Revalorization of Broccoli By-Products for Cosmetic Uses Using Supercritical Fluid Extraction.

The agri-food industry is currently one of the main engines of economic development worldwide. The region of Murcia is a reference area in Europe for the cultivation of fruits and vegetables and produces the bulk of Spanish exports of broccoli (Brassica oleracea var. italica). The processing of fresh produce generates a huge number of by-products that represent an important economic and environmental problem when discarded. In this work, an advanced extraction technique using environmentally friendly solvents was applied to assess the revalorization of broccoli by-products, by performing a comparative analysis with conventional extraction. To achieve this goal, supercritical fluid extraction based on response surface methodology was performed using CO2 and ethanol as solvents. The results obtained showed that the supercritical fluid extracts were rich in ß-carotene, phenolic compounds, chlorophylls and phytosterols. Moreover, in bioactivity assays, the supercritical fluid extracts exhibited a high antioxidant activity and a cytoprotective effect in a non-tumorigenic keratinocyte cell line exposed to ultraviolet B light. The results indicate that supercritical fluid extracts from broccoli by-products could potentially serve as an ingredient for cosmetic purposes.

A rational approach to improving the biotechnological production of taxanes in plant cell cultures of Taxus spp.

Taxol is a complex diterpene alkaloid scarcely produced in nature and with a high anticancer activity. Biotechnological systems for taxol production based on cell cultures of Taxus spp. have been developed, but the growing commercial demand for taxol and its precursors requires the optimization of these procedures. In order to increase the biotechnological production of taxol and related taxanes in Taxus spp. cell cultures, it is necessary not only to take an empirical approach that strives to optimize in-put factors (cell line selection, culture conditions, elicitation, up-scaling, etc.) and out-put factors (growth, production, yields, etc.), but also to carry out molecular biological studies. The latter can provide valuable insight into how the enhancement of taxane biosynthesis and accumulation affects metabolic profiles and gene expression in Taxus spp. cell cultures. Several rational approaches have focused on studying the transcriptomic profiles of key genes in the taxol biosynthetic pathway in Taxus spp. cell cultures treated with elicitors such as methyl jasmonate, coronatine and cyclodextrins in relation with the taxane pattern, production and excretion to the culture medium. These studies have provided new insights into the taxol biosynthetic pathway and its regulation. Additionally, identifying genes with low levels of expression even in the presence of elicitors, together with metabolomics studies, has shed light on the limiting steps in taxol biosynthesis and could help define suitable metabolic targets for engineering with the main aim of obtaining highly productive Taxus cultured cells. In this review, we have summarized the latest endeavors to enhance the molecular understanding of the action mechanism of elicitors in Taxus spp. cell cultures. Developments in the ongoing search for new and more effective elicitation treatments and the application of metabolic engineering to design new transgenic cell lines of Taxus with an improved capacity for taxane production are described.

Induction of extracellular defense-related proteins in suspension cultured-cells of Daucus carota elicited with cyclodextrins and methyl jasmonate.

Suspension cultured-cells (SCC) of Daucus carota were used to evaluate the effect of methyl jasmonate and cyclodextrins, separately or in combination, on the induction of defense responses, particularly the accumulation of pathogenesis-related proteins. A comparative study of the extracellular proteome (secretome) between control and elicited carrot SCC pointed to the presence of amino acid sequences homologous to glycoproteins which have inhibitory activity against the cell-wall-degrading enzymes secreted by pathogens and/or are induced when carrot cells are exposed to a pathogen elicitor. Other amino acid sequences were homologous to Leucine-Rich Repeat domain-containing proteins, which play an essential role in defense against pathogens, as well as in the recognition of microorganisms, making them important players in the innate immunity of this plant. Also, some tryptic peptides were shown to be homologous to a thaumatin-like protein, showing high specificity to abiotic stress and to different reticuline oxidase-like proteins that displayed high levels of antifungal activity, suggesting that methyl jasmonate and cyclodextrins could play a role in mediating defense-related gene product expression in SCC of D. carota. Apart from these elicitor-inducible proteins, we observed the presence of PR-proteins in both control and elicited carrot SCC, suggesting that their expression is mainly constitutive. These PR-proteins are putative class IV chitinases, which also have inhibitory activity against pathogen growth and the class III peroxidases that participate in response to environmental stress (e.g. pathogen attack and oxidative), meaning that they are involved in defense responses triggered by both biotic and abiotic factors.

Suspension-cultured plant cells as a tool to analyze the extracellular proteome.

Suspension-cultured cells (SCC) are generally considered the most suitable cell systems to carry out scientific studies, including the extracellular proteome (secretome). SCC are initiated by transferring friable callus fragments into flasks containing liquid culture medium for cell biomass growth, and they are maintained in an orbital shaker to supply the sufficient oxygen that allows cell growth. SCC increase rapidly during the exponential phase and after 10-20 days (depending on the cell culture nature), the growth rate starts to decrease due to limitation of nutrients, and to maintain for decades these kinds of cell cultures is needed to transfer a portion of these SCC into a fresh culture medium. Despite the central role played by extracellular proteins in most processes that control growth and development, the secretome has been less well characterized than other subcellular compartments, meaning that our understanding of the cell wall physiology is still very limited. Useful proteomic tools have emerged in recent years to unravel metabolic network that occurs in cell walls. With the recent progress made in mass spectrometry technology, it has become feasible to identify proteins from a given organ, tissue, cells, or even a subcellular compartment. Compared with other methods used to isolate cell wall proteins, the spent medium of SCC provides a convenient, continuous, and reliable and unique source of extracellular proteins. Therefore, this biological system could be used as a large-scale cell culture from which these proteins can be secreted, easily separated from cells without cell disruption, and so, without any cytosolic contamination, easily recovered from the extracellular medium. This nondestructive cell wall proteome approach discloses a set of proteins that are specifically expressed in the remodelling of the cell wall architecture and stress defense.

Enhancement of phytosterols, taraxasterol and induction of extracellular pathogenesis-related proteins in cell cultures of Solanum lycopersicum cv Micro-Tom elicited with cyclodextrins and methyl jasmonate.

Suspension-cultured cells of Solanum lycopersicum cv Micro-Tom were used to evaluate the effect of methyl jasmonate and cyclodextrins, separately or in combination, on the induction of defense responses. An extracellular accumulation of two sterols (isofucosterol and ß-sitosterol) and taraxasterol, a common tomato fruit cuticular triterpene, were observed. Their levels were higher in Micro-Tom tomato suspension cultured cells elicited with cyclodextrins than in control and methyl jasmonate-treated cells. Also, their accumulation profiles during the cell growth phase were markedly different. The most striking feature in response to cyclodextrin treatments was the observed enhancement of taraxasterol accumulation. Likewise, the exogenous application of methyl jasmonate and cyclodextrins induced the accumulation of pathogenesis-related proteins. Analysis of the extracellular proteome showed the presence of amino acid sequences homologous to pathogenesis-related 1 and 5 proteins, a cationic peroxidase and a biotic cell death-associated protein, which suggests that methyl jasmonate and cyclodextrins could play a role in mediating defense-related gene product expression in S. lycopersicum cv Micro-Tom.

Kinetic characterization of a basic peroxidase from garlic (Allium sativum L.) cloves.

Peroxidases catalyze the reduction of H(2)O(2) by taking electrons from a variety of compounds from the secondary metabolism including flavonoids and lignin precursors. This work describes the purification and kinetic characterization of a basic peroxidase from garlic cloves using quercetin and p-coumaric acid, flavonoid and phenolic compounds found in garlic cloves. The high catalytic efficiency shown by this basic peroxidase in the oxidation of quercetin at acidic pH suggests good adaptation of this enzyme, involved in quercetin catabolism in the acidic physiological pH conditions of the vacuoles, where it is presumably located. Likewise, garlic peroxidase showed similar oxidation rates for hydroxycinnamyl (p-coumaric) and sinapyl-type structures, which suggests its involvement in the cross-coupling reactions that occur in the cell wall during lignification. On the other hand, the high affinity of this enzyme for H(2)O(2) would be in accordance with the oxidation of both flavonoid and phenolic compounds to regulate H(2)O(2) levels in tissues/organelles, where this peroxidase is expressed.