Shikha Saha

Isothiocyanate concentrations and interconversion of sulforaphane to erucin in human subjects after consumption of commercial frozen broccoli compared to fresh broccoli.

SCOPEnSulforaphane (a potent anticarcinogenic isothiocyanate derived from glucoraphanin) is widely considered responsible for the protective effects of broccoli consumption. Broccoli is typically purchased fresh or frozen and cooked before consumption. We compared the bioavailability and metabolism of sulforaphane from portions of lightly cooked fresh or frozen broccoli, and investigated the bioconversion of sulforaphane to erucin.nnnMETHODS AND RESULTSnEighteen healthy volunteers consumed broccoli soups produced from fresh or frozen broccoli florets that had been lightly cooked and sulforaphane thio-conjugates quantified in plasma and urine. Sulforaphane bioavailability was about tenfold higher for the soups made from fresh compared to frozen broccoli, and the reduction was shown to be due to destruction of myrosinase activity by the commercial blanching-freezing process. Sulforaphane appeared in plasma and urine in its free form and as several thio-conjugates forms. Erucin N-acetyl-cysteine conjugate was a significant urinary metabolite, and it was shown that human gut microflora can produce sulforaphane, erucin, and their nitriles from glucoraphanin.nnnCONCLUSIONnThe short period of blanching used to produce commercial frozen broccoli destroys myrosinase and substantially reduces sulforaphane bioavailability. Sulforaphane was converted to erucin and excreted in urine, and it was shown that human colonic flora were capable of this conversion.

Genetic regulation of glucoraphanin accumulation in Beneforté® broccoli

Diets rich in broccoli (Brassica oleracea var italica) have been associated with maintenance of cardiovascular health and reduction in risk of cancer. These health benefits have been attributed to glucoraphanin that specifically accumulates in broccoli. The development of broccoli with enhanced concentrations of glucoraphanin may deliver greater health benefits. Three high-glucoraphanin F1 broccoli hybrids were developed in independent programmes through genome introgression from the wild species Brassica villosa. Glucoraphanin and other metabolites were quantified in experimental field trials. Global SNP analyses quantified the differential extent of B. villosa introgression The high-glucoraphanin broccoli hybrids contained 2.5–3 times the glucoraphanin content of standard hybrids due to enhanced sulphate assimilation and modifications in sulphur partitioning between sulphur-containing metabolites. All of the high-glucoraphanin hybrids possessed an introgressed B. villosa segment which contained a B. villosa Myb28 allele. Myb28 expression was increased in all of the high-glucoraphanin hybrids. Two high-glucoraphanin hybrids have been commercialised as Beneforté® broccoli. The study illustrates the translation of research on glucosinolate genetics from Arabidopsis to broccoli, the use of wild Brassica species to develop cultivars with potential consumer benefits, and the development of cultivars with contrasting concentrations of glucoraphanin for use in blinded human intervention studies.

Diurnal and light regulation of sulphur assimilation and glucosinolate biosynthesis in Arabidopsis

Glucosinolates are a major class of sulphur-containing secondary metabolites involved in plant defence against pathogens. Recently many regulatory links between glucosinolate biosynthesis and sulphate assimilation were established. Since sulphate assimilation undergoes diurnal rhythm and is light regulated, this study analysed whether the same is true for glucosinolate biosynthesis. The levels of glucosinolates and glutathione were found to be higher during the day than during the night. This agreed with variation in sulphate uptake as well as activity of the key enzyme of the sulphate assimilation pathway, adenosine 5’-phosphosulphate reductase. Correspondingly, the flux through sulphate assimilation was higher during the day than during the night, with the maximum flux through primary assimilation preceding maximal incorporation into glucosinolates. Prolonged darkness resulted in a strong reduction in glucosinolate content. Re-illumination of such dark-adapted plants induced accumulation of mRNA for many genes of glucosinolate biosynthesis, leading to increased glucosinolate biosynthesis. The light regulation of the glucosinolate synthesis genes as well as many genes of primary sulphate assimilation was controlled at least partly by the LONG HYPOCOTYL5 (HY5) transcription regulator. Thus, glucosinolate biosynthesis is highly co-regulated with sulphate assimilation.

Effects of fou8/fry1 Mutation on Sulfur Metabolism: Is Decreased Internal Sulfate the Trigger of Sulfate Starvation Response?

The fou8 loss of function allele of adenosine bisphosphate phosphatase FIERY1 results in numerous phenotypes including the increased enzymatic oxygenation of fatty acids and increased jasmonate synthesis. Here we show that the mutation causes also profound alterations of sulfur metabolism. The fou8 mutants possess lower levels of sulfated secondary compounds, glucosinolates, and accumulate the desulfo-precursors similar to previously described mutants in adenosine 5′phosphosulfate kinase. Transcript levels of genes involved in sulfate assimilation differ in fou8 compared to wild type Col-0 plants and are similar to plants subjected to sulfate deficiency. Indeed, independent microarray analyses of various alleles of mutants in FIERY1 showed similar patterns of gene expression as in sulfate deficient plants. This was not caused by alterations in signalling, as the fou8 mutants contained significantly lower levels of sulfate and glutathione and, consequently, of total elemental sulfur. Analysis of mutants with altered levels of sulfate and glutathione confirmed the correlation of sulfate deficiency-like gene expression pattern with low internal sulfate but not low glutathione. The changes in sulfur metabolism in fou8 correlated with massive increases in 3′-phosphoadenosine 5′-phosphate levels. The analysis of fou8 thus revealed that sulfate starvation response is triggered by a decrease in internal sulfate as opposed to external sulfate availability and that the presence of desulfo-glucosinolates does not induce the glucosinolate synthesis network. However, as well as resolving these important questions on the regulation of sulfate assimilation in plants, fou8 has also opened an array of new questions on the links between jasmonate synthesis and sulfur metabolism.

Pro-oxidant effect of α-tocopherol in patients with Type 2 Diabetes after an oral glucose tolerance test – a randomised controlled trial

BackgroundAs a part of a larger study investigating the effects of α-tocopherol on gene expression in type 2 diabetics we observed a pro-oxidant effect of α-tocopherol which we believe may be useful in interpreting outcomes of large intervention trials of α-tocopherol.Methods19 type 2 diabetes subjects were randomised into two groups taking either 1200 IU/day of α-tocopherol or a matched placebo for 4 weeks. On day 0 and 29 of this study oxidative DNA damage was assessed in mononuclear cells from fasted blood samples and following a 2 h glucose tolerance test (GTT).ResultsOn day 0 there was no significant difference in oxidative DNA damage between the two groups or following a GTT. On day 29 there was no significant difference in oxidative DNA damage in fasted blood samples, however following a GTT there was a significant increase in oxidative DNA damage in the α-tocopherol treatment group.ConclusionHigh dose supplementation with α-tocopherol primes mononuclear cells from patients with type 2 diabetes for a potentially damaging response to acute hyperglycaemia.

Human O-sulfated metabolites of (−)-epicatechin and methyl-(−)-epicatechin are poor substrates for commercial aryl-sulfatases: Implications for studies concerned with quantifying epicatechin bioavailability

Epicatechin is a widely consumed dietary flavonoid and there is substantial evidence that it contributes to the health benefits reported for flavanol-rich cocoa products including dark chocolate. Numerous reports have described the appearance of epicatechin and epicatechin phase-2 conjugates (sulfates and glucuronides of epicatechin and methylepicatechin) in blood and urine samples of subjects following ingestion of epicatechin. The most widely reported method of quantifying total epicatechin in plasma and urine samples involves hydrolysis with a mixture of β-glucuronidase and sulfatase to convert the conjugates to epicatechin aglycone which is subsequently quantified. We observed a lack of hydrolysis of epicatechin sulfates and methylepicatechin sulfates using commercial sulfatases and investigated this further. Samples of urine or plasma from subjects who had consumed epicatechin were subjected to enzyme hydrolysis and then analysed using LC-MS/MS, or analysed without enzyme hydrolysis. Attempts to increase the extent of hydrolysis of epicatechin conjugates were made by increasing the amount of enzyme, hydrolysis pH and length of incubations, and using alternative sources of enzyme. The standard hydrolysis conditions failed to hydrolyse the majority of epicatechin sulfates and methylepicatechin sulfates. Even when the quantity of enzyme and incubation period was increased, the pH optimised, or alternative sources of sulfatases were used, epicatechin monosulfates and methylepicatechin monosulfates remained as major peaks in the chromatograms of the samples. An assessment of literature data strongly suggested that the majority of reports where enzyme hydrolysis was used had significantly underestimated epicatechin bioavailability in humans. Methods for quantifying epicatechin concentrations in blood and urine need to take account of the lack of hydrolysis of (methyl)epicatechin-sulfates, for example by quantifying these directly using LC-MS/MS.

Flavan-3-ol-enriched dark chocolate and white chocolate improve acute measures of platelet function in a gender-specific way—a randomized-controlled human intervention trial

SCOPEnWe examined whether flavan-3-ol-enriched dark chocolate, compared with standard dark and white chocolate, beneficially affects platelet function in healthy subjects, and whether this relates to flavan-3-ol bioavailability.nnnMETHODS AND RESULTSnA total of 42 healthy subjects received an acute dose of flavan-3-ol-enriched dark, standard dark or white chocolate, in random order. Blood and urine samples were obtained just before and 2 and 6 h after consumption for measurements of platelet function, and bioavailability and excretion of flavan-3-ols. Flavan-3-ol-enriched dark chocolate significantly decreased adenosine diphosphate-induced platelet aggregation and P-selectin expression in men (all p ≤ 0.020), decreased thrombin receptor-activating peptide-induced platelet aggregation and increased thrombin receptor-activating peptide-induced fibrinogen binding in women (both p ≤ 0.041), and increased collagen/epinephrine-induced ex vivo bleeding time in men and women (p ≤ 0.042). White chocolate significantly decreased adenosine diphosphate-induced platelet P-selectin expression (p = 0.002) and increased collagen/epinephrine-induced ex vivo bleeding time (p = 0.042) in men only. Differences in efficacy by which flavan-3-ols affect platelet function were only partially explained by concentrations of flavan-3-ols and their metabolites in plasma or urine.nnnCONCLUSIONnFlavan-3-ols in dark chocolate, but also compounds in white chocolate, can improve platelet function, dependent on gender, and may thus beneficially affect atherogenesis.

In vivo biological activity of rocket extracts (Eruca vesicaria subsp. sativa (Miller) Thell) and sulforaphane

Eruca is thought to be an excellent source of antioxidants like phenolic compounds, carotenoids, glucosinolates and their degradation products, such as isothiocyanates. Sulforaphane is one of the most potent indirect antioxidants of Eruca isolated until the date. In this work we investigate: (i) the safety and DNA protective activity of Eruca extracts and sulforaphane (under and without oxidative stress) in Drosophila melanogaster; and (ii) the influence on D. melanogaster life span treated with Eruca extracts and sulforaphane. Our results showed that among the four concentrations of Eruca extracts tested (from 0.625 to 5mg/ml), intermediate concentrations of the Es2 accession (1.25 and 2.5mg/ml) exhibited no genotoxic activity, as well as antigenotoxic activity (inhibition rate of 0.2-0.6) and the lowest concentration of Es2 and Es4 accessions (0.625 mg/ml) also enhanced the health span portion of the live span curves. Sulforaphane presented a high antigenotoxic activity in the SMART test of D. melanogaster and intermediate concentrations of this compound (3.75 μM) enhanced average healthspan. The results of this study indicate the presence of potent antigenotoxic factors in rocket, which are being explored further for their mechanism of action.

Consumption of both low and high (-)-epicatechin apple puree attenuates platelet reactivity and increases plasma concentrations of nitric oxide metabolites: A randomized controlled trial

We hypothesised that consumption of flavanol-containing apple puree would modulate platelet activity and increase nitric oxide metabolite status, and that high flavanol apple puree would exert a greater effect than low flavanol apple puree. 25 subjects consumed 230 g of apple puree containing 25 and 100mg epicatechin (low and high flavanol apple puree, respectively) and aspirin (75 mg) in random order. Measurements were made at baseline, acutely after treatment (2, 6 and 24 h), and after 14 d of treatment. Low flavanol apple puree significantly attenuated ADP and epinephrine-induced integrin-β3 expression 2 h and 6 h after consumption and ADP and epinephrine-induced P-selectin expression within 2h of consumption. High flavanol apple puree attenuated epinephrine and ADP-induced integrin-β3 expression after 2 and 6h. ADP and epinephrine-induced integrin-β3 expression was significantly attenuated 2, 6 and 24 h after consumption of aspirin, whilst 14 d aspirin consumption attenuated collagen-induced P-selectin expression only. The plasma total nitric oxide metabolite conc. was significantly increased 6h after consumption of both low and high flavanol apple purees. In conclusion, consumption of apple purees containing ⩾25 or 100 mg flavanols transiently attenuated ex vivo integrin-β3 and P-selectin expression and increased plasma nitric oxide metabolite conc. in healthy subjects, but the effect was not enhanced for the high flavanol apple puree.

An approach to the phytochemical profiling of rocket [Eruca sativa (Mill.) Thell].

BACKGROUNDnEruca sativa (rocket) contains a wide range of compounds with nutraceutical and organoleptical properties. This research aimed to characterise the nutraceutical interest of four rocket accessions by analysis of glucosinolates, isothiocyanates, phenolics, carotenoids and carbohydrates. Different methods based on chromatographic separation with ultraviolet absorbance or mass spectrometry detection were used.nnnRESULTSnThe total content of glucosinolates ranged from 14.02 to 28.24 µmol g(-1) of dry weight. Glucoraphanin represented up to 52% of the total glucosinolates in leaves of one accession. Accessions showed differences in the hydrolysis of glucoraphanin to the isothiocyanate sulforaphane. No correlation between these compounds was observed, which insisted differences in the myrosinase activity within accessions. Rocket leaves had variable phenolic profiles represented by quercetin-3-glucoside, rutin, myricetin, quercetin and ferulic and p-coumaric acids. A high variability was observed for the total carotenoids ranged from 16.2 to 275 µg g(-1) with lutein as the main carotenoid. Glucose was the predominant sugar, representing >70% of the total soluble carbohydrates.nnnCONCLUSIONSnSome accessions could be candidates for future breeding programmes because of their pattern of beneficial compounds for human health. However, further research is essential to evaluate the biological activity of these accessions before designing functional food.