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.

Kinetic and structural characterization of therapeutic albumin chemical functionalization using complementary mass spectrometry techniques.

Protein conjugates such as antibody-drugs conjugates (ADCs) represents the next generation of therapeutic proteins. They allow to combine the biological properties of the protein format with the characteristics of the conjugated ligands. The reaction implemented to couple ligands to the peptide backbone represents a crucial aspect of the production of protein conjugates, influencing the nature and the heterogeneity of the conjugates obtained. Here, we report the concomitant use of MALDI-TOF MS and LC-MS/MS analysis to investigate the chemical functionalization of human serum albumin (HSA) by the intermediate of lysine residues, previously used to generate biopharmaceutical agents for medical imaging. A kinetic was performed by collecting samples after different reaction times and analyzing them using the two techniques. MALDI-TOF MS analyses allowed estimating the number of conjugated ligands in a robust manner and assess the global functionalization kinetic on the intact protein level. Results demonstrated a maximum of 38 modified residues out of the 59 lysines available showing the limitation of the chemical functionalization. Consequently, LC-MS/MS analysis provided a site-specific characterization of the residues undergoing chemical modification. Data exhibited unique properties due to the presence of the ligands which allowed to identify without ambiguity the residues exhibiting different modification rate and enabled the identification of the unmodified lysine. Results were compared to the structure of HSA described from crystallography data. The comparison strongly suggested that accessibility is influencing the residues respective reactivity. The relevant complementarity of the different techniques could be emphasized in order to perform an extensive characterization concerning the evolution of the primary structure of the protein during the chemical reaction, providing an improved insight on the conjugation process and offering the potentiality to tune the reaction.

Effects of α-, ß- and maltosyl-ß-cyclodextrins use on the glucoraphanin-sulforaphane system of broccoli juice.

Cyclodextrins (CDs) are macromolecules with several industrial applications, being particularly used in the food industry as health-promoting compounds protection agents, as flavour stabilizers, or to eliminate undesired tastes and browning reactions, among others. This study shows the effects of α- (10, 30 and 40 mmol L-1 ), ß- (3, 6 and 10 mmol L-1 ) and maltosyl-ß-CDs (30, 60 and 90 mmol L-1 ) use on the health-promoting glucoraphanin-sulforaphane system of a broccoli juice up to 24 h at 22 °C. Maltosyl-ß-CD (90 mmol L-1 ) highly retained glucoraphanin content after 24 h at 22 °C, showing better effectiveness than ß-CD (10 mmol L-1 ). Sulforaphane was efficiently encapsulated with ß-CD at just 3 mmol L-1 , and the sulforaphane formed was stable during 3 h at 22 °C. On the other hand, 40 mmol L-1 α-CD retained a high glucoraphanin content in broccoli juice. In contrast, glucoraphanin levels in juice without CDs decreased by 71% after 24 h. Consequently, CDs addition may potentially preserve glucoraphanin in this broccoli juice during industrial processing with the possibility to be later transformed by endogenous myrosinase after ingestion to the health-promoting sulforaphane. © 2018 Society of Chemical Industry.

Synthesis of Two Tetrasaccharide Pentenyl Glycosides Related to the Pectic Rhamnogalacturonan I Polysaccharide.

The synthesis of two protected tetrasaccharide pentenyl glycosides with diarabinan and digalactan branching related to the pectic polysaccharide rhamnogalacturonan I is reported. The strategy relies on the coupling of N-phenyl trifluoroacetimidate disaccharide donors to a common rhamnosyl acceptor. The resulting trisaccharide thioglycosides were finally coupled to an n-pentenyl galactoside acceptor to access the two protected branched tetrasaccharides.

Biofortification of oilseed Brassica juncea with the anti-cancer compound glucoraphanin by suppressing GSL-ALK gene family.

Glucosinolates are amino acids derived secondary metabolites, invariably present in Brassicales, which have huge health and agricultural benefits. Sulphoraphane, the breakdown product of glucosinolate glucoraphanin is known to posses anti-cancer properties. AOP (2-oxoglutarate-dependent dioxygenases) or GSL-ALK enzyme catalyzes the conversion of desirable glucoraphanin to deleterious gluconapin and progoitrin, which are present in very high amounts in most of the cultivable Brassica species including Brassica juncea. In this study we showed that B. juncea encodes four functional homologs of GSL-ALK gene and constitutive silencing of GSL-ALK homologs resulted in accumulation of glucoraphanin up to 43.11 µmoles g(-1) DW in the seeds with a concomitant reduction in the anti-nutritional glucosinolates. Glucoraphanin content was found remarkably high in leaves as well as sprouts of the transgenic lines. Transcript quantification of high glucoraphanin lines confirmed significant down-regulation of GSL-ALK homologs. Growth and other seed quality parameters of the transgenic lines did not show drastic difference, compared to the untransformed control. High glucoraphanin lines also showed higher resistance towards stem rot pathogen Sclerotinia sclerotiorum. Our results suggest that metabolic engineering of GSL-ALK has huge potential for enriching glucoraphanin content, and improve the oil quality and vegetable value of Brassica crops.

(RS)-glucoraphanin purified from Tuscan black kale and bioactivated with myrosinase enzyme protects against cerebral ischemia/reperfusion injury in rats.

Ischemic stroke is the result of a transient or permanent reduction in cerebral blood flow caused by the occlusion of a cerebral artery via an embolus or local thrombosis. Restoration of blood supply to ischemic tissues can cause additional damage known as reperfusion injury that can be more damaging than the initial ischemia. This study was aimed to examine the possible neuroprotective role of (RS)-glucoraphanin, bioactivated with myrosinase enzyme (bioactive RS-GRA), in an experimental rat model of brain ischemia/reperfusion injury (I/R). RS-GRA is a thiosaccharidic compound found in Brassicaceae, notably in Tuscan black kale (Brassica oleracea L. var. acephala sabellica). The mechanism underlying the inhibitory effects of bioactive RS-GRA on inflammatory and apoptotic responses, induced by carotid artery occlusion in rats, was carefully examined. Cerebral I/R was induced by the clamping of carotid artery for 1h, followed by 40 min of reperfusion through the release of clamp. Our results have clearly shown that administration of bioactive RS-GRA (10 mg/kg, i.p.) 15 min after ischemia, significantly reduces proinflammatory parameters, such as inducible nitric oxide synthase expression (iNOS), intercellular adhesion molecule 1 (ICAM-1), nuclear factor (NF)-kB traslocation as well as the triggering of the apoptotic pathway (TUNEL and Caspase 3 expression). Taken together our data have shown that bioactive RS-GRA possesses beneficial neuroprotective effects in counteracting the brain damage associated to I/R. Therefore, bioactive RS-GRA, could be a useful treatment in the cerebral ischemic stroke.

Impact of selenium supply on Se-methylselenocysteine and glucosinolate accumulation in selenium-biofortified Brassica sprouts.

Brassica sprouts are widely marketed as functional foods. Here we examined the effects of Se treatment on the accumulation of anticancer compound Se-methylselenocysteine (SeMSCys) and glucosinolates in Brassica sprouts. Cultivars from the six most extensively consumed Brassica vegetables (broccoli, cauliflower, green cabbage, Chinese cabbage, kale, and Brussels sprouts) were used. We found that Se-biofortified Brassica sprouts all were able to synthesize significant amounts of SeMSCys. Analysis of glucosinolate profiles revealed that each Brassica crop accumulated different types and amounts of glucosinolates. Cauliflower sprouts had high total glucosinolate content. Broccoli sprouts contained high levels of glucoraphanin, a precursor for potent anticancer compound. Although studies have reported an inverse relationship between accumulation of Se and glucosinolates in mature Brassica plants, Se supply generally did not affect glucosinolate accumulation in Brassica sprouts. Thus, Brassica vegetable sprouts can be biofortified with Se for the accumulation of SeMSCys without negative effects on chemopreventive glucosinolate contents.

Synthesis of oligosaccharide fragments of the rhamnogalacturonan of Nerium indicum.

Three trisaccharides, one pentasaccharide, and one heptasaccharide, namely α-D-GalA-(1→2)-α-L-Rha-(1→4)-ß-D-GalA-OC3H7 (1), α-L-Rha-(1→4)-α-D-GalA-(1→4)-ß-D-GalA-OC3H7 (2), α-D-GalA-(1→4)-α-D-GalA-(1→2)-α-L-Rha-OC3H7 (3), α-D-GalA-(1→2)-α-L-Rha-(1→4)-α-D-GalA-(1→2)-α-L-Rha-(1→4)-ß-D-GalA-OC3H7 (4), and α-D-GalA-(1→2)-α-L-Rha-(1→4)-α-D-GalA-(1→2)-α-L-Rha-(1→4)-α-D-GalA-(1→2)-α-L-Rha-(1→4)-ß-D-GalA-OC3H7 (5), which are relevant to the fragments of the rhamnogalacturonan of Nerium indicum, were concisely synthesized. The syntheses feature highly stereoselective formation of the α-D-GalA-linkage with GalA N-phenyltrifluoroacetimidates as donors.

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.

Development and characterization of immuno-nanocarriers targeting the cancer stem cell marker AC133.

In the context of targeted therapy, we addressed the possibility of developing a drug delivery nanocarrier capable to specifically reach cancer cells that express the most prominent marker associated with cancer stem cell (CSC) phenotype, AC133. For this purpose, 100nm lipid nanocapsules (LNCs) were functionalized with a monoclonal antibody (mAb) directed against AC133 according to two distinct methods: firstly, post-insertion within 100nm LNCs of a lipid poly(ethylene glycol) functionalized with reactive-sulfhydryl maleimide groups (DSPE-PEG(2000)-maleimide) followed by thiolated mAb coupling, and, secondly, creation of a thiolated lipo-immunoglobulin between DSPE-PEG(2000)-maleimide and AC133, then post-inserted within LNCs. Due to the reduced number of purification steps, lower amounts of DSPE-PEG(2000)-maleimide that were necessary as well as lower number of free maleimide functions present onto the surface of immuno-LNC, the second method was found to be more appropriate. Thus, 126nm AC133-LNC with a zeta potential of -22mV while keeping a narrow distribution were developed. Use of the IgG1κ isotype control-immunoglobulins produced similar control IgG1-LNCs. Micro-Bradford colorimetric assay indicated a fixation of about 40 immunoglobulins per LNC. Use of human Caco-2 cells that constitutively express AC133 (Caco-2-AC133(high)) allowed addressing the behavior of the newly functionalized immuno-LNCs. siRNA knockown strategy permitted to obtain Caco-2-AC133(low) for comparison. Immunofluorescence-combined flow cytometry analysis demonstrated that the epitope-recognition function of AC133 antibody was preserved when present on immuno-LNCs. Although grafting of immunoglobulins onto the surface of LNCs repressed their internalization within Caco-2 cells as evaluated by flow cytometry, AC133-specific cellular binding was obtained with AC133-LNC as assessed by computer-assisted fluorescence microscopy. In conclusion, interest of AC133-LNCs as niche carriers is discussed toward the development of CSC targeted chemo- or radio-nanomedicines.

Enhancing sulforaphane absorption and excretion in healthy men through the combined consumption of fresh broccoli sprouts and a glucoraphanin-rich powder.

Sulforaphane (SF) is a chemopreventive isothiocyanate (ITC) derived from glucoraphanin (GRP) hydrolysis by myrosinase, a thioglucoside present in broccoli. The ability of broccoli powders sold as supplements to provide dietary SF is often of concern as many supplements contain GRP, but lack myrosinase. In a previous study, biomarkers of SF bioavailability from a powder rich in GRP, but lacking myrosinase, were enhanced by co-consumption of a myrosinase-containing air-dried broccoli sprout powder. Here, we studied the absorption of SF from the GRP-rich powder used in the previous study, but in combination with fresh broccoli sprouts, which are commercially available and more applicable to the human diet than air-dried sprout powder. A total of four participants each consumed four meals (separated by 1 week) consisting of dry cereal and yogurt with sprouts equivalent to 70 µmol SF, GRP powder equivalent to 120 µmol SF, both or neither. Metabolites of SF were analysed in blood and urine. The 24 h urinary SF-N-acetylcysteine recovery was 65, 60 and 24 % of the dose ingested from combination, broccoli sprout and GRP powder meals, respectively. In urine and plasma, ITC appearance was delayed following the GRP powder meal compared with the sprout and combination meals. Compared with the GRP powder or sprouts alone, combining broccoli sprouts with the GRP powder synergistically enhanced the early appearance of SF, offering insight into the combination of foods for improved health benefits of foods that reduce the risk for cancer.

Dicationic ionic liquid stationary phase for GC-MS analysis of volatile compounds in herbal plants.

The seeming "dual nature" of ionic liquids (ILs) for separating both apolar and polar compounds suggests that ILs may have a great potential for complex samples like essential oils from herbal plants that contain a great variety of compounds. In the present work, a geminal dicationic IL, 1,9-di(3-vinylimidazolium)nonane bis[(trifluoromethyl)sulfonyl]imidate, was investigated for this purpose. To find the best way to achieve satisfactory separations simultaneously for the compounds in essential oils, the dicationic IL was used as the stationary phase for capillary gas chromatography (GC) in two ways, either in its pure state or as a mixed stationary phase with monocationic ILs and a polysiloxane diluent. Interestingly, it was found that the mixed stationary phase exhibited a much better selectivity for polar and nonpolar compounds than either the dicationic IL or the polysiloxane, suggesting that a kind of synergistic effect occurred when these stationary phases were combined in the way described. A comparison with two commercial stationary phases (polar and nonpolar) indicated that this novel mixed stationary phase behaved in a way closer to a polar stationary phase in terms of selectivity and elution order. The present work demonstrates that the mixed stationary phase is efficient and selective and can be an alternative choice for the GC analysis of samples of complex composition.

Oral insulin delivery: the potential of thiolated chitosan-insulin tablets on non-diabetic rats.

It was the aim of this study to develop a delivery system providing an improved efficacy of orally administered insulin utilizing a thiolated polymer. 2-Iminothiolane was covalently linked to chitosan. The resulting chitosan-TBA (chitosan-4-thiobutylamidine) conjugate exhibited 453.5+/-64.1 micromol thiol groups per gram polymer. 3.1% of these thiol groups were oxidised. Additionally, the enzyme inhibitors BBI (Bowman-Birk-Inhibitor) and elastatinal were covalently linked to chitosan representing 3.5+/-0.1% and 0.5+/-0.03% of the total weight of the resulting polymer conjugate, respectively. Chitosan-TBA conjugate (5 mg), insulin (2.75 mg), the permeation mediator reduced glutathione (0.75 mg) and the two inhibitor conjugates (in each case 0.75 mg) were compressed to so-called chitosan-TBA-insulin tablets. Control tablets consisted of unmodified chitosan (7.25 mg) and insulin (2.75 mg). Chitosan-TBA-insulin tablets showed a controlled release of insulin over 8 h. In vitro mucoadhesion studies showed that the mucoadhesive/cohesive properties of chitosan were at least 60-fold improved by the immobilisation of thiol groups on the polymer. After oral administration of chitosan-TBA-insulin tablets to non-diabetic conscious rats, the blood glucose level decreased significantly for 24 h corresponding to a pharmacological efficacy of 1.69+/-0.42% (means+/-S.D.; n=6) versus s.c. injection. In contrast, neither control tablets nor insulin given in solution showed a comparable effect. According to these results the combination of chitosan-TBA, chitosan-enzyme-inhibitor conjugates and reduced glutathione seems to represent a promising strategy for the oral application of insulin.

Mucoadhesive thiolated chitosans as platforms for oral controlled drug delivery: synthesis and in vitro evaluation.

The aim of the present study was to evaluate the influence of the degree of modification and the polymer chain length on the mucoadhesive properties and the swelling behavior of thiolated chitosan derivatives obtained via a simple one-step reaction between the polymer and 2-iminothiolane. The conjugates differing in molecular mass of the polymer backbone and in the amount of immobilized thiol groups were compressed into tablets. They were investigated for their mucoadhesive properties on freshly excised porcine mucosa via tensile studies and the rotating cylinder method. Moreover, the swelling behavior of these tablets in aqueous solutions was studied by a simple gravimetric method. The obtained results demonstrated that the total work of adhesion of chitosan-TBA (=4-thio-butyl-amidine) conjugates can be improved by an increasing number of covalently attached thiol groups; a 100-fold increase compared to unmodified chitosan was observed for a medium molecular mass chitosan-TBA conjugate exhibiting 264 microM thiol groups per gram polymer. Also, the polymer chain length had an influence on the mucoadhesive properties of the polymer. The medium molecular mass polymer displayed a fourfold improved adhesion on the rotating cylinder compared to the derivative of low molecular mass. These results contribute to the development of new delivery systems exhibiting improved mucoadhesive properties.