Potential of Sulforaphane and Broccoli Membrane Vesicles as Regulators of M1/M2 Human Macrophage Activity.

Macrophages have emerged as important therapeutic targets in many human diseases. The aim of this study was to analyze the effect of broccoli membrane vesicles and sulphoraphane (SFN), either free or encapsulated, on the activity of human monocyte-derived M1 and M2 macrophage primary culture. Our results show that exposure for 24 h to SFN 25 µM, free and encapsulated, induced a potent reduction on the activity of human M1 and M2 macrophages, downregulating proinflammatory and anti-inflammatory cytokines and phagocytic capability on C. albicans. The broccoli membrane vesicles do not represent inert nanocarriers, as they have low amounts of bioactive compounds, being able to modulate the cytokine production, depending on the inflammatory state of the cells. They could induce opposite effects to that of higher doses of SFN, reflecting its hormetic effect. These data reinforce the potential use of broccoli compounds as therapeutic agents not only for inflammatory diseases, but they also open new clinical possibilities for applications in other diseases related to immunodeficiency, autoimmunity, or in cancer therapy. Considering the variability of their biological effects in different scenarios, a proper therapeutic strategy with Brassica bioactive compounds should be designed for each pathology.

Nanoencapsulation of Bimi® extracts increases its bioaccessibility after in vitro digestion and evaluation of its activity in hepatocyte metabolism.

Isothiocyanates (ITCs) have low stability in aqueous conditions, reducing their bioavailability when used as food ingredients. Therefore, the aim of this work was to increase the stability of the ITCs present in extracts of Bimi® edible parts by nanoencapsulation using cauliflower-derived plasma membrane vesicles. The bioactivity of these nanoencapsulates was evaluated in a HepG2 hepatocyte cell line in a model for low-grade chronic inflammation. The vesicles showed a higher capacity of retention in the in vitro gastrointestinal digestion for 3,3-diindolylmethane (DIM), indole-3-carbinol (I3C) and sulforaphane (SFN). Furthermore, Transmission Electron Microscopy (TEM) analysis of the vesicles revealed a decreased size under acidic pH and a release of their cargo after the intestinal digestion. The HepG2 experiments revealed differences in metabolism under the condition of chronic inflammation. The cauliflower-derived plasma membrane vesicles are able to enhance the stability of ITCs through the in vitro gastrointestinal digestion, improving their bioaccesibility and potential bioavailability.

Membrane Vesicles for Nanoencapsulated Sulforaphane Increased Their Anti-Inflammatory Role on an In Vitro Human Macrophage Model.

At present, there is a growing interest in finding new non-toxic anti-inflammatory drugs to treat inflammation, which is a key pathology in the development of several diseases with considerable mortality. Sulforaphane (SFN), a bioactive compound derived from Brassica plants, was shown to be promising due to its anti-inflammatory properties and great potential, though its actual clinical use is limited due to its poor stability and bioavailability. In this sense, the use of nanocarriers could solve stability-related problems. In the current study, sulforaphane loaded into membrane vesicles derived from broccoli plants was studied to determine the anti-inflammatory potential in a human-macrophage-like in vitro cell model under both normal and inflammatory conditions. On the one hand, the release of SFN from membrane vesicles was modeled in vitro, and two release phases were stabilized, one faster and the other slower due to the interaction between SFN and membrane proteins, such as aquaporins. Furthermore, the anti-inflammatory action of sulforaphane-loaded membrane vesicles was demonstrated, as a decrease in interleukins crucial for the development of inflammation, such as TNF-α, IL-1ß and IL-6, was observed. Furthermore, these results also showed that membrane vesicles by themselves had anti-inflammatory properties, opening the possibility of new lines of research to study these vesicles, not only as carriers but also as active compounds.

Bioactive peptides from broccoli stems strongly enhance regenerative keratinocytes by stimulating controlled proliferation.

CONTEXT: As the interest on the research of plant derived bioactive peptides (BPs) for nutraceutical, cosmeceutical and medical applications is increasing, in this work, the application of peptide derived from broccoli to keratinocytes was studied. OBJECTIVE: We focussed on the characterization of different peptides hydrolysates from broccoli stems [extracted from total protein (E) and from membrane protein (MF)], and their activity when applied to human keratinocytes. MATERIALS AND METHODS: Peptide mixtures from broccoli stems (E and MF) were characterized by proteomics. They were applied to HaCaT cells in order to study cytotoxicity in a concentration range between 20 and 0.15625 µg of protein/mL and wound healing was studied after 24 and 48 h of treatment application. Also, proteomic and gene expression of keratinocytes were analysed. RESULTS: Depending on the source, proteins varied in peptide and amino acid composition. An increased proliferation of keratinocytes was shown after the application of the E peptides mixtures, reaching 190% with the lowest concentrations, but enhanced wound healing repair with E and MF appeared, reaching 59% of wound closure after 48 h. At the gene expression and protein levels of keratinocytes, the upregulation of anti-oncogene p53 and keratinization factors were observed. DISCUSSION: These results suggest that peptide mixtures obtained from broccoli augmented cell proliferation and prevented the carcinogenic, uncontrolled growth of the cells, with different mechanisms depending on the protein source. CONCLUSIONS: The results encourage the opening of new lines of research involving the use of Brassica peptides for pharmaceutic or cosmetic use.

Nanoencapsulation of sulforaphane in broccoli membrane vesicles and their in vitro antiproliferative activity.

CONTEXT: The development of nanocarriers of plant origin, such as plant cell membranes, has recently been investigated. Also, plant bioactive compounds as sulforaphane (SFN) from broccoli have recognized antioxidant or anticancer properties. OBJECTIVE: To investigate the capacity of membrane vesicles from broccoli (BM-vesicles) to encapsulate SFN and their application in the cancer cell line. MATERIALS AND METHODS: Physicochemical analysis was carried out to characterize BM-vesicles through different approaches: dynamic light scattering, transmission electron microscopy, stopped-flow analysis, and proteomic analysis. They were applied at different concentrations (BM-vesicles at 0.04-0.00315% of protein and SFN at 5, 25, and 100 µM) in SK-MEL-28 cells during 24 h for studying cytotoxicity and gene expression. RESULTS: The entrapment efficiency was 41%. The anticancer activity tested in cells showed a decrease in proliferation when SFN in BM-vesicles was utilized. Expression patterns when SFN was applied in an encapsulated form showed a reduction of cancer markers and an increase of AQP3. Also, the metabolism of SFN occurred inside of cells, and higher SFN penetrated when it was encapsulated. DISCUSSION: The results showed that encapsulated SFN was better absorbed by melanoma cells providing metabolism products and a reduction of cancer molecular markers. Also aquaporin, AQP3 was pointed to as an important marker since it appeared to play a key role in homeostasis due to the importance of water transport in biological processes. CONCLUSION: These results indicate that SFN and SFN encapsulated in BM-vesicles have a high activity for the inhibition of melanocyte development. Therefore, BM-vesicles could serve as nanocarriers for drugs.

Halophytes of the Mediterranean Basin-Underutilized Species with the Potential to Be Nutritious Crops in the Scenario of the Climate Change.

Halophyte plants are adapted to saline environments and represent a novel type of crops given their possible uses at both culinary and industrial levels. In this work, the nutritional quality of different Mediterranean halophyte species, Atriplex halimus, Salicornia fruticosa, and Cakile maritima, was evaluated under conditions of high salinity. For this, plants were grown at different NaCl concentrations (0, 100, 200, and 300 mM) and the contents of proteins, total lipids, polyphenols, and mineral elements were analyzed as well as growth. Of the three species, C. maritima was the most sensitive to salt stress and therefore showed the highest phenolic compounds content. By contrast, whereas salinity increased the amounts of proteins and phenolics with respect to the control in A. halimus and S. fruticosa, it decreased them in C. maritima. Plants of A. halimus accumulated higher amounts of Na+ in their leaves, but the level of this ion, considering human consumption, was below that of other culinary halophyte species. In conclusion, all the results indicate that these three halophyte species grown at high salt levels represent optimal crops for-new foodstuff-production as green salt or spice due to their nutritional potential.

Genotype influences sulfur metabolism in broccoli (Brassica oleracea L.) under elevated CO2 and NaCl stress.

Climatic change predicts elevated salinity in soils as well as increased carbon dioxide dioxide [CO2] in the atmosphere. The present study aims to determine the effect of combined salinity and elevated [CO2] on sulfur (S) metabolism and S-derived phytochemicals in green and purple broccoli (cv. Naxos and cv. Viola, respectively). Elevated [CO2] involved the amelioration of salt stress, especially in cv. Viola, where a lower biomass reduction by salinity was accompanied by higher sodium (Na(+)) and chloride (Cl(-)) compartmentation in the vacuole. Moreover, salinity and elevated [CO2] affected the mineral and glucosinolate contents and the activity of biosynthetic enzymes of S-derived compounds and the degradative enzyme of glucosinolate metabolism, myrosinase, as well as the related amino acids and the antioxidant glutathione (GSH). In cv. Naxos, elevated [CO2] may trigger the antioxidant response to saline stress by means of increased GSH concentration. Also, in cv. Naxos, indolic glucosinolates were more influenced by the NaCl×CO2 interaction whereas in cv. Viola the aliphatic glucosinolates were significantly increased by these conditions. Salinity and elevated [CO2] enhanced the S cellular partitioning and metabolism affecting the myrosinase-glucosinolate system.

The Acinetobacter baumannii Omp33-36 porin is a virulence factor that induces apoptosis and modulates autophagy in human cells.

Acinetobacter baumannii is an extracellular opportunistic human pathogen that is becoming increasingly problematic in hospitals. In the present study, we demonstrate that the A. baumannii Omp 33- to 36-kDa protein (Omp33-36) is a porin that acts as a channel for the passage of water. The protein is found on the cell surface and is released along with other porins in the outer membrane vesicles (OMVs). In immune and connective cell tissue, this protein induced apoptosis by activation of caspases and modulation of autophagy, with the consequent accumulation of p62/SQSTM1 (sequestosome 1) and LC3B-II (confirmed by use of autophagy inhibitors). Blockage of autophagy enables the bacterium to persist intracellularly (inside autophagosomes), with the subsequent development of cytotoxicity. Finally, we used macrophages and a mouse model of systemic infection to confirm that Omp33-36 is a virulence factor in A. baumannii. Overall, the study findings show that Omp33-36 plays an important role in the pathogenesis of A. baumannii infections.

Natural antioxidants in purple sprouting broccoli under Mediterranean climate.

UNLABELLED: Broccoli, (Brassica oleracea var. Italica), is generally recognized as a nutritive food rich in natural antioxidants, including vitamins and phenolics compounds. Phytochemical composition of 3 different purple sprouting broccoli varieties according with their production cycle: EEP (Extra Early), EP (Early), and LP (Late) were determined. The 3 cultivars tested were grown under the same conditions in an experimental field (SE Spain) where they were not previously cultivated since these cultivars are typical of cold, Northern-European areas. EP variety showed greater growth according to the photosynthetic rate and C assimilation. However, it was not possible to obtain inflorescences for variety LP. The health-promoting compounds (vitamin C, phenolic compounds, and glucosinolates) were higher in purple broccolis than in traditionally grown green broccolis and showed differences according to the variety. With respect to the use of broccoli byproducts as source of antioxidants, any variety would be suitable although the leaves of EP variety seems the richest in total phenolic acids, whereas the amount of anthocyanins was higher in LP variety, followed by EP and EEP varieties. Under our experimental conditions, the levels of glucosinolates were higher in purple broccoli than in green cultivars and also higher than other purple broccolis grown under different climate conditions. Therefore, the results reported data for production of the EEP and EP varieties in the Mediterranean climate with potential to obtain vegetables with improved nutritional quality. PRACTICAL APPLICATION: The results of this study reported data of the health-promoting nutrients and natural antioxidants of EEP, EP, and LP purple sprouting broccoli varieties grown under the Mediterranean climate. The vitamin C, phenolic compounds, and glucosinolates in these purple varieties were higher than in traditionally grown green broccolis and other purple broccolis grown under different climate conditions.

Changes in plasma membrane lipids, aquaporins and proton pump of broccoli roots, as an adaptation mechanism to salinity.

Salinity stress is known to modify the plasma membrane lipid and protein composition of plant cells. In this work, we determined the effects of salt stress on the lipid composition of broccoli root plasma membrane vesicles and investigated how these changes could affect water transport via aquaporins. Brassica oleracea L. var. Italica plants treated with different levels of NaCl (0, 40 or 80mM) showed significant differences in sterol and fatty acid levels. Salinity increased linoleic (18:2) and linolenic (18:3) acids and stigmasterol, but decreased palmitoleic (16:1) and oleic (18:1) acids and sitosterol. Also, the unsaturation index increased with salinity. Salinity increased the expression of aquaporins of the PIP1 and PIP2 subfamilies and the activity of the plasma membrane H(+)-ATPase. However, there was no effect of NaCl on water permeability (P(f)) values of root plasma membrane vesicles, as determined by stopped-flow light scattering. The counteracting changes in lipid composition and aquaporin expression observed in NaCl-treated plants could allow to maintain the membrane permeability to water and a higher H(+)-ATPase activity, thereby helping to reduce partially the Na(+) concentration in the cytoplasm of the cell while maintaining water uptake via cell-to-cell pathways. We propose that the modification of lipid composition could affect membrane stability and the abundance or activity of plasma membrane proteins such as aquaporins or H(+)-ATPase. This would provide a mechanism for controlling water permeability and for acclimation to salinity stress.

Boric acid and salinity effects on maize roots. Response of aquaporins ZmPIP1 and ZmPIP2, and plasma membrane H+-ATPase, in relation to water and nutrient uptake.

Under saline conditions, an optimal cell water balance, possibly mediated by aquaporins, is important to maintain the whole-plant water status. Furthermore, excessive accumulation of boric acid in the soil solution can be observed in saline soils. In this work, the interaction between salinity and excess boron with respect to the root hydraulic conductance (L(0)), abundance of aquaporins (ZmPIP1 and ZmPIP2), ATPase activity and root sap nutrient content, in the highly boron- and salt-tolerant Zea mays L. cv. amylacea, was evaluated. A downregulation of root ZmPIP1 and ZmPIP2 aquaporin contents were observed in NaCl-treated plants in agreement with the L(0) measurements. However, in the H3BO3-treated plants differences in the ZmPIP1 and ZmPIP2 abundance were observed. The ATPase activity was related directly to the amount of ATPase protein and Na+ concentration in the roots, for which an increase in NaCl- and H3BO3+ NaCl-treated plants was observed with respect to untreated and H3BO3-treated plants. Although nutrient imbalance may result from the effect of salinity or H3BO3 alone, an ameliorative effect was observed when both treatments were applied together. In conclusion, our results suggest that under salt stress, the activity of specific membrane components can be influenced directly by boric acid, regulating the functions of certain aquaporin isoforms and ATPase as possible components of the salinity tolerance mechanism.

Different cation stresses affect specifically osmotic root hydraulic conductance, involving aquaporins, ATPase and xylem loading of ions in Capsicum annuum, L. plants.

In order to study the effect of nutrient stress on water uptake in pepper plants (Capsicum annuum L.), the excess or deficiency of the main cations involved in plant nutrition (K(+), Mg(2+), Ca(2+)) and two different degrees of salinity were related to the activity of plasma membrane H(+)-ATPase, the pH of the xylem sap, nutrient flux into the xylem (J(s)) and to a number of parameters related to water relations, such as root hydraulic conductance (L(0)), stomatal conductance (g(s)) and aquaporin activity. Excess of K(+), Ca(+) and NaCl produced a toxic effect on L(0) while Mg(2+) starvation produced a positive effect, which was in agreement with aquaporin functionality, but not with ATPase activity. The xylem pH was altered only by Ca treatments. The results obtained with each treatment could suggest that detection of the quality of the nutrient supply being received by roots can be related to aquaporins functionality, but also that each cation stress triggers specific responses that have to be assessed individually.

New evidence about the relationship between water channel activity and calcium in salinity-stressed pepper plants.

This study, of how Ca2+ availability (intracellular, extracellular or linked to the membrane) influences the functionality of aquaporins of pepper (Capsicum annuum L.) plants grown under salinity stress, was carried out in plants treated with NaCl (50 mM), CaCl2 (10 mM), and CaCl2 (10 mM) + NaCl (50 mM). For this, water transport through the plasma membrane of isolated protoplasts, and the involvement of aquaporins and calcium (extracellular, intracellular and linked to the membrane) has been determined. After these treatments, it could be seen that the calcium concentration was reduced in the apoplast, in the cells and on the plasma membrane of roots of pepper plants grown under saline conditions; these concentrations were increased or restored when extra calcium was added to the nutrient solution. Protoplasts extracted from plants grown under Ca2+ starvation showed no aquaporin functionality. However, for the protoplasts to which calcium was added, an increase of aquaporin functionality of the plasma membrane was observed [osmotic water permeability (Pf) inhibition after Hg addition]. Interestingly, when verapamil (a Ca2+ channel blocker) was added, no functionality was observed, even when Ca2+ was added with verapamil. Therefore, calcium seems to be involved in plasma membrane aquaporin regulation via a chain of processes within the cell but not by alteration of the stability of the plasma membrane.