Natural compounds are a promising source to treat several pathologies. The present study shows the in vivo pharmacological beneficial effect of 4(alpha-L-rhamnosyloxy)-benzyl isothiocyanate (glucomoringin isothiocyanate; GMG-ITC) obtained from glucomoringin (GMG; 4(alpha-L-rhamnosyloxy)benzyl glucosinolate), purified from Moringa oleifera seeds and hydrolyzed by myrosinase enzyme (beta-thioglucoside glucohydrolase; E.C. 3.2.1.147). Cerebral ischemia/reperfusion (CIR) was induced in rats according to a classic model of carotid artery occlusion for a time period of 1 h and the reperfusion time was prolonged for seven days. GMG-ITC (3.5 mg GMG/ml plus 30 mu l enzyme/rat; one ml i.p./rat) was administered 15 min after the beginning of ischemia and daily. The results clearly show that GMG-ITC possesses the capability to counteract the CIR-induced damage reducing TNF-alpha release, I kappa B-alpha cytosolic degradation/NF kappa Bp65 nuclear translocation, as well as several other direct or indirect markers of inflammation (phospho-ERK p42/44, p-selectin) and oxidative stress (inducible Nitric Oxide Synthase (iNOS), MMP-9). GMG-ITC was shown to exert neuroprotective properties in preventing CIR-induced damage and the related cascade of inflammatory and oxidative mediators that exacerbate the progression of this disease in an experimental rat model. Our results clearly show that the tested phytochemical GMG-ITC possesses the capability to counteract CIR-induced damage.

OBJECTIVES:Alterations in blood-brain barrier (BBB) permeability are due to the disruption of the Tight Junctions (TJs), large multiprotein complexes important for the maintenance of structural integrity and for permeability of the barrier. In this experimental study we evaluated the neuroprotective role of (RS)-glucoraphanin, a glucosinolate present in Brassicaceae, notably in Tuscan black kale, and bioactivated with myrosinase enzyme (bioactive RS-GRA) (10 mg/kg/d intraperitoneally), to prevent the dysfunction of BBB, in an experimental autoimmune encephalomyelitis (EAE), a model of multiple sclerosis (MS).MATERIALS AND METHODS:EAE was induced by immunization with myelin oligodendroglial glycoprotein peptide (MOG)35-55 in mice. By western blot analysis of brain tissues, we evaluated expression and distribution of the TJ-associated proteins, claudin-1, -3, -5 and ZO-1. Additionally, in order to gain a better insight into the mechanisms of action of bioactive RS-GRA, we investigated Foxp3, ERK1/2 and caspase 3 expression associated both to inflammatory response as well as to apoptotic pathway.RESULTS:Our results demonstrated that treatment with bioactive RS-GRA counteracts the alteration of all these parameters and preserves TJ integrity through an antinflammatory and antiapoptotic activity during MS.CONCLUSIONS:Bioactive RS-GRA, could be a therapeutic perspective helpful in preventing dysfunction of the BBB.

Woad (Isatis tinctoria L.) is a wild plant widely diffused throughout Europe, Italy included. In ancient times it was exploited as a dye-plant, to obtain the indigo colour. Recently woad was rediscovered also for its cosmetic and anti-inflammatory properties. Belonging to Brassicaceae family, woad contains glucosinolates, which are precursor of isothiocyanates, which possess chemopreventive antioxidant properties. Although woad is not considered as an edible vegetable worldwide, rural people living around Mount Etna in Sicily, Italy, are accustomed to collect the flower buds from wild plants in the late winter, to be consumed after boiling as ingredients for salads or omelets. To investigate the nutraceutical value of woad, fresh flower buds were collected on Mount Etna slopes in Sicily over two years and analysed for their bio-active compound content. Woad samples contained considerable amounts of glucosinolates, ranging from 80 up to over 100 mu mol/g d.w., which are values higher than those found in other Brassica vegetables. Only gluconapin and the most interesting glucobrassicin, which is the natural precursor of indole-3-carbinol, a multiple anti-tumorigenic compound, were found. High mean values were also recorded for total polyphenols and flavonoids (25 GAE mg/g d.w. and 7 CE mg/g d.w., respectively). Consequently, also the antioxidant activity (H-ORAC) of the woad flower buds resulted among the highest reported for other Brassica vegetables, being about 300 mu mol TE/g d.w. Since processing can partly reduce the glucosinolate content of vegetables, different cooking ways, such as boiling and microwaving, were compared. Boiling for 10 min caused losses of 34% of total glucosinolates, while microwaving (10 min, 800 W) only reduced the glucosinolate amount by 3%. The results obtained suggest that woad flower buds could be considered as a functional food with possible chemopreventive properties.

Plants of the Brassicales order, and, in particular, the species belonging to the Brassicaceae family, are characterized by the presence of the glucosinolate (GLs)-myrosinase (MYR) system, an efficient internal defensive system that plays a role in the control of several types of pathogens. After wounding or a pathogen attack, GLs are hydrolyzed by MYR and release isothiocyanates (ITCs) harmful to pathogens or pests. The exploitation of this system has led to the definition of the "biofumigation" technique arising from the use of the biocidal properties of ITCs released from GL-containing plants and materials. In addition, Brassicaceae seeds are characterized by an oil content ranging from 10 to 45 % of their dry matter. The oils contain different fatty acids that confer different tribological properties for the production of bioenergy, biolubricants and molecules for lipochemistry. In order to better exploit and increase our knowledge of the Brassicaceae biodiversity, our research group has collected seeds from several wild and cultivated non-food species. They were characterized for their GL and fatty acid composition and the most interesting species were tested for their adaptability to full-field cultivation. This study characterizes a collection of seeds of 66 Brassicaceae species, preserved and available at CRA-CIN. The results confirm a wide variation in their profile that could open some application perspectives.

Les Crucifères, ou Brassicacées, constituent une famille importante de plantes — moutarde, chou, radis, navet, cresson, roquette, wasabi, colza, etc. — qui sont couramment utilisées en alimentation humaine et animale, mais aussi dans des applications pharmaceutiques, cosmétiques et tinctoriales. Du point de vue phytochimique, cette famille végétale est caractérisée par des produits naturels soufrés appelés glucosinolates (GLs). Lorsque les cellules de ces plantes sont endommagées, les glucosinolates sont dégradés par la myrosinase, une enzyme présente dans des compartiments cellulaires séparés, libérant ainsi de nombreuses molécules. Des isothiocyanates (ITCs) sont majoritairement formés, mais aussi des nitriles, thiocyanates et oxazolidinethiones, selon la structure du glucosinolate (GL) de départ et les conditions physicochimiques de l’hydrolyse. Nous présentons quelques exemples d’utilisation des extraits végétaux de Brassicacées, en évoquant certaines avancées, notamment sur la mise en évidence de bioactivités des ITCs et des glucosinolates.

Primary cultures of rat hepatocytes were used to investigate whether and how eight isothiocynates (ITCs) with different chemical structures (the aromatic benzyl, 4-hydroxybenzyl, phenethyl isothiocyanates and the aliphatic allyl, napin, iberin, raphasatin isothiocyanates and sulforaphane) derived from hydrolyzed glucosinolates, were able to modulate cytochrome P450 (CYP) and antioxidant/detoxifying enzymes and to activate the Nrf2 transcription factor. The aromatic ITCs at 40 μM markedly increased the transcription of CYP1A1 and 1A2 mRNA and increased the associated ethoxyresorufin O-deethylase (EROD) activity after 24 h of treatment. By contrast, the aliphatic ITCs (40 μM) decreased CYP1A1 and 1A2 transcription, together with the corresponding EROD activity. The same treatment also caused a striking and similar transcriptional repression of CYP3A2, and the corresponding benzyloxyquinoline debenzylase activity in response to all the ITCs tested. In the same culture conditions, most of the antioxidant/detoxifying enzymes were significantly up-regulated by 40μM ITCs. In particular, NAD(P)H:quinone oxidoreductase and heme oxygenase-1 were induced, although to different levels, at transcriptional, protein and/or activity levels by all the ITCs. However, glutathione S-transferase activity was not induced by the allyl, benzyl, and 4-hydroxybenzyl ITCs, glutathione reductase activity was not induced by benzyl, and 4-hydroxybenzyl ITCs and catalase activity was not induced by allyl ITC. As for the Nrf2 transcription factor, a partial translocation of its protein from the cytosol to the nucleus was revealed by immunoblotting after 1h of treatment for all the ITCs tested. The ability of ITCs to induce the antioxidant and phase II enzymes did not appear to be affected by their hydrophilicity or other structural factors. Taken together, these results show that these ITCs are effective inducers of ARE/Nrf2-regulated antioxidant/detoxifying genes and have the potential to inhibit, at least in rat liver, the bioactivation of carcinogens dependent on CYP3A2 catalysis.

Reduction of lipid absorption has been recognized as an attractive approach for the discovery of new drugs to treat obesity and overweight. The leave extract of Ginkgo biloba has been widely used for the treatment of metabolic diseases (such as hyperlipidemia) in both eastern and western countries, but the bioactive compounds in Ginkgo biloba and the underlying mechanism have not been fully characterized. This study aimed to investigate the inhibition potentials and mechanism of major biflavones from G. biloba on pancreatic lipase (PL), a key target regulating lipid absorption. The results clearly demonstrated that all tested biflavones in G. biloba including isoginkgetin, bilobetin, ginkgetin and sciadopitysin, displayed strong to moderate inhibitory effects on PL with the IC50 values ranging from 2.90 μM to 12.78 μM. Further investigations on both inhibition kinetic analyses and docking simulations demonstrated that isoginkgetin, bilobetin and ginkgetin were potent PL inhibitors (Ki < 2.5 μM), which could create strong interactions with the catalytic triad of PL via hydrogen bonding. These findings provided a new powerful evidence for explaining the hypolipidemic effects of G. biloba, while these newly identified PL inhibitors from G. biloba could serve as lead compounds for the development of biflavonoid-type PL inhibitors.

The present study investigates the antimicrobial activity of various solvent extracts of Eruca sativa (aerial and root) and seed oil against-antibiotic resistant Gram-negative (Escherichia coli, Pseudomoms aeruginosa and Shigella flexneri) and Gram-positive (Staphylococcus aureus and Bacillus subtilis) bacteria. Among the various preparations, seed oil was the most active, exhibiting a maximum zone inhibition of 97% for Gram-positive bacteria and of 74–97% for Gram-negative bacteria. The MIC of the seed oil was found to be 65–75 and 60–70μg/ml for Gram-negative and Gram-positive bacteria, respectively. Analytical investigation on main volatile and non-volatile components was performed on seed oil. Among the formers allyl isothiocyanate (40μg/g), 3-butenyl isothiocyanate (260μg/g), 4-methylsulfinybutyl isothiocyanate (sulforaphane 743μg/g), 2-phenylethyl isothiocyanate (159μg/g) and bis(isothiocyanatobutyl)disulphide (∼5000μg/g) were determined by head space/SPME/GC–MS analysis. Free fatty acids were 1.6% w/w of the oil and overall 25 fatty acids were identified. Erucic and oleic acids were the main fatty acids both in the free (7.8 and 2.1mg/ml) and esterified forms (50.6% w/w and 14.9% w/w of total fatty acids). Unsaponifiable fraction was 1.8% w/w.

The most promising among glucosinolates (GLs) are those bearing in their aglycon an extra sulfur function, such as glucoraphasatin (4-methylthio-3-butenyl GL; GRH) and glucoraphenin (4-methylsulfinyl-3-butenyl GL; GRE). The GRE/GRH redox couple is typically met among secondary metabolites of Raphanus sativus L. and, whereas GRE prevails in seeds, GRH is the major GL in full-grown roots. During the 10 days of sprouting of R. sativus seeds, the GRE and GRH contents were determined according to the Eurpean Union official method (ISO 9167-1). In comparison to the seeds, the GRE content in sprouts decreased from about 90 to about 12 mu mol g(-1) of dry weight (dw), whereas a 25-fold increase-from about 3 to 76 mu mol g(-1) of dw-of the GRH content was measured. An efficient pure GRH gram-scale production process from R. sativus (kaiware daikon) sprouts resulted in significant yield improvement of up to 2.2% (dw basis). The reaction of GRH with both H2O2 and ABTS(center dot+) radical cation was investigated. Whereas H2O2 oxidation of GRH readily resulted in complete transformation into GRE, ABTS(center dot+) caused complete decay of the GL. Even though not directly related to its radical scavenging activity, the assessed reducing capacity of GRH suggests that R. sativus sprouts might possess potential for health benefits.

Rocket (Eruca sativa Mill. or Eruca vesicaria L.) is widely distributed all over the world and is usually consumed fresh (leafs or sprouts) for its typical spicy taste. Nevertheless, it is mentioned in traditional pharmacopoeia and ancient literature for several therapeutic properties, and it does contain a number of health promoting agents including carotenoids, vitamin C, fibers, flavonoids, and glucosinolates (GLs). The latter phytochemicals have recently gained attention as being the precursors of isothiocyanates (ITCs), which are released by myrosinase hydrolysis during cutting, chewing, or processing of the vegetable. ITCs are recognized as potent inducers of phase II enzymes (e.g., glutathione transferases, NAD(P)H:quinone reductase, epoxide hydrolase, etc.), which are important in the detoxification of electrophiles and protection against oxidative stress. The major GL found in rocket seeds is glucoerucin, GER (108 +/- 5 micromol g(-)(1) d.w.) that represents 95% of total GLs. The content is largely conserved in sprouts (79% of total GLs), and GER is still present to some extent in adult leaves. Unlike other GLs (e.g., glucoraphanin, the bio-precursor of sulforaphane), GER possesses good direct as well as indirect antioxidant activity. GER (and its metabolite erucin, ERN) effectively decomposes hydrogen peroxide and alkyl hydroperoxides with second-order rate constants of k(2) = 6.9 +/- 0.1 x 10(-)(2) M(-)(1) s(-)(1) and 4.5 +/- 0.2 x 10(-)(3) M(-)(1) s(-) , respectively, in water at 37 degrees C, thereby acting as a peroxide-scavenging preventive antioxidant. Interestingly, upon removal of H(2)O(2) or hydroperoxides, ERN is converted into sulforaphane, the most effective inducer of phase II enzymes among ITCs. On the other hand, ERN (and conceivably GER), like other ITCs, does not possess any chain-breaking antioxidant activity, being unable to protect styrene from its thermally (37 degrees C) initiated autoxidation in the presence of AMVN. The mechanism and relevance of the antioxidant activity of GER and ERN are discussed.

It is thought that induction of detoxifying phase-II drug metabolizing enzymes or inhibition of bioactivating phase-I by phytoalexins could protect against mutagens and neoplasia. In the search for potential naturally occurring molecular chemoprevention agents, particular attention has been devoted to isothiocyanates, which are breakdown products-via myrosinase-of glucosinolates such as gluconasturtiin (GNST), a natural constituent of cruciferae. Here, we first investigated the ability of GNST to modulate metabolizing enzymes in male Swiss Albino CD1 mice injected by gavage (24 mg/kg or 48 mg/kg b.w.) with GNST either in single or repeated (daily for four consecutive days) dose. Using selected probes to various cytochrome P450 (CYP) isoforms, a marked and generalized decrease of CYP content, NADPH-(CYP)-c-reductase and various CYP-linked monooxygenases (measuring CYP1A1, CYP2B1/2, CYP3A1/2, CYP1A2 and CYP2E1), was observed in hepatic, renal and pulmonary subcellular preparations (up to approximately 66% loss, liver). Similar behavior was recorded using the regio- and stereo-selective hydroxylation of testosterone as multibiomarker (CYP2A1 and CYP2B9, up to approximately 96% loss), as well as with the phase-II marker glutathione S-transferase (up to approximately 50% loss, liver). We also performed genotoxicity investigations, using the diploid D7 strain of yeast Saccharomyces cerevisiae as a biological test system. GNST was able to significantly induce point reverse mutation in growing cells without myrosinase, thus suggesting either a direct GNST or a CYP-linked metabolite role in the genotoxic response. On the contrary, in suspension test, the addition of myrosinase significantly increased mitotic gene conversion, probably due to the formation of GNST-derived phenylethyl isothiocyanate (PEITC) breakdown product. Taken together, our data suggest that GNST exerts a dual effect: while strongly inhibiting the microsomal (bioactivating) metabolism, GNST also possesses genotoxic activity. This concomitant mutagenic activity underlines the necessity of overall toxicological characterization of this (or any other molecule) prior to mass chemopreventive use.

Abstract: Isothiocyanates 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 isothiocyanates, we studied proliferation, apoptosis induction and p53, bcl‐2 and bax protein expression in Jurkat T‐leukemia cells by the isothiocyanate sulforaphane. Sulforaphane caused G2/M‐phase delay and increase of apoptotic cell fraction 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. Since selective targeting and low toxicity for normal host tissues are fundamental requisites for proposed chemopreventive agents such as sulforaphane, we tested sulforaphane on non‐transformed phytohemagglutinin‐stimulated human T‐lymphocytes. We demonstrated that sulforaphane arrested cell‐cycle progression in G1 phase by a significant down‐modulation of cyclin D3. Moreover, sulforaphane induced apoptosis (and also necrosis), mediated by an increase in the expression of p53, whereas it exerted little effect on bcl‐2 and bax levels. These findings indicate that sulforaphane can exert protective effects inhibiting leukemic cell growth. Moreover, sulforaphane is active not only in transformed lymphocytes but also in their normal counterpart. Although in vitro studies do not necessarily predict in vivo outcomes, our findings raise important questions regarding the suitability of sulforaphane for cancer chemoprevention.

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.

Despite experimental evidence that sulforaphane can exert chemopreventive effects, whether these effects are specific for neoplastic cells is not known. Following our previous demonstration that sulforaphane induces cell cycle arrest and apoptosis in human T lymphoblastoid Jurkat leukemia cells and increases p53 and bax protein expression, we tested sulforaphane on non-transformed phytohemagglutinin-stimulated human lymphocytes. Here, we demonstrate that sulforaphane arrested cell cycle progression in G1 phase, through a decrease in the protein expression of cyclin D3. Moreover, sulforaphane induced apoptosis (and also necrosis), mediated by an increase in the expression of p53. These findings suggest that sulforaphane is a growth modulator for T cells. Our in vitro evidence that sulforaphane is active and even cytotoxic in normal as well as transformed lymphocytes raises important questions regarding its suitability for cancer chemoprevention.

The alpha-L-rhamnopyranosyl derivative of glucosinalbin (1) was isolated from Moringa oleifera seeds, in which it constitutes the major glucosinolate. Its structure was established through a seven-step chemical synthesis starting from L-rhamnose.

Cytotoxic and cell-transforming activity of gluconasturtiin (GNST), a promising chemopreventive agent commonly found in human diet, was studied in a medium-term bioassay utilizing BALB/c 3T3 cells. We also assessed whether GNST coupled with myrosinase, thus yielding product phenylethyl isothiocyanate (as shown by gas chromatography-mass spectral analysis), can affect the transforming potential of benzo(a)pyrene (B(a)P). Neither cytotoxicity nor cell-transforming activity was recorded. On the contrary, a marked increase (up to sevenfold) of the transforming activity of B(a)P was seen. This cocarcinogenic potential could be ascribed to an imbalance among bioactivation/detoxication during cell growth. These results indicate the need for an overall toxicological characterization of a chemopreventive agent prior to large-scale use.

The fungitoxicity of allyl‐isothiocyanate (AITC) vapour against Penicillium expansum, the causal agent of blue mould on pears (cvs Conference and Kaiser), was evaluated. The best control of blue mould was obtained by exposing fruits for 24 h in a 5 mg L−1 AITC‐enriched atmosphere, the extent of control depending on the inoculum density. Lesion diameter was inversely related to AITC concentration. In treated fruits the percentage of infected wounds increased with conidial concentration, with fewer than 20% affected at 1 × 103 conidia mL−1 to almost 80% at 1 × 106 conidia mL−1. In comparison, >98% of wounds were infected in untreated fruits irrespective of conidial concentration. AITC treatments were effective up to 24 h after inoculation for Conference and 48 h for Kaiser. AITC treatments also controlled a thiabendazole‐resistant strain of P. expansum, reducing the incidence of blue mould by 90% in both cultivars. The use of AITC produced from pure sinigrin or from Brassica juncea defatted meal may be an economically viable alternative to synthetic fungicides against P. expansum.

Plants of the family Brassicaceae are receiving increased attention within the policy of reducing pesticides towards a more friendly agricultural system. The effect of these plants has been attributed to glucosinolates and to their hydrolysis derivatives, particularly the isothiocyanates. Potatoes, the largest cultivated horticultural crop, is susceptible to the nematode Globodera rostochiensis which is controlled by the use of synthetic compounds, with subsequent environmental impact. The search for alternative control methods prompted this study in which is reported the effect of several concentrations (0.05, 0.3 and 1.0 mg/ml) of 2-phenylethyl glucosinolate on the mortality of juveniles of G. rostochiensis. The study was conducted over a period of 72 h with evaluations at every 8 h. The results showed no effect with the pure glucosinolate, whilst a 100% mortality occurred within 8 h for 2-phenylethyl glucosinolate (1 mg/ml) with added myrosinase (25%).