Enzyme Myrosinase Research Articles

Chemoprotective effects of broccoli and other Brassica vegetables

Numerous epidemiological studies indicate that Brassica vegetables protect humans against cancer. The family Cruciferae genus Brassica consists of the species Brassica oleracea (e.g., broccoli, cabbage, cauliflower, Brussels sprouts, kale, turnips, collards), which are the most frequently consumed vegetables worldwide. Brassica vegetables are the richest sources of glucosinolates (p-thioglycoside-N-hydroxysulfates), which have considerable anticarcinogenic properties. Glucosinolates are hydrolyzed to biologically active isothiocyanates by the plant enzyme myrosinase when the cells in the plant are damaged (e.g., cut, ground or chewed). Glucoraphanin, which accounts for 35-50% of broccoli glucosinolates, is converted to sulforaphane (1-isothiocyannato-4-(methylsulfinyl)butane), a potent monofunctional phase II enzyme inducer. Several animal studies demonstrated that Brassica vegetables are protective against various classes of DNA-reactive carcinogens. They can reduce chemically induced tumor formation, attenuate the effects of polycyclic aromatic hydrocarbons and nitrosoamines and have a protective effect against heterocyclic amines. This review outlines Brassica vegetables and their metabolism, chemoprotective mechanisms of action, pharmacogenomics and adverse effects. In addition, the overview of recently conducted epidemiological studies on risk reduction of different cancers due to consumption of Brassica vegetables is provided.

Targeting cell cycle machinery as a molecular mechanism of sulforaphane in prostate cancer prevention

Epidemiological studies recently concluded that consumption of cruciferous vegetables such as broccoli, cabbage, and cauliflower, etc. is inversely related to prostate cancer risk, although the mechanism of prevention and the responsible phytochemicals are unknown. Since clinically significant prostate cancer eventually can grow independent of androgen, the association of the growth and tumorigenesis of such prostate cancer cells with sulforaphane (SFN) which is a predominant isothiocyanate in cruciferous vegetables, investigated. These vegetables contain high concentrations of glucosinolate glucoraphanin, which yield sulforaphane when hydrolyzed by the plant enzyme myrosinase. This study showed that exposure of human androgen-independent DU-145 prostate cancer cells to SFN resulted in the inhibition of growth and tumorigenesis, as revealed by a reduction in cell density, DNA synthesis, and clonogenesis. Analyses of the mechanism revealed that SFN mediated cell cycle arrest by modulating the expression and functions of cell cycle regulators. SFN induced signals that inhibited the activity of cyclin-dependent kinase cdk4 with an up-stream induction of cdk inhibitor p21WAF-1/Cip-1, and reduced cyclin D1. The inhibition of cdk kinase activity could be affected with <1 micro M SFN within 24 h. As a result, phosphorylation of Rb proteins, which activates the transition from G1- to S-phase, was significantly decreased and the cell cycle progression retarded. SFN also down-regulated the expression of bcl-2, a suppressor of apoptosis, and activated caspases to execute apoptosis in the prostate cancer cells. The regulators of cell cycle have thus been revealed as targets of sulforaphane for growth arrest and apoptosis induction. The potential of SFN, as an active dietary factor to inhibit initiation and post-initiation of prostate cancer carcinogenesis is discussed.

Effects of Brassica vegetable juice on the induction of apoptosis and aberrant crypt foci in rat colonic mucosal crypts in vivo.

Glucosinolates are sulfur-containing glycosides found in the Brassica vegetables. Their breakdown products include isothiocyanates, which are produced following exposure to the endogenous plant enzyme myrosinase. Isothiocyanates are pungent, biologically active compounds that suppress carcinogenesis in vivo, and induce apoptosis in vitro. We have shown previously that oral administration of the isolated glucosinolate sinigrin induces apoptosis, and suppresses aberrant crypt foci in the colonic mucosa of rats treated previously with 1,2-dimethylhydrazine (DMH). In this study we explored the effects of both raw and thermally processed Brussels sprout tissue on the modulation of crypt cell apoptosis and mitosis, and the frequency of aberrant crypt foci in the colon. Freeze-dried raw and microwave-cooked Brussels sprouts contained high levels of intact glucosinolates, but they were absent from freshly prepared sprout juice. Oral administration of uncooked Brussels sprouts, whether as a juice, or as a freeze-dried powder, was associated with significantly enhanced levels of apoptosis and reduced mitosis in the colonic crypts. However, this effect was confined to rats previously injected (48 h) with DMH, in which levels of apoptosis and mitosis following DNA damage were already high. There was no effect of treatment in control animals. There was also little evidence of these effects when intact glucosinolates were administered in blanched sprout tissue, which lacked active myrosinase. We conclude that glucosinolate breakdown products derived from Brassica vegetables can exert a profound effect on the balance of colorectal cell proliferation and death in an animal model of colorectal neoplasia.

A SIMPLE METHOD FOR ASSAYING TOTAL GLUCOSINOLATE CONTENT OF PLANT MATERIALS VIA SULFATE-ION-RELEASE USING ION CHROMATOGRAPHY

Naturally occurring phytochemicals possessing unique medicinal properties have become a matter of high interest in recent years. A good example is the enormous increase in the use of Saint John's Wort in Europe to treat depression. The anticancerous property of the vegetables of the family cruciferae, e.g., cauliflower, kale, broccoli, cabbage, mustard, and Brussels sprouts, has been attributed to a class of sulfur-containing phytochemicals known as glucosinolates. From a practical standpoint, the total glucosinolate content is generally considered the important parameter and hence that is what would generally be needed as a marker of product quality. The most frequently used method for the assay of total glucosinolates so far has been by assaying glucose which is produced stoichiometrically by the action of enzyme myrosinase. Various approaches for glucose assay have been employed and most are time-consuming and expensive. Fortunately, all glucosinolates, as a group of compounds, posses a sulfate group along with glucose which can be easily and stoichiometrically released by the action of enzymes myrosinase or sulfatase. The resulting sulfate can be easily and conveniently assayed by the use of ion chromatography. This paper reports the results of a study undertaken to develop a simple method for assaying total glucosinolates in plant materials by sulfate-ions-release and using Suppressed Ion Chromatography. Since the method is based on the sulfate ions released from the hydrolysis of glucosinolates, endogenous sulfate ions first had to be removed from the sample extracts. This was successfully accomplished via precipitation of sulfate ions by treating the extracts with 20 mM barium ions at 40 C for 60 minutes. Further, the residual (excess) barium ions were removed via precipitation by treatment of extracts with 25 mM chromate ions at 40 C for 15 minutes. Following these steps, glucosinolates were hydrolyzed by using myrosinase enzyme at a pH of 6.0 resulting in the release of sulfate moiety. Nearly 100% recovery of added sinigrin was achieved by assaying sulfate. However, the time needed for the complete hydrolysis of sinigrin by enzyme myrosinase in the presence of other salts needed for the procedure, was much longer than in their absence. Consequently, either a greater quantity of enzyme and/or a greater incubation time was necessary to achieve complete hydrolysis. Results of nine broccoli samples analyzed for total glucosinolates using the sulfate ion release method reported here were in agreement with those using glucose release method. Various parameters of the method are discussed.

Broccoli

Broccoli

Complex formation of myrosinase isoenzymes in oilseed rape seeds are dependent on the presence of myrosinase-binding proteins.

The enzyme myrosinase (EC 3.2.3.1) degrades the secondary compounds glucosinolates upon wounding and serves as a defense to generalist pests in Capparales. Certain myrosinases are present in complexes together with other proteins such as myrosinase-binding proteins (MBP) in extracts of oilseed rape (Brassica napus) seeds. Immunhistochemical analysis of wild-type seeds showed that MBPs were present in most cells but not in the myrosin cells, indicating that the complex formation observed in extracts is initiated upon tissue disruption. To study the role of MBP in complex formation and defense, oilseed rape antisense plants lacking the seed MBPs were produced. Western blotting and immunohistochemical staining confirmed depletion of MBP in the transgenic seeds. The exclusive expression of myrosinase in idioblasts (myrosin cells) of the seed was not affected by the down-regulation of MBP. Using size-exclusion chromatography, we have shown that myrosinases with subunit molecular masses of 62 to 70 kD were present as free dimers from the antisense seed extract, whereas in the wild type, they formed complexes. In accordance with this, MBPs are necessary for myrosinase complex formation of the 62- to 70-kD myrosinases. The product formed from sinalbin hydrolysis by myrosinase was the same whether MBP was present or not. The performance of a common beetle generalist (Tenebrio molitor) fed with seeds, herbivory by flea beetles (Phyllotreta undulata) on cotyledons, or growth rate of the Brassica fungal pathogens Alternaria brassicae or Lepthosphaeria maculans in the presence of seed extracts were not affected by the down-regulation of MBP, leaving the physiological function of this protein family open.

Metabolism of sinigrin (2-propenyl glucosinolate) by the human colonic microflora in a dynamic in vitro large-intestinal model.

Cruciferous vegetables, such as Brassica, which contain substantial quantities of glucosinolates, have been suggested to possess anticarcinogenic activity. Cutting and chewing of cruciferous vegetables releases the thioglucosidase enzyme myrosinase, which degrades glucosinolates to isothiocyanates and other minor metabolites. Cooking of cruciferous vegetables inactivates the myrosinase enzyme, allowing intact glucosinolates to reach the large intestine, where they can be degraded by the indigenous microflora into isothiocyanates. This local release of isothiocyanates may explain the protective effect of cruciferous vegetables on the colon epithelium. However, little is known about the amounts and identities of glucosinolate metabolites produced by the human microflora. The production of allyl isothiocyanate from sinigrin was investigated in a dynamic in vitro large-intestinal model, after inoculation with a complex microflora of human origin. Sinigrin and allyl isothiocyanate concentrations were analysed in the lumen and dialysis fluid of the model. Peak levels of allyl isothiocyanate were observed between 9 and 12 h after the addition of sinigrin. The model was first set up with a pooled and cultured human microflora, in which 1 and 4% of, respectively, 1 and 15 mM sinigrin, was converted into AITC. However, the conversion rate was remarkably higher if different individual human microflora were used. Between 10% and 30% (mean 19%) of the sinigrin was converted into allyl isothiocyanate. The results of this study suggest that allyl isothiocyanate is converted further into other, yet unknown, metabolites.

Glucosinolates in the human diet. Bioavailability and implications for health

The glucosinolates are a large group of sulphur-containing glucosides found in brassica vegetables. After physical damage to the plant tissue, glucosinolates are broken down, by the endogenous enzyme myrosinase, releasing glucose and a complex variety of biologically active products. The most important and extensively studied of these compounds are the isothiocyanates. Glucosinolates can be degraded or leached from vegetable tissue during food processing, but thermal inactivation of myrosinase preserves some intact glucosinolates in cooked vegetables. Once ingested, any remaining intact glucosinolates may be broken down by plant myrosinase in the small intestine, or by bacterial myrosinase in the colon. Isothiocyanates are absorbed from the small bowel and colon, and the metabolites are detectable in human urine 2–3 h after consumption of brassica vegetables. Isothiocyanates are potent inducers of Phase II enzymes in vitro, and they have been shown to increase the metabolism and detoxification of chemical carcinogens in vitro and in animal models. Some of these compounds also inhibit mitosis and stimulate apoptosis in human tumour cells, in vitro and in vivo. This second effect raises the possibility that in addition to blocking DNA damage, isothiocyanates may selectively inhibit the growth of tumour cells even after initiation by chemical carcinogens. Epidemiological evidence supports the possibility that glucosinolate breakdown products derived from brassica vegetables may protect against human cancers, especially those of the gastrointestinal tract and lung. To define and exploit these potentially anticarcinogenic effects it is important to understand and manipulate glucosinolate chemistry and metabolism across the whole food-chain, from production and processing to consumption.

Guard cell- and phloem idioblast-specific expression of thioglucoside glucohydrolase 1 (myrosinase) in Arabidopsis.

Thioglucoside glucohydrolase 1 (TGG1) is one of two known functional myrosinase enzymes in Arabidopsis. The enzyme catalyzes the hydrolysis of glucosinolates into compounds that are toxic to various microbes and herbivores. Transgenic Arabidopsis plants carrying beta-glucuronidase and green fluorescent protein reporter genes fused to 0.5 or 2.5 kb of the TGG1 promoter region were used to study spatial promoter activity. Promoter activity was found to be highly specific and restricted to guard cells and distinct cells of the phloem. No promoter activity was detected in the root or seed. All guard cells show promoter activity. Positive phloem cells are distributed in a discontinuous pattern and occur more frequent in young tissues. Immunocytochemical localization of myrosinase in transverse and longitudinal sections of embedded material show that the TGG1 promoter activity reflects the position of the myrosinase enzyme. In the flower stalk, the myrosinase-containing phloem cells are located between phloem sieve elements and glucosinolate-rich S cells. Our results suggest a cellular separation of myrosinase enzyme and glucosinolate substrate, and that myrosinase is contained in distinct cells. We discuss the potential advantages of locating defense and communication systems to only a few specific cell types.

Growth inhibition, cell-cycle arrest and apoptosis in human T-cell leukemia by the isothiocyanate sulforaphane

Glucosinolates (GL) can inhibit, retard or reverse experimental multistage carcinogenesis. When brassica plant tissue is broken, GLs are hydrolyzed by the endogenous enzyme myrosinase (Myr), releasing many products including isothiocyanates (ITC). Synthetic ITCs like sulforaphane exert chemopreventive effects against chemically induced tumors in animals, modulating enzymes required for carcinogens' activation/detoxification and/or the induction of cell-cycle arrest and apoptosis in tumor cell lines. To investigate the chemopreventive potential of ITCs while reproducing the circumstances of dietary contact with sulforaphane, we studied proliferation, apoptosis induction and p53, bcl-2 and bax protein expression in Jurkat T-leukemia cells by sulforaphane, the ITC generated in situ in a quantitative manner by Myr starting from glucoraphanin (GRA). Jurkat cells were treated with different doses of GRA-Myr mixture. Effects on cell growth or survival were evaluated by counting trypan blue-excluding cells. Cell-cycle progression, apoptosis and expression of p53, bax and bcl-2 proteins were analyzed by flow cytometry. Results were analyzed by two-sided Fisher's exact test. Sulforaphane, but not GRA, caused G(2)/M-phase arrest (P = 0.028) and increase of apoptotic cell fraction (P < 0.0001) in a time- and dose-dependent manner. Necrosis was observed after prolonged exposure to elevated sulforaphane doses. Moreover, it markedly increased p53 and bax protein expression, and slightly affected bcl-2 expression. These findings indicate that sulforaphane but not the native GL GRA can exert both protective and toxic effects inhibiting leukemic cell growth. Sulforaphane therefore deserves study as a potential chemopreventive/chemotherapeutic antileukemic agent.

Composition and content of glucosinolates in developing Arabidopsis thaliana.

The glucosinolate composition and content in various tissues of Arabidopsis thaliana (L.) Heynh. ecotype Columbia during development from seeds to bolting plants were determined in detail by high-performance liquid chromatography. Comparison of the glucosinolate profiles of leaves, roots and stems from mature plants with those of green siliques and mature seeds indicated that a majority of the seed glucosinolates were synthesized de novo in the silique. A comparison of the glucosinolate profile of mature seeds with that of cotyledons indicated that a major part of seed glucosinolates was retained in the cotyledons. Turnover of glucosinolates was studied by germination of seeds containing radiolabelled p-hydroxybenzylglucosinolate (p-OHBG). Approximately 70% of the content of [14C]p-OHBG in the seeds was detected in seedlings at the cotyledon stage and [14C]p-OHBG was barely detectable in young plants with rosettes of six to eight leaves. The turn-over of p-OHBG was found to coincide with the expression of the glucosinolate-degrading enzyme myrosinase, which was detectable at very low levels in seedlings at the cotyledon stage, but which dramatically increased in leaves from plants at later developmental stages. This indicates that there is a continuous turnover of glucosinolates during development and not only upon tissue disruption.

Effects of intact glucosinolates and products produced from glucosinolates in myrosinase-catalyzed hydrolysis on the potato cyst nematode (Globodera rostochiensis Cv. Woll).

The potato cyst nematode (Globodera rostochiensis cv. Woll) is responsible for large yield losses in the potato crop, and opportunities for reducing the attack of these plant nematode species are, therefore, important. This study has been devoted to the testing of the in vitro effects on the potato cyst nematode of eight glucosinolates [prop-2-enyl-, but-3-enyl-, (R)-4-methylsulfinylbut-3-enyl-, benzyl-, phenethyl-, 4-hydroxybenzyl-, (2S)-2-hydroxybut-3-enyl-, and (2R)-2-hydroxy-2-phenylethylglucosinolate] as well as the effects of the products of this myrosinase-catalyzed hydrolysis. The glucosinolates were used at three concentrations, 0.05, 0.3, and 1.0 mg/mL, in the presence or absence of the enzyme myrosinase. The effects of the compounds on the mortality were monitored every 8 h for a 72 h period. No effects were found for any of the intact glucosinolates. However, when active myrosinase was included with 1 mg/mL phenethylglucosinolate at pH 6.5, 100% mortality was observed within just 16 h. A similar effect was achieved at the same concentration of benzyl- and prop-2-enylglucosinolates in the myrosinase-containing solutions, although longer exposures were required (24 and 40 h, respectively). The main aglucone products released from the glucosinolates with pronounced effects on the nematodes were shown to be the corresponding isothiocyanates. The results suggest that mixtures of these specific glucosinolates and active myrosinase or autolysis of plant materials containing these enzymes and glucosinolates might be used to control the potato cyst nematode in the soil.

Glucosinolates: bioavailability and importance to health.

Epidemiological studies suggest that brassica vegetables are protective against cancers of the lungs and alimentary tract. Cruciferous vegetables are the dietary source of glucosinolates, a large group of sulfur-containing glucosides. These compounds remain intact unless brought into contact with the enzyme myrosinase by pests, food processing, or chewing. Myrosinase releases glucose and breakdown products, including isothiocyanates. These highly reactive compounds are potent inducers of Phase II enzymes in vitro. Isothiocyanates also inhibit mitosis and stimulate apoptosis in human tumor cells, in vitro and in vivo. To understand and exploit such effects it is important to determine the routes of absorption of glucosinolate breakdown products, their metabolism, and delivery to systemic tissues. Glucosinolates can be gained or lost by vegetables during storage. They may be degraded or leached during processing, or preserved by thermal inactivation of myrosinase. Glucosinolates are broken down by plant myrosinase in the small intestine or by bacterial myrosinase in the colon. Isothiocyanates are absorbed from the small bowel and colon, and metabolites are detectable in human urine two to three hours after consumption of brassica vegetables. Interpretation of epidemiological data and exploitation of brassica vegetables for human health requires an understanding of glucosinolate chemistry and metabolism, across the whole food chain, from production and processing to the consumer.

The ecological genetics of aliphatic glucosinolates

Glucosinolates are plant secondary metabolites composed of a thioglucose group and an amino acid side-chain. They occur in the Brassicaceae and related families. A wide variety of glucosinolates exists owing to modification of the side-chain structure. Following tissue damage, myrosinase enzymes catalyse the decomposition of glucosinolates to a variety of volatile and nonvolatile products. The genetic control of concentration and side-chain modification of aliphatic glucosinolates, which have side-chains derived from methionine, are simple and well known from work on Arabidopsis and Brassica crops. In controlled conditions in the laboratory or in field trials, many aliphatic glucosinolates, or their degradation products, affect the behaviour of herbivores. For these reasons, we suggest that polymorphism for aliphatic glucosinolates in natural populations offers an attractive system for the study of ecological genetics of plant-herbivore interactions.

The myrosinase‐glucosinolate system in the interaction between Leptosphaeria maculans and Brassica napus

summary Leptosphaeria maculans causes blackleg disease, and resistance to this fungal pathogen is an important trait in the breeding of oilseed rape. A better comprehension of the role of the myrosinase-glucosinolate system in this context is of great value. The present study is the first to address effects on multiple components of this complex system, including concentrations of individual glucosinolates, product formation, myrosinase isoform distribution and activity, and levels of myrosinase binding proteins during the infection process. One resistant B. napus cultivar (Maluka) and one susceptible cultivar (Westar) were compared in the investigation. Our results show that the two cultivars had the same histological distribution, isoform expression, and activity of the myrosinase enzymes. The glucosinolate levels were also similar, with the exception of glucobrassicin and neoglucobrassicin, which were significantly lower in the resistant cultivar at 11 days post-infection. Growth of the fungus on the plant tissues did not alter glucosinolate levels, suggesting that L. maculans does not degrade these compounds. When the plants were starved of sulphur, and thereby depleted of glucosinolates, no increased susceptibility was observed. Hence, we suggest that the myrosinase-glucosinolate system does not determine the outcome of the interaction between B. napus and L. maculans.

Purification and characterization of myrosinase from the cabbage aphid (Brevicoryne brassicae), a brassica herbivore.

Aphids are among the most serious insect pests of agricultural crops in the world. They often have specific hosts, and the cabbage aphid (Brevicoryne brassicae) is a specialist on Cruciferae. It has previously been described that certain insects contain the enzyme myrosinase (EC 3.2.3.1), which is considered an important defence enzyme of crucifers. Myrosinase was purified to homogeneity from cabbage aphid soluble extracts using anion-exchange and phenyl-Sepharose chromatography. The protein has an apparent subunit molecular mass of 57-58 kDa and is a dimer. The isoelectric point is 4.9 and the enzyme has a temperature optimum around 40 degrees C. The enzyme was active towards the glucosinolates tested, sinigrin and glucotropaeolin, but was inhibited by ascorbate at concentrations that normally activate plant myrosinases. Using sinigrin as the substrate Km was determined as 0.41 mM, and the kcat as 36 s(-1). With glucotropaeolin the Km and kcat values were determined as 0.52 mM and 22.8 s(-1), respectively. The enzyme was stable upon storage at 4 degrees C for many months, but lost some activity upon freezing. The insect myrosinase did not cross-react with antibodies raised to plant myrosinase. Peptide sequencing of a tryptic digest of the protein showed homology to beta-glucosidases. The presence of myrosinase in an insect pest specialist may be an example of a coevolution process that facilitates host specialization.

Disposition of Glucosinolates and Sulforaphane in Humans After Ingestion of Steamed and Fresh Broccoli

The cancer-chemopreventive effects of broccoli may be attributed, in part, to isothiocyanates (ITCs), hydrolysis products of glucosinolates. Glucosinolates are hydrolyzed to their respective ITCs by the enzyme myrosinase, which is inactivated by heat. In this study, the metabolic fate of glucosinolates after ingestion of steamed and fresh broccoli was compared in 12 male subjects in a crossover design. During each 48-hour baseline period, no foods containing glucosinolates or ITCs were allowed. The subjects then consumed 200 g of fresh or steamed broccoli; all other dietary sources of ITCs were excluded. Blood and urine samples were collected during the 24-hour period after broccoli consumption. Total ITC equivalents in broccoli and total ITC equivalents in plasma and urine were assayed by high-performance liquid chromatography as the cyclocondensation product of 1,2-benzenedithiol. The content of ITCs in fresh and steamed broccoli after myrosinase treatment was found to be virtually identical (1.1 vs. 1.0 μmol/g wet wt). The average 24-hour urinary excretion of ITC equivalents amounted to 32.3 ± 12.7% and 10.2 ± 5.9% of the amounts ingested for fresh and steamed broccoli, respectively. Approximately 40% of total ITC equivalents in urine, 25.8 ± 13.9 and 6.9 ± 2.5 μmol for fresh and steamed broccoli, respectively, occurred as the N-acetyl-L-cysteine conjugate of sulforaphane (SFN-NAC). Total ITC metabolites in plasma peaked between 0 and 8 hours, whereas urinary excretion of total ITC equivalents and SFN-NAC occurred primarily between 2 and 12 hours. Results of this study indicate that the bioavailability of ITCs from fresh broccoli is approximately three times greater than that from cooked broccoli, in which myrosinase is inactivated. Considering the cancer-chemopreventive potential of ITCs, cooking broccoli may markedly reduce its beneficial effects on health.

The Activity of Myrosinase from Broccoli (Brassica oleracea L. cv. Italica): Influence of Intrinsic and Extrinsic Factors

The potential of some intrinsic (MgCl2, ascorbic acid, pH) and extrinsic (temperature, pressure) factors for controlling/altering activity of myrosinase from broccoli was investigated in this paper. A combination of MgCl2 and ascorbic acid was found to enhance enzyme activity. Concentrations resulting in optimal activity were determined as 0.1 g/liter and 2 g/liter, respectively. Both in the absence and presence of this enzyme activator, the optimal pH was situated between 6.5 and 7, corresponding to the natural pH of fresh broccoli juice. At atmospheric pressure, the enzyme was optimally active at a temperature about 30°C. Application of low pressure (50 to 100 MPa) slightly enhanced the activity while at higher pressure (300 MPa), the activity was largely reduced. Future work should focus on the extension of this work to real food products in order to take cellular disruption into account. In intact vegetable tissues, the enzyme myrosinase is present in compartments separated from its substrate, the glucosinolates. Hence, enzymatic hydrolysis can merely occur after cellular disruption. In this respect, processes such as cutting, cooking, freezing, or pressurizing of the vegetables will have a large effect on the glucosinolate hydrolysis by myrosinase. This work could then be the basis for controlling glucosinolate hydrolysis in food preparation and processing.

Indole derivatives in vegetables of the cruciferae family

The chemical background of the biological activities of vegetables of the Cruciferae family is considered. These vegetables contain alkaloids of the glucobrassicin group that are decomposed by the enzyme myrosinase (thioglucosidase, EC 3.2.3.1) released upon damage to the plant cells. This results in several indole derivatives, with ascorbigen and indole-3-carbinol predominating. In the gastrointestinal tract, these compounds form 5H,11H-indolo[3,2-b]carbazole, a natural ligand of the aromatic hydrocarbon receptor (Ah receptor) and a functional analogue of 2,3,7,8-tetrachlorodibenzo-p-dioxin, a dangerous xenobiotic. The indolocarbazole—Ah receptor complex activates the gene of CYP1A1, an isoenzyme of cytochrome P450-dependent monoamine oxidase, which enhances the 2-hydroxylation (inactivation) of estrogens. In its turn, the resulting lowered level of estrogens inhibits the growth of hormone-dependent tumors or prevents their appearance. The mechanism of xenobiotic inactivation, underlying the anticarcinogenic action of food products including vegetables of the Cruciferae family and some homogeneous indole compounds, is similar. Some other effects of nutrient indole compounds, e.g., the inhibition of expression of the cyclin-dependent kinase 6 (CDK6) by indole-3-carbinol that leads to the cell cycle arrest in G1 phase, are also considered. Analysis of the biological effects of the Cruciferae diet has helped start clinical studies of indole-3-carbinol as an antitumor and anticarcinogenic remedy for patients with a high risk of tumor diseases.

The first synthesis of C-glucotropaeolin

Starting from tetra- O-benzyl- d-glucose ( 2), the first synthesis of a C-analogue 7 of glucotropaeolin, the major glucosinolate of the genus Nasturtium was performed. Compound 7 constitutes a potential non-hydrolyzable inhibitor of the enzyme myrosinase.