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.     

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.     

Application of high-speed countercurrent chromatography for the isolation of sulforaphane from broccoli seed meal

In order to produce large amounts of pure sulforaphane for research purposes, a novel method using high-speed countercurrent chromatography (HSCCC) was developed. Without any initial cleanup steps, sulforaphane was successfully purified from the ethyl acetate extract of the broccoli seed meal by HSCCC. The separation was performed with two-phase solvent systems: n-hexane/ethyl acetate/methanol/water (1:5:1:5, v/v/v/v). From 850 mg of the ethyl acetate extract, 186 mg of sulforaphane was isolated with the solvent system. The purified compound was over 97% purity as determined by HPLC analysis, and the chemical structure was confirmed by MS and (1)H and (13)C NMR.     

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.     

Separation and purification of sulforaphane from broccoli seeds by solid phase extraction and preparative high-performance liquid chromatography

A novel, rapid, and economical method to isolate and purify natural sulforaphane from broccoli seeds is described. The procedure involves solvent extraction of autolyzed seed meal, followed by separation by solid phase extraction (SPE) and purification by preparative high-performance liquid chromatography (HPLC). The SPE method provides higher yield of sulforaphane from crude extracts compared to conventional liquid-liquid extraction. High purity and recovery of sulforaphane product can be obtained by preparative HPLC with a C 18 column and 30% methanol in water as the mobile phase. The purified compound was characterized by MS and (1)H and (13)C NMR. The techniques described here are useful tools in the preparative-scale isolation of sulforaphane in a fast, cost-effective, and waste-conscious manner.     

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.     

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.     

Epithiospecifier protein from broccoli (Brassica oleracea L. ssp. italica) inhibits formation of the anticancer agent sulforaphane

In some cruciferous plants, epithiospecifier protein (ESP) directs myrosinase (EC 3.2.3.1)-catalyzed hydrolysis of alkenyl glucosinolates toward epithionitrile formation. Here, for the first time, we show that ESP activity is negatively correlated with the extent of formation of the health-promoting phytochemical sulforaphane in broccoli (Brassica oleracea L. ssp. italica). A 43 kDa protein with ESP activity and sequence homology to the ESP of Arabidopsis thaliana was cloned from the broccoli cv. Packman and expressed in Escherichia coli. In a model system, the recombinant protein not only directed myrosinase-dependent metabolism of the alkenyl glucosinolate epi-progoitrin [(2S)-2-hydroxy-3-butenyl glucosinolate] toward formation of an epithionitrile but also directed myrosinase-dependent hydrolysis of the glucosinolate glucoraphanin [4-(methylsulfinyl)butyl glucosinolate] to form sulforaphane nitrile, in place of the isothiocyanate sulforaphane. The importance of this finding is that, whereas sulforaphane has been shown to have anticarcinogenic properties, sulforaphane nitrile has not. Genetic manipulation designed to attenuate or eliminate expression of ESP in broccoli could increase the fractional conversion of glucoraphanin to sulforaphane, enhancing potential health benefits.     

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.     

Thermal degradation of sulforaphane in aqueous solution.

Sulforaphane, a cancer chemopreventive agent identified from broccoli, was degraded in an aqueous solution at 50 and 100 degrees C. The reaction mixtures were extracted with methylene chloride and analyzed by gas chromatography (GC) and gas chromatography/mass spectrometry (GC/MS). Dimethyl disulfide, S-methyl methylthiosulfinate, S-methyl methylthiosulfonate, methyl (methylthio)methyl disulfide, 1,2,4-trithiolane, 4-isothiocyanato-1-(methylthio)-1-butene, and 3-butenyl isothiocyanate were identified as volatile decomposition products. After methylene chloride extraction, the aqueous layer was dried and silica gel column chromatography was used to separate and purify the nonvolatile decomposition products. The major thermal degradation compound was determined by (1)H NMR, (13)C NMR, and FAB-MS as N, N'-di(4-methylsulfinyl)butyl thiourea. A possible mechanism for the formation of these products is proposed.     

Presence of 2-furoylmethyl derivatives in hydrolysates of processed tomato products

Acid hydrolysis of Amadori compounds yields the corresponding 2-furoylmethylamino acids (2-FM-AA) that can be analyzed by ion-pair HPLC. The relative proportions of the different 2-FM-AA present in the hydrolysates of tomato products were determined to assess their usefulness as indicators of quality. In the lyophilized tomato samples stored at 50 degrees C and a(w) = 0.44 the formation of 2-FM derivatives of alanine, gamma-aminobutyric acid (GABA), asparagine, aspartic acid, glutamic acid, lysine, serine, and threonine was detected. In commercial tomato products the most abundant 2-FM-AA was 2-FM-GABA (from traces to 26.4 mg/100 g of dry matter) followed by 2-FM-lysine (furosine). Differences in 2-FM-AA contents among samples may be related to processing and storage conditions.     

Cancer-protective properties of high-selenium broccoli

Selenium (Se) from high-Se garlic reduces the incidence of chemically induced mammary tumors, and Se from high-Se broccoli reduces colon cancer. However, the ability of Se from high-Se broccoli to protect against mammary cancer has not been tested. Also, the sprout form of broccoli contains many secondary plant compounds that are known to reduce cancer risk, but the anticarcinogenic activity of broccoli sprouts has not been investigated. The present studies examined the ability of high-Se broccoli or high-Se broccoli sprouts to protect against chemically induced mammary or colon cancer. In one experiment, Sprague--Dawley rats that consumed diets containing 3.0 microg of Se/g supplied as high-Se broccoli had significantly fewer mammary tumors than rats fed 0.1 microg of Se as selenite with or without the addition of regular broccoli. In the second experiment, Fisher F-344 rats fed 2.0 microg of Se/g of diet supplied as either high-Se broccoli florets or high-Se broccoli sprouts had significantly fewer aberrant colon crypts than rats fed 0.1 or 2 microg of Se/g of diet supplied as selenite with or without the addition of low-Se broccoli. These data demonstrate that the cancer-protective effect of Se in high-Se broccoli extends to mammary cancer and the protective forms of broccoli against colon cancer include high-Se broccoli sprouts.     

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.     

Plant-derived biomolecules in fermented cabbage

The formation of plant-derived biomolecules during sauerkraut fermentation was studied. Cabbage was fermented with a starter culture, and the results were compared to the results of spontaneous fermentation. The concentration of flavonoids and glucosinolates was analyzed by HPLC, and that of the glucosinolate breakdown products, by GC-MS. Of the 20 different flavonoids tested, only kaempferol was found (0.9 mg/ kg FW, fresh weight). The content of kaempferol remained constant in the cabbage fiber matrix over the fermentation process. The nitrite concentration was below the detection limit in both fermentations. The total glucosinolate content in the raw material was 3.71 micro mol/g DW, dry weight. Glucosinolates were totally decomposed in both fermentations during two weeks, and different types of breakdown products were formed. Isothiocyanates, indole-3-carbinol, goitrin, allyl cyanide, and nitriles were determined in the fermented cabbage. Isothiocyanates and allyl cyanide were the predominant breakdown products in both fermentations. Sulforaphane nitrile and goitrin were found only in small quantities in the end products.     

西兰花叶中生物活性成分的测定

为探究西兰花叶中生物活性成分的具体含量,以西兰花叶为原材料,对西兰花叶中萝卜硫素、总酚、总黄酮、总蛋白质的含量和组成进行了测定与分析。结果表明,西兰花废弃叶中萝卜硫素含量为92.50 μg·g^-1 DW,总酚含量为3.15 mg·g^-1 DW,总黄酮含量为2.48 mg·g^-1 DW,总蛋白质含量为89.38 mg·g^-1 DW,蛋白质组成中核酮糖-1,5-二磷酸羧化酶/加氧酶(Rubisco)所占比例最大。本研究明确了西兰花叶片中4种生物活性成分的具体含量,为进一步开发利用西兰花废弃物提供了一定的数据支持。     

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.     

Formation of Amadori compounds in dehydrated fruits.

The presence of Amadori compounds in commercial dehydrated fruits has been shown through HPLC analysis of the corresponding 2-furoylmethyl-amino acids obtained by acid hydrolysis. Furosine (2-furoylmethyl-lysine) was the main 2-furoylmethyl derivative observed in dried figs and apricot samples, whereas in prunes and dates similar amounts of furosine and 2-furoylmethyl-gamma-aminobutyric acid were detected. A considerable variation of 2-furoylmethyl-amino acid contents among commercial raisin samples was observed. 2-Furoylmethyl-gamma-aminobutyric acid and 2-furoylmethyl-arginine, the most abundant 2-furoylmethyl-amino acids, ranged between 9.9 and 75.8 mg/100 g sample and 10.0 and 62.5 mg/100 g sample, respectively. Most of the Amadori compounds present in raisins seem to have originated during the commercial shelf life period rather than during processing. Determination of 2-furoylmethyl-amino acids could be used as a method of controlling commercial dehydrated fruit and selecting storage conditions.     

Kinetics of the stability of broccoli (Brassica oleracea Cv. Italica) myrosinase and isothiocyanates in broccoli juice during pressure/temperature treatments

The Brassicaceae plant family contains high concentrations of glucosinolates, which can be hydrolyzed by myrosinase yielding products having an anticarcinogenic activity. The pressure and temperature stabilities of endogenous broccoli myrosinase, as well as of the synthetic isothiocyanates sulforaphane and phenylethyl isothiocyanate, were studied in broccoli juice on a kinetic basis. At atmospheric pressure, kinetics of thermal (45-60 degrees C) myrosinase inactivation could be described by a consecutive step model. In contrast, only one phase of myrosinase inactivation was observed at elevated pressure (100-600 MPa) combined with temperatures from 10 up to 60 degrees C, indicating inactivation according to first-order kinetics. An antagonistic effect of pressure (up to 200 MPa) on thermal inactivation (50 degrees C and above) of myrosinase was observed indicating that pressure retarded the thermal inactivation. The kinetic parameters of myrosinase inactivation were described as inactivation rate constants (k values), activation energy (Ea values), and activation volume (Va values). On the basis of the kinetic data, a mathematical model describing the pressure and temperature dependence of myrosinase inactivation rate constants was constructed. The stability of isothiocyanates was studied at atmospheric pressure in the temperature range from 60 to 90 degrees C and at elevated pressures in the combined pressure-temperature range from 600 to 800 MPa and from 30 to 60 degrees C. It was found that isothiocyanates were relatively thermolabile and pressure stable. The kinetics of HP/T isothiocyanate degradation could be adequately described by a first-order kinetic model. The obtained kinetic information can be used for process evaluation and optimization to increase the health effect of Brassicaceae.