Estudio de posibles aplicaciones farmacológicas de extractos de especies de bromeliáceas y su comparación con bromelina

Es propósito del presente trabajo aportar al conocimiento en relación a los posibles usos medicinales de especies de bromeliáceas autóctonas de nuestro país no estudiadas aún para dichos fines. Bajo la suposición de que las proteasas de Bromelia hieronymi, B balansae y Pseudananas macrodontes, por pertenecer al mismo tipo catalítico y a la misma subfamilia vegetal, podrían mostrar actividades biológicas similares a las proteasas de Ananas comosus, nos planteamos como objetivo general de la presente tesis la búsqueda de posibles efectos farmacológicos de extractos ricos en cisteínendopeptidasas obtenidos de frutos de Bromelia hieronymi, B. balansae y Pseudananas macrodontes y su comparación con bromelina. Y como objetivos específicos: - Evaluar el efecto antiinflamatorio de los extractos de B. hieronymi, B. balansae y P. macrodontes. - Evaluar los efectos de los extractos de B. hieronymi, B. balansae y P. macrodontes sobre algunos de los componentes de la hemostasia. - Evaluar los extractos de B. hieronymi, B. balansae y P. macrodontes como posibles agentes antitumorales. - Tratar de establecer la posible relación entre los efectos medidos y la actividad proteolítica de las cisteínendopeptidasas estudiadas.Facultad de Ciencias Exacta

Proteasas vegetales: potenciales usos en la curtiembre como alternativa eco-compatible y para el aprovechamiento de residuos proteicos

Dos problemas medioambientales asociados con el proceso de transformación de piel en cuero en las curtiembres son: el uso de sulfuro de sodio e hidróxido de calcio (Na 2 S/Ca(OH) 2 ) durante el paso de depilado y la generación de residuos proteicos sólidos. Éste último, a su vez, está relacionado con el bajo rendimiento del proceso: sólo alrededor del 25% de piel vacuna en estado salado es aprovechada como cuero, mientras que el 50% de colágeno (proteína base del cuero) es desechado. Las proteasas son una alternativa eco-compatible para reemplazar el depilado convencional mediante Na 2 S/Ca(OH) 2, pero además una herramienta para el aprovechamiento de residuos proteicos mediante la obtención de hidrolizados que muestren propiedades de interés para otras industrias. Numerosas proteasas extraídas de especies vegetales que crecen en el país han sido caracterizadas bioquímicamente en el CIProVe (UNLP - Asociado CIC). En el presente trabajo se muestran los resultados de ensayos realizados en el CITEC (INTI – CIC) para caracterizar extractos proteolíticos obtenidos de 5 especies vegetales frente a diferentes sustratos representativos de la piel, así como para evaluar el depilado enzimático sobre pieles vacunas y de carpincho en comparación con una enzima comercial

Proteolytic extracts of three bromeliaceae species as eco-compatible tools for leather industry

In addition to the large quantities of solid waste rich in protein, most tanneries use high proportions of Na2S and CaO during the dehairing step, resulting in effluents of high alkalinity and large amounts of suspended solid, besides the risk of liberating the toxic hydrogen sulphide. The current worldwide legislation on environmental requires tanneries to reduce pollution and to replace conventional processes by greener technologies. Enzymes are a technological tool of interest for industry because are able to achieve a high reaction rate under soft pH, temperature, and pressure conditions, besides a high specificity of reaction, biodegradability, non-toxic nature and non-polluting effluent generation. In leather industry enzymes are principally used in pre-tanning operations (soaking, dehairing, bating, and degreasing) and waste treatment. Particularly, proteases have been chosen as a promising eco-friendly alternative to lime and sodium sulphide dehairing. Extracts rich in cysteine proteases with high proteolytic activity (CU) have been obtained from fruits of Bromeliaceae species: Bromelia balansae (Bb), B. hieronymi (Bh), and Pseudananas macrodontes (Pm). In this work, Bb, Bh, and Pm have been studied for application in leather industry compared with commercial enzyme, focusing in their dehairing properties. Enzymatic activities against representative substrates of skin proteins were spectrophotometrically measured at 25, 35, and 55ºC (Tris-HCl, 0.1 M, pH 8, Cys 20 mM). Keratin Azure (KA), Elastin-Congo Red (E), epidermis substrate (EP), and Hide Powder Azure (HPA) were used as representative substrates of keratin, elastin, epidermis, and collagen, respectively. Ability to dehairing was evaluated by incubating soaked cow skins with different concentrations of extracts at 25ºC and pH 8 during 24 h. Grain surface and cross section of skins were observed by scanning electron microscopy. Extracts were able to degrade representative substrates of skin proteins and when compared to the same CU showed similar activity on collagen and epidermis; however, Bh and Pm were the most actives against keratin, while Bh was the only active against elastin. Extracts showing different proteolytic activity (Bb required 1 CU/ml, Bh 1.5 CU/ml, and Pm 0.5 CU/ml) were able to depilate cow skin after a gentle scraping. Although depilated skins with Bb, Bh, and Pm showed different surface aspects, desirable characteristics of dehairing were observed for all extracts since hair pores did not show residual hair, grain surface were clean and intact, and collagen fiber bundles of dermis were not damaged. In conclusion, results here presented show that proteolytic extracts of Bromeliaceae species are promising eco-compatible tools for leather industry, principally in treatment of their waste and dehairing process.Centro de Investigación de Proteínas VegetalesComisión de Investigaciones Científicas de la provincia de Buenos Aire

Proteolytic extracts of three bromeliaceae species as eco-compatible tools for leather industry

In addition to the large quantities of solid waste rich in protein, most tanneries use high proportions of Na2S and CaO during the dehairing step, resulting in effluents of high alkalinity and large amounts of suspended solid, besides the risk of liberating the toxic hydrogen sulphide. The current worldwide legislation on environmental requires tanneries to reduce pollution and to replace conventional processes by greener technologies. Enzymes are a technological tool of interest for industry because are able to achieve a high reaction rate under soft pH, temperature, and pressure conditions, besides a high specificity of reaction, biodegradability, non-toxic nature and non-polluting effluent generation. In leather industry enzymes are principally used in pre-tanning operations (soaking, dehairing, bating, and degreasing) and waste treatment. Particularly, proteases have been chosen as a promising eco-friendly alternative to lime and sodium sulphide dehairing. Extracts rich in cysteine proteases with high proteolytic activity (CU) have been obtained from fruits of Bromeliaceae species: Bromelia balansae (Bb), B. hieronymi (Bh), and Pseudananas macrodontes (Pm). In this work, Bb, Bh, and Pm have been studied for application in leather industry compared with commercial enzyme, focusing in their dehairing properties. Enzymatic activities against representative substrates of skin proteins were spectrophotometrically measured at 25, 35, and 55ºC (Tris-HCl, 0.1 M, pH 8, Cys 20 mM). Keratin Azure (KA), Elastin-Congo Red (E), epidermis substrate (EP), and Hide Powder Azure (HPA) were used as representative substrates of keratin, elastin, epidermis, and collagen, respectively. Ability to dehairing was evaluated by incubating soaked cow skins with different concentrations of extracts at 25ºC and pH 8 during 24 h. Grain surface and cross section of skins were observed by scanning electron microscopy. Extracts were able to degrade representative substrates of skin proteins and when compared to the same CU showed similar activity on collagen and epidermis; however, Bh and Pm were the most actives against keratin, while Bh was the only active against elastin. Extracts showing different proteolytic activity (Bb required 1 CU/ml, Bh 1.5 CU/ml, and Pm 0.5 CU/ml) were able to depilate cow skin after a gentle scraping. Although depilated skins with Bb, Bh, and Pm showed different surface aspects, desirable characteristics of dehairing were observed for all extracts since hair pores did not show residual hair, grain surface were clean and intact, and collagen fiber bundles of dermis were not damaged. In conclusion, results here presented show that proteolytic extracts of Bromeliaceae species are promising eco-compatible tools for leather industry, principally in treatment of their waste and dehairing process.Centro de Investigación de Proteínas VegetalesComisión de Investigaciones Científicas de la provincia de Buenos Aire

Proteolytic extracts of three bromeliaceae species as eco-compatible tools for leather industry

In addition to the large quantities of solid waste rich in protein, most tanneries use high proportions of Na2S and CaO during the dehairing step, resulting in effluents of high alkalinity and large amounts of suspended solid, besides the risk of liberating the toxic hydrogen sulphide. The current worldwide legislation on environmental requires tanneries to reduce pollution and to replace conventional processes by greener technologies. Enzymes are a technological tool of interest for industry because are able to achieve a high reaction rate under soft pH, temperature, and pressure conditions, besides a high specificity of reaction, biodegradability, non-toxic nature and non-polluting effluent generation. In leather industry enzymes are principally used in pre-tanning operations (soaking, dehairing, bating, and degreasing) and waste treatment. Particularly, proteases have been chosen as a promising eco-friendly alternative to lime and sodium sulphide dehairing. Extracts rich in cysteine proteases with high proteolytic activity (CU) have been obtained from fruits of Bromeliaceae species: Bromelia balansae (Bb), B. hieronymi (Bh), and Pseudananas macrodontes (Pm). In this work, Bb, Bh, and Pm have been studied for application in leather industry compared with commercial enzyme, focusing in their dehairing properties. Enzymatic activities against representative substrates of skin proteins were spectrophotometrically measured at 25, 35, and 55ºC (Tris-HCl, 0.1 M, pH 8, Cys 20 mM). Keratin Azure (KA), Elastin-Congo Red (E), epidermis substrate (EP), and Hide Powder Azure (HPA) were used as representative substrates of keratin, elastin, epidermis, and collagen, respectively. Ability to dehairing was evaluated by incubating soaked cow skins with different concentrations of extracts at 25ºC and pH 8 during 24 h. Grain surface and cross section of skins were observed by scanning electron microscopy. Extracts were able to degrade representative substrates of skin proteins and when compared to the same CU showed similar activity on collagen and epidermis; however, Bh and Pm were the most actives against keratin, while Bh was the only active against elastin. Extracts showing different proteolytic activity (Bb required 1 CU/ml, Bh 1.5 CU/ml, and Pm 0.5 CU/ml) were able to depilate cow skin after a gentle scraping. Although depilated skins with Bb, Bh, and Pm showed different surface aspects, desirable characteristics of dehairing were observed for all extracts since hair pores did not show residual hair, grain surface were clean and intact, and collagen fiber bundles of dermis were not damaged. In conclusion, results here presented show that proteolytic extracts of Bromeliaceae species are promising eco-compatible tools for leather industry, principally in treatment of their waste and dehairing process.Centro de Investigación de Proteínas VegetalesComisión de Investigaciones Científicas de la provincia de Buenos Aire

Evaluation of Anti-Inflammatory Activity of Pseudananas macrodontes (Morr.) Harms (Bromeliaceae) Fruit Extract in Rats

Several species of the family Bromeliaceae are characterized by the production of proteases in unusual amounts, especially in fruits. Bromelain, an extract rich in cysteine endo-peptidases obtained from Ananas comosus L., and a few other proteases have been used as anti-infl ammatory agents for some years, but bromelain is still mainly being used as alternative and/or complementary therapy to the treatment with glucocorticoids, nonsteroi-dal antirheumatics, and immunomodulators. In this study, the anti-infl ammatory action of a partially purifi ed extract from Pseudananas macrodontes (Morr.) Harms fruits (PPEPm) is presented, whose main components are cysteine endopeptidases. The effect of PPEPm was assessed in carrageenan-induced and serotonin-induced rat paw edema, as well as in the cotton pellet granuloma model. Doses with equal proteolytic activity of PPEPm and bromelain produced signifi cantly similar anti-infl ammatory responses in the acute infl ammatory models assayed, supporting the hypothesis that proteolytic activity could be responsible for the anti-infl ammatory action. On the contrary, comparable anti-infl ammatory effects of PPEPm and bromelain in the chronic infl ammatory assay required a much lower proteolytic activity content of PPEPm, which could be due to a differential affi nity for the protein target involved in this process.Facultad de Ciencias Exacta

Preparación proteolítica de Carica papaya como insumo eco-compatible para la industria del cuero

El impacto negativo de la actividad industrial sobre el medioambiente puede minimizarse tratando los desechos peligrosos generados antes de ser eliminados o usando procesos y/o insumos que eviten la producción de los mismos. Durante la transformación de piel en cuero en las curtiembres, grandes cantidades de desechos son producidos. Uno de los procesos más contaminantes aplicados en la piel es el depilado mediante Na2S/CaO (depilado convencional). El depilado enzimático mediante proteasas es propuesto como una alternativa ecocompatible al depilado convencional por mostrar una significativa reducción en los residuos tóxicos generados. Por otro lado, las proteasas pueden ser utilizadas para tratar los residuos proteicos de forma que puedan ser reaprovechados. El objetivo del presente trabajo fue obtener y caracterizar un preparado proteolítico a partir de Carica papaya L. para evaluar su posible aplicación en la industria del cuero. De este modo, látex obtenido a partir de frutos verdes de C. papaya fue secado en condiciones controladas, resuspendido en tampón fosfatos pH 6,0 (con EDTA y Cys 5 mM) y centrifugado para eliminar gomas y otros materiales insolubles. El sobrenadante fue liofilizado constituyendo el producto enzimático PCp, el cual fue caracterizado por métodos electroforéticos (SDS-PAGE, IEFzimograma), su actividad proteolítica total fue medida usando caseína como sustrato (UCas) y la concentración de proteínas mediante el método de Bradford. Se evaluó la actividad enzimática de PCp a diferentes temperaturas (25, 35 y 55°C) sobre un sustrato epidermis así como sobre sustratos representativos de proteínas de la piel: azul de queratina, HPA y elastina rojo congo (representativos de queratina, colágeno y elastina, respectivamente). La acción depilante fue ensayada sobre muestras de piel vacuna (4g) luego de incubarla con diferentes concentraciones de PCp en buffer Tris-HCl (pH 8,0) a 25°C durante 24 h. La calidad del depilado fue evaluada por observación directa y mediante microscopía electrónica (SEM). En todos los ensayos se usó una enzima comercial (New1875, Cergen) con fines comparativos. La concentración de proteínas de PCp fue 180±30 µg/ml y la actividad proteolítica específica 6UCas/mg. Por SDS-PAGE se detectaron dos bandas mayoritarias de PM cercano a 26 y 14KDa las cuales coincidieron con las de papaína comercial. Mediante IEF-zimograma, dos bandas de pI alcalino (pI>9 y pI=8,7) mostraron actividad proteolítica. PCp tuvo actividad enzimática sobre queratina, colágeno, elastina y sustrato epidermis, las cuales aumentaron con la temperatura. Dichas actividades, al ser normalizadas respecto a la actividad proteolítica total (1UCas), fueron similares a las de New1875. Sin embargo, al compararlas por mg de producto fueron 1 orden de magnitud superior a las de la enzima comercial. PCp fue capaz de depilar piel vacuna para una concentración de 1mg/ml, mientras que New1875 requirió 2mg/ml para depilar en las mismas condiciones. Mediante SEM se confirmó que los pelos así como la epidermis fueron eliminados completamente, mostrando poros limpios y una superficie sin daño. Se concluye que PCp es un preparado proteolítico con potenciales aplicaciones en la curtiembre, como alternativa eco-compatible al depilado convencional, así como para el tratamiento de los residuos proteicos generados en dicha industria.Centro de Investigación de Proteínas Vegetale

Desarrollo de procesos destinados a minimizar el impacto ambiental de las curtiembres de la provincia de Buenos Aires

La industria curtidora es una de las más tradicionales de nuestro país y al mismo tiempo genera, a través del curtido, una variedad de desechos que resultan contaminantes para el medioambiente. La implementación de tecnologías limpias y el tratamiento integral de los residuos sólidos y líquidos resulta fundamental para minimizar el impacto ambiental y cumplir con la legislación vigente. Mediante la obtención, producción y caracterización de extractos enzimáticos vegetales y fúngicos se logró depilar la piel bovina a escala laboratorio, sin daño apreciable en la capa reticular colagénica. Además, el residuo sólido pelo bovino actuó como sustrato para el crecimiento de especies fúngicas y para la producción de un pool enzimático aplicable en la curtiembre. Por medio del tratamiento biológico de los efluentes líquidos se logró disminuir la demanda química de oxígeno (DQO) hasta valores aceptables para su descarga, con un tiempo de retención hidráulico de 60 h para una relación alimento/microorganismo (F/M= 0,47/día), DQO inicial 5000 mg O2/L y DQO final 250 mg O2/L.Trabajo presentado por el Centro de Investigación de tecnología del Cuero (CITEC)

Evaluation of Anti-Inflammatory Activity of Pseudananas macrodontes (Morr.) Harms (Bromeliaceae) Fruit Extract in Rats

Several species of the family Bromeliaceae are characterized by the production of proteases in unusual amounts, especially in fruits. Bromelain, an extract rich in cysteine endo-peptidases obtained from Ananas comosus L., and a few other proteases have been used as anti-infl ammatory agents for some years, but bromelain is still mainly being used as alternative and/or complementary therapy to the treatment with glucocorticoids, nonsteroi-dal antirheumatics, and immunomodulators. In this study, the anti-infl ammatory action of a partially purifi ed extract from Pseudananas macrodontes (Morr.) Harms fruits (PPEPm) is presented, whose main components are cysteine endopeptidases. The effect of PPEPm was assessed in carrageenan-induced and serotonin-induced rat paw edema, as well as in the cotton pellet granuloma model. Doses with equal proteolytic activity of PPEPm and bromelain produced signifi cantly similar anti-infl ammatory responses in the acute infl ammatory models assayed, supporting the hypothesis that proteolytic activity could be responsible for the anti-infl ammatory action. On the contrary, comparable anti-infl ammatory effects of PPEPm and bromelain in the chronic infl ammatory assay required a much lower proteolytic activity content of PPEPm, which could be due to a differential affi nity for the protein target involved in this process.Facultad de Ciencias Exacta

Latex peptidases of Calotropis procera for dehairing of leather as an alternative to environmentally toxic sodium sulfide treatment

Dehairing of crude leather is a critical stage performed at the beginning of its processing to obtain industrially useful pieces. Tanneries traditionally apply a chemical process based on sodium sulfide. Since this chemical reactive is environmentally toxic and inefficiently recycled, innovative protocols for reducing or eliminating its use in leather depilation are welcomed. Therefore, latex peptidases from Calotropis procera (CpLP) and Cryptostegia grandiflora (CgLP) were assayed for this purpose. Enzyme activity on substrates representative of skin such as hide powder azure (UHPA), elastin (UE), azocollagen (UAZOCOL), keratin (UK), and epidermis (UEP) was determined, while depilation activity was assayed on cow hide. Only CpLP was active against keratin (13.4 UK) and only CgLP was active against elastin (0.12 UE). CpLP (93.0 UHPA, 403.6 UAZOCOL, 36.3 UEP) showed higher activity against the other substrates than CgLP (47.6 UHPA, 261.5 UAZOCOL, 8.5 UEP). In pilot assays, CpLP (0.05% w/v with sodium sulfite 0.6% w/v as activator) released hairs from cow hide pieces. Macroscopic and microscopic analyses of the hide revealed that the dehairing process was complete and the leather structure was preserved. The proteolytic system of C. procera is a suitable bioresources to be exploited by tanneries.Fil: Lopez, Laura Maria Isabel. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina. Instituto Nacional de Tecnología Industrial. Centro de Investigación y Desarrollo del Cuero. Provincia de Buenos Aires. Gobernación. Comision de Investigaciones Centíficas. Centro de Investigación y Desarrollo del Cuero; ArgentinaFil: Viana, Carolina A.. Universidade Estadual Do Ceara; BrasilFil: Errasti, María Eugenia. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina. Instituto Nacional de Tecnología Industrial. Centro de Investigación y Desarrollo del Cuero. Provincia de Buenos Aires. Gobernación. Comision de Investigaciones Centíficas. Centro de Investigación y Desarrollo del Cuero; ArgentinaFil: Garro, María Laura. Instituto Nacional de Tecnología Industrial. Centro de Investigación y Desarrollo del Cuero. Provincia de Buenos Aires. Gobernación. Comision de Investigaciones Centíficas. Centro de Investigación y Desarrollo del Cuero; ArgentinaFil: Martegani, José Eduardo. Instituto Nacional de Tecnología Industrial. Centro de Investigación y Desarrollo del Cuero. Provincia de Buenos Aires. Gobernación. Comision de Investigaciones Centíficas. Centro de Investigación y Desarrollo del Cuero; ArgentinaFil: Mazzilli, German Ariel. Instituto Nacional de Tecnología Industrial. Centro de Investigación y Desarrollo del Cuero. Provincia de Buenos Aires. Gobernación. Comision de Investigaciones Centíficas. Centro de Investigación y Desarrollo del Cuero; ArgentinaFil: Freitas, Cléverson D. T.. Universidade Estadual Do Ceara; BrasilFil: Araújo, Ídila M. S.. EMBRAPA Agrotropical; BrasilFil: da Silva, Rafaela O.. Universidade Estadual Do Ceara; BrasilFil: Ramos, Márcio V.. Universidade Estadual Do Ceara; Brasi