Desarrollo preclínico de una formulación farmacéutica cicatrizante para uso tópico a base de extractos vegetales.
Loading...
Date
2025
Authors
Journal Title
Journal ISSN
Volume Title
Publisher
Universidad de Concepción
Abstract
Las heridas crónicas suponen un reto clínico significativo debido a su elevada incidencia de complicaciones inflamatorias e infecciosas y a la limitada eficacia de los tratamientos convencionales. En este contexto, las especies Quercus robur y Opuntia ficus-indica han sido empleadas tradicionalmente por sus propiedades astringentes, antiinflamatorias y antioxidantes, posicionándolas como candidatas prometedoras para el desarrollo de terapias tópicas innovadoras.
El objetivo general de esta tesis consistió en desarrollar una formulación farmacéutica piloto basada en la combinación de extractos acuosos de Q. robur y O. ficus-indica, y evaluar su potencial cicatrizante frente al gel comercial Reoxcare®, en modelos in vitro. Para ello, se plantearon objetivos específicos dirigidos a caracterizar la citotoxicidad y el mecanismo pro-migratorio de los extractos en queratinocitos HaCaT, determinar el perfil proteómico tras el tratamiento con Q. robur, y diseñar formulaciones de hidrogel y crema que mantuvieran estabilidad físico-química y aceptabilidad sensorial.
Para alcanzar estos fines, se recolectaron hojas de Q. robur y cladodios de O. ficus-indica en la Región del Biobío, Chile, entre enero de 2021 y enero de 2023. Los extractos se obtuvieron por infusión en agua caliente y maceración en frío con agitación seguida de liofilización. Su caracterización incluyó análisis fitoquímicos cualitativos y cuantitativos, determinación de actividad antioxidante y evaluación de parámetros físico-químicos (pH, densidad). En ensayos in vitro, se realizó perfil de citotoxicidad (MTT), cierre de brecha y proliferación celular en HaCaT, seguido de un análisis proteómico LC-MS/MS con bioinformática de rutas celulares. Finalmente, se formularon hidrogeles de carbómero y cremas oil in water (O/W), sometidos a almacenamiento acelerado y panel organoléptico para evaluar estabilidad y aceptación.
Los extractos de Q. robur presentaron un rendimiento del 11,1 % y un perfil rico en proteínas (14,9 %) y fenoles (3,2 mg GAE·g⁻¹) con actividad antioxidante (22 % DPPH), mientras que el extracto de O. ficus‐indica destacó por su elevado contenido de azúcares (88,8 %) y ácidos urónicos (79,9 %), indicativo de un fuerte efecto humectante. En la línea celular HaCaT ambos extractos resultaron no citotóxicos (IC50 de Q. robur = 943 μg·mL⁻¹) y, especialmente, Q. robur aceleró la migración celular, alcanzando el cierre completo de la brecha a las 24 h y reduciendo el área libre entre un 81% y 83 % tras 12 h, un efecto comparable al del gel comercial Reoxcare®. El análisis proteómico reveló más de 100 proteínas diferencialmente expresadas, destacando la regulación al alza de enzimas de la vía de biosíntesis de colesterol y de procesos de desgranulación plaquetaria. Tanto el hidrogel como las cremas conservaron integridad físico-química y pH adecuado tras tres meses de estrés térmico, y alcanzaron alta aceptabilidad sensorial en textura, olor y color,
Estos hallazgos respaldan la viabilidad de formulaciones tópicas basadas en extractos de Q. robur y O. ficus-indica como estrategia segura, eficaz y de bajo costo para el tratamiento de heridas crónicas. Se recomienda avanzar hacia estudios in vivo en modelos animales, optimizar dosis y vehículos galénicos, y diseñar ensayos clínicos que permitan validar su aplicación terapéutica en humanos.
Chronic wounds pose a significant clinical challenge due to their high incidence of inflammatory and infectious complications and the limited efficacy of conventional treatments. In this context, Quercus robur and Opuntia ficus-indica have been traditionally used for their astringent, anti-inflammatory and antioxidant properties, positioning them as promising candidates for the development of innovative topical therapies. The primary objective of this thesis was to develop a pilot pharmaceutical formulation based on the combination of aqueous extracts of Q. robur and O. ficus-indica, and to evaluate its wound-healing potential against the commercial gel Reoxcare® in in vitro models. Specific aims included characterizing the cytotoxicity and pro-migratory mechanism of the extracts in HaCaT keratinocytes, determining the proteomic profile following Q. robur treatment, and designing hydrogel and cream formulations that maintain physicochemical stability and sensory acceptability. Leaves of Q. robur and cladodes of O. ficus-indica were collected in the Biobío Region, Chile, between January 2021 and January 2023. Extracts were obtained by hot-water infusion and cold maceration with agitation, followed by freeze-drying. Characterization comprised qualitative and quantitative phytochemical analyses, antioxidant activity assays, and evaluation of physicochemical parameters (pH, density). In vitro studies included MTT cytotoxicity assays, wound-closure and proliferation assays in HaCaT cells, and LC-MS/MS proteomic analysis with subsequent pathway bioinformatics. Finally, carbomer hydrogels and oil in water (O/W) creams were formulated, subjected to accelerated storage, and assessed by an organoleptic panel for stability and sensory acceptance. Q. robur extracts yielded 11.1 % (w/w) and exhibited a profile rich in proteins (14.9 %) and phenols (3.2 mg GAE·g⁻¹) with 22 % DPPH scavenging activity, while the O. ficus-indica extract contained high levels of sugars (88.8 %) and uronic acids (79.9 %), indicative of strong humectant capacity. Both extracts were non-cytotoxic in HaCaT cells (IC₅₀ of Q. robur = 943 μg·mL⁻¹). Notably, Q. robur accelerated cell migration, achieving complete gap closure at 24 h and reducing the free area by 81–83 % after 12 h—an effect comparable to Reoxcare®. Proteomic analysis identified over 100 differentially expressed proteins, including upregulation of enzymes in the cholesterol biosynthesis pathway and factors involved in platelet degranulation. Both the hydrogel and creams preserved physicochemical integrity and suitable pH after three months of thermal stress and achieved high sensory acceptability in texture, odor and color. These results support the feasibility of topical formulations based on Q. robur and O. ficus-indica extracts as a safe, effective and low-cost strategy for chronic wound treatment. Future work should advance to in vivo animal models, optimize dosing and galenic vehicles, and design clinical trials to validate therapeutic application in humans.
Chronic wounds pose a significant clinical challenge due to their high incidence of inflammatory and infectious complications and the limited efficacy of conventional treatments. In this context, Quercus robur and Opuntia ficus-indica have been traditionally used for their astringent, anti-inflammatory and antioxidant properties, positioning them as promising candidates for the development of innovative topical therapies. The primary objective of this thesis was to develop a pilot pharmaceutical formulation based on the combination of aqueous extracts of Q. robur and O. ficus-indica, and to evaluate its wound-healing potential against the commercial gel Reoxcare® in in vitro models. Specific aims included characterizing the cytotoxicity and pro-migratory mechanism of the extracts in HaCaT keratinocytes, determining the proteomic profile following Q. robur treatment, and designing hydrogel and cream formulations that maintain physicochemical stability and sensory acceptability. Leaves of Q. robur and cladodes of O. ficus-indica were collected in the Biobío Region, Chile, between January 2021 and January 2023. Extracts were obtained by hot-water infusion and cold maceration with agitation, followed by freeze-drying. Characterization comprised qualitative and quantitative phytochemical analyses, antioxidant activity assays, and evaluation of physicochemical parameters (pH, density). In vitro studies included MTT cytotoxicity assays, wound-closure and proliferation assays in HaCaT cells, and LC-MS/MS proteomic analysis with subsequent pathway bioinformatics. Finally, carbomer hydrogels and oil in water (O/W) creams were formulated, subjected to accelerated storage, and assessed by an organoleptic panel for stability and sensory acceptance. Q. robur extracts yielded 11.1 % (w/w) and exhibited a profile rich in proteins (14.9 %) and phenols (3.2 mg GAE·g⁻¹) with 22 % DPPH scavenging activity, while the O. ficus-indica extract contained high levels of sugars (88.8 %) and uronic acids (79.9 %), indicative of strong humectant capacity. Both extracts were non-cytotoxic in HaCaT cells (IC₅₀ of Q. robur = 943 μg·mL⁻¹). Notably, Q. robur accelerated cell migration, achieving complete gap closure at 24 h and reducing the free area by 81–83 % after 12 h—an effect comparable to Reoxcare®. Proteomic analysis identified over 100 differentially expressed proteins, including upregulation of enzymes in the cholesterol biosynthesis pathway and factors involved in platelet degranulation. Both the hydrogel and creams preserved physicochemical integrity and suitable pH after three months of thermal stress and achieved high sensory acceptability in texture, odor and color. These results support the feasibility of topical formulations based on Q. robur and O. ficus-indica extracts as a safe, effective and low-cost strategy for chronic wound treatment. Future work should advance to in vivo animal models, optimize dosing and galenic vehicles, and design clinical trials to validate therapeutic application in humans.
Description
Tesis presentada para optar al grado de Doctor en Biotecnología Molecular.
Keywords
Cicatrización de heridas, Heridas crónicas, Antioxidantes