A monocentric, randomized, double-blind, controlled crossover trial of nasturtium (Tropaeolum majus) on the lipid regulator prostaglandin E2.

Scope: As prostaglandin E2 (PGE2) has important roles in physiological and inflammatory functions, a double-blind randomized controlled crossover study to investigate the potential of nasturtium (Tropaeolum majus) for modulating PGE2 was conducted, aiming at clarifying the role of benzyl isothiocyanate (BITC). As secondary parameters leukotriene 4 (LTB4), and cytokine release (tumor necrosis factor alpha, TNF-α; interleukins IL-1ß, IL-10, and IL-12) were quantified. Methods and results: Thirty-four healthy female participants consumed 1.5 g nasturtium containing BITC, (verum) or no BITC (control) twice a day for 2 weeks each. Nasturtium intervention resulted in an increase in mean PGE2 levels in serum samples (verum: 1.76-fold, p ≤ 0.05; control: 1.78-fold, p ≤ 0.01), and ex vivo stimulated peripheral blood mononuclear cells (PBMC) (verum: 1.71-fold, p ≤ 0.01; control: 1.43-fold). Using a pre-to-post responder analysis approach, 18 of 34 subjects showed a > 25% PGE2 increase in serum, while it was >25% decreased for 9 subjects (stimulated PBMC: 14 and 8 of 28, respectively). Under the selected conditions, the BITC content of nasturtium did not affect the observed changes in PGE2. Verum intervention also increased mean LTB4 serum level (1.24-fold, p ≤ 0.01), but not in LPS stimulated PBMC, and significantly increased TNF-α release in stimulated PBMC after 3 h (verum: 1.65-fold, p = 0.0032; control: 1.22-fold, p = 0.7818). No change was seen in the anti-inflammatory cytokine IL-10, or the pro-inflammatory cytokines IL-1ß, and IL-12. Conclusion: In contrast to the previously reported in vitro results, on average, LPS activated PBMC and serum from both groups showed increased PGE2 levels. Further analyses suggest that PGE2 release after intervention could possibly depend on the baseline PGE2 level. Identification of phenotypes that respond differently to the nasturtium intervention could be useful to establish personalized approaches for dosing phytopharmaceuticals medicines.

Characterization of Conjugates between α-Lactalbumin and Benzyl Isothiocyanate-Effects on Molecular Structure and Proteolytic Stability.

In complex foods, bioactive secondary plant metabolites (SPM) can bind to food proteins. Especially when being covalently bound, such modifications can alter the structure and, thus, the functional and biological properties of the proteins. Additionally, the bioactivity of the SPM can be affected as well. Consequently, knowledge of the influence of chemical modifications on these properties is particularly important for food processing, food safety, and nutritional physiology. As a model, the molecular structure of conjugates between the bioactive metabolite benzyl isothiocyanate (BITC, a hydrolysis product of the glucosinolate glucotropaeolin) and the whey protein α-lactalbumin (α-LA) was investigated using circular dichroism spectroscopy, anilino-1-naphthalenesulfonic acid fluorescence, and dynamic light scattering. Free amino groups were determined before and after the BITC conjugation. Finally, mass spectrometric analysis of the BITC-α-LA protein hydrolysates was performed. As a result of the chemical modifications, a change in the secondary structure of α-LA and an increase in surface hydrophobicity and hydrodynamic radii were documented. BITC modification at the ε-amino group of certain lysine side chains inhibited tryptic hydrolysis. Furthermore, two BITC-modified amino acids were identified, located at two lysine side chains (K32 and K113) in the amino acid sequence of α-LA.

Benzyl isothiocyanate-modified α-lactalbumin - Two-dimensional high-performance thin-layer chromatography for analyzing modified peptides.

In complex food matrices, non-directed reactions between food proteins and secondary plant metabolites (SPM) are conceivable. In this study, the interaction between the bioactive metabolite from garden cress (Lepidium sativum) and selected Brassicaceae - benzyl isothiocyanate (BITC) - and the dairy protein α-lactalbumin (α-LA) was investigated. It was focused on monitoring the proteolytic degradation behaviour of unmodified and BITC-modified α-LA with two-dimensional high-performance thin-layer chromatography (2D-HPTLC). The two-dimensional approach of HPTLC offers high resolution in the separation of complex peptide mixtures and might enable differentiation of protein modifications. Based on the specific peptide patterns of native and modified peptides, conclusions can be drawn about differences in protein/peptide polarity, location of a modification, and digestibility. The aim was to characterize tryptically hydrolyzed unmodified and BITC-modified peptides using the 2D method and to investigate the influence of BITC modification of α-LA on polarity and digestibility. To determine the repeatability of peptide separation by 2D-HPTLC, the unmodified and BITC-modified protein hydrolyzates were separated six times. The absolute standard deviations between the retardation factors of the individual peptide spots varied between 0.52 and 4.79 mm for the x-coordinates and between 0.41 and 6.47 mm for the y-coordinates for all three samples. Here, the mean relative standard deviations ranged from 5.80 to 10.4% for the x-coordinates and from 5.91 to 18.3% for the y-coordinates. The results of the tryptic hydrolysis indicated that, depending on the concentration of BITC used, the modification sterically hinders the cleavage sites for the enzyme, resulting in a reduced digestibility. Covalent binding of the hydrophobic BITC altered the digestibility and polarity of the protein, leading to a difference in peptide patterns between the unmodified and modified α-LA. It was concluded that the reaction was undirected, resulting in a mixture of unmodified and modified peptides, and that elongated modified peptides were formed by BITC blocking of trypsin cleavage sites.

Allyl Isothiocyanate: A TAS2R38 Receptor-Dependent Immune Modulator at the Interface Between Personalized Medicine and Nutrition.

Understanding individual responses to nutrition and medicine is of growing interest and importance. There is evidence that differences in bitter taste receptor (TAS2R) genes which give rise to two frequent haplotypes, TAS2R38-PAV (functional) and TAS2R38-AVI (non-functional), may impact inter-individual differences in health status. We here analyzed the relevance of the TAS2R38 receptor in the regulation of the human immune response using the TAS2R38 agonist allyl isothiocyanate (AITC) from Brassica plants. A differential response in calcium mobilization upon AITC treatment in leucocytes from healthy humans confirmed a relevance of TAS2R38 functionality, independent from cation channel TRPV1 or TRPA1 activation. We further identified a TAS2R38-dependence of MAPK and AKT signaling activity, bactericidal (toxicity against E. coli) and anti-inflammatory activity (TNF-alpha inhibition upon cell stimulation). These in vitro results were derived at relevant human plasma levels in the low micro molar range as shown here in a human intervention trial with AITC-containing food.

Immunological Analysis of Isothiocyanate-Modified α-Lactalbumin Using High-Performance Thin Layer Chromatography.

Undirected modifications between food proteins and secondary plant metabolites can occur during food processing. The results of covalent interactions can alter the functional and biological properties of the proteins. The present work studied the extent of which covalent conjugation of the bioactive metabolite benzyl isothiocyanate (BITC; a glucosinolate breakdown product) to the whey protein α-lactalbumin affects the protein's allergenicity. Additional to the immunological analysis of native untreated and BITC-modified α-lactalbumin, the analysis of antigenic properties of proteolytically digested protein derivatives was also performed by high performance thin layer chromatography and immunostaining. As a result of the chemical modifications, structural changes in the protein molecule affected the allergenic properties. In this process, epitopes are destroyed or inactivated, but at the same time, buried epitopes can be exposed or newly formed, so that the net effect was an increase in allergenicity, in this case. Results from the tryptic hydrolysis suggest that BITC conjugation sterically hindered the cleavage sites for the enzyme, resulting in reduced digestibility and allergenicity. Residual antigenicity can be still present as short peptide fragments that provide epitopes. The desire to make food safer for allergy sufferers and to protect sensitized individuals from an allergenic reaction makes it clear that the detection of food antigens is mandatory; especially by considering protein interactions.