Persistence Enhancement of a Promising Tick Repellent, Benzyl Isothiocyanate, by Yeast Microcarriers

Phenethyl isothiocyanate isolated from Armoracia rusticana root oil and its derivatives were tested at different doses in a bioassay designed to evaluate repellency against individual Haemaphysalis longicornis nymphs. Among the tested compounds, benzyl isothiocyanate exhibited repellency against H. longicornis nymphs at the lowest dose of 0.00625 mg/cm2, followed by phenethyl isothiocyanate (0.0125 mg/cm2) and phenyl isothiocyanate (0.025 mg/cm2). The behavioral responses of H. longicornis nymphs exposed to benzyl isothiocyanate and phenethyl isothiocyanate indicated that the mode of action of these compounds can be mainly attributed to the vapor phase. Encapsulated benzyl isothiocyanate showed repellency up to 120 min post-application at 0.1 mg/cm2, whereas pure benzyl isothiocyanate showed repellency up to 60 min post-application at 0.1 mg/cm2. The present study suggests that benzyl isothiocyanate is a potential repellent for protection against H. longicornis nymphs, and encapsulation in yeast cells may enhance the repellency effect.

Benzyl Isothiocyanate, a Vegetable-Derived Compound, Induces Apoptosis via ROS Accumulation and DNA Damage in Canine Lymphoma and Leukemia Cells

Treatment of neoplastic diseases in companion animals is one of the most important problems of modern veterinary medicine. Given the growing interest in substances of natural origin as potential anti-cancer drugs, our goal was to examine the effectiveness of benzyl isothiocyanate (BITC), a compound found in cruciferous vegetables, against canine lymphoma and leukemia. These are the one of the most common canine cancer types, and chemotherapy is the only treatment option. The study involved established cell lines originating from various hematopoietic malignancies: CLBL-1, GL-1, CLB70 and CNK-89, immortalized noncancerous cell lines: MDCK and NIH-3T3 and canine peripheral blood mononuclear cells (PBMCs). The cytotoxic activity of BITC, apoptosis induction, caspase activity and ROS generation were evaluated by flow cytometry. H2AX phosphorylation was assessed by western blot. The study showed that the compound was especially active against B lymphocyte-derived malignant cells. Their death resulted from caspase-dependent apoptosis. BITC induced ROS accumulation, and glutathione precursor N-acetyl-l-cysteine reversed the effect of the compound, thus proving the role of oxidative stress in BITC activity. In addition, exposure to the compound induced DNA damage in the tested cells. This is the first study that provides information on the activity of BITC in canine hematopoietic malignancies and suggests that the compound may be particularly useful in B-cell neoplasms treatment.

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.

Bacteriostatic effects of benzyl isothiocyanate on Vibrio parahaemolyticus: Transcriptomic analysis and morphological verification

Background Foodborne illness caused by Vibrio parahaemolyticus (V. parahaemolyticus) is generally associated with the consumption of seafood. Fish and other seafood can be contaminated with V. parahaemolyticus, natural inhabitants of the marine, estuarine, and freshwater environment. In this study, the antibacterial activities of benzyl isothiocyanate (BITC) against V. parahaemolyticus were investigated by both transcriptomic analysis and morphological verification. Results Treatment with 1/8 minimum inhibitory concentration (1/8 MIC) BITC resulted in 234 upregulated genes and 273 downregulated genes. The results validated by quantitative real-time polymerase chain reaction (qRT-PCR) revealed that the relative expression levels of the six genes VP0820, VP0548, VP2233, VPA2362, fliA and fliG were only 31.0%, 31.1%, 55.8%, 57.0%, 75.3%, and 79.9% of the control group, respectively. Among them, genes VP2233, fliA and fliG are related to flagella and VP2362 can regulate a protein relevant to biofilm formation. Morphologically, we verified that the swimming diffusion diameter of V. parahaemolyticus was significantly reduced by 14.9% by bacterial swimming ability, and biofilm formation was significantly inhibited by treatment with 1/8 MIC BITC by crystal violet quantification assay. Conclusions These results indicated that 1/8 MIC BITC had antibacterial effect on V. parahaemolyticus by inhibiting virulence gene expression related to flagella and biofilm.

The Effect of Benzyl Isothiocyanate On The Expression of Genes Encoding NADH Oxidase And Fibronectin-Binding Protein In Oral Streptococcal Biofilms

Background: Recent studies have shown that antibiotic treatment results in up- or down regulation of several virulence-associated genes. The genes encoding NADH oxidase (nox) and fibronectin-binding protein (fbp) are known to play important roles in biofilms of some oral bacterial species. The objective was to study the effect of benzyl isothiocyanate (BITC), an antimicrobial agent from Miswak plant, on the expression of nox and fbp genes in some oral streptococci.Methods: Bacterial strains were grown as biofilms in brucella broth. The crystal violet stained biofilms were quantified by optical density measurements at 590 nm. The biofilms were treated with an antimicrobial agent (BITC) for 2 h and fold change in mRNA expression of nox and fbp genes in BITC treated selected oral streptococci was measured by comparative ∆∆Ct method on Real-Time PCR machine.Results: The highest amount of biofilm mass was produced by A. defectiva, followed by S. gordonii, S. mutans, G. elegans and G. adiacens. Upon treatment with BITC, S. gordonii biofilms showed highest mRNA expression for both fbp and nox genes. Mean (SE) folds increase in the expression of nox mRNA: S. gordonii 2 (0.30), followed by S. mutans 1.25 (0.18), A. defectiva 1.03 (0.09), G. adiacens 0.7 (0.03). Similarly for fbp, folds increase in mRNA expression was: S. gordonii 2.65 (0.03), followed by A. defectiva 2.09 (0.60), G. elegans 1.61 (0.40), S. mutans 1.57 (0.20), and G. adiacens 0.58 (0.06). G. elegans mRNA levels for nox were extremely low (0.006-fold). Conclusion: BITC treatment of the biofilms caused an upregulation of biofilm-associated genes fbp and nox genes in most of the tested species suggesting the significance of these genes in biofilm lifestyle of these oral bacteria. Increased expression of nox and fbp genes in the biofilm lifestyle of these species needs further investigation to understand if it contributes to antimicrobial resistance.