Efeito do extrato do parênquima clorofiliano proveniente da Aloe barbadensis Miller em células animais

Dissertação (mestrado) - Universidade Federal de Santa Catarina, Centro Tecnológico, Programa de Pós-Graduação em Engenharia Química, Florianópolis, 2016.Diversas culturas se baseiam na utilização de plantas para o tratamento de doenças. Plantas do gênero Aloe vem sendo utilizadas a séculos na medicina tradicional para cicatrização de feridas, como agente antibacteriano, como laxante e até mesmo para o tratamento do câncer. O extrato do parênquima clorofiliano (EPC) da Aloe barbadensis Miller é excretado quando a folha é cortada ou quebrada, este líquido de cor amarelada é rico em antronas, cromonas e seus derivados. Estudos com compostos isolados do EPC, aloína e Aloe emodina, mostram uma promissora atividade antitumoral dos mesmos. A incidência do melanoma cutâneo vem aumentando nos últimos anos, o que gera uma grande preocupação, uma vez que esse câncer é altamente agressivo apresentando alta possibilidade de metástase. Neste trabalho o EPC foi padronizado por HPLC, apresentando 5,6% em massa seca do isômero aloína B. A citotoxicidade foi avaliada do EPC em linhagens de fibroblastos L-929, assim como o efeito do EPC sobre a viabilidade, proliferação, morfologia e migração de duas linhagens de melanoma (SK-MEL-28 e B16-F10). De acordo com a ISO 10993-5, concentrações superiores a 185 µg·mL-1 de EPC são citotóxicas (decréscimo na viabilidade maior que 30%), sendo esse valor maior que o valor de IC50 de ambas as linhagens de melanoma, 125 e 165 µg·mL-1 para a SK-MEL-28 e B16-F10, respectivamente. Análise morfológica mostrou ponto de alta fluorescência, indicando uma reorganização dos filamentos de actina presentes no citoesqueleto. O resultado do ensaio de migração para ambas linhagens de melanoma mostrou que o EPC é mais eficaz em inibir a migração do melanoma humano SK-MEL-28. Células endoteliais (HUVEC) foram expostas ao EPC e os parâmetros de viabilidade celular, proliferação, morfologia, migração e formação de tubos foram avaliados. O EPC inibiu a migração de HUVECs mesmo em concentrações que não apresentaram efeito de decréscimo na atividade metabólica e proliferação celular. Pôde-se observar a retração das fibras de estresse em células tratadas com as concentrações de 10 e 100 µg·mL-1 de EPC. A formação de tubos em células endoteliais foi inibida na maior concentração testada (100 µg·mL-1), contudo ainda se observa a formação de brotos vasculares. Em suma o EPC proveniente de Aloe barbadensis Miller causou um decréscimo na viabilidade e proliferação celular assim como uma diminuição da capacidade de migração e alterações morfológicas de células de melanoma e endoteliais, influenciando também na formação de tubos vasculares por células endoteliais.Abstract : Several cultures are based on the use of plants for the treatment of diseases. Plants of the genus Aloe has been used for centuries in traditional medicine, in wound healing, such as antibacterial agent, laxative and even for the treatment of cancer. The chlorophyll parenchyma extract (EPC) from Aloe barbadensis Miller is excreted when the sheet is cut or broken, this yellowish liquid is rich in anthrones, chromones and their derivatives. Studies on compounds isolated EPC, aloin and Aloe emodin, showed promising antitumor activity. The incidence of melanoma has increased in recent years, creating a great concern, since this cancer is highly aggressive and presents high possibility of metastasis. In this work the EPC was standardized by HPLC, showing 5.6% of dry weight of aloin B isomer. The EPC cytotoxicity was evaluated on fibroblast L-929 cell line, as well as the effect of EPC in cell viability, proliferation, morphology alterations and migration on two melanoma cell lines (SK-MEL-28 and B16-F10). According to ISO 10993-5, concentrations higher than 185 µg·mL-1 EPC are cytotoxic (decrease in viability was greater than 30%), this value being higher than the IC50 value of both melanoma cell lines, 125 and 165 µg·mL- 1 to SK-MEL-28 and B16-F10, respectively. Morphological analysis showed high fluorescence spots, indicating a reorganization of actin filaments present in the cytoskeleton. The result of the migration test for both melanoma cell lines showed that the EPC is more effective to inhibit the migration of human melanoma SK-MEL-28. Endothelial cells (HUVEC) were exposed to the EPC and parameters of cell viability, proliferation, morphology alterations, migration and tube formation was evaluated. The EPC inhibited HUVEC migration even at concentrations that has not shown effect in decrease of metabolic activity and cellular proliferation. It was observed the decrease of stress fibers in cells after treated with concentrations of 10 and 100 µg·mL-1of EPC. The tube formation on endothelial cells was inhibited at the highest concentration tested (100 µg·mL-1), however it was still possible to observe the formation of vascular sprouts. In short, the EPC from Aloe barbadensis Miller caused a decrease in cell viability and proliferation as well as reduced migration ability and morphological changes on melanoma and endothelial cells, also influencing the vascular tube formation by endothelial cells

Optimization of low-cost nutritional supplementation for lignocellulosic ethanol fermentation

[Excerpt] Bioethanol from lignocellulosic materials (LCM), also called second generation bioethanol, is considered a promising alternative to first generation bioethanol. An efficient production process of lignocellulosic bioethanol involves an effective pretreatment of LCM to improve the accessibility of cellulose and thus enhance the enzymatic saccharification. One interesting approach is to use the whole slurry from treatment, since allows economical and industrial benefits: washing steps are avoided, water consumption is lower and the sugars from liquid phase can be used, increasing ethanol concentration [1]. However, during the pretreatment step some compounds (such as furans, phenolic compounds and weak acids) are produced. These compounds have an inhibitory effect on the microorganisms used for hydrolysate fermentation [2]. To overcome this, the use of a robust industrial strain together with agro-industrial by-products as nutritional supplementation was proposed to increase the ethanol productivities and yields. (...

Simultaneous saccharification and fermentation of hydrothermal pretreated lignocellulosic biomass: evaluation of process performance under multiple stress conditions

Industrial lignocellulosic bioethanol processes are exposed to different environmental stresses (such as inhibitor compounds, high temperature, and high solid loadings). In this study, a systematic approach was followed where the liquid and solid fractions were mixed to evaluate the influence of varied solid loadings, and different percentages of liquor were used as liquid fraction to determine inhibitor effect. Ethanol production by simultaneous saccharification and fermentation (SSF) of hydrothermally pretreated Eucalyptus globulus wood (EGW) was studied under combined diverse stress operating conditions (3038 °C, 6080 g of liquor from hydrothermal treatment or autohydrolysis (containing inhibitor compounds)/100 g of liquid and liquid to solid ratio between 4 and 6.4 g liquid in SSF/g unwashed pretreated EGW) using an industrial Saccharomyces cerevisiae strain supplemented with low-cost byproducts derived from agro-food industry. Evaluation of these variables revealed that the combination of temperature and higher solid loadings was the most significant variable affecting final ethanol concentration and cellulose to ethanol conversion, whereas solid and autohydrolysis liquor loadings had the most significant impact on ethanol productivity. After optimization, an ethanol concentration of 54 g/L (corresponding to 85 % of conversion and 0.51 g/Lh of productivity at 96 h) was obtained at 37 °C using 60 % of autohydrolysis liquor and 16 % solid loading (liquid to solid ratio of 6.4 g/g). The selection of a suitable strain along with nutritional supplementation enabled to produce noticeable ethanol titers in quite restrictive SSF operating conditions, which can reduce operating cost and boost the economic feasibility of lignocellulose-to-ethanol processes.The authors thank the financial support from the Strategic Project of UID/BIO/04469/2013 CEB Unit and A Romaní postdoctoral grant funded by Xunta of Galicia (Plan I2C, 2014)

Antineoplastic drugs: effect of doxorubicin on enriched archaea culture from anaerobic digestion and potential degradation via an enzymatic process

Tese (doutorado) - Universidade Federal de Santa Catarina, Centro Tecnológico, Programa de Pós-Graduação em Engenharia Química, Florianópolis, 2022.Fármacos antineoplásicos vem sendo detectados em efluentes e águas superficiais. Devido a sua toxicidade, estes fármacos apresentam grande risco ao meio ambiente, mesmo em baixas concentrações. Sabe-se que estes não são removidos completamente pelo sistema convencional de tratamento de efluentes. Deste modo, o desenvolvimento de novas estratégias para a sua remoção são cruciais. Enzimas como a lacase apresentam grande potencial para degradar diversos poluentes, devido a não especificidade pelo substrato. Além disso, os produtos de degradação gerados por uma degradação enzimática apresentam menor toxicidade em comparação com as moléculas originais. Também não se sabe ao certo como estes fármacos influenciam a comunidade microbiana presente em uma estação de tratamento de efluentes. Alguns grupos de microrganismos representam papel chave no tratamento de efluentes. Por exemplo, as arqueas metanogênicas (que compõem a última etapa da digestão anaeróbica), se estas forem inibidas o processo com um todo será comprometido. Neste sentido, este trabalho tem como objetivo estudar o potencial efeito inibitório do fármaco antineoplásico doxorubicina em arqueas metanogênicas, assim como avaliar o potencial de aplicação da lacase na degradação destes fármacos. Para isso, o efeito inibitório da doxorrubicina sobre a produção de biogás foi avaliado em ensaios de exposição em batelada e em longo prazo. Nos ensaios em batelada, o valor de IC50 de 648 ± 50 µg?L-1 foi obtido para a doxorrubicina. Além disso, verificou-se que a inibição causada pela exposição a 10 × 103 µg?L-1 foi reversível após subsequentes bateladas sem a presença do fármaco no meio e alimentação. A doxorrubicina foi rapidamente adsorvida pela biomassa (apesar do baixo KOW), o que pode ter contribuído para o efeito inibitório sobre os microrganismos. Os resultados dos ensaios de exposição de longo prazo mostraram que quando a quantidade do fármaco foi aumentada de 100 µg?L-1?dia-1 para 200 µg?L-1?dia-1, a produção de biogás e a remoção da carga orgânica diminuíram drasticamente. No entanto, as archaeas metanogênicas foram capazes de se adaptar à condição inibitória, corroborando os resultados encontrados nos ensaios em bateladas sequenciais. Em suma, a doxorrubicina pode desempenhar um papel fundamental na inibição de processos de tratamento biológico de efluentes, se sua concentração em estações de tratamento aumentarem abruptamente. Os ensaios de exposição à doxorrubicina mostraram que esta pode inibir a velocidade de produção de biogás. Desta forma, decidiu-se estudar a degradação desse fármaco por via enzimática. A lacase proveniente de Trametes versicolor foi utilizada para realização dos estudos cinéticos de degradação. A citotoxicidade do fármaco degradado pela lacase foi avaliada e comparada com a doxorrubicina in natura. As melhores condições de degradação foram em pH 7 e em 30ºC, o que se assemelha a parâmetros operacionais de estações de tratamento de efluentes. Os parâmetros cinéticos de Michaelis?Menten foram v_max de 769,2 µg?h-1?L-1 e KM de 4,60 µM indicando uma boa afinidade pelo substrato. Já os ensaios de citotoxicidade demostraram que a lacase reduziu a toxicidade da doxorrubicina, após degradação, apresentando potencial para aplicação na degradação de fármacos antineoplásicos presentes em efluentes, o que abre novos horizontes para estudos sobre o tema.Abstract: Antineoplastic drugs have been detected in effluents and surface waters. Due to their toxicity, these drugs present a high risk to the environment, even at low concentrations. In addition, it is known that the conventional wastewater treatment system does not completely remove these drugs. Thus, it is crucial the development of new strategies for their removal. Enzymes such as laccase have a great potential to degrade several pollutants due to their non-specificity for the substrate. Besides, the degradation products generated by an enzymatic degradation show less toxicity compared with the original molecule. Furthermore, it is unknown how these drugs influence the microbial community from a wastewater treatment plant. Some groups of microorganisms play a key role in effluent treatment. For example, the whole process will be compromised if microorganisms such as methanogenic archaea are inhibited (which constitutes the last step of anaerobic digestion). In this sense, the main objective of this thesis is to study the inhibitory effect of the antineoplastic drugs doxorubicin on methanogenic archaeas and evaluate the potential application of enzymes (more specifically laccase) in the degradation of these drugs. For this, the inhibitory effect of doxorubicin on biogas production was evaluated in batch and long-term exposure assays. An IC50 value of 648 ± 50 µg?L-1 was obtained for doxorubicin in batch assays. Besides, it was found that the inhibition caused by exposure to 10×103 µg?L-1 was reversible after subsequent batches without the drug in the synthetic medium. Doxorubicin was rapidly adsorbed by biomass (despite the low KOW value), which may have contributed to the inhibitory effect on microorganisms. The long-term exposure assays showed that when the amount of drug was increased from 100 µg?L-1?day-1 to 200µg?L-1?day-1, the biogas production and COD removal drastically decreased. However, the methanogenic archaeas were able to adapt to the inhibitory condition, corroborating the results found in the sequential batch tests. In summary, doxorubicin can play a key role in inhibiting biological processes if its concentration in wastewater treatment plants increases abruptly. Doxorubicin exposure trials have shown that it can inhibit the biogas production rate. Therefore, it was decided to study the enzymatic degradation of this drug. The laccase from Trametes versicolor was used to carry out the kinetic degradation studies. The cytotoxicity of the degraded doxorubicin (by laccase) was evaluated and compared to doxorubicin. The best degradation conditions were at pH 7 and 30ºC, which resembles effluent characteristics from wastewater treatment plants. The Michaelis?Menten kinetic parameters were Vmax of 769.2 µg?h-1?L-1 and KM of 4.60 µM, which showed a good affinity for the substrate. Cytotoxicity showed that laccase reduces the toxicity of doxorubicin after degradation. Laccase presents potential for application for degradation of effluents that contain antineoplastic drugs opens new directions for studies on the subject

Lignocellulosic bioethanol production with revalorization of low-cost agroindustrial by-products as nutritional supplements

Accepted 27 October 2014. "In press"During the pretreatment of lignocellulosic biomass for second generation bioethanol production, fermentation inhibitors are released. To overcome this, the use of a robust industrial strain together with agro-industrial by-products as nutritional supplementation was proposed to increase ethanol productivity and yields. Two factorial experimental designs were carried out to optimize fermentation of hydrolysate from autohydrolysis of Eucalyptus globulus. The mostinfluential variables on ethanol production were cheese whey and K2O5S2 (potassium metabisulfite) supplementation. Nutrient addition effect was demonstrated using the whole slurry from autohydrolysis in two process configurations (separate hydrolysis and fermentation, SHF and simultaneous saccharification and fermentation, SSF). Comparing the supplemented SHF and SSF assays with non-supplemented, 2.3 and 7.4 fold higher ethanol concentrations were obtained, respectively. In the case of SSF, 50.4 g L−1 of ethanol concentration and 92.2% of ethanol conversion were attained, demonstrating an improved fermentation performance in industrial lignocellulose fermentations.The authors, A. Romani and F.B. Pereira, thank Portuguese Foundation for Science and Technology (FCT, Portugal) for their fellowships (grant numbers, SFRH/BPD/77995/2011 and SFRH/BD/64776/2009, respectively). Research described in this article was financially supported by FEDER and "Fundacao para a Ciencia e a Tecnologia" (FCT): Strategic Project PEstOE/EQB/LA0023/2013, Project "BioInd - Biotechnology and Bioengineering for improved Industrial and Agro-Food processes, REF. NORTE-07-0124-FEDER-000028" Co-funded by the Programa Operacional Regional do Norte (ON.2-O Novo Norte) QREN, FEDER

Evaluation of the addition of glycerol to Cupriavidus necator culture medium over Poly(3-hydroxybutyrate) production

<div><p>ABSTRACT Glycerol was used as a source of additional carbon in the production of Poly(3-hydroxybutyrate) (P(3HB)). The inverted sugar and glycerol concentrations and the temperature of the Cupriavidus necator culture medium were evaluated using a Central Composite Rotational Design (CCRD). The results showed that the increase in temperature and sugar concentration led to an increase in production and P(3HB) accumulation and when 15 g L-1 of glycerol was added better results were obtained, however these were not considered statistically significant. The best results were obtained at 38 °C and with 30 g L-1 of inverted sugar. Although not considered statistically significant, the addition of 15 g L-1 of glycerol increased the P(3HB) accumulation percentage by 15 %, thus in kinetic terms, greater productivity was obtained in 0.32 g L-1 h-1 polymer. Larger scale assays are being conducted to verify if the addition of glycerol improves the thermal and mechanical properties of the synthesized polymer.</p></div