cells.

Protective effect of kavain in meristematic cells of Allium cepa L.

Kavain is one of the main kavalactones of Piper methysticum (Piperaceae) with anxiolytic, analgesic, and antioxidant activities. Therefore, the aim of the study was to evaluate the cytotoxic, mutagenic, and antimutagenic potential of kavain in Allium cepa cells. Roots of A. cepa were transferred to the negative (2% acetone) and positive (10 µg/mL of Methylmethanesulfonate, MMS) controls and to the concentrations of kavain (32, 64 and 128 µg/mL) for 48 h. A total of 5,000 meristematic cells were analyzed under an optical microscope to determine the mitotic index, mean number of chromosomal alterations and percentage of damage reduction. Data were analyzed by Kruskal-Wallis test (p <0.05). All concentrations of kavain were not cytotoxic and did not show significant chromosomal changes when compared to 2% acetone. Kavain showed a cytoprotective effect in the pre (128 µg/mL) and in the post-treatment (32 and 64 µg/mL) and reduced damage against the mutagenic action of MMS in all concentrations of the pre and simultaneous and at the highest of post (128 µg/mL). Kavain promoted a significant reduction in micronuclei, nuclear buds and chromosomal losses in relation to MMS. The observed data indicate the importance of kavain for the inhibition of damage and chemoprevention.

Modulating effect of DL-kavain on the mutagenicity and carcinogenicity induced by doxorubicin in Drosophila melanogaster.

Kavain, kavalactone, present in Piper methysticum exhibits anticonvulsive, analgesic, anxiolytic, antiepileptic, antithrombotic, anti-inflammatory and antioxidant properties. Given its importance, the aim of the present study was to assess (1) the mutagenic and carcinogenicity of kavain administered alone and (2) the antimutagenic and anticarcinogenic potential when administered simultaneously with the chemotherapeutic drug doxorubicin (DXR) using the Somatic Mutation and Recombination Test (SMART) and Epithelial Tumor Test (ETT) using Drosophila melanogaster as a model system. Third-stage larvae from a standard (ST) and high metabolic bioactivation (HB) crosses were treated with different kavain concentrations (32, 64 or 128 µg/ml), alone or in conjunction with DXR (0.125 mg/ml). In ST descendants, kavain produced no significant mutagenic or recombinogenic effects. In the HB cross, mutagenic activity was observed at kavain concentrations of 64 and 128 µg/ml. In the DXR and kavain co-treatment, a modulating effect of the DXR-mediated mutagenic response dependent upon the concentration was detected in both crosses. In ETT, no marked carcinogenic or anticarcinogenic activity was noted for kavain. However, when kavain was combined with DXR synergistic induction of tumors by the chemotherapeutic drug occurred indicating that kavain enhanced the carcinogenic action of DXR.

Phytochemical prospecting, isolation, and protective effect of the ethanolic extract of the leaves of Jatropha mollissima (Pohl) Baill.

Jatropha mollissima is used in folk medicine as antimicrobial, antiparasitic, and larvicidal. However, few toxicogenetic studies have been carried out. Therefore, the aim of this study was to determine the phytochemical profile of ethanolic leaf extract of J. mollissima (EEJM) as well as potential cytotoxic, mutagenic, and antimutagenic properties. The EEJM was subjected to successive fractionation for the isolation of secondary metabolites, and five concentrations (0.01; 0.1; 1; 10 and 100 mg/ml) of extract were investigated using Allium cepa assay and the Somatic Mutation and Recombination (SMART) test. The mitotic index and % damage reduction were analyzed for A. cepa and the frequency of mutant hair for SMART. The presence of coumarins, alkaloids, flavonoids, saponins, and tannins was detected, while spinasterol and n-triacontane were the isolates identified for the first time for this species. EEJM did not exhibit cytotoxicity and was not mutagenic at 1 or 10 mg/ml using A. cepa and all concentrations of EEJM were not mutagenic in the SMART test. A cytoprotective effect was found at all concentrations. At 1 or 10 mg/ml EEJM exhibited antimutagenicity in A. cepa. In SMART, the protective effect was observed at 0.1 to 100 mg/ml EEJM. Our results demonstrate the important chemopreventive activity of EEJM, a desired quality in the search for natural anticarcinogenic compounds.

Phytochemical screening, phenolic and flavonoid contents, antioxidant and cytogenotoxicity activities of Combretum leprosum Mart. (Combretaceae).

Combretum leprosum Mart. (Combretaceae), a shrub popularly known as mofumbo, is used in folk medicine for treatment of uterine bleeding, pertussis, gastric pain, and as a sedative. The aim of this study was to (1) determine the phytochemical profile,(2) identify chemical constituents and (3) examine antioxidant and cytogenotoxic activity of ethanolic extracts and fractions of stem bark and leaves. The plant material (leaf and stem bark) was submitted to extraction with ethanol, followed by partition using hexane, chloroform, and ethyl acetate. It was possible to identify and quantify the epicatechin in the ethanolic stem bark extract (0.065 mg/g extract) and rutin in the leaf extract (3.33 mg/g extract). Based upon in vitro tests a significant relationship was noted between findings from antioxidant tests and levels of total phenolic and flavonoid. Comparing all samples (extracts and fractions), the ethyl acetate fractions of stem bark (411.40 ± 15.38 GAE/g) and leaves (225.49 ± 9.47 GAE/g) exhibited higher phenolic content, whereas hexanic fraction of stem bark (124.28 ± 56 mg/g sample) and ethyl acetate fraction of leaves (238.91 ± 1.73 mg/g sample) demonstrated a higher content of flavonoids. Among the antioxidant tests, the intermediate fraction of stem bark (28.5 ± 0.60 µg/ml) and ethyl acetate fraction of leaves (40 ± 0.56 µg/ml) displayed a higher % inhibition of free radical DPPH activity, whereas intermediate fraction of stem bark (27.5 ± 0.9 µg/ml) and hydromethanol fraction of leaves (81 ± 1.4 µg/ml) demonstrated inhibition of the free radical ABTS. In biological tests (Allium cepa and micronucleus in peripheral blood), data showed that none of the tested concentrations of ethanolic extracts of leaves and stem bark produced significant cytotoxicity, genotoxicity, and mutagenic activity.Abbreviations AA%: percentage of antioxidant activity; ABTS: 2,2'-azinobis-(3-ethylbenzothiazoline-6-sulfonic acid); CEUA: Ethics Committee in the Use of Animals; TLC: Thin Layer Chromatography; DNA: deoxyribonucleic acid; DPPH: 1,1-diphenyl-2-picrylhydrazyl; ROS: Reactive oxygen species; EEB: ethanol extract of the stem bark; HFB: Hexanic fraction of stem bark; IFB: Intermediate fraction of stem bark; CFB: Chloroform fraction of stem bark; EAFB: Ethyl acetate fraction of stem bark; HMFB: Hydromethanol fraction of the stem bark; EEL: Ethanol extract from leaves; HFL: Hexane fraction of leaves; CFL: Chloroform fraction of leaves; EAFL: Ethyl acetate fraction of leaves; HMFL: Hydromethanol fraction of leaves; GAE: Gallic Acid Equivalent; IC50: 50% inhibition concentration; HCOOH: Formic acid; HCl: hydrochloric acid; HPLC: High-performance liquid chromatography; MN: micronucleus; WHO: World Health Organization; UFLC: Ultra-Fast Liquid Chromatography; UESPI: State University of Piauí.

Phytochemical and antioxidant characterization, cytogenotoxicity and antigenotoxicity of the fractions of the ethanolic extract of in Poincianella bracteosa (Tul.) L.P. Queiroz.

POINCIANELLA BRACTEOSA: has been widely used in folk medicine to treat catarrhal infections, diarrhea, and anemia; however, phytochemical and toxicogenetic data are still lacking. The objective of this study was to examine the phytochemical and antioxidant characteristics as well as assess cytogenotoxicity and antigenotoxicity in hexane (HF), ether (EF) and ethyl acetate (AF) fractions of P. bracteosa leaves using Allium cepa bioassay. Phytochemical analysis revealed the presence of saponins and phenolic groups. EF fraction contained a higher content of total phenolics (441.23 ± 1.82 mg GAE/g), while HF fraction showed a higher content of total flavonoids (84.77 ± 5.33 mg QE/g). Higher antioxidant activity was observed in EF (EC50 25.06 ± 0.07 µg/ml). Cytotoxic effect was verified for all fractions, but no chromosomal alterations were observed in the A. cepa assay. With respect to antigenotoxicity, the protective effect of EF and AF fractions was attributed to as evidenced by the modulation of mutagenic action of methyl methanesulfonate (MMS), mainly by inhibiting the development of micronuclei. Among the fractions, EF was considered the most promising, as it exhibited higher antioxidant activity, was not genotoxic, exerted protective activity against the damage induced by MMS and also presented cytotoxic activity, a desired quality in the search for natural anticarcinogenic compounds.

Actividad antimutagénica e identificación de compuestos antioxidantes en la planta Poincianella bracteosa (Fabaceae)

Introducción: Poincianella bracteosa (Tul.) L.P. Queiroz. (Fabaceae), conocida como catingueira, es tradicionalmente utilizada en la medicina para tratar diarrea, hepatitis y anemia. Sin embargo, no hay estudios sobre los efectos tóxico genéticos de la P. bracteosa. Objetivo: En el presente estudio se tuvo como objetivo investigar el perfil fitoquímico y el potencial mutagénico y antimutagénico del extracto acuoso de la cáscara de P. bracteosa en Allium cepa y Mus musculus. Métodos: El extracto de la cáscara fue diluido en agua destilada para fornecer las cuatro concentraciones (2, 4, 8 y 16 mg/ml) utilizadas en el bioensayo A. cepa y las tres dosis (10, 20 y 40 mg / Kg) fueron administradas a los ratones (5 animales por grupo). El perfil fito-químico fue realizado por el test colorimétrico para identificar los principales metabólitos secundarios en el extracto de la cáscara. Tras el tratamiento, 5 000 células meristemáticas fueron analizadas para determinar el índice mitótico, el promedio de alteraciones cromosómicas y el porcentaje de reducción de daños. Para ratones, tras 24, 48 y 72 h, la sangre de la cola de cada animal fue recolectado para la preparación de dos láminas por animal. Para cada animal, 2 000 eritrocitos normocromáticos por ratón fueron evaluados para establecer el número de micronúcleos y el efecto protector. Se analizaron los dados por el test de Kruskal-Wallis (P < 0.05). El estudio fito-químico del extracto detectó azúcares reductores y taninos. Resultados: Ninguna de las concentraciones del extracto fue citotóxica y en todos los tratamientos (pre, simultáneo y después) fue observado el efecto citoprotetor en A. cepa. El promedio total de las alteraciones cromosómicas en todas las concentraciones apuntó actividad no mutagénica de la cáscara. El porcentaje de reducción del daño fue observada en los tratamientos pre (de 77.6 al 90.5 %), simultáneo (del 95.6 al 114.7 %) y tras (de 84.8 al 117.7 %). En los ratones, ninguna de las dosis del extracto presentó efecto mutagénico y el porcentaje de reducción del daño osciló de -21.2 al 78.6 % (pre); de 27.5 al 101.3 % (simultánea) y de 85.5 al 120.6 % (tras-tratamiento). Probablemente, los fito-químicos presentes en el extracto no interfirieron en el ciclo celular (A. cepa), tampoco causaron daños al DNA (A. cepa y ratones) y presentaron efecto protector en las dos especies estudiadas. Los datos observados apuntan la importancia del extracto de la cáscara de P. bracteosa para inhibición del daño y quimio prevención. Sin embargo, más estudios deben ser realizados para garantizar su efecto protector sobre el material genético.

Introduction: Poincianella bracteosa (Tul.) L.P. Queiroz. (Fabaceae), known as catingueira, is traditionally used in medicine to treat diarrhea, hepatitis and anemia. However, there are no studies on their toxicogenetic effects. Objective: The present study aimed to investigate the phytochemical profile as well as the mutagenic and antimutagenic potential of P. bracteosa aqueous bark extract in Allium cepa and Mus musculus. Methods: The extract from barks was diluted in distilled water to yield the four concentrations (2, 4, 8 and 16 mg/ml) used in the A. cepa bioassay and the three doses (10, 20 and 40 mg/Kg) administered to the mice (five animals per group). The phytochemical profile was performed by the colorimetric test to identify the main secondary metabolites in the bark extract. After treatment, five-thousand meristematic cells were analyzed to determine the mitotic index, the mean number of chromosome alterations and the percentage of damage reduction. For mice, after 24, 48 and 72 h, tail blood was collected from each animal for the preparation of two slides per animal. For each animal, 2 000 normochromatic erythrocytes per mice were evaluated to establish the number of micronuclei and the protective effect. Data were analyzed by Kruskal-Wallis test (P < 0.05). Results: The phytochemical analysis of the extract detected reducing sugars and tannins. None of the concentrations of extract was cytotoxic and the cytoprotective effect was observed in A. cepa for all treatments (pre-, simultaneous and post-). The total mean of chromosome alterations in all concentrations indicated a non-mutagenic activity of the bark. The percentage of damage reduction was observed in the pre- (77.6 to 90.5 %), simultaneous (95.6 to 114.7 %) and post- (84.8 to 117.7 %) treatments. In mice, none of the dosages of extract presented mutagenic effect and the percentage of damage reduction varied from -21.16 to 78.63 % (pre-); from 27.51 to 101.28 % (simultaneous) and from 85.47 to 120.63 % (post-treatment). Conclusions: Probably, the phytochemicals in the extract did not interfere with the cell cycle (A. cepa) nor caused damage to the DNA (A. cepa and mice), and exhibited protective effect in both studied species. The observed data indicate the importance of P. bracteosa bark extract for the inhibition of damage and chemoprevention. However, more studies should be carried out to ensure its protective effect on the genetic material.

Larvicidal, cytotoxic and genotoxic effects of aqueous leaf extract ofJatropha mollissima (Pohl) Baill / Efeitos larvicida, citotóxico e genotóxico do extrato aquoso das folhas de Jatrophamollissima (Pohl) Baill

The present study aimed to evaluate the larvicidal effect of aqueous leaf extract fromJatropha mollissima on the larvae of Aedes aegypti and analyze its cytotoxic and genotoxic activity in the Alliumcepa test. Larvae of the mosquito were exposed to the negative and positive controls (distilled water anddiflubenzuron, 0.003 mg mL-1, respectively) and to leaf extract concentrations of 0.001, 0.005, 0.01, 0.02,0.04, 0.06, 0.08 and 0.1 mg mL-1. The mortality rate was evaluated every 24 hours over five days. For thecytotoxic and genotoxic analyses, roots of A. cepa were exposed to the negative (distilled water) and positivecontrol (trifluralin, 0.84 ppm) and to different leaf extract concentrations (0.01, 0.1, 1 and 10 mg mL-1) for24 hours. The statistical analyses were performed by Kruskal-Wallis test (p < 0.05). The leaf extractpresented promising larvicidal activity at the concentrations of 0.08 and 0.1 mg mL-1, and none of theconcentrations evaluated in A. cepa exhibited cytotoxic or genotoxic effect. Since the larvicidal action of J.mollissima and the absence of cellular toxicity have been demonstrated, further studies are recommended todetermine the mechanism of action of the extract as a possible natural larvicide.

O presente estudo teve como objetivo avaliar o efeito larvicida do extrato aquoso das folhas deJatropha mollissima sobre as larvas de Aedes aegypti e analisar sua atividade citotóxica e genotóxica no testeAllium cepa. As larvas do mosquito foram expostas aos controles negativo e positivo (água destilada ediflubenzuron, 0,003 mg mL-1, respectivamente) e ao extrato foliar nas concentrações de 0,001; 0,005; 0,01;0,02; 0,04; 0,06; 0,08 e 0,1 mg mL-1. A taxa de mortalidade foi avaliada a cada 24horas durante cinco dias.Para as análises citotóxica e genotóxica, as raízes de A. cepa foram expostas ao controle negativo (águadestilada) e positivo (trifluralina, 0,84 ppm) e nas concentrações (0,01; 0,1; 1 e 10 mg mL-1) do extrato foliarpor 24 horas. Análises estatísticas foram realizadas pelo teste de Kruskal-Wallis (p < 0,05). O extrato foliarapresentou atividade larvicida promissora nas concentrações de 0,08 e 0,1 mg mL-1, e nenhuma dasconcentrações avaliadas em A. cepa exibiu efeito citotóxico ou genotóxico. Uma vez demonstrada a açãolarvicida de J. mollissima e a ausência de toxicidade celular, mais estudos são recomendados para determinaro mecanismo de ação do extrato como um possível larvicida natural.

Cytotoxic and genotoxic effects of ethanolic extract of Euphorbia hyssopifolia L. on HepG2 cells.

ETHNOPHARMACOLOGICAL RELEVANCE: Euphorbia hyssopifolia L. is a weed with recognized antimicrobial potential employed in Indian, Asian and Latin-American popular medicine. However, little is known with regard to its toxic potential. The present study aimed to investigate the cytotoxic and genotoxic effects of ethanolic extract of E. hyssopifolia in HepG2 cell culture. MATERIALS AND METHODS: Phytochemical screening of ethanolic extract was carried out to determine the presence of active secondary plant metabolites. Six concentrations (0.00001, 0.0001, 0.001, 0.01, 0.1 and 1.0mg/mL) of ethanolic extract were tested by the MTT assay to verify cytotoxicity. Then, genotoxic evaluations (alkaline comet assay and cytokinesis-block micronucleus assay - CBMN) were carried out in HepG2 cells with extract concentrations of 0.01, 0.1 and 1.0mg/mL. RESULTS: Mono and sesquiterpenes, triterpenes and steroids, and flavonoids were the main classes found in the phytochemical screening. Extract concentrations used in the MTT assay showed no cytotoxic activity. On the other hand, genotoxic activity was verified at 0.1 and 1.0mg/mL in the alkaline comet assay. Additionally, the 1.0mg/mL concentration induced severe cell damage leading to death in the CBMN assay, indicating a cytotoxic effect for this concentration in the latter method. CONCLUSION: The use of E. hyssopifolia extract for medicinal purposes should be avoided, because concentrations above 0.01mg/mL may pose risk to human health due to cytotoxic and/or genotoxic effects.

Genotoxic potential of the latex from cotton-leaf physicnut (Jatropha gossypiifolia L.).

Jatropha gossypiifolia L. (Euphorbiaceae), popularly known as cotton-leaf physicnut, is a milky shrub notable for its medicinal properties. The present study aimed to evaluate the toxic, cytotoxic and genotoxic effects of the latex of J. gossypiifolia, using Allium cepa L. as test system. Seeds of A. cepa were exposed to five concentrations of the latex (1.25; 2.5; 5; 10 and 20 mL/L) in order to evaluate parameters of toxicity (evaluation of root growth), cytotoxicity (mitotic index frequency) and genotoxicity (frequency of chromosome alterations). The latex showed a significant decrease in root mean growth value as well as mitotic index for the tested concentrations, except for 1.25 mL/L, when compared to results from the negative control. The 1.25, 2.5 and 5 mL/L concentrations induced significant chromo-some adherences, C-metaphases and/or chromosome bridges, as genotoxic effects. The significant frequency of chromosome bridges also indicated mutagenic potential for chromosomes of J. gossypiifolia as discussed in the paper. Considering that the latex is used in popular therapies, and that the test system A. cepa presents good correlation with tests carried out in mammals, it can be pointed out that its use for medicinal purposes may be harmful to human health especially if ingested.