Bacterial nanocellulose loaded with bromelain and nisin as a promising bioactive material for wound debridement.

The bacterial nanocellulose (BnC) membranes were produced extracellularly by a novel aerobic acetic acid bacterium Komagataeibacter melomenusus. The BnC was modified in situ by adding carboxymethyl cellulose (CMC) into the culture media, obtaining a BnC-CMC product with denser fibril arrangement, improved rehydration ratio and elasticity in comparison to BnC. The proteolytic enzyme bromelain (Br) and antimicrobial peptide nisin (N) were immobilized to BnC matrix by ex situ covalent binding and/or adsorption. The optimal Br immobilization conditions towards the maximized specific proteolytic activity were investigated by response surface methodology as factor variables. At optimal conditions, i.e., 8.8 mg/mL CMC and 10 mg/mL Br, hyperactivation of the enzyme was achieved, leading to the specific proteolytic activity of 2.3 U/mg and immobilization efficiency of 39.1 %. The antimicrobial activity was observed against Gram-positive bacteria (S. epidermidis, S. aureus and E. faecalis) for membranes with immobilized N and was superior when in situ modified BnC membranes were used. N immobilized on the BnC or BnC-CMC membranes was cytocompatible and did not cause changes in normal human dermal fibroblast cell morphology. BnC membranes perform as an efficient carrier for Br or N immobilization, holding promise in wound debridement and providing antimicrobial action against Gram-positive bacteria, respectively.

Peptides with biological and technofunctional properties produced by bromelain hydrolysis of proteins from different sources: A review.

Bromelains are cysteine peptidases with endopeptidase action (a subfamily of papains), obtained from different parts of vegetable belonging to the Bromeliaceae family. They have some intrinsic medical activity, but this review is focused on their application (individually or mixed with other proteases) to produce bioactive peptides. When compared to other proteases, perhaps due to the fact that they are commercialized as an extract containing several proteases, the hydrolysates produced by this enzyme tends to have higher bioactivities than other common proteases. The peptides and the intensity of their final properties depend on the substrate protein and reaction conditions, being the degree of hydrolysis a determining parameter (but not always positive or negative). The produced peptides may have diverse activities such as antioxidant, antitumoral, antihypertensive or antimicrobial ones, among others or they may be utilized to improve the organoleptic properties of foods and feeds. Evolution of the use of this enzyme in this application is proposed to be based on a more intense direct application of Bromeliaceae extract, without the cost associated to enzyme purification, and the use of immobilized biocatalysts of the enzyme by simplifying the enzyme recovery and reuse, and also making the sequential hydrolysis using diverse proteases possible.

Bromelain: An Enzyme Expanding its Horizon from Food to Pharmaceutical Industry.

Bromelain is a protein digestive enzyme obtained from the extract of pineapple (steam, fruit, and leaves). It is a cocktail of several thiol endopeptidases and other components like peroxidase, cellulase, phosphatase, and several protease inhibitors. It is a glycoprotein with an oligosaccharide in its molecular structure that contains xylose, fucose, mannose, and N-acetyl glucosamine. Many approaches have been used in the extraction and purification of bromelain like filtration, membrane filtration, INT filtration, precipitation, aqueous two-phase system, ion-exchange chromatography, etc. This enzyme is widely used in the food industry for meat tenderization, baking, cheese processing, seafood processing, etc. However, this enzyme also expands its applicability in the food industry. It is reported to have the potential for the treatment of bronchitis, surgical trauma, sinusitis, etc. The in vitro and in vivo studies showed that it possesses fibrinolytic, antiinflammatory, antithrombotic, anti-edematous activity, etc. The human body absorbed bromelain without any side effects or reduction in its activity. However, in some cases, it shows side effects in those patients who are allergic to pineapple. To minimize such adverse effects bromelain is immobilized inside the nanoparticles. This paper gives an overview of the production, purification, and application of this industrially important enzyme in the food and pharmaceutical industry. It also discusses the various immobilization strategies used to enhance its efficiency.

Multifunctional bioactive properties of hydrolysates from colocynth (Citrullus colocynthis) seeds derived proteins: Characterization and biological properties.

Citrullus colocynthis (Colocynth) has gained a great deal of interest in their applications as indigenous nutraceutical and as a functional food ingredient. The intact colocynth seed protein was enzymatically hydrolyzed using proteolytic enzymes (alcalase, bromelain, and chymotrypsin) at different time intervals of 3, 6, and 9 h. The highest degree of hydrolysis (87.82%) was observed in chymotrypsin derived colocynth seed protein hydrolysates (CSPH) for 9 h. The CSPHs was further investigated through in-vitro assay to explore its potential biological activity such as antioxidant, inhibition of enzymatic marker related to diabetes (DPP-IV, α-glucosidase and α-amylase) and hyperlipidaemia (cholesteryl esterase and pancreatic lipase). Chymotrypsin hydrolysate showed the strongest DPPH (65.7 mM TEAC) and ABTS (525.2 mM TEAC) radical scavenging activity after 6 h of hydrolysis. Moreover, chymotrypsin-treated CSPH for 6 h inhibited cholesteryl esterase (IC50 = 13.68 µg/mL) and pancreatic lipase (IC50 = 14.12 µg/mL) significantly when compared to native protein. Whereas, bromelain and alcalase treated hydrolysate for 6 h effectively inhibited α-glucosidase and α-amylase at an inhibitory concentration of IC50 = 13.27 µg/mL and of IC50 = 17 µg/mL. Overall, the findings indicated that protein hydrolysates exhibited superior biological activity than intact colocynth seed proteins isolate (CSPI) and could be a sustainable source of bioactive peptides.

Heterologous Expression and Catalytic Properties of Codon-Optimized Small-Sized Bromelain from MD2 Pineapple.

Bromelain is a unique enzyme-based bioactive complex containing a mixture of cysteine proteases specifically found in the stems and fruits of pineapple (Ananas comosus) with a wide range of applications. MD2 pineapple harbors a gene encoding a small bromelain cysteine protease with the size of about 19 kDa, which might possess unique properties compared to the other cysteine protease bromelain. This study aims to determine the expressibility and catalytic properties of small-sized (19 kDa) bromelain from MD2 pineapple (MD2-SBro). Accordingly, the gene encoding MD2-SBro was firstly optimized in its codon profile, synthesized, and inserted into the pGS-21a vector. The insolubly expressed MD2-SBro was then resolubilized and refolded using urea treatment, followed by purification by glutathione S-transferase (GST) affinity chromatography, yielding 14 mg of pure MD2-SBro from 1 L of culture. The specific activity and catalytic efficiency (kcat/Km) of MD2-SBro were 3.56 ± 0.08 U mg-1 and 4.75 ± 0.23 × 10-3 µM-1 s-1, respectively, where optimally active at 50 °C and pH 8.0, and modulated by divalent ions. The MD2-SBro also exhibited the ability to scavenge the 2,2-diphenyl-1-picryl-hydrazyl-hydrate (DPPH) with an IC50 of 0.022 mg mL-1. Altogether, this study provides the production feasibility of active and functional MD2-Bro as a bioactive compound.

Fruit Bromelain-Derived Peptide Potentially Restrains the Attachment of SARS-CoV-2 Variants to hACE2: A Pharmacoinformatics Approach.

Before entering the cell, the SARS-CoV-2 spike glycoprotein receptor-binding domain (RBD) binds to the human angiotensin-converting enzyme 2 (hACE2) receptor. Hence, this RBD is a critical target for the development of antiviral agents. Recent studies have discovered that SARS-CoV-2 variants with mutations in the RBD have spread globally. The purpose of this in silico study was to determine the potential of a fruit bromelain-derived peptide. DYGAVNEVK. to inhibit the entry of various SARS-CoV-2 variants into human cells by targeting the hACE binding site within the RBD. Molecular docking analysis revealed that DYGAVNEVK interacts with several critical RBD binding residues responsible for the adhesion of the RBD to hACE2. Moreover, 100 ns MD simulations revealed stable interactions between DYGAVNEVK and RBD variants derived from the trajectory of root-mean-square deviation (RMSD), radius of gyration (Rg), and root-mean-square fluctuation (RMSF) analysis, as well as free binding energy calculations. Overall, our computational results indicate that DYGAVNEVK warrants further investigation as a candidate for preventing SARS-CoV-2 due to its interaction with the RBD of SARS-CoV-2 variants.

Beneficial Properties of Bromelain.

Bromelain is a major sulfhydryl proteolytic enzyme found in pineapple plants, having multiple activities in many areas of medicine. Due to its low toxicity, high efficiency, high availability, and relative simplicity of acquisition, it is the object of inexhaustible interest of scientists. This review summarizes scientific reports concerning the possible application of bromelain in treating cardiovascular diseases, blood coagulation and fibrinolysis disorders, infectious diseases, inflammation-associated diseases, and many types of cancer. However, for the proper application of such multi-action activities of bromelain, further exploration of the mechanism of its action is needed. It is supposed that the anti-viral, anti-inflammatory, cardioprotective and anti-coagulatory activity of bromelain may become a complementary therapy for COVID-19 and post-COVID-19 patients. During the irrepressible spread of novel variants of the SARS-CoV-2 virus, such beneficial properties of this biomolecule might help prevent escalation and the progression of the COVID-19 disease.

Exploring Effects of Protease Choice and Protease Combinations in Enzymatic Protein Hydrolysis of Poultry By-Products.

A study of the effects of single and combined protease hydrolysis on myofibrillar versus collagenous proteins of poultry by-products has been conducted. The aim was to contribute with knowledge for increased value creation of all constituents of these complex by-products. A rational approach was implemented for selecting proteases exhibiting the most different activity towards the major protein-rich constituents of mechanically deboned chicken residue (MDCR). An initial activity screening of 18 proteases on chicken meat, turkey tendons and MDCR was conducted. Based on weight yield, size exclusion chromatography (SEC) and SDS-PAGE, stem Bromelain and Endocut-02 were selected. Studies on hydrolysis of four different poultry by-products at 40 °C, evaluated by protein yield, SEC, and SDS-PAGE, indicate that the proteases' selectivity difference can be utilized in tailor-making hydrolysates, enriched in either meat- and collagen-derived peptides or gelatin. Three modes of stem Bromelain and Endocut-02 combinations during hydrolysis of MDCR were performed and compared with single protease hydrolysis. All modes of the protease combinations resulted in a similar approximately 15% increase in product yield, with products exhibiting similar SEC and SDS-PAGE profiles. This shows that irrespective of the modes of combination, the use of more than one enzyme in hydrolysis of collagen-rich material can provide means to increase the total protein yield and ultimately contribute to increased value creation of poultry by-products.

Anti-Inflammatory Effect of Pineapple Rhizome Bromelain through Downregulation of the NF-κB- and MAPKs-Signaling Pathways in Lipopolysaccharide (LPS)-Stimulated RAW264.7 Cells.

Bromelain is a mixture of proteolytic enzymes derived from pineapple (Ananas comosus) fruit and stem possessing several beneficial properties, particularly anti-inflammatory activity. However, the molecular mechanisms underlying the anti-inflammatory effects of bromelain are unclear. This study investigated the anti-inflammatory effects and inhibitory molecular mechanisms of crude and purified rhizome bromelains on lipopolysaccharide (LPS)-induced inflammation in RAW 264.7 macrophage cells. RAW264.7 cells were pre-treated with various concentrations of crude bromelain (CB) or purified bromelain (PB), and then treated with LPS. The production levels of pro-inflammatory cytokines and mediators, including nitric oxide (NO), interleukin (IL)-6, and tumor necrosis factor (TNF)-α were determined by Griess and ELISA assays. The expressions of inducible nitric oxide synthetase (iNOS), cyclooxygenase (COX)-2, nuclear factor kappa B (NF-κB), and mitogen-activated protein kinases (MAPKs)-signaling pathway-related proteins were examined by western blot analysis. The pre-treatment of bromelain dose-dependently reduced LPS-induced pro-inflammatory cytokines and mediators, which correlated with downregulation of iNOS and COX-2 expressions. The inhibitory potency of PB was stronger than that of CB. PB also suppressed phosphorylated NF-κB (p65), nuclear factor of kappa light polypeptide gene enhancer in B-cells inhibitor alpha, extracellular signal-regulated kinases, c-Jun amino-terminal kinases, and p38 proteins in LPS-treated cells. PB then exhibited potent anti-inflammatory effects on LPS-induced inflammatory responses in RAW264.7 cells by inhibiting the NF-κB and MAPKs-signaling pathways.

In vitro performance of free and encapsulated bromelain.

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.

Physicochemical characteristics and textural parameters of restructured pork steaks hydrolysed with bromelain.

Enzymatic tenderisation including bromelain enhances underused cuts of meat in emerged restructuring technology. Physicochemical and textural characteristics of restructured pork steak hydrolysed with bromelain for masticatory dysfunction people were evaluated. Restructured pork steak treated with bromelain at 0.05 and 0.1% (w/w) was hydrolysed at 50 °C for 0, 3, 6, 9 and 12 min. The cooking losses of 0.05% (w/w) bromelain for 0, 3 and 6 min were lower than 0.1% (w/w) bromelain samples. The ΔE increased after increasing the enzyme concentration and hydrolysis time. Bromelain-treated samples at higher concentrations showed lower WBSF, KSF and TPA parameters, but cohesiveness of 0.05% (w/w) had higher than 0.1% (w/w) bromelain samples. Total protein, sarcoplasmic protein solubility, TCA-soluble peptide, total collagen and soluble collagen contents were the highest in 0.1% (w/w) bromelain-treated samples for 12 min (P < 0.05). According to SDS-PAGE and SEM, various proteins in the enzyme-treated samples were degraded.

Novel biotechnological formulations of cysteine proteases, immobilized on chitosan. Structure, stability and activity.

Bromelain, papain, and ficin are studied the most for meat tenderization, but have limited application due to their short lifetime. The aim of this work is to identify the adsorption mechanisms of these cysteine proteases on chitosan to improve the enzymes' stability. It is known that immobilization can lead to a significant loss of enzyme activity, which we observed during the sorption of bromelain (protease activity compared to soluble enzyme is 49% for medium and 64% for high molecular weight chitosan), papain (34 and 28% respectively) and ficin (69 and 70% respectively). Immobilization on the chitosan matrix leads to a partial destruction of protein helical structure (from 5 to 19%). Using computer modelling, we have shown that the sorption of cysteine proteases on chitosan is carried out by molecule regions located on the border of domains L and R, including active cites of the enzymes, which explains the decrease in their catalytic activity upon immobilization. The immobilization on chitosan does not shift the optimal range of pH (7.5) and temperature values (60 °C for bromelain and papain, 37-60 °C for ficin), but significantly increases the stability of biocatalysts (from 5.8 times for bromelain to 7.6 times for papain).

Cytotoxic Effect of Bromelain on HepG2 Hepatocellular Carcinoma Cell Line.

Cancer is a complicated long-term disease due to computable key molecular players involved in aggravating the disease. Among various kinds of cancer, hepatocellular carcinoma (HCC) is the ninth leading cause of cancer. Recently, plant-based products are gaining a lot of attention in the field of research because of their anti-tumor properties. In our previous study, we reported based on in-silico method that bromelain, a cysteine protease extracted from the stem of the pineapple, has high binding affinity with the transcription factors p53 and ß-catenin proteins which are key players in controlling the progression of hepatocellular carcinoma. Bromelain, isolated mainly from the stem of Pineapple (Ananas comosus), belongs to the family Bromeliaceae. The present study deals with preclinical analysis of bromelain as an anti-cancer agent and its intracellular effect on the expression of p53 and ß-catenin protein. Our study reports cytotoxic activity, cell proliferation, migration, invasion, arrest in the S-phase, and G2/M phase in cell cycle analysis by treating with bromelain in HepG2 cell lines. We also report up-regulation of p53 protein by drug-induced impediment leading to apoptotic process in HepG2 cells and down-regulation of ß-catenin protein in HepG2 cells which interferes in ß-catenin/TCF-DNA interaction further, down-regulating Wnt genes and suppressing the canonical pathway. Finally, we conclude that bromelain inhibits tumorigenic potential in HepG2 cell lines.

Soluble Expression and Catalytic Properties of Codon-Optimized Recombinant Bromelain from MD2 Pineapple in Escherichia coli.

Bromelain, a member of cysteine proteases, is found abundantly in pineapple (Ananas comosus), and it has a myriad of versatile applications. However, attempts to produce recombinant bromelain for commercialization purposes are challenging due to its expressibility and solubility. This study aims to express recombinant fruit bromelain from MD2 pineapple (MD2Bro; accession no: OAY85858.1) in soluble and active forms using Escherichia coli host cell. The gene encoding MD2Bro was codon-optimized, synthesized, and subsequently ligated into pET-32b( +) for further transformation into Escherichia coli BL21-CodonPlus(DE3). Under this strategy, the expressed MD2Bro was in a fusion form with thioredoxin (Trx) tag at its N-terminal (Trx-MD2Bro). The result showed that Trx-MD2Bro was successfully expressed in fully soluble form. The protein was successfully purified using single-step Ni2+-NTA chromatography and confirmed to be in proper folds based on the circular dichroism spectroscopy analysis. The purified Trx-MD2Bro was confirmed to be catalytically active against N-carbobenzoxyglycine p-nitrophenyl ester (N-CBZ-Gly-pNP) with a specific activity of 6.13 ± 0.01 U mg-1 and inhibited by a cysteine protease inhibitor, E-64 (IC50 of 74.38 ± 1.65 nM). Furthermore, the catalytic efficiency (kcat/KM) Trx-MD2Bro was calculated to be at 5.64 ± 0.02 × 10-2 µM-1 s-1 while the optimum temperature and pH were at 50 °C and pH 6.0, respectively. Furthermore, the catalytic activity of Trx-MD2Bro was also affected by ethylenediaminetetraacetic acid (EDTA) or metal ions. Altogether it is proposed that the combination of codon optimization and the use of an appropriate vector are important in the production of a soluble and actively stable recombinant bromelain.

Structures of the free and inhibitors-bound forms of bromelain and ananain from Ananas comosus stem and in vitro study of their cytotoxicity.

The Ananas comosus stem extract is a complex mixture containing various cysteine ​​proteases of the C1A subfamily, such as bromelain and ananain. This mixture used for centuries in Chinese medicine, has several potential therapeutic applications as anti-cancer, anti-inflammatory and ecchymosis degradation agent. In the present work we determined the structures of bromelain and ananain, both in their free forms and in complex with the inhibitors E64 and TLCK. These structures combined with protease-substrate complexes modeling clearly identified the Glu68 as responsible for the high discrimination of bromelain in favor of substrates with positively charged residues at P2, and unveil the reasons for its weak inhibition by cystatins and E64. Our results with purified and fully active bromelain, ananain and papain show a strong reduction of cell proliferation with MDA-MB231 and A2058 cancer cell lines at a concentration of about 1 µM, control experiments clearly emphasizing the need for proteolytic activity. In contrast, while bromelain and ananain had a strong effect on the proliferation of the OCI-LY19 and HL-60 non-adherent cell lines, papain, the archetypal member of the C1A subfamily, had none. This indicates that, in this case, sequence/structure identity beyond the active site of bromelain and ananain is more important than substrate specificity.

Excellency of pyrimidinyl moieties containing α-aminophosphonates over benzthiazolyl moieties for thermal and structural stability of stem bromelain.

An efficient approach has been made for the synthesis of a series of novel di α-aminophosphonates by the reaction of terephthalaldehyde with various pyrimidine/benzthiazole amines and diethyl phosphite using sulfonated graphitic carbon nitride - SA@g-C3N4 as catalyst under room temperature and solvent free conditions. Later, the different effects of these newly synthesized α-aminophosphonates as a function of concentration gradient has been scrutinized on the thermal and structural stability of stem bromelain (SBM) through combining the results of various spectroscopic techniques like UV-vis, steady state fluorescence and circular dichroism (CD). Lastly the competitive and distinctive behaviour of α-aminophosphonates towards the stability of SBM has been envisaged using molecular docking simulations which suggest that nature of α-aminophosphonates plays a crucial role for their interactions with SBM. Molecular docking results clearly show that α-aminophosphonates with pyrimidine ring are having more number of hydrogen bonding interaction with amino acid residues of SBM than α-aminophosphonates with benzthiazolyl ring. Sequentially for thermal and structure stability of SBM, concentration of α-aminophosphonates plays an inexorable role and through these results it must be concluded that most of the α-aminophosphonates are stabilizing the SBM upto the 0. 1 mM concentration.

Investigating the calcium binding characteristics of black bean protein hydrolysate.

The black bean protein has been widely utilized to prepare hydrolysates with different bioactive properties. Herein, we hydrolyzed the black bean protein to prepare hydrolysate with calcium binding activity and characterized its behavior. Our results showed that ficin was superior in obtaining hydrolysate with calcium binding capacity in comparison with trypsin, alcalase and bromelain. In particular, the optimal capacity of ficin hydrolysate reached 77.54 ± 1.61 µg mg-1, where the optimal hydrolysis conditions of ficin were a temperature of 70 °C, a pH value of 6.2, an enzyme concentration of 1.61% and a time of 3 h. This might be due to high proportions of aspartic acid and glutamic acid (35.59%). Further spectral analysis evidenced the formation of hydrolysate-calcium complexes, demonstrating that the interaction between hydrolysate and calcium ions primarily occur on carboxyl oxygen atoms and amino nitrogen atoms. These findings provide a possible utilization of black bean hydrolysate to serve as a calcium supplement nutraceutical to enhance the absorption and bioavailability.

Bromelain from Ananas comosus stem attenuates oxidative toxicity and testicular dysfunction caused by aluminum in rats.

BACKGROUND: Aluminum (Al) has been reported to induce testicular injury via oxidative stress. Ananas comosus stem extract is an inexpensive byproduct waste rich in bromelain which is a group of sulfur-containing enzymes known for its biological activities and medicinal applications. So, the current investigation aims to evaluate the efficacy of bromelain in counteracting oxidative injury and testicular dysfunction stimulated by aluminum in rats. METHODS: Male adult Wistar rats were divided into four groups. The first group used as control, however, the second and third groups were received bromelain (250 mg/kg) and AlCl3 (34 mg/Kg, 1/25 LD50), and the fourth group supplemented with bromelain one hour before AlCl3 intoxication, respectively. Bromelain was administered daily while AlCl3 was given every other day by oral gavages for one month. RESULTS: Al intoxicated animals revealed an elevation in lipid peroxidation (TBARS and H2O2) level and lactate dehydrogenase (LDH) activity. However, reduced glutathione (GSH) and protein contents, antioxidant enzymes (SOD, CAT, GPx, GR, GST), phosphatases (ALP, AcP) and aminotransferases (AST, ALT) activities were significantly reduced. Additionally, considerable amendments in hormonal levels (testosterone, luteinizing and follicle-stimulating hormone) and sperm characteristics were spotted. Further, histological variations in the testes section were detected and this supports the biochemical observations. Otherwise, rats supplemented with bromelain alone diminished TBARS and H2O2 and augmented mostly other parameters. Furthermore, supplementation with bromelain before Al intoxication in rats exhibited worthy betterment in oxidative stress markers, hormones, and sperm quality compared to Al treated group. CONCLUSION: In conclusion, bromelain had a powerful protective role against Al-induced testicular dysfunction so, it represents a novel approach in metal toxicity processing.

pH-sensitive bromelain nanoparticles by ortho ester crosslinkage for enhanced doxorubicin penetration in solid tumor.

Dense extracellular matrix (ECM) is a primary obstacle that restrains the permeation of therapeutic drugs in tumor tissues. Degrading ECM with bromelain (Br) to increase drug penetration is an attractive strategy to enhance antitumor effects. However, the poor stability in circulation and potential immunogenicity severely limit their applications. In this work, a novel pH-sensitive nanocarrier was prepared by crosslinking Br with an ortho ester-based crosslink agent, and Br still retained a certain ability to degrade ECM after crosslinking. The nanoparticles showed higher DOX release rate than non-sensitive nanoparticles, and DOX release amount reached to 86% at pH 5.5 within 120 h. In vivo experiments revealed that the pH-sensitive nanoparticles could be degraded in mildly acidic condition, and the released Br further promoted nanoparticles penetration in tumor parenchyma via in situ hydrolysis of ECM. Furthermore, Br itself could inhibit the proliferation of tumor cells at high concentration, and produce synergistic antitumor effects with DOX. Finally, tumor growth inhibition of these nanoparticles reached to 62.5%. Overall, the bromelain-based pH-sensitive nanoparticles can be potential drug carriers for efficient drug delivery and tumor treatment.

Stem Bromelain Proteolytic Machinery: Study of the Effects of its Components on Fibrin (ogen) and Blood Coagulation.

BACKGROUND: Antiplatelet, anticoagulant and fibrinolytic activities of stem bromelain (EC 3.4.22.4) are well described, but more studies are still required to clearly define its usefulness as an antithrombotic agent. Besides, although some effects of bromelain are linked to its proteolytic activity, few studies were performed taking into account this relationship. OBJECTIVE: We aimed at comparing the effects of stem bromelain total extract (ET) and of its major proteolytic compounds on fibrinogen, fibrin, and blood coagulation considering the proteolytic activity. METHODS: Proteolytic fractions chromatographically separated from ET (acidic bromelains, basic bromelains, and ananains) and their irreversibly inhibited counterparts were assayed. Effects on fibrinogen were electrophoretically and spectrophotometrically evaluated. Fibrinolytic activity was measured by the fibrin plate assay. The effect on blood coagulation was evaluated by the prothrombin time (PT) and activated partial thromboplastin time (APTT) tests. Effects were compared with those of thrombin and plasmin. RESULTS: Acidic bromelains and ananains showed thrombin-type activity and low fibrinolytic activity, with acidic bromelains being the least effective as anticoagulants and fibrinolytics; while basic bromelains, without thrombin-like activity, were the best anticoagulant and fibrinolytic proteases present in ET. Procoagulant action was detected for ET and its proteolytic compounds by the APTT test at low concentrations. The measured effects were dependent on proteolytic activity. CONCLUSION: Two sub-populations of cysteine proteases exhibiting different effects on fibrin (ogen) and blood coagulation are present in ET. Using well characterized stem bromelain regarding its proteolytic system is a prerequisite for a better understanding of the mechanisms underlying the bromelain action.