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Abstract
Concurrent with the increase in our knowledge of the genetic and environmental factors that lead to glucosinolate accumulation
in plants, and the role of these compounds and their derivatives in mediating plant–herbivore interactions, there has been
significant advances in our understanding of how glucosinolates and their products may contribute to a reduction in risk of
carcinogenesis and heart disease when consumed as part of the diet. In this paper, we review the epidemiological evidence
for the health promoting effects of cruciferous vegetables, the processes by which glucosinolates and isothiocyanates are
absorbed and metabolised by humans, with particular regard to the role of glutathione S-transferases, and the biological activity
of isothiocyanates towards mammalian cells and tissues.
... The broad therapeutic spectrum of T. majus can be linked to a group of compounds known as glucosinolates [15][16][17][18]. Benzyl glucosinolate is a metabolite found in every part of the T. majus plant, especially in the seeds [15][16][17][18][19]. When hydrolyzed by the endogenous enzyme myrosinase (Thioglucoside hydrolase, EC 3.2.3.1), it generates a variety of breakdown products, including benzyl isothiocyanate (BITC) [16,17,19], which has been reported to have antimicrobial [20][21][22][23], larvicidal [24], anthelmintic [25], and anticancer [26] activities. ...
... Benzyl glucosinolate is a metabolite found in every part of the T. majus plant, especially in the seeds [15][16][17][18][19]. When hydrolyzed by the endogenous enzyme myrosinase (Thioglucoside hydrolase, EC 3.2.3.1), it generates a variety of breakdown products, including benzyl isothiocyanate (BITC) [16,17,19], which has been reported to have antimicrobial [20][21][22][23], larvicidal [24], anthelmintic [25], and anticancer [26] activities. ...
Malaria remains an important and challenging infectious disease, and novel antimalarials are required. Benzyl isothiocyanate (BITC), the main breakdown product of benzyl glucosinolate, is present in all parts of Tropaeolum majus L. (T. majus) and has antibacterial and antiparasitic activities. To our knowledge, there is no information on the effects of BITC against malaria. The present study evaluates the antimalarial activity of BITC and T. majus seeds, leaves, and stems aqueous extracts. We used flow cytometry to calculate the growth inhibition (GI) percentage of the extracts and BITC against unsynchronized cultures of chloroquine-susceptible Plasmodium falciparum (P. falciparum) 3D7-GFP strain. Extracts and/or compounds with at least 70% GI were validated by IC50 estimation against P. falciparum 3D7-GFP and Dd2 (chloroquine-resistant strain) unsynchronized cultures by flow cytometry, and the resistance index (RI) was determined. T. majus aqueous extracts showed some antimalarial activity higher in seeds than in leaves or stems. BITC’s GI was comparable to chloroquine’s. BITC’s IC50 was similar in both strains, thus a cross-resistance absence with aminoquinolines was found (RI < 1). BITC presented features that could open new avenues for malaria drug discovery.
... Among the hydrolysis products, isothiocyanates have a direct positive effect on human health. Some isothiocyanates activate phase 2 detoxification enzymes, resulting in the inhibition of cancer growth and carcinogens [10]. Additionally, in Chinese cabbage, it is known that glucosinolate degradation products play a role in defense against other insects [11]. ...
... The decomposition byproducts of indole glucosinolates, such as GBS, have been recognized as phytoalexins in several species of Brassica, and they are acknowledged to have a significant role in plants defense metabolism [23]. In addition, sulforaphane, indole-3-carbinol, and glucobrassin (GBS), produced by myrosinase, are known to have anticancer functions [10].. They inhibit phase 1 detoxification enzyme activity and induce the activity of phase 2 detoxification enzymes to suppress cancer development [24,25]. ...
The genebank at the National Agrobiodiversity Center (RDA-Genebank, Jeonju, Republic of Korea), conserves approximately 8000 germplasms of Brassica spp., of which Chinese cabbage (Brassica rapa L. ssp. pekinensis) is one of the major crops actively used as food in Northeast Asia, including Korea, as the main ingredient for kimchi. Glucosinolates are a major class of compounds in Chinese cabbage that are responsible for their distinctive flavor, and RDA-Genebank is constantly building a related database (DB) to select suitable germplasms required by consumers and provide resources for breeding programs. In this study, ten glucosinolates were analyzed in sixty Chinese cabbage germplasms. Six aliphatic glucosinolates were the major components, accounting for 85.00% to 91.98% of total glucosinolates in each germplasm. Among them, gluconapin (333.26 to 23,501.58 μmol∙kg−1 DW) was highly represented, followed by glucobrassicanapin (545.60 to 10,344.70 μmol∙kg−1 DW) and progoitrin (155.28 to 8536.51 μmol∙kg−1 DW). In addition, we selected germplasms with a high content of each studied glucosinolate. To analyze the diversity and distribution of glucosinolates among the studied germplasms, Pearson’s correlation was performed, and the related results were interpreted through their biosynthetic pathways. The k-means clustering indicated four optimal clusters, which were confirmed through principal component analysis. Orthogonal projection to latent structure discriminant analysis (OPLS-DA) was also performed on the status (landrace and cultivar) and origin (Korea, China, Taiwan, and Japan) passport data of the germplasms, followed by the calculation of variable importance in the projection (VIP) values. These results are part of a continuous series of studies to analyze the glucosinolates of Brassica germplasms that are being conserved at RDA-Genebank. We aim to provide related results through a public platform accessible to everyone and thereby improve the distribution of Brassica germplasms.
... The increased consumer interest in the consumption of safe and healthy food has resulted in growing demand for locally grown products such as kale. Due to known kale characteristic sensory and chemical properties, screening of kale landraces for agronomic and nutrient use efficiency traits was done (Batelja et al., 2009;Urlic et al., 2016) (Traka et al., 2009;Orouji et al., 2023), reduce or stimulate insect attack, inhibit the growth of nematodes and fungi and the growth of neighbouring plants (Brennan et al., 2020), as also affect mostly specific flavor of Brassica vegetables. ...
... Many of these phytochemicals have the ability to neutralise cobalt ions through electron transfer or to form complex molecules with the cobalt ions in the precursor solution. These neutral ions or complex molecules can be easily converted into Co 3 O 4 -NPs through a heat treatment process [21][22][23][24][25][26][27][28][29][30][31]. This phytochemical-based approach offers several advantages, including low power requirements, simplicity, eco-friendliness, rapidity, cost-effectiveness, and high efficiency [32-41]. ...
In this study, we investigated the structural and optical properties of garlic extract-based green-synthesised tricobalt tetroxide nanoparticles. Transmission electron microscopy revealed a particle size range of 8–22 nm for the prepared powder sample. Powder x-ray diffraction data and Rietveld refinement results confirmed the spinel cubic crystal structure of the tricobalt tetroxide nanoparticles, with an average crystallite size of 11.23 nm. This crystal structure corresponds to the Fd3̅m space group and has an average lattice constant of 0.791 nm. The bond lengths of Co³⁺–O²⁻ and Co²⁺–O²⁻ are measured to be 0.188 nm and 0.190 nm, respectively. The FTIR data provided evidence of the presence of various functional bands, which helped qualitatively determine the purity of the sample. The UV–vis spectrum estimated two direct energy band gap values (3.7 eV and 2.2 eV) that may be useful for efficient interaction with a wide range of ray spectra to create more electron–hole pairs for various photo-responsive applications, such as dye degradation, solar cells, and optoelectronic components.
... The benzyl glucosinolate metabolite is found in every part of the T. majus plant, especially in the seeds [15][16][17][18]20]. When hydrolyzed by the endogenous enzyme myrosinase (Thioglucoside hydrolase, EC 3.2.3.1), it generates a variety of breakdown products, including benzyl isothiocyanate [19]. ...
Malaria remains an important and challenging infectious disease, and novel antimalarials are required. Benzyl isothiocyanate (BITC), the main breakdown product of benzyl glucosinolate, is present in all parts of Tropaeolum majus L. (T. majus) and has antibacterial and antiparasitic activities. To our knowledge, there is no information on the effects of BITC against malaria. The present study evaluates the antimalarial activity of aqueous extracts of BITC and T. majus seeds, leaves, and stems. We used flow cytometry to calculate the growth inhibition (GI) percentage of the extracts and BITC against unsynchronized cultures of the chloroquine-susceptible Plasmodium falciparum 3D7 − GFP strain. Extracts and/or compounds with at least 70% GI were validated by IC50 estimation against P. falciparum 3D7 − GFP and Dd2 (chloroquine-resistant strain) unsynchronized cultures by flow cytometry, and the resistance index (RI) was determined. T. majus aqueous extracts showed some antimalarial activity that was higher in seeds than in leaves or stems. BITC’s GI was comparable to chloroquine’s. BITC’s IC50 was similar in both strains; thus, a cross-resistance absence with aminoquinolines was found (RI < 1). BITC presented features that could open new avenues for malaria drug discovery.
... Here are a few herbal remedies that have been studied for their potential to cure metabolic disorders, along with an overview of the clinical evidence that is currently available. Table 3 offers a selection of herbal remedies together with clinical information to support their application in the management of metabolic diseases [68][69][70][71][72][73][74][75][76][77][78][79][80]. ...
Comprehensive and effective care techniques have become essential due to the global epidemic dimensions of metabolic disorders, including diabetes, obesity, and cardiovascular ailments. Recent research highlights the potential of dietary supplements, herbal extracts, and phytochemicals in treating metabolic diseases. This abstract conveys the current state of the science in this field by highlighting these findings' underlying mechanisms and potential therapeutic applications. Plant-based diets contain naturally occurring bioactive molecules termed phytochemicals, which have shown promise in treating various metabolic illnesses. Examples include curcumin, flavonoids, and polyphenols' insulin-sensitizing, antioxidant, and antiinflammatory properties. Herbal extracts, derived from ancient medicinal herbs, have been used by people for years to treat a wide range of ailments. Recent studies have shown the efficacy of these strategies in improving lipid profiles, glucose metabolism, and overall cardiovascular health. Omega-3 fatty acids, vitamins, and minerals are just a few of the numerous nutritional supplements that are critical to metabolic health. These vitamins improve insulin sensitivity, regulate blood sugar, and decrease inflammation. Probiotics and prebiotics also affect the gut flora, which significantly affects metabolic function. These natural medicines' ability to treat metabolic diseases either by themselves or in combination with conventional medical interventions. However, when using it therapeutically, one must consider the differences in doses, individual responses, and bioavailability. The article concludes that phytochemicals, plant extracts, and food supplements offer a promising avenue for the management of metabolic illnesses. Comprehensive research, including clinical studies, is needed to ascertain their safety and efficacy characteristics. When added to treatment strategies, these natural therapies could be helpful supplements that improve overall health and the quality of life among individuals with metabolic diseases. Naringenin, a citrus flavonoid, can potentially prevent kidney injury in hyperuricemia by reducing uric acid, inflammation, apoptosis, DNA damage, and activating antioxidants. Further research and professional consultation are essential. Factors contributing to metabolic diseases, current approaches to management nutritional approaches for managing obesityassociated metabolic impairments in the liver and small intestine, and nutritional approaches for managing obesity-associated metabolic dysregulation are also explained briefly.
... From a nutritional point of view, they are unique as they contain a group of phytochemicals called glucosinolates which, when they are hydrolysed in the digestive tract, produce, amongst other compounds, isothiocyanates. These compounds stimulate anticarcinogenic phase II human enzyme activities [7][8][9]. Furthermore, previous studies [10][11][12] have shown that unlike other green leafy vegetables (such as spinach or Swiss chard), the trace element bioaccessibility present in cruciferous vegetables is notably high, similar in some cases to that of powdered milk. This is due to a low content of anti-nutritional compounds such as oxalates. ...
The objective of this work was to study the influence of three dietary fibre fractions (pectin, gum arabic and cellulose) and three protein fractions (casein, lactalbumin and soy) on the trace element bioaccessibility (Fe, Mn, Ni, Se and Zn) of turnip tops (B. rapa subsp. Rapa) growing under Mediterranean conditions. Then, it aimed to promote the use of this vegetable not only for direct fresh consumption but also as a main ingredient in the development of food mixtures. The results showed that soluble fibre fractions, such as pectin and gum arabic, can enhance the bioaccessibility of trace elements, such as Fe, Mn, Se and Zn. This effect was not proved for cellulose (an insoluble fibre fraction), in which, at best, no bioaccessibility effect was observed. Regarding the protein fractions, with the exception of Se, caseins and lactalbumin had a neutral effect on improving the trace element bioaccessibility. This did not hold true for soy protein, in which a considerable improvement in the bioaccessibility of Fe, Mn, Se and Zn was determined.
... As a result of the presence of a wide range of variability in the crop such as its morphological features, there are diverse accessions of pak choy found in many major Genebanks across the globe [9]. As a cruciferous vegetable, pak choy contains glucosinolates (GSL), a category of sulfur-containing secondary plant metabolites that have come under intense scrutiny for their bioactive properties and potential health-enhancing effects [11,12]. GSLs are famous based on their linkage with a broad range of health benefits such as their antioxidant, antimicrobial, and anti-inflammatory activities, and perhaps most notably their anticancer properties [12][13][14]. ...
Glucosinolates are sulfur-containing phytochemicals generally abundant in cruciferous vegetables such as pak choy. Glucosinolates participate in a range of biological activities essential for promoting a healthy human body. In this study, we aimed to elucidate glucosinolate variability present in pak choy germplasm that are under conservation at the Rural Development Administration Genebank, Jeonju, Republic of Korea. The Acquity Ultra-Performance Liquid Chromatography (UHPLC) analytical system was used in profiling the glucosinolate content in leaf samples of various accessions. We identified a total of 17 glucosinolates in the germplasm. Based on principal compoment analysis performed, three separate groups of the accessions were obtained. Group 1 contained the cultivar cheongsacholong which recorded high content of glucobrassicin (an indole), glucoerucin (aliphatic), gluconasturtiin (aromatic) and glucoberteroin (aliphatic). Group 2 consisted of six accessions, BRA77/72, Lu ling gaogengbai, 9041, Wuyueman, RP-75 and DH-10, predominatly high in aliphatic compounds including glucoiberin, glucocheirolin, and sinigrin. Group 3 comprised the majority of the accessions which were characterized by high content of glucoraphanin, epiprogoitrin, progoitrin, and glucotropaeolin. These results revealed the presence of variability among the pak choy germplasm based on their glucosinolate content, providing an excellent opprtunity for future breeding for improved glucosinolate content in the crop.
... italica), in which several enzymecatalyzed reactions can occur impacting the nutritional and sensorial characteristics of the end product. The hydrolysis of glucosinolates (GSLs) by the enzyme myrosinase (MYR) can result in possible flavoraffecting volatile compounds (e.g., isothiocyanates (ITCs), thiocyanates, nitriles, epithionitrilles) (Bongoni et al., 2014;Kissen et al., 2009;Marcinkowska & Jeleń, 2020;Nintemann et al., 2018;Pan et al., 2022;Shirakawa & Hara-nishimura, 2018;Traka & Mithen, 2009;Wieczorek et al., 2022). Another prominent enzymatic pathway in Brassicaceae is the conversion of S-methyl-L-cysteine sulfoxides, catalyzed by cysteine (sulfoxide) lyase (C-S lyase), which can contribute to the formation of sulfur-containing flavor-affecting compounds in broccoli (e.g., dimethyl disulfide and dimethyl trisulfide) (Tulio, et al., 2002). ...
... The beneficial effects of selenium on GSL biosynthesis are likely due to the shared transporters of selenium and sulfur and the application of selenium can influence the expression of genes encoding for these transporters, thereby increasing the accumulation of GSLs. Moreover, the amino acids methionine and tryptophan, precursors of aliphatic and indolic GSLs respectively, could increase GSLs content in broccoli (Traka & Mithen, 2008). Aliphatic GSL levels were dramatically increased by 32% when the sulfur-containing amino acid methionine was administered at a concentration of 5 mM. ...
Background
Cruciferous sprout is a new form of vegetable product that is gaining attention due to its high content of bioactive compounds such as glucosinolates. As a diverse class of phytochemicals, glucosinolates and glucosinolate-derived products such as isothiocyanates are popular targets for scientific research because of their health associated benefits.
Scope and approach
The present review highlights the unique properties of beneficial glucosinolates and bioactive derivatives, the biosynthetic and metabolic regulatory networks of glucosinolates, and their role in cruciferous sprouts for producing health-promoting glucosinolates biofortification benefits along the entire agro-food chain.
Key findings and conclusions
Glucosinolates and their bioactive derivatives have been extensively studied for their beneficial effects on human health. Among various plant products, cruciferous sprouts are more suitable as a functional food due to their high content of health-promoting glucosinolates and bioactive derivatives. This review describes the unique properties of glucosinolates and their bioactive derivatives contained in cruciferous sprouts with potential health benefits. We discussed the glucosinolate metabolic pathway as well as their regulatory mechanisms. Furthermore, we proposed to apply the integrative and sustainable treatment approaches in cruciferous sprout industry including breeding for glucosinolates-biofortified cultivars, pre-harvest treatment to improve glucosinolates accumulation, followed by postharvest handlings and processing methods for benefit glucosinolates retention along the whole agro-food chain to fight health-promoting glucosinolates deficiency in human daily diet. This review presents a comprehensive overview of effective health-promoting glucosinolates biofortification in cruciferous sprouts along the entire agro-food chain, which is potential in the sustainable cruciferous sprouts industry as well as significance in human nutrition.
... These compounds are highly volatile and are responsible for the biological activity and specific taste of horseradish. ITCs have been intensively researched recently, as they are recognized to have antitumor, bactericidal and fungicidal effects [28]. Zhang [29] reported the inhibition of tumor cells proliferation by extracts from horseradish roots. ...
... Consumption of Brassica vegetables has been consistently shown to be beneficial to human health as they contain health-promoting compounds, including glucosinolates (GSLs) and phenolic compounds, such as flavonoids and hydroxycinnamic acids [1][2][3]. GSLs are associated with risk reduction in many kinds of cancer, such as colorectal, lung and prostate cancers [4,5]. As Brassica vegetables contain high levels of antioxidants, it has been claimed that they could additionally prevent other chronic diseases, such as diabetes and cardiovascular disease [6]. ...
Brassica vegetables are bitter, predominantly because they contain bitter-tasting glucosinolates. Individuals with high bitter taste sensitivity are reported to have lower consumption of bitter vegetables. Studies reported that cooking methods can alter the sensory characteristics of vegetables, increasing acceptability. This study investigated consumer liking of turnip cooked by four methods (boiled-pureed, roasted, steamed-pureed and stir-fried) and related this to sensory characteristics. Additionally, this study examined the effect of the bitter taste genotype on taste perception and liking of the cooked turnip samples. Participants (n = 74) were recruited and the TAS2R38 genotype was measured. Liking, consumption intent, perception of bitterness and sweetness of turnip were evaluated. A sensory profile of the cooked turnip variants was also determined by a trained sensory panel. There were significant differences in the overall (p = 0.001) and taste (p = 0.002) liking between cooking methods. Turnip liking was increased when preparation led to sweeter taste profiles. The TAS2R38 genotype had a significant effect on bitter perception (p = 0.02) but did not significantly affect taste liking. In conclusion, the cooking method affected turnip liking, and the bitter perception in turnip was influenced by the TAS2R38 genotype. However, taste sensitivity did not predict turnip liking in this UK adult cohort.
... This study identified eight ITCs in zhacai, with allyl isothiocyanate (E1) expressed as the dominant component, which was consistent with previous studies (Liu, 2009). Allyl isothiocyanate elicits strong pungent smells, and also has anti-inflammatory, anticancer, antibacterial, and other biological activities (Traka & Mithen, 2009), affording zhacai a variety of physiological and health benefits. ...
Abstract The salt‐reducing pickling method has been applied to the industrial production of zhacai. In order to reveal the succession of the microbial community structure and flavor components during the pickling process, this study used PacBio Sequel to sequence the full length of 16S rRNA (bacteria, 1400 bp) and ITS (fungi, 1200 bp) genes, and detected flavor components simultaneously, including organic acids, volatile flavor components (VFC), monosaccharides, and amino acids. Eleven phyla and 148 genera were identified in the bacterial community, and 2 phyla and 60 genera in the fungal community. During the four stages of pickling, the dominant bacterial genera were Leuconostoc, Lactobacillus, Leuconostoc, and Lactobacillus, while the dominant fungal genera were Aspergillus, Kazachstania, Debaryomyces, and Debaryomyces, respectively. There were 32 main flavor components (5 organic acids, 19 VFCs, 3 monosaccharides, and 5 amino acids). Correlation heat mapping and bidirectional orthogonal partial least squares (O2PLS) analysis showed that the flora having close relation to flavor components included 14 genera of bacteria (Leuconostoc, Clostridium, Devosia, Lactococcus, Pectobacterium, Sphingobacterium, Serratia, Stenotrophomonas, Halanaerobium, Tetragenococcus, Chromohalobacter, Klebsiella, Acidovorax, and Acinetobacter) and 3 genera of fungi (Filobasidium, Malassezia, and Aspergillus). This study provides detailed data regarding the microbial community and flavor components during the salt‐reducing pickling process of zhacai, which can be used as a reference for the development and improvement of salt‐reducing pickling methods.
... Currently, several families are candidates to be exploited in microgreen production. Among these, many species of the Brassicaceae family present high amounts of micronutrients such as polyphenols, ascorbic acid, carotenoids, tocopherols [15][16][17][18], selenium, sulfur, calcium, and glycosylates [19][20][21], which are very effective in neutralizing radiation-induced oxidative damage and the risk of the occurrence of several types of cancer [7,20,[22][23][24]. These secondary metabolites are tightly linked to plant protection and defense pathways, and may be influenced by environmental conditions and agricultural practices [25][26][27]. ...
An adequate and balanced diet is fundamental in preserving the health of astronauts from several space-induced diseases. Therefore, the integration of a diet with fresh food, rich in bioactive compounds such as microgreens produced directly onboard, may be useful in space for human nutrition. However, ionizing radiation (IR) in space represents a significant hindrance for organisms, with potential critical outcomes on plant morpho-anatomical, eco-physiological, and biochemical aspects, depending on the plant and IR features (e.g., species, developmental stage, IR dose, and type). In this study, we analyzed the effect of different doses of X-rays (0-control, 0.3, 1, 10, 20, and 30 Gy) on the morpho-anatomical and nutritional traits of microgreens of Brassica rapa L., irradiated at the stage of germinated seeds. After the irradiation, microgreens were cultivated in controlled conditions. At harvest, the morpho-biometric traits were analyzed, along with the leaf functional anatomical traits and the phytochemical content of the aboveground biomass. The results showed that X-ray exposure does not induce detrimental effects on growth, while it stimulates the production of antioxidants, improving plant defense and nutritional value. The overall results support the idea of using this species in space as a supplemental functional food.
... Cruciferous crops are economically important as vegetables, edible and industrial oil sources, animal feeds, manure and biofuel [1]. Cruciferous vegetables are rich in nutrients and also have medical benefits for human health [2,3]. With growing market demands for yield and quality, the global needs for efficient and sustainable cultivation of cruciferous crops are increasing. ...
Clubroot, caused by Plasmodiophora brassicae, is a severe soil-borne disease that restricts the production of cruciferous crops worldwide. A better understanding of biotic and abiotic factors regulating germination of P. brassicae resting spores in the soil is significant for developing novel control methods. Previous studies reported that root exudates can trigger P. brassicae resting spore germination, thus enabling a targeted attack of P. brassicae on host plant roots. However, we found that native root exudates collected under sterile conditions from host or non-host plants cannot stimulate the germination of sterile spores, indicating that root exudates may not be direct stimulation factors. Instead, our studies demonstrate that soil bacteria are essential for triggering germination. Through 16s rRNA amplicon sequencing analysis, we found that certain carbon sources and nitrate can reshape the initial microbial community to an inducing community leading to the germination of P. brassicae resting spores. The stimulating communities significantly differed in composition and abundance of bacterial taxa compared to the non-stimulating ones. Several enriched bacterial taxa in stimulating community were significantly correlated with spore germination rates and may be involved as stimulation factors. Based on our findings, a multi-factorial ‘pathobiome’ model comprising abiotic and biotic factors is proposed to represent the putative plant-microbiome-pathogen interactions associated with breaking spore dormancy of P. brassicae in soil. This study presents novel views on P. brassicae pathogenicity and lays the foundation for novel sustainable control strategies of clubroot.
... Particularly, aromatic isothiocyanates containing phenethyl-, benzyl-, and benzoyl-groups exhibit a wide range of antimicrobial activity due to their ability to cross and depolarize bacterial membranes [21]. ITCs are also known to possess anticancer activity by inducing apoptosis and cytokine production, as well as anti-inflammatory and cardio-protective properties [22]. It was also found that the MLE contained megastigmatrienone (also known as tabanone), one of the main terpene components. ...
In this study, we report the green synthesis of silver nanoparticles (AgNPs) from Morus alba or white mulberry leaf extract (MLE) and assess their antibacterial and anticancer potential. The GC–MS analysis of MLE confirmed the existence of phenolic compounds, serving as reducing, capping, and stabilizing agents in the biosynthesis of AgNPs. The MLE-AgNPs were spherical, with an average particle size of 20–44.5 nm and a face-centered cubic structure. EDX spectra confirmed the formation of AgNPs, and a negative zeta potential value (−14.5 mV) suggested their physicochemical stability. Excellent antibacterial activity was demonstrated by MLE-AgNPs against Acinetobacter baumannii strains with a MIC of 2 μg/mL, while good activity was observed against other Gram-negative (Escherichia coli and Salmonella typhimurium) and Gram-positive (Bacillus subtilis and Staphylococcus aureus) bacteria with a MIC of 32 μg/mL. In vitro cytotoxic effects on MCF-7 (human breast cancer cells) and MCF-10A (normal human mammary epithelial cells) were investigated by the MTT assay. The half-maximal inhibitory concentrations (IC50) against MCF-7 cells were 18 and 33 μg/mL for MLE-AgNPs and MLE, respectively, with no effect on normal MCF-10A cells. Altogether, the results support the high antibacterial and anticancer potential of biosynthesized AgNPs by white mulberry leaf extract.
... Glucosinolates are plant-specialized metabolites of the order Brassicales, which includes agriculturally and nutritionally important crops of Brassicaceae, such as cabbage, broccoli, kale, mustard and oilseed rape [1][2][3][4]. The presence of glucosinolates in these crops has attracted considerable interest, as they play a role in plant chemical defense against herbivores, and microbial pathogens and are of interest as health-beneficial components in the human diet (e.g., [5][6][7][8][9]). Most of the biological activities associated with glucosinolates are due to their breakdown products, which are formed when glucosinolates are mixed with their hydrolytic enzymes, thioglucoside glucohydrolases known as myrosinases (EC 3.2.1.147) ...
Glucosinolates, specialized metabolites of the Brassicales including Brassica crops and Arabidopsis thaliana, have attracted considerable interest as chemical defenses and health-promoting compounds. Their biological activities are mostly due to breakdown products formed upon mixing with co-occurring myrosinases and specifier proteins, which can result in multiple products with differing properties, even from a single glucosinolate. Whereas product profiles of aliphatic glucosinolates have frequently been reported, indole glucosinolate breakdown may result in complex mixtures, the analysis of which challenging. The aim of this study was to assess the breakdown of indole glucosinolates in A. thaliana root and rosette homogenates and to test the impact of nitrile-specifier proteins (NSPs) on product profiles. To develop a GC-MS-method for quantification of carbinols and nitriles derived from three prominent indole glucosinolates, we synthesized standards, established derivatization conditions, determined relative response factors and evaluated applicability of the method to plant homogenates. We show that carbinols are more dominant among the detected products in rosette than in root homogenates of wild-type and NSP1- or NSP3-deficient mutants. NSP1 is solely responsible for nitrile formation in rosette homogenates and is the major NSP for indolic nitrile formation in root homogenates, with no contribution from NSP3. These results will contribute to the understanding of the roles of NSPs in plants.
In an aging society, there is a growing interest in functional foods that offer anti-aging benefits. Food-derived bioactive compounds such as carotenoids and polyphenols can enhance skin elasticity and delay aging. However, the mechanisms by which these orally ingested compounds directly impact the skin are not fully understood. Recent studies on exosomes have suggested significant physiological functions, including their potential for intercellular communication. Similar to mammalian exosomes, plant-derived exosomes are known for their functional roles, including cross-kingdom communication, and their ability to target specific organs in animal models as delivery vehicles. The authors have been investigating the anti-aging effects of kale and have previously reported its benefits on cognitive function and skin aging in mouse models. Long-term oral administration of glucoraphanin-enriched kale suppresses the senescence symptoms in skin and hair and increases type I collagen and antioxidant enzyme expression in skin tissues, indicating its role in promoting skin health. Exosome-like nanoparticles (ELNs) from glucoraphanin-enriched kale appear to modulate the expression of extracellular matrix-related genes. Kale-derived ELNs exhibit great potential for ameliorating skin aging, suggesting their ability to promote skin health through targeted cellular mechanisms and supporting their use as an active natural compound in nutraceuticals and functional beverages.
Bok-choy is a green leafy vegetable packed with impressive health benefits. Bok choy is rich in many nutritious, such as vitamins, minerals, and dietary fiber, as well as non-nutritive bioactive compounds, such as flavonoids, total glucosinolates, anthocyanins, kaempferol, and quercetin. These bioactive compounds protect chronic diseases, such as inflammation, cancer, Alzheimer's, cardiovascular disease, and other diseases. Bok chow is the best source of nutritional and non-nutrient compounds and should be taken as a part of the diet regularly.
Naturally occurring isothiocyanates (ITCs) found in cruciferous vegetables, such as benzyl isothiocyanate (BITC), phenethyl isothiocyanate (PEITC), and sulforaphane (SFN), have attracted significant research interest for their promising anti-cancer activity in vitro and in vivo. While the induction of apoptosis is recognized to play a key role in the anti-cancer effects of ITCs, the specific protein targets and associated upstream events underlying ITC-induced apoptosis remain unknown. In this study, we present a set of chemical probes that are derived from BITC, PEITC, and SFN and equipped with bioorthogonal alkynyl handles to systematically profile the target proteins of ITCs in live cancer cells. Using a competition-based quantitative chemical proteomics approach, we identify a range of candidate target proteins of ITCs enriched in biological processes such as apoptosis. We show that BID, an apoptosis regulator of the Bcl-2 family, is covalently modified by ITCs on its N -terminal cysteines. Functional characterization demonstrates that covalent binding to N -terminal cysteines of BID by PEITC results in conformational changes of the protein and disruption of the self-inhibitory interaction between N - and C -terminal regions of BID, thus unleashing the highly active C -terminal segment to exert downstream pro-apoptotic effects. Consistently, PEITC promotes the cleavage and mitochondrial translocation of BID, leading to a strong induction of apoptosis. We further show that mutation of N -terminal cysteines impairs the N - and C -terminal interaction of BID, relieving the self-inhibition and enhancing its apoptotic activity. Overall, our chemical proteomics profiling and functional studies not only reveal BID as the principal target of PEITC in mediating upstream events for the induction of apoptosis, but also uncover a novel molecular mechanism involving N -terminal cysteines within the first helix of BID in regulating its pro-apoptotic potential.
Health reflects the ability of an organism to adapt to stress. Stresses—metabolic, proteotoxic, mitotic, oxidative, and DNA damage—not only contribute to the etiology of cancer and other chronic degenerative diseases but are also hallmarks of the cancer phenotype. Activation of the Kelch-like ECH-associated protein 1 (KEAP1)-NF-E2-related factor 2 (NRF2)-signaling pathway is an adaptive response to environmental and endogenous stresses. It renders animals resistant to chemical carcinogenesis and other forms of toxicity. In contrast, disruption of the pathway exacerbates the outcomes. The pathway can be induced by thiol-reactive small molecules that demonstrate protective efficacy in preclinical chemoprevention models and clinical trials. However, mutations and epigenetic modifications affecting the regulation and fate of NRF2 can lead to constitutive dominant hyperactivation of signaling that preserves rather than attenuates cancer phenotypes by providing selective resistance to stresses.
Microgreens, also called superfoods, emerge because of their high levels of nutrients, diverse flavour profiles, and sustainable cultivation methods, which make them culinary delights and valuable to a healthy and flavorful diet. The present study investigated Brassicaceae family microgreens, proposing a novel system (quality indices) that allows scoring among them. Fourteen Brassica microgreen species were morphological, phytochemical, and sensorial investigated. The morphological assessment revealed that radish microgreens exhibited the highest leaf area (p < 0.05), while red mizuna demonstrated superior yield. Cauliflower microgreens contained the highest concentrations of ascorbic acid (HPLC-DAD) and total phenolic content (p < 0.05). Phytochemical analysis using HPLC-MS/MS identified over 18 glucosinolates and phenolic compounds. Red mustard and red cabbage showed the highest glucosinolate content (p < 0.05). Watercress exhibited the highest phenolic compound content (p < 0.05), primarily flavonoids, while broccoli and radish contained the highest isothiocyanate levels. Cauliflower microgreens resulted in the most consumer-accepted variety. Appling quality indices scoring system identified radish, cauliflower, and broccoli microgreens as the most promising species. This study underscores the potential of Brassica microgreens as an excellent source of health-promoting phytochemicals with favorable market acceptance, providing valuable insights for both nutritional research and commercial applications.
Leaf mustard (Brassica juncea L.) is explored for its biofumigant properties, derived from its secondary metabolites, particularly allyl isothiocyanate (AITC), produced during the enzymatic breakdown of glucosinolates like sinigrin. The research examines eight leaf mustard cultivars developed in Yeosu city, South Korea, focusing on their genetic characteristics, AITC concentration and nitriles formation rates from glucosinolates. Results indicate that the allelopathic effects, largely dependent on AITC concentration and enzymatic activity, vary across cultivar. Sinigrin and AITC constitute 79% and 36%, respectively, of glucosinolate and its hydrolysis products. The cultivar 'Nuttongii' demonstrates significant potential for inhibiting weeds, exhibiting the highest AITC concentration at 27.47 ± 6.46 µmole g⁻¹ These outcomes highlight the importance of selecting mustard cultivars for biofumigation based on their glucosinolate profiles and hydrolysis product yields. The study also identifies a significant genetic influence on AITC and nitrile formation, suggesting that epithiospecifier protein modulation could enhance both allelopathic and other beneficial effects. Collectively, the research underscores the promise of mustard as a sustainable, environmentally friendly alternative to traditional herbicides.
We describe ‘Boldrewood’, a new accession of watercress (Nasturtium officinale R. Br.) that was initially found to be of short stature with high antioxidant capacity (Payne et al. 2015). This was of particular commercial interest because it offered the potential to develop a novel watercress product with fork-friendly size and improved health-benefits. In two commercial trials comparing Boldrewood to a control, we confirmed that Boldrewood exhibits a dwarf phenotype with a significantly shorter stem and consistently produced more leaves per stem area alongside comparable crop biomass. The antioxidant and chemopreventive capacity of Boldrewood were comparable to the commercial crop. For the first time, we observed a novel increase in glucosinolate concentrations and cytotoxicity to cancer cells, characterised as decreased IC50 (half-maximal concentration of an inhibitor), associated with increased crop age at harvest. This suggests that a slower-growing and longer to harvest crop provides a significant improvement in health benefits gained in this leafy crop which is already known to be highly nutrient dense and with considerable chemopreventive ability.
Sulfur (S) is an essential element for life on Earth. Plants are able to take up and utilize sulfate (SO42-), the most oxidized inorganic form of S compounds on Earth, through the reductive S assimilatory pathway that couples with the photosynthetic energy conversion. Organic S compounds are subsequently synthesized in plants and made accessible to animals, primarily as the amino acid methionine. Thus, the plant S metabolism clearly has nutritional importance in the global food chain. The S metabolites may be part of the redox regulation and drivers of essential metabolic pathways as cofactors and prosthetic groups, such as Fe-S centers, coenzyme A, thiamine, and lipoic acid. The evolution of the S metabolic pathways and enzymes reflects critical importance of the functional innovation and diversifications. Here we review the major evolutionary alterations that took place in the S metabolism across different scales and outline research directions that may take advantage of understanding the evolutionary adaptations.
Cabbage (Brassica oleracea var. capitata L.) holds significant agricultural and nutritional importance in Ethiopia; yet, its production faces challenges, including suboptimal nitrogen fertilizer management. The aim of this review was to review the possible effect of nitrogen fertilizer levels on the production of cabbage in Ethiopia. Nitrogen fertilization significantly influences cabbage yield and quality. Moderate to high levels of nitrogen application enhance plant growth, leaf area, head weight, and yield. However, excessive nitrogen levels can lead to adverse effects such as delayed maturity, increased susceptibility to pests and diseases, and reduced postharvest quality. In Ethiopia, small-scale farmers use different nitrogen levels for cabbage cultivation. In Ethiopia, NPSB or NPSBZN fertilizers are widely employed for the growing of various crops such as cabbage. 242 kg of NPS and 79 kg of urea are the blanket recommendation for the current production of cabbage in Ethiopia. The existing rate is not conducive for farmers. Therefore, small-scale farmers ought to utilize an optimal and cost-effective nitrogen rate to boost the cabbage yield. Furthermore, the effectiveness of nitrogen fertilization is influenced by various factors including the soil type, climate, cabbage variety, and agronomic practices. Integrated nutrient management approaches, combining nitrogen fertilizers with organic amendments or other nutrients, have shown promise in optimizing cabbage production while minimizing environmental impacts. The government ought to heed suggestions concerning soil characteristics such as the soil type, fertility, and additional factors such as the soil pH level and soil moisture contents.
Phenethyl isothiocyanate (PEITC), a compound derived from cruciferous vegetables, has garnered attention for its anticancer properties. This review synthesizes existing research on PEITC, focusing on its mechanisms of action in combatting cancer. PEITC has been found to be effective against various cancer types, such as breast, prostate, lung, colon, and pancreatic cancers. Its anticancer activities are mediated through several mechanisms, including the induction of apoptosis (programmed cell death), inhibition of cell proliferation, suppression of angiogenesis (formation of new blood vessels that feed tumors), and reduction of metastasis (spread of cancer cells to new areas). PEITC targets crucial cellular signaling pathways involved in cancer progression, notably the Nuclear Factor kappa‐light‐chain‐enhancer of activated B cells (NF‐κB), Protein Kinase B (Akt), and Mitogen‐Activated Protein Kinase (MAPK) pathways. These findings suggest PEITC's potential as a therapeutic agent against cancer. However, further research is necessary to determine the optimal dosage, understand its bioavailability, and assess potential side effects. This will be crucial for developing PEITC‐based treatments that are both effective and safe for clinical use in cancer therapy.
A káposztarepce (Brassica napus L.) sokoldalú felhasználása miatt kiemelkedő szerepet játszik a mezőgazdasági termesztésben. DK Exstrom repcehibridddel végzett lombtrágyázási kísérlet beállítására Regölyben került sor, 1 ha-os parcellákon, sávos elhelyezésben, a kezeléseket a virágzás kezdeti állapotában végeztük. A lombtrágyaként alkalmazott kezelések az adott elemre számítva a következők voltak: Ca=3kg/ha (CaCO3); Zn=1kg/ha (ZnCO3); Zn+Ca=1kg/ha+3kg/ha (ZnCO3 + CaCO3); C-komplex (réz+cink komplex)=0,5 kg/ha réz és 0,8 kg/ha cinktartalommal. A betakarítást hozam térképpel ellátott kombájnnal végeztük. A magvak metanolos kivonatából spektrofotometriás úton az összes polifenoltartalmat (TPC) és az antioxidáns/redukáló kapacitást (FRAP) határoztuk meg. A kontrollhoz viszonyítva mindegyik kezelés növekedést eredményezett az összes polifenoltartalomban és az antioxidáns kapacitásban is. A legjobb eredményeket a C-komplex-el (réz+cink komplex) értük el.
Proanthocyanidins (PACs), the predominant constituents within Grape Seed Extract (GSE), are intricate compounds composed of interconnected flavan-3-ol units. Renowned for their health-affirming properties, PACs offer a shield against a spectrum of inflammation associated diseases, such as diabetes, obesity, degenerations and possibly cancer. While monomeric and dimeric PACs undergo some absorption within the gastrointestinal tract, their larger oligomeric and polymeric counterparts are not bioavailable. However, higher molecular weight PACs engage with the colonic microbiota, fostering the production of bioavailable metabolites that undergo metabolic processes, culminating in the emergence of bioactive agents capable of modulating physiological processes.
Within this investigation, a GSE enriched with polymeric PACs was employed to explore in detail their impact. Through comprehensive analysis, the present study unequivocally verified the gastrointestinal-mediated transformation of medium to high molecular weight polymeric PACs, thereby establishing the bioaccessibility of a principal catabolite termed 5-(3′,4′-dihydroxyphenyl)-γ-valerolactone (VL). Notably, our findings, encompassing cell biology, chemistry and proteomics, converge to the proposal of the notion of the capacity of VL to activate, upon oxidation to the corresponding quinone, the nuclear factor E2-related factor 2 (Nrf2) pathway—an intricate process that incites cellular defenses and mitigates stress-induced responses, such as a challenge brought by TNFα. This mechanistic paradigm seamlessly aligns with the concept of para-hormesis, ultimately orchestrating the resilience to stress and the preservation of cellular redox equilibrium and homeostasis as benchmarks of health.
Cruciferous-rich diets, particularly broccoli, have been associated with reduced risk of developing cancers of various sites, cardiovascular disease and type-2 diabetes. Sulforaphane (SF), a sulfur-containing broccoli-derived metabolite, has been identified as the major bioactive compound mediating these health benefits. Sulforaphane is a potent dietary activator of the transcription factor Nuclear factor erythroid-like 2 (NRF2), the master regulator of antioxidant cell capacity responsible for inducing cytoprotective genes, but its role in glucose homeostasis remains unclear. In this study, we set to test the hypothesis that SF regulates glucose metabolism and ameliorates glucose overload and its resulting oxidative stress by inducing NRF2 in human hepatoma HepG2 cells. HepG2 cells were exposed to varying glucose concentrations: basal (5.5 mM) and high glucose (25 mM), in the presence of physiological concentrations of SF (10 μM). SF upregulated the expression of glutathione (GSH) biosynthetic genes and significantly increased levels of reduced GSH. Labelled glucose and glutamine experiments to measure metabolic fluxes identified that SF increased intracellular utilisation of glycine and glutamate by redirecting the latter away from the TCA cycle and increased the import of cysteine from the media, likely to support glutathione synthesis. Furthermore, SF altered pathways generating NADPH, the necessary cofactor for oxidoreductase reactions, namely pentose phosphate pathway and 1C-metabolism, leading to the redirection of glucose away from glycolysis and towards PPP and of methionine towards methylation substrates. Finally, transcriptomic and targeted metabolomics LC-MS analysis of NRF2-KD HepG2 cells generated using CRISPR-Cas9 genome editing revealed that the above metabolic effects are mediated through NRF2. These results suggest that the antioxidant properties of cruciferous diets are intricately connected to their metabolic benefits.
The amino acid L-serine (Ser) is indispensable for several cellular processes. In addition to forming part of proteins, Ser is required for the synthesis of other amino acids, nitrogen bases, and lipids and is a source of one-carbon units (1C) for methylation reactions. Unlike animals, plants synthesize Ser through different pathways, which has complicated the understanding of the amino acid homeostasis. Three Ser biosynthesis pathways have been described in plants: the glycolate pathway, associated with photorespiration, and two non-photorespiratory pathways, the phosphorylated and the glycerate pathway of Ser biosynthesis. In recent years, the specific contribution of each pathway to Ser homeostasis and how they are coordinated to participate in plant metabolism and development have started to be elucidated. Current knowledge on serine biosynthesis and functions in plants is reviewed here and, whenever possible, compared with current knowledge in mammals. We focus on recent advances that link Ser with other metabolic processes, such as the Ser-glycine-1C network, and nitrogen and sulfur metabolism. We also discuss the role of Ser metabolic reprogramming in the plant response to biotic and abiotic stresses and how it can be affected by climate change.KeywordsGlycineNitrogen metabolismOne-carbon metabolismSerineSulfur metabolism
Orange Chinese cabbage (Brassica rapa L. ssp. pekinensis) is an excellent source of health-promoting nutrients that could reduce the risk of chronic diseases. This study mainly investigated the accumulation patterns of eight lines of orange Chinese cabbage for indolic glucosinolates (GLSs) and pigment content from representative plant organs across multiple developmental stages. The indolic GLSs were highly accumulated at the rosette stage (S2), especially in inner and middle leaves, and the order of indolic GLSs accumulation in non-edible organs was flower > seed > stem > silique. The expression levels of biosynthetic genes in light signaling, MEP, carotenoids, and GLS pathways were consistent with the metabolic accumulation patterns. The results of a principal component analysis show a clear separation of high indolic GLS lines (15S1094 and 18BC6) from low indolic GLS lines (20S530). A negative correlation between the accumulation of indolic GLS and carotenoids was identified in our study. Our work contributes to providing valuable knowledge required to breed, grow, and select orange Chinese cabbage varieties and their eatable organs with higher nutritional value.
Nowadays, the yeast Saccharomyces cerevisiae is the platform of choice for demonstrating the proof of concept of the production of metabolites with a complex structure. However, introducing heterologous genes and rewiring the endogenous metabolism is still not standardized enough, affecting negatively the readiness-to-market of such metabolites. We developed the Easy Modular Integrative fuSion-ready Expression (Easy-MISE) toolkit, which is a novel combination of synthetic biology tools based on a single Golden Gate multiplasmid assembly meant to further ameliorate the rational predictability and flexibility of the process of yeast engineering. Thanks to an improved cloning screening strategy, double and independent transcription units are easily assembled and subsequently integrated into previously characterized loci. Moreover, the devices can be tagged for localization. This design allows for a higher degree of modularity and increases the flexibility of the engineering strategy. We show with a case study how the developed toolkit accelerates the construction and the analysis of the intermediate and the final engineered yeast strains, leaving space to better characterize the heterologous biosynthetic pathway in the final host and, overall, to improve the fermentation performances. Different S. cerevisiae strains were built harboring different versions of the biochemical pathway toward glucobrassicin (GLB) production, an indolyl-methyl glucosinolate. In the end, we could demonstrate that in the tested conditions the best-producing strain leads to a final concentration of GLB of 9.80 ± 0.267 mg/L, a titer 10-fold higher than the best result previously reported in the literature.
Production of plant-derived nutraceuticals using mutation, elicitation, precursor feeding, genetic transformation and metabolic engineering.
The objective was to study the phytochemical profile of yellow (Sinapis alba) and oriental mustard (Brassica juncea) flour and bran fractions. The HS-SPME-GC/MS technique was used to determine the volatile profile. Up to 53 volatiles, including isothiocyanates, alkanes, alcohols, ketones and esters were identified in the mustard powders. In addition, the free and bound bioactive fraction was studied and the determination was performed by UHPLC-MS/MS. A total of 26 compounds (phenolic acids, flavonoids and glucosinolates) were identified in the mustard samples. Sinapis alba, including the bran fraction, was richer in p-hydroxybenzoic, ferulic and p-coumaric acids compared to Brassica juncea. In addition, the volatile profile in yellow mustard seeds, as analysed by HS-SPME-GC/MS, has been described for the first time in this work. The study of the principal components showed that according to the bioactive compounds profile it is possible to differentiate between the two mustard species analysed. Moreover, the bran fraction, as a by-product of the seed processing, is a matrix rich in bioactive compounds and could be applied for the recovery of polyphenols for agro-industrial or pharmaceutical application.
The role of underground starchy crops in global food security is widely recognized. These are subsistence crops considered as neglected and underutilized. However, these have become the new focus of international community for providing income opportunities and presenting different potential uses. The social and economic trajectories of most South American countries are linked to these crops, with emphasis on cassava, yams, potatoes, and sweet potatoes, responsible for 93% of the root and tuber species consumed worldwide. However, there are several other neglected plant species that are cultivated by traditional communities with commercial-scale potential. These were not widely recognized in the literature and mainly include contain phosphorus, mucilage, and high levels of phenolic compounds. This chapter highlights the role of South America in the origin, evolution, and domestication of underground starchy species and how local communities have shaped the genetic architecture and biodiversity of these plants.
Cytochrome b5 (CB5) is a small heme-binding protein, known as an electron donor delivering reducing power to the terminal enzymes involved in oxidative reactions. In plants, the CB5 protein family is substantially expanded both in its isoform numbers and cellular functions, compared to its yeast and mammalian counterparts. As an electron carrier, plant CB5 proteins function not only in fatty acid desaturation, hydroxylation and elongation, but also in the formation of specialized metabolites such as flavonoids, phenolic esters, and heteropolymer lignin. Furthermore, plant CB5s are found to interact with different non-catalytic proteins such as ethylene signaling regulator, cell death inhibitor, and sugar transporters, implicating their versatile regulatory roles in coordinating different metabolic and cellular processes, presumably in respect to the cellular redox status and/or carbon availability. Compared to the plentiful studies on biochemistry and cellular functions of mammalian CB5 proteins, the cellular and metabolic roles of plant CB5 proteins have received far less attention. This article summarizes the fragmentary information pertaining to the discovery of plant CB5 proteins, and discusses the conventional and peculiar functions that plant CB5s might play in different metabolic and cellular processes. Gaining comprehensive insight into the biological functions of CB5 proteins could offer effective biotechnological solutions to tailor plant chemodiversity and cellular responses to environment stimuli.
Isothiocyanates are the highly reactive organo-sulphur phytochemicals and are product of hydrolysis of glucosinolates which are present mainly in the cruciferous vegetables. These compounds due to their unique chemical reactivity possess anti-cancer, anti-inflammatory, and neuroprotective properties. Epidemiological and experimental evidences suggest that isothiocyanates reduce oxidative stress and act as indirect antioxidants as well as antimicrobials, therefore, have received attention from the researchers for their possible application in pharmacological and food industry. However, due to high volatility and heat sensitivity of these bioactive compounds, their extraction is very challenging and requires the application of various innovative technologies. In addition to that, their fate during the processing conditions also needs to be considered as these processes tend to affect their bioavailability. Isothiocyanates exhibit wide range of antimicrobial activity due to their ability of reducing oxygen consumption and depolarizing the mitochondrial membrane in bacterial cells. They are generally regarded as safe (GRAS) compounds and hence are allowed to be added to the food as preservatives. Due to their antimicrobial properties, isothiocyanates incorporated food packaging films have become popular in the last decade. They are known to act as substrates to activate lactoperoxidases (LPO) for extension of shelf life of dairy products due to its bactericidal and bacteriostatic properties. This review addresses the detailed evidences supporting the biological activities, bioavailability and stability, methods of extraction, and explanation for their taste perception, as well as utilization of these isothiocyanates in food packaging as natural antimicrobials or natural preservatives to improve shelf life of foodstuffs.
Numerous herbivores orally secrete defense compounds to detoxify plant toxins. However, little is known about the role of orally secreted enzymes by a specialized pest, Plutella xylostella, in the detoxification of plant defense compounds. Three glucosinolate sulfatases (GSSs) or two sulfatase-modifying factors (SUMF1s) mutant strains were established on the basis of CRISPR/Cas9 technology to validate the existence of a species-specific GSSs−SUMF1s system. In comparison to the bioassay data from mutant strains of GSS1/GSS2 or SUMF1a/SUMF1b, GSS3 had a minimal role because no significant change was found in GSS3−/− under different feeding contexts. Antibody-based technologies were used to examine GSSs-related deficient strains, and the results showed that the GSS1 protein was primarily released through larval oral secretion. On the basis of high-performance liquid chromatography, we found that GSS1 was secreted to pre-desulfate the typical plant defensive glucosinolates known as 4-(methylsulfinyl)butyl glucosinolate (4MSOB-GL) to suppress the production of the toxic substance, which is referred to as predetoxification strategy. These findings highlighted that the GSSs−SUMF1s system is the key factor for counteradaptation of P.
xylostella to cruciferous plants, which strengthens the concept that herbivores deploy pre-detoxification strategies to disrupt the plant
chemical defenses to facilitate the colonization process.
Brassicaceae vegetables are an important traditional daily food in China and around the world, which provide nutrients and phytochemicals that are beneficial for human health. Among them, Brassica and Raphanus are widely cultivated and eaten, have been evolved and/or bred for special characteristics during the long history of cultivation. Epidemiological studies suggest that the health benefits of Brassicaceae vegetables are mainly associated with glucosinolates (GSLs) and their hydrolytic products. In this review, we discuss the diversity of common consumed Brassicaceae vegetables and their GSL composition in edible parts. We also discuss the diversity factors affecting GSL content, and the diversity roles and functions of GSL. The information in this review provides guidance for consumers to select vegetables with a high GSL content, optimum edible stages, suitable edible methods, and provides a theoretical basis for crop molecular breeding and market development of GSL products.
Gnotobiotic growing rats harbouring either a whole human faecal flora or single human strains of Escherichia coli (EM0) or Bacteroides vulgatus (BV8H1) were fed for 7 weeks on semi-synthetic diets in which the protein source was either soya-bean meal (SM) or rape-seed meal (RM). For each bacterial status the RM-diet group was compared with the control group fed on the SM diet. The association of human faecal flora with the RM diet was responsible for reduced feed intake and reduced weight gain, an enlargement of the liver and thyroid and a decrease in both thyroxine and triiodothyronine plasma levels. The association of the B. vulgatus BV8H1 strain with the RM diet reproduced all these effects, except that triiodothyronine plasma levels were not significantly modified. Rats inoculated with the E. coli EM0 strain and fed on the RM diet exhibited a goitre and lowered thyroxine and triiodothyronine plasma levels. These results show that the human intestinal microflora may be involved in glucosinolate metabolism when cruciferous vegetables are consumed by man. The specificity of the symptoms observed according to the rat bacterial status supports the hypothesis that bacteria yield specific toxic glucosinolate derivatives according to their enzymic potential.
Sulforaphane (SUL), an isothiocyanate found in broccoli and other cruciferous vegetables, has been shown to induce phase II detoxification enzymes, inhibit chemically induced mammary tumors in rats, and more recently to induce cell cycle arrest and apoptosis in cancer cells of the colon. Here, we provide evidence that SUL also acts as a breast cancer anti-proliferative agent. The BALB/c mouse mammary carcinoma cell line F3II was treated with SUL at concentrations up to 15 microM and examined for markers of cell cycle arrest and apoptosis. Treatment of asynchronous F3II cells with 15 microM SUL resulted in G2/M cell cycle arrest, elevated p34cdc2 (cdc2) kinase activity, Bcl-2 down-regulation, evidence of caspase activation, and aggregation of condensed nuclear chromatin. Subsequent exposure of synchronized cells to 15 microM SUL resulted in elevated numbers of prophase/prometaphase mitotic figures, indicating cell cycle progression beyond G2 and arrest early within mitosis. Moreover, cells treated with 15 microM SUL displayed aberrant mitotic spindles, and higher doses of SUL inhibited tubulin polymerization in vitro. In addition, BALB/c mice injected s.c. with F3II cells and subsequently injected daily i.v. with SUL (15 nmol/day for 13 days) developed significantly smaller tumors (approximately 60% less in mass) than vehicle-treated controls. Western blot analysis of tumor proteins demonstrated significantly (P<0.05) reduced PCNA and elevated PARP fragmentation in samples from animals dosed with SUL. Taken together, these results indicate that SUL has mammary cancer suppressive actions both in cell culture and in the whole animal. Inhibition of mammary carcinogenesis appears in part to involve perturbation of mitotic microtubules and early M-phase block associated with cdc2 kinase activation, indicating that cells arrest prior to metaphase exit.
The development of hybrid broccoli genotypes with enhanced levels of 4-methylsulphinylbutyl glucosinolate, the precursor of anticarcinogenic isothiocyanate sulforaphane (SF), by introgressing genomic segments from the wild ancestor Brassica villosa is described. We demonstrate that to obtain enhanced levels of either 3-methylsulphinylpropyl or 4-methylsulphinylbutyl glucosinolate it is necessary to have B. villosa alleles in either a homozygous or heterozygous state at a single quantitative trait locus (QTL) on O2. The ratio of these two glucosinolates, and thus whether iberin or SF is generated upon hydrolysis, is determined by the presence or absence of B. villosa alleles at this QTL, but also at an additional QTL2 on O5. We further demonstrate that following mild cooking high glucosinolate broccoli lines generate about three fold higher levels of SF than conventional varieties. Commercial freezing processes and storage of high glucosinolate broccoli maintains the high level of glucosinolates compared to standard cultivars, although the blanching process denatures the endogenous myrosinase activity.
Cruciferous vegetables contain glucosinolates that, after conversion to isothiocyanates (ITC), are capable of inducing cytoprotective genes. We examined whether broccoli seeds can elicit a chemoprotective response in mouse organs and rodent cell lines and investigated whether this response requires nuclear factor-erythroid 2 p45-related factor 2 (Nrf2). The seeds studied contained glucosinolate at 40 mmol/kg, of which 59% comprised glucoiberin, 19% sinigrin, 8% glucoraphanin, and 7% progoitrin. Dietary administration of broccoli seeds to nrf2+/+ and nrf2-/- mice produced a [~]1.5-fold increase in NAD(P)H:quinone oxidoreductase 1 (NQO1) and glutathione S-transferase (GST) activities in stomach, small intestine, and liver of wild-type mice but not in mutant mice; increased transferase activity was associated with elevated levels of GSTA1/2, GSTA3, and GSTM1/2 subunits. These seeds also increased significantly the level of glutamate cysteine ligase catalytic (GCLC) subunit in the stomach and the small int
The glucosinolates (GSs) were estimated in the normally eaten portions of 72 cultivars of Oriental brassica vegetables including mustard greens ( Brassica juncea L.), Chinese kale ( B. oleracea L. Alboglabra Group Bail.), Chinese cabbage ( B. rapa L. Pekinensis Group Bail.), pak choy ( B. rapa Chinensis Group Bail.), tendergreen ( B. rapa Perviridis Group Bail.), turnip ( B. rapa L. Rapifera Group Bail., B. narinosa Bail., and B. nipposinica Bail.). Variation in GS profiles was complex. There was variation in percentages of major GSs and total GS among B. juncea, B. oleracea , and the combination B. rapa plus narinosa and nipposinica and among four subspecific groups of rapa plus the two species closely related to rapa: narinosa and nipposinica. B. juncea had distinctively high proportions of allyl-GS, ranging from 81% to 94%, whereas B. oleracea had distinctively high proportions of 4-methylsulfinylbutyl-GS, ranging from 9% to 68%. Differences in GS profiles among the rapa groups, narinosa and nipposinica , were less distinctive. Cultivars of pak choy from China differed in percentages of three minor GSs from cultivars from Japan and elsewhere. There was also variation among cultivars of Chinese kale and between turnip foliage and roots.
Fourteen cultivars of turnip [ Brassica rapa, rapifera group, also B. campestris L. ssp. rapifera (Metzg.) Sinsk.] recommended for human consumption of either tops or tops and roots and five cultivars recommended for consumption of roots were selected to compare glucosinolate (GS) levels in tops and roots. Also, two cultivars used for animal feed were included. The study revealed significantly lower levels of 1-methylpropyl-GS and 2-hydroxy-3-butenyl-GS in tops and roots of cultivars grown for greens, compared to those used for animal feed. Contents of 1-methylpropyl-, 3-butenyl-, and 4-pentenyl-GSs were higher in turnip tops than in roots, while 2-hydroxy-3-butenyl-, 4-(methylthio)butyl-, 4-(methylsulfinyl)butyl-, 2-hydroxy-4-pentenyl-, 5-(methylthio)pentyl-, 2-phenylethyl-, 3-indolylmethyl-GSs and total GS were all higher in the roots. GS patterns for seeds tended to correlate with those of the tops.
Correlation coefficients based on relative concentrations of 13 glucosinolates in the edible parts of 30 cultivars were determined. Brussels sprouts ( Brassica oleracea L. gemmifera group), cauliflower ( B. oleracea L. botrytis group), and either marrow-stem or smooth-leafed kale ( B. oleracea L. acephala group) had similar glucosinolate patterns based on significant correlations ( P < 0.01). The glucosinolates of ‘Morris Heading’ collards [( B. oleracea L. acephala group (var. sabellica )] were highly correlated with those of curly kale [ B. oleracea L. acephala group (var. selensia )]. Mustard greens [ B. juncea (L.) Czern. & Coss. var. rugosa Bailey] and the corresponding seeds were the most highly correlated of the 17 cultivars for which the edible parts and seeds were compared. Seed analyses indicated relationships among the cultivars somewhat similar to those seen for the edible portions.
The effects of two aromatic thiocyanates, benzyl thiocyanate (BTC) and benzyl isothiocyanate (BITC), on methylazoxymethanol (MAM) acetate-induced intestinal carcinogenesis were examined using female ACI/N rats. Starting at 5 weeks of age, animals were fed diets containing 100 or 400 p.p.m. BTC, 400 p.p.m. BITC or a control diet At 6 weeks of age, all animals were treated with i.p. injections of MAM acetate (25 mg/kg body wt, once weekly for 3 weeks) or saline. Animals fed experimental diets were changed to the control diet from a week after the last carcinogen treatment Three groups of animals fed the control diet were switched to 100 p.p.m. BTC, 400 p.p.m. BTC or 400 p.p.m. BITC diet from a week after carcinogen treatment Animals given the high-dose BTC diet at the initiation and the post-initiation phase showed smaller incidence (5% and 17%) and multiplicity (0.05 ± 0.21 and 0.17 ± 0.37) of tumors in small intestine compared with those of rats exposed to the carcinogen alone (61% and 1.06 ± 1.18). The incidence and multiplicity of tumors in small intestine (21% and 032 ± 0.73) and the incidence of colon tumors of rats given BITC in the initiation phase (47%) were significantly lower than those of animals treated with carcinogen alone (61%, 1.06 ± 1.18 and 83%). Bromodeoxyuridine labeling indices of the intestinal mucosal cells were measured. The labeling indices were reduced by BTC and BITC exposure at initiation phase in both small intestine and colon. The results of measurement of labeling indices correlated with the decreased tumor incidence and multiplicity in the intestine. These data suggest that BTC and BITC could be promising chemopreventive agents for human intestinal neoplasia.
The catalytic properties of four human glutathione transferases (GSTs), A1-1, M1-1, M4-4 and P1-1, were examined with 14 isothiocyanate (R-NCS) substrates. The compounds include aliphatic and aromatic homologues, some of which are natural constituents of human food, namely sulphoraphane [1-isothiocyanato-4-(methylsulphinyl)butane], erucin [1-isothiocyanato-4-(methylthio)butane], erysolin [1-isothiocyanato-4-(methylsulphonyl)butane], benzyl-NCS, phenethyl-NCS and allyl-NCS. All isothiocyanates investigated were substrates for the four GSTs. The enzymes promote addition of the thiol group of GSH to the electrophilic central carbon of the isothiocyanate group to form dithiocarbamates [R-NH-C(=S)-SG] which have high UV absorption at 274 nm. Molar absorption coefficients and non-enzymic rate constants as well as standardized enzyme assay conditions for all compounds were established. Of the four isoenzymes investigated, GSTs M1-1 and P1-1 were generally the most efficient catalysts, whereas GST M4-4 was the least efficient. Isothiocyanates are among the GST substrates that are most rapidly conjugated. On the basis of rate-enhancement data and binding energies, the isothiocyanates were compared with 4-hydroxyalkenals, another class of natural GST substrates previously subjected to systematic kinetic analysis. The incremental transition-state stabilization attributable to an increased number of methylene groups in homologous alkyl isothiocyanates is similar to that previously noted for homologous 4-hydroxyalkenals.
The effect of consumption of Brussels sprouts on levels of 8-oxo-7,8-dihydro-2'-deoxyguanosine (8-oxodG) in human urine was
investigated in 10 healthy, male, non-smoking volunteers. Following a 3 week run-in period, five volunteers continued on a
diet free of cruciferous vegetables for a subsequent 3 week intervention period (control group), while the other five (sprouts
group) consumed 300 g of cooked Brussels sprouts per day, at the expense of 300 g of a glucosinolate-free vegetable. Levels
of 8-oxodG in 24 h urine samples were measured by HPLC. In the control group there was no difference between the two periods
in levels of 8-oxodG (P = 0.72). In contrast, in the sprouts group the levels of 8-oxodG were decreased by 28% during the intervention period (P = 0.039). The present findings support the results of epidemiologic studies that consumption of cruciferous vegetables may
result in a decreased cancer risk.
The broccoli constituent sulforaphane (1-isothiocyanate-4-methylsulfinylbutane) has previously been shown to protect rats
against 9, 10-dimethyl-1, 2-benz[a]anthracene tumori-genesis, thought to be due, at least in part, to induction of phase II detoxification. We investigated
the ability of sulforaphane to also inhibit the phase I enzyme cytochrome P450 isoenzyme 2E1 (CYP2E1), which is responsible
for activation of several carcinogens, including dialkylnitros-amines. Using the p-nitrophenol hydroxylation assay in microsomes from livers of acetone-treated Sprague-Dawley rats, sulforaphane was shown
to be a potent competitive inhibitor of CYP2E1 with a K1pg of 347.0 ± 4.5 μM. In view of this result, we studied the capacity of sulforaphane to inhibit the genotoxicity of N-nitrosodimethylamine (NDMA). Sulforaphane at concentrations of >0.8 μM inhibited the mutagenicity of NDMA (4.4 mg/plate)
in Salmonella typhimurium strainDTA100 after pre-incubation for 45 min with cytosol extract from livers of Balb/c mice pre-treated with acetone. Unsecheduled
DNA synthesis induced by NDMA (33.5 μM)in mouse hepatocytes was inhibited in a dose-dependent manner by sulforaphane at 0.064–20
μM. Sulforaphane was unable to inhibit mutagenicity of sodium azide (5 μg/plate), a direct acting mutagen, in the Salmonella assay. It was not itself genotoxic in hepatocytes, as measured by unscheduled DNA synthesis, or mutagenic in the strain of
Salmonella employed and cytotoxic only at high concentrations (≧0.5 mM). These findings suggest that inhibition of CYP2E1 by sulforaphane
may offer chemoprotection against carcinogenic substrates of this enzyme.
Chemoprevention involves the use of natural or synthetic substances to reduce the risk of developing cancer. Two dietary components capable of mediating chemopreventive activity in animal models by modulation of drug-metabolizing enzymes are sulforaphane, an aliphatic isothiocyanate, and brassinin, an indole-based dithiocarbamate, both found in cruciferous vegetables. We currently report the synthesis and activity of a novel cancer chemopreventive agent, (+/-)-4-methylsulfinyl-1-(S-methyldithiocarbamyl)-butane (trivial name, sulforamate), an aliphatic analogue of brassinin with structural similarities to sulforaphane. This compound was shown to be a monofunctional inducer of NAD(P)H:quinone oxidoreductase [quinone reductase (QR)], a Phase II enzyme, in murine Hepa 1c1c7 cell culture and two mutants thereof. Induction potential was comparable to that observed with sulforaphane (concentration required to double the specific activity of QR, approximately 0.2 microM), but cytotoxicity was reduced by about 3-fold (IC50 approximately 30 microm). In addition, sulforaphane, as well as the analogue, increased glutathione levels about 2-fold in cultured Hepa 1c1c7 cells. Induction of QR was regulated at the transcriptional level. Using Northern blotting techniques, time- and dose-dependent induction of QR mRNA levels were demonstrated in Hepa 1c1c7 cell culture. To further investigate the mechanism of induction, HepG2 human hepatoma cells were transiently transfected with QR-chloramphenicol acetyltransferase plasmid constructs containing various portions of the 5'-region of the QR gene. Sulforaphane and the analogue significantly induced (P < 0.0001) CAT activity at a concentration of 12.5 microM by interaction with the antioxidant responsive element (5-14-fold induction) without interacting with the xenobiotic responsive element. Moreover, both compounds significantly induced mouse mammary QR and glutathione S-transferase activity (feeding of 3 mg/mouse intragastric for 4 days), whereas the elevation of hepatic enzyme activities was less pronounced. Both sulforaphane and the analogue were identified as potent inhibitors of preneoplastic lesion formation in carcinogen-treated mouse mammary glands in organ culture (84 and 78% inhibition at 1 microm, respectively). On the basis of these results, the sulforaphane analogue can be regarded as a readily available promising new cancer chemopreventive agent.
The isothiocyanate sulforaphane (SF) is thought to be a potential chemoprotective agent. Its effects on Phase I and Phase II enzymes of carcinogen metabolism in primary cultures of rat and human hepatocytes have been investigated. Northern blot analyses of rat hepatocytes showed a dose-dependent induction of mRNAs for rat glutathione S-transferases (rGSTs) A1/A2 and P1 but not M1. This was associated with enhanced levels of not only rGSTA1, A2, A4, A5, and P1 but also of rGSTs M1 and M2. On the other hand, the enzyme activities in rat hepatocytes associated with cytochromes P-450 (CYPs) 1A1 and 2B1/2, namely ethoxyresorufin-O-deethylase and pentoxyresorufin-O-dealkylase, respectively, were decreased in a dose-dependent manner. In SF-treated human hepatocytes, hGSTA1/2 but not hGSTM1 mRNAs were induced, and the expression of CYP1A2 was unaffected, whereas the expression of CYP3A4, the major CYP in human liver, was markedly decreased at both mRNA and activity levels. These observations demonstrate that in intact human and rat hepatocytes, SF may both induce a number of GSTs and cause enzyme inhibition of some but not all CYPs and, in the case of CYP3A4, inhibit both its enzyme activity and its expression.
Isothiocyanates (ITCs) are a family of biologically active compounds that are distributed widely in cruciferous vegetables. Although studies in rodents have shown that these compounds are effective and versatile inhibitors of tumorigenesis, the role of dietary ITCs in the protection against human cancers remains to be established. A prerequisite of human studies is to develop an uptake biomarker for dietary ITCs. In this study, we describe a rapid high-performance liquid chromatography-based assay to measure the total ITC level in human urine. This assay is based on a previously described reaction of ITCs or their thiol conjugates with 1,2-benzenedithiol to yield a cyclocondensation product, 1,3-benzodithiole-2-thione, which then can be quantified by reverse phase high-performance liquid chromatography with UV detection. This new assay was validated by analyzing urine samples from 14 subjects who had consumed a known amount of watercress or brown mustard in a controlled experiment. The N-acetylcysteine conjugates of phenethyl ITC and allyl ITC from watercress and brown mustard, respectively, were quantified and compared with the results obtained from the current assay. Results of the two methods were highly correlated (r = 0.978), indicating the specificity of this new assay for dietary ITCs. The feasibility of this assay for population-based studies was examined using stored urine samples collected from nine participants of a prospective cohort study in Shanghai, China, who indicated that they were daily consumers of dark green vegetables. There was a 10-fold variation in urinary ITC contents among these samples, ranging from 0.7 to 7.0 micromol/g creatinine. These results show the potential use of this uptake biomarker in epidemiological studies to identify the role of dietary ITCs in modifying cancer risks in humans.
The putative anticarcinogenic activity of Brassica vegetables has been associated with the presence of certain glucosinolates. 4-Methylsulphinylbutyl isothiocyanate (sulphoraphane), derived from the corresponding glucosinolate found in broccoli, has previously been identified as a potent inducer of the anticarcinogenic marker enzyme quinone reductase [NADP(H):quinone-acceptor oxidoreductase] in murine hepatoma Hepa 1c1c7 cells. We have therefore produced a broccoli hybrid with increased levels of this anticarcinogenic glucosinolate and tested the ability of extracts to induce quinone reductase. A 10-fold increase in the level of 4-methylsulphinylbutyl glucosinolate was obtained by crossing broccoli cultivars with selected wild taxa of the Brassica oleracea (chromosome number, n = 9) complex. Tissue from these hybrids exhibited a >100-fold increase in the ability to induce quinone reductase in Hepa 1c1c7 cells over broccoli cultivars, due to both an increase in 4-methylsulphinylbutyl glucosinolate content and increased percentage conversion to sulphoraphane.
Transcription factor Nrf2 is essential for the antioxidant responsive element (ARE)-mediated induction of phase II detoxifying and oxidative stress enzyme genes. Detailed analysis of differential Nrf2 activity displayed in transfected cell lines ultimately led to the identification of a new protein, which we named Keap1, that suppresses Nrf2 transcriptional activity by specific binding to its evolutionarily conserved amino-terminal regulatory domain. The closest homolog of Keap1 is a Drosophila actin-binding protein called Kelch, implying that Keap1 might be a Nrf2 cytoplasmic effector. We then showed that electrophilic agents antagonize Keap1 inhibition of Nrf2 activity in vivo, allowing Nrf2 to traverse from the cytoplasm to the nucleus and potentiate the ARE response. We postulate that Keap1 and Nrf2 constitute a crucial cellular sensor for oxidative stress, and together mediate a key step in the signaling pathway that leads to transcriptional activation by this novel Nrf2 nuclear shuttling mechanism. The activation of Nrf2 leads in turn to the induction of phase II enzyme and antioxidative stress genes in response to electrophiles and reactive oxygen species.
The genes glutathione S-transferase M1 (GSTM1) (chromosome 1p13.3) and glutathione S-transferase T1 (GSTT1) (22q11.2) code for cytosolic enzymes glutathione S-transferase (GST)-μ and GST-θ, respectively, which are involved in phase 2 metabolism. Both genes may be deleted. There is
geographic and ethnic variation in genotype frequencies for both genes. In developed countries, colorectal cancer is the second
most common cancer. Colorectal cancer has been inconsistently associated with polycyclic aromatic hydrocarbons in diet and
tobacco. Because GST enzymes are involved in polycyclic aromatic hydrocarbon metabolism, it has been postulated that genotype
may modify colorectal cancer risk associated with polycyclic aromatic hydrocarbon exposure. No consistent associations between
GSTM1 or GSTT1 genotype and colorectal cancer have been observed. However, most studies have methodological limitations. Few have investigated
gene-environment interactions. No interactions between GSTM1 or GSTT1 genotype and smoking and colorectal cancer risk have been reported. One polyp study suggests an interaction between GSTM1 genotype and smoking. Two studies suggest increased disease risk in subjects with high meat intake and GST nonnull genotype,
contrary to the underlying hypothesis. One study suggests a strong inverse relation between colorectal adenomas and broccoli
consumption, particularly in subjects who are GSTM1 null. These finding require confirmation. Methods for determining GSTM1 and GSTT1 genotype are well established. Population testing is not currently justified. Am J Epidemiol 2000; 151:7–32.
Sulforaphane is an isothiocyanate that is present naturally in widely consumed vegetables and has a particularly high concentration in broccoli. This compound has been shown to block the formation of tumors initiated by chemicals in the rat. Although sulforaphane has been proposed to modulate the metabolism of carcinogens, its mechanism of action remains poorly understood. We have previously demonstrated that sulforaphane inhibits the reinitiation of growth and decreases the cellular viability of quiescent human colon carcinoma cells (HT29). Moreover, the weak effect observed on differentiated CaCo2 cells suggests a specific anticancer activity for this compound. Here we investigated the effect of sulforaphane on the growth and viability of HT29 cells during their exponentially growing phase. We observed that sulforaphane induced a cell cycle arrest in a dose-dependent manner, followed by cell death. This sulforaphane-induced cell cycle arrest was correlated with an increased expression of cyclins A and B1. Moreover, we clearly demonstrated that sulforaphane induced cell death via an apoptotic process. Indeed, a large proportion of treated cells display the following: (a) translocation of phosphatidylserine from the inner layer to the outer layer of the plasma membrane; (b) typical chromatin condensation; and (c) ultrastructural modifications related to apoptotic cell death. We also showed that the expression of p53 was not changed in sulforaphane-treated cells. In contrast, whereas bcl-2 was not detected, we observed increased expression of the proapoptotic protein bax, the release of cytochrome c from the mitochondria to the cytosol, and the proteolytic cleavage of poly(ADP-ribose) polymerase. In conclusion, our results strongly suggest that in addition to the activation of detoxifying enzymes, induction of apoptosis is also involved in the sulforaphane-associated chemoprevention of cancer.
Gibberellins (GAs) are key modulators of plant growth and development. PsGA3ox1 (LE) encodes a GA 3β-hydroxylase that catalyzes the conversion of GA20 to biologically active GA1. To further clarify the role of GA3ox expression during pea (Pisum sativum) plant growth and development, we generated transgenic pea lines (in a lele background) with cauliflower mosaic virus-35S-driven expression of PsGA3ox1 (LE). PsGA3ox1 transgene expression led to higher GA1 concentrations in a tissue-specific and development-specific manner, altering GA biosynthesis and catabolism gene expression and plant phenotype. PsGA3ox1 transgenic plants had longer internodes, tendrils, and fruits, larger stipules, and displayed delayed flowering, increased apical meristem life, and altered vascular development relative to the null controls. Transgenic PsGA3ox1 overexpression lines were then compared with lines where endogenous PsGA3ox1 (LE) was introduced, by a series of backcrosses, into the same genetic background (BC LEle). Most notably, the BC LEle plants had substantially longer internodes containing much greater GA1 levels than the transgenic PsGA3ox1 plants. Induction of expression of the GA deactivation gene PsGA2ox1 appears to make an important contribution to limiting the increase of internode GA1 to modest levels for the transgenic lines. In contrast, PsGA3ox1 (LE) expression driven by its endogenous promoter was coordinated within the internode tissue to avoid feed-forward regulation of PsGA2ox1, resulting in much greater GA1 accumulation. These studies further our fundamental understanding of the regulation of GA biosynthesis and catabolism at the tissue and organ level and demonstrate that the timing/localization of GA3ox expression within an organ affects both GA homeostasis and GA1 levels, and thereby growth.
Myrosinase in cell-free extract of Enterobacter cloacae, no. 506, was purified about 1, 000 fold by precipitation with ammonium sulfate, chromatography on CM-Sephadex and gelfiltration on Sephadex G-200 and Sephadex G-100. The enzyme was shown to be homogeneous by chromatography and by ultracentrifuge. Molecular weight obtained by gelfiltration was 61, 000 and the sedimentation coefficient was 4.5S. Maximum activity occurred at pH 6.8. The enzyme was stable in a pH range of 5.0 to 7.0 at temperature below 40°C and for 24 hr. Copper(I) and (II), mercury (II) and ferrous(II) ions strongly inhibited the activity. Sulfhydryl reagents had little effect but EDTA was a strong inhibitor. In contrast to plant myrosinase, this enzyme was inhibited by L-ascorbic acid. Many glucosides and sugars inhibited the enzyme. The relation between bacterial myrosinase and β-glucosidase is discussed in comparison to plant and fungous myrosinases. Some comparative properties of bacterial, fungous and plant myrosinases are discussed.
Epidemiological studies have linked consumption of broccoli to a reduced risk of colon cancer in individuals with the glutathione S-transferase M1 (GSTM1) null genotype. GSTs are involved in excretion and elimination of isothiocyanates (ITCs), which are major constituents of broccoli and other cruciferous vegetables and have cancer chemopreventive potential, so it is speculated that ITCs may play a role in protection against human colon cancer. However, there is a lack of data from animal studies to support this. We carried out a bioassay to examine whether sulforaphane (SFN) and phenethyl isothiocyanate (PEITC), major ITCs in broccoli and watercress, respectively, and their corresponding N-acetylcysteine (NAC) conjugates, show any chemopreventive activity towards azoxymethane (AOM)-induced colonic aberrant crypt foci (ACF) in F344 rats. Groups of six male F344 rats were treated with AOM subcutaneously (15 mg/kg body wt) once weekly for 2 weeks. SFN and PEITC and their NAC conjugates were administered by gavage either three times weekly for 8 weeks (5 and 20 μmol, respectively) after AOM dosing (post-initiation stage) or once daily for 3 days (20 and 50 μmol, respectively) before AOM treatment (initiation stage). The bioassay was terminated on week 10 after the second AOM dosing and ACF were quantified. SFN, SFN-NAC, PEITC and PEITC-NAC all significantly reduced the formation of total ACF from 153 to 100-116 (P < 0.01) and multicrypt foci from 52 to 27-38 (more than four crypts/focus; P < 0.05) during the post-initiation treatment. However, only SFN and PEITC were effective during the initiation phase, reducing the total ACF from 153 to 109-115 (P < 0.01) and multicrypt foci from 52 to 35 (more than four crypts/focus; P < 0.05). The NAC conjugates were inactive as anti-initiators against AOM-induced ACF. These findings provide important laboratory evidence for a potential role of SFN and PEITC in the protection against colon cancer.
Screening tests were undertaken to obtain microorganisms which produce myrosinase. One strain of microorganism indicated a strong capasity for producing myrosinase. The morphological and physiological characteristics of this strain were studied. The organism was identified as Enterobacter cloacae, no. 506.
Enzyme production was induced by the addition of 0.01% sinigrin and 6% mustard extract*2 to the culture medium. The highest production was obtained after 36_??_40 hr cultivation when the strain was cultivated at 28°C in a medium containing 0.01% sinigrin, 6% Mustard ext., 0.1% KH2PO4, 0.1% NH4Cl, 0.1% NaCl and 0.1% MgSO4•7aq, (pH 7.0).
Glucoraphanin in Brassica vegetables breaks down to either sulforaphane or sulforaphane nitrile depending on the conditions, and sulforaphane can be further conjugated with glutathione. Using a high-throughput microtitre plate assay and TaqMan real time quantitative RT-PCR to measure mRNA, we show that sulforaphane and its glutathione conjugate, but not the nitrile, increased significantly (P < 0.05) both UGT1A1 and GSTA1 mRNA levels in HepG2 and HT29 cells. These changes were accompanied by an increase in UGT1A1 protein, as assessed by immunoblotting, and a 2‐8-fold increase in bilirubin glucuronidation. When treated together, the nitrile derivative did not affect sulforaphane induction. The induction of UGT1A1 and GSTA1 mRNA by sulforaphane was time and concentration dependent. The results show a functional induction of glucuronidation by sulforaphane but not sulforaphane nitrile, and show that the pathway of metabolism of glucosinolates in Brassica vegetables is important in determining the resulting biological and anticarcinogenic activities.
Dietary isothiocyanates (ITCs) are highly effective in affording protection against chemically induced cancers in laboratory animals. In the present study, we demonstrate that allyl isothiocyanate (AITC), a constituent of cruciferous vegetables, significantly inhibits proliferation of cultured PC-3 (androgen-independent) and LNCaP (androgen-dependent) human prostate cancer cells in a dose-dependent manner with an IC50 of ~15‐17 mM. On the other hand, survival of a normal prostate epithelial cell line (PrEC) was minimally affected by AITC even at concentrations that were highly cytotoxic to the prostate cancer cells. Reduced proliferation of PC-3 as well as LNCaP cells in the presence of AITC correlated with accumulation of cells in G2/M phase and induction of apoptosis. In contrast, AITC treatment failed to induce apoptosis or cause G2/M phase arrest in PrEC cells. A 24 h treatment of PC-3 and LNCaP cells with 20 mM AITC caused a significant decrease in the levels of proteins that regulate G2/M progression, including Cdk1 (32‐50% reduction), Cdc25B (44‐48% reduction) and Cdc25C (490% reduction). A significant reduction in the expression of cyclin B1 protein (~45%) was observed only in LNCaP cells. A 24 h exposure of PC-3 and LNCaP cells to an apoptosis-inducing concentration of AITC (20 mM) resulted in a significant decrease (31‐68%) in the levels of anti-apoptotic protein Bcl-2 in both cell lines, and ~58% reduction in Bcl-XL protein expression in LNCaP cells. In conclusion, it seems reasonable to hypothesize that AITC, and possibly other ITCs, may find use in the treatment of human prostate cancers.
The putative anticarcinogenic activity of Brassica vegetables has been associated with the presence of certain glucosinolates. 4-Methylsulphinylbutyl isothiocyanate (sulphoraphane), derived from the corresponding glucosinolate found in broccoli, has previously been identified as a potent inducer of the anticarcinogenic marker enzyme quinone reductase [NADP(H):quinone-acceptor oxidoreductase] in murine hepatoma Hepa lclc7 cells. We have therefore produced a broccoli hybrid with increased levels of this anticarcinogenic glucosinolate and tested the ability of extracts to induce quinone reductase. A 10-fold increase in the level of 4-methylsulphinylbutyl glucosinolate was obtained by crossing broccoli cultivars with selected wild taxa of the Brassica oleracea (chromosome number, n = 9) complex. Tissue from these hybrids exhibited a >100-fold increase in the ability to induce quinone reductase in Hepa lclc7 cells over broccoli cultivars, due to both an increase in 4-methylsulphinylbutyl glucosinolate content and increased percentage conversion to sulphoraphane.
We report the RFLP mapping of quantitative trait loci (QTLs) which regulate the total seed aliphaticglucosinolate content in Brassica napus L. A population of 99 F1-derived doubled-haploid (DH) recombinant lines from a cross between the cultivars Stellar (low-glucosinolate) and Major (high-glucosinolate) was used for singlemarker analysis and the interval mapping of QTLs associated with total seed glucosinolates. Two major loci, GSL-1 and GSL-2, with the largest influence on total seed aliphatic-glucosinolates, were mapped onto LG 20 and LG 1, respectively. Three loci with smaller effects, GSL-3, GSL-4 and GSL-5, were tentatively mapped to LG 18, LG 4 and LG 13, respectively. The QTLs acted in an additive manner and accounted for 71 % of the variation in total seed glucosinolates, with GSL-1 and GSL-2 accounting for 33% and 17%, respectively. The recombinant population had aliphatic-glucosinolate levels of between 6 and 160 μmoles per g(-1) dry wt of seed. Transgressive segregation for high seed glucosinolate content was apparent in 25 individuals. These phenotypes possessed Stellar alleles at GSL-3 and Major alleles at the four other GSL loci demonstrating that low-glucosinolate genotypes (i.e. Stellar) may possess alleles for high glucosinolates which are only expressed in particular genetic backgrounds. Gsl-elong and Gsl-alk, loci which regulate the ratio of individual aliphatic glucosinolates, were also mapped. Gsl-elong-1 and Gsl-elong-2, which control elongation of the α-amino-acid precursors, mapped to LG 18 and LG 20 and were coincident with GSL loci which regulate total seed aliphatic glucosinolates. A third tentative QTL, which regulates side-chain elongation, was tentatively mapped to LG 12. Gsl-alk, which regulates H3CS-removal and side-chain de-saturation, mapped to LG 20.
Glucosinolates were evaluated in 5 groups and 65 accessions of Brassica oleracea (50 broccoli, 4 Brussels sprouts, 6 cabbage, 3 cauliflower, and 2 kale) grown under uniform cultural conditions. Glucosinolates and their concentrations varied among the different groups and within each group. The predominant glucosinolates in broccoli were 4-methylsulfinylbutyl glucosinolate (glucoraphanin), 3-butenyl glucosinolate (gluconapin), and 3-indolylmethyl glucosinoate (glucobrassicin). Glucoraphanin concentration in broccoli ranged from 0.8 mu mol g(-1) DW in EV6-1 to 21.7 mu mol g(-1) DW in Brigadier. Concentrations of the other glucosinolates in broccoli varied similarly over a wide range. In Brussels sprouts, cabbage, cauliflower, and kale, the predominant glucosinolates were sinigrin (8.9, 7.8, 9.3, and 10.4 mu mol g(-1) DW, respectively) and glucobrassicin (3.2, 0.9, 1.3, and 1.2 mu mol g(-1) DW, respectively). Brussels sprouts also had significant amounts of gluconapin (6.9 mu mol g(-1) DW). Wide variations in glucosinolate content among genotypes suggest differences in their health-promoting properties and the opportunity for enhancement of their levels through genetic manipulation.
The effect of consumption of glucosinolate-containing Brussels sprouts on α-class glutathione S-transferase levels in human blood plasma was investigated in 10 healthy, male, non-smoking volunteers. Following a 3-week run-in period, five volunteers continued on a glucosinolate-free diet during a subsequent 3-week intervention period (control group), while the other five (sprouts group) consumed 300 g of cooked Brussels sprouts per day, at the expense of 300 g of a glucosinolate-free vegetable. α-Class glutathione S-transferases were measured by radioimmunoassay. In the control group, similar α-class glutathione S-transferase levels were observed in both periods (P=0.814), while in the sprouts group the α-class glutathione S-transferase levels were elevated by a factor of 1.4 P=0.002)
Phenethyl isothiocyanate (PEITC) is a natural product that is among the most effective cancer chemopreventive agents known. Mechanistic studies indicate that the chemopreventive activity of PEITC is associated with its favorable modification of carcinogen metabolism and its induction of apoptosis. Here, we found that PEITC blocks tumor promoter (12-0- tetradecanoylphorbol-13-acetate or epidermal growth factor)-induced cell transformation in mouse epidermal JB6 cells, and this inhibitory activity on cell transformation is correlated with induction of apoptosis. Most importantly, apoptosis induction by PEITC occurs through a p53-depend- ent pathway. This was demonstrated not only by results that PEITC induction of p53 protein expression and p53-dependent transactivation but also by PEITC-induced apoptosis inp53 +/+ cells but not inp53 -/- cells. In contrast, PEITC induced apoptosis in cells with both normal or deficient sphingomyelinase activity. Our results demonstrate for the first time that p53 elevation is required for PEITC-induced apoptosis, which may be involved in its cancer chemopreventive activity.
A case-control study to evaluate risk factors of gastric cancer was carried out in areas with contrasting incidence rates in Sweden. Face-to-face interviews were conducted with 338 of 456 eligible histologically confirmed gastric-cancer cases and 669 of 880 eligible control subjects, sampled from population registers and frequency-matched by age and gender. We focused on 2 periods, adolescence and 20 years prior to interview. The association of gastric-cancer risk with dietary habits during adolescence were similar to that found for the period 20 years before interview; high consumption of wholemeal bread, fruit and vegetables was associated with reduced gastric-cancer risk. In addition, cheese, fish and tea had a protective effect during adolescence. Increased gastric-cancer risk was related to whole-milk consumption, but this association decreased substantially in a multivariate analysis including vegetables. There was a positive relationship between gastric-cancer risk and the age at which the interviewees started using refrigerators. This population-based study confirmed the protective effect of a high consumption of vegetables and fruit in the development of gastric cancer, but failed to find any association between intake of meat, sausage, cold cuts, liver, salt, coffee, the habit of frying, smoking or grilling foods, and risk of gastric cancer.
Total aliphatic and indole/aromatic glucosinolates and vitamin C content (ascorbic+dehydroascorbic acid) were evaluated in the edible portions of fresh harvested broccoli (Brassica oleracea L. var. italica) florets (Marathon cv.) before and after cooking and in the cooking water. High pressure boiling, steam cooking, microwaving and low pressure boiling (conventional) were the four domestic cooking processes used in this work. Results showed great differences among the four cooking processes and their influence on the content of glucosinolates and vitamin C. Thus, clear disadvantages were detected when cooking in a microwave due to the high loss of vitamin C (40%) and total glucosinolates (74%) in comparison with the rest of treatments. High pressure and conventional boiling had a significant loss rate of total glucosinolates (33% and 55% respectively) regarding fresh raw broccoli, due to the leaching into the cooking water. On the other hand, steaming had minimal effects on glucosinolates and vitamin C. Therefore, we can conclude that a large quantity of glucosinolates and vitamin C will be consumed in steamed broccoli when compared to the other cooking processes.
The mechanisms by which brassica vegetables might decrease the risk of cancer are reviewed in this paper. Brassicas, including all types of cabbages, broccoli, cauliflower and Brussels sprouts, may be protective against cancer due to their relatively high glucosinolate content. Glucosinolates are usually broken down through hydrolysis catalyzed by myrosinase, an enzyme that is released from damaged plant cells. Some of the hydrolysis products, viz. indoles and isothiocyanates, are able to influence phase 1 and phase 2 biotransformation enzyme activities, thereby possibly influencing several processes related to chemical carcinogenesis, e.g. the metabolism, DNA-binding and mutagenic activity of promutagens. A reducing effect on tumor formation has been shown in rats and mice. The anticarcinogenic action of isothiocyanates and indoles depends upon many factors, such as the test system, the target tissue, the type of carcinogen challenge and the anticarcinogenic compound, their dosage, as well as the timing of the treatment. Most evidence concerning anticarcinogenic effects of glucosinolate hydrolysis products and brassica vegetables has come from studies in animals. Animal studies are invaluable in identifying and testing potential anticarcinogens. In addition, studies carried out in humans using high but still realistic human consumption levels of indoles and brassica vegetables have shown putative positive effects on health.
The consumption of cruciferous vegetables has long been associated with a reduced risk in the occurrence of cancer at various sites, including the prostate, lung, breast and colon. This protective effect is attributed to isothiocyanates present in these vegetables, and sulforaphane (SF), present in broccoli, is by far the most extensively studied to uncover the mechanisms behind this chemoprotection. The major mechanism by which SF protects cells was traditionally thought to be through Nrf2-mediated induction of phase 2 detoxification enzymes that elevate cell defense against oxidative damage and promote the removal of carcinogens. However, it is becoming clear that there are multiple mechanisms activated in response to SF, including suppression of cytochrome P450 enzymes, induction of apoptotic pathways, suppression of cell cycle progression, inhibition of angiogenesis and anti-inflammatory activity. Moreover, these mechanisms seem to have some degree of interaction to synergistically afford chemoprevention.
Benzyl isothiocyanate and phenethyl isothiocyanate, two aromatic phytochemicals present in substantial concentrations in edible vegetables of the genus Brassica, were investigated for their effects on Caco-2 cell proliferation. Benzyl and phenethyl isothiocyanate inhibited DNA synthesis, with 50% inhibitory concentrations of 5.1 and 2.4 micromol/L, respectively, and significantly increased the doubling times of Caco-2 cells from 32 h to 220 and 120 h, respectively. There was no adverse effect of either chemical on cell viability in the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay, but benzyl isothiocyanate and phenethyl isothiocyanate both caused an accumulation of cells in the G(2)/M phase of the cell cycle, which was maintained for at least 48 h in cells synchronized at prometaphase with nocodazole and subsequently treated with 10 micromol/L benzyl isothiocyanate or phenethyl isothiocyanate. Both benzyl and phenethyl isothiocyanate increased DNA strand breakage, increased phosphorylation of the G(2)/M checkpoint enforcer Chk2, and induced p21 expression. These results suggest that the antiproliferative effects of benzyl and phenethyl isothiocyanates toward Caco-2 cells are due at least in part to the activation of the G(2)/M DNA damage checkpoint, and that sustained G(2)/M phase cell cycle arrest in response to benzyl and phenethyl isothiocyanates may be maintained through upregulation of p21. This study indicates that some dietary isothiocyanates may exert an antiproliferative effect through activation of the G(2)/M DNA damage checkpoint.
Four glucosinolate derivatives were evaluated individually and as a mixture for their effects on hepatic P4501A (CYP1A), glutathione S-transferase (GST), quinone reductase (QR), glutathione reductase (G-Rd), and GSH levels. Doses of the derivatives were chosen to represent their relative abundance in Brussels sprouts. Adult male F344 rats received either corn oil (vehicle); one of the agents: indole-3-carbinol (I3C, 56 mg/kg), iberin (38 mg/kg), phenylethylisothiocyanate (PEITC, 0.1 mg/kg), or cyanohydroxybutene (crambene, 50 mg/kg); or all of the agents at the doses shown (as a mixture) given by gavage daily for 7 days. The mixture and I3C caused an 11- and 9.4-fold induction of CYP1A, respectively. Crambene and I3C each caused a 1.4-fold increase in GST, while the mixture caused a 2.5-fold increase. Crambene and I3C caused a 2.5- and 1.9-fold increase in QR, respectively. The mixture caused a 6.2-fold increase. Crambene, PEITC, and the mixture caused a 1.8-, 1.6-, and 2.0-fold increase in hepatic GSH levels, respectively. Crambene, I3C, iberin, and the mixture caused 1.3-, 1.4-, 1.2-, and 1.7-fold increases in G-Rd, respectively. In a second study the mixture was given at 60 and 20% of the original dose. CYP 1A, QR, G-Rd, and GST elevations were dose-dependent; GSH levels were not elevated. It is concluded that I3C and crambene are responsible for the majority of enzyme increases seen. A synergistic effect of I3C and crambene was evident on induction of GST and QR, but not on GSH, G-Rd, or P4501A.
1. The corresponding cysteine conjugate was formed when the GSH (reduced glutathione) or cysteinylglycine conjugates of benzyl isothiocyanate were incubated with rat liver or kidney homogenates. When the cysteine conjugate of benzyl isothiocyanate was similarly incubated in the presence of acetyl-CoA, the corresponding N-acetylcysteine conjugate (mercapturic acid) was formed. 2. The non-enzymic reaction of GSH with benzyl isothiocyanate was rapid and was catalysed by rat liver cytosol. 3. The mercapturic acid was excreted in the urine of rats dosed with benzyl isothiocyanate or its GSH, cysteinyl-glycine or cysteine conjugate, and was isolated as the dicyclohexylamine salt. 4. An oral dose of the cysteine conjugate of [14C]benzyl isothiocyanate was rapidly absorbed and excreted by rats and dogs. After 3 days, rats had excreted a mean of 92.4 and 5.6% of the dose in the urine and faeces respectively, and dogs had excreted a mean of 86.3 and 13.2% respectively. 5. After an oral dose of the cystein conjugate of [C]benzyl isothiocyanate, the major 14C-labelled metabolite in rat urine was the corresponding mercapturic acid (62% of the dose), whereas in dog urine it was hippuric acid (40% of the dose). 5. Mercapturic acid biosynthesis may be an important route of metabolism of certain isothiocyanates in some mammalian species.
Four compounds commonly found in the human diet, allyl isothiocyanate (AITC), phenethyl isothiocyanate (PEITC) and their parent glucosinolates sinigrin and gluconasturtiin, were tested for cytotoxic and genotoxic effects in a Chinese hamster ovary cell line (CHO). The isothiocyanates were found to be more than one thousand times more cytotoxic than the glucosinolates, showing significant cytotoxic activity at concentrations below 1.0 microgram/ml. AITC was unable to induce either chromosome aberrations or sister chromatid exchanges (SCEs) even at highly cytotoxic doses. In contrast, PEITC was found to induce both aberrations and SCE at concentrations of 0.9-1.2 micrograms/ml whilst sinigrin and gluconasturtiin induced aberrations at concentrations above 2 mg/ml.
Forty-two lactobacilli were screened for their potential to degrade glucosinolate sinigrin. One of them, strain R16, demonstrated a high level of sinigrin degradation; it was identified as Lactobacillus agilis. The sinigrin degrading activity of L. agilis R16 could only be demonstrated when intact cells were used. The products of sinigrin degradation are allyl-isothiocyanate (AITC) and glucose (which is further fermented to DL-lactic acid), suggesting that myrosinase activity is involved. The activity was induced by the presence of sinigrin. Glucose inhibited the myrosinase activity, even in induced cells. Lactobacillus agilis R16 was able to grow on an extract of brown mustard seed and caused glucosinolate degradation.
Rates were determined for the nonezymatic (second order rate constants) and enzyme-catalyzed conjugations with glutathione of four isothiocyanates that are present in edible plants (allyl-, benzyl-, phenethyl-isothiocyanates, and sulforaphane). Of four cloned human glutathione transferases studied, GSTP1-1 and GSTM1-1 were the most efficient catalysts. GSTA1-1 was less efficient, and GSTM2-2 was the least efficient. Conjugation of benzyl-NCS is the most rapid and that of sulforaphane [CH3S(O)(CH2)4-NCS] is the slowest. The large enzymatic rate enhancements and the abundance of the enzymes suggest that the glutathione transferases play important roles in the metabolic disposition of isothiocyanates in humans. Enzymatic cleavage of the GSH conjugates of isothiocyanates (dithiocarbamates) is catalyzed by glutathione transferases. The importance of these reverse reactions is probably limited because they are slow and inhibited by high intracellular concentrations of glutathione.
The reversible reaction of GSH with two dietary anticarcinogens, benzyl isothiocyanate (BITC) and phenethyl isothiocyanate (PEITC), has been studied in the absence and presence of human glutathione S-transferases (GSTs). The spontaneous reaction at pH 7.4 and 37 degrees C yielded values for k2 of 17.9 and 6.0 M-1.s-1 for GSH conjugation of BITC and PEITC respectively (forward reaction), and k1 values of 6.9 x 10(-4) and 2.4 x 10(-4) s-1 for dissociation of the respective GSH conjugates, BITC-SG and PEITC-SG (reverse reaction). GSTs A1-1, A2-2, M1a-1a and P1-1 catalysed both the forward and reverse reactions with specific activities (mumol/min per mg at 30 microM isothiocyanate or GSH conjugate) ranging from 23.1 for the GSH conjugation of BITC by GST P1-1 to 0.03 for the dissociation of BITC-SG by GST A1-1. When present at similar concentration to substrates (12 microM), GSTs A1-1 and A2-2 but not GST M1a-1a shifted the equilibrium in favour of BITC-SG or PEITC-SG. Kinetic studies confirmed that GST A1-1 interacted selectively with the GSH conjugates in the micromolar range (Km 6.9 microM, Ki 4.3 microM), whereas GST M1a-1a interacted with BITC-SG and PEITC-SG with approx. 5-fold lower affinity. In conclusion, GSTs are true catalysts; at high intracellular concentration they also sequester GSH conjugates, promoting GSH conjugation, whereas trace extracellular GSTs promote dissociation of effluxed organic isothiocyanate-GSH conjugates.