Organ transplantations have an increasing medical relevance. It is becoming a regular procedure with an increase in individuals waiting for organs. The increase in the number of discarded organs is mostly due to the donor bacterial and/or viral infection. In this article, we are demonstrating the feasibility of reduction of the bacterial load in kidney model by using ultraviolet-C as a germicidal agent in circulating liquids. Using Staphylococcus aureus as a bacteria model, we were able to demonstrate that in less than 30 min of liquid circulation and associated to irradiation, the bacterial load of the perfusate Custodiol HTK, histidine-tryptophan-ketoglutarate (solution with 5 log CFU mL À1 ), was fully eliminated. A modeling approach was created to verify the possibility of bacterial load decrease, when an organ (here, a renal experimental model) is present in the circuit, releasing a varied rate of microorganisms over time, while the solution is irradiated. Finally, we use an ex vivo model with swine kidney, circulating in the preservation solution with a Lifeport Kidney Transporter machine, to demonstrate that we can contaminate the organ and then promote the elimination of the microbiological load. The results show the feasibility of the technique.
Background:Excoecaria lucida Sw. (Euphorbiaceae) is a plant conventionally used throughout the Caribbean in the treatment of infectious diseases.Objective:To evaluate, using bioassay-guided fractionation, the in vitro cytotoxicity and antimicrobial activity of E. lucida leaves.Materials and Methods:A 95% ethanol crude extract was dried and fractionated by solid-liquid separation in four phases (hexane, dichloromethane, ethyl acetate, and butanol). Antimicrobial activity (3 bacteria, 6 yeasts, and 2 fungi) was evaluated by the dilution method with resazurin (2048, 512, 128, 32, and 8 μg/mL). The cytotoxicity assays were evaluated in two cell lines: MRC-5 and RAW 264.7; calculating the selectivity index. Assays were performed for the total extract, the isolated compound with the highest yield, and the ethyl acetate and butanol phases. Isolated compounds were characterized by nuclear magnetic resonance and mass spectrometry techniques.Results:Fractionation process led to the isolation of ellagic acid (784.29 mg), 3,3',4'-tri-O-methyl ellagic 4-O-β-D-glucopyranoside acid (6.1 mg), and corilagin (6.91 mg). The most active were ethyl acetate phase and ellagic acid with IC50= 128 μg/mL against seven and five different species of microorganisms, respectively. The total extract (IC50=512 μg/mL) and the ethyl acetate phase (IC50=128 μg/mL) were cytotoxic in both cell lines, while butanol phase and ellagic acid both with IC50>2048 μg/mL seemed to be safer.Conclusions:The results obtained indicate that the Excoecaria leaves can be conventionally used as antimicrobial, but it should be present that some cytotoxicity could appear. In addition, the three identified compounds were reported for the first time in the species.SUMMARYExcoecaria lucida leaves (Euphorbiaceae) are used by the Cuban population due to their antimicrobial activity. This ethnopharmacological knowledge is confirmed by the integrated antibacterial and antifungal in vitro screening developed, using the bioassay-guided fractionation method.Abbreviations Used: MRC-5-SV2: Diploid human lung fibroblasts cells, RAW 264.7: Murine macrophages cells, IC50: Inhibitory Concentration 50%, ATCC: American Type Culture Collection, CCEBI: Culture Collection of Industrial Biotechnology Center, CECT: Spanish Culture Collection Type, CFU: Colony forming units, CC50: 50% cytotoxic concentration, CO2: Carbon dioxide, SI: Selectivity index, IR: Infrared spectroscopy, 1H NMR: Nuclear Magnetic Resonance of hydrogen, 13C NMR: Nuclear Magnetic Resonance of carbon, HMQC: Heteronuclear Multiple-Quantum Correlation, HMBC: Heteronuclear Multiple Bond Correlation, COSY: Correlation Spectroscopy, NOESY: Nuclear Overhauser Effect Spectroscopy, KBr: Potassium bromide, DMSO-D6: Deuterated dimethyl sulfoxide, LC.MS: Liquid Chromatography-Mass Spectrometry, [α]D: Optical rotation, EL1: ellagic acid, EL2: 3,3’,4’-tri-O-methyl ellagic 4-O-β-D-glucopyranoside acid, EL3: corilagin, Active (+), inactive (-).
Agradezco a Dios todo poderoso, en su infinita bondad, por mi vida, por guiarme e iluminar mi camino, por darme fuerzas para seguir adelante cuando creía que todo estaba perdido.A toda mi familia, en especial a mis padres Mirna y Riquildo y hermana Yoandra, por su apoyo incondicional, dedicación y amor. A mi princesita Tábatha por brindarme tanto amor y ser el motor impulsor que me da fuerzas para seguir adelante y sobrepasar todos los obstáculos.A mi esposo Luis Carlos por su apoyo incondicional, amor y comprensión. Por ser mi compañero y amigo, por ayudarme a levantar cuando estaba perdiendo las fuerzas para continuar adelante con mis proyectos.A mis amigos Loraine, Erika, Camilinha, Johan y mi madrina Eunice, los cuales forman parte de mi familia; que siempre han estado conmigo en los buenos y malos momentos, dándome su apoyo incondicional.A Jose Dirceu, por sus consejos y ayuda durante todo el doctorado, mostrándome que siempre es posible, que después de la tormenta, llega la calma.A la profesora Cristina Kurachi por ser mi orientadora y por darme la oportunidad de formar parte del grupo de pesquisa; por el soporte científico, análisis y sugerencias que fueron de extrema importancia para el desenvolvimiento de la pesquisa y por ser, además de orientadora, amiga.Al profesor Vanderlei y Natalia por su ayuda, contribución científica e incentivo a mi crecimiento profesional.A los integrantes del grupo de descontaminación de órganos: José Dirceu, Caroline, Loraine y de forma general a todos los colegas del grupo de Biofotónica que de una forma u otra contribuyeron al desenvolvimiento de la pesquisa.
Organ transplantations have an increasing medical relevance. It is becoming a regular procedure with an increase in individuals waiting for organs. The increase in the number of discarded organs is mostly due to the donor's bacterial and/or viral infection. In this article, we are demonstrating the feasibility of reduction of the bacterial load in the kidney model by using Ultraviolet-C (UV-C) as a germicidal agent in circulating liquids. Using Staphylococcus aureus as a bacteria model, we were able to demonstrate that in less than 30 min of liquid circulation and associated to irradiation, the bacterial load of the perfusate Custodiol ® HTK, histidine-tryptophan-ketoglutarate (solution with 5 log CFU ml À1 ), was fully eliminated. A modeling approach was created to verify the possibility of bacterial load decrease, when an organ (here, a renal experimental model) is present in the circuit, releasing a varied rate of microorganisms over time, while the solution is irradiated. Finally, we use an ex vivo model with a swine kidney, circulating in the preservation solution with a Lifeport ® Kidney Transporter machine, to demonstrate that we can contaminate the organ and then promote the elimination of the microbiological load. The results show the feasibility of the technique.Abbreviations: ABTO, associação brasileira de transplante de orgãos; BHI, brain heart infusion; CFU, colony-forming units; CKD, chronic kidney disease; EVLP, ex vivo lung perfusion; FBS, fetal bovine serum; HTK, histidine-tryptophan-ketoglutarate; LKT, lifeport kidney transporter; PBS, phosphate buffer saline; UNOS, united network for organ sharing; UV-C, ultraviolet-c. This article corrects JBIO.202100319.
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