Isolation and Modification of Plant Glucosinolates and their Role in the Prevention of Pathologies of the Central Nervous System

The glucosinolate-myrosinase system is present in a large number of plants, mainly of the order Brassicales, including several common vegetables like broccoli, cabbage, kale, radish and rocket consumed worldwide. The knowledge about this fascinating biochemical system is still evolving and attracting the combined efforts of scientists of several disciplines. The research focused on the multigram-scale isolation of selected glucosinolates, the setting-up of their enzymatic transformation, and the in-vivo investigation of their activity in the protection of the central nervous system from neurodegenerative disorders. One of the main task was the screening of different Brassicaceae to identify the most convenient source to isolate glucoraphanin, which pointed towards Tuscan black kale seeds that resulted to be a multifunctional source to achieve several purposes. The procedure for extraction and purification was set-up by combining preparative anion exchange chromatography and gel filtration chromatography. Further, a set of other four glucosinolates, namely thiofunctionalized glucoerucin, glucoraphasatin, glucoraphenin and the atypical glycosylated glucomoringin were isolated and purified using similar experimental approach starting from suitable plant sources. An efficient protocol for the enzymatic transformation of the selected glucosinolates and isolation of the corresponding isothiocyanates and dithiocarbamates was set up and optimized using a biphasic system. The pharmacological studies focused on glucoraphanin and glucomoringin that are the precursors of dietary isothiocyanates R-sulforaphane and moringin, respectively, recognized for their chemopreventive and medicinal properties. In contrast to the well-known R-sulforaphane, little is known about the molecular pathways targeted by moringin. The neuroprotective effects of R-sulforaphane and moringin freshly prepared by the action of myrosinase on highly pure glucoraphanin and glucomoringin, was tested in in vivo pharmacological investigations. Overall, results could contribute to a better understanding of the potential neuroprotection of R-sulforaphane and moringin in several pathologies of the central nervous system

A glucosinolate-rich extract of Japanese Daikon perturbs carcinogen-metabolizing enzyme systems in rat, being a potent inducer of hepatic glutathione S-transferase.

Purpose: Glucosinolates/isothiocyanates are an established class of naturally occurring chemopreventive agents, a principal mechanism of action being to limit the generation of genotoxic metabolites of chemical carcinogens, as a result of modulation of cytochrome P450 and phase II detoxification enzymes. The objective of this study was to assess whether a glucosinolate-rich extract from Daikon sprouts, containing glucroraphasatin and glucoraphenin, is a potential chemopreventive agent by modulating such enzymes in the liver and lung of rats. Methods: Rats were exposed to the glucosinolate-rich Daikon extract through the diet, at three dose levels, for 14 days, so that the low dose simulates dietary intake. Results: At the low dose only, a modest increase was noted in the hepatic dealkylations of methoxy-, ethoxy-, pentoxyresorufin and benzyloxyquinoline that was accompanied by elevated expression of CYP1 and CYP3A2 apoprotein levels. In lung, only a modest increase in the dealkylation of pentoxyresorufin was observed. At higher doses, in both tissues, these increases were abolished. At the same low dietary dose, the Daikon extract elevated markedly glutathione S-transferase activity paralleled by rises in GSTα, GSTμ and GSTπ protein expression. An increase was also noted in quinone reductase activity and expression. Finally, glucuronosyl transferase and epoxide hydrolase activities and expression were also up-regulated, but necessitated higher doses. Conclusion: Considering the ability of Daikon glucosinolates to effectively enhance detoxification enzymes, in particular glutathione S-transferase, it may be inferred that consumption of this vegetable may possess significant chemopreventive activity and warrants further evaluation through epidemiology and studies in animal models of cancer

Glucosinolate Profile of Croatian Stenoendemic Plant Fibigia triquetra (DC.) Boiss. ex Prantl.

As part of our ongoing investigation of the stenoendemic plants belonging to the Brassicaceae family, we report on the chemistry of Fibigia triquetra (DC.) Boiss. ex Prantl for the first time. Different plant parts (flower, leaf, stem, and seed) of F. triquetra were characterized and quantified for glucosinolates (GLs) according to the ISO 9167-1 EU official method based on the HPLC analysis of desulfo-GLs. A taxonomic screening showed that F. triquetra contained relatively high levels of C-4 GLs, namely but-3-enyl GL (gluconapin, 1a), 4-methylsulfanylbutyl GL (glucoerucin, 3a), and 4-methylsulfinylbutyl GL (glucoraphanin, 5a). GC-MS analysis of the volatile fractions obtained after enzyme hydrolysis and/or HPLC-ESI-MS of intact GLs confirmed the GL profile. Four minor GLs, namely isopropyl GL (glucoputranjivin, 6a), sec-butyl GL (glucocochlearin, 7a), pent-4-enyl GL (glucobrassicanapin, 2a), and 5-methylsulfanylpentyl GL (glucoberteroin, 4a) were also identified and quantified while 4-methylpentyl GL, 5-methylhexyl GL, and n-heptyl GL, were tentatively identified by GC-MS of their degradation products. Based on the major, as well as the minor GLs, this study shows differences in chemotaxonomy between F. triquetra and the related Degenia velebitica (Degen) Hayek as well as other investigated species of the genus Fibigia

Novel Gram-Scale Production of Enantiopure R-Sulforaphane from Tuscan Black Kale Seeds

Dietary R-sulforaphane is a highly potent inducer of the Keap1/Nrf2/ARE pathway. Furthermore, sulforaphane is currently being used in clinical trials to assess its effects against different tumour processes. This study reports an efficient preparation of enantiopure R-sulforaphane based on the enzymatic hydrolysis of its natural precursor glucoraphanin. As an alternative to broccoli seeds, we have exploited Tuscan black kale seeds as a suitable source for gram-scale production of glucoraphanin. The defatted seed meal contained 5.1% (w/w) of glucoraphanin that was first isolated through an anion exchange chromatographic process, and then purified by gel filtration. The availability of glucoraphanin (purity ≈ 95%, weight basis) has allowed us to develop a novel simple hydrolytic process involving myrosinase (EC 3.2.1.147) in a biphasic system to directly produce R-sulforaphane. In a typical experiment, 1.09 g of enantiopure R-sulforaphane was obtained from 150 g of defatted Tuscan black kale seed meal

Evaluation of the activity of dhurrin and sorghum towards Meloidogyne incognita

The epidermal cells of leaf and roots of sorghum (Sorghum bicolor), sudangrass (5. sudanensis) and sorghum-sudangrass hybrids (5. bicolor \uc3\u97 S. sudanensis) contain the cyanogenic glucoside dhurrin, which can degrade into hydrogen cyanide (HCN), well known for its toxicity to many organisms, including nematodes. In vitro bioassays were carried out with the aim of evaluating the nematicidal and the nematistatic effects of dhurrin concentrations on second-stage juveniles (J2) of Meloidogyne incognita, and the effectiveness of dhurrin in decreasing or preventing hatching of M. incognita. Furthermore, Sorghum hybrid c v. Super Dolce 10, selected for its dhurrin content, and Eruca sativa sel. Nemat were used in two glasshouse experiments to study the host-nematode relationship and the development of M. incognita life cycle in the roots. In the in vitro bioassays, Sorghum hybrid cv. Super Dolce 10 showed an inhibitory effect on hatching, a slow nematicidal effect on J2 and no nematistatic activity. In the glasshouse experiments, both Sorghum hybrid cv. Super Dolce 10 and E. sativa sel. Nemat proved to be a poor to non-host of M. incognita, with reproduction factor (R) always <1, compared with Solanum lycopersicum cv. UC82, an excellent host (R = 43). In both bioactive accessions two generations were completed in 15 weeks (four cycles in S. lycopersicum), but with the production of very few egg masses, while root infestation was also always very low, with gall index (G.I.) < 1, compared with S. lycopersicum (G.I. = 4). In this study, the effectiveness of E. sativa sel. Nemat in controlling M. incognita confirmed data from previous work. \uc2\ua9 Koninklijke Brill NV., Leiden, 2012

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 by1H and13C 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 10 mg/kg, ip +5 μl/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·40 mg/kg MPTP) and a sub-acute (5 injections·20 mg/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-1β 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. © 2013 Elsevier Ltd. All rights reserved

A simple analytical method for dhurrin content evaluation in cyanogenic plants for their utilization in fodder and biofumigation

Cyanogenic plants have some potential as biocidal green manure crops in limiting several soilborne pests and pathogens. Sorghum (Sorghum bicolor (L.) Moench) and Sudangrass (Sorghum bicolor subsp. sudanense (P.) Stapf), in fact, contain the cyanogenic glucoside p-hydroxy-(S)-mandelonitrile-\uce\ub2-d-glucoside (dhurrin) as a substrate of its secondary defensive system able to release hydrogen cyanide following tissue lesions due to biotic or abiotic factors. Given that dhurrin content is correlated with the biofumigant efficacy of the plants, a high dhurrin content could be a positive character for utilization of sorghum and Sudangrass as biocidal green manure plants. For chemical characterization of the available germplasm, a simple, safe, and accurate method is necessary. In this paper, a new method for dhurrin analysis, based on methanol extraction and high-performance liquid chromatography, is reported and discussed. The feasibility of this analytical procedure was tested by evaluating dhurrin level in roots and stems during cultivation of four different sorghum and Sudangrass varieties in agronomic trials performed in 2008 in the Po valley (Italy). The dhurrin content ranged from 0.16 \uc2\ub1 0.04 to 7.14 \uc2\ub1 0.32 mg g -1 on dried matter (DM) in stems and from 1.38 \uc2\ub1 0.02 to 6.57 \uc2\ub1 0.09 mg g -1 on DM in roots, showing statistical differences among the tested germplasms that could be linked to the efficacy of their utilization as biofumigant plants. The method also opens new perspectives for the characterization of sorgum plants as fodder, for which the presence of dhurrin is considered to be negative for its well-known toxicity. \uc2\ua9 2011 American Chemical Society

(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 1 h, 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

RS-Glucoraphanin bioactivated with myrosinase treatment counteracts proinflammatory cascade and apoptosis associated to spinal cord injury in an experimental mouse model

Spinal cord injury (SCI) is a highly debilitating pathology. Although innovative medical care has been improved, drug therapies to counteract neuronal damage and promote regeneration are limited. An experimental mouse model of SCI was designed to examine the possible neuroprotective role of the glucosinolate (RS)-glucoraphanin (RS-GRA), bioactivated with myrosinase enzyme (MYR-activated RS-GRA). Methodologically, the injury was induced by application of an aneurysm clip (force of 24g) for 1 min via four-level T5-T8 after laminectomy. MYR-activated RS-GRA was administered in mice (10 mg/kg i.p.) 1 and 6 h after the trauma, identified as the therapeutic intervention window. The treatment with MYR-activated RS-GRA significantly decreased histological damage resulted by proinflammatory events as well as by apoptosis cascade. Overall, by quantitative analysis of immunohistochemical images, the neuroprotection has been quite evident. MYR-activated RS-GRA has given a histological quantification around zero in all determinations. Particularly, looking at the strongest data obtained, regarding the glial fibrillary acidic protein (GFAP), result the high tissue localization of this damage marker mediated by astrocyte activity, estimated as about 80% of positive staining, was shot down by MYR-activated RS-GRA treatment. Taken together, our results show that MYR-activated RS-GRA could represent an interesting approach for the management of secondary damage following SCI. \uc2\ua9 2013 Elsevier B.V

Tuscan black kale sprout extract bioactivated with myrosinase: a novel natural product for neuroprotection by inflammatory and oxidative response during cerebral ischemia/reperfusion injury in rat

Background: Cerebral ischemia and reperfusion (CIR) is a pathological condition characterized by a first blood supply restriction to brain followed by the consequent restoration of blood flow and simultaneous reoxygenation.The aim of this study was to evaluate the neuroprotective effects of Tuscan black kale sprout extract (TBK-SE) bioactivated with myrosinase enzyme, assessing its capability to preserve blood-brain barrier (BBB), in a rat model of CIR.Methods: CIR was induced in rats according to a classic model of carotid artery occlusion for a time period of 1 h and the reperfusion time was prolonged for seven days.Results: By immunohistochemical evaluation and western blot analysis of brain and cerebellum tissues, our data have clearly shown that administration of bioactive TBK-SE is able to restore alterations of tight junction components (claudin-5 immunolocalization). Also, bioactive TBK-SE reduces some inflammatory key-markers (p-selectin, GFAP, Iba-1, ERK1/2 and TNF-alpha), as well as the triggering of neuronal apoptotic death pathway (data about Bax/Bcl-2 balance, p53 and cleaved-caspase 3) and the generation of radicalic species by oxidative stress (results focused on iNOS, nitrotyrosine and Nrf2).Conclusion: Taken together, our findings lead to believe that bioactive TBK-SE exerts pharmacological properties in protecting BBB integrity through a mechanism of action that involves a modulation of inflammatory and oxidative pathway as well into control of neuronal death