Heating decreases epithiospecifier protein activity and increases sulforaphane formation in broccoli

Sulforaphane, an isothiocyanate from broccoli, is one of the most potent food-derived anticarcinogens. This compound is not present in the intact vegetable, rather it is formed from its glucosinolate precursor, glucoraphanin, by the action of myrosinase, a thioglucosidase enzyme, when broccoli tissue is crushed or chewed. However, a number of studies have demonstrated that sulforaphane yield from glucoraphanin is low, and that a non-bioactive nitrile analog, sulforaphane nitrile, is the primary hydrolysis product when plant tissue is crushed at room temperature. Recent evidence suggests that in Arabidopsis, nitrile formation from glucosinolates is controlled by a heat-sensitive protein, epithiospecifier protein (ESP), a non-catalytic cofactor of myrosinase. Our objectives were to examine the effects of heating broccoli florets and sprouts on sulforaphane and sulforaphane nitrile formation, to determine if broccoli contains ESP activity, then to correlate heat-dependent changes in ESP activity, sulforaphane content and bioactivity, as measured by induction of the phase II detoxification enzyme quinone reductase (QR) in cell culture. Heating fresh broccoli florets or broccoli sprouts to 60 degrees C prior to homogenization simultaneously increased sulforaphane formation and decreased sulforaphane nitrile formation. A significant loss of ESP activity paralleled the decrease in sulforaphane nitrile formation. Heating to 70 degrees C and above decreased the formation of both products in broccoli florets, but not in broccoli sprouts. The induction of QR in cultured mouse hepatoma Hepa lclc7 cells paralleled increases in sulforaphane formation.     

Sulforaphane Bioavailability from Glucoraphanin-Rich Broccoli: Control by Active Endogenous Myrosinase

Glucoraphanin from broccoli and its sprouts and seeds is a water soluble and relatively inert precursor of sulforaphane, the reactive isothiocyanate that potently inhibits neoplastic cellular processes and prevents a number of disease states. Sulforaphane is difficult to deliver in an enriched and stable form for purposes of direct human consumption. We have focused upon evaluating the bioavailability of sulforaphane, either by direct administration of glucoraphanin (a glucosinolate, or β-thioglucoside-N-hydroxysulfate), or by co-administering glucoraphanin and the enzyme myrosinase to catalyze its conversion to sulforaphane at economic, reproducible and sustainable yields. We show that following administration of glucoraphanin in a commercially prepared dietary supplement to a small number of human volunteers, the volunteers had equivalent output of sulforaphane metabolites in their urine to that which they produced when given an equimolar dose of glucoraphanin in a simple boiled and lyophilized extract of broccoli sprouts. Furthermore, when either broccoli sprouts or seeds are administered directly to subjects without prior extraction and consequent inactivation of endogenous myrosinase, regardless of the delivery matrix or dose, the sulforaphane in those preparations is 3- to 4-fold more bioavailable than sulforaphane from glucoraphanin delivered without active plant myrosinase. These data expand upon earlier reports of inter- and intra-individual variability, when glucoraphanin was delivered in either teas, juices, or gelatin capsules, and they confirm that a variety of delivery matrices may be equally suitable for glucoraphanin supplementation (e.g. fruit juices, water, or various types of capsules and tablets).     

High-performance liquid chromatography-based method to evaluate kinetics of glucosinolate hydrolysis by Sinapis alba myrosinase

Isothiocyanates (ITCs) are one of several hydrolysis products of glucosinolates, plant secondary metabolites that are substrates for the thioglucohydrolase myrosinase. Recent pursuits toward the development of synthetic non-natural ITCs have consequently led to an exploration of generating these compounds from non-natural glucosinolate precursors. Evaluation of the myrosinase-dependent conversion of select non-natural glucosinolates to non-natural ITCs cannot be accomplished using established ultraviolet–visible (UV–Vis) spectroscopic methods. To overcome this limitation, an alternative high-performance liquid chromatography (HPLC)-based analytical approach was developed where initial reaction velocities were generated from nonlinear reaction progress curves. Validation of this HPLC method was accomplished through parallel evaluation of three glucosinolates with UV–Vis methodology. The results of this study demonstrate that kinetic data are consistent between both analytical methods and that the tested glucosinolates respond similarly to both Michaelis–Menten and specific activity analyses. Consequently, this work resulted in the complete kinetic characterization of three glucosinolates with Sinapis alba myrosinase, with results that were consistent with previous reports.     

Reducing progoitrin and enriching glucoraphanin in Braasica napus seeds through silencing of the GSL-ALK gene family

The hydrolytic products of glucosinolates in brassica crops are bioactive compounds. Some glucosinolate derivatives such as oxazolidine-2-thione from progoitrin in brassica oilseed meal are toxic and detrimental to animals, but some isothiocyanates such as sulforaphane are potent anti-carcinogens that have preventive effects on several human cancers. In most B. rapa, B. napus and B. juncea vegetables and oilseeds, there is no or only trace amount of glucoraphanin that is the precursor to sulforaphane. In this paper, RNA interference (RNAi) of the GSL-ALK gene family was used to down-regulate the expression of GSL-ALK genes in B. napus. The detrimental glucosinolate progoitrin was reduced by 65 %, and the beneficial glucosinolate glucoraphanin was increased to a relatively high concentration (42.6 μmol g(-1) seed) in seeds of B. napus transgenic plants through silencing of the GSL-ALK gene family. Therefore, there is potential application of the new germplasm with reduced detrimental glucosinolates and increased beneficial glucosinolates for producing improved brassica vegetables.     

Micronucleus formation and induction of apoptosis by different isothiocyanates and a mixture of isothiocyanates in human lymphocyte cultures

Isothiocyanates (ITCs) are the main sulfur-containing metabolites found in cruciferous vegetables. There is evidence that some ITCs may act as chemopreventive agents against different tumor types and induce apoptosis and modulate cell-cycle progression of highly proliferative cancer cells. However, there are also studies reporting genotoxic or co-carcinogenic effects for some ITCs, such as benzyl ITC and phenyl ITC. Since selectivity for transformed cells and absence of genotoxicity for healthy cells are important pre-requisites for new chemopreventive agents, we investigated micronucleus formation and induction of apoptosis by 4-(methylthio)butylisothiocyanate (MTBITC), sulforaphane and a mixture of ITCs in human T-lymphocyte cultures. We demonstrate that MTBITC, sulforaphane and the mixture of ITCs did not induce micronuclei. Moreover, sulforaphane induced a dose-dependent increase in the number of apoptotic cells, which was significant at the highest concentration tested (30 microM) (41% versus 18% in the untreated samples, P<0.05). The mixture of ITCs presented a trend similar to that found for sulforaphane. In fact, the mixture of ITCs was able to induce a dose-dependent increase in the percentage of apoptotic cells, which reached a maximum value at the concentration of 13 microg/ml (46% versus 19% in control samples, P<0.05). Induction of apoptosis was not observed in cultures treated with MTBITC. Our results suggest that different ITCs can have different effects. Moreover, although the mixture of glucosinolates (GLs) used in the present study does not reflect the exact composition of broccoli, our findings demonstrate that the quantitative effects of a single, specific ITC can be significantly different from those of an ITC mixture, where other ITCs of the mixture contribute to the outcome observed.     

Determination of sialic acids in biological fluids using reversed-phase ion-pair high-performance liquid chromatography

A simple, rapid and sensitive reversed-phase ion-pair high-performance liquid chromatographic method for the determination of N-acetylneuraminic acid and 2-deoxy-2,3-dehydro-N-acetylneuraminic acid in biological fluids is described. Determination of N-acetylneuraminic acid released by acidic hydrolysis, in serum, urine and saliva, and 2-deoxy-2,3-dehydro-N-acetylneuraminic acid in urine, without hydrolysis, was accomplished by injecting the sample without derivatization, into the chromatograph. Measurements were carried out isocratically within 6 min using a C₁₈ column and a mobile phase of aqueous solution of triisopropanolamine, as ion-pair reagent, 60 mM, pH 3.5 at room temperature with UV absorbance detection. The present method is reported for the first time for the determination of sialic acids in biological fluids. Recoveries in serum, urine and saliva ranged from 90 to 102% and the limits of detection were 60 nM and 20 nM for the two sialic acids, respectively. The method has been applied to normal and pathological sera from patients with breast, stomach, colon, ovarian and cervix cancers, to normal urine and urine from patient with sialuria and to normal saliva.     

HPLC-based kinetics assay facilitates analysis of systems with multiple reaction products and thermal enzyme denaturation

Glucosinolates are plant secondary metabolites abundant in Brassica vegetables that are substrates for the enzyme myrosinase, a thioglucoside hydrolase. Enzyme-mediated hydrolysis of glucosinolates forms several organic products, including isothiocyanates (ITCs) that have been explored for their beneficial effects in humans. Myrosinase has been shown to be tolerant of non-natural glucosinolates, such as 2,2-diphenylethyl glucosinolate, and can facilitate their conversion to non-natural ITCs, some of which are leads for drug development. An HPLC-based method capable of analyzing this transformation for non-natural systems has been described. This current study describes (1) the Michaelis–Menten characterization of 2,2-diphenyethyl glucosinolate and (2) a parallel evaluation of this analogue and the natural analogue glucotropaeolin to evaluate effects of pH and temperature on rates of hydrolysis and product(s) formed. Methods described in this study provide the ability to simultaneously and independently analyze the kinetics of multiple reaction components. An unintended outcome of this work was the development of a modified Lambert W(x) which includes a parameter to account for the thermal denaturation of enzyme. The results of this study demonstrate that the action of Sinapis alba myrosinase on natural and non-natural glucosinolates is consistent under the explored range of experimental conditions and in relation to previous accounts.     

Variation of glucosinolates in vegetable crops of Brassica rapa

Glucosinolate levels in leaves were determined in a collection of 113 varieties of turnip greens (Brassica rapa L.) from northwestern Spain grown at two sites. Sensorial attributes were also assessed by a consumer panel. The objectives were to determine the diversity among varieties in total glucosinolate content and glucosinolate profile and to evaluate their sensory attributes in relation to glucosinolate content for breeding purposes. Sixteen glucosinolates were identified, being the aliphatic glucosinolates, gluconapin and glucobrassicanapin the most abundant. Other aliphatic glucosinolates, such as progoitrin, glucoalyssin, and gluconapoleiferin were relatively abundant in varieties with a different glucosinolate profile. Indolic and aromatic glucosinolate concentrations were low and showed few differences among varieties. Differences in total glucosinolate content, glucosinolate profile and bitterness were found among varieties, with a total glucosinolate content ranging from 11.8 to 74.0micromolg(-1) dw at one site and from 7.5 to 56.9micromolg(-1) dw at the other site. Sensory analysis comparing bitterness with variation in glucosinolate, gluconapin and glucobrassicanapin concentrations suggested that these compounds and their breakdown products are not the only determinants of the characteristic flavour of this vegetable. Other phytochemicals are probably involved on the characteristic bitter flavour. The varieties MBG-BRS0132, MBG-BRS0082, MBG-BRS0173, and MBG-BRS0184 could be good candidates for future breeding programs since they had high total glucosinolate content and good agronomic performance. The presence of glucoraphanin in some varieties should be studied more extensively, because this aliphatic glucosinolate is the precursor of sulforaphane, a potent anti-cancer isothiocyanate.     

Formation of glucoraphanin by chemoselective oxidation of natural glucoerucin: a chemoenzymatic route to sulforaphane

A new semi-synthetic way to produce glucoraphanin (2), the bio-precursor of the potential anticarcinogen sulforaphane (3), has been developed. Starting from glucoerucin (1), isolated from ripe seeds of Eruca sativa, glucoraphanin was obtained through chemoselective oxidation. Controlled myrosinase-catalysed hydrolysis of this precursor quantitatively afforded sulforaphane.     

Quantitative determination of intact glucosinolates in broccoli, broccoli sprouts, Brussels sprouts, and cauliflower by high-performance liquid chromatography-electrospray ionization-tandem mass spectrometry

Many methods have been proposed to analyze glucosinolates, a class of phytochemicals whose breakdown products are thought to be responsible for an improvement in health; however, few are quantitative and many are time consuming. A selective and sensitive quantitative method for direct determination of intact glucosinolates was developed using liquid chromatography coupled to electrospray ionization tandem mass spectrometry with selected reaction monitoring detection. Detection limits for glucoiberin, sinigrin, progoitrin, glucoerucin, and glucotropaeolin were 1.75, 1.38, 1.36, 0.6, and 0.63 pmol, respectively. Intraassay precision of the method was within 10% for each compound. The method was successfully applied to quantify 10 individual glucosinolates in broccoli, broccoli sprouts, Brussels sprouts, and cauliflower. The advantage of the proposed method includes analysis of individual intact glucosinolates rather than the conversion to desulfoglucosinolates, an increased selectivity through the use of mass spectrometry, and a 10-fold improvement in detection sensitivity over conventionally used HPLC techniques.     

DNA Extraction from Protozoan Oocysts/Cysts in Feces for Diagnostic PCR

PCR detection of intestinal protozoa is often restrained by a poor DNA recovery or by inhibitors present in feces. The need for an extraction protocol that can overcome these obstacles is therefore clear. QIAamp® DNA Stool Mini Kit (Qiagen) was evaluated for its ability to recover DNA from oocysts/cysts directly from feces. Twenty-five Giardia-positive, 15 Cryptosporidium-positive, 15 Entamoeba histolytica-positive, and 45 protozoa-free samples were processed as control by microscopy and immunoassay tests. DNA extracts were amplified using 3 sets of published primers. Following the manufacturer''s protocol, the kit showed sensitivity and specificity of 100% towards Giardia and Entamoeba. However, for Cryptosporidium, the sensitivity and specificity were 60% (9/15) and 100%, respectively. A series of optimization experiments involving various steps of the kit''s protocol were conducted using Cryptosporidium-positive samples. The best DNA recoveries were gained by raising the lysis temperature to the boiling point for 10 min and the incubation time of the InhibitEX tablet to 5 min. Also, using a pre-cooled ethanol for nucleic acid precipitation and small elution volume (50-100 µl) were valuable. The sensitivity of the amended protocol to Cryptosporidium was raised to 100%. Cryptosporidium DNA was successfully amplified by either the first or the second primer set. When applied on parasite-free feces spiked with variable oocysts/cysts counts, ≈ 2 oocysts/cysts were theoretically enough for detection by PCR. To conclude, the Qiagen kit with the amended protocol was proved to be suitable for protozoan DNA extraction directly from feces and support PCR diagnosis.     

Physiological effects of broccoli consumption

Epidemiological studies suggest that broccoli can decrease risk for cancer. Broccoli contains many bioactives, including vitamins C and E, quercetin and kaempferol glycosides and, like other members of the Brassicaceae, several glucosinolates, including glucobrassicin (3-indolylmethyl glucosinolate) and glucoraphanin (4-methylsulphinylbutyl glucosinolate). A key bioactive component responsible for much of this activity may be sulforaphane (1-isothiocyanato-4-methylsulfinylbutane), a hydrolysis product of glucoraphanin. Sulforaphane not only upregulates a number of phase II detoxification enzymes involved in clearance of chemical carcinogens and reactive oxygen species, but has anti-tumorigenic properties, causing cell cycle arrest and apoptosis of cancer cells. The bioequivalency of sulforaphane and whole broccoli have not been fully evaluated, leaving it unclear whether whole broccoli provides a similar effect to purified sulforaphane, or whether the presence of other components in broccoli, such as indole-3-carbinol from glucobrassicin, is an added health benefit. Dietary indole-3-carbinol is known to alter estrogen metabolism, to cause cell cycle arrest and apoptosis of cancer cells and, in animals, to decrease risk for breast cancer. Recent research suggests that both dietary broccoli and the individual components sulforaphane and indole-3-carbinol may offer protection from a far broader array of diseases than cancer, including cardiovascular and neurodegenerative diseases. A common link between these oxidative degenerative diseases and cancer may be aggravation by inflammation. A small body of literature is forming suggesting that both indole-3-carbinol and sulforaphane may protect against inflammation, inhibiting cytokine production. It remains to be seen whether cancer, cardiovascular disease, dementia and other diseases of aging can all benefit from a diet rich in broccoli and other crucifers.     

Pharmacokinetics, tissue distribution and excretion study of dl-praeruptorin A of Peucedanum praeruptorum in rats by liquid chromatography tandem mass spectrometry

dl-Praeruptorin A (Pd-Ia), isolated from Chinese traditional herbal medicine Peucedanum praeruptorum Dunn, has been proved to be a novel Ca²⁺-influx blocker and K⁺-channel opener, and displayed bright prospects in prevention and therapy of cardiac diseases. The aim of this study was to investigate the pharmacokinetics, tissue distribution and excretion of Pd-Ia in rats following a single intravenous (i.v.) administration. The levels of Pd-Ia in plasma, tissues, bile, urine and feces were measured by a rapid and sensitive liquid chromatography-tandem mass spectrometry (LC-MS/MS) method. The results showed that Pd-Ia was rapidly distributed and then eliminated from rat plasma and manifested linear dynamics in dose range of 5-20 mg/kg. The mean elimination half-life (t_1/2) of Pd-Ia for 5, 10 and 20 mg/kg dose were 57.46, 60.87 and 59.01 min, respectively. The major distribution tissues of Pd-Ia in rats were spleen, heart and lung, and low polarity enabled Pd-Ia to cross the blood-brain barrier. There was no long-term accumulation of Pd-Ia in rat tissues. Total recoveries of Pd-Ia within 24 h were low (0.097% in bile, 0.120% in urine and 0.009% in feces), which might be resulted from liver first pass effect.     

Epithiospecifier protein activity in broccoli: the link between terminal alkenyl glucosinolates and sulphoraphane nitrile

The chemical nature of the hydrolysis products from the glucosinolate-myrosinase system depends on the presence or absence of supplementary proteins, such as epithiospecifier proteins (ESPs). ESPs (non-catalytic cofactors of myrosinase) promote the formation of epithionitriles from terminal alkenyl glucosinolates and as recent evidence suggests, simple nitriles at the expense of isothiocyanates. The ratio of ESP activity to myrosinase activity is crucial in determining the proportion of these nitriles produced on hydrolysis. Sulphoraphane, a major isothiocyanate produced in broccoli seedlings, has been found to be a potent inducer of phase 2 detoxification enzymes. However, ESP may also support the formation of the non-inductive sulphoraphane nitrile. Our objective was to monitor changes in ESP activity during the development of broccoli seedlings and link these activity changes with myrosinase activity, the level of terminal alkenyl glucosinolates and sulphoraphane nitrile formed. Here, for the first time, we show ESP activity increases up to day 2 after germination before decreasing again to seed activity levels at day 5. These activity changes paralleled changes in myrosinase activity and terminal alkenyl glucosinolate content. There is a significant relationship between ESP activity and the formation of sulforaphane nitrile in broccoli seedlings. The significance of these findings for the health benefits conferred by eating broccoli seedlings is briefly discussed.     

Effects of metal ions on myrosinase activity and the formation of sulforaphane in broccoli seed

Effects of six metal ions on the formation of sulforaphane and the liberation of glucose upon hydrolysis of glucoraphanin by myrosinase at neutral pH were studied. The yields of sulforaphane and glucose were determined by HPLC. Copper ion and magnesium ion decreased the yields of sulforaphane and glucose. Ferrous ion and ferric ion inhibited the formation of sulforaphane, but had no effects on the liberation of glucose. Calcium ion increased the yield of glucose liberation, but inhibited the formation of sulforaphane. Only zinc ion was beneficial to the liberation of glucose and accelerated the formation of sulforaphane at initial reaction intervals.     

Ultrasensitive assay for three polyphenols (catechin,quercetin and resveratrol) and their conjugates in biological fluids utilizing gas chromatography with mass selective detection

The concentrations of three polyphenols ((+)-catechin, quercetin and trans-resveratrol) in blood serum, plasma and urine, as well as whole blood, have been measured after their oral and intragastric administration, respectively, to humans and rats. The method developed for this purpose utilized ethyl acetate extraction of 100 μl samples and their derivatization with bis(trimethylsilyl)trifluoroacetamide (BSTFA) followed by gas-chromatographic analysis on a DB-5 column followed by mass selective detection employing two target ions and one qualifier ion for each compound. Total run time was 17 min with excellent resolution and linearity. The limits of detection (LOD) and quantitation (LOQ) were an order of magnitude less than for any previously published method, being 0.01 μg/l and 0.1 μg/l, respectively, for all compounds. Recovery at 1 μg/l and 10 μg/l was >80% in all instances but one, and was >90% in 50%. Imprecision was acceptable at 0.25 and 1.0 μg/l, concentrations below the LOQ of previous methods. Aglycones released from conjugates after hydrolysis were easily measurable. Optimal conditions for hydrolysis were established. After oral administration of the three polyphenols to humans, their conjugates vastly exceeded the concentrations of the aglycones in both plasma and urine. Concentrations peaked within 0.5–1.0 h in plasma and within 8 h in urine. During the first 24 h, 5.1% of the (+)-catechin and 24.6% of the trans-resveratrol given were recovered in the urine (free plus conjugated). This method can be proposed as the method of choice to assay these polyphenols and their conjugates in biological fluids.     

Time-resolved fluorescence-based assay for rapid detection of Escherichia coli

Fast and simple detection of pathogens is of utmost importance in health care and the food industry. In this article, a novel technology for the detection of pathogenic bacteria is presented. The technology uses lytic-specific bacteriophages and a nonspecific interaction of cellular components with a luminescent lanthanide chelate. As a proof of principle, Escherichia coli-specific T4 bacteriophage was used to infect the bacteria, and the cell lysis was detected. In the absence of E. coli, luminescent Eu³⁺–chelate complex cannot be formed and low time-resolved luminescence signal is monitored. In the presence of E. coli, increased luminescence signal is observed as the cellular contents are leached to the surrounding medium. The luminescence signal is observed as a function of the number of bacteria in the sample. The homogeneous assay can detect living E. coli in bacterial cultures and simulated urine samples within 25 min with a detection limit of 1000 or 10,000 bacterial cells/ml in buffer or urine, respectively. The detection limit is at the clinically relevant level, which indicates that the method could also be applicable to clinical settings for fast detection of urine bacteria.     

Monoclonal Antibody-Based Dipstick Assay: A Reliable Field Applicable Technique for Diagnosis of Schistosoma mansoni Infection Using Human Serum and Urine Samples

A field applicable diagnostic technique, the dipstick assay, was evaluated for its sensitivity and specificity in diagnosing human Schistosoma mansoni infection. A monoclonal antibody (mAb) against S. mansoni adult worm tegumental antigen (AWTA) was employed in dipstick and sandwich ELISA for detection of circulating schistosome antigen (CSA) in both serum and urine samples. Based on clinical and parasitological examinations, 60 S. mansoni-infected patients, 30 patients infected with parasites other than schistosomiasis, and 30 uninfected healthy individuals were selected. The sensitivity and specificity of dipstick assay in urine samples were 86.7% and 90.0%, respectively, compared to 90.0% sensitivity and 91.7% specificity of sandwich ELISA. In serum samples, the sensitivity and specificity were 88.3% and 91.7% for dipstick assay vs. 91.7% and 95.0% for sandwich ELISA, respectively. The diagnostic efficacy of dipstick assay in urine and serum samples was 88.3% and 90.0%, while it was 90.8% and 93.3% for sandwich ELISA, respectively. The diagnostic indices of dipstick assay and ELISA either in serum or in urine were statistically comparable (P>0.05). In conclusion, the dipstick assay offers an alternative simple, rapid, non-invasive technique in detecting CSA or complement to stool examinations especially in field studies.     

Glucosinolate profiling of Brassica rapa cultivars after infection by Leptosphaeria maculans and Fusarium oxysporum

The glucosinolate contents of two different cultivars of Brassica rapa (Herfstraap and Oleifera) infected with Leptosphaeria maculans and Fusarium oxysporum were determined. Infection triggered the accumulation of aliphatic glucosinolates (gluconapin, progoitrin, glucobrassicanapin and gluconapoleiferin) and indole glucosinolate (4-hydroxy-glucobrassicin) in Herfstraap and of two indole glucosinolates (glucobrassicin and 4-hydroxy-glucobrassicin) in Oleifera. While total and aliphatic glucosinolates decreased significantly in Oleifera, a large increase was observed in Herfstraap after fungal infection. The indole glucosinolate glucobrassicin accumulated in Oleifera at a higher rate than Herfstraap especially after infection with F. oxysporum. Apparently the interaction between fungus and B. rapa is cultivar and fungal species specific.