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.     

Exogenous Methyl Jasmonate Treatment Increases Glucosinolate Biosynthesis and Quinone Reductase Activity in Kale Leaf Tissue

Methyl jasmonate (MeJA) spray treatments were applied to the kale varieties ‘Dwarf Blue Curled Vates’ and ‘Red Winter’ in replicated field plantings in 2010 and 2011 to investigate alteration of glucosinolate (GS) composition in harvested leaf tissue. Aqueous solutions of 250 µM MeJA were sprayed to saturation on aerial plant tissues four days prior to harvest at commercial maturity. The MeJA treatment significantly increased gluconasturtiin (56%), glucobrassicin (98%), and neoglucobrassicin (150%) concentrations in the apical leaf tissue of these genotypes over two seasons. Induction of quinone reductase (QR) activity, a biomarker for anti-carcinogenesis, was significantly increased by the extracts from the leaf tissue of these two cultivars. Extracts of apical leaf tissues had greater MeJA mediated increases in phenolics, glucosinolate concentrations, GS hydrolysis products, and QR activity than extracts from basal leaf tissue samples. The concentration of the hydrolysis product of glucoraphanin, sulforphane was significantly increased in apical leaf tissue of the cultivar ‘Red Winter’ in both 2010 and 2011. There was interaction between exogenous MeJA treatment and environmental conditions to induce endogenous JA. Correlation analysis revealed that indole-3-carbanol (I3C) generated from the hydrolysis of glucobrassicin significantly correlated with QR activity (r = 0.800, P<0.001). Concentrations required to double the specific QR activity (CD values) of I3C was calculated at 230 µM, which is considerably weaker at induction than other isothiocyanates like sulforphane. To confirm relationships between GS hydrolysis products and QR activity, a range of concentrations of MeJA sprays were applied to kale leaf tissues of both cultivars in 2011. Correlation analysis of these results indicated that sulforaphane, NI3C, neoascorbigen, I3C, and diindolylmethane were all significantly correlated with QR activity. Thus, increased QR activity may be due to combined increases in phenolics (quercetin and kaempferol) and GS hydrolysis product concentrations rather than by individual products alone.     

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).     

Proteomic analysis of stress-related proteins in transgenic broccoli harboring a gene for cytokinin production during postharvest senescence

Our previous study revealed a cytokinin-related retardation of post-harvest floret yellowing in transgenic broccoli (Brassica oleracea var. italica) that harbored the bacterial isopentenyltransferase (ipt) gene. We aimed to investigate the underlining mechanism of this delayed post-harvest senescence. We used 2D electrophoresis and liquid chromatography-electrospray ionization-mass spectrometry/mass spectrometry for a proteomics analysis of heads of ipt-transgenic and non-transgenic inbred lines of broccoli at harvest and after four days post-harvest storage. At harvest, we found an accumulation of stress-responsive proteins involved in maintenance of protein folding (putative protein disulfide isomerase, peptidyl-prolyl cis-trans isomerase and chaperonins), scavenging of reactive oxygen species (Mn superoxide dismutase), and stress protection [myrosinase-binding protein, jasmonate inducible protein, dynamin-like protein, NADH dehydrogenase (ubiquinone) Fe-S protein 1 and stress-inducible tetratricopeptide repeat-containing protein]. After four days’ post-harvest storage of non-transgenic broccoli florets, the levels of proteins involved in protein folding and carbon fixation were decreased, which indicates cellular degradation and a change in metabolism toward senescence. In addition, staining for antioxidant enzyme activity of non-transgenic plants after post-harvest storage revealed a marked decrease in activity of Fe-superoxide dismutase and ascorbate peroxidase. Thus, the accumulation of stress-responsive proteins and antioxidant enzyme activity in ipt-transgenic broccoli are most likely associated with retardation of post-harvest senescence.     

Effect of ethylene and 1-methylcyclopropene on chlorophyll catabolism of broccoli florets

Branchlets of broccoli (Brassica oleracea L.) were used to examine ethylene-stimulated chlorophyll catabolism. Branchlets treated with: 1) air (CK); 2) 1 µL·L^–1 1-methylcyclopropene (1-MCP) for 14 hr at 20 °C; 3) 1000 µL·L^–1 ethylene (C₂H₄) for 5 hr at 20 °C; or 4) 1-MCP then C₂H₄, were stored in the dark at 20 °C for up to 3 d. Chlorophyll (Chl) content and branchlet hue angle decreased during the storage period and 1-MCP treatment delayed this change. Chl degradation in broccoli was accelerated by exposure to C₂H₄, especially for Chl a. Prior treatment with 1-MCP prevented degreening stimulated by C₂H₄. Lipoxygenase activity was not altered by any of the treatments, however, 1-MCP with or without ethylene resulted in reduced activity of chlorophyllase (Chlase) and peroxidase (POD). Exposure to C₂H₄ stimulated Chlase activity and extended the duration of high POD activity. Treatment with 1-MCP followed by C₂H₄ resulted in reduced POD activity and delayed the increase in Chlase activity. The results suggest chlorophyll in broccoli can be degraded via the POD – hydrogen peroxide system. Exposure to C₂H₄ enhances activity of Chlase and extends the duration of high POD activity, and these responses may accelerate degreening. Treatment with 1-MCP delays yellowing of broccoli, an effect that may be due to the 1-MCP-induced reduction in POD and Chlase activities.     

Glucoraphanin, the bioprecursor of the widely extolled chemopreventive agent sulforaphane found in broccoli, induces phase-I xenobiotic metabolizing enzymes and increases free radical generation in rat liver

Epidemiological and animal studies linking high fruit and vegetable consumption to lower cancer risk have strengthened the belief that long-term administration of isolated naturally occurring dietary constituents could reduce the risk of cancer. In recent years, metabolites derived from phytoalexins, such as glucoraphanin found in broccoli and other cruciferous vegetables (Brassicaceae), have gained much attention as potential cancer chemopreventive agents. The protective effect of these micronutrients is assumed to be due to the inhibition of Phase-I carcinogen-bioactivating enzymes and/or induction of Phase-II detoxifying enzymes, an assumption that still remains uncertain. The protective effect of glucoraphanin is thought to be due to sulforaphane, an isothiocyanate metabolite produced from glucoraphanin by myrosinase. Here we show, in rat liver, that while glucoraphanin slightly induces Phase-II enzymes, it powerfully boosts Phase-I enzymes, including activators of polycyclic aromatic hydrocarbons (PAHs), nitrosamines and olefins. Induction of the cytochrome P450 (CYP) isoforms CYP1A1/2, CYP3A1/2 and CYP2E1 was confirmed by Western immunoblotting. CYP induction was paralleled by an increase in the corresponding mRNA levels. Concomitant with this Phase-I induction, we also found that glucoraphanin generated large amount of various reactive radical species, as determined by electron paramagnetic resonance (EPR) spectrometry coupled to a radical-probe technique. This suggests that long-term uncontrolled administration of glucoraphanin could actually pose a potential health hazard.     

A derivatization method for the simultaneous detection of glucosinolates and isothiocyanates in biological samples

Various analytical methods have been established to quantify isothiocyanates (ITCs) that derive from glucosinolate hydrolysis. However, to date there is no valid method applicable to pharmacokinetic studies that detects both glucosinolates and ITCs. A specific derivatization procedure was developed for the determination of ITCs based on the formation of a stable N-(tert-butoxycarbonyl)-l-cysteine methyl ester derivative, which can be measured by high-performance liquid chromatography with ultraviolet detection after extraction with ethylacetate. The novel method, which is also applicable to the indirect determination of glucosinolates after their hydrolysis by myrosinase, was established for the simultaneous determination of glucoraphanin and sulforaphane. By derivatization, the sensitivity of ITC detection was increased 2.5-fold. Analytical recoveries from urine and plasma were greater than 75% and from feces were approximately 50%. The method showed intra- and interday variations of less than 11 and 13%, respectively. Applicability of the method was demonstrated in mice that received various doses of glucoraphanin or that were fed a glucoraphanin-rich diet. Besides glucoraphanin and sulforaphane, glucoerucin and erucin were detected in urine and feces of mice. The novel method provides an essential tool for the analysis of bioactive glucosinolates and their hydrolysis products and, thus, will contribute to the elucidation of their bioavailability.     

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.     

High glucosinolate broccoli: a delivery system for sulforaphane

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.     

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.     

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.     

Identification of dehydration-responsive cysteine proteases during post-harvest senescence of broccoli florets

Harvest-induced senescence of broccoli results in tissue wilting and sepal chlorosis. As senescence progresses, chlorophyll and protein levels in floret tissues decline and endo-protease activity (measured with azo-casein) increases. Protease activity increased from 24 h after harvest for tissues held in air at 20 degrees C. Activity was lower in floret tissues from branchlets that had been held in solutions of sucrose (2% w/v) or under high carbon dioxide, low oxygen (10% CO(2), 5% O(2)) conditions. Four protease-active protein bands were identified in senescing floret tissue by zymography, and the use of chemical inhibitors of protease action suggests that some 44% of protease activity in senescing floret tissue 72 h after harvest is due to the action of cysteine and serine proteases. Four putative cysteine protease cDNAs have been isolated from broccoli floret tissue (BoCP1, BoCP2, BoCP3, BoCP4). The cDNAs are most similar (73-89% at the amino acid level) to dehydration-responsive cysteine proteases previously isolated from Arabidopsis thaliana (RD19, RD21). The mRNAs encoded by the broccoli cDNAs are expressed in floret tissue during harvest-induced senescence with mRNA accumulating within 6 h of harvest for BoCP1, 12 h of harvest for BoCP4 and within 24 h of harvest for BoCP2 and BoCP3. Induction of the cDNAs is differentially delayed when broccoli branchlets are held in solutions of water or sucrose. In addition, the expression of BoCP1 and BoCP3 is inhibited in tissue held in atmospheres of high carbon dioxide/low oxygen (10% CO(2), 5% O(2)). The putative cysteine protease mRNAs are expressed before measurable increases in endo-protease activity, loss of protein, chlorophyll or tissue chlorosis.     

Suppression of the cysteine protease,aleurain, delays floret senescence in <Emphasis Type="Italic">Brassica oleracea</Emphasis>

An aleurain-like protein, BoCP5, is up-regulated during harvest-induced senescence in broccoli floret and leaf tissue. BoCP5 is most closely related to an Arabidopsis protein (91%, AAF43041) and has 71% identity to barley aleurain (P05167). The mRNA for this gene accumulates within 6 h after harvest in broccoli florets, and its expression is reduced in tissue that has been held in senescence-delaying treatments (e.g. water, sucrose feeding, controlled atmosphere). The gene is also expressed in leaves during aging-related and harvest-induced senescence. Analysis of protein bands that cross-react with antibodies raised to the bacterial BoCP5 fusion protein, revealed prominent immunoreactive bands at ca. 26, 28, 31, and 38 kD in floret tissue. The 31 kD band was absent in protein extracts from leaf tissue. Agrobacterium-mediated transformation was used to produce transgenic broccoli plants with down-regulated BoCP5. A reduction in the postharvest expression of BoCP5 in floret tissue was achieved for four transgenic lines in the current study. In three of these lines postharvest floret senescence (yellowing) was delayed, and florets contained significantly greater chlorophyll levels during postharvest storage at 20 °C than wild-type plants. Line 4 showed the greatest down-regulation of BoCP5, and in this line postharvest protease activity remained at pre-harvest levels, and the yield of soluble proteins extracted from florets after harvest was significantly greater than that of wild-type tissue.     

Involvement of 1-Methylcyclopropene in Plant Growth,Ethylene Production,and Synthase Activity of Inferior Spikelets in Hybrid Rice Differing in Panicle Architectures

Chlorophyll content (SPAD values) of leaves, 1-aminocyclopropane-1-carboxylic acid synthase (ACS) activities and ethylene production of inferior spikelets, aboveground biomass, grain yield, and yield components were studied to compare the effects of 1-methylcyclopropene (1-MCP) on plant growth, and inferior spikelet development in hybrid rice (Oryza sativa L.) differing in panicle architectures. There were four hybrid rice cultivars in this trial, including two lax-panicled cultivars, Liangyoupeijiu (LYPJ) and Guodao 6 (GD6), and two compact-panicled cultivars, Yongyou 9 (YY9) and Yongyou 12 (YY12). Results showed that chlorophyll content of leaves in 1-MCP treatment was higher than control (CK) after application of 1-MCP. ACS activities and ethylene production in 1-MCP treatment for LYPJ, GD6, and YY9 were significantly decreased compared to their corresponding CK. Aboveground biomass and grain yield of cultivars were increased with the increase of chlorophyll content of leaves, but decreased with the increase of ACS activities and ethylene production of inferior spikelets. On average, grain yield in 1-MCP treatment for LYPJ was increased by 7.3 % compared to CK, GD6 was increased by 2.6 %, YY9 was increased by 3.2 %, and YY12 was increased by 0.8 %, respectively. Application of 1-MCP showed marked effects on increasing spikelet fertility and harvest index of cultivars, but exhibited little effects on improving sink capacity and 1,000-grain weight. Our results indicate that 1-MCP could play a positive role in regulating the growth and development of hybrid rice with lax panicles or with compact panicles.     

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.     

1-MCP对室温贮藏下不同成熟度猕猴桃的生理效应

以适期采收(成熟度Ⅰ)和晚采收(成熟度Ⅱ)的'海沃德'猕猴桃果实为材料,研究0.5 μL·L~(-1) 1-甲基环丙烯(1-MCP)处理对室温贮藏(20℃)条件下两种果实的生理生化指标的影响.结果显示:1-甲基环丙烯(1-MCP)处理能有效延缓贮藏期间两种成熟度果实的硬度、可滴定酸含量、维生素C含量的下降,显著降低二者的乙烯释放速率和呼吸强度及其峰值,提高果实的POD、CAT和SOD活性.与成熟度Ⅰ相比,成熟度Ⅱ果实的硬度下降较快,且丙二醛含量和呼吸速率却明显较高,呼吸速率和乙烯释放量高峰时间相对提前.研究表明,在相同的贮藏条件下,晚采收的'海沃德'猕猴桃果实成熟衰老进程明显快于适期采收果实,贮藏效果较差;1-MCP能够明显延长猕猴桃果实的贮藏时间,表现出显著的保鲜效果.     

Suppressing expression of a soluble acid invertase (BoINV2) in broccoli (Brassica oleracea) delays postharvest floret senescence and downregulates cysteine protease (BoCP5) transcription

We report on the production and selection of transgenic Brassica oleracea var. Italica lines with a downregulated soluble acid invertase ( BoINV2 ). Explants of broccoli (cv. Triathlon) were transformed with an antisense construct of BoINV2 under the control of an Asparagus officinalis -derived harvest-induced promoter using Agrobacterium tumefaciens -mediated transformation. BoINV2 is upregulated in wild-type broccoli floret tissue after harvest. Transgenic broccoli lines showed reduced BoINV2 mRNA accumulation immediately after harvest compared with wild-type. Downregulation of BoINV2 had no significant impact on the expression of a second broccoli acid invertase gene ( BoINV1 ), but plants with downregulated BoINV2 also had lower expression of a senescence-associated cysteine protease ( BoCP5 ) compared with wild-type. The total soluble sugar levels in floret tissue of antisense BoINV2 lines were greater than wild-type tissue after harvest (up to 1.5 times higher). Soluble protein content of wild-type tissue decreased from 48 h after harvest with an increase in protease activity. In comparison, two antisense BoINV2 lines retained at-harvest levels of soluble protein until 72 and 96 h after harvest and had lower postharvest endoprotease activity compared with wild-type. Antisense BoINV2 lines also had a slower rate of floret sepal chlorosis after harvest compared with wild-type.