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Esquamosan, a new furofuran lignan, has been isolated by bio-guided assays from the methanolic extract of the leaves of Annona squamosa L., and its structure was elucidated by spectroscopic methods. Esquamosan inhibited the rat aortic ring contraction evoked by phenylephrine in a concentration-dependent manner and showed an inhibitory effect on vasocontraction of the depolarized aorta with high-concentration potassium. The vasorelaxant effect by esquamosan could be attributed mainly to the inhibition of calcium influx from extracellular space through voltage-dependent calcium channels or receptor-operated Ca2+ channels and also partly mediated through the increased release of NO from endothelial cells. The ability of esquamosan to modify the vascular reactivity of rat aortic rings incubated with high glucose (D-glucose 55 mM) was then evaluated, and this furofuran lignan reverted the endothelium-dependent impairment effect of high glucose in rat aortic rings. The antioxidant capacity of esquamosan was assessed using DPPH and FRAP assays. Esquamosan exhibited a similar antioxidant capacity compared to ascorbic acid, which was used as a positive control. In conclusion, this lignan showed a vasorelaxant effect, free radical scavenging capacity, and potential reductive power, suggesting its potential beneficial use to treat complex cardiometabolic diseases due to free radical-mediated diseases and its calcium antagonist effect.

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Aim: The dose response in growth inhibition of Staphylococcus aureus treated with colloidal copper oxide nanoparticles (CuO-NP) was evaluated. Methods: An in vitro microbial viability assay was conducted with CuO-NP concentrations spreading over the 0.4-848.0 µg/ml range. The dose-response curve was modeled with a double Hill equation. UV-Visible absorption and photoluminescence spectroscopies allowed tracking concentration-dependent modifications in CuO-NP. Results: Two specific phases separated by the critical concentration of 26.5 µg/ml were observed in the dose-response curve, with each exhibiting proper IC50 parameters, Hill coefficients and relative amplitudes. Spectroscopy techniques reveal the occurrence of a concentration-triggered aggregation of CuO-NP starting from this critical concentration. Conclusion: The findings demonstrate a dose-related change in S. aureus sensitivity to CuO-NP, which probably arises from the aggregation of this agent.

Antibacterial agents are often used to stop the growth of bacteria such as Staphylococcus aureus (S. aureus). Copper oxide nanoparticles (CuO-NP) stand as a promising candidate for this purpose. Generally, the agent´s effectiveness is inspected following a dose-response curve in which de agent´s antibacterial response is plotted against its dose (concentration). In this work, employing an extended mathematical interpretation we were capable of discerning experimentally the existence of two stages of dose-response (biphasic dose-response) in the treatment of S. aureus with CuO-NP. These results in combination with insights from spectroscopic techniques lead to the understanding that the biphasic behavior arises from the aggregation of CuO-NP at high concentrations. Therefore, according to the adopted concentration to treat S. aureus, the agent can behave as a dispersed nanoparticle or as an aggregated nanoparticle. In summary, understanding whether antibacterial agents transform as a function of concentration is important in determining their practical applications.

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L-asparaginase is an important enzyme in the pharmaceutical field used as treatment for acute lymphoblastic leukemia due to its ability to hydrolyze L-asparagine, an essential amino acid synthesized by normal cells, but not by neoplastic cells. Adverse effects of L-asparaginase formulations are associated with its glutaminase activity and bacterial origin; therefore, it is important to find new sources of L-asparaginase produced by eukaryotic microorganisms with low glutaminase activity. This work aimed to identify the L-asparaginase gene sequence from Penicillium sizovae, a filamentous fungus isolated from the Brazilian Savanna (Cerrado) soil with low glutaminase activity, and to biosynthesize higher yields of this enzyme in the yeast Komagataella phaffii. The L-asparaginase gene sequence of P. sizovae was identified by homology to L-asparaginases from species of Penicillium of the section Citrina: P. citrinum and P. steckii. Partial L-asparaginase from P. sizovae, lacking the periplasmic signaling sequence, was cloned, and expressed intracellularly with highest enzymatic activity achieved by a MUT+ clone cultured in BMM expression medium; a value 5-fold greater than that obtained by native L-asparaginase in P. sizovae cells. To the best of our knowledge, this is the first literature report of the heterologous production of an L-asparaginase from a filamentous fungus by a yeast.

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The enzyme L-asparaginase (L-ASNase) is used in the treatment of Acute Lymphoblastic Leukemia. The preparations of this enzyme for clinical use are derived from bacterial sources and its use is associated with serious adverse reactions. In this context, it is important to find new sources of L-ASNase. In this work, the Placket-Burman Experimental Design (PBD) was used to determine the influence of the variables on the L-ASNase production then it was followed by a 28-4 Factorial Fractional Design (FFD). The results obtained from PBD have shown a range of L-ASNase activity, from 0.47 to 1.77 U/gcell and the results obtained from FFD have showed a range of L-ASNase activity, from 1.10 to 2.36 U/gcell. L-proline and ammonium sulfate were identified as of significant positive variables on this production enzyme by Penicillium cerradense sp. nov. The precise identification of this new species was confirmed by morphological characteristics and sequence comparisons of the nuclear 18S-5.8S-28S partial nrDNA including the ITS1 and ITS2 regions, RNA polymerase II, ß-tubulin and calmodulin genomic regions. The genetic sequence coding for the L-ASNase was obtained after carrying out a full genome sequencing. The L-ASNase expressed by P. cerradense sp. nov may have promising antineoplastic properties.

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Since 1961, L-asparaginase has been used to treat patients with acute lymphocytic leukemia. It rapidly depletes the plasma asparagine and deprives the blood cells of this circulating amino acid, essential for the metabolic cycles of cells. In the search for viable alternatives to produce L-asparaginase, this work aimed to produce this enzyme from Escherichia coli in a shaker and in a 3 L bioreactor. Three culture media were tested: defined, semi-defined and complex medium. L-asparaginase activity was quantified using the ß-hydroxamate aspartic acid method. The defined medium provided the highest L-asparaginase activity. In induction studies, two inducers, lactose and its analog IPTG, were compared. Lactose was chosen as an inducer for the experiments conducted in the bioreactor due to its natural source, lower cost and lower toxicity. Batch and fed-batch cultures were carried out to reach high cell density and then start the induction. Batch cultivation provided a final cell concentration of 11 g L-1 and fed-batch cultivation produced 69.90 g L-1 of cells, which produced a volumetric activity of 43,954.79 U L-1 after lactose induction. L-asparaginase was produced in a shaker and scaled up to a bioreactor, increasing 23-fold the cell concentration and thus, the enzyme productivity.

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L-asparaginase has been used in the remission of malignant neoplasms such as acute lymphoblastic leukemia. The search for new sources of this enzyme has become attractive for therapeutics. Traditional methods for biomolecule purification involve several steps. A two-phase system may be a good strategy to anticipate one of these stages. This study aimed to produce and purify a fungal L-asparaginase through an aqueous two-phase micellar system (ATPMS) using Triton X-114. The fungus Penicillium sp.-encoded 2DSST1 was isolated from Cerrado soil. Plackett-Burman design followed by a 24 full factorial design was used to determine the best conditions to produce L-asparaginase. The evaluated variables were L-asparagine, L-proline, wheat bran, potato dextrose broth, ammonium sulfate, yeast extract, sucrose and glucose concentrations, incubation temperature, incubation period, and initial pH of the culture medium. L-asparaginase quantification was valued by the formation of ß-aspartyl hydroxamate. The significant positive variables, L-asparagine, L-proline, potato dextrose broth, and sucrose concentrations, were evaluated at 2 levels (+ 1 and - 1) with triplicate of the central point. After 34 runs, maximum activity (2.33 IU/mL) was achieved at the factorial design central point. A central composite design was performed in ATPMS at two levels (+ 1 and - 1) varying Triton X-114 concentration (w/v), separation phase temperature, and crude extract concentration (w/v). The L-asparaginase partition coefficient (K) was considered the experimental design response. Out of the 16 systems that were examined, the most promising presented a purification factor of 1.4 and a yield of 100%.

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Eugenia dysenterica ex DC Mart. (Myrtaceae) is a Brazilian tree with pharmacological and biological properties. The aqueous leaf extract, rich in polyphenols, was tested in the human neuroblastoma cell line SH-SY5Y to evaluate its effect on cell viability. The extract and two isolated compounds were also assessed for the potential inhibitory activity on acetylcholinesterase, an enzyme related to Alzheimer's disease. A simple chromatographic method using Sephadex LH-20 was developed to separate catechin and quercetin from the aqueous leaf extract of E. dysenterica. Identification was carried out by spectroscopic techniques IR, UV, and 1H and 13C NMR. The IC50 values were obtained by constructing dose-response curves on a graph with percentage inhibition versus log of inhibitor concentration and compared with physostigmine, a well-known AChE inhibitor. The extract was toxic for SH-SY5Y cells at concentrations higher than 7.8 µg/ml given for 24 h. The decline in SH-SY5Y cell viability appears to be related to its antiproliferative activity. The extract also showed relatively moderate acetylcholinesterase inhibitory activity of 66.33% ± 0.52% at 1.0 mg/ml with an IC50 value of 155.20 ± 2.09 µg/ml. Physostigmine, quercetin, and catechin showed IC50 values of 18.69 ± 0.07, 46.59 ± 0.49, and 42.39 ± 0.67 µg/ml, respectively.

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The mouth and oropharynx cancer is the 6th most common type of cancer in the world. The treatment may involve surgery, chemotherapy and radiotherapy. More than 50% of drugs against cancer were isolated from natural sources, such as Catharanthus roseus and epipodophyllotoxin, isolated from Podophyllum. The biggest challenge is to maximize the control of the disease, while minimizing morbidity and toxicity to the surrounding normal tissues. The Erythroxylum suberosum is a common plant in the Brazilian Cerrado biome and is popularly known as "cabelo-de-negro". The objective of this study was to evaluate the cytotoxic activity of Erythroxylum suberosum plant extracts of the Brazilian Cerrado biome associated with radiotherapy in human cell lines of oral and hypopharynx carcinomas. Cells were treated with aqueous, ethanolic and hexanic extracts of Erythroxylum suberosum and irradiated at 4 Gy, 6 Gy and 8 Gy. Cytotoxicity was evaluated by MTT assay and the absorbance was measured at 570 nm in a Beckman Counter reader. Cisplatin, standard chemotherapy, was used as positive control. The use of Erythroxylum suberosum extracts showed a possible radiosensitizing effect in vitro for head and neck cancer. The cytotoxicity effect in the cell lines was not selective and it is very similar to the effect of standard chemotherapy. The aqueous extract of Erythroxylum suberosum, combined with radiotherapy was the most cytotoxic extract to oral and hypopharynx carcinomas.

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Plant-derived molecules showing antineoplastic effects have recently gained increased attention as potential adjuvants to traditional therapies for various cancers. Cerrado biome in Brazil contains high floral biodiversity, but knowledge about the potential therapeutic effects of compounds derived from that flora is still limited. The present study investigated the antineoplastic activity of Erythroxylum daphnites Mart., a Brazilian native plant from Cerrado biome, in the SCC-9 oral squamous cell carcinoma cell line. Cells were treated with various concentrations of hexane extract of Erythroxylum daphnites leaves (EDH) and assessed for cytotoxicity, proliferation, and apoptosis. Thin layer chromatography was conducted to characterize the substances present in EDH. Our results showed that EDH exerted anti-proliferative effects in SCC-9 cells by stabilizing the cell cycle at G1 phase in association with reduced intracellular levels of cyclins D and E and increased level of p21. EDH also demonstrated pro-apoptotic properties, as shown by an increased expression of caspase-3. Triterpenes were the major constituents of EDH. Our findings demonstrated a cytotoxic effect of EDH against SCC-9 cells in vitro mediated by the restraint of cellular proliferation and induction of apoptosis. Taken together, these findings support EDH constituents as potential therapeutic adjuvants for oral cancer.

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Abstract The mouth and oropharynx cancer is the 6th most common type of cancer in the world. The treatment may involve surgery, chemotherapy and radiotherapy. More than 50% of drugs against cancer were isolated from natural sources, such as Catharanthus roseus and epipodophyllotoxin, isolated from Podophyllum. The biggest challenge is to maximize the control of the disease, while minimizing morbidity and toxicity to the surrounding normal tissues. The Erythroxylum suberosum is a common plant in the Brazilian Cerrado biome and is popularly known as "cabelo-de-negro". The objective of this study was to evaluate the cytotoxic activity of Erythroxylum suberosum plant extracts of the Brazilian Cerrado biome associated with radiotherapy in human cell lines of oral and hypopharynx carcinomas. Cells were treated with aqueous, ethanolic and hexanic extracts of Erythroxylum suberosum and irradiated at 4 Gy, 6 Gy and 8 Gy. Cytotoxicity was evaluated by MTT assay and the absorbance was measured at 570 nm in a Beckman Counter reader. Cisplatin, standard chemotherapy, was used as positive control. The use of Erythroxylum suberosum extracts showed a possible radiosensitizing effect in vitro for head and neck cancer. The cytotoxicity effect in the cell lines was not selective and it is very similar to the effect of standard chemotherapy. The aqueous extract of Erythroxylum suberosum, combined with radiotherapy was the most cytotoxic extract to oral and hypopharynx carcinomas.

Resumo O câncer de boca e de orofaringe emerge como o 6º tipo de câncer mais comum no mundo. O tratamento pode envolver cirurgia, quimioterapia e radioterapia. Mais de 50% das drogas com atividade de combate ao câncer foram isoladas de fontes naturais, tais como a Catharanthus roseus e a epipodofilotoxina, isolada de Podophyllum. O maior desafio é maximizar o controle da doença, enquanto minimiza a morbidade e toxicidade para os tecidos normais circundantes. O Erythroxylum suberosum é uma planta comum no bioma Cerrado brasileiro e é popularmente conhecida como "cabelo-de-negro". O objetivo deste estudo foi avaliar a citotoxicidade dos extratos da planta Erythroxylum suberosum do bioma Cerrado brasileiro, associados à radioterapia em linhagens celulares humanas de carcinomas de língua e de hipofaringe. As células foram tratadas com os extratos aquoso, etanólico e hexânico do Erythroxylum suberosum e irradiadas com 4 Gy, 6 Gy e 8 Gy. A citotoxidade foi avaliada pelo ensaio de MTT e a absorvância foi medida a 570 nm em uma leitora Beckman. A cisplatina, quimioterápico padrão, foi utilizada como controle positivo. O uso de extratos de Erythroxylum suberosum mostrou potencial efeito radiosensibilizante in vitro no câncer de cabeça e pescoço. O efeito da citotoxicidade nas linhagens foi de forma não seletiva e muito semelhante ao efeito da quimioterapia padrão. O extrato aquoso de Erythroxylum suberosum, combinado com radioterapia, foi o extrato mais citotóxico para os carcinomas de língua e hipofaringe, associados à radioterapia.

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OBJECTIVES: Antineoplastic effects of molecules derived from plants have recently gained increasing attention as an additive to traditional therapies. The aim of this study was to evaluate the cytotoxic activity of plant extracts from the Brazilian Cerrado biome associated with radiotherapy in head and neck carcinoma cells (HNSCC). MATERIALS AND METHODS: Fifteen extracts derived from five Cerrado plants were tested in HNSCC cell lines (SCC-25, SCC-9, FaDu) and keratinocyte cells (HaCat). Cell cytotoxicity of extracts and association extract/radiation (2Gy/min) was assessed by MTT assay. Cisplatin (50 µg/mL) was used as a positive control. Extracts with the major cytotoxic activity were selected and their IC50 concentrations were defined. Apoptosis was assessed using flow cytometric analysis. RESULTS: Ten isolated extracts resulted in moderate cytotoxicity (>20 and ≤ 50 % of viable cells), while three extracts induced severe cytotoxic effects (≤ 20 % of viable cells). Plant extracts treatment improved radiotherapy cytotoxicity in all cell lines. Although plant extracts are not as potent as cisplatin plus radiation, in FaDu cells, seven extracts associated with irradiation showed cytotoxic activity similar or better than the association of cisplatin and radiation. Hexanic extract of Erythroxylum daphinites could induce apoptosis in oral cancer cells; however, necrosis was the prevalent kind of death in FaDu cells treated with hexanic extract of Erythroxylum suberosum. CONCLUSIONS: Pre-treatment of HNSCC cells with the extract derived from Cerrado plants followed by irradiation induced a supra-additive cytotoxic effect. CLINICAL RELEVANCE: This study highlights the potential biological relevance of the Cerrado biome when associated with traditional therapy for cancer.

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The partitioning of protease expressed by Penicillium fellutanum from the Brazilian savanna in a novel inexpensive and stable aqueous two-phase system (ATPS) composed of poly(ethylene glycol) (PEG) and sodium polyacrylate (NaPA) was studied in this work using factorial design. The ATPS is formed by mixing both polymers with a salt (NaCl) and fermented broth of P. fellutanum. The effects of molar mass (2,000, 4,000, and 6,000 g ⋅ mol(-1)) and concentration (6, 8, and 10 wt%) of PEG and that of NaPA concentration (6, 8, and 10 wt%) on protease partitioning (K) at 25 °C were studied. A two-level factorial design (2(3)) was implemented. The effect of Na2 SO4 concentration (5, 10, and 15 wt%) on the reextraction of the enzyme was also analyzed. The partition coefficient K ranged from 77.51 to 1.21, indicating the versatility of the method. The reextraction was achieved with the addition of 5% Na2 SO4 , allowing the partitioning of the protease to the upper phase, whereas total proteins were directed to the bottom phase. The results of partitioning using the PEG/NaPA/NaCl system and that of the subsequent reextraction with Na2 SO4 suggest that this method can be used to purify proteases from fermented broth of P. fellutanum.

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In biotechnology, endotoxin (LPS) removal from recombinant proteins is a critical and challenging step in the preparation of injectable therapeutics, as endotoxin is a natural component of bacterial expression systems widely used to manufacture therapeutic proteins. The viability of large-scale industrial production of recombinant biomolecules of pharmaceutical interest significantly depends on the separation and purification techniques used. The aim of this work was to evaluate the use of aqueous two-phase micellar system (ATPMS) for endotoxin removal from preparations containing recombinant proteins of pharmaceutical interest, such as green fluorescent protein (GFPuv). Partition assays were carried out initially using pure LPS, and afterwards in the presence of E. coli cell lysate. The ATPMS technology proved to be effective in GFPuv recovery, preferentially into the micelle-poor phase (K(GFPuv) < 1.00), and LPS removal into the micelle-rich phase (%REM(LPS) > 98.00%). Therefore, this system can be exploited as the first step for purification in biotechnology processes for removal of higher LPS concentrations.

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