Extraction of metabolites from Calendula officinalis and evaluation of their colorant and antibacterial capacity / Extracción de metabolitos a partir de Calendula officinalis y evaluación de su capacidad colorante y antibacterial

ABSTRACT In this work, different bioactive compounds were obtained by different extractions from Calendula officinalis. The effect of different extraction time and temperature on the quantity of bioactive compounds was investigated. The extracts were quantified by UV-visible spectrometric analysis. The effect of extraction technique on both the colorant power and antibacterial capacity of metabolites obtained was evaluated. Colorant power was evaluated by UV-visible spectrometric and dyeing ability in dairy product. Antibacterial activity of extracts was evaluated using Escherichia coli and Salmonella typhimurium by diffusion technique in well. Outstanding extraction results were obtained by ultrasound extraction technique performed during 2 hours, at 30 °C and ethanol 96% as solvent. The same treatment showed the best result in colorant power and dyeing ability. The extracts obtained by ultrasound had a slightly inhibitory effect of growth on E. coli in comparison with the other techniques. None of the extracts obtained from the different techniques reported a significant inhibition on the growth of S. typhimurium. Therefore, ultrasound-assisted extraction is considered as an alternative process for obtaining the bioactive compounds from C. officinalis with high concentration, colorant power and antibacterial activity.

RESUMEN En este trabajo, diferentes compuestos bioactivos fueron obtenidos a partir de extracciones de las flores de Caléndula officinalis. Se investigó el efecto que tiene el tiempo de extracción y la temperatura sobre la cantidad de compuestos bioactivos. Los extractos obtenidos fueron cuantificados mediante espectrofotometría UV-visible. Se evaluó el efecto de la técnica de extracción sobre el poder colorante y la capacidad antibacterial de los metabolitos obtenidos. El poder colorante se evaluó mediante espectrofotometría UV-visible y la capacidad de teñido se evaluó en un producto lácteo. La actividad antibacterial de los extractos fue evaluada usando Escherichia coli y Salmonella typhimurium mediante la técnica de difusión en pocillos. Se obtuvieron buenas cantidades de metabolitos evaluados mediante la técnica por ultrasonido realizada durante 2 horas, a 30 °C, con etanol a una concentración de 96%. El mismo tratamiento mostro los mejores resultados en cuanto al poder colorante y la capacidad de teñido en una matriz láctea. Los extractos obtenidos por ultrasonido tuvieron un ligero efecto inhibitorio del crecimiento sobre E. coli en comparación con las otras técnicas. Ninguno de los extractos obtenidos mediante las diferentes técnicas mostró una inhibición significativa en el crecimiento de S. typhimurium. Por lo tanto, la extracción asistida por ultrasonido se considera como un proceso alternativo para obtener los compuestos bioactivos de C. officinalis con alta concentración, poder colorante y actividad antibacteriana.

Different Organization of Type I Collagen Immobilized on Silanized and Nonsilanized Titanium Surfaces Affects Fibroblast Adhesion and Fibronectin Secretion.

Silanization has emerged in recent years as a way to obtain a stronger and more stable attachment of biomolecules to metallic substrates. However, its impact on protein conformation, a key aspect that influences cell response, has hardly been studied. In this work, we analyzed by atomic force microscopy (AFM) the distribution and conformation of type I collagen on plasma-treated surfaces before and after silanization. Subsequently, we investigated the effect of the different collagen conformations on fibroblasts adhesion and fibronectin secretion by immunofluorescence analyses. Two different organosilanes were used on plasma-treated titanium surfaces, either 3-chloropropyl-triethoxy-silane (CPTES) or 3-glycidyloxypropyl-triethoxy-silane (GPTES). The properties and amount of the adsorbed collagen were assessed by contact angle, X-ray photoelectron spectroscopy, optical waveguide lightmode spectroscopy, and AFM. AFM studies revealed different conformations of type I collagen depending on the silane employed. Collagen was organized in fibrillar networks over very hydrophilic (plasma treated titanium) or hydrophobic (silanized with CPTES) surfaces, the latter forming little globules with a beads-on-a-string appearance, whereas over surfaces presenting an intermediate hydrophobic character (silanized with GPTES), collagen was organized into clusters with a size increasing at higher protein concentration in solution. Cell response was strongly affected by collagen conformation, especially at low collagen density. The samples exhibiting collagen organized in globular clusters (GPTES-functionalized samples) favored a faster and better fibroblast adhesion as well as better cell spreading, focal adhesions formation, and more pronounced fibronectin fibrillogenesis. In contrast, when a certain protein concentration was reached at the material surface, the effect of collagen conformation was masked, and similar fibroblast response was observed in all samples.

Collagen-functionalised titanium surfaces for biological sealing of dental implants: effect of immobilisation process on fibroblasts response.

The clinical success of a dental implant requires not only an optimum osseointegration, but also the development of a biological sealing; i.e., a soft tissue seal around the transmucosal part of the implant. A promising approach to improve the biological seal of dental implants is the biomimetic modification of titanium surfaces with proteins or peptides that have specific cell-binding moieties. In this work we investigated the process of immobilising collagen on smooth and rough titanium surfaces and its effect on human dermal fibroblast (HDF) cell response. Titanium samples were activated by either oxygen plasma or acid etching to generate a smooth or nanorough surface, respectively. Subsequently, collagen grafting was achieved by either physisorption or covalent bonding through organosilane chemistry. The biofunctionalised titanium samples were then tested for stability and characterised by fluorescent labelling, wettability, OWLS and XPS studies. Biological characterisation was also performed through HDF adhesion, proliferation and gene expression. Covalent-bonded collagen showed higher stability than physisorbed collagen. A significant overexpression of the genes involved in fibroblast activation and extracellular matrix remodelling was observed in the collagen-coated surfaces. This effect was more pronounced on smooth than on rough surfaces. Immobilised collagen on the smooth plasma-treated surfaces favoured both fibroblast adhesion and activation. This study provides essential information for the design of implants with optimal biological sealing, a key aspect to avoid peri-implantitis and ensure long-lasting implant fixation.