No de novo sulforaphane biosynthesis in broccoli seedlings

The isothiocyanate sulforaphane, present in significant amounts in broccoli (Brassica oleracea L.) seedlings in the form of its precursor glucoraphanin, has been identified as an inducer of quinine reductase, a phase-II detoxification enzyme known for its anticarcinogenic properties. Its concentration in broccoli seedlings usually decreases during the first 7–14 days after germination. No conclusive data on sulforaphane metabolism in seedlings are available in the literature. Here, we unambiguously demonstrate in ¹²C/¹³C-cross experiments that sulforaphane is not biosynthesised de novo during the first week of seedling development. Both ¹²C (99 atom% ¹²C) and ¹³C (98 atom% ¹³C) broccoli seeds were produced and subsequently germinated and grown either in a ¹³CO₂ or a ¹²CO₂ environment. Afterwards, the labelling degree of sulforaphane in seeds and in seedlings was analysed by HPLC–MS. We conclude that sulforaphane exclusively originates from seed reserves and that de novo biosynthesis is not detectable (<1%) in broccoli seedlings.     

Induction of Detoxification Enzymes by Feeding Unblanched Brussels Sprouts Containing Active Myrosinase to Mice for 2 Wk

Abstract: In cruciferous vegetables, myrosinase metabolizes the relatively inactive glucosinolates into isothiocyanates and other products that have the ability to increase detoxification enzyme expression. Thus, maintaining myrosinase activity during food preparation may be critical to receiving the maximum benefit of consumption of Brussels sprouts or other cruciferous vegetables. To test the importance of maintaining myrosinase activity for maximizing bioactivity, experimental diets containing 20% unblanched (active myrosinase) or 20% blanched (inactivated myrosinase) freeze-dried Brussels sprouts and a nutrient-matched control diet were evaluated in vitro and in vivo for their ability to induce detoxification enzymes. Treatment of immortalized HepG2 human hepatoma cells with the unblanched Brussels sprout diet caused a greater increase quinone activity compared to the blanched Brussels sprout diet. C3H/HeJ mice fed the unblanched Brussels sprout diets for 2 wk had significantly higher plasma sulforaphane concentrations. Liver expression of CYP1A1 and epoxide hydrolase, measured using real-time PCR, was correlated with the plasma concentration of sulforaphane. In the lung, expression of epoxide hydrolase, thioredoxin reductase, UDP glucuronosyltransferase, quinone reductase, heme oxygenase, CYP1A1, CYP1A2, and CYP1B1 were also correlated with the plasma concentration of sulforaphane. Together these data demonstrate that, as predicted by the in vitro experiment, in vivo exposure to Brussels sprouts with active myrosinase resulted in greater induction of both phase I and phase II detoxification enzymes in the liver and the lungs that correlated with plasma sulforaphane concentrations.     

Extraction and on-line concentration of flavonoids in Brassica oleracea by capillary electrophoresis using large volume sample stacking

Flavonoids are bioactive compounds found in plants. Studies indicate consumption of food containing these compounds may reduce the incidences of cancer and cardiovascular diseases. In broccoli, the flavonoids are present at variable concentrations and so far have mainly been determined using high performance liquid chromatography (HPLC). This paper describes a rapid capillary electrophoresis method, involving large volume sample stacking (LVSS), suitable for the analysis of flavonoids in broccoli. Following acid hydrolysis, the two key flavonoids (kaempferol and quercetin) in a broccoli extract were concentrated on-line by LVSS prior to separation by capillary zone electrophoresis (CZE). Using an optimised method, the extract was injected for 50 s into a 50 μm (internal diameter) × 85 cm (total length) capillary followed by stacking/matrix removal at −5 kV for 83 s. The two analytes were then separated in less than 8 min by CZE using a 10 mM sodium borate buffer (pH 8.40) and a separation voltage of +30 kV at 30 °C. A linear relationship in the range 1–20 ppm was observed for the method (r² = 0.9991–0.9995) with detection limits of 0.9 and 0.6 mg/kg of broccoli for kaempferol and quercetin, respectively. This method demonstrated good repeatability for the standard and extract with relative standard deviations of less than 5% for both peak area and migration time measured over five different days (n = 5). The method was successfully applied to quantitatively determine kaempferol and quercetin contents in a commercial broccoli sample as 11.8 and 14.6 mg/kg fresh weight, respectively. This result was validated by HPLC analysis and is within the ranges reported in the literature.     

Retention of Phytochemicals in Fresh and Processed Broccoli

Our objective was to determine whether steam blanching, storage and preparation affected concentrations of sulforaphane (SF), sulforaphane nitrile (SFN), cyanohydroxybutene (CHB), iberin (I) or iberin nitrile (IN) in fresh and frozen broccoli. Broccoli (var. “Arcadia”) was grown in St. Charles, IL over three seasons. Samples were steam blanched (2 min at 93 ± 5°C) within 24h of harvest, frozen and stored at ?20°C up to 90 days, and fresh broccoli was stored at 4°C up to 21 days. Samples were analyzed uncooked or microwave cooked. SF, SFN, I, IN and CHB were determined by GC in dichloromethane extracts from lyophilized samples. Rates of loss for CHB and SF were similar during storage of fresh broccoli. Blanching, storage, and microwave cooking decreased (p < 0.01) concentrations of each compound in fresh and frozen broccoli.     

Time dependence of bioactive compounds and antioxidant capacity during germination of different cultivars of broccoli and radish seeds

Optimisation of the germination process of different cultivars of broccoli (Brassica oleracea var. italica cv. Lucky, cv. Tiburon and cv. Belstar) and radish (Raphanus sativus cv. Rebel and cv. Bolide) seeds in relation to the content of glucosinolates (GLS), vitamin C and total antioxidant capacity was carried out in order to maximise the health-promoting properties of Brassica sprouts. The content of total and individual GLS varied between species, among cultivars, and germination time. Glucoraphanin in broccoli and glucoraphenin in radish were the predominant GLS in raw seeds (61–77 and 63–129 μmol/g DM, respectively) and, although their content decreased during germination, they were maintained in rather large proportions in sprouts. Vitamin C was not detected in raw seeds and its content increased sharply in broccoli and radish sprouts (162–350 and 84–113 mg/100 g DM, respectively). Raw brassica seeds are an excellent source of antioxidant capacity (64–90 and 103–162 μmol Trolox/g DM in broccoli and radish, respectively) and germination led to a sharp increase. Germination of broccoli cv. Belstar and radish cv. Rebel for 4 days provided the largest glucoraphanin and glucoraphenin content, respectively, and also brought about large amounts of vitamin C and antioxidant capacity.     

Development of a Novel Functional Soup Rich in Bioactive Sulforaphane Using Broccoli (Brassica oleracea L. ssp. italica) Florets and Byproducts

Broccoli florets are rich in health-promoting compounds such as glucoraphanin, the precursor of the bioactive compound sulforaphane. In addition, broccoli byproducts such as stalk also contain health-promoting compounds and represent attractive ingredients in the development of functional foods. The bioconversion of glucosinolates into bioactive isothiocyanates is affected by many factors including heat and therefore cooking of Brassica such as broccoli may result in significant loss of sulforaphane production. The aim of this study was to develop a suitable food system as a vehicle for the delivery of sulforaphane in the human diet in adequate quantities. To this end, the feasibility of dry-mix ready soup as a food matrix for the delivery of broccoli floret and byproducts was evaluated. In particular, this study investigated the bioconversion of glucosinolates into bioactive isothiocyanates during the cooking process of this novel food product by microwave heating. In addition to total isothiocyanate and sulforaphane content, other key physical and biochemical quality attributes of the broccoli floret- and byproduct-enriched soups were investigated. Total isothiocyanate and sulforaphane content in floret- and stalk-enriched soups was high in both cases and increased in the order stalk<floret. The overall acceptability of stalk containing soups was not significantly different compared with the control soups, whereas floret containing soups received significantly lower acceptability scores. These results suggest that ready soups prepared by microwave heating represent a feasible food product for the delivery of broccoli florets and byproducts which is compatible with the bioconversion of glucosinolates into bioactive isothiocyanates.     

Identification and analysis of isothiocyanates and new acylated anthocyanins in the juice of Raphanus sativus cv. Sango sprouts

The freeze-dried sprouts’ juice of Raphanus sativus (L.) cv. Sango was prepared and analysed for the first time. HPLC analysis of total isothiocyanates, after protein displacement, resulted in 77.8 ± 3.0 μmol/g of dry juice while GC–MS analysis of hexane and acetone extracts showed E- and Z-raphasatin (8.9 and 0.11 μmol/g, respectively) and sulforaphene (11.7 μmol/g), summing up to 20.7 ± 1.7 μmol/g of free isothiocyanates. Sprouts’ juice contained an unprecedented wealth of anthocyanins and a new fractionation methodology allowed us to isolate 34 mg/g of acylated anthocyanins (28.3 ± 1.9 μmol/g), belonging selectively to the cyanidin family. Analysis was performed by HPLC–PDA–ESI–MSⁿ and extended to deacylated anthocyanins and aglycones, obtained, respectively, by alkaline and acid hydrolysis. This study identified 70 anthocyanins, 19 of which have never been described before and 32 of which are reported here in R. sativus for the first time. Sango radish sprouts are exceptional dietary sources of heath-promoting micronutrients.     

Detection of alkaline phosphatase by competitive indirect ELISA using immunoglobulin in yolk (IgY) specific against bovine milk alkaline phosphatase

Immunoglobulin in yolk (IgY) (with a titer of 1.3 × 10⁶) specific against bovine milk (BM) alkaline phosphatase (ALP) was obtained by intramuscularly immunizing hens on the thigh and was used as the primary antibody to conduct competitive indirect enzyme-linked immunosorbent assay (CI-ELISA) to determine BMALP in ALPs from BM and Escherichia coli sources. A relationship between the ELISA value and the BMALP level (0.01–10 μg/mL) in whole milk (R² = 0.9019) or in skimmed milk (R² = 0.9402) was observed. The maximal inhibition (%) of BMALP on the microtiter plate by free BMALP at 10 μg/mL whole milk (3.89 mU/μg BMALP) was about 50%, while no inhibition (%) of BMALP by free E. coli ALP at concentrations between 0.01 to 10 μg/mL (60 mU/μg E. coli ALP) was determined. At BMALP levels higher than 0.1 μg/mL, CI-ELISA was proved to be effective in differentiating between BMALP and E. coli ALP and quantifying BMALP in whole milk or skimmed milk in the presence of E. coli ALP with an activity of 0.6 U/mL. Higher inhibition (about 70%) of BMALP on the microtiter plate by free BMALP in diluted (10¹–10⁴ fold) milk samples was observed. The optimal conditions for CI-ELISA in determining BMALP (0.1–10 μg/mL) from ALPs in milk samples were using 10³-fold diluted crude IgY specific against BMALP as primary antibody and 10³-fold diluted goat anti-chicken IgG–ALP conjugate as the secondary antibody.     

Improving the phytochemical composition of broccoli sprouts by elicitation

In order to increase the concentrations of bioactive compounds in broccoli sprouts, exogenous elicitors: methionine, tryptophan, chitosan, salicylic acid (SA) and methyl jasmonate (MeJA) were sprayed at different concentrations during germination. The effects of the elicitors on vitamin C, phenolic compounds and glucosinolate levels were assessed in 3, 5 and 7-days old seedlings. The application of 200, 300 μM SA and 0.01% chitosan induced increases, by 26%, 18% and 54%, respectively in the content of vitamin C in 5 days old broccoli sprouts. Flavonoid concentration was also increased by 31% and 33% after 10 μM MeJA and 100 μM SA treatments, respectively in 7 days old broccoli sprouts. In contrast, methionine or tryptophan solutions did not positively affect the vitamin C or the phenolic compounds in the sprouts. The individual classes of glucosinolates respond differently to the elicitor treatment. Methionine, a sulphur-containing amino acid, applied at 5 mM, significantly increased, by 32%, the levels of aliphatic glucosinolates. However, indole glucosinolates presented a significant response to the induction with tryptophan, SA or MeJA treatments. Tryptophan at 10 mM concentration augmented by 1.8-fold the indole glucosinolate concentration in 7 days old broccoli sprouts, and the treatments of 100 μM SA and 25 μM MeJA also induced significantly higher amounts of indole glucosinolates (by 33% and 51%, respectively) in 7 days old treated broccoli sprouts. Therefore, the application of elicitors could be a useful tool for improving the bioactive metabolites in broccoli sprouts for fresh consumption or functional ingredients for nutraceuticals foods.     

Cancer preventive effect of Thai rat-tailed radish (Raphanus sativus L. var. caudatus Alef)

The cancer preventive effect of Raphanus sativus L. var. caudatus Alef (Thai rat-tailed radish) extract was evaluated against the HCT116 colon cancer cell line. HPLC and GC–MS were used to identify the phytoconstituents in Thai rat-tailed radish extract. Thai rat-tailed radish extract showed high cytotoxicity against HCT116 with an IC₅₀ at 9.42 ± 0.46 μg/ml. Apoptosis induced by Thai rat-tailed radish extract was confirmed (a) by DAPI staining which demonstrated nuclei morphological changes, chromatin condensation and nuclear fragmentation and (b) by Annexin V/PI flow cytometry. GC–MS analysis of Thai rat-tailed radish extract revealed two isothiocyanates; sulforaphane and sulforaphene, whose cytotoxicity and apoptosis induction effect were evident, giving Thai rat-tailed radish extract its anticancer attributes. This is the first report on the cancer preventive effect of Thai rat-tailed radish extract against HCT116 colon cancer cell line, through apoptosis induction.     

Occurrence of selenoprotein enzyme activities and mRNA in bovine mammary tissue

To elucidate the possible role of selenoproteins for milk formation and mammary gland physiology, the activities of selenoprotein enzymes and the expression of selenoprotein genes were studied in the bovine mammary gland. Messenger RNA was demonstrated for selenoprotein P, thioredoxin reductase 1, and for glutathione peroxidase (GPx) 1, 3, and 4. Significant differences in mRNA expression between the cows were seen for GPx 1 and GPx 3. The enzyme activity of glutathione peroxidase varied approximately 16-fold among cows, and the activity of thioredoxin reductase and the concentration of soluble Se varied approximately 6-fold among cows. There were positive correlations between glutathione peroxidase activity, thioredoxin reductase activity, and soluble Se, the correlation between glutathione peroxidase activity and soluble Se being the strongest. Furthermore, selenoprotein P expression correlated with GPx 1 mRNA expression and with soluble Se. There was also a correlation between glutathione peroxidase activity and the mRNA expression of GPx 1. The general conclusion from the data was that the activity of glutathione peroxidase and thioredoxin reductase and the mRNA expression of selenoprotein P and GPx 1 and 3 were influenced by Se status, but the expression of GPx 4 and thioredoxin reductase 1 were not. These results indicate that the Se status in mammary tissue is an important regulator of selenoprotein activity and expression, but that other factors are also in operation.     

HPLC screening of anti-cancer sulforaphane from important European Brassica species

Natural sulforaphane has been of increasing interest for nutraceutical and pharmaceutical industries due to its anti-cancer effect. The sulforaphane contents of 14 different major European Brassica oleracea L. varieties (cauliflower Alverda, cauliflower di Jesi, cauliflower Minaret, broccoli Ramoso calabrese, white cabbage, palm cabbage Cuor di Bue, broccoli Primor, savoy cabbage riccio d’Asti, broccoli Eolo, Delicato, cauliflower Nuvola, cauliflower Palla di neve, San martino, Velox) and four different Brassica rapa L. varieties (turnip Senza Testa, cima di rapa 40, cima di rapa 90, cima di rapa 120) which are cultivated in Italy, were analysed during two different developmental stages (3rd and 9th day after germination) of the seedlings. A quantitative determination of sulforaphane in Brassica species seedlings was established by HPLC. The highest amount of sulforaphane (2.21 mg/g d w) was found in San martino 3-day old seedlings. Seedlings of this European Brassica variety may be considered as a potential new source of natural sulforaphane.     

Accumulation and species distribution of selenium in Se-enriched bacterial cells of the Bifidobacterium animalis 01

Bifidobacterium animalis 01 (B. animalis 01) could absorb 16.7–39.6% of inorganic selenium in the medium and transform most of it into organic selenium. Most of the organic selenium (50.7–63.0%) was found in the protein fraction, 9.62–18.7% in the polysaccharide fraction, 0.273–0.754% in the nucleic acid fraction, and 20.8–30.9% in other components. Furthermore, the selenium content of different protein extractions was in the following order: Alkaline-soluble protein-bound Se (46.5–53.4%) > Water-soluble protein-bound Se (27.4–30.8%) > Salt-soluble protein-bound Se (7.79–11.9%) > Alcohol-soluble protein-bound Se (not detected). Additionally, the molecular mass of most proteins or protein subunits containing selenium was about 10–20 kDa. Analysis by LC–MS showed that selenomethionine (SeMet) is the major selenocompound in protein.     

Limited contribution of isoflavones to hepatocellular phase II enzyme-inducing activity of soybean (Glycine max) extracts

Soy flour was successively extracted with ethyl acetate, ethanol, and water and tested for quinone reductase (QR) induction in the Hepa 1c1c7 cell line. The ethanol extract was found to be the most potent, with a CD value (concentration required to double QR specific activity) of 460 μg/ml. Phase separation of the ethanol extract and a phospholipid removal procedure yielded a fraction that doubled QR at 80 μg/ml. Reverse phase medium pressure chromatography of the purified ethanol extract yielded 13 fractions. Constituents of selected fractions were identified using mass spectrometry and NMR analysis. Fractions 2, 8, and 10 contained 47% of the recovered mass and were the least active of the isolates. These three fractions contained isoflavone glycosides. The latest eluting fractions 11–13 were found to be the most potent, and contained the majority of recovered inducing units (IUs), a measure of potency.     

Syzygium aqueum leaf extract and its bioactive compounds enhances pre-adipocyte differentiation and 2-NBDG uptake in 3T3-L1 cells

The insulin-like and/or insulin-sensitising effects of Syzygium aqueum leaf extract and its six bioactive compounds; 4-hydroxybenzaldehyde, myricetin-3-O-rhamnoside, europetin-3-O-rhamnoside, phloretin, myrigalone-G and myrigalone-B were investigated in 3T3-L1 adipocytes. We observed that, S. aqueum leaf extract (0.04–5 μg/ml) and its six bioactive compounds (0.08–10 μM) at non-cytotoxic concentrations were effectively enhance adipogenesis, stimulate glucose uptake and increase adiponectin secretion in 3T3-L1 adipocytes. Clearly, the compounds myricetin-3-O-rhamnoside and europetin-3-O-rhamnoside showed insulin-like and insulin-sensitising effects on adipocytes from a concentration of 0.08 μM. These compounds were far better than rosiglitazone and the other isolated compounds in enhancing adipogenesis, stimulating 2-NBDG uptake and increasing adiponectin secretion at all the concentrations tested. These suggest the antidiabetic potential of S. aqueum leaf extract and its six bioactive compounds. However, further molecular interaction studies to explain the mechanisms of action are highly warranted.     

Mate (Ilex paraguariensis St. Hilaire) saponins induce caspase-3-dependent apoptosis in human colon cancer cells in vitro

Saponins are naturally occurring metabolites associated with several health benefits. The objective was to quantify and purify saponins from mate dry leaves, and to assess their anti inflammatory and apoptotic mechanisms in human colon cancer cells in vitro. Matesaponins were extracted with methanol from dry leaves, partially purified and quantified. Leaves contained 10–15 mg/g dry weight total saponins, predominantly matesaponins 1 and 2. HPLC and LC/ESI-MS-MS identified saponins in six preparative chromatographic fractions (A, B, C, D, E, and F). Major matesaponins were identified as 1 [M–H]⁻ = 911 and 2 [M–H]⁻ = 1057, with trace amounts of 3 [M–H]⁻ = 1073, 4 [M–H]⁻ = 1219, and 5 [M–H]⁻ = 1383. Fractions D, E, and F significantly inhibited iNOS (IC₃₅ = 36.3, 29.5, 43.7 μM), PGE₂ (IC₃₅ = 23.1, 22.3, 11.7 μM) and COX-2 (IC₃₅ = 45.7, 32.4, 17.0 μM). Fraction F reduced nuclear translocation of nuclear factor-κB subunits p50 (49.8%) and p65 (49.0%) and induced apoptosis through suppression of Bcl-2 and increased Bax protein expressions and activated caspase-3 activity. Saponins in leaves of mate prevent inflammation and colon cancer in vitro.     

Inhibition of cooked food-induced mutagenesis by dietary constituents: Comparison of two natural isothiocyanates

Sulforaphane(1-isothiocyanato-(4R)-(methylsulphinyl)butane), a major constituent of broccoli (Brassica oleracea, var. italica) and a structurally related natural aliphatic isothiocyanate, sulforaphen (4-isothiocyanato-(1R)-(methylsulphinyl)-1-(E)-butene), found in radish (Raphanus sativus L., Cruciferae) were investigated for their antimutagenic potential against different classes of cooked food mutagens (heterocyclic amines) in the Ames assay using Salmonella typhimurium TA98 and TA100 strains in the presence of Aroclor 1254-induced rat liver S9. Results of the in vitro antimutagenicity studies in the TA100 strain strongly suggest that both isothiocyanates were potent inhibitors of the mutagenicity induced by all the tested mutagens. Sulforaphen, possessing unsaturation in the alkyl chain of its structure, was, however, found to be 1.3–1.5 times more active than sulforaphane. These studies strongly warrant further investigations of sulforaphen for its potential as a chemopreventive agent.     

Different postharvest strategies to preserve broccoli quality during storage and shelf life: Controlled atmosphere and 1-MCP

Broccoli (Brassica oleracea var. italica) is a vegetable that requires the application of postharvest techniques to extend its marketability. Controlled atmosphere and 1-MCP treatments are most used to extend the shelf life of broccoli and reduce post-harvest deterioration. The aim of this study was to evaluate the visual, physicochemical and functional changes of broccoli head samples stored at 1–2 °C and 85–90% relative humidity (RH) in air (Control samples), under controlled atmospheres (10% O₂ and 5% CO₂) (CA samples) and treated with 1-MCP (0.6 μL/L). After storage all samples were maintained at 20 °C for 2 and 4 days, in order to assess their shelf life. The most suitable postharvest treatment to extend broccoli quality during storage and shelf life, in terms of maintaining the visual quality and reducing loss of health-promoting compounds, was achieved by storage under controlled atmosphere conditions. The use of 1-MCP reduced the loss of green colour and chlorophyll pigments, but only during cold storage not during shelf life at 20 °C.