Preparation of Poly(glycidyl methacrylate) (PGMA) and Amine Modified PGMA Adsorbents for Purification of Glucosinolates from Cruciferous Plants.

Glucosinolates (GLs) are of great interest for their potential as antioxidant and anticancer compounds. In this study, macroporous crosslinked copolymer adsorbents of poly (glycidyl methacrylate) (PGMA) and its amine (ethylenediamine, diethylamine, triethylamine)-modified derivatives were prepared and used to purify the GLS glucoerucin in a crude extract obtained from a cruciferous plant. These four adsorbents were evaluated by comparing their adsorption/desorption and decolorization performance for the purification of glucoerucin from crude plant extracts. According to the results, the strongly basic triethylamine modified PGMA (PGMA-III) adsorbent showed the best adsorption and desorption capacity of glucoerucin, and its adsorption data was a good fit to the Freundlich isotherm model and pseudo-second-order kinetics; the PGMA adsorbent gave the optimum decolorization performance. Furthermore, dynamic adsorption/desorption experiments were carried out to optimize the purification process. Two glass columns were serially connected and respectively wet-packed with PGMA and PGMA-III adsorbents so that glucoerucin could be decolorized and isolated from crude extracts in one process. Compared with KCl solution, aqueous ammonia was a preferable desorption solvent for the purification of glucoerucin and overcame the challenges of desalination efficiency, residual methanol and high operation costs. The results showed that after desorption with 10% aqueous ammonia, the purity of isolated glucoerucin was 74.39% with a recovery of 80.63%; after decolorization with PGMA adsorbent, the appearance of glucoerucin was improved and the purity increased by 11.30%. The process of using serially connected glass columns, wet-packed with PGMA and PGMA-III, may provide a simple, low-cost, and efficient method for the purification of GLs from cruciferous plants.

Ferroelectric Polarization and Second Harmonic Generation in Supramolecular Cocrystals with Two Axes of Charge-Transfer.

Ferroelectricity in organic materials remains a subject of great interest, given its potential impact as lightweight information storage media. Here we report supramolecular charge-transfer cocrystals formed by electron acceptor and donor molecules that exhibit ferroelectric behavior along two distinct crystallographic axes. The solid-state superstructure of the cocrystals reveals that a 2:1 ratio of acceptor to donor molecules assemble into nearly orthogonal mixed stacks in which the molecules are positioned for charge-transfer in face-to-face and edge-to-face orientations, held together by an extended hydrogen-bonding network. Polarization hysteresis was observed along the face-to-face and edge-to-face axes at room temperature. The noncentrosymmetric nature of the cocrystals, required to observe ferroelectric behavior, is demonstrated using second harmonic generation measurements. This finding suggests the possibility of designing supramolecular arrays in which organic molecules support multidimensional information storage.

Anti-inflammatory and anti-apoptotic effects of (RS)-glucoraphanin bioactivated with myrosinase in murine sub-acute and acute MPTP-induced Parkinson's disease.

This study was focused on the possible neuroprotective role of (RS)-glucoraphanin, bioactivated with myrosinase enzyme (bioactive RS-GRA), in an experimental mouse model of Parkinson's disease (PD). RS-GRA is one of the most important glucosinolates, a thiosaccharidic compound found in Brassicaceae, notably in Tuscan black kale seeds. RS-GRA was extracted by one-step anion exchange chromatography, further purified by gel-filtration and analyzed by HPLC. Following, pure RS-GRA was characterized by (1)H and (13)C NMR spectrometry and the purity was assayed by HPLC analysis of the desulfo-derivative according to the ISO 9167-1 method. The obtained purity has been of 99%. To evaluate the possible pharmacological efficacy of bioactive RS-GRA (administrated at the dose of 10mg/kg, ip +5µl/mouse myrosinase enzyme), C57BL/6 mice were used in two different sets of experiment (in order to evaluate the neuroprotective effects in different phases of the disease), according to an acute (2 injections·40mg/kg MPTP) and a sub-acute (5 injections·20mg/kg MPTP) model of PD. Behavioural test, body weight changes measures and immunohistochemical localization of the main PD markers were performed and post-hoc analysis has shown as bioactive RS-GRA is able to reduce dopamine transporter degradation, tyrosine hydroxylase expression, IL-1ß release, as well as the triggering of neuronal apoptotic death pathway (data about Bax/Bcl-2 balance and dendrite spines loss) and the generation of radicalic species by oxidative stress (results focused on nitrotyrosine, Nrf2 and GFAP immunolocalization). These effects have been correlated with the release of neurotrophic factors, such as GAP-43, NGF and BDNF, that, probably, play a supporting role in the neuroprotective action of bioactive RS-GRA. Moreover, after PD-induction mice treated with bioactive RS-GRA are appeared more in health than animals that did not received the treatment both for phenotypic behaviour and for general condition (movement coordination, presence of tremors, nutrition). Overall, our results suggest that bioactive RS-GRA can protect neurons against the neurotoxicity involved in PD via an anti-apoptotic/anti-inflammatory action.

Multi-phase extraction of glycoraphanin from broccoli using aminium ionic liquid-based silica.

INTRODUCTION: Glucosinolates, a class of phytochemicals found in broccoli, have attracted recent interest due to the potential health benefits associated with their dietary intake. Glucoraphanin, the most common glucosinolate in broccoli can be converted to a known cancer chemopreventive agent. Multi-phase extraction in solid-phase extraction cartridges was developed to simultaneously extract and separate this compound. OBJECTIVE: Multi-phase extraction with functionalised ionic liquid-based silica as a sorbent was used to simultaneously extract and separate glucoraphanin from broccoli. METHODOLOGY: The sorbent and broccoli sample were packed into a single cartridge, and a fixed volume of water was then used to extract and remove the target compound from the sample to the sorbent over 15 repetitions. The sorbent was then washed with n-hexane to remove any interference and the target compound was eluted with water-1% acetic acid (vol.). RESULTS: Under the optimised condition, 0.038 mg/g of glucoraphanin was obtained by multi-phase extraction with 0.2 g of sorbent. CONCLUSION: The adsorption isotherm allowed investigation of the interactions between the sorbent and target compound and provided evidence for the accuracy of this method. The low deviation error, small amount of solvents required, highly selective separation and stability of the method justify further research.

Rapid profiling of intact glucosinolates in Arabidopsis leaves by UHPLC-QTOFMS using a charged surface hybrid column.

INTRODUCTION: The analysis of glucosinolates (GS) is traditionally performed by reverse-phase liquid chromatography coupled to ultraviolet detection after a time-consuming desulphation step, which is required for increased retention. Simpler and more efficient alternative methods that can shorten both sample preparation and analysis are much needed. OBJECTIVE: To evaluate the feasibility of using ultrahigh-pressure liquid chromatography coupled to quadrupole time-of-flight mass spectrometry (UHPLC-QTOFMS) for the rapid profiling of intact GS. METHODOLOGY: A simple and short extraction of GS from Arabidopsis thaliana leaves was developed. Four sub-2 µm reverse-phase columns were tested for the rapid separation of these polar compounds using formic acid as the chromatographic additive. High-resolution QTOFMS was used to detect and identify GS. RESULTS: A novel charged surface hybrid (CSH) column was found to provide excellent retention and separation of GS within a total running time of 11 min. Twenty-one GS could be identified based on their accurate mass as well as isotopic and fragmentation patterns. The method was applied to determine the changes in GS content that occur after herbivory in Arabidopsis. In addition, we evaluated its applicability to the profiling of other Brassicaceae species. CONCLUSION: The method developed can profile the full range of GS, including the most polar ones, in a shorter time than previous methods, and is highly compatible with mass spectrometric detection.

Bioavailability of Sulforaphane Following Ingestion of Glucoraphanin-Rich Broccoli Sprout and Seed Extracts with Active Myrosinase: A Pilot Study of the Effects of Proton Pump Inhibitor Administration.

We examined whether gastric acidity would affect the activity of myrosinase, co-delivered with glucoraphanin (GR), to convert GR to sulforaphane (SF). A broccoli seed and sprout extract (BSE) rich in GR and active myrosinase was delivered before and after participants began taking the anti-acid omeprazole, a potent proton pump inhibitor. Gastric acidity appears to attenuate GR bioavailability, as evidenced by more SF and its metabolites being excreted after participants started taking omeprazole. Enteric coating enhanced conversion of GR to SF, perhaps by sparing myrosinase from the acidity of the stomach. There were negligible effects of age, sex, ethnicity, BMI, vegetable consumption, and bowel movement frequency and quality. Greater body mass correlated with reduced conversion efficiency. Changes in the expression of 20 genes in peripheral blood mononuclear cells were evaluated as possible pharmacodynamic indicators. When grouped by their primary functions based on a priori knowledge, expression of genes associated with inflammation decreased non-significantly, and those genes associated with cytoprotection, detoxification and antioxidant functions increased significantly with bioavailability. Using principal components analysis, component loadings of the changes in gene expression confirmed these groupings in a sensitivity analysis.

Evaluation of the safety and bioactivity of purified and semi-purified glucoraphanin.

The anti-carcinogenic effects of broccoli have been attributed to sulforaphane, the hydrolysis product of glucoraphanin (GRP). Here we determined if purified GRP, in the absence of the plant-derived hydrolyzing enzyme myrosinase, could affect pulmonary and hepatic ethoxyresorufin O-deethylase (EROD) and/or NAD(P)H-quinone oxidoreductase 1 (NQO1) activity. Male F344 rats were administered semi-synthetic, semi-purified or purified GRP (240 mg/kg: 550 micromol/kg rat daily for 4 days) by gavage. Hepatic and pulmonary NQO1 activity increased ( approximately 20%), but not EROD. Varying doses of semi-purified GRP (30, 60, or 120 mg/kg rat daily for 4 days) again caused no change in EROD activity, although a dose-dependent increase in NQO1 was seen. Urinary excretion of mercapturic acids showed no difference between preparations, and recovery increased with decreasing dose. Histopathologic examination revealed no abnormal tissues other than cecum, where inflammation was dose dependent; mild at 120 mg/kg and severe at 240 mg/kg, a greatly supra-physiological dose. We conclude that GRP 30-60 mg/kg p.o. is safe and effectively enhances NQO1 in all tissues evaluated.

Glucoraphanin: a broccoli sprout extract that ameliorates obesity-induced inflammation and insulin resistance.

Obesity is a low-grade sustained inflammatory state that causes oxidative stress in different metabolic tissues, which leads to insulin resistance and nonalcoholic fatty liver disease (NAFLD). Particularly, obesity-induced metabolic endotoxemia plays an important role in the pathogenesis of insulin resistance and inflammation. Nuclear factor erythroid 2-related factor 2 (Nrf2) is a key regulator of antioxidant signaling that serves as a primary cellular defense against the cytotoxic effects of oxidative stress. Pharmacological stimulation of Nrf2 mitigates obesity and insulin resistance in mice; however, Nrf2 activators are not clinically available due to biosafety concerns. A recent study demonstrated that glucoraphanin, a precursor of the Nrf2 activator sulforaphane, ameliorates obesity by enhancing energy expenditure and browning of white adipose tissue, and attenuates obesity-related inflammation and insulin resistance by polarizing M2 macrophages and reducing metabolic endotoxemia. Thus, this review focuses on the efficiency and safety of glucoraphanin in alleviating obesity, insulin resistance, and NAFLD. Abbreviations: ALT, Alanine aminotransferase; AMPK, AMP-activated protein kinase; ATMs, Adipose tissue macrophages; BAT, Brown adipose tissue; CDDO-Im, 2-cyano-3,12-dioxoolean-1,9-dien-28-oic acid-imidazolide; CDDO-Me, CDDO-methyl ester; DIO, High-fat-diet-induced obese; FFA, Free fatty acid; FGF, Fibroblast growth factor; GTP, Glutamyl transpeptidase; HFD, High-fat diet; IKKß, Inhibitor of κB-kinase ß; IL, Interleukin; JNK, C-Jun N-terminal kinase; KD, Knockdown; Keap1, Kelch-like ECH-associated protein 1; KO, Knockout; LPS, Lipopolysaccharide; NADPH, Nicotinamide adenine dinucleotide phosphate; NAFLD, Non-alcoholic fatty liver disease; NF-κB, Nuclear factor-κB; Nrf2, Nuclear factor E2-related factor 2; ROS, Reactive oxygen species; T2D, Type 2 diabetes; TLR, Toll-like receptor; TNF, tumor necrosis factor; UCP, Uncoupling protein; WAT, White adipose tissue.

Strong ion-exchange centrifugal partition chromatography as an efficient method for the large-scale purification of glucosinolates.

The glucosinolates sinalbin and glucoraphanin were purified by strong ion-exchange displacement centrifugal partition chromatography (SIXCPC). The optimized conditions involved the biphasic solvent system ethyl acetate/n-butanol/water (3:2:5, v/v), the lipophilic anion-exchanger Aliquat 336 (trioctylmethylammonium chloride, 160 and 408 mM) and a sodium iodide solution (80 and 272 mM) as displacer. Amounts as high as 2.4 g of sinalbin and 2.6g of glucoraphanin were obtained in one step in 2.5 and 3.5h respectively, starting from 12 and 25 g of mustard and broccoli seed aqueous extracts, using a laboratory scale CPC column (200 mL inner volume).

The isolation and purification of glucoraphanin from broccoli seeds by solid phase extraction and preparative high performance liquid chromatography.

Plant foods contain not only essential nutrients, e.g. protein, amino acids, vitamins and minerals, but also phytochemicals that have added health benefits. One such class of phytochemicals are the glucosinolates. Glucosinolates, particularly glucoraphanin, are predominant in plants of the Brassica genus, most notably in vegetables such as broccoli. There is a growing interest in the role glucoraphanin plays in chemoprotection and as a result there is a requirement to accurately determine the levels of glucoraphanin in vegetable products. Reverse phase ion pair high performance liquid chromatography (HPLC) is the method of choice; however, this work has been hindered by the lack of available standard reference materials. Broccoli seeds, which are particularly rich in glucoraphanin (20-50 mg/g), have proved to be ideal for the isolation of glucoraphanin on the preparative scale. A novel preparative scale HPLC method with simple compound recovery has been developed to meet the need for a glucoraphanin standard.

Direct antioxidant activity of purified glucoerucin, the dietary secondary metabolite contained in rocket (Eruca sativa Mill.) seeds and sprouts.

Rocket (Eruca sativa Mill. or Eruca vesicaria L.) is widely distributed all over the world and is usually consumed fresh (leafs or sprouts) for its typical spicy taste. Nevertheless, it is mentioned in traditional pharmacopoeia and ancient literature for several therapeutic properties, and it does contain a number of health promoting agents including carotenoids, vitamin C, fibers, flavonoids, and glucosinolates (GLs). The latter phytochemicals have recently gained attention as being the precursors of isothiocyanates (ITCs), which are released by myrosinase hydrolysis during cutting, chewing, or processing of the vegetable. ITCs are recognized as potent inducers of phase II enzymes (e.g., glutathione transferases, NAD(P)H:quinone reductase, epoxide hydrolase, etc.), which are important in the detoxification of electrophiles and protection against oxidative stress. The major GL found in rocket seeds is glucoerucin, GER (108 +/- 5 micromol g(-)(1) d.w.) that represents 95% of total GLs. The content is largely conserved in sprouts (79% of total GLs), and GER is still present to some extent in adult leaves. Unlike other GLs (e.g., glucoraphanin, the bio-precursor of sulforaphane), GER possesses good direct as well as indirect antioxidant activity. GER (and its metabolite erucin, ERN) effectively decomposes hydrogen peroxide and alkyl hydroperoxides with second-order rate constants of k(2) = 6.9 +/- 0.1 x 10(-)(2) M(-)(1) s(-)(1) and 4.5 +/- 0.2 x 10(-)(3) M(-)(1) s(-) , respectively, in water at 37 degrees C, thereby acting as a peroxide-scavenging preventive antioxidant. Interestingly, upon removal of H(2)O(2) or hydroperoxides, ERN is converted into sulforaphane, the most effective inducer of phase II enzymes among ITCs. On the other hand, ERN (and conceivably GER), like other ITCs, does not possess any chain-breaking antioxidant activity, being unable to protect styrene from its thermally (37 degrees C) initiated autoxidation in the presence of AMVN. The mechanism and relevance of the antioxidant activity of GER and ERN are discussed.

Simultaneous determination of glucoraphanin and sulforaphane in Brassica oleracea seeds by high-performance liquid chromatography with evaporative light-scattering detector.

In this article, a novel and efficient analysis method is described to simultaneously and quantitatively determine glucoraphanin and its major degradation product sulforaphane by reversed-phase high-performance liquid chromatography (HPLC) coupled with an evaporative light-scattering detector (ELSD). This method was validated according to the regulatory guidelines with respect to precision, accuracy and linearity. The HPLC-ELSD method was successfully used to assess the contents of glucoraphanin and sulforaphane in varieties of Brassica oleracea seeds. The developed analytical method not only avoids the underestimation of total glucoraphanin content, but also provides a reduction in analysis time and good efficiency and sensitivity compared to with conventional HPLC methods.

Protective role of (RS )-glucoraphanin bioactivated with myrosinase in an experimental model of multiple sclerosis.

AIM: The discovery of new natural compounds with pharmacological properties is a field of interest widely growing. Recent literature shows that Brassica vegetables (Cruciferae) possess therapeutic effects particularly ascribed due to their content in glucosinolates, which upon myrosinase hydrolysis release the corresponding isothiocyanates. This study examines the potential neuroprotective and immunomodulatory effects of (RS )-glucoraphanin from Tuscan black kale (Brassica oleracea L. var. acephala sabellica) bioactivated with myrosinase (bioactive RS -GRA) (10 mg/kg/day intraperitoneally), in an experimental autoimmune encephalomyelitis (EAE), a model of multiple sclerosis. METHODS: EAE was induced by immunization with myelin oligodendroglial glycoprotein peptide (MOG35-55 ) in mice. After immunization, mice were observed daily for signs of EAE and weight loss. Clinical score was evaluated using a standardized scoring system. RESULTS: By Western blot analysis of spinal cord tissues, we have demonstrated that treatment with bioactive RS -GRA significantly decreased nuclear factor (NF)-kB translocation, pro-inflammatory cytokine production such as interleukin-1ß (IL-1ß), and apoptosis (Bax and caspase 3 expression). CONCLUSION: Our results clearly demonstrate that bioactive RS -GRA treatment may represent a useful therapeutic perspective in the treatment of this disease.

Glucosinolates in the subantarctic crucifer Kerguelen cabbage (Pringlea antiscorbutica).

Glucosinolates in the subantarctic Kerguelen cabbage (Pringlea antiscorbutica) were determined by HPLC. Glucoerucin (6) was present only in the seeds, whereas sinigrin (2), gluconapin (3), n-butyl glucosinolate (4), glucoraphanin (1), and glucotropaeolin (5) were present in both the seeds and leaves. High concentrations of glucosinolates, precursors of bioactive isothiocyanates, were found in the leaves of Kerguelen cabbage. In particular, the lack of unhealthy beta-hydroxylated aliphatic side-chain glucosinolates is supportive of this vegetable being a possible dietary source with a high nutritional value.

Preparation and reactions of an iodinated imidoester reagent with actin and alpha-actinin.

The chemical iodination of an imidoester (methyl-p-hydroxybenzimidate, Wood et al. (1975) Anal. Biochem. 68, 339) and subsequent coupling of iodinated imidoester (IIE) to protein is an indirect method of iodinating proteins that is specific for the epsilon amino group of lysine residues and maintains the positive charge on the amino group at physiological pH. Purification of the IIE from chloramine-T and free iodine by benzene extraction eliminates the need for isoelectric precipitation and produces a more time- and cost-efficient IIE preparation and purification protocol. The separation of free from protein-bound label by chromatography, using centrifugal elution, provides a separation method that is rapid and efficient, without the generation of large volumes of radioactive wastes characteristic of conventional chromatographic and dialysis methods. To optimize the parameters of labeling protein with IIE, a systematic assessment of the effects of pH, reactant concentrations, and reaction time was made using purified cardiac actin and gizzard alpha-actinin. The parameters were defined to achieve an average labeling ratio of one IIE per protein polypeptide. The data demonstrate that both proteins appear to be labeled at the same rate and define several determining factors that limit the rate and extent of IIE incorporation into protein.