Novel Oleanolic and Maslinic Acids derivatives as a promising treatment against bacterial biofilm in nosocomial infections: An in Vitro and in Vivo study.

Oleanolic acid (OA) and maslinic acid (MA) are pentacyclic triterpenic compounds that abound in industrial olive oil waste. These compounds have renowned antimicrobial properties and lack cytotoxicity in eukaryotic cells as well as resistance mechanisms in bacteria. Despite these advantages, their antimicrobial activity has only been tested in vitro, and derivatives improving this activity have not been reported. In this work, a set of 14 OA and MA C-28 amide derivatives have been synthesized. Two of these derivatives, MA-HDA and OA-HDA, increase the in vitro antimicrobial activity of the parent compounds while reducing their toxicity in most of the Gram-positive bacteria tested, including a methicillin-resistant Staphylococcus aureus-MRSA. MA-HDA also shows an enhanced in vivo efficacy in a Galleria mellonella invertebrate animal model of infection. A preliminary attempt to elucidate their mechanism of action revealed that these compounds are able to penetrate and damage the bacterial cell membrane. More significantly, their capacity to reduce antibiofilm formation in catheters has also been demonstrated in two sets of conditions: a static and a more challenged continuous-flow S. aureus biofilm.This study was partially supported by grants from the Ministerio de Economiá , Industria y Competitividad, MINECO, and Agencia Estatal de Investigación, AEI, Spain, cofunded by Fondo Europeo de Desarrollo Regional, FEDER, European Union (BIO2015−63557-R, FIS2017−85954-R and RTI2018−098573-B-100)

Novel Oleanolic and Maslinic Acid Derivatives as a Promising Treatment against Bacterial Biofilm in Nosocomial Infections: An in Vitro and in Vivo Study

Oleanolic acid (OA) and maslinic acid (MA) are pentacyclic triterpenic compounds that abound in industrial olive oil waste. These compounds have renowned antimicrobial properties and lack cytotoxicity in eukaryotic cells as well as resistance mechanisms in bacteria. Despite these advantages, their antimicrobial activity has only been tested in vitro, and derivatives improving this activity have not been reported. In this work, a set of 14 OA and MA C-28 amide derivatives have been synthesized. Two of these derivatives, MA-HDA and OA-HDA, increase the in vitro antimicrobial activity of the parent compounds while reducing their toxicity in most of the Gram-positive bacteria tested, including a methicillin-resistant Staphylococcus aureus-MRSA. MA-HDA also shows an enhanced in vivo efficacy in a Galleria mellonella invertebrate animal model of infection. A preliminary attempt to elucidate their mechanism of action revealed that these compounds are able to penetrate and damage the bacterial cell membrane. More significantly, their capacity to reduce antibiofilm formation in catheters has also been demonstrated in two sets of conditions: a static and a more challenged continuous-flow S. aureus biofilm

Synthesis, Optical Properties, and Antiproliferative Evaluation of NBD-Triterpene Fluorescent Probes

A fluorescent labeling protocol for hydroxylated natural compounds with promising antitumor properties has been used to synthesize 12 derivatives having fluorescent properties and biological activity. The reagent used for the synthesis of these fluorescent derivatives was 7-nitrobenzo-2-oxa-1,3-diazole chloride (NBD-Cl). The linkers employed to bind the NBD-Cl reagent to the natural compounds were ω-amino acids of different chain lengths. The natural triterpene compounds chosen were oleanolic and maslinic acid, as their corresponding 28-benzylated derivatives. Thus, triterpene conjugates with NBD have been studied for their optical fluorescence properties and their biological activities against cell proliferation in three cancer cell lines (B16-F10, HT-29, and HepG2), compared with three nontumor cell lines (HPF, IEC-18, and WRL68) from different tissues. The results of the fluorescence study have shown that the best fluorescent labels are those in which the ω-amino acid chain is shorter, and the carboxylic group is not benzylated. Analysis by confocal microscopy showed that these compounds were rapidly incorporated into cells in all three cancer cell lines, with these same derivatives showing the highest toxicity against the cancer cell lines tested. Then, the fluorescent labeling of these triterpene conjugates with NBD enabled their uptake and subcellular distribution to be followed to probe in detail their biological properties at the cellular and molecular level.Grupo de Investigación "Biotecnología y Química de Productos Naturales" (grupo FQM-139 del PAIDI de la Junta de Andalucía

Synthesis and Biological Activity of Triterpene-Coumarin Conjugates.

A set of 12 maslinic acid-coumarin conjugates was synthesized, with 9 being maslinic acid-diamine-coumarin conjugates at the C-28 carboxylic acid group of triterpene acid and the other three being maslinic acid-coumarin conjugates at C-2/C-3 and/or C-28 of the triterpene skeleton. The cytotoxic effects of these 12 triterpene conjugates were evaluated in three cancer cell lines (B16-F10, HT29, and Hep G2) and compared, respectively, with three nontumor cell lines from the same or similar tissue (HPF, IEC-18, and WRL68). The most potent cytotoxic results were achieved by a conjugate with two molecules of coumarin-3-carboxylic acid coupled through the C-2 and C-3 hydroxy groups of maslinic acid. This conjugate showed submicromolar IC50 values in two of the three cancer cell lines tested (0.6, 1.1, and 0.9 μM), being between 110- and 30-fold more effective than its corresponding precursor. Furthermore, this conjugate (10) showed percentages of cell viability for the three nontumor lines of 90%. Four maslinic acid-coumarin conjugates displayed apoptotic effects in the treated cells, with total apoptosis rates of between 40 and 85%, relative to the control. Almost all the compounds assayed caused cell-cycle arrest in all cancer cell lines, increasing the number of these cells in the G0/G1 phase

Funcionalización de derivados terpénicos (ácido oleanólico y ácido maslínico) con aplicaciones

El trabajo de investigación que se expone en esta Memoria se encuadra dentro del campo de los Productos Naturales en el área de Química Orgánica, por cuanto la materia prima utilizada procede de los desechos de molturación de la aceituna, con lo cual, además de evitar su contaminación del medio ambiente, se consigue un óptimo aprovechamiento y un valor añadido de los mismos. Los ácidos oleanólico (AO) y maslínico (AM), principales componentes de los residuos de molturación de la aceituna han sido aislados a partir de ellos mediante un procedimiento de extracción recogido en una patente titularizada por la Universidad de Granada. Además, los propios ácidos triterpénicos y algunos derivados de ellos, han manifestado numerosas y variadas actividades biológicas, ampliamente descritas en bibliografía como agentes anticancerígenos, antimicrobianos, antivirales, etc. En primer lugar, los esfuerzos del presente trabajo se centraron en el aislamiento, purificación y caracterización de los ácidos oleanólico y maslínico a partir de los mencionados residuos sólidos o líquidos de la industria oleica mediante procedimientos de extracción con disolventes de diferente polaridad y las distintas técnicas cromatográficas. La derivatización de productos naturales es un procedimiento ampliamente empleado en disciplinas como la Biología, la Farmacia, la Bioquímica, La Medicina, etc., para obtener compuestos con actividad biológica mejorada respecto a sus precursores, pero aprovechando el esqueleto carbonado de la sustancia natural. Una vez que se dispone de la materia prima adecuada en pureza y cantidad se han preparados diferentes derivados del esqueleto triterpénico que han sido estudiado por sus propiedades biológicas o posibles aplicaciones en otros ámbitos de la Ciencia y Tecnología Químicas. El primer objetivo estriba en la preparación de un conjunto de derivado de AO y AM acoplando 4 dialquilaminas de distinta longitud en el carboxilo de C-28 del esqueleto triterpénico mediante un enlace amida. Estos derivados aminados de AO y AM fueron testeados por sus actividades antimicrobianas resultando que dos de ellos presentan una actividad antimicrobiana importante en la mayoría de las bacterias Gram-positivas testeadas. • El segundo objetivo consiste en la semisíntesis de derivados de AO y AM que presentaran propiedades gelificantes por formación de geles de bajo peso molecular. Así prepararon un derivado de AO y otro de AM por tratamiento de estos productos naturales con una sal de uronio (TBTU) que presentan las propiedades gelificantes deseadas por captación lenta de agua desde sus disoluciones de DMSO y DMF. • El tercer objetivo de este trabajo es la preparación de sondas fluorescentes por derivatización de AO y AM con una etiqueta fluorescente que permita su seguimiento en tejidos biológicos mediante su propiedad de fluorescencia. Se obtiene así un conjunto de 15 derivados por una reacción entre un hidroxilo del anillo A de AO y AM con una etiqueta fluorescente como el NBD-Cl empleando como linker entre ambos un w-aminoácido de distinta longitud. Dichos compuestos son estudiados por sus propiedades ópticas y su actuación sobre tres líneas celulares cancerígenas con resultados prometedores. En cuarto y último lugar, el objetivo se logra al preparar derivados de los triterpenos naturales antes citas con cumarina a través de los dos puntos de funcionalización del esqueleto triterpénico, a saber, los hidroxilos del anillo A y el carboxilo de C-28. De nuevo se obtienen 12 derivados triterpeno-cumarina mediante enlaces de tipo éster o enlace tipo amida. Estos compuestos también son estudiados por su actuación sobre las mismas tres líneas celulares y se obtuvieron resultados en algunos de estos ensayos que se acercan a la escala nanomolar lo cual les atribuye potenciales utilizaciones como agentes anticancerígenos.Tesis Univ. Granada.Becas al Extranjero del Consejo Nacional de Ciencia y Tecnología (CONACYT)- Gobierno del Estado de Baja California México 2015.Proyectos de Excelencia de la Junta de Andalucía, convocatoria 2012: Geles supramoleculares para su empleo en ingeniería tisular, en medicina, en catálisis y en cristalización: FQM2012-2721Proyectos de Excelencia de la Junta de Andalucía, convocatoria 2013: Incremento de la Biodisponibilidad y la Actividad Biológica de Ácido Maslínico e Hidroxitirosol, dos Compuestos Procedentes de los Residuos de Molturación de la Aceituna, por Acilación y Pegilación mediante Técnicas de Síntesis Orgánica en Fase Sólida: FQM0737

Novel Oleanolic and Maslinic Acid Derivatives as a Promising Treatment against Bacterial Biofilm in Nosocomial Infections: An in Vitro and in Vivo Study

Oleanolic acid (OA) and maslinic acid (MA) are pentacyclic triterpenic compounds that abound in industrial olive oil waste. These compounds have renowned antimicrobial properties and lack cytotoxicity in eukaryotic cells as well as resistance mechanisms in bacteria. Despite these advantages, their antimicrobial activity has only been tested in vitro, and derivatives improving this activity have not been reported. In this work, a set of 14 OA and MA C-28 amide derivatives have been synthesized. Two of these derivatives, MA-HDA and OA-HDA, increase the in vitro antimicrobial activity of the parent compounds while reducing their toxicity in most of the Gram-positive bacteria tested, including a methicillin-resistant Staphylococcus aureus-MRSA. MA-HDA also shows an enhanced in vivo efficacy in a Galleria mellonella invertebrate animal model of infection. A preliminary attempt to elucidate their mechanism of action revealed that these compounds are able to penetrate and damage the bacterial cell membrane. More significantly, their capacity to reduce antibiofilm formation in catheters has also been demonstrated in two sets of conditions: a static and a more challenged continuous-flow S. aureus biofilm.This study was partially supported by grants from the Ministerio de Economiá , Industria y Competitividad, MINECO, and Agencia Estatal de Investigación, AEI, Spain, cofunded by Fondo Europeo de Desarrollo Regional, FEDER, European Union (BIO2015−63557-R, FIS2017−85954-R and RTI2018−098573-B-100)

Synthesis and Biological Activity of Triterpene-Coumarin Conjugates

A set of 12 maslinic acid−coumarin conjugates was synthesized, with 9 being maslinic acid−diamine−coumarin conjugates at the C-28 carboxylic acid group of triterpene acid and the other three being maslinic acid−coumarin conjugates at C- 2/C-3 and/or C-28 of the triterpene skeleton. The cytotoxic effects of these 12 triterpene conjugates were evaluated in three cancer cell lines (B16-F10, HT29, and Hep G2) and compared, respectively, with three nontumor cell lines from the same or similar tissue (HPF, IEC-18, and WRL68). The most potent cytotoxic results were achieved by a conjugate with two molecules of coumarin-3-carboxylic acid coupled through the C-2 and C-3 hydroxy groups of maslinic acid. This conjugate showed submicromolar IC50 values in two of the three cancer cell lines tested (0.6, 1.1, and 0.9 μM), being between 110- and 30-fold more effective than its corresponding precursor. Furthermore, this conjugate (10) showed percentages of cell viability for the three nontumor lines of 90%. Four maslinic acid−coumarin conjugates displayed apoptotic effects in the treated cells, with total apoptosis rates of between 40 and 85%, relative to the control. Almost all the compounds assayed caused cell-cycle arrest in all cancer cell lines, increasing the number of these cells in the G0/G1 phase.Junta de Andalucia B1-FQM-217-UGR18 B1-BIO-281-UGR1