Glucosinolates and Cytotoxic Activity of Collard Volatiles Obtained Using Microwave-Assisted Extraction.

Glucosinolates (GSLs) in Brassica oleracea L. convar. acephala var. viridis (collard) flower, leaf, stem, and root were analyzed qualitatively and quantitatively via their desulfo-counterparts using UHPLC-DAD-MS/MS. Twelve GSLs were identified, including Met-derived GSLs (sinigrin, glucoibervirin, glucoerucin, glucoiberin, glucoraphanin, progoitrin), Trp-derived GSLs (4-hydroxyglucobrassicin, glucobrassicin, 4-methoxyglucobrassicin, and neoglucobrassicin), and Phe-derived GSLs (glucotropaeolin and gluconasturtiin). Total GSL content was highest in the root, having 63.40 µmol/g dried weight (DW), with gluconasturtiin (34.02 µmol/g DW) as the major GSL, followed by sinigrin and glucoibervirin (12.43 and 7.65 µmol/g DW, respectively). Total GSL contents in the flower, leaf, and stem were lower than in root, having 6.27, 2.64, and 1.84 µmol/g DW, respectively, with Trp and/or Met-derived GSLs as the predominant ones. GSL breakdown products were obtained via microwave hydrodiffusion and gravity (MHG) and volatile breakdown products were analyzed using GC-MS techniques. Volatile isolates were tested for their cytotoxic activity using MTT assay. MHG volatile extract from the root demonstrated the best cytotoxic activity against human bladder cancer cell line T24 and breast cancer cell line MDA-MB-231 during an incubation time of 72 h (IC50 21.58, and 11.62 µg/mL, respectively). The activity of the root extract can be attributed to its major volatile, 2-phenylethyl isothiocyanate (gluconasturtiin breakdown product).

Microwave-Assisted versus Conventional Isolation of Glucosinolate Degradation Products from Lunaria annua L. and Their Cytotoxic Activity.

Glucosinolates (GSLs) from Lunaria annua L. seeds were analyzed qualitatively and quantitatively by their desulfo counterparts using UHPLC-DAD-MS/MS technique and by their volatile breakdown products, isothiocyanates (ITCs), using GC-MS technique. GSL breakdown products were obtained by conventional techniques (hydrodistillation in a Clevenger type apparatus (HD), CH2Cl2 extraction after myrosinase hydrolysis (EXT) for 24 h) as well as by modern techniques, microwave-assisted distillation (MAD) and microwave hydrodiffusion and gravity (MHG). Seven GSLs were identified as follows: isopropyl GSL (1), sec-butyl GSL (2), 5-(methylsulfinyl)pentyl GSL (3), 6-(methylsulfinyl)hexyl GSL (4), 5-(methylsulfanyl)pentyl GSL (5), 6-(methylsulfanyl)hexyl GSL (6), and benzyl GSL (7). Additionally, pent-4-enyl- and hex-5-enyl ITCs were detected in the volatile extracts. However, their corresponding GSLs were not detected using UHPLC-DAD-MS/MS. Thus, they are suggested to be formed during GC-MS analysis via thermolysis of 5-(methylsulfinyl)pentyl- and 6-(methylsulfinyl)hexyl ITCs, respectively. Volatile isolates were tested for their cytotoxic activity using MTT assay. EXT and MHG showed the best cytotoxic activity against human lung cancer cell line A549 during an incubation time of 72 h (IC50 18.8, and 33.5 µg/mL, respectively), and against breast cancer cell line MDA-MB-231 after 48 h (IC50 6.0 and 11.8 µg/mL, respectively). These activities can be attributed to the ITCs originating from 3 and 4.

Investigation of the glucosinolates in Hesperis matronalis L. and Hesperis laciniata All.: Unveiling 4'-O-ß-d-apiofuranosylglucomatronalin.

The glucosinolate (GSL) profiles of wild-growing plants from the genus Hesperis, i.e. Hesperis laciniata All. (leaf, stem, flower, and root) from Croatia and Hesperis matronalis L. (leaf, stem, flower, seed, and root) from Canada, were established by LC-MS. During this investigation, 5-(methylsulfanyl)pentyl- (3), 6-(methylsulfanyl)hexyl- (4), 6-(methylsulfinyl)hexyl- (6), and 4'-α-l-rhamnopyranosyloxybenzyl- (17) GSLs were identified. In addition, the presence of 7-(methylsulfinyl)heptyl GSL (18), hydroxy-(α-l-rhamnopyranosyloxy)benzyl GSL, and of one d-apiosylated analogue of 17 were suggested. Moreover, one new GSL, 4'-O-ß-d-apiofuranosylglucomatronalin (19) was isolated from H. laciniata (flower, steam and leaf) and characterized by spectroscopic data interpretation. Finally, we report the presence of 3, 4, 6, 19, glucosinalbin (12), and 4-hydroxyglucobrassicin (20) in H. matronalis and hypothesize the presence of glucomatronalin (13) and 3-hydroxy-6-(methylsulfanyl)hexyl GSL (21).

Neuroprotective Potential of Secondary Metabolites from Melicope lunu-ankenda (Rutaceae).

Plant natural compounds have great potential as alternative medicines for preventing and treating diseases. Melicope lunu-ankenda is one Melicope species (family Rutaceae), which is widely used in traditional medicine, consumed as a salad and a food seasoning. Consumption of different parts of this plant has been reported to exert different biological activities such as antioxidant and anti-inflammatory qualities, resulting in a protective effect against several health disorders including neurodegenerative diseases. Various secondary metabolites such as phenolic acid derivatives, flavonoids, coumarins and alkaloids, isolated from the M. lunu-ankenda plant, were demonstrated to have neuroprotective activities and also exert many other beneficial biological effects. A number of studies have revealed different neuroprotective mechanisms for these secondary metabolites. This review summarizes the most significant and recent studies for neuroprotective activity of M. lunu-ankenda major secondary metabolites in neurodegenerative diseases.

Antimicrobial and Cytotoxic Activities of Lepidium latifolium L. Hydrodistillate, Extract and Its Major Sulfur Volatile Allyl Isothiocyanate.

The cultivated Lepidium latifolium L. was investigated to decipher its glucosinolate profile, antimicrobial, and cytotoxic activities. HPLC/ESI-MS analyses of the intact glucosinolates and GC/MS analysis of their hydrolysis products showed the presence of sinigrin (1), glucocochlearin (2), glucotropaeolin (3), and 4-methoxyglucobrassicin (4). Hydrodistillate, extract, and allyl isothiocyanate, the main volatile resulting from sinigrin degradation, showed antimicrobial activity against all eleven tested pathogenic and food spoilage bacteria and fungi, with highest effect observed against Candida albicans with MIC50 8 and 16 µg/mL. Hydrodistillate and extract showed the best cytotoxic activity on bladder cancer UM-UC-3 cell line during an incubation time of 24 h (IC50 192.9 and 133.8 µg/mL, respectively), while the best effect on glioblastoma LN229 cell line was observed after 48 h (IC50 110.8 and 30.9 µg/mL, respectively). Pure allyl isothiocyanate displayed a similar trend in cytotoxic effect on both cell lines (IC50 23.3 and 36.5 µg/mL after 24 h and 48 h, respectively).

Applying the hydrodistillation process to Pentadiplandra brazzeana Baill. root: a chemical assessment.

A chemical study of the volatile components obtained by applying the hydrodistillation and reflux processes to Pentadiplandra brazzeana roots was performed by GC-FID and GC-MS. The hydrodistillation process showed a total yield of 0.97% with 0.11% of essential oil and 0.86% of volatile compounds from the aqueous reaction medium; in the reflux process, the volatile extract yield was 1.03%. Benzylic-type isothiocyanates were the major degradation products of glucosinolates in the essential oil (95.0%); the CH2Cl2 extracts obtained from the aqueous solutions were characterised by alcohols and amines in both processes. This study has shown that during hydrodistillation, only 10% of the glucosinolate degradation products are recovered in the essential oil whereas 90% remain in the aqueous medium, being converted into alcohols and amines. The relative percentages of the different chemical classes recovered in our experimental conditions are discussed in relation with the glucosinolate composition in the raw material.

Moringa isothiocyanate complexed with α-cyclodextrin: a new perspective in neuroblastoma treatment.

BACKGROUND: Several lines of evidence suggest the consume of natural products for cancer prevention or treatment. In particular, isothiocyanates (ITCs) exerting anti-cancer properties, have received great interest as potential chemotherapeutic agents. This study was designed to assess the anti-proliferative activities of a new preparation of Moringa oleifera-derived 4-(α-L-rhamnopyranosyloxy)benzyl ITC (moringin) complexed with alpha-cyclodextrin (moringin + α-CD; MAC) on SH-SY5Y human neuroblastoma cells. This new formulation arises in the attempt to overcome the poor solubility and stability of moringin alone in aqueous media. METHODS: SH-SY5Y cells were cultured and exposed to increasing concentrations of MAC (1.0, 2.5 and 5.0 µg). Cell proliferation was examined by MTT and cell count assays. The cytotoxic activity of the MAC complex was assessed by lactate dehydrogenase (LDH) assay and trypan blue exclusion test. In addition, western blotting analyses for the main apoptosis-related proteins were performed. RESULTS: Treatment of SH-SY5Y cells with the MAC complex reduced cell growth in concentration dependent manner. Specifically, MAC exhibited a potent action in inhibiting the PI3K/Akt/mTOR pathway, whose aberrant activation was found in many types of cancer. MAC was also found to induce the nuclear factor-κB (NF-κB) p65 activation by phosphorylation and its translocation into the nucleus. Moreover, treatment with MAC was able to down-regulate MAPK pathway (results focused on JNK and p38 expression). Finally, MAC was found to trigger apoptotic death pathway (based on expression levels of cleaved-caspase 3, Bax/Bcl-2 balance, p53 and p21). CONCLUSION: These findings suggest that use of MAC complex may open novel perspectives to improve the poor prognosis of patients with neuroblastoma.

Identification of Glucosinolates in Seeds of Three Brassicaceae Species Known to Hyperaccumulate Heavy Metals.

Plants from the Brassicaceae family are known to contain secondary metabolites called glucosinolates. Our goal was to establish by LC/MS the glucosinolate profile of seeds of three Brassicaceae species known to hyperaccumulate heavy metals. We investigated Alyssum fallacinum auct. non Hausskn., Iberis intermedia Guers., and Noccaea caerulescens (J. Presl & C. Presl) F. K. Mey. Our results indicate that A. fallacinum seeds contain glucoiberin and glucoibervirin, which had not been previously identified in this plant. Furthermore, we report for the first time the presence of glucoiberin, glucoibervirin, glucotropaeolin, and sinigrin in I. intermedia. We have detected for the first time glucoconringiin in N. caerulescens. In addition, glucosinalbin, 4-hydroxyglucobrassicin, and glucomoringin were also detected.

Probing for the presence of glucosinolates in three Drypetes spp. (Drypetes euryodes (Hiern) Hutch., Drypetes gossweileri S. Moore, Drypetes laciniata Hutch.) and two Rinorea spp. (Rinorea subintegrifolia O. Ktze and Rinorea woermanniana (Büttner) Engl.) from Gabon.

Drypetes euryodes (Hiern) Hutch., Drypetes gossweileri S. Moore, Drypetes laciniata Hutch. (Putranjivaceae), Rinorea subintegrifolia O. Ktze, and Rinorea woermanniana (Büttner) Engl. (Violaceae) from Gabon were probed for the presence of glucosinolates (GLs). When present, the GLs were identified and quantified by HPLC analysis. 2-Hydroxy-2-methyl GL (1) was the major GL in the cork of D. euryodes. Moreover, 4-hydroxybenzyl GL (2) was the major GL in the seed of D. gossweileri whereas the bark contained 2 as the minor GL and benzyl GL (3) was the major one. In addition, 4-methoxybenzyl GL (4), 3-methoxybenzyl GL (5), and 3 were found in the root of R. subintegrifolia. However, no GL was detected in D. laciniata (leaf and stem), D. euryodes (leaf and stem), and R. woermanniana (leaf and stem-branch). Our results support the hypothesis of the existence of GLs in plants of the Putranjivaceae and Violaceae families (order Malpighiales).

LC-MS profiling of glucosinolates in the seeds of Brassica elongata Ehrh., and of the two stenoendemic B. botteri Vis and B. cazzae Ginzb. & Teyber.

The glucosinolates (GLs) present in seed extracts of Brassica elongata Ehrh., B. botteri Vis and B. cazzae Ginzb. & Teyber from Croatia were identified by LC-MS. 4-Hydroxyindol-3-ylmethyl GL (3) was the major GL in the seeds of B. elongata, along with the four minor GLs 2-(R)-hydroxy-3-butenyl- (1), 3-butenyl- (2), 4-pentenyl- (4) and indol-3-ylmethyl (5). The seeds of B. botteri (Vis island) and B. cazzae (Susac island) contained 2 as the major GL as well as 1, 3, 5 and 4-methoxyindol-3-ylmethyl GL (6). However, the GLs in B. botteri (Palagruza island) differed from other varieties having 2-propenyl GL (7) as the major GL in the seeds, and the four minor GLs 2, 3, 5 and 6. This first report of the GL content in the seeds of B. elongata, B. botteri and B. cazzae indicates that the unique GL profiles could be specific to the geographical origin of the plant.

Anticancer activity of glucomoringin isothiocyanate in human malignant astrocytoma cells.

Isothiocyanates (ITCs) released from their glucosinolate precursors have been shown to inhibit tumorigenesis and they have received significant attention as potential chemotherapeutic agents against cancer. Astrocytoma grade IV is the most frequent and most malignant primary brain tumor in adults without any curative treatment. New therapeutic drugs are therefore urgently required. In the present study, we investigated the in vitro antitumor activity of the glycosylated isothiocyanate moringin [4-(α-l-rhamnopyranosyloxy)benzyl isothiocyanate] produced from quantitative myrosinase-induced hydrolysis of glucomoringin (GMG) under neutral pH value. We have evaluated the potency of moringin on apoptosis induction and cell death in human astrocytoma grade IV CCF-STTG1 cells. Moringin showed to be effective in inducing apoptosis through p53 and Bax activation and Bcl-2 inhibition. In addition, oxidative stress related Nrf2 transcription factor and its upstream regulator CK2 alpha expressions were modulated at higher doses, which indicated the involvement of oxidative stress-mediated apoptosis induced by moringin. Moreover, significant reduction in 5S rRNA was noticed with moringin treatment. Our in vitro results demonstrated the antitumor efficacy of moringin derived from myrosinase-hydrolysis of GMG in human malignant astrocytoma cells.

An overview on neuroprotective effects of isothiocyanates for the treatment of neurodegenerative diseases.

The discovery of new natural compounds with pharmacological properties is a field of interest widely growing, especially for the management of neurodegenerative diseases. As no pharmacological treatment is available to prevent the development of these disorders, dietary intake of foods or plant-based extracts with antioxidant properties might have beneficial effects on human health and improve brain functions. Isothiocyanates (ITCs), derived from the hydrolysis of the corresponding glucosinolates (GLs), mainly found in Brassica vegetables (Brassicaceae) and, to a lesser extent, in Moringaceae plants, have demonstrated to exert neuroprotective properties. Specifically, strong evidences suggest that antioxidant effects may be ascribed mainly to their peculiar ability to activate the Nrf2/ARE pathway, but alternative mechanisms of action have also been suggested. This review summarizes the current knowledge about the neuroprotective effects of ITCs in counteracting oxidative stress as well as inflammatory and apoptotic mechanisms, using in vitro and in vivo models of acute and chronic neurodegenerative disease. Therefore, ITCs could be regarded as a promising source of alternative medicine for the prevention and/or treatment of neurodegenerative diseases.

Glucosinolate Diversity in Bretschneidera sinensis of Chinese Origin.

The glucosinolate (GL) profile in several plant parts (leaf, branch, bark, root, and fruit) of Bretschneidera sinensis from three geographical regions of the People's Republic of China was established for the first time by HPLC. During this investigation, benzyl GL (1), 4-hydroxybenzyl GL (2), 2-hydroxy-2-methylpropyl GL (3), and 4-methoxybenzyl GL (4) were identified. In addition, one new GL, 3-hydroxy-4-methoxybenzyl GL (5), was isolated in a minor amount from the fruit and characterized by spectroscopic data interpretation. Furthermore, traces of 4-hydroxy-3-methoxyphenylacetonitrile were detected by GC-MS analysis in the fruits, thus confirming the presence of the regioisomeric 4-hydroxy-3-methoxybenzyl GL (6). GLs 1-5 were also quantified for the first time by HPLC in the various plant organs.

Long-chain Glucosinolates from Arabis turrita: Enzymatic and Non-enzymatic Degradations.

C8-C10 methylsulfinylalkyl glucosinolates (GLs), and C8-C10 methylsulfonylalkyl GLs were identified in the seed of Arabis turrita L. by HPLC-MS/ESI analysis of intact GLs. Enzymatic (with myrosinase) and non-enzymatic (thermal at 100 degrees C, and chemical at different pH) hydrolyses were performed and the volatile isolates were analyzed by GC-MS. Only the enzymatic and chemical (pH 10) degradations produced volatiles which are originating from GL degradation. GC-MS analysis showed the presence of long-chain olefinic isothiocyanates (ITCs) along with other the long-chain thiofunctionalized GL breakdown products.

Glucosinolates in two endemic plants of the Aurinia genus and their chemotaxonomic significance.

Glucosinolates (GLs) were characterized in the seed and root of Aurinia leucadea (Guss.) C. Koch and A. sinuata (L.) Griseb., and quantified according to the ISO 9167-1 official method based on the HPLC analysis of desulfo-GLs. Glucoalyssin (GAL, 1), glucobrassicanapin (GBN, 2) and glucoberteroin (GBE, 3) were the major GLs identified in A. leucadea and A. sinuata. GC/MS analysis of the volatile fractions obtained after enzyme hydrolysis showed that they mostly contain isothiocyanates (ITCs) originating from the parent GLs. On this basis and from previous reports, C-5 alkyl GLs 1, 2, and 3 can be considered as chemotaxonomic markers of the Aurinia genus.

Comparison of bioactive phytochemical content and release of isothiocyanates in selected brassica sprouts.

The consumption of brassica sprouts as raw vegetables provides a fair amount of glucosinolates (GLs) and active plant myrosinase, which enables the breakdown of GLs into health-promoting isothiocyanates (ITCs). This study reports the determination of the main constituents related to human health found in edible sprouts of two Brassica oleracea varieties, broccoli and Tuscan black kale, and two Raphanus sativus varieties, Daikon and Sango. Radish sprouts exhibited the highest ability to produce ITCs, with Daikon showing the greatest level of conversion of GLs into bioactive ITCs (96.5%), followed by Sango (90.0%). Tuscan black kale gave a value of 68.5%, whereas broccoli displayed the lowest with 18.7%. ITCs were not the exclusive GL breakdown products in the two B. oleracea varieties, since nitriles were also produced, thus accounting for the lower conversion observed. Measuring the release of plant ITCs is a valuable tool in predicting the potential level of exposure to these bioactive compounds after the consumption of raw brassica sprouts.

Updated glucosinolate profile of Dithyrea wislizenii.

Fruit extracts of Dithyrea wislizenii were analyzed for desulfoglucosinolates and intact glucosinolates using HPLC-APCI-MS and HPLC-ESI-MS, respectively. 2-Propenylglucosinolate (sinigrin) was shown to be present in the extracts. 6-Methylsulfanylhexyl- (glucolesquerellin 9), 6-methylsulfinylhexyl- (glucohesperin 10), 7-methylsulfanylheptyl- (11), and 5-methylsulfanylpentylglucosinolate (glucoberteroin 12) were isolated from the extracts and characterized by NMR and MS data. 7-Methoxyglucobrassicin was not detected in D. wislizenii extracts.