Genotoxic evaluation of infusions of Urera baccifera leaves and roots in Allium cepa cells

Context: The aqueous extracts of Urera baccifera Wedd. leaves and roots are used to inflammatory and infectious diseases in Brazilian folk medicine. Oxalic acid, a substance co-related with toxicity and stinging, was already quantified in this plant. Aims: To evaluate the action of leaves and roots infusions (1, 30, 75 g/L) and the oxalic acid standard on mitosis as indicative of presumably antimitotic and genotoxic actions, using the Allium cepa test. Methods: Oxalic acid was quantified in the roots and leaves infusions by High-performance liquid chromatography (HPLC-DAD), with the mobile phase of 25 mM phosphate buffer (pH 2.5): acetonitrile at 95:5 (v/v). To the genotoxicity test, onion bulbs were used. After the rootlets germination, each bulb was submitted for 24 h of the individual treatments. Were analyzed 1000 cells per bulb, in a total of 5000 cells per treatment. Results: Results showed that all concentrations of roots infusions induced chromosomes abnormalities, except for the highest, that caused a substantial inhibition in the mitosis, precluding to be observed abnormalities. In the leaves infusions, only the two higher concentrations caused the highest values of damage in the cellular cycle. The oxalic acid also caused abnormalities in the mitosis, and may be considered responsible by part of the genotoxic action of U. baccifera. Conclusions: Oxalic acid can be responsible by part of the chromosomal abnormalities caused by U. baccifera, although, there must have more metabolites that evoke the same effect promoting the genotoxic effect of this nettle

Efeito anti-proliferativo das infusões de Achyrocline satureioides DC (Asteraceae) sobre o ciclo celular de Allium cepa

Achyrocline satureioides (marcela) é utilizada na medicina popular brasileira, na forma de chá, como tratamento de patologias digestivas e inflamatórias. O efeito anti-proliferativo de infusões de marcela sobre o ciclo celular da cebola foi avaliado, utilizando-se inflorescências de marcela recém coletadas (2005) e após armazenamento por 30 meses (2003). Preparou-se as infusões em duas concentrações: 5,0 mg/mL (concentração usual como chá) e 20 mg/mL. Utilizaram-se 3 grupos de 6 bulbos de cebola para cada população de marcela. Retirou-se um grupo de bulbos controle de cada população. Todos os bulbos enraizados em água destilada foram transferidos para os extratos de marcela e permaneceram por 24 horas, (os bulbos controle permaneceram em água). As radículas foram coletadas, fixadas em etanol-ácido acético (3:1) por 24 h e estocadas em álcool 70%. Foram analisadas 6000 células por grupo de bulbos, e os índices mitóticos calculados submetidos a análise estatística pelo teste chi2 a 5%. Conclui-se que as infusões de marcela possuem ação antiproliferativa sobre o ciclo celular da cebola e que essa ação inibitória da divisão celular aumenta conforme aumento da concentração, bem como após o armazenamento

Tretinoin-loaded polymeric nanocapsules: evaluation of the potential toimprove the antiproliferative activities on Allium cepa root-tip compared to the free drug

Se prepararon nanopartículas poliméricas conteniendo tretinoína por el método de depósito interfacial de polímero preformado. Para el ensayo de actividad antiproliferativa se utilizaron: tretinoína en forma de solución y contenida en nanocápsulas poliméricas, que fueron ensayadas usando el modelo de raíz de Allium cepa. Las nanocápsulas mostraron un tamaño promedio en el rango de los nanómetros, con un bajo índice de polidispersidad, pH ácido, y una eficiencia de encapsulación mayor al 99%. Las nanocápsulas ensayadas mostraron una disminución significativa del índice mitótico (1,36%) comparado con el control-agua (8,20%), así como respecto al fármaco libre (2,76%). Esta mejora en la actividad antiproliferativa no conllevó a un aumento en la expresión de aberraciones cromosómicas. En conclusión, la encapsulación en nanocápsulas poliméricas mejora la eficacia antiproliferativa de la tretinoína en el modelo ensayado.Tretinoin-loaded nanocapsules were prepared by the interfacial deposition of preformed polymer method. For the antiproliferative activity assay a tretinoin solution and tretinoin-loaded nanocapsules were tested on Allium cepa root-tip model. Tretinoin-loaded nanocapsules presented nanometric mean size, low polydispersity index, acidic pH value and encapsulation efficiency higher than 99%. Tretinoin-loaded nanocapsules presented a significant decrease in the mitotic index (1.36%) compared to the control-water (8.20%), as well as to the free drug (2.76%). This improvement in the antiproliferative activity did not lead to an increase in the frequency of chromosome aberrations. Encapsulation in polymeric nanocapsule suspensions improves the in vivo antiproliferative efficacy of tretinoin on the tested model.Colegio de Farmacéuticos de la Provincia de Buenos Aire

Genotoxic and chromatographic analyses of aqueous extracts of Peltodon longipes Kunth ex Benth. (hortelã-do-campo)

Peltodon longipes is used as a stimulant and emmenagogue. The objective of this study was to perform genotoxic and chromatographic analyses of the extracts of two samples of P. longipes, collected from the cities of Santa Maria and Tupanciretã, RS, Brazil. The Allium cepa assay was used to analyze genotoxicity while high-performance liquid chromatography was employed to determine phenolic compounds. The genotoxicity experiment consisted of nine groups each comprising four A. cepa bulbs. Bulb roots were developed in distilled water and then transferred for the treatments, for 24 hours, and the negative control remained in water. The treatments were: aqueous extracts at concentrations of 5 and 15 g L -1 for each sample, plus four groups treated with 1% glyphosate, one of which was used as a positive control and the other three for testing DNA damage recovery using water and the extracts of P. longipes from Santa Maria. All extracts of P. longipes exhibited anti-proliferative potential, although the effect was significantly greater for the extracts from the Tupanciretã sample. This sample also contained the highest amount of rosmarinic acid and kaempferol, which may confer the effects found in these extracts. Only extracts from the Santa Maria sample exhibited genotoxic potential. Uniterms: P. longipes/antiproliferative effect. P. longipes/genotoxic potential. P. longipes/chromatographic analyses. Allium cepa test/genotoxicity. Peltodon longipes é utilizada como estimulante e emenagoga. Objetivou-se realizar análises genotóxica e cromatográfica dos extratos de duas amostras de P. longipes, coletadas nos municípios de Santa Maria e Tupanciretã, RS, Brasil. O teste de Allium cepa foi utilizado para análise da genotoxicidade e a cromatografia líquida de alta eficiência, para determinação dos compostos fenólicos. O experimento de genotoxicidade constou de nove grupos de quatro bulbos de A. cepa. Os bulbos foram enraizados em água destilada e após transferidos para os tratamentos, por 24 horas, permanecendo o controle negativo em água. Os tratamentos foram: extratos aquosos nas concentrações de 5 e 15 g L -1 de cada amostra, além de quatro grupos tratados com glifosato 1%, um deles usado como controle positivo e outros três para testar a recuperação de danos ao DNA, utilizando água e os extratos de P. longipes da amostra de Santa Maria. Todos os extratos de P. longipes demonstraram potencial antiproliferativo, porém o efeito foi significativamente maior para os extratos da amostra de Tupanciretã. Essa amostra também apresentou maior quantidade de ácido rosmarínico e canferol, o que pode estar relacionado com os efeitos encontrados nesses extratos. Somente extratos da amostra de Santa Maria demonstraram potencial genotóxico. Unitermos INTRODUCTION The species Peltodon longipes Kunth ex Benth., belonging to the Lamiaceae family, is found in the Southern region of Brazil and is also referred to as P. comaroides Briq. (Briquet, 1989). The plant is known popularly as hortelã-do-campo (wild mint) Often in ethnic communities and groups, the only resource available for the treatment and prevention of diseases is knowledge of medicinal plants. In some regions of Brazil, even in large cities, plants used in alternative folk medicine are sold in local street markets and stores. This resource is used by the population at large, validating therapeutic information gathered over centuries, even though their chemical constituents remain unknown and little studied Many laboratory studies have found a large number of antimutagenic and anticarcinogenic compounds in plant species The majority of toxicity testing systems depend on small animals, rendering them slow, expensive and the target of much criticism (Fatima, Ahmad, 2006; Besides toxicity tests, the chromatographic profile of a plant extract is also essential in that it can be considered representative of the chemical complexity of the sample, allowing assessment of the relationship between the chemical information and the characteristics of each plant sample, such as the differentiation between botanically similar species, variability among plants collected from different geographical locations and under different climatic and growing conditions Against this background, the objective of the present study was to perform genotoxic and chromatographic analyses of leaf extracts of two samples of P. longipes, collected from the cities of Santa Maria and Tupanciretã, Rio Grande do Sul state, Brazil. MATERIAL AND METHODS Genotoxic analysis by the Allium cepa test The leaves of two samples of P. longipes were collected from two different cities, Santa Maria and Tupanciretã, in Rio Grande do Sul state, Brazil at the geographical locations 29°42'19.8"S 53°43'44.6"W and 29°03'56.0"S 53°50'33.8"W, respectively. Collection was carried out in the summer (December 2013). In February 2014, after drying the plant material, the experimental procedures commenced. The plants were identified by Prof. Dr. Thais do Canto-Dorow and a voucher specimen of each access was deposited at the SMDB (Santa Maria Department of Biology), UFSM, under registration numbers 15406 and 15412. The aqueous extracts were prepared at the two concentrations 5 g L -1 and 15 g L -1 , where the lower concentration is generally used by the population for preparing medicinal tea infusions. The dried leaves were placed in boiling water and infused for 10 minutes. The extracts were then strained and left to cool at room temperature. The experimental set-up consisted of 36 A. cepa bulbs comprising nine groups each with four repetitions. Bulb roots were developed in distilled water and after emergence of the roots, each group of onions was transferred for respective treatment. The first group served as the negative control, remaining in distilled water, while the others were transferred for the following treatments: aqueous extracts Genotoxic and chromatographic analyses of aqueous extracts of Peltodon longipes Kunth ex Benth (hortelã-do-campo) 535 of P. longipes at concentrations of 5 g L -1 (Santa Maria sample), 5 g L -1 (Tupanciretã sample), 15 g L -1 (Santa Maria sample) and 15 g L -1 (Tupanciretã sample). Four further groups were treated with 1% glyphosate (Glyphosate 480 AKB Herbicide), one of which served as the positive control and the remaining three to test possible recovery from DNA damage in distilled water, in aqueous extract of P. longipes at the lower concentration, and in aqueous extract of P. longipes at the higher concentration, with both the latter prepared with leaves from the Santa Maria sample. The bulbs were subjected to the treatments for 24 hours and roots subsequently collected and fixed in ethanol: acetic acid (3:1) for 24 h. The roots were then refrigerated in 70% alcohol until slide preparation. Two slides were produced per bulb for each treatment and control. For slide preparation, one root per slide was used, i.e. a total of two roots per bulb were analyzed. These were hydrolyzed in 1 mol/L HCl for five minutes and then washed in distilled water and stained with 2% acetic orcein. The meristematic region of the roots was fragmented with the aid of histological needles, crushed according to the technique of Guerra and Souza (2002), and coverslips placed over the material. The analysis included 500 cells per root, 1000 per bulb, 4000 cells per treatment, giving a total of 36000 cells at experiment endpoint. The slides were assessed using an optical light microscope (LEICA) with a 40X objective by observing cells in interphase, prophase, metaphase, anaphase, telophase and possible occurrence of chromosome changes during the cellular cycle. After analysis of slides, the Mitotic Index (MI) was determined by calculating the number of cells in division / total number of cells analyzed x 100. High performance liquid chromatography (HPLC-DAD) High performance liquid chromatography was employed for the determination and quantification of the phenolic compounds present in the aqueous extracts of P. longipes leaves. The analysis was performed at the Phytochemistry Laboratory of the Department of Industrial Pharmacy of the Federal University of Santa Maria, Santa Maria, Rio Grande Sul state. Chemicals, apparatus and general procedures All chemical were analytical grade. Acetonitrile, formic acid, gallic acid, chlorogenic acid, caffeic acid, ellagic acid and rosmarinic acid were purchased from Merck (Darmstadt, Germany). Quercetin and kaempferol were acquired from Sigma Chemical Co. (St. Louis, MO, USA). High performance liquid chromatography (HPLC-DAD) was performed with a Shimadzu Prominence Auto Sampler (SIL-20A) HPLC system (Shimadzu, Kyoto, Japan), equipped with Shimadzu LC-20AT reciprocating pumps connected to a DGU 20A5 degasser with a CBM 20A integrator, SPD-M20A diode array detector and running LC solution 1.22 SP1 software. Quantification of compounds by HPLC-DAD Reversed phase chromatographic analyses were carried out under gradient conditions using a C 18 column (4.6 mm x 150 mm) packed with 5 μm diameter particles; the mobile phase was water containing 1% formic acid (A) and acetonitrile (B), and the composition gradient was: 13% of B up to 10 min and changed thereafter to obtain 20%, 30%, 50%, 60%, 70%, 20% and 10% B at 20, 30, 40, 50, 60, 70 and 80 min, respectively, following the method described by The limit of detection (LOD) and limit of quantification (LOQ) were calculated based on the standard deviation of the responses and the slope using three independent analytical curves. LOD and LOQ were calculated as 3.3 and 10 σ/S, respectively, where σ is the standard deviation of the response and S is the slope of the calibration curve Statistical analysis The data were submitted to analysis of variance (ANOVA) and the means were compared by the ScottKnott test at 5% probability using the Assistat 7.7 beta software program. RESULTS AND DISCUSSION Genotoxic analysis by the Allium cepa test According to the results obtained in the genotoxicity analysis Given the fact that the extracts studied were from plants collected from different cities, the results of treatments at the same concentration involving different samples differed significantly in mitotic index. By contrast, treatments with extracts from the same sample, even at different concentrations, showed similar effects on the cellular division of A. cepa. The anti-proliferative effect of the treatments derived from leaves collected at the Tupanciretã sample was found to be significantly greater (MI = 1.32 and 0.87%). These results may be explained by possible variations in the levels of production of secondary metabolites of the plants studied, since it is known that such metabolites constitute a chemical interface between the plants and also that their synthesis is often affected by the surrounding environment and environmental conditions With regard to the mitotic indexes of the positive controls (1% glyphosate) and of treatments with glyphosate testing the possibility of recovery from damage to genetic material by subsequent use of distilled water or P. longipes extracts (Santa Maria sample), no significant differences between them were evident, with similar rates of cellular division being observed between the positive control and these three recovery treatments. Regarding the percentage alterations found Among the treatments with extracts of P. longipes at the standard (5 g L -1 ) and higher (15 g L -1 ) concentrations, a statistically significant difference in percentage chromosomal alterations was observed between the aqueous extracts of plants from different samples. Treatments with extracts of leaves from Santa Maria were associated with a significantly higher percentage of chromosome alterations compared to the control in distilled water, confirming genotoxic potential. By contrast, the treatments with aqueous extracts of plants collected from Tupanciretã showed no significant difference compared to the negative control. Thus, the treatments with the extracts from the Tupanciretã sample, besides displaying good Genotoxic and chromatographic analyses of aqueous extracts of Peltodon longipes Kunth ex Benth (hortelã-do-campo) 537 anti-proliferative potential showed no genotoxic activity. This presence of an anti-proliferative effect and absence of genotoxicity was also observed by Frescura et al. (2013) ) concentrations studied as well as very few chromosomal alterations, thereby confirming the absence of genotoxic potential. Extracts of Pterocaulum polystachyum DC. For treatments aimed at detecting a possible antigenotoxic effect by recovery using distilled water and extracts of P. longipes (Santa Maria sample) at both lower and higher concentrations, the three treatments were associated with significantly lower manifestation of chromosomal alterations in meristematic cells of A. cepa than the positive control. Use of distilled water led to good recovery of cell division, with a 0.65% reduction in chromosomal alterations compared to the positive control which had 0.72% chromosomal changes. Similar results were found by Frescura et al. (2013) who also assessed the recovery of onion roots through the application of distilled water after the use of glyphosate. In this case, water was also shown to be effective for recovering from damage to DNA, with a decrease in chromosomal changes from 102 (3% glyphosate) to 41 (glyphosate following the application of water). In the recovery treatments based on the application of P. longipes extracts, only recovery with the 5 g L -1 extract (Santa Maria sample) differed significantly from recovery in water, but proved less effective for reducing the damage caused by glyphosate. On the other hand, application of the 15 g L -1 extract after glyphosate treatment did not differ significantly to recovery in water, showing the same effect. Although the number of chromosomal alterations was significantly higher in some treatments compared to the negative control, it is should be noted that all values were relatively low, representing less than 1% of the total cells analyzed per treatment High Performance Liquid Chromatography (HPLC-DAD) Despite the great importance of medicinal plants for pharmacological research and in the development of drugs, studies elucidating their constituents remain scarce. HPLC fingerprinting of P. longipes (Santa Maria and Tupanciretã) extracts revealed the presence of gallic acid (t R = 9.86 min; peak 1), chlorogenic acid (t R = 19.47 min; peak 2), caffeic acid (t R = 24.98 min; peak 3), ellagic acid (t R = 33.17; peak 4), rosmarinic acid (t R = 38.06 min; peak 5), quercetin (t R = 41.25 min; peak 6) and kaempferol (t R = 56.61 min; peak 7

Assessment of the antiproliferative and antigenotoxic activity and phytochemical screening of aqueous extracts of Sambucus australis Cham. & Schltdl. (ADOXACEAE)

<div><p>ABSTRACT The purpose of this study was to evaluate the antiproliferative and antigenotoxic activity of Sambucus australis Cham. & Schltdl. aqueous extracts on the cell cycle of Allium cepa L. as well as determine the phenolic compounds in such extracts. S. australis inflorescences and leaves of two accessions were used for aqueous extract preparation at concentrations: 0.003 g/ml and 0.012 g/ml. A. cepa bulbs were rooted in distilled water and, subsequently, placed in treatments for 24 hours. Rootlets were collected and fixed in modified Carnoy’s solution for 24 hours and kept. The squash technique was performed for slide preparation. Root tips were smashed and stained with 2% acetic orcein, and a total of 4000 cells per treatment were analyzed. The phenolic compounds were determined using high-performance liquid chromatography and data was analyzed using the Scott-Knott test. The results show that S. australis aqueous extracts have antiproliferative potential. Besides, the extracts prepared from S. australis leaves of both accessions at a concentration of 0.012 g/ml have shown antigenotoxic activity. The phytochemical analysis allowed us to determine the presence of flavonoids and phenolic acids, of which kaempferol and chrologenic acid were the most predominant compounds in the extracts from the inflorescences and leaves, respectively.</p></div

Cytogenotoxicity of rice crop water after application of the tricyclazole fungicide

ABSTRACT Tricyclazole is currently one of the fungicides recommended for the treatment of diseases in irrigated rice. However, there is relatively little information on its cytotoxic and genotoxic potential. The objective of this study was to evaluate the cytotoxicity and genotoxicity of rice crop water after apllication of the tricyclazole fungicide through the Allium cepa L. test. The rice crop water samplings were collected before and 1, 15 and 30 days after application of the fungicide in rice plant shoots. The Allium cepa roots were placed in contact with the rice crop water to check for possible chromosomal abnormalities and mitotic index of the bioindicators meristematic cells. The data obtained by the Allium cepa test indicates that the application of the tricyclazole fungicide leads to an increase in the genotoxic activity in the rice crop water, through the appearance of chromosomal abnormalities, without, however, causing significant effects on the mitotic index. The major chromosomal alterations observed were anaphasic and telophasic bridges and laggard chromosomes

Mode of reproduction of Brazilian species of Adesmia (Leguminosae)

Mode of reproduction was studied in 15 species of Adesmia DC. (Leguminosae). In six species, three treatments were used: mutual pollination, mechanical stimulation and control. Fifty-four plants of these six species were grown in a greenhouse, individually isolated in nylon screen boxes. Flowers were labelled and submitted to the different treatments. In addition, the frequency of spontaneous self-pollination in the absence of pollinators was studied in 200 plants of nine other species. These 200 plants were kept in a greenhouse, which avoided contact with any possible pollinator. Adesmia bicolor, A. muricata, A. punctata and A. riograndensis produced seed both by cross- and self-pollination. Adesmia punctata and A. riograndensis need mechanical stimulation for self-pollination. Adesmia incana reproduced by self-pollination; however, the possibility of cross-pollination cannot be totally ruled out. Adesmia tristis reproduced mainly by cross-pollination and a mechanism of self-incompatibility is suggested. Among the nine species that were not exposed to pollinators, A. securigerifolia produced a large amount of seed, indicating that it is a self-pollinating species. Adesmia arillata, A. araujoi, A. ciliata, A. psoraleoides, A. rocinhensis, A. reitziana, A. sulina and A. vallsii did not produce any seed under the experimental conditions, suggesting that they are cross-pollinated or that they need mechanical stimulation to reproduce.<br>Foram estudadas 15 espécies do gênero Adesmia DC. (Leguminosae), quanto ao modo de reprodução. Em seis espécies do gênero Adesmia, o modo de reprodução foi determinado através de três tratamentos: polinização mútua, estímulo mecânico e controle. As 54 plantas submetidas aos tratamentos foram cultivadas em casa de vegetação e mantidas isoladas individualmente, através de armações de tela de náilon. As flores foram marcadas e submetidas aos distintos tratamentos. Adicionalmente, foram observadas 200 plantas de outras 9 espécies do mesmo gênero quanto à ocorrência de autofecundação na ausência de polinizadores. As 200 plantas apenas isoladas e observadas foram cultivadas em vasos e mantidas em casa de vegetação telada, sem acesso de polinizadores. Os resultados mostraram que, das espécies investigadas pelos três tratamentos, A. bicolor, A. muricata, A. punctata e A. riograndensis são versáteis, pois permitiram a reprodução por fecundação cruzada e autofecundação. As duas últimas, para se autofecundarem, necessitaram de estímulo mecânico. Adesmia incana se reproduziu por autofecundação, mas não se descarta a possibilidade de ocorrer fecundação cruzada. Adesmia tristis se reproduziu quase que totalmente por fecundação cruzada e é possível a ocorrência de mecanismos de autoincompatibilidade. Das nove espécies apenas observadas, Adesmia securigerifolia se reproduziu por autofecundação, formando elevado número de sementes por planta. A. arillata, A. araujoi, A. ciliata, A. psoraleoides, A. rocinhensis, A. reitziana, A. sulina e A. vallsii não produziram sementes por autofecundação espontânea. Estas espécies são de fecundação cruzada ou necessitam do estímulo do polinizador

Alterations in the mitotic index of Allium cepa induced by infusions of Pluchea sagittalis submitted to three different cultivation systems

We evaluated the antiproliferative effect of infusions from Pluchea sagittalis using the Allium cepa test. Infusions in three concentrations (2.5, 5, and 25 g dm-3) of leaves cultivated in three environments (in vitro, acclimatized growth chamber, and field) were used. Six onion bulbs were used for each of the eight treatments, and the mitotic index was obtained from 6000 cells per treatment. In conclusion, leaf infusions of P. sagittalis cultivated in the field have a high antiproliferative activity, as well as the cultivation system influences the antiproliferative potential.<br>Avaliou-se o efeito antiproliferativo de infusões de Pluchea sagittalis usando o teste de Allium cepa. Foram usadas infusões em três concentrações (2,5, 5 e 25g dm-3) de folhas cultivadas em três ambientes (in vitro, sala de crescimento climatizada e em campo). Foram usados seis grupos de bulbos para cada um dos 8 tratamentos e o os índices mitóticos foram obtidos a partir de 6000 células por tratamento. Concluiu-se que a infusão de folhas de P. sagittalis cultivadas em campo possui grande atividade antiproliferativa, bem como o sistema de cultivo de plantas influencia o potencial antiproliferativo