Levantamento de extratos vegetais com ação anti-Candida no período de 2005-2013 / Survey of plant extracts with anti-Candida activity in the 2005-2013 period

Entre as micoses relevantes em saúde pública destaca-se a candidíase, infecção oportunista que acomete o homem e animais. A enfermidade era considerada pouco frequente na medicina veterinária, porém relatos demonstram um aumento considerável, assim como a resistência aos antifúngicos. Com isso, pesquisas têm sido desenvolvidas visando encontrar substâncias bioativas frente ao gênero Candida. Desta forma, objetivou-se reunir dados das bases Scielo e ScienceDirect com informações entre os anos de 2005-2013 referentes à ação anti-Candida de diferentes extratos vegetais. Foi encontrado um total de 78 famílias e 208 espécies de plantas com atividade frente à Candida spp., destacando-se as famílias Asteraceae, Geraniaceae, Myrtaceae, Fabaceae, Lamiaceae, Rubiaceae, Verbenaceae e Anacardiaceae, com extratos diclorometânicos, aquosos, etanólicos, metanólico, frações e subfrações, sendo as folhas a parte vegetal mais utilizada. As plantas descritas apresentaram ação anti-Candida, porém algumas necessitam concentrações muito altas dos extratos com pequena inibição de crescimento/eliminação destas leveduras, ocorrendo variações, principalmente, quanto ao método de avaliação, tipo de extrato, parte vegetal, e procedência dos isolados fúngicos. Chama a atenção a raridade dos estudos com isolados de animais, principalmente de casos clínicos. Por fim, destacam-se as famílias Asteraceae e Geraniaceae que apresentaram maior número de espécies vegetais com atividade, podendo ser uma fonte de investigação frente à Candida spp.

Among the relevant mycoses in public health, one that stands out is candidiasis, an opportunistic infection that affects humans and animals. The disease was considered uncommon in veterinary medicine, but reports show a significant increase, as well as resistance, to conventional antifungal agents. Therefore, research has been undertaken aimed at finding bioactive substances from plants that fight against Candida. Thus, the objective of this work was to gather the databases SciELO and ScienceDirect with information between the years 2005 and 2013 concerning the anti-Candida activity of different plant extracts. A total of 78 families and 208 species of plants with activity against Candida spp. was found highlighting the Asteraceae, Geraniaceae, Myrtaceae, Fabaceae, Lamiaceae, Rubiaceae, Verbenaceae and Anacardiaceae families, with dichloromethane, aqueous, ethanol and methanol extracts and fractions and subfractions, being the leaf the most used plant part. The plants described showed anti-Candida activity, but some require very high concentrations of the extracts with little growth inhibition / elimination of these yeasts, with variations related mainly to the method of assessment, type of extract, plant parts and origin of the fungal isolates. The rarity of studies with isolates from animals, mainly clinical cases, draws attention. Finally, we highlight the Asteraceae and Geraniaceae families, which had a greater number of plant species with activity and which may be a source of research against Candida spp.

Atividade antifúngica do óleo essencial de Origanum vulgare frente a Malassezia pachydermatis / Antifungal activity of Origanum vulgare essential oil against Malassezia pachydermatis

Objetivou-se com este estudo avaliar a atividade antifúngica in vitro do óleo essencial de Origanum vulgare frente a isolados clínicos de Malassezia pachydermatis. As folhas secas de O. vulgare foram adquiridas de distribuidor comercial com certificado de qualidade e origem e encaminhadas para extração do óleo essencial e cromatografia. Para realização do teste in vitro, foi utilizada a técnica de microdiluição em caldo (CLSI M27A3) com modificações para fitofármacos e M. pachydermatis. O óleo essencial de orégano foi testado nas concentrações de 28 a 0,87mg/mL diluído em caldo Sabouraud com 1% de tween 80. Todos os isolados foram testados em duplicata. Na análise cromatográfica do óleo essencial, foram identificados 12 compostos, sendo timol, a-terpineno e 4-terpineol os compostos majoritários. A CIM e a CFM dos 42 isolados de M. pachydermatis variaram de <0,87 a 7mg/mL, com valores de CIM50 e CIM90 de 1,18 e 3,28mg/mL, respectivamente. Com este estudo foi possível concluir que M. pachydermatis é sensível ao óleo essencial de orégano mesmo em concentrações baixas. Dessa maneira, o óleo essencial de orégano apresenta-se como promissor na bioprospecção de novos fármacos para o tratamento das otites e dermatites na clínica de pequenos animais...

The aim of this study was to evaluate the in vitro antifungal activity of essential oil of Origanum vulgare against clinical isolates of Malassezia pachydermatis. The dried leaves of O. vulgare were purchased from a commercial distributor with certified quality and origin and referred for essential oil extraction and chromatography. The technique for in vitro testing was microdilution (CLSI M27A3) with modifications to phytochemicals and M. pachydermatis. The essential oil of O. vulgare was tested at concentrations from 28 to 0.87mg/mL in Sabouraud broth diluted with 1% of tween 80. All isolates were tested in duplicate. In the chromatographic analysis of the essential oil 12 compounds were identified, and thymol, α-terpinene, 4-terpineol were the major compounds. The MIC and the MFC of the 42 isolates of M. pachydermatis ranged from <0.87 to 7mg/mL with MIC50 and MIC90 values of 1.18 and 3.28 mg/mL, respectively. With this study it was concluded that M. pachydermatis is sensible to O. vulgare essential oil even at low concentrations. Thus, the essential oil of O. vulgare is presented as bioprospecting in the promising new drugs for the treatment of otitis and dermatitis in small animal clinic...

Pharmacogenetics of immunosuppressant polymorphism of CYP3A5 in renal transplant recipients.

The tacrolimus is metabolized primarily by CYP3A5, a member of the single nucleotide polymorphism family. It shows cytochrome P450 (SNP) in intron 3, which consists of a change of base, G for A, producing a stop codon. The result is a nonfunctional protein (allele *3). Allele *1 is the wild type. The patients that show the allelic variant *3 in homozygosis (G/G) are slow metabolizers of the immunosuppressant, increasing its concentration in blood. In contrast, heterozygote A/G alleles *1/*3 are intermediate metabolizers, whereas those of allele *1 in homozygosis (A/A) are normal metabolizers. The aim of this study was to determine CYP 3A5 polymorphism among adult renal transplant recipients and the general Argentinean population. We analyzed 21 recipients and 36 healthy controls. All subjects gave written informed consent approved by the local committee. To determine the polymorphism, we extracted DNA from peripheral blood and used polymerase chain reaction (PCR) to amplify intron 3 of the CYP 3A5. The presence of variant was confirmed by direct sequencing. Among the controls the CYP3A5 genotype *3/*3 (G/G) was detected in 32 individuals, 4 showed *1/*3 (A/G), and none had *1/*1 (A/A); among the recipients, the results were as follows: 18, 2, and 1, respectively. The frequencies of polymorphism in both groups were similar, although they differed from those published for other populations. These results are the basis for the development of a pharmacogenomic program applied to organ transplantation. The genetic polymorphisms can determine responses to drugs. The molecular diagnosis must be transferred to clinical practice so as to guide selection of medicine and drug doses to be optimal for each individual.

Exercise-induced effects on growth hormone levels are associated with ghrelin changes only in presence of prolonged exercise bouts in male athletes.

AIM: The aim of this study was to evaluate growth hormone (GH) and ghrelin levels in response to physical exercise in athletes. METHODS: Two different exercise workloads were administered in two different groups of athletes. Group A athletes (19 males, 18 females; mean age +/- standard deviation: 25+/-6.7 years), performing a 60-90 min training session at approximately 80% of VO2max, were sampled for GH and ghrelin determinations before and immediately at the end of a training session on-the-field. Group B athletes (4 males; mean age: 28.2+/-7.2 years) performed two consecutive 30-min cycling sessions at 80% of individual VO2max at different time intervals between bouts (2 and 6 h) in two different days. GH and ghrelin concentrations were determined in blood samples collected at 15-min intervals during exercise and following 1 h of recovery. RESULTS: In group A athletes, GH levels increased after the training session (P<0.0001), with no differences between males and females. In male athletes, ghrelin levels significantly decreased after the training session (from 1 506.4+/-859 to 1 254.8+/-661.7 pg/mL, P<0.05), while no significant changes were found in females. No correlations were observed between GH and ghrelin levels at rest and after training. In group B athletes, GH levels significantly increased after the first exercise bouts (peak: 26.8+/-11.2 and 17.3+/-3.5 ng/mL, respectively), while the pattern of GH response was different after the second bout of exercise performed at 2-h or 6-h interval. In fact, peak GH concentration in response to the second bout (4.3+/-1.6 ng/mL) was lower (P<0.01) than that of the first bout when the interval elapsed was only 2 h, while a recovery of GH responsiveness was evident after the 6-h interval between the two exercise bouts (11.9+/-3.3 ng/mL). As far as ghrelin levels are concerned, no significant changes were observed during and after the two exercise bouts performed at the different time intervals. CONCLUSION: GH responses to prolonged exercise bouts (60-90 min) are associated with changes in ghrelin levels only in male athletes, while repeated exercise bouts of lower duration (30 min), capable to determine marked GH responses, are divorced from changes in ghrelin concentrations.

Serial protocol biopsies to quantify the progression of chronic transplant nephropathy in stable renal allografts.

AIM: To evaluate the utility of intimal thickness and interstitial width as a primary efficacy variable in the design of clinical trials aimed to modify the natural history of chronic allograft nephropathy. METHODS: A donor and a 4-month protocol biopsy were evaluated in 40 stable grafts according to the Banff schema. In 27 patients, a second protocol biopsy was done at 1 yr. Arterial intimal volume fraction (Vvintima/artery) and cortical interstitial volume fraction (Vvinterstitium/cortex) were estimated with a point counting technique. RESULTS: Chronic Banff scores increased during follow-up, while acute scores reached its peak at 4 months. Vvintima/artery and Vvinterstitium/cortex significantly increased at 4 months, but not at 1 yr. Vvintima/artery at 4 months correlated with donor Vvintima/artery (r = 0.57, p < 0.001), histocompatibility (r = 0.38, p = 0.01) and serum cholesterol (r = 0.31, p = 0.047). Vvinterstitium/cortex at 4 months correlated with recipient body surface area (r = 0.44, p = 0.004) and delayed graft function (p = 0.016). Power calculations showed that Vvintima/artery and Vvinterstitium/cortex allow an important reduction in minimum sample size of a hypothetical trial aimed to prevent chronic allograft nephropathy. CONCLUSIONS: Intimal thickening and interstitial widening progresses rapidly during the first 4 months after transplantation and slowly thereafter. These parameters can be considered as a primary efficacy variable in trials aimed to prevent chronic allograft nephropathy.

Evidence for genomic changes in transgenic rice (Oryza sativa L.) recovered from protoplasts.

The occurrence of genomic modifications in transgenic rice plants recovered from protoplasts and their transmission to the self-pollination progeny has been verified with the random amplified polymorphic DNA (RAPD) approach. The plant was the Indica-type rice (Oryza sativa L.) cultivar Chinsurah Boro II. The analysed material was: (1) microspore-derived embryogenic rice cells grown in suspension culture, (2) transgenic plants recovered from protoplasts produced from the cultured cells and (3) the self-pollination progeny (two successive generations) of the transgenic plants. DNA purified from samples of these materials was PCR-amplified with different random oligonucleotide primers and the amplification products were analysed by agarose gel electrophoresis. Band polymorphism was scored and used in band-sharing analyses to produce a similarity matrix. Relationships among the analysed genomes were expressed in a dendrogram. The extensive DNA changes evidenced in cultured cells demonstrate the occurrence of somaclonal variation in the material used to produce protoplasts for gene transfer. Quantitatively reduced DNA changes were also found in the resulting transgenic plants and in their self-pollination progenies. While confirming the stability of the foreign gene in transgenic plants, this work gives molecular evidence for the occurrence of stable genomic changes in transgenic plants and points to in vitro cell culture as the causative agent. RAPDs are shown to be a convenient tool to detect and estimate the phenomenon at the molecular level. The methodology is also proposed as a fast tool to select those transgenic individuals that retain the most balanced genomic structure and to control the result of back-crosses planned to restore the original genome.

Presence of a chloroplast DNA sequence in an autonomous circular DNA molecule in cultured rice cells (Oryza sativa L).

Sequence analysis of twelve DNA fragments, which had previously been found to be extensively amplified in suspension-cultured rice cells, revealed that two of them, isolated on plasmids designated pE10 and pE11, have sequences identical to distinct regions of chloroplast DNA (ct-DNA). Both sequences are part of an extrachromosomal circular DNA molecule (ECD). The molecular structure of the ECD was investigated by a combination of restriction analysis, standard and pulsed-field gel electrophoresis, hybridization with ct-DNA probes and amplification by the polymerase chain reaction in the presence of oligonucleotide primers homologous to selected regions of rice ct-DNA. The results showed that a continuous and unrearranged stretch of ct-DNA from the long single-copy region, of at least 28 kbp in length, is present in the ECD. It was estimated that the number of copies of the ECD in cultured cells was almost equivalent to that of ct-DNA molecules in rice leaves, while the ratio of ECD to ct-DNA molecules in the cultured cells was approximately 200:1.

State of the foreign gene and of the genome in transgenic rice (Oryza sativa L.).

PCR with random primers (RAPD analysis) performed on the DNA of embryogenic and non-embryogenic suspension cultured rice and of transformed rice plants allows the evaluation of the extent of DNA changes in the different biological materials. This is thus suggested as a convenient approach, in combination with restriction analysis and Southerns blotting, to evaluate the integrity of the foreign gene, the stability of the insertion site and the stability of the whole genome.

State of the foreign gene and of the genome in transgenic rice (Oryza sativa L.).

PCR with random primers (RAPD analysis) performed on the DNA of embryogenic and non-embryogenic suspension cultured rice and of transformed rice plants allows the evaluation of the extent of DNA changes in the different biological materials. This is thus suggested as a convenient approach, in combination with restriction analysis and Southerns blotting, to evaluate the integrity of the foreign gene, the stability of the insertion site and the stability of the whole genome.

A repeated chromosomal DNA sequence is amplified as a circular extrachromosomal molecule in rice (Oryza sativa L.).

The plasmid pE10 is a pBR322-derived plasmid carrying a 4.5 kb rice (Oryza sativa L.) repeated DNA sequence. The cloned sequence has been shown to be amplified in cultured rice cells. The analysis of practically intact chromosomal rice DNA molecules by pulsed field gel electrophoresis has now shown that the amplification is associated with the appearance of extrachromosomal molecules. In fact, pE10 hybridizes exclusively with unfractionated DNA from leaf protoplasts, while it recognizes predominantly an extrachromosomal DNA molecule (ECD) of about 45 kb and its multiples in the case of protoplasts from cultured cells. Insensitivity to the action of the exonuclease Bal31 suggests that the molecule is circular. Analysis of restriction endonuclease products with both standard horizontal and pulsed field gel electrophoresis suggest that the extrachromosomal DNA, and its chromosomal counterpart, is composed of tandemly repeated units of about 7 kb. Thus, the smaller extrachromosomal circle should contain 6-7 repeats, while the sequence cloned in pE10 is a subset of this repeat. The extrachromosomal DNA represents about 1% of total rice DNA and its level of amplification is not affected by the different phases of growth in culture.