Isolation and characterization of antihypertensive peptides from soy bean protein

Proteins and peptides are the most diverse biomolecules found in nature and make our interest due to their wide applications in food and pharmaceutical industry. Angiotensin Converting Enzyme (ACE) plays a major role in controlling blood pressure. The inhibition of ACE with peptides is a main target in the regulation of hypertension. The objective of the present study was to investigate the therapeutic potential of soy bean. This was accomplished by isolation of ACE inhibitory peptides using response surface methodology (RSM) and characterization of these bioactive peptides by mass spectrometry. 31 hydrolyzed fractions were isolated and evaluated for their ACE inhibition potential. Hydrolyzed fraction having highest ACE inhibitory activity was characterized by liquid chromatography-mass spectrometry (LC-MS) technique. RSM results showed maximum ACE inhibition potential (64%) by hydrolyzate was obtained at 45 ºC temperature, pH 8.0, E/S 0.2 in 2 hours hydrolysis time. Results of LC-MS analysis revealed Ser-Gly, Ser-Pro, Met-Ala, His-Ala, Lys-Pro, Phe-Thr, Met-Leu, Pro-Arg, Ala-Pro-Val, Pro-Ala-Leu, Val-Met-Gly, Pro-Leu-Val, Pro-Pro-Gln, His-Arg-Gly, Ser-Phe-Val-Leu, Ala-Val-His-Try, Arg-Thr-Val-Arg, His-His-Tyr-Leu-Val, Asp-Gly-Ala-Cys-Ser-Ala-Asn and MetVal-Thr-Gly-Pro-Gly-Cys-His bioactive peptides in hydrolyzed fraction of soy bean. Our data provide evidence that response surface methodology is a good approach for isolation of antihypertensive bioactive peptides with more potent activity as nutraceuticals or pharmaceuticals. Therefore soy bean can be use for industrial production of pharmaceutical grade natural medicines for handling high blood pressure.

Desenvolvimento de métodos microbiológicos rápidos (MMRs) para avaliação da potência de agentes antimicrobianos e suas incertezas de medição / Development of rapid microbiological methods (RMMs) for evaluation of potency of antimicrobial agents and their measurement uncertainties

O método de difusão em ágar tem sido utilizado na avaliação da atividade antimicrobiana desde a descoberta da penicilina. Apesar disso, pouco avanço ocorreu no sentido de reduzir o tempo necessário para a determinação dos halos de inibição de crescimento. O objetivo deste projeto foi desenvolver, otimizar e validar métodos microbiológicos rápidos (MMRs) para a avaliação da potência de agentes antimicrobianos, além de identificar, quantificar e avaliar as principais fontes de incerteza associadas à determinação da potência. O projeto foi dividido em quatro etapas: 1) influência da composição do meio de cultura na formação dos halos de inibição; 2) estudo da incerteza de medição associada à determinação da potência de agentes antimicrobianos; 3) desenvolvimento, otimização e validação de métodos microbiológicos rápidos (MMRs) para determinação da potência de agentes antimicrobianos e 4) determinação dos parâmetros envolvidos na formação dos halos de inibição de crescimento e estudo dos mecanismos de difusão e crescimento microbiano. Os resultados deste projeto possibilitaram a redução do tempo necessário para a determinação do tamanho dos halos de inibição. Adicionalmente, contribuiu com a elucidação dos mecanismos de difusão e crescimento microbiano, possibilitando identificar e quantificar as principais fontes de incerteza de medição associadas à formação dos halos de inibição

Agar diffusion method has been used in the evaluation of antimicrobial activity since the discovery of penicillin. Nevertheless, little progress has occurred in order to reduce the time required for the determination of growth inhibition zones. The goal of this project was to develop, optimize and validate rapid microbiological methods (RMMs) for evaluation of potency of antimicrobials, as well as to identify, quantify and assess the main sources of uncertainty associated with potency. The project was divided into four steps: 1) influence of culture medium composition on inhibition zones; 2) study of measurement uncertainty associated with antimicrobials potencies; 3) development, optimization and validation of rapid microbiological methods (RMMs) for the determination of antimicrobials potencies and 4) determination of the parameters involved in the formation of inhibition zones and study of mechanisms of diffusion and microbial growth. The results of this project allowed the reduction of the time required for the determination of inhibition zone sizes. Additionally, it contributed to the elucidation of the mechanisms of diffusion and microbial growth, making it possible to identify and quantify the main sources of measurement uncertainty associated with formation of inhibition zone sizes

Optimization of self-nanoemulsifying formulations for weakly basic lipophilic drugs: role of acidification and experimental design

ABSTRACT Formulators face great challenges in adopting systematic approaches for designing self-nanoemulsifying formulations (SNEFs) for different drug categories. In this study, we aimed to build-up an advanced SNEF development framework for weakly basic lipophilic drugs, such as cinnarizine (CN). First, the influence of formulation acidification on CN solubility was investigated. Second, formulation self-emulsification in media with different pH was assessed. Experimentally designed phase diagrams were also utilized for advanced optimization of CN-SNEF. Finally, the optimized formulation was examined using cross polarizing light microscopy for the presence of liquid crystals. CN solubility was significantly enhanced upon external and internal acidification. Among the various fatty acids, oleic acid-based formulations showed superior self-emulsification in all the tested media. Surprisingly, formulation turbidity and droplet size significantly decreased upon equilibration with CN. The design was validated using oleic acid/Imwitor308/Cremophor El (25/25/50), which showed excellent self-nanoemulsification, 43-nm droplet size (for CN-equilibrated formulations), and 88 mg/g CN solubility. In contrast to CN-free formulations, CN-loaded SNEF presented lamellar liquid crystals upon 50% aqueous dilution. These findings confirmed that CN-SNEF efficiency was greatly enhanced upon drug incorporation. The adopted strategy offers fast and accurate development of SNEFs and could be extrapolated for other weakly basic lipophilic drugs.

Utilização de planejamento fatorial para a determinação da capacidade antioxidante e doseamento de flavonoides totais em Verbena minutiflora Briq. ex Moldenke (gervai) / The use of factorial design for determining the antioxidant capacity and dosing the total flavonoids in Verbena minutiflora Briq. Ex Moldenke (Gervai)

RESUMO Verbena minutiflora Briq. ex Moldenke (gervai) tem seu uso medicinal relatado popularmente para tratamento de doenças hepáticas, diarreia e outros problemas de saúde. Entretanto, pouco se conhece a respeito de seus componentes químicos e estudos que comprovem suas propriedades medicinais são escassos. O objetivo desse estudo foi avaliar a composição química dos extratos aquosos e etanólicos de flores de V. minutiflora e otimizar processos de obtenção de extratos com maiores capacidades antioxidantes e maiores concentrações de flavonoides. O método de extração foi desenhado por planejamento fatorial, onde as variáveis para a determinação da capacidade antioxidante foram: pH, extração líquida, método e tempo de extração. Para a determinação de flavonoides totais as variáveis avaliadas por planejamento fatorial foram: concentração de hexametilenotetramina, tipo de ácido, volume de ácido e tempo de aquecimento. Os resultados das análises químicas dos extratos mostraram: aminogrupos, taninos e ácidos fixos (extrato aquoso) aminogrupos, flavonoides, triterpenos, esteroides, alcaloides e cumarinas (extrado hidroetanólico). Os resultados dos planejamentos fatoriais mostraram que o melhor método de extração para a capacidade antioxidante foi o que usou vórtex, por 35 min, com água:etanol 50:50, com pH1, obtendo 0,1899± 5,8.10-3 mmol expressos em ácido ascórbico g-1 nos extratos de V. minutiflora. Enquanto, para as dosagens de flavonoides totais as variáveis significantes foram: tipo de ácido e volume de ácido. A melhor extração obtida foi: 6,748. 10-2± 2,085 10-3% expressos em quercetina. Os resultados mostraram que o planejamento fatorial é uma importante ferramenta para a otimização de extração de componentes químicos em produtos naturais.

ABSTRACT Verbena minutiflora Briq. ex Moldenke (gervai) has its popular use reported for liver disorders treatments, diarrhea, and other health problems. However, little is known about its chemical components and studies that proves its medicinal properties are rare. The aim of this study was to evaluate the chemical composition of aqueous and ethanolic extracts from flowers of V. minutiflora and to optimize processes to obtain extracts with higher antioxidant capacity and greater concentration of flavonoids. The methods of extraction were designed by factorial planning, where the variables to determine the antioxidant capacity were: pH; extraction liquid; method and extraction time. To determinate the total flavonoids the variables evaluated by factorial design were: concentration of hexamethylenetetramine; type of acid; volume of acid and warming time. The results of chemical analysis of the extracts showed: amino groups, tannins and fixed acids (aqueous extract) amino groups, flavonoids, triterpenes, steroids, alkaloids and coumarins (hydroalcoholic extract). The factorial designs results showed that the best extraction method for the antioxidant capacity was the one that uses vortex, for 35 min, with water: ethanol 50:50, at pH 1, getting 0,1899 ± 5,8.10-3 mmol expressed in ascorbic acid g-1 in extracts of V. minutiflora . While, for dosages of total flavonoids the significant variables were the type of acid and volume of acid. The best extraction obtained was: 6,748. 10-2± 2,085 10-3% expressed in quercetin. These data showed that the factorial design is an important tool in optimizing the extraction of chemical components in natural products.

Optimización de las condiciones de inoculación por biobalística de un Begomovirus en tomate y tabaco / Optimizing conditions for biolistic inoculation of Begomovirus in tomato and tobacco

La transmisión experimental de Begomovirus es problemática. La mayoría de estos virus se pueden transmitir de planta a planta por su vector biológico, Bemisia tabaci. Las inoculaciones experimentales con mosca blanca son problemáticas debido a sus hábitos de alimentación, requerimiento de una planta viva infectada e instalaciones de contención para el vector. Por su parte la inoculación mecánica de Begomovirus es posible, pero generalmente a tasas bajas y no en todos los casos. Por esta razón el bombardeo de partículas (biobalística) de DNA viral como una estrategia de inoculación fue desarrollada. La posibilidad de utilizar el dispositivo de mano Helios Gen Gun System (Biorad®), un equipo de biobalística, para la transmisión de un Begomovirus bipartita a plantas de tomate y tabaco fue ensayado y optimizado. Los parámetros evaluados fueron: número de disparos (1-2), presión de helio (220 y 320 psi) y diámetro de las partículas de oro (0.6 y 1.6µm). Los síntomas característicos de la enfermedad viral (clorosis, mosaico y deformación de la hoja) aparecieron 3 semanas después del bombardeo en las hojas jóvenes no inoculadas. La replicación del DNA viral en las plantas se confirmó por Reacción en cadena de la polimerasa. Plantas infectadas en un 100 se obtuvieron cuando en el bombardeo se emplearon partículas de oro de 1.6 µm recubiertas con DNA viral a una presión de 320psi. A nuestro entender este es el primer reporte en Colombia de la inoculación directa de plantas de tomate y tabaco con un Begomovirus bipartita usando un dispositivo portátil de biobalística.

Experimental transmission of Begomovirus is problematic. Most Begomoviruses can be transmitted readily from plant to plant by the whitefly vector, but this also requires a live infected plant and extensive facilities to maintain the insect. Whitefly inoculations can also be problematic because of their preferential feeding habits on certain plants. Mechanical inoculation of Begomovirus is possible but generally at low rates and for others not at all. For this reason particle bombardment (biolistic) of DNA viral as an inoculum was developed. The possibility of using the Helios Gen Gun System (Biorad®), a biolistic hand-held device, for transmitting Begomovirus bipartite to tomato and tobacco plants was assayed and optimized. Biolistic inoculation was carried out with the hand held device at 220 or 320 psi, applying 1 or 2 shots /plant and using gold particles of 0.6 or 1.6µm in size. Characteristic symptoms of viral disease (chlorosis, mosaic and leaf deformation) appeared 3 weeks post-inoculation in the newly developing leaves. Replication of the viral DNA in plants was confirmed by Polymerase Chain Reaction. All bombarded plants became infected when biolistic inoculation was carried out with the hand held device at 320psi and using 1.6 µm gold particles in size. To our knowledge this is the first report in Colombia of successful direct inoculation of tomato and tobacco plants with Begomovirus bipartite geminivirus using a biolistic hand-held device.

Floating tablets of hydralazine hydrochloride: optimization and evaluation

Hydralazine hydrochloride has a half-life of 2 to 4 hours with an oral bioavailability of 26-50%. Since hydralazine has a demethylating effect on various suppressor genes, it can be used in various types of cancer to support chemotherapy. The purpose of this study was to optimize and evaluate floating tablets of hydralazine hydrochloride designed to prolong the gastric residence time and to provide controlled release of the drug for 14 h. The floating tablets of hydralazine hydrochloride were prepared by the wet granulation method. Semi-synthetic polymers of hydroxy propyl methyl cellulose (HPMC K100M) and ethyl cellulose were used as the release retarding agents. A 2² factorial design was applied to systematically optimize the drug release profile. The concentrations of HPMC K100M and ethyl cellulose were optimized to provide controlled release of hydralazine for 14h. Non-Fickian diffusion release transport was confirmed as the release mechanism for the optimized formulation and the predicted values agreed well with the experimental values. Drug excipient compatibility studies were investigated by FTIR, DSC and XRD. These data indicate that there were no chemical interactions between the drug and the polymer. In vivo X-ray imaging showed floating tablet performance in rabbits.

O cloridrato de hidralazina apresenta meia-vida de 2 a 4 horas, com biodisponibilidade oral de 26-50%. Uma vez que a hidralazina possui efeito desmetilante em vários genes supressores, ela pode ser utilizada para diversos tipos de câncer, em apoio à quimioterapia. O objetivo deste estudo foi o de avaliar e otimizar comprimidos flutuantes de cloridrato de hidralazina, planejados para prolongar o tempo de residência gástrica e proporcionar liberação controlada do fármaco por 14 h. Os comprimidos flutuantes de cloridrato de hidralazina foram preparados pelo método de granulação úmida. Polímeros semi-sintéticos de hidroxipropiletil celulose (HPMCK100M) e acetato de celulose foram utilizados como agente de retardamento de liberação. Aplicou-se planejamento fatorial 2² para otimizar sistematicamente o perfil de liberação do fármaco. As concentrações de HPMCK100M e de etilcelulose foram otimizadas para se obter liberação controlada de hidralazina durante 14 h. O transporte de liberação de difusão não-Fickiana foi confirmado como o mecanismo de liberação para a formulação otimizada e os valores previstos estiveram de acordo com os valores experimentais. Estudos de compatibilidade entre fármaco e excipiente foram realizados por FTIR, DSC e DRX. Estes dados indicaram que não havia interação química entre o fármaco e o polímero. Imagens de raios-X in vivo mostraram o desempenho dos comprimidos flutuantes em coelhos.