RESUMO
In vitro culture of ovarian tissue containing primordial follicles is an important tool to study the initiation of follicular populations and to develop efficient culture systems to support in vitro follicle growth. Considering that in vitro culture favours oxidative stress, it is very important to supplement culture medium with antioxidant substances such as Aloe vera extract. This study aims to evaluate the effects of different concentrations of Aloe vera on the distribution of collagen fibres in the extracellular matrix, follicular activation, development and survival in bovine ovarian cortical tissues cultured in vitro, as well as on expression of mRNAs for antioxidant enzymes [superoxide dismutase (SOD), catalase (CAT), peroxiredoxin 6 (PRDX6) and glutathione peroxidase 1 (GPX1)]. To this end, ovarian cortical tissues were cultured for 6 days in α-MEM alone or supplemented with different concentrations of Aloe vera extract (1.0, 5.0, 10.0 or 50.0%). After culture, fragments were fixed and processed histologically to evaluate follicular morphology and activation, as well as the extracellular matrix by staining with picrosirius red. The levels of mRNA for SOD, CAT, PRDX6 and GPX1 in cultured ovarian tissues were evaluated by real-time polymerase chain reaction (PCR). Ovarian tissues cultured with 10.0 or 50.0% Aloe vera had higher percentages of collagen fibres than tissues cultured in control medium. A significant increase in developing follicles was observed in ovarian tissues cultured in α-MEM alone or supplemented with 10% Aloe vera when compared with fresh control or tissues cultured with 1.0% Aloe vera. Presence of Aloe vera did not influence the percentage of morphologically normal follicles when compared with control medium. Ovarian tissues cultured with 50.0% Aloe vera had higher percentages of morphologically normal follicles than those cultured with 10.0% Aloe vera. Furthermore, 10% Aloe vera significantly increased mRNA levels for PRDX6. In conclusion, 10.0% Aloe vera improves extracellular matrix distribution in cultured tissues and increases the expression of mRNA for PRDX6 after 6 days in vitro.
Assuntos
Aloe , Aloe/genética , Animais , Antioxidantes , Bovinos , Colágeno/genética , Matriz Extracelular , Peroxirredoxina VI , Extratos Vegetais , RNA Mensageiro/genética , Superóxido Dismutase , Técnicas de Cultura de TecidosRESUMO
The main polysaccharide of the gel present in the leaves of or Aloe vera Burm.F., (Aloe barbadensis Miller) a xerophytic crassulacean acid metabolism (CAM) plant, is an acetylated glucomannan named acemannan. This polysaccharide is responsible for the succulence of the plant, helping it to retain water. In this study we determined using polysaccharide analysis by carbohydrate gel electrophoresis (PACE) that the acemannan is a glucomannan without galactose side branches. We also investigated the expression of the gene responsible for acemannan backbone synthesis, encoding a glucomannan mannosyltransferase (GMMT, EC 2.4.1.32), since there are no previous reports on GMMT expression under water stress in general and specifically in Aloe vera. It was found by in silico analyses that the GMMT gene belongs to the cellulose synthase-like A type-9 (CSLA9) subfamily. Using RT-qPCR it was found that the expression of GMMT increased significantly in Aloe vera plants subjected to water stress. This expression correlates with an increase of endogenous ABA levels, suggesting that the gene expression could be regulated by ABA. To corroborate this hypothesis, exogenous ABA was applied to non-water-stressed plants, resulting in a significant increase of GMMT expression after 48â¯h of ABA treatment.
Assuntos
Ácido Abscísico/farmacologia , Aloe/genética , Regulação Enzimológica da Expressão Gênica/efeitos dos fármacos , Regulação da Expressão Gênica de Plantas/efeitos dos fármacos , Genes de Plantas , Mananas/metabolismo , Metiltransferases/genética , Estresse Fisiológico , Água/metabolismo , Aloe/enzimologia , Aloe/metabolismo , Sequência de Aminoácidos , Sequência de Bases , Domínio Catalítico , DNA Complementar/genética , Secas , Eletroforese em Gel de Amido/métodos , Cromatografia Gasosa-Espectrometria de Massas , Metiltransferases/química , Metiltransferases/metabolismo , Homologia de Sequência de AminoácidosRESUMO
Aloe (Aloe spp), containing abundant polysaccharides and numerous bioactive ingredients, has remarkable medical, ornamental, calleidic, and edible values. In the present study, the total RNA was extracted from aloe leaf tissue. The isolated high-quality RNA was further used to clone actin gene by using reverse transcription-polymerase chain reaction (RT-PCR). The result of sequence analysis for the amplified fragment revealed that the cloned actin gene was 1012 bp in length (GenBank accession No. KC751541.1) and contained a 924-bp coding region and encoded a protein consisting of 307 amino acids. Homologous alignment showed that it shared over 80 and 96% identity with the nucleotide and amino acid sequences of actin from other plants, respectively. In addition, the cloned gene was used for phylogenetic analyses based on the deduced amino acid sequences, and the results suggested that the actin gene is highly conserved in evolution. The findings of this study will be useful for investigating the expression patterns of other genes in Aloe.
Assuntos
Actinas/genética , Aloe/genética , Filogenia , Folhas de Planta/genética , Proteínas de Plantas/genética , Actinas/química , Aloe/classificação , Sequência de Aminoácidos , Sequência de Bases , Evolução Biológica , Clonagem Molecular , Sequência Conservada , Expressão Gênica , Dados de Sequência Molecular , Folhas de Planta/classificação , Proteínas de Plantas/química , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Alinhamento de SequênciaRESUMO
KEY MESSAGE : The study determined the tolerance of Aloe vera to high temperature, focusing on the expression of hsp70 , hsp100 and ubiquitin genes. These were highly expressed in plants acclimated at 35 °C prior to a heat shock of 45 °C. Aloe barbadensis Miller (Aloe vera), a CAM plant, was introduced into Chile in the semiarid IV and III Regions, which has summer diurnal temperature fluctuations of 25 to 40 °C and annual precipitation of 40 mm (dry years) to 170 mm (rainy years). The aim of this study was to investigate how Aloe vera responds to water and heat stress, focusing on the expression of heat shock genes (hsp70, hsp100) and ubiquitin, which not studied before in Aloe vera. The LT(50) of Aloe vera was determined as 53.2 °C. To study gene expression by semi-quantitative RT-PCR, primers were designed against conserved regions of these genes. Sequencing the cDNA fragments for hsp70 and ubiquitin showed a high identity, over 95 %, with the genes from cereals. The protein sequence of hsp70 deduced from the sequence of the cDNA encloses partial domains for binding ATP and the substrate. The protein sequence of ubiquitin deduced from the cDNA encloses a domain for interaction with the enzymes E2, UCH and CUE. The expression increased with temperature and water deficit. Hsp70 expression at 40-45 °C increased 50 % over the controls, while the expression increased by 150 % over the controls under a water deficit of 50 % FC. The expression of all three genes was also studied under 2 h of acclimation at 35 or 40 °C prior to a heat shock at 45 °C. Under these conditions, the plants showed greater expression of all genes than when they were subjected to direct heat stress.
Assuntos
Aclimatação , Aloe/genética , Regulação da Expressão Gênica de Plantas , Proteínas de Choque Térmico/genética , Proteínas de Plantas/genética , Ubiquitina/genética , Aloe/fisiologia , Sequência de Aminoácidos , Sequência de Bases , Primers do DNA/genética , DNA Complementar/genética , Desidratação , Proteínas de Choque Térmico HSP70/genética , Proteínas de Choque Térmico HSP70/metabolismo , Proteínas de Choque Térmico/metabolismo , Resposta ao Choque Térmico , Temperatura Alta , Dados de Sequência Molecular , Filogenia , Folhas de Planta/genética , Folhas de Planta/fisiologia , Proteínas de Plantas/metabolismo , Estrutura Terciária de Proteína , RNA Mensageiro/genética , RNA de Plantas/genética , Análise de Sequência de DNA , Ubiquitina/metabolismoRESUMO
The ethanol, methanol and acetone extracts of Aloe vera gel were studied for their antimicrobial activity against four Gram-positive and Gram-negative bacteria using agar well diffusion method. The extracts showed varied levels of antimicrobial activity against the tested pathogens. The ethanol and methanol extracts showed higher activity while acetone extract, showed least or no activity against most of the tested pathogens. Fractions obtained from the extracts by Thin Layer and Column Chromatography were studied for their antagonistic properties using Spot Assay Technique. Compounds with maximum antibacterial activity isolated from the ethanol and methanol extracts were identified as p - coumaric acid (Mol. wt.165), ascorbic acid (Mol. wt.177 ), pyrocatechol (Mol. wt.110 ) and cinnamic acid (Mol. wt.148), on the basis of Gas Chromatography Mass Spectrometry. The study suggests the antimicrobial activity of the A. vera gel extract to be dependant on the synergistic effect of different compounds. With the broad spectral antimicrobial effect of A. vera gel, it could be further recommended in the treatment of various bacterial diseases.
Assuntos
Ágar , Acetona/análise , Antibacterianos/isolamento & purificação , Aloe/genética , Bactérias Gram-Negativas/isolamento & purificação , Bactérias Gram-Positivas/isolamento & purificação , Etanol/análise , Extratos Vegetais/análise , Extratos Vegetais/isolamento & purificação , Técnicas In Vitro , Metanol/análise , Meios de Cultura , Difusão , Cromatografia Gasosa-Espectrometria de Massas , Métodos , VirulênciaRESUMO
Con el objetivo de esclarecer la posible existencia de anomalías citogenéticas que aminoren la fertilidad del polen de Aloe vera, se analizó la etapa de proliferación celular que lleva a la formación de células madres del polen (CMPs). Se recolectaron botones florales (BF) en 25 plantas de una población ubicada a 10°3415 N, 64°1208 W, los cuales fueron fijados en Carnoy I por 24 h y almacenados en etanol (70 % v/v). Las observaciones se realizaron en preparaciones temporales obtenidas por la tinción del contenido de las anteras suspendidas en orceína acética (1.5 % p/v) por 5 minutos. De las 9 411 células analizadas, 17 % mostraron 1-8 puentes entre cromátidas hermanas, 13 % 1-7 micronúcleos de 0.9-4.8 µm, 8.1 % estaban unidas por puentes y 0.1 % no contenían cromatina. El resto de las células (61.8 %) presentó configuraciones aparentemente normales y sin variaciones morfométricas. La proliferación irregular de una fracción de CMPs (39.2 %) sugiere que las condiciones ambientales de la zona árida donde se realizaron los muestreos inducen inestabilidad cromosómica y cambios fisiológicos que afectan el normal desarrollo de la mitosis premeiótica, generando pérdida o adición de fragmentos, asociados a deficiencias y duplicaciones génicas.
Assuntos
Aloe/citologia , Instabilidade Cromossômica/genética , Cromossomos de Plantas/genética , Mitose/genética , Pólen/citologia , Células-Tronco/citologia , Aloe/genética , Pólen/genética , VenezuelaRESUMO
In order to clarify possible cytogenetic anomalies that reduce pollen fertility, premeiotic mitosis was studied in Aloe vera plants from a naturalized population in the northeast of Venezuela (10 degrees 34' 15" N and 64 degrees 12' 08" W). Karyological configurations were evaluated during the stage of cell proliferation leading to the formation of pollen mother cells (PMCs). The sampling was carried out in March 2005, choosing inflorescences without mechanical or biological damage from 25 plants selected at random. Flower buds (FB) 2 to 6 mm in length were collected from 7:00 AM through 6:00 PM, their perianths removed, and fixed in Carnoy I (3:1 ethanol/glacial acetic acid) for 24 h and stored in ethanol (70% v/v) until observation. Light microscope observations were done on temporary preparations obtained by overflowing anther content suspended in acetic orcein (1.5% w/v) for 5 min and softly squashing with the cover slip. A total of 9 411 cells were analyzed. Upper mitotic activity was observed in FB from 3.8 +/- 0.09 mm long, collected at 11:00 AM through 1:00 PM; 17% of PMCs showed one to eight sister chromatid bridges from anaphase to telophase; 13%, one to seven micronucleus of variable diameter (0.9 to 4.8 microm); 8.1% were united by thin chromatin filaments, and 0.1% lacked a nucleus. Other evaluated cells (61.8%) had apparently normal mitotic configurations, without considerable morphometric variations. The evident irregular proliferation of a PMCs fraction (39.2%) suggests that environmental stress conditions (day temperatures ranging 32.7 to 39.8 degrees C, high solar radiation and low humidity) induce chromosome instability and physiologic changes that affect the normal development during premeiotic mitosis. As a consequence, loss or addition of chromosome fragments can occur in association with deficiencies and gene duplications.
Assuntos
Aloe/citologia , Instabilidade Cromossômica/genética , Cromossomos de Plantas/genética , Mitose/genética , Pólen/citologia , Células-Tronco/citologia , Aloe/genética , Pólen/genética , VenezuelaRESUMO
Con el objetivo de conocer la posible actividad mutagénica del extracto fluido de Cassia grandis L. y el gel de Aloe vera L., que puedan representar algún efecto adverso para su uso en la fitoterapia, se llevó a cabo un estudio toxicogenético empleando 2 sistemas de ensayo a corto plazo uno in vitro y otro in vivo; el modelo Aspergillus nidulans D-30 que detecta daño primario al ADN y el ensayo de inducción de micronúcleos en médula ósea de ratón el cual determina daño clastogénico y aneugénico. En el ensayo in vitro con el hongo Aspergillus nidulans D-30 (segregación mitótica) se evaluaron concentraciones del extracto fluido de Cassia grandis L., desde 0,067 a 1,675 mg de sólidos totales/mL y para el gel de Aloe vera L., concentraciones de 0,09 a 1,00 mg de sólidos totales/mL. En la prueba in vivo de inducción de micronúcleos se ensayaron para la Cassia grandis L. y para el gel de Aloe vera L., dosis de 500, 1 000 y 2 000 mg/kg de peso corporal (pc). En ambas baterías de ensayos genotóxicos ninguno de los 2 fitofármacos mostraron ni daño celular ni actividad genotóxica
Assuntos
Aloe/genética , Aloe/toxicidade , Aspergillus nidulans , Medicina Herbária , Testes de Mutagenicidade , Extratos Vegetais/genética , Extratos Vegetais/toxicidade , Plantas Medicinais , Testes para Micronúcleos , CamundongosRESUMO
Se realizo la evaluacion mutagenica de un extracto acuoso liofilizado de hojas de Aloe vera L., para lo cual se emplearon tres ensayos a corto plazo: induccion de mutaciones puntuales (supresores) en el locus meth G1 de Aspergillus nidulans, segregacion mitotica en un diploide heterocigotico de Aspergillus nidulans y el test de micronucleos en medula osea de raton. Para los ensayos in itro se eavaluaron concentraciones entre 0,05 y 5 mg de extracto de aloe/mL en medio de cultivo (mutaciones puntuales) y de 0,04 a 1 mg/mL (segregacion mitotica); se empleo el metodo de incorporacion en placa. No se detectaron aumentos significativos para la frecuencia de mutantes supresores en el primer ensayo, ni de sectores segregantes homocigoticos en el segundo, que son indicadores de genotoxicidad para estas pruebas. En el ensayo in vivo se emplearon ratones de la linea isogenica suizo, a los que se hicieron dos administraciones del extracto por via intragastrica, en dosis de 0,5 1,0 y 2,0 g/kg/dia, con sacrificio 24 horas despues de la ultima aplicacion. En ningun caso se detecto efecto citotoxico sobre la proliferacion celular en la medula osea, ni aumentos significativos en la frecuencia de eritrocitos policromaticos micronucleados 2(mPCE), indicador de mutagenicidad para este ensayo