RESUMO
For centuries, bromelain has been used to treat a range of ailments, even though its mechanism of action is not fully understood. Its therapeutic benefits include enzymatic debridement of the necrotic tissues of ulcers and burn wounds, besides anti-inflammatory, anti-tumor, and antioxidant properties. However, the protease is unstable and susceptible to self-hydrolysis over time. To overcome the stability issues of bromelain, a previous study formulated chitosan-bromelain nanoparticles (C-B-NP). We evaluated the optimized nanoformulation for in vitro antioxidant, cell antiproliferative activities and cell migration/proliferation in the scratch assay, comparing it with free bromelain. The antioxidant activity of free bromelain was concentration and time-dependent; after encapsulation, the activity level dropped, probably due to the slow release of protein from the nanoparticles. In vitro antiproliferative activity was observed in six tumor cell lines for free protein after 48 h of treatment (glioma, breast, ovarian, prostate, colon adenocarcinoma and chronic myeloid leukemia), but not for keratinocyte cells, enabling its use as an active topical treatment. In turn, C-B-NP only inhibited one cell line (chronic myeloid leukemia) and required higher concentrations for inhibition. After 144 h treatment of glioma cells with C-B-NP, growth inhibition was equivalent to that promoted by the free protein. This last result confirmed the delayed-release kinetics of the optimized formulation and bromelain integrity. Finally, a scratch assay with keratinocyte cells showed that C-B-NP achieved more than 90% wound retraction after 24 h, compared to no retraction with the free bromelain. Therefore, nanoencapsulation of bromelain with chitosan conferred physical protection, delayed release, and wound retraction activity to the formulation, properties that favor topical formulations with a modified release. In addition, the promising results with the glioma cell line point to further studies of C-B-NP for anti-tumor treatments.
Assuntos
Bromelaínas/química , Bromelaínas/metabolismo , Bromelaínas/farmacologia , Antioxidantes , Linhagem Celular , Linhagem Celular Tumoral , Movimento Celular/efeitos dos fármacos , Proliferação de Células/efeitos dos fármacos , Quitosana/química , Quitosana/farmacologia , Sistemas de Liberação de Medicamentos , Humanos , Nanopartículas/química , Cicatrização/efeitos dos fármacosRESUMO
An ideal wound dressing ensures a moist environment around the wound area and absorbs exudates from the wound surface. Topical application of bromelain to incised wounds has been shown to reprogram the wound microenvironment to promote effective tissue repair. Combining the characteristics of hydrogels and bromelain is therefore of great interest. Herein, we describe the development of a hydrogel, formulated using alginate and Arabic gum, for bromelain loading and release. The hydrogel formulation was evaluated using response surface methodology, considering the pH value and the concentration of alginate and Arabic gum. Bromelain loading and release were evaluated based on passive diffusion. Differential scanning calorimetry and Fourier transform infrared spectroscopy were performed to confirm bromelain immobilization in the hydrogel. The final hydrogel formulation had a swelling ratio of 227â% and incorporated 19â% of bromelain from a bromelain solution. Bromelain immobilization in the hydrogel was the result of hydrogen bond formation and was optimal at 4â°C after 4 h of contact. This evidence suggests that bromelain entrapment into a hydrogel is a promising strategy for the development of wound dressings that support the debridement of burns and wounds.
Assuntos
Alginatos , Bromelaínas/administração & dosagem , Liberação Controlada de Fármacos , Goma Arábica , Bromelaínas/metabolismo , Composição de Medicamentos , Ácido Glucurônico , Ácidos Hexurônicos , Hidrogel de Polietilenoglicol-DimetacrilatoRESUMO
BACKGROUND: Bromelain is a mixture of proteolytic enzymes found in various tissues of the pineapple plant (Ananas comosus) and other species of Bromeliaceae. Owing to its proteolytic activity, bromelain has been used in the food, medical, pharmaceutical and cosmetic industries, for its cell renewal, anti-ageing, whitening and anti-cellulite properties. OBJECTIVE: This study evaluated the stability of bromelain (commercial powder) incorporated in topical formulations. METHODS: Bromelain was incorporated at three concentrations, 0.5%, 1.0% and 2.0%, in oil-in-water emulsion and gel, and stored for six months at varying stress conditions. Stability was accessed by measuring the changes in the protein content, enzymatic activity, viscosity, rheology, pH and colour of the selected formulations. RESULTS: The colour of all the samples changed after 180 days of incubation, indicating the concentration-dependence and temperature-sensitive nature of these formulations. No relationship was observed between the changes in the pH, temperature and luminosity exposure in all the samples. Gels proved to be the least preferred base for incorporation of bromelain for use as a topical formulation, owing to its inability to maintain the integrity of bromelain, thereby affecting the formulation characteristics. CONCLUSION: The emulsion-based formulations at all the concentrations of bromelain were more stable than the gel-based formulation over 180 days of evaluation, at a temperature of 5°C, protected from light.
Assuntos
Bromelaínas/química , Administração Tópica , Bromelaínas/metabolismo , Cor , Cosméticos , Reologia , Temperatura , ViscosidadeRESUMO
The partitioning patterns of papain (PAP) and bromelain (BR), two well-known cysteine-proteases, in polyethyleneglycol/sodium citrate aqueous two-phase systems (ATPSs) were determined. Polyethyleneglycols of different molecular weight (600, 1000, 2000, 4600 and 8000) were assayed. Thermodynamic characterization of partitioning process, spectroscopy measurements and computational calculations of protein surface properties were also carried out in order to explain their differential partitioning behavior. PAP was observed to be displaced to the salt-enriched phase in all the assayed systems with partition coefficients (KpPAP) values between 0.2 and 0.9, while BR exhibited a high affinity for the polymer phase in systems formed by PEGs of low molecular weight (600 and 1000) with partition coefficients (KpBR) values close to 3. KpBR values resulted higher than KpPAP in all the cases. This difference could be assigned neither to the charge nor to the size of the partitioned biomolecules since PAP and BR possess similar molecular weight (23,000) and isoelectric point (9.60). The presence of highly exposed tryptophans and positively charged residues (Lys, Arg and His) in BR molecule would be responsible for a charge transfer interaction between PEG and the protein and, therefore, the uneven distribution of BR in these systems.
Assuntos
Bromelaínas/química , Extração Líquido-Líquido , Papaína/química , Bromelaínas/metabolismo , Dicroísmo Circular , Ativação Enzimática , Papaína/metabolismo , Polietilenoglicóis/química , TermodinâmicaRESUMO
Blackheart is a physiological disorder induced by postharvest chilling storage during pineapple fruit export shipping. The aim of this study was to check the involvement of bromelain, the cysteine protease protein family abundantly present in pineapple fruits, and AcCYS1, an endogenous inhibitor of bromelain, in the development of blackheart. For this we checked the response to postharvest chilling treatment of two pineapple varieties (MD2 and Smooth Cayenne) differing in their resistance to blackheart. Quantitative RT-PCR analyses showed that postharvest chilling treatment induced a down-regulation of bromelain transcript accumulation in both varieties with the most dramatic drop in the resistant variety. Regarding AcCYS1 transcript accumulation, the varieties showed opposite trends with an up-regulation in the case of the resistant variety and a down-regulation in the susceptible one. Taken together our results suggest that the control of bromelain and AcCYS1 expression levels directly correlates to the resistance to blackheart development in pineapple fruits.
Assuntos
Ananas/fisiologia , Bromelaínas/genética , Cistatinas/genética , Regulação da Expressão Gênica de Plantas , Proteínas de Plantas/genética , Ananas/genética , Bromelaínas/metabolismo , Temperatura Baixa , Cistatinas/metabolismo , Frutas/genética , Frutas/fisiologia , Dados de Sequência Molecular , Proteínas de Plantas/metabolismo , Reação em Cadeia da Polimerase , Análise de Sequência de DNARESUMO
The aim of this work was to evaluate the activity of bromelain in pineapple plants (Ananas comosus var. Comosus), Pérola cultivar, produced in vitro in different culture conditions. This enzyme, besides its pharmacological effects, is also employed in food industries, such as breweries and meat processing. In this work, the enzymatic activity was evaluated in the tissues of leaves and stems of plants grown in culture medium without plant growth regulator. The most significant levels of bromelain were observed in leaf tissue after 4 months of culture in vitro in medium with a filter paper bridge, followed by medium gelled by the agar. The results of this study, regarding the different structures of the pineapple (leaves and stems) in vitro showed that the activity of bromelain varied depending on the culture conditions, the time and structure of which was quantified, ensuring a viable strategy in the production of seedlings with high levels of bromelain in subsequent phases of micropropagation.
Assuntos
Ananas/enzimologia , Ananas/crescimento & desenvolvimento , Bromelaínas/metabolismo , Técnicas de Cultura/métodos , Ensaios Enzimáticos/métodos , Ananas/metabolismo , Bromelaínas/biossíntese , Bromelaínas/isolamento & purificação , Meios de Cultura/química , Folhas de Planta/enzimologia , Caules de Planta/enzimologia , ProteóliseRESUMO
Two trypsin inhibitors (called PdKI-3.1 and PdKI-3.2) were purified from the seeds of the Pithecellobium dumosum tree. Inhibitors were obtained by TCA precipitation, affinity chromatography on Trypsin-Sepharose and reversed-phase-HPLC. SDS-PAGE analysis with or without reducing agent showed that they are a single polypeptide chain, and MALDI-TOF analysis determined molecular masses of 19696.96 and 19696.36 Da, respectively. The N-terminal sequence of both inhibitors showed strong identity to the Kunitz family trypsin inhibitors. They were stable over a wide pH (2-9) and temperature (37 to 100 degrees C) range. These inhibitors reduced over 84% of trypsin activity with inhibition constant (Ki) of 4.20 x 10(-8) and 2.88 x 10(-8) M, and also moderately inhibited papain activity, a cysteine proteinase. PdKI-3.1 and PdKI-3.2 mainly inhibited digestive enzymes from Plodia interpunctella, Zabrotes subfasciatus and Ceratitis capitata guts. Results show that both inhibitors are members of the Kunitz-inhibitor family and that they affect the digestive enzyme larvae of diverse orders, indicating a potential insect antifeedant.
Assuntos
Fabaceae/química , Lepidópteros/efeitos dos fármacos , Papaína/antagonistas & inibidores , Peptídeos/farmacologia , Proteínas de Plantas/farmacologia , Tripsina/metabolismo , Sequência de Aminoácidos , Animais , Bromelaínas/antagonistas & inibidores , Bromelaínas/metabolismo , Bovinos , Quimotripsina/antagonistas & inibidores , Larva/efeitos dos fármacos , Larva/enzimologia , Lepidópteros/enzimologia , Dados de Sequência Molecular , Elastase Pancreática/antagonistas & inibidores , Peptídeos/química , Proteínas de Plantas/química , Sementes/química , Alinhamento de Sequência , Análise de SequênciaRESUMO
Proteinaceous inhibitors with high inhibitory activities against human neutrophil elastase (HNE) were found in seeds of the Tamarind tree (Tamarindus indica). A serine proteinase inhibitor denoted PG50 was purified using ammonium sulphate and acetone precipitation followed by Sephacryl S-300 and Sephadex G-50 gel filtration chromatographies. Inhibitor PG50 showed a Mr of 14.9 K on Sephadex G-50 calibrated column and a Mr of 11.6 kDa on sodium dodecyl sulfate-polyacrylamide gel electrophoresis. PG50 had selective activity while cysteine proteinases (papain and bromelain) and serine proteinases (porcine pancreatic elastase and bovine chymotrypsin) were not inhibited, it was strongly effective against serine proteinases such as bovine trypsin and isolated human neutrophil elastase. The IC50 value was determined to be 55.96 microg.mL-1. PG50 showed neither cytotoxic nor haemolytic activity on human blood cells. After pre-incubation of PG50 with cytochalasin B, the exocytosis of elastase was initiated using PAF and fMLP. PG50 exhibited different inhibition on elastase release by PAF, at 44.6% and on release by fMLP, at 28.4%. These results showed that PG50 preferentially affected elastase release by PAF stimuli and this may indicate selective inhibition on PAF receptors.
Assuntos
Elastase de Leucócito/antagonistas & inibidores , Sementes/química , Inibidores de Serina Proteinase/isolamento & purificação , Tamarindus/química , Resinas Acrílicas , Células Sanguíneas/fisiologia , Bromelaínas/metabolismo , Sobrevivência Celular/fisiologia , Cromatografia em Gel , Quimotripsina/metabolismo , Dextranos , Eletroforese em Gel de Poliacrilamida , Citometria de Fluxo , Humanos , Concentração Inibidora 50 , Elastase Pancreática/metabolismo , Papaína/metabolismo , Glicoproteínas da Membrana de Plaquetas/metabolismo , Receptores Acoplados a Proteínas G/metabolismo , Inibidores de Serina Proteinase/metabolismoRESUMO
Microbial fish silage was produced from ground low commercial fish species added with 15% mollase and stored at 35 degrees C. Experiment were conducted to evaluate the effect of the addition of a commercial preparation of bromeline (0.20-0.90%) to the fish silage. Liquefaction was evaluated measuring both the product consistency and its non protein nitrogen content. Results indicate that bromeline addition (0.7 or higher), increases dramatically the rate of proteolysis and decreases the time required for liquefaction from 15 days (no enzyme control) to only 12 hours. Therefore, the use of the commercial preparation of bromeline is highly recommended for silage production.
Assuntos
Bromelaínas/metabolismo , Peixes/metabolismo , Papaína/metabolismo , Peptídeo Hidrolases/metabolismo , Silagem , Animais , Concentração de Íons de Hidrogênio , Melaço , Nitrogênio/análiseRESUMO
The nonenzymic glucation of most proteins occurs only at epsilon-amino groups of lysine residues. Hydrolysis catalyzed by bovine trypsin, porcine trypsin and pineapple bromelian were studied using native and glucated proteins as substrates. Glucosylated ovalbumin, human serum albumin, gamma-globulin and myoglobin show reduced susceptibility to degradation by trypsin as compared to the nonglucated proteins, apparently by direct modification of lysine residues. Trypsin cleaves at substrate arginine and lysine peptides bonds. Bromelian, a less specific enzyme, shows similar hydrolysis rates for native casein, ovalbumin and myoglobin, and identical rates for glucated hemoglobin and myoglobin. Bovine trypsin showed 100% decrease in enzymatic activity with glucated human serum albumin and gamma-globulin and pineapple bromelian with human serum albumin.