Comparison of the bioactivity of two glucoraphanin hydrolysis products found in broccoli, sulforaphane and sulforaphane nitrile.

Epidemiological and laboratory studies suggest that dietary broccoli may prevent or delay a variety of cancers. Broccoli and other crucifers contain a relatively unique family of secondary metabolites called glucosinolates. Glucoraphanin, the major glucosinolate in broccoli, is hydrolyzed by an endogenous plant myrosinase to form either the potent anticarcinogen sulforaphane (SF) or sulforaphane nitrile (SF nitrile). The bioactivities of SF and SF nitrile were compared in rats and in mouse hepatoma cells. Male, 4-week-old, Fischer 344 rats were administered SF or SF nitrile (200, 500, or 1000 micromol/kg) by gavage daily for 5 days. Hepatic, colonic mucosal, and pancreatic quinone reductase and glutathione S-transferase activities were induced by high doses of SF, but not by SF nitrile. When Hepa 1c1c7 cells were exposed to increasing levels of each compound for 24 h, quinone reductase showed a 3-fold maximal induction over control at 2.5 microM SF and a 3.5-fold maximal induction over control at 2000 microM SF nitrile, the highest concentration tested. These results demonstrate that SF nitrile is substantially less potent than SF as an inducing agent of phase II detoxification enzymes. Therefore, glucoraphanin hydrolysis directed toward the production of SF rather than SF nitrile could increase the potential chemoprotective effects of broccoli.     

The metabolic fate of purified glucoraphanin in F344 rats

Dietary broccoli is commonly eaten cooked, exposing individuals to intact glucoraphanin rather than to its hydrolysis product, the anticarcinogenic isothiocyanate sulforaphane, since cooking destroys the hydrolyzing enzyme myrosinase. There is little information on the absorption and metabolism of glucoraphanin, due partly to the lack of purified compound. In this study, glucoraphanin was purified from broccoli seed and 150 mumol/kg was administered to male F344 rats. Glucoraphanin (5% of an oral dose) was recovered intact in urine, showing that it is absorbed intact, and no glucoraphanin or metabolites were found in feces. Total urinary products accounted for 20 and 45% of oral and intraperitonneal doses, respectively, including sulforaphane N-acetyl cysteine conjugate (12.5 and 2%), free sulforaphane (0.65 and 0.77%), sulforaphane nitrile (2 and 1.4%), and erucin (0.1 and 0.1%), respectively. Both glucoraphanin and its reduced form glucoerucin were identified in bile following intravenous glucoraphanin administration. We conclude that orally administered glucoraphanin is absorbed intact, undergoes enterohepatic circulation, and is hydrolyzed in the gut in F344 rats.     

The effect of post-harvest and packaging treatments on glucoraphanin concentration in broccoli (Brassica oleracea var. italica)

The effects of post-harvest and packaging treatments on glucoraphanin (4-methylsulfinylbutyl glucosinolate), the glucosinolate precursor of anticancer isothiocyanate sulforaphane [4-methylsulfinylbutyl isothiocyanate], were examined in broccoli (Brassica oleracea var. italica) during storage times. The results showed that at 20 degrees C, 55% loss of glucoraphanin concentration occurred in broccoli stored in open boxes during the first 3 days of the treatment and 56% loss was found in broccoli stored in plastic bags by day 7. Under both air and controlled atmosphere (CA) storage, glucoraphanin concentration appeared to fluctuate slightly during 25 days of storage and the concentrations under CA was significantly higher than those stored under air treatment. In modified atmosphere packaging (MAP) treatments, glucoraphanin concentration in air control packaging decreased significantly whereas there were no significant changes in glucoraphanin concentration in MAP with no holes at 4 degrees C and two microholes at 20 degrees C for up to 10 days. Decreases in glucoraphanin concentration occurred when the broccoli heads deteriorated. In the present study, the best method for preserving glucoraphanin concentration in broccoli heads after harvest was storage of broccoli in MAP and refrigeration at 4 degrees C. This condition maintained the glucoraphanin concentration for at least 10 days and also maintained the visual quality of the broccoli heads.     

Bioavailability and kinetics of sulforaphane in humans after consumption of cooked versus raw broccoli

The aim of this study was to determine the bioavailability and kinetics of the supposed anticarcinogen sulforaphane, the hydrolysis product of glucoraphanin, from raw and cooked broccoli. Eight men consumed 200 g of crushed broccoli, raw or cooked, with a warm meal in a randomized, free-living, open cross-over trial. Higher amounts of sulforaphane were found in the blood and urine when broccoli was eaten raw (bioavailability of 37%) versus cooked (3.4%, p ) 0.002). Absorption of sulforaphane was delayed when cooked broccoli was consumed (peak plasma time ) 6 h) versus raw broccoli (1.6 h, p ) 0.001). Excretion half-lives were comparable, 2.6 and 2.4 h on average, for raw and cooked broccoli, respectively (p ) 0.5). This study gives complete kinetic data and shows that consumption of raw broccoli results in faster absorption, higher bioavailability, and higher peak plasma amounts of sulforaphane, compared to cooked broccoli.     

Influence of temperature and ontogeny on the levels of glucosinolates in broccoli (Brassica oleracea Var. italica) sprouts and their effect on the induction of mammalian phase 2 enzymes

Broccoli inflorescences have been recognized as components of healthy diets on the basis of their high content of fiber, vitamin C, carotenoids, and glucosinolates/isothiocyanates. Broccoli sprouts have been recently shown to have high levels of glucoraphanin (4-methylsulfinylbutyl glucosinolate), the precursor of the chemoprotective isothiocyanate, sulforaphane. This study evaluated the effects of temperature and developmental stage on the glucosinolate content of broccoli sprouts. Seedlings cultivated using a 30/15 degrees C (day/night) temperature regime had significantly higher glucosinolate levels (measured at six consecutive days postemergence) than did sprouts cultivated at lower temperatures (22/15 and 18/12 degrees C; p < 0.001). Both higher (33.1 degrees C) and lower (11.3 degrees C) constant temperatures induced higher glucosinolate levels in sprouts grown to a uniform size. Glucosinolate levels were highest in cotyledons and lowest in roots of sprouts dissected both early and late in the 11 day developmental span investigated. Nongerminated seeds have the highest glucosinolate levels and concordantly greater induction of mammalian phase 2 detoxication enzymes. Levels decline as sprouts germinate and develop, with consistently higher glucosinolate content in younger developmental stages, independent of the temperature regime. Temperature stress or its associated developmental anomalies induce higher glucosinolate levels, specific elevations in glucoraphanin content, and parallel induction of phase 2 chemoprotective enzymes.     

6-苄氨基嘌呤处理对鲜切西兰花硫代葡萄糖苷代谢的影响

为探讨6-苄氨基嘌呤（6-Benzylaminopurine,6-BA）处理对鲜切西兰花中硫代葡萄糖苷（简称硫苷）代谢的作用机理,该研究首先鉴定得出西兰花花球中分别以萝卜硫苷和萝卜硫素为主的12种硫苷和6种水解产物,然后以30 mg/L的6-BA溶液对鲜切西兰花进行处理,研究外源6-BA处理对其硫苷含量、黑芥子酶活性、硫苷水解产物及其代谢途径关键基因表达水平等的影响。结果表明,6-BA处理通过延缓西兰花花球叶绿素含量下降从而延迟其黄化进程;同时,显著提高了脂肪族硫苷合成相关基因MYB28、CYP79F1、CYP83A1、ST5b、FMOGS-OX1,和吲哚族硫苷合成关键基因MYB51、CYP79B2及CYP83B1的表达水平（P<0.05）,其中6-BA处理组FMOGS-OX1的表达量在贮藏第6天时为对照组的1.93倍,MYB51的表达量为对照组1.41～1.91倍,CYP79B2的表达量在4 d后高出对照组41.90%～93.75%;由此,6-BA处理保持了其组织较高的萝卜硫苷、3-甲基亚磺酰丙基硫苷、吲哚甲基硫苷和4-甲氧基吲哚甲基硫苷的含量,总硫苷含量为对照组的2.46～4.79倍;此外,6-BA处理的西兰花花球组织黑芥子酶活性和其基因MYR的表达水平在4～8 d期间分别高出对照组15.32%～90.22%和25.86%～33.73%,并显著提高了其组织萝卜硫素等异硫氰酸酯水解产物的含量（P<0.05）,其中萝卜硫素含量高于对照组17.58%～25.39%,但对硫苷水解关键基因ESP的表达量并无显著影响（P>0.05）。因此,6-BA处理可通过提高硫苷合成相关基因和水解关键基因来保持鲜切西兰花花球中硫苷的含量和异硫氰酸酯的水平,为西兰花功能活性成分的保持提供理论和技术支持。     

Comparison of isothiocyanate metabolite levels and histone deacetylase activity in human subjects consuming broccoli sprouts or broccoli supplement

Increased consumption of cruciferous vegetables such as broccoli may reduce the risk of various cancers. Myrosinase is required to convert dietary glucosinolates from broccoli into bioactive isothiocyanates. We evaluated isothiocyanate excretion profiles in healthy subjects who consumed broccoli sprouts or broccoli supplement (no myrosinase) with equivalent glucosinolate content. Urinary metabolites of two major isothiocyanates, sulforaphane and erucin, were measured by liquid chromatography coupled with tandem mass spectrometry. Peak excretion of sulforaphane and erucin was higher and occurred sooner in subjects who consumed broccoli sprouts as compared to subjects who consumed the supplement. A subject-dependent shift in the ratio of urinary sulforaphane to erucin metabolites was observed in both groups, indicating conversion of sulforaphane to erucin. Lower histone deacetylase activity was observed in the peripheral blood mononuclear cells only in subjects consuming sprouts. Fresh broccoli sprouts differ from broccoli supplements in regards to excretion of isothiocyanates and bioactivity in human subjects.     

6-Benzylaminopurine delays senescence and enhances health-promoting compounds of harvested broccoli

The effect of 6-benzylaminopurine (6-BA) on the color, antioxidant activity, and contents of total phenols, glucosinolate, and sulforaphane in broccoli florets was investigated. The results showed that 6-BA treatment markedly inhibited the increase of the L* value and malondialdehyde (MDA) content and retarded the decrease of the H value. 6-BA treatment reduced the rate of chlorophyll degradation by regulating the activities of chlorophyllase and Mg-dechelatase. When compared to control florets, the 1,1-diphenyl-2-picrylhydrazyl (DPPH) radical-scavenging activity and the activities of superoxide dismutase (SOD), ascorbate peroxidase (APX), and catalase (CAT) were enhanced in florets treated with 6-BA, whereas the activity of peroxidase (POD) was significantly reduced. The contents of total phenols, glucosinolate, and sulforaphane in broccoli florets were also profoundly increased after treatment with 6-BA. These results indicated that 6-BA could maintain the quality, delay senescence, and improve the nutritional value of broccoli.     

Preparative HPLC method for the purification of sulforaphane and sulforaphane nitrile from Brassica oleracea

An extraction and preparative HPLC method has been devised to simultaneously purify sulforaphane and sulforaphane nitrile from the seed of Brassica oleracea var. italica cv. Brigadier. The seed was defatted with hexane, dried, and hydrolyzed in deionized water (1:9) for 8 h. The hydrolyzed seed meal was salted and extracted with methylene chloride. The dried residue was redissolved in a 5% acetonitrile solution and washed with excess hexane to remove nonpolar contaminants. The aqueous phase was filtered through a 0.22-microm cellulose filter and separated by HPLC using a Waters Prep Nova-Pak HR C-18 reverse-phase column. Refractive index was used to detect sulforaphane nitrile, and absorbance at 254 nm was used to detect sulforaphane. Peak identification was confirmed using gas chromatography and electron-impact mass spectrometry. Each kilogram of extracted seed yielded approximately 4.8 g of sulforaphane and 3.8 g of sulforaphane nitrile. Standard curves were developed using the purified compounds to allow quantification of sulforaphane and sulforaphane nitrile in broccoli tissue using a rapid GC method. The methodology was used to compare sulforaphane and sulforaphane nitrile content of autolyzed samples of several broccoli varieties.     

Variation of glucosinolates in vegetable crops of Brassica oleracea

Glucosinolates were evaluated in 5 groups and 65 accessions of Brassica oleracea (50 broccoli, 4 Brussels sprouts, 6 cabbage, 3 cauliflower, and 2 kale) grown under uniform cultural conditions. Glucosinolates and their concentrations varied among the different groups and within each group. The predominant glucosinolates in broccoli were 4-methylsulfinylbutyl glucosinolate (glucoraphanin), 3-butenyl glucosinolate (gluconapin), and 3-indolylmethyl glucosinoate (glucobrassicin). Glucoraphanin concentration in broccoli ranged from 0.8 micromol g(-1) DW in EV6-1 to 21.7 micromol g(-1) DW in Brigadier. Concentrations of the other glucosinolates in broccoli varied similarly over a wide range. In Brussels sprouts, cabbage, cauliflower, and kale, the predominant glucosinolates were sinigrin (8.9, 7.8, 9.3, and 10.4 micromol g(-1) DW, respectively) and glucobrassicin (3.2, 0.9, 1.3, and 1.2 micromol g(-1) DW, respectively). Brussels sprouts also had significant amounts of gluconapin (6.9 micromol g(-1) DW). Wide variations in glucosinolate content among genotypes suggest differences in their health-promoting properties and the opportunity for enhancement of their levels through genetic manipulation.     

Glucoraphanin and 4-hydroxyglucobrassicin contents in seeds of 59 cultivars of broccoli, raab, kohlrabi, radish, cauliflower, brussels sprouts, kale, and cabbage

The importance of dietary sulforaphane in helping maintain good health continues to gain support within the health-care community and awareness among U.S. consumers. In addition to the traditional avenue for obtaining sulforaphane, namely, the consumption of appropriate cruciferous vegetables, other consumer products containing added glucoraphanin, the natural precursor to sulforaphane, are now appearing in the United States. Crucifer seeds are a likely source for obtaining glucoraphanin, owing to a higher concentration of glucoraphanin and the relative ease of processing seeds as compared to vegetative parts. Seeds of several commonly consumed crucifers were analyzed not only for glucoraphanin but also for components that might have negative health implications, such as certain indole-containing glucosinolates and erucic acid-containing lipids. Glucoraphanin, 4-hydroxyglucobrassicin, other glucosinolates, and lipid erucic acid were quantified in seeds of 33 commercially available cultivars of broccoli, 4 cultivars each of kohlrabi, radish, cauliflower, Brussels sprouts, kale, and cabbage, and 2 cultivars of raab.     

The effect of sulfur fertilizer on glucoraphanin levels in broccoli (B. oleracea L. var. italica) at different growth stages

Three sulfur (S) treatements were imposed by applying gypsum to three broccoli cultivars (Claudia, Marathon, and TB-234) known to differ in glucoraphanin content of mature seeds. The S treatments were control (very low added S), low S (23 kg S ha(-)(1)), and high S (92 kg S ha(-)(1)). The gypsum applications during the early vegetative phase of the three broccoli cultivars increased S uptake and the glucoraphanin content in each plant organ. There were significant genotypic differences for the content of both S and glucoraphanin in all plant organs at different growth stages with gypsum applications. A large increase in S and glucoraphanin content was found in the green heads of broccoli and mature seeds. S present in glucoraphanin accounted for only 4-10% of total S content in broccoli heads. However, S present in glucoraphanin in mature seeds accounted for 40-46% of the total S in the seeds of moderate and high glucoraphanin cultivars (Marathon and TB-234). The partitioning of S into glucoraphanin also increased with gypsum applications. Differences in S uptake, S distribution between organs, and partitioning of S into glucoraphanin largely explained the differences in glucoraphanin content in the green heads and mature seeds for the three broccoli cultivars and three S treatments.     

Food matrix effects on bioactivity of broccoli-derived sulforaphane in liver and colon of F344 rats

Sulforaphane (SF) is considered to be the major anticarcinogenic component in broccoli. The effects of feeding rats purified SF (5 mmol/kg of diet), broccoli containing SF formed in situ during laboratory hydrolysis (broccoli-HP; 20% freeze-dried broccoli diet, 0.16 mmol of SF/kg of diet), and broccoli containing intact glucosinolates (broccoli-GS; 20% freeze-dried broccoli diet, 2.2 mmol of glucoraphanin/kg of diet) were compared. Rats (male F344 rats, five per group) were fed control (modified AIN-76 B-40), SF, broccoli-HP, or broccoli-GS for 5 days. In rats fed broccoli-GS, quinone reductase activities (QR) in the colon and liver were greater (4.5- and 1.4-fold over control, respectively) than in rats fed broccoli-HP (3.2- and 1.1-fold over control, respectively). Broccoli-GS and SF diets increased QR to the same extent, even though the broccoli-GS diet contained far less SF (as the unhydrolyzed glucosinolate, glucoraphanin) than the purified SF diet. In a second experiment, rats were fed one of six diets for 5 days: (1) control; (2) 20% broccoli-GS; (3) diet 2 + low SF (0.16 mmol/kg of diet); (4) diet 2 + high SF (5 mmol/kg of diet); (5) low SF (0.32 mmol/kg of diet); or (6) high SF (5.16 mmol/kg of diet). In both liver and colon, QR was increased most by high SF plus broccoli-GS; individually, high SF and broccoli-GS had similar effects, and adding the low-dose SF to broccoli-GS had either no effect or a negative effect. In both experiments, urinary SF-mercapturic acid correlated with QR activity, not with dietary intake. It was concluded that all diets were substantially more effective in the colon than in the liver and that broccoli-GS was more potent than SF or broccoli-HP.     

Physiological and biochemical metabolism of germinating broccoli seeds and sprouts

Changes in physiological and biochemical metabolism as well as glucoraphanin and sulforaphane contents of germinating broccoli seeds and sprouts were investigated in this study. Sprout length, root length, and fresh weight increased with germination time. Dry weight varied from 2.5 to 3.0 mg per sprout. A rapid increase in respiratory rate of sprouts occurred between 24 and 36 h of germination and then stayed at a high level. HPLC analysis found that glucoraphanin content increased at the early stage (0-12 h) of germination, decreased to a low value of 3.02 mg/g at 48 h, and then reached the highest value of 6.30 mg/g at 72 h of germination. Sulforaphane content decreased dramatically during the first day of germination, then increased slowly, and reached a high value of 3.38 mg/g at 48 h before declining again.     

Evaporative light-scattering analysis of sulforaphane in broccoli samples: Quality of broccoli products regarding sulforaphane contents

Broccoli sulforaphane has received attention as a possible anticarcinogen. Sulforaphane analysis is difficult due to the lack of a chromophore for spectrometric detection. Hence, we developed a method for determining sulforaphane by using high-performance liquid chromatography (HPLC) coupled with an evaporative light-scattering detector (ELSD). Sulforaphane was extracted from acid-hydrolyzed broccoli samples, followed by solid-phase extraction and reversed-phase HPLC. Sulforaphane was detected by ELSD and concurrently identified by electrospray ionization time-of-flight mass spectrometry. The recovery of sulforaphane from broccoli samples was above 95%. The detection limit was 0.5 mug. The present method was sensitive enough to determine sulforaphane in mature broccoli, broccoli sprouts, and commercial broccoli products. Sulforaphane concentration in broccoli sprout (1153 mg/100 g dry weight) was about 10 times higher than that of mature broccoli (44-171 mg/100 g dry weight). Therefore, the broccoli sprout is recommended as a source of sulforaphane-rich products. In contrast, we found that sulforaphane could not be detected in most of broccoli products, suggesting present commercial broccoli products having low quality.     

LC-MS/MS quantification of sulforaphane and indole-3-carbinol metabolites in human plasma and urine after dietary intake of selenium-fortified broccoli

This study aimed at developing a sensitive LC-MS/MS method for the quantification of sulforaphane (SFN) and indole-3-carbinol metabolites in plasma and urine after dietary intake of regular and selenium-fertilized broccoli using stable isotope dilution analysis. In a three-armed, placebo-controlled, randomized human intervention study with 76 healthy volunteers, 200 g of regular (485 μg of total glucosinolates and <0.01 μg of selenium per gram fresh weight) or selenium-fertilized broccoli (589 μg of total glucosinolates and 0.25 μg of selenium per gram fresh weight) was administered daily for 4 weeks. Glucoraphanin and glucobrassicin metabolites quantified in plasma and urine were SFN-glutathione, SFN-cysteine, SFN-cysteinylglycine, SFN-acetylcysteine, and indole-3-carboxaldehyde, indole-3-carboxylic acid, and ascorbigen, respectively. Dietary intake of selenium-fertilized broccoli increased serum selenium concentration analyzed by means of atomic absorption spectroscopy by up to 25% (p < 0.001), but affected neither glucosinolate concentrations in broccoli nor their metabolite concentrations in plasma and urine compared to regular broccoli.     

Sulfur and nitrogen supply influence growth,product appearance,and glucosinolate concentration of broccoli

The effects of insufficient and optimal sulfur (S) and nitrogen (N) supply on plant growth and glucosinolate formation were studied under controlled experimental conditions in broccoli “Monaco”. Here, we report on the interaction between S and N supply, plant growth, and quality parameters and discuss the relevance of this interaction in relation to crop‐management strategies. Broccoli plants supplied with insufficient amounts of S or N showed typical deficiency symptoms and yield decreases. In contrast, total glucosinolate concentrations were high at insufficient N supply, independent of the S level, and low at insufficient S supply in combination with an optimal N supply. This was mainly due to the presence of the alkyl glucosinolates glucoraphanin and glucoiberin. Furthermore, with S concentrations above 6 g (kg DM)^–1 and an N : S ratio lower than 10:1, the glucosinolate concentrations were on average around 0.33 g (kg fresh matter)^–1 and differed significantly from those plants characterized by an S concentration below 6 g (kg DM)^–1 and an N : S ratio above 10:1. In addition, N : S ratios between 7:1 and 10:1 promoted plant yield and enhanced overall appearance. Therefore, to produce broccoli (and potentially other Brassicaceae) with higher crop yields and enhanced product quality in the field, it is vital to establish the optimal S and N nutritional status of the plant and to integrate this information into crop‐management strategy programs.     

Characterization of products from the reaction of glucosinolate-derived isothiocyanates with cysteine and lysine derivatives formed in either model systems or broccoli sprouts

Glucosinolates, present in Brassica vegetables, are thought to contribute to human health prevention because of their enzymatically induced breakdown products, primarily isothiocyanates (ITCs). ITCs are reactive substances that readily react with nucleophilic (food) compounds. The reactivity of allyl-ITC and 4-(methylsulfinyl)butyl-ITC (sulforaphane) toward thiol and amino groups of cysteine and lysine derivatives was studied in buffered model systems as well as broccoli sprouts. The thiol group is the preferred reaction site, and it was demonstrated that even endogenously released sulforaphane is able to react very fast with cysteine in broccoli sprouts. Amino groups reacted slower and only under basic conditions. However, great differences in the reactivity between the different amino compounds were revealed. The aliphatic allylamine reacted very fast with allyl-ITC, forming N,N'-diallylthiourea, a compound identified as a main thermal degradation product of allyl-ITC.     

高温胁迫下硒硫互作调控西兰花芽苗生理及萝卜硫素代谢研究

为探讨高温胁迫下硒硫互作对西兰花芽苗生理及萝卜硫素代谢的影响,以西兰花籽粒为试验材料,经单独喷施硫酸锌(ZnSO₄)、亚硒酸钠(Na₂SeO₃)、二者联合喷施、以及结合高温胁迫下喷施处理,分析发芽期间西兰花芽苗主要生理生化指标并利用荧光定量PCR技术分析萝卜硫素代谢关键酶基因表达的变化。结果表明,单独施用Na₂SeO₃可显著增加芽长和芽苗单株鲜重(P<0.05),有效缓解高温及ZnSO₄对西兰花芽苗生长发育的抑制作用;发芽期间,相较单独喷施ZnSO₄处理,西兰花芽苗经高温后联合喷施ZnSO₄及Na₂SeO₃处理,其硒元素含量、总抗氧化能力、硫代葡萄糖苷含量、黑介子酶活性、异硫氰酸酯含量、萝卜硫素含量均显著提高(P<0.05),其中萝卜硫素含量相较单独喷施ZnSO₄处理增加了39%。高温联合ZnSO₄及Na₂SeO₃处理4 d的芽苗中MYB28、UGT74B1及ST5b基因表达量均较对照显著上调,而BoHMT1显著下调(P<0.05)。综上所述,西兰花芽苗经高温联合硒硫处理是富集萝卜硫素的有效方式,该研究结果为生产富含萝卜硫素的功能性芽苗菜提供了理论依据和技术支撑。