Enzyme Myrosinase Research Articles

A new source of bacterial myrosinase isolated from endophytic Bacillus sp. NGB-B10, and its relevance in biological control activity

Plant metabolism interacts strongly with the plant microbiome. Glucosinolates, secondary metabolites synthesized by Brassica plants, are hydrolyzed by myrosinase into bioactive compounds of great importance in human health and plant protection. Compared with myrosinase from plant sources, myrosinase enzymes of microbial origin have not been extensively investigated. Therefore, seven endophytic strains corresponding to Bacillus sp. were isolated from Eruca vesicaria ssp. sativa plants that could hydrolyse glucosinolates (sinigrin) in the culture medium and showed myrosinase activity (0.08–19.92 U mL−1). The bglA myrosinase-related gene encoding the 6-phospho-β-glucosidase (GH 1) from Bacillus sp. NGB-B10, the most active myrosinase-producing bacterium, was successfully identified. Response surface methodology (RSM) was applied to statistically optimize culture conditions for myrosinase production from Bacillus sp. strain NGB-B10. The Plackett–Burman design indicated that nitrogen concentration, incubation period, and agitation speed were the significant parameters in myrosinase production. The application of the Box–Behnken design of RSM resulted in a 10.03-fold increase in enzyme activity as compared to the non-optimized culture conditions. The myrosinase was partially purified by 40% fractionation followed by SDS-PAGE analysis which yielded two subunits that had a molecular weight of 38.6 and 35.0 KDa. The purified enzyme was stable under a broad range of pH (5.5–10) and temperatures (10–65 °C). The hydrolysis products released by bacterial myrosinase from some glucosinolate extracts had higher and/or equivalent in vitro antagonistic activity against several phytopathogenic fungi compared to the nystatin (a broad-spectrum antifungal agent). This study provides original information about a new source of bacterial myrosinase and affords an optimized method to enhance myrosinase production.

Kinetic Study and Modeling of Wild-Type and Recombinant Broccoli Myrosinase Produced in E. coli and S. cerevisiae as a Function of Substrate Concentration, Temperature, and pH

The myrosinase enzyme hydrolyzes glucosinolates, among which is glucoraphanin, the precursor of the anticancer isothiocyanate sulforaphane (SFN). The main source of glucoraphanin is Brassicaceae; however, its natural concentration is relatively low, limiting the availability of SFN. An option to obtain SFN is its exogenous production, through enzymatic processes and under controlled conditions, allowing complete conversion of glucoraphanin to SFN. We characterized the kinetics of wild-type (BMYR) and recombinant broccoli myrosinases produced in E. coli (EMYR) and S. cerevisiae (SMYR) in terms of the reaction conditions. Kinetics was adjusted using empirical and mechanistic models that describe reaction rate as a function of substrate concentration, temperature, and pH, resulting in R2 values higher than 90%. EMYR kinetics differed significantly from those of BMYR and SMYR probably due to the absence of glycosylations in the enzyme produced in E. coli. BMYR and SMYR were subjected to substrate inhibition but followed different kinetic mechanisms attributed to different glycosylation patterns. EMYR (inactivation Ea = 76.1 kJ/mol) was more thermolabile than BMYR and SMYR. BMYR showed the highest thermostability (inactivation Ea = 52.8 kJ/mol). BMYR and EMYR showed similar behavior regarding pH, with similar pK1 (3.4 and 3.1, respectively) and pK2 (5.4 and 5.0, respectively), but differed considerably from SMYR.

Consumption of Glucosinolate-Rich Nasturtium officinale and Brassica oleracea L. var. sabellica May Reduce the Risk of Cancer Occurrence in Humans

Vegetables containing glucosinolates, commonly referred to as crucifers, are gaining popularity due to their anticancer benefits in human. Glucosinolates are secondary metabolites that are usually found in cruciferous vegetables belonging to the family Brassicaceae. Several studies pointed out that glucosinolates could undergo hydrolysis and produce bioactive isothiocyanates, via the action of enzyme myrosinase. In this study, the total glucosinolates in watercress (Nasturtium officinale) and kale (Brassica oleracea L. var. sabellica) were quantified using high-performance liquid chromatography (HPLC). HPLC analysis revealed that watercress contains 228.93 ± 26.39 μmol/g, while kale contains 87.60 ± 80.12 μmol/g of total glucosinolates. Analysis of cyclocondensation products via HPLC directly correlates the amount of isothiocyanates present in the extracts, which were found to be 0.398 ± 0.018 and 0.378 ± 0.014 for unhydrolyzed watercress and kale, respectively. An increase in isothiocyanate content was observed for both watercress and kale after subjecting them to hydrolysis in the presence of myrosinase enzyme. Moreover, the biological activity of the extracts derived from the plants was determined using standard assay methods. Free radical DPPH assay showed that methanolic extracts of both watercress and kale exhibit antioxidant properties with the IC50 values of 13.04 ± 1.11 µg/mL and 33.14 ± 1.44 µg/mL, respectively. On the other hand, the hydrolysates showed higher scavenging activity values reported at the IC50 values of 7.69 ± 0.93 µg/mL and 20.40 ± 1.26 µg/mL, for watercress and kale, respectively. PrestoBlue™ assay also revealed the ability of the aqueous extracts to selectively target cancer cells. Watercress showed anticancer potential towards breast adenocarcinoma (MCF-7, IC50 = 17.67 ± 1.64 µg/mL). On the other hand, kale showed anticancer potential towards small lung cancer cell (H69PR, with IC50 = 19.35 ± 1.54 µg/mL) and colon cancer cell (HT-29, IC50 = 18.32 ± 2.41 µg/mL). Moreover, the extracts were found to be non-cytotoxic to normal cells (HDFn, human dermal fibroblast neonatal, IC50 >100 µg/mL), which indicates that the extracts are selective towards targeting cancer cells.

A Specific Broccoli Sprout Preparation Reduces Chemically-Induced Colitis Via Gut Microbiota

ObjectivesSulforaphane is a bioactive metabolite with anti-inflammatory activity that is derived from glucosinolates, which are highly abundant in cruciferous vegetables including broccoli sprouts. There have been limited studies to investigate a whole foods approach to increase sulforaphane levels with therapeutic effect and reduce inflammation. In the current study, using a mouse model of inflammatory bowel disease, we aimed to investigate the ability of steamed broccoli sprouts to ameliorate colitis and the role of the microbiota in mediating any effects. MethodsC57BL/6J male mice were fed base diet or 5% steamed broccoli sprout diet for 2 weeks prior to induction of colitis with 2–3% DSS (5–7 days) and kept on their respective diets until sacrifice. Body weight and disease activity index scores were recorded daily, and histological assessment was performed on H&E sections of colon. Urine, feces, blood, and GI tract tissues and intestinal content were collected for LC-MS/MS quantification of sulforaphane and glucoraphanin. The 16S rRNA sequencing was performed to analyze gut microbiome. Germ-free mice were fed base diet or 5% steamed broccoli sprout diet for 2 weeks followed by treatment with 5 days of DSS to determine the role of gut microbiota in mediating the anti-inflammatory effects of the steamed broccoli sprout diet. ResultsWe observed that despite inactivation of the plant myrosinase enzyme responsible for the generation of sulforaphane via steaming, measurable levels of sulforaphane were detectable in the colon tissue and feces of mice after ingestion of steamed broccoli sprouts. In addition, this preparation of broccoli sprouts was also capable of reducing chemically-induced colitis. This protective effect was dependent on the presence of an intact gut microbiota, highlighting an important role for the microbiota in the metabolism of cruciferous vegetables to generate bioactive metabolites and promote their anti-inflammatory effects. ConclusionsAltogether, these results suggest that the incorporation of a steamed broccoli sprouts diet is a viable dietary intervention to complement current medical therapies for patients with inflammatory bowel disease. Funding SourcesThis work was supported by USDA-NIFA-AFRI Foundational Program [Grant No. 2018–67,017-27,520].

IDF21-0354 Glucoraphanin has the therapeutic potential of resolving hepatic steatosis in a murine model of diet-induced obesity

Background: Glucoraphanin (GR) is a natural glucosinolate abundantly found in broccoli sprouts. GR is converted to sulforaphane by myrosinase enzyme and the metabolite is a potent activator of a nuclear factor (erythroid-derived 2)-like 2 (Nrf2). Activation of Nrf2 induces antioxidant and detoxication-related genes, thus leading to cellular protection against oxidative stress. We have demonstrated that GR has preventive effects on impairment of glucose homeostasis in diet-induced obese (DIO) mice via an increment in energy expenditure, shifting macrophage polarization in liver and adipose tissue, and changing microbiota characteristics.

Sulphoraphane Affinity-Based Chromatography for the Purification of Myrosinase from Lepidium sativum Seeds.

Sulforaphane and other natural isothiocyanates released from the respective plant glucosinolates by the plant enzyme myrosinase (β-thioglucoside glucohydrolase) show extensive anticancer and antimicrobial effects. In this study, myrosinase from garden cress (Lepidium sativum) seeds was purified to electrophoretic homogeneity by a fast and easy strategy consisting of fractionation by isoelectric precipitation with ammonium sulphate (AS) and affinity chromatography using sulforaphane (SFN) attached to cellulose resin. The overall purification of enzyme with respect to crude extract was 169-fold and recovery of 37%. Under non-reducing conditions, two protein bands exhibiting myrosinase activity with masses of about 114 and 122 kDa, respectively, and a 58 kDa protein band with no activity were detected by SDS-PAGE and zymography on polyacrylamide gel. MALDI-Tof/Tof of tryptic fragments obtained from the respective protein bands detected sequence motifs homologous to the regions responsible for glycoside-substrate binding and similarities to members of the enzyme subfamilies β-glucosidases and myrosinases GH. The enzyme hydrolyzed both the natural (sinigrin, sinalbin, glucoraphanin) and the synthetic (p-nitrophenol-β-D-glucopyranoside (pNPG)) substrates. The highest catalytic activity of purified enzyme was achieved against sinigrin. The KM and Vmax values of the enzyme for sinigrin were found to be 0.57 mM, and 1.3 mM/s, respectively. The enzyme was strongly activated by 30 μM ascorbic acid. The optimum temperature and pH for enzyme was 50 °C and pH 6.0, respectively. The purified enzyme could be stored at 4 °C and slightly acidic pH for at least 45 days without a significant decrease in specific activity.

Broccoli Myrosinase cDNA Expression in Escherichia coli and Saccharomyces cerevisiae.

Myrosinases (EC 3.2.1.147) are enzymes known for the generation of hydrolysis products that have a potential beneficial effect on human health. Their reaction mechanisms are widely studied, in order to improve and optimize secondary metabolite production processes. In this work, kinetic and biochemical properties of the broccoli myrosinase enzyme produced from its cDNA cloned in Escherichia coli and Saccharomyces cerevisiae were investigated. The results revealed that the thermal stability of the enzyme produced in S. cerevisiae was slightly higher (30 to 60 °C) than that of myrosinase produced in E. coli (20 to 50 °C). The effect of pH on the enzymatic activity was similar in both enzymes, with pH 3 being the optimum value under the reaction conditions used. The kinetic behavior of both enzymes was adjusted to the Michaelis–Menten model. The catalytic efficiency was up to 4 times higher in myrosinase produced in S. cerevisiae, compared to myrosinase produced in E. coli. The glycosylations present in the enzyme would be related to the formation of a dimeric quaternary structure and would not play an essential role in enzymatic activity, since both enzymes were biologically active. These results will probably allow the development of strategies for the production of bioactive metabolites of medical interest.

Influence of Cabbage (Brassica oleracea) Accession and Growing Conditions on Myrosinase Activity, Glucosinolates and Their Hydrolysis Products.

Glucosinolates are secondary plant metabolites present in Brassica vegetables. The endogenous enzyme myrosinase is responsible for the hydrolysis of glucosinolates, yielding a variety of compounds, including health-promoting isothiocyanates. The influence of cabbage accession and growing conditions on myrosinase activity, glucosinolates (GSL) and their hydrolysis products (GHPs) of 18 gene-bank cabbage accessions was studied. Growing conditions, cabbage morphotype and accession all significantly affected myrosinase activity and concentration of glucosinolates and their hydrolysis products. In general, cabbages grown in the field with lower growth temperatures had significantly higher myrosinase activity than glasshouse samples. Profile and concentration of glucosinolates and their hydrolysis products differed across the accessions studied. Aliphatic glucosinolates accounted for more than 60 % of total glucosinolates in most of the samples assessed. Nitriles and epithionitriles were the most abundant hydrolysis products formed. The results obtained showed that consumption of raw cabbages might reduce the amount of beneficial hydrolysis products available to the consumer, as more nitriles were produced from hydrolysis compared to beneficial isothiocyanates. However, red and white cabbages contained high concentrations of glucoraphanin and its isothiocyanate, sulforaphane. This implies that careful selection of accessions with ample concentrations of certain glucosinolates can improve the health benefits derived from raw cabbage consumption.

A Physiological-Based Model for Simulating the Bioavailability and Kinetics of Sulforaphane from Broccoli Products.

There are no known physiological-based digestion models that depict glucoraphanin (GR) to sulforaphane (SR) conversion and subsequent absorption. The aim of this research was to make a physiological-based digestion model that includes SR formation, both by endogenous myrosinase and gut bacterial enzymes, and to simulate the SR bioavailability. An 18-compartment model (mouth, two stomach, seven small intestine, seven large intestine, and blood compartments) describing transit, reactions and absorption was made. The model, consisting of differential equations, was fit to data from a human intervention study using Mathwork’s Simulink and Matlab software. SR urine metabolite data from participants who consumed different broccoli products were used to estimate several model parameters and validate the model. The products had high, medium, low, and zero myrosinase content. The model’s predicted values fit the experimental values very well. Parity plots showed that the predicted values closely matched experimental values for the high (r2 = 0.95), and low (r2 = 0.93) products, but less so for the medium (r2 = 0.85) and zero (r2 = 0.78) myrosinase products. This is the first physiological-based model to depict the unique bioconversion processes of bioactive SR from broccoli. This model represents a preliminary step in creating a predictive model for the biological effect of SR, which can be used in the growing field of personalized nutrition.

Sulforaphane: A Broccoli Bioactive Phytocompound with Cancer Preventive Potential.

Simple SummaryAs of the past decade, phytochemicals have become a major target of interest in cancer chemopreventive and chemotherapeutic research. Sulforaphane (SFN) is a metabolite of the phytochemical glucoraphanin, which is found in high abundance in cruciferous vegetables, such as broccoli, watercress, Brussels sprouts, and cabbage. In both distant and recent research, SFN has been shown to have a multitude of anticancer effects, increasing the need for a comprehensive review of the literature. In this review, we critically evaluate SFN as an anticancer agent and its mechanisms of action based on an impressive number of in vitro, in vivo, and clinical studies.There is substantial and promising evidence on the health benefits of consuming broccoli and other cruciferous vegetables. The most important compound in broccoli, glucoraphanin, is metabolized to SFN by the thioglucosidase enzyme myrosinase. SFN is the major mediator of the health benefits that have been recognized for broccoli consumption. SFN represents a phytochemical of high interest as it may be useful in preventing the occurrence and/or mitigating the progression of cancer. Although several prior publications provide an excellent overview of the effect of SFN in cancer, these reports represent narrative reviews that focused mainly on SFN’s source, biosynthesis, and mechanisms of action in modulating specific pathways involved in cancer without a comprehensive review of SFN’s role or value for prevention of various human malignancies. This review evaluates the most recent state of knowledge concerning SFN’s efficacy in preventing or reversing a variety of neoplasms. In this work, we have analyzed published reports based on in vitro, in vivo, and clinical studies to determine SFN’s potential as a chemopreventive agent. Furthermore, we have discussed the current limitations and challenges associated with SFN research and suggested future research directions before broccoli-derived products, especially SFN, can be used for human cancer prevention and intervention.

Transcriptomic Reprograming of Xanthomonas campestris pv. campestris after Treatment with Hydrolytic Products Derived from Glucosinolates.

The bacterium Xanthomonas campestris pv. campestris (Xcc) causes black rot disease in Brassica crops. Glucosinolates are known to be part of the defence system of Brassica crops against Xcc infection. They are activated upon pathogen attack by myrosinase enzymes. Their hydrolytic products (GHPs) inhibit the growth of Xcc in vitro. However, the mechanisms underlying this inhibition and the way Xcc can overcome it are not well understood. We studied the transcriptomic reprogramming of Xcc after being supplemented with two chemically different GHPs, one aliphatic isothiocyanate (allyl-ITC) and one indole (indol-3-carbinol), by RNA-seq. Based on our results, the arrest in Xcc growth is related to the need to stop cell division to repair damaged DNA and cell envelope components. Otherwise, GHPs modify energy metabolism by inhibiting aerobic respiration and increasing the synthesis of glycogen. Xcc induces detoxification mechanisms such as the antioxidant defence system and the multidrug efflux system to cope with the toxic effects driven by GHPs. This is the first time that the transcriptomic reprogramming of a plant pathogenic bacterium treated with GHPs has been studied. This information will allow a better understanding of the interaction of a plant pathogen mediated by GSLs.

The Metabolism of Glucosinolates by Gut Microbiota.

Glucosinolates (GLS) and their derivatives are secondary plant metabolites abundant in Brassicaceae. Due to the enzymatic reaction between GLS and myrosinase enzyme, characteristic compounds with a pungent taste are formed, used by plants to defend themselves against insect herbivores. These GLS derivatives have an important impact on human health, including anti-inflammation and anti-cancer effects. However, GLS derivatives’ formation needs previous enzymatic reactions catalyzed by myrosinase enzyme. Many of the brassica-based foods are processed at a high temperature that inactivates enzymes, hindering its bioavailability. In the last decade, several studies showed that the human gut microbiome can provide myrosinase activity that potentially can raise the beneficial effects of consumption of vegetables rich in GLS. The variability of the human gut microbiome (HGM) in human populations and the diverse intake of GLS through the diet may lead to greater variability of the real dose of pro-healthy compounds absorbed by the human body. The exploitation of the genetic and biochemical potential of HGM and correct ecological studies of both isolated strains and mixed population are of great interest. This review focuses on the most recent advances in this field.

Hijacking the Mustard-Oil Bomb: How a Glucosinolate-Sequestering Flea Beetle Copes With Plant Myrosinases

Myrosinase enzymes play a key role in the chemical defense of plants of the order Brassicales. Upon herbivory, myrosinases hydrolyze the β-S-linked glucose moiety of glucosinolates, the characteristic secondary metabolites of brassicaceous plants, which leads to the formation of different toxic hydrolysis products. The specialist flea beetle, Phyllotreta armoraciae, is capable of accumulating high levels of glucosinolates in the body and can thus at least partially avoid plant myrosinase activity. In feeding experiments with the myrosinase-deficient Arabidopsis thaliana tgg1 × tgg2 (tgg) mutant and the corresponding Arabidopsis Col-0 wild type, we investigated the influence of plant myrosinase activity on the metabolic fate of ingested glucosinolates in adult P. armoraciae beetles. Arabidopsis myrosinases hydrolyzed a fraction of ingested glucosinolates and thereby reduced the glucosinolate sequestration rate by up to 50% in adult beetles. These results show that P. armoraciae cannot fully prevent glucosinolate hydrolysis; however, the exposure of adult beetles to glucosinolate hydrolysis products had no impact on the beetle’s energy budget under our experimental conditions. To understand how P. armoraciae can partially prevent glucosinolate hydrolysis, we analyzed the short-term fate of ingested glucosinolates and found them to be rapidly absorbed from the gut. In addition, we determined the fate of ingested Arabidopsis myrosinase enzymes in P. armoraciae. Although we detected Arabidopsis myrosinase protein in the feces, we found only traces of myrosinase activity, suggesting that P. armoraciae can inactivate plant myrosinases in the gut. Based on our findings, we propose that the ability to tolerate plant myrosinase activity and a fast glucosinolate uptake mechanism represent key adaptations of P. armoraciae to their brassicaceous host plants.

The selective sequestration of glucosinolates by the cabbage aphid severely impacts a predatory lacewing

The cabbage aphid Brevicoryne brassicae is a notorious agricultural pest that specializes on plants of the Brassicaceae family, which are chemically defended by glucosinolates. By sequestering glucosinolates from its host plants and producing its own activating enzyme (myrosinase), this aphid employs a self-defense system against enemies paralleling that in plants. However, we know little about the metabolic fate of individual glucosinolates during aphid sequestration and activation and about the biochemical effects of this defense on aphid enemies. Here, we probed these questions focusing on B. brassicae and a predatory lacewing, Chrysoperla carnea. We found that distinct glucosinolates were accumulated by B. brassicae at different rates, with aliphatic glucosinolates being taken up more quickly than indolic ones. B. brassicae myrosinase enzymatic activities toward different glucosinolates were strongly correlated to their rates of accumulation in vivo. Surprisingly, after simulated predation, the production of toxic isothiocyanate products (ITCs) was quantitatively outweighed by less toxic products such as nitriles and ITC-conjugates. Nevertheless, the defensive cocktails significantly impaired C. carnea development. Tissue-specific quantification of glucosinolate metabolites revealed that the lacewings employ both conjugation and mobilization to reduce the toxicity of aliphatic ITCs, but these strategies were only partially effective. These results clarify the metabolic fates of glucosinolates after sequestration by an aphid herbivore and further in a higher trophic level, as well as the consequences for predator survival and development, and might be instructive for integrative pest management approaches targeting the cabbage aphid.

Efficacy of Sulforaphane in Neurodegenerative Diseases.

Sulforaphane (SFN) is a phytocompound belonging to the isothiocyanate family. Although it was also found in seeds and mature plants, SFN is mainly present in sprouts of many cruciferous vegetables, including cabbage, broccoli, cauliflower, and Brussels sprouts. SFN is produced by the conversion of glucoraphanin through the enzyme myrosinase, which leads to the formation of this isothiocyanate. SFN is especially characterized by antioxidant, anti-inflammatory, and anti-apoptotic properties, and for this reason, it aroused the interest of researchers. The aim of this review is to summarize the experimental studies present on Pubmed that report the efficacy of SFN in the treatment of neurodegenerative disease, including Alzheimer’s disease (AD), Parkinson’s disease (PD), and multiple sclerosis (MS). Therefore, thanks to its beneficial effects, SFN could be useful as a supplement to counteracting neurodegenerative diseases.

Biomedical application of Indole-3-carbinol: A mini-review

Abstract Indole-3-carbinol (I3C) is a plant-derived phytochemical produced when raw cruciferous vegetables are chopped, and their plant cells are damaged. I3C is an orally bioactive compound formed upon the hydrolysis of glucosinolate by the plant enzyme myrosinase. Cruciferous vegetables belong to the Brassicaceae family and include many genera and species with potential biomedical applications. I3C is more abundant in certain cruciferous vegetables, such as broccoli, cabbage, green peas, cauliflower, and brussels sprouts, and has multiple biological activities, including antimicrobial, antioxidant, antiviral, anti-inflammatory, and anti-cancer activities. Most recent studies indicate that regular consumption of I3C-containing vegetables decreases the rate of various types of cancer. This review focuses on the general biomedical applications of I3C and its role in human wellness.

Selection and microencapsulation of myrosinase enzyme from broccoli sprouts of different varieties and characteristics evaluation

AbstractMyrosinase (MYR) can hydrolyze glucosinolates rich in crucifer to produce sulforaphane, which has significant anticancer effect. In this study, MYR activity in broccoli seeds and sprouts of different varieties were firstly compared. Then, after optimization for the microencapsulation condition of MYR, the characteristics of free and encapsulated MYR enzyme were evaluated and compared. Broccoli variety and germination time both significantly affected the specific MYR activity, total protein content and total MYR activity in sprouts. The optimum condition for encapsulating MYR was the sodium alginate addition of 5.29 ml, CaCl2 concentration of 0.75%, the hardening time of 29.98 min, and the crosslinking time of 47.20 min. Encapsulated MYR with the specific activity of 2.25 U/mg was obtained (54.6% of free MYR). Encapsulated MYR could retain its activity under wider pH range and higher temperature than free MYR. Encapsulated MYR also kept higher activity during storage at room temperature. Supplement of encapsulated MYR was favorable for SF production in broccoli sprouts during storage. This research would be beneficial for producing more sulforaphane from broccoli sprouts as raw materials of functional foods.Practical ApplicationsThe function of sulforaphane has been widely accepted. However, products currently on the market contain only its precursor of glucosinolate, which is extremely inefficient in converting to SF in vivo. Encapsulated myrosinase have better temperature and pH tolerance, which can ensure the conversion of Gls to SF before oral intake. It is a good choice to provide encapsulated myrosinase and glucosinolates together to consumer.

Protective Effect of Isothiocyanates from Cruciferous Vegetables on Breast Cancer: Epidemiological and Preclinical Perspectives.

The effect of cruciferous vegetable intake on breast cancer survival is controversial at present. Glucosinolates are the naturally occurring constituents found across the cruciferous vegetables. Isothiocyanates are produced from the hydrolysis of glucosinolates and this reaction is catalysed by the plant-derived enzyme myrosinase. The main Isothiocyanates (ITCs) from cruciferous vegetables are sulforaphane, benzyl ITC, and phenethyl ITC, which had been intensively investigated over the last decade for their anti-breast cancer effects. The aim of this article is to systematically review the evidence from all types of studies, which examined the protective effect of cruciferous vegetables and/or their isothiocyanate constituents on breast cancer. A systematic review was conducted in Pubmed, EMBASE, and the Cochrane Library from inception to 27 April 2020. Peer-reviewed studies of all types (in vitro studies, animal studies, and human studies) were selected. The systematic literature search identified 16 human studies, 4 animal studies, and 65 in vitro studies. The effect of cruciferous vegetables and/or their ITCs intake on breast cancer survival was found to be controversial and varied greatly across human studies. Most of these trials were observational studies conducted in specific regions, mainly in the US and China. Substantial evidence from in vitro and animal studies was obtained, which strongly supported the protective effect of sulforaphane and other ITCs against breast cancer. Evidence from in vitro studies showed that sulforaphane and other ITCs reduced cancer cell viability and proliferation via multiple mechanisms and pathways. Isothiocyanates inhibited cell cycle, angiogenesis and epithelial mesenchymal transition, as well as induced apoptosis and altered the expression of phase II carcinogen detoxifying enzymes. These are the essential pathways that promote the growth and metastasis of breast cancer. Noticeably, benzyl ITC showed a significant inhibitory effect on breast cancer stem cells, a new dimension of chemo-resistance in breast cancer treatment. Sulforaphane and other ITCs displayed anti-breast cancer effects at variable range of concentrations and benzyl isothiocyanate appeared to have a relatively lower inhibitory concentration IC50. The mechanisms underlying the cancer protective effect of sulforaphane and other ITCs have also been highlighted in this article. Current preclinical evidence strongly supports the role of sulforaphane and other ITCs as potential therapeutic agents for breast cancer, either as adjunct therapy or combined therapy with current anti-breast cancer drugs, with sulforaphane displaying the greatest potential.

Inactivation of Thioglucosidase from Sinapis alba (White Mustard) Seed by Metal Salts.

The glucosinolates which are specialized plant metabolites of Brassica vegetables are prone to hydrolysis catalyzed by an endogenous enzyme myrosinase (thioglycoside hydrolase, thioglucosidase) that exists in Brassica plant tissue causing volatile isothiocyanates release. Currently existing literature data on the inactivation of myrosinase is insufficient in particular for use in the analysis of volatile and odor compounds in vegetables rich in glucosinolates. In this study, the impact of different metal salts in effective inactivation of enzyme activity was investigated by solid-phase microextraction (SPME) and GC/MS system in aqueous samples and kohlrabi matrix. A saturated solution of calcium chloride which is commonly used to stop enzyme activity in plant tissue inactivates the myrosinase–glucosinolate system. However, even without the participation of myrosinase, it changes the reaction pathway towards nitrile formation. The model experiment shows that optimum efficiency in inhibition of the enzyme system shows iron(III) ions, silver ions, and anhydride sodium sulfate resulting in no volatile products derived from glucosinolates. However, in the kohlrabi matrix, the strongest enzyme inhibition effect was observed for silver salt resulting in no volatile products, also both anhydrous Na2SO4 and saturated CaCl2 solution seem to be useful inhibitors in flavor studies.

Mechanisms Underlying Biological Effects of Cruciferous Glucosinolate-Derived Isothiocyanates/Indoles: A Focus on Metabolic Syndrome.

An inverse correlation between vegetable consumption and the incidence of cancer has long been described. This protective effect is stronger when cruciferous vegetables are specifically consumed. The beneficial properties of vegetables are attributed to their bioactive components like fiber, antioxidants vitamins, antioxidants, minerals, and phenolic compounds. Cruciferous vegetables contain all these molecules; however, what makes them different are their sulfurous components, called glucosinolates, responsible for their special smell and taste. Glucosinolates are inactive biologically in the organism but are hydrolyzed by the enzyme myrosinase released as a result of chewing, leading to the formation of active derivatives such as isothiocyanates and indoles. A considerable number of in vitro and in vivo studies have reported that isothiocyanates and indoles elicit chemopreventive potency through multiple mechanisms that include modulation of phases I and II detoxification pathway enzymes, regulation of cell cycle arrest, and control of cell growth, induction of apoptosis, antioxidant activity, anti-angiogenic effects, and epigenetic regulation. Nuclear erythroid 2-related factor 2 (Nrf2) and Nuclear factor-κB (NF-κB) are key and central regulators in all these processes with a main role in oxidative stress and inflammation control. It has been described that isothiocyanates and indoles regulate their activity directly and indirectly. Today, the metabolic syndrome (central obesity, insulin resistance, hyperlipidemia, and hypertension) is responsible for a majority of deaths worldwide. All components of metabolic syndrome are characterized by chronic inflammation with deregulation of the PI3K/AKT/mTOR, MAPK/EKR/JNK, Nrf2, and NF-κB signaling pathways. The effects of GLSs derivatives controlling these pathways have been widely described in relation to cancer. Changes in food consumption patterns observed in the last decades to higher consumption of ultra-processed foods, with elevation in simple sugar and saturated fat contents and lower consumption of vegetables and fruits have been directly correlated with metabolic syndrome prevalence. In this review, it is summarized the knowledge regarding the mechanisms by which cruciferous glucosinolate derivatives (isothiocyanates and indoles) directly and indirectly regulate these pathways. However, the review places a special focus on the knowledge of the effects of glucosinolates derivatives in metabolic syndrome, since this has not been reviewed before.