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Abstract The study has proved the stimulating effects of different strategies of treatments with elicitors on the production of glucosinolates (GSLs), flavonoids, polyphenols, saccharides, and photosynthetic pigments in watercress (Nasturtium officinale) microshoot cultures. The study also assessed antioxidant and anti-melanin activities. The following elicitors were tested: ethephon (ETH), methyl jasmonate (MeJA), sodium salicylate (NaSA), and yeast extract (YeE) and were added on day 10 of the growth period. Cultures not treated with the elicitor were used as control. The total GSL content estimations and UHPLC-DAD-MS/MS analyses showed that elicitation influenced the qualitative and quantitative profiles of GSLs. MeJA stimulated the production of gluconasturtiin (68.34 mg/100 g dried weight (DW)) and glucobrassicin (65.95 mg/100 g DW). The elicitation also increased flavonoid accumulation (max. 1131.33 mg/100 g DW, for 100 μM NaSA, collection after 24 h). The elicitors did not boost the total polyphenol content. NaSA at 100 μM increased the production of total chlorophyll a and b (5.7 times after 24 h of treatment), and 50 μM NaSA caused a 6.5 times higher production of carotenoids after 8 days of treatment. The antioxidant potential (assessed with the CUPRAC FRAP and DPPH assays) increased most after 24 h of treatment with 100 μM MeJA. The assessment of anti-melanin activities showed that the microshoot extracts were able to cause inhibition of tyrosinase (max. 27.84% for 1250 µg/mL). Key points • Elicitation stimulated of the metabolite production in N. officinale microshoots. • High production of pro-health glucosinolates and polyphenols was obtained. • N. officinale microshoots have got tyrosinase inhibition potential. • The antioxidant potential of N. officinale microshoots was evaluated. Graphical abstract

Glucosinolates (GSLs) from

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).

The effects of copper addition, from various adsorbents, on the accumulation ability and glucosinolate content of cultivated rocket were studied. Different adsorbents (zeolite NaX, egg shells, substrate, fly ash) were treated with copper(II) solution with an adsorption efficiency of 98.36, 96.67, 51.82 and 39.13%, respectively. The lowest copper content and the highest total glucosinolate content (44.37 μg/g DW and 4269.31 µg/g DW, respectively) were detected in the rocket grown in the substrate with the addition of a substrate spiked with copper(II) ions. Rocket grown in the fly ash-substrate mixture showed an increase in copper content (84.98 μg/g DW) and the lowest total glucosinolate content (2545.71 µg/g DW). On the other hand, when using the egg shells-substrate mixture, the rocket copper content increased (113.34 μg/g DW) along with the total GSLs content (3780.03 µg/g DW), indicating the influence of an adsorbent type in addition to the copper uptake. The highest copper content of 498.56 μg/g DW was detected in the rocket watered with copper(II) solution with a notable decrease in the glucosinolate content, i.e., 2699.29 µg/g DW. According to these results rocket can be considered as a copper accumulator plant.

Glucosinolates (GSLs) from Lepidium graminifolium L. 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 analysis. Thirteen GSLs were identified with arylaliphatic as the major ones in the following order: 3-hydroxybenzyl GSL (glucolepigramin, 7), benzyl GSL (glucotropaeolin, 9), 3,4,5-trimethoxybenzyl GSL (11), 3-methoxybenzyl GSL (glucolimnanthin, 12), 4-hydroxy-3,5-dimethoxybenzyl GSL (3,5-dimethoxysinalbin, 8), 4-hydroxybenzyl GSL (glucosinalbin, 6), 3,4-dimethoxybenzyl GSL (10) and 2-phenylethyl GSL (gluconasturtiin, 13). GSL breakdown products obtained by hydrodistillation (HD) and CH2Cl2 extraction after hydrolysis by myrosinase for 24 h (EXT) as well as benzyl ITC were tested for their cytotoxic activity using MTT assay. Generally, EXT showed noticeable antiproliferative activity against human bladder cancer cell line UM-UC-3 and human glioblastoma cell line LN229, and can be considered as moderately active, while IC50 of benzyl ITC was 12.3 μg/mL, which can be considered as highly active.

The study demonstrated the effects of precursor feeding on the production of glucosinolates (GSLs), flavonoids, polyphenols, saccharides, and photosynthetic pigments in Nasturtium officinale microshoot cultures grown in Plantform bioreactors. It also evaluated the antioxidant and antimicrobial activities of extracts. L-phenylalanine (Phe) and L-tryptophan (Trp) as precursors were tested at 0.05, 0.1, 0.5, 1.0, and 3.0 mM. They were added at the beginning (day 0) or on day 10 of the culture. Microshoots were harvested after 20 days. Microshoots treated with 3.0 mM Phe (day 0) had the highest total GSL content (269.20 mg/100 g DW). The qualitative and quantitative profiles of the GSLs (UHPLC-DAD-MS/MS) were influenced by precursor feeding. Phe at 3.0 mM stimulated the best production of 4-methoxyglucobrassicin (149.99 mg/100 g DW) and gluconasturtiin (36.17 mg/100 g DW). Total flavonoids increased to a maximum of 1364.38 mg/100 g DW with 3.0 mM Phe (day 0), and polyphenols to a maximum of 1062.76 mg/100 g DW with 3.0 mM Trp (day 0). The precursors also increased the amounts of p-coumaric and ferulic acids, and rutoside, and generally increased the production of active photosynthetic pigments. Antioxidant potential increased the most with 0.1 mM Phe (day 0) (CUPRAC, FRAP), and with 0.5 mM Trp (day 10) (DPPH). The extracts of microshoots treated with 3.0 mM Phe (day 0) showed the most promising bacteriostatic activity against microaerobic Gram-positive acne strains (MIC 250–500 µg/mL, 20–21 mm inhibition zones). No extract was cytotoxic to normal human fibroblasts over the tested concentration range (up to 250 μg/mL).

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-β- 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).

The copper accumulation ability and its impact on the glucosinolate content of the garden candytuft were studied. Different copper sources (adsorbents or solution) were used. Generally, the seedlings copper uptake from the adsorbents was in the amount needed for its growth and development with the beneficial or no impact on the glucosinolate content. The lowest copper concentration was detected in the total seedlings biomass which grew in the humus with the addition of Cu-exchanged zeolite NaX (27.88 &mu, g g&minus, 1 DW) having glucosinolate content of 9757.81 &micro, 1 DW (23.86 &micro, mol g&minus, 1 DW). The highest copper concentration among all the garden candytuft samples was detected in the seedlings watered with CuSO4∙5H2O solution (514.63 &mu, 1 DW) with a sharp decrease of the glucosinolate content 3103.33 &micro, 1 DW (7.59 &micro, 1 DW). Based on the results obtained, the garden candytuft can be considered as a copper accumulator plant.

The glucosinolates (GSLs) of Matthiola incana were characterized and quantified as desulfoGSLs in different plant tissues (seeds, siliquae, stems, leaves, and flowers) using ultra-high performance liquid chromatography coupled with tandem mass spectrometry (UHPLC-DAD-MS/MS). The major GSL detected in all plant parts was 4-(methylsulfinyl)but-3-enyl GSL (glucoraphenin, 1), followed by 4-(methylsulfanyl)but-3-enyl GSL (glucoraphasatin, 2), while indol-3-ylmethyl GSL (glucobrassicin, 3) was also found in the flowers. GSL 2 was found to be the dominant in the plants analysed after only 2 days of drying, contrary to all analyses performed after longer storage of plant material, thus suggesting an oxidation of 2 into 1. The stability of GSLs was monitored over a one-month period. The desulfo-counterpart of 1 was unstable in water, showing a dramatic decrease (ca 80%) during the observation time, with spontaneous conversion into cyclic thioimidate N-oxides. Bioaccessibility of GSLs through the simulated ex vivo gastrointestinal digestion was determined using human gastrointestinal juices. Glucoraphenin 1 showed high bioaccessibility in the gastric phase (>90%), while both GSLs 1 and 2 were transformed in the intestinal phase.

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 &mu, g/mL, respectively), and against breast cancer cell line MDA-MB-231 after 48 h (IC50 6.0 and 11.8 &mu, g/mL, respectively). These activities can be attributed to the ITCs originating from 3 and 4.

U radu je prikazan doprinos istraživanju glukozinolata u razdoblju 2005. – 2020. koji je ostvaren radom na Kemijsko-tehnološkom fakultetu, Sveučilišta u Splitu. Pregledom sveoubuhvatne dokumentacije utvrđeno je da znatnom broju predloženih struktura nedostaju važne strukturne informacije koje bi osigurala NMR i MS spektroskopija, pa je danas broj glukozinolata još uvijek varijabilan. U najnovijem istraživanju, u rodu Hesperis, identificiran je 4-O-D-apiozilglukomatronalin, čime ukupni broj glukozinolata danas broji između 89 i 136 struktura. U svrhu identificiranja izvora specifičnih glukozinolata koji bi mogli djelovati kao prekursori biološki aktivnih izotiocijanata, istraživan je sadržaj glukozinolata u biljkama. Istraživanja uključuju i razgradnju glukozinolata (enzimska, termička i kemijska) kao i biološku aktivnost (citotoksična, antimikrobna, protuupalna i dr.). Ovo djelo je dano na korištenje pod licencom Creative Commons Imenovanje 4.0 međunarodna., This review paper presents a contribution to the research of glucosinolates over the period 2005–2020, which was obtained during work at the Faculty of Chemical Technology, University of Split. After review of the comprehensive documentation, it was determined that a significant number of proposed structures lack important structural information that would be provided by NMR and MS spectroscopy, and today the number of glucosinolates is still variable. In the recent study of Hesperis genus, 4-O-D-apiosylglucomatronalin was identified, which gives the total number of glucosinolates today ranging from 89 to 136 structures. In order to identify the sources of specific glucosinolates that could act as precursors of biologically active isothiocyanates, the content of glucosinolates in plants was investigated. The study also includes the degradation of glucosinolates (enzymatic, thermal, and chemical), as well as biological activity (cytotoxic, antimicrobial, anti-inflammatory, etc.). This work is licensed under a Creative Commons Attribution 4.0 International License.

Essential oils constituents are a diverse family of low molecular weight organic compounds with comprehensive biological activity. According to their chemical structure, these active compounds can be divided into four major groups: terpenes, terpenoids, phenylpropenes, and "others". In addition, they may contain diverse functional groups according to which they can be classified as hydrocarbons (monoterpenes, sesquiterpenes, and aliphatic hydrocarbons); oxygenated compounds (monoterpene and sesquiterpene alcohols, aldehydes, ketones, esters, and other oxygenated compounds); and sulfur and/or nitrogen containing compounds (thioesters, sulfides, isothiocyanates, nitriles, and others). : Compounds that act as cholinesterase inhibitors still represent the only pharmacological treatment of Alzheimer´s disease. Numerous in vitro studies showed that some compounds, found in essential oils, have a promising cholinesterase inhibitory activity, such as α-pinene, δ-3-carene, 1,8-cineole, carvacrol, thymohydroquinone, α- and β-asarone, anethole, etc. : Essential oils constituents are a diverse family of low molecular weight organic compounds with comprehensive biological activity. According to their chemical structure, these active compounds can be divided into four major groups: terpenes, terpenoids, phenylpropenes, and "others". In addition, they may contain diverse functional groups according to which they can be classified as hydrocarbons (monoterpenes, sesquiterpenes, and aliphatic hydrocarbons); oxygenated compounds (monoterpene and sesquiterpene alcohols, aldehydes, ketones, esters, and other oxygenated compounds); and sulfur and/or nitrogen containing compounds (thioesters, sulfides, isothiocyanates, nitriles, and others).

Finding a new type of cholinesterase inhibitor that would overcome the brain availability and pharmacokinetic parameters or hepatotoxic liability has been a focus of investigations dealing with the treatment of Alzheimer’s disease. Isothiocyanates have not been previously investigated as potential cholinesterase inhibitors. These compounds can be naturally produced from their glucosinolate precursors, secondary metabolites widely distributed in our daily Brassica vegetables. Among eleven tested compounds, phenyl isothiocyanate and its derivatives showed the most promising inhibitory activity. 2-Methoxyphenyl ITC showed best inhibition on acetylcholinesterase with IC50 of 0.57 mM, while 3-methoxyphenyl ITC showed the best inhibition on butyrylcholinesterase having 49.2% at 1.14 mM. Assessment of the antioxidant efficacy using different methods led to a similar conclusion. The anti-inflammatory activity was also tested using human COX-2 enzyme, ranking phenyl isothiocyanate and 3-methoxyphenyl isothiocyanate as most active, with ~99% inhibition at 50 μM.

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.

The glucosinolate (GSL) profiles (inflorescence, stem, root, and fruit) of the wild-growing plant

Bunias erucago belongs to the Brassicaceae family, which represents a forgotten crop of the Euro-Mediterranean area. The aim of the present study was to determine the glucosinolate profile in different plant parts and biological properties (antioxidant, anticholinesterase, and cytotoxic activities) of the isolates containing glucosinolate breakdown products. The chemical profiles were determined by using HPLC-PDA-MS/MS of desulfoglucosinolates and GC-MS of glucosinolate degradation products. The analysis of B. erucago showed the presence of seven glucosinolates: gluconapin (1), glucoraphasatin (2), glucoraphenin (3), glucoerucin (4), glucoraphanin (5), glucotropaeolin (6), and glucosinalbin (7). The total glucosinolate content ranged from 7.0 to 14.6 &micro, mol/g of dry weight, with the major glucosinolate glucosinalbin in all parts. The antioxidant activity of all volatile isolates was not notable. At a tested concentration of 227 &mu, g/mL, flower hydro-distillate (FH) showed good AChE inhibition, i.e., 40.9%, while root hydro-distillate (RH) had good activity against BChE, i.e., 54.3%. FH showed the best activity against both tested human bladder cancer cell lines, i.e., against T24 after 72 h, which have IC50 of 16.0 &mu, g/mL, and against TCCSUP after 48 h with IC50 of 7.8 &mu, g/mL, and can be considered as highly active. On the other hand, RH showed weak activity against tested cancer cells.

The glucosinolate (GSL) profiles (inflorescence, stem, root, and fruit) of the wild-growing plant Lepidium graminifolium L. (Brassicaceae) from Croatia was established by LC-MS analysis. During this investigation, we confirmed the presence of benzyl- (1), 3-methoxybenzyl- (2), 4-hydroxybenzyl- (4), 4-methoxyindol-3-ylmethyl- (7) GSLs and reported for the first time in the plant the presence of (2 R)-hydroxybut-3-enyl- (11), (2S)-hydroxybut-3-enyl- (12), but-3-enyl- (13), and 2-phenylethyl- (14) GSLs. Finally, 3-hydroxybenzyl GSL (3) was isolated for the first time from L. graminifolium inflorescence and characterised by spectroscopic data interpretation.

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.

Horseradish degradation products, mainly isothiocyanates (ITC) and nitriles, along with their precursors glucosinolates, were characterized by GC-MS and UHPLC-MS/MS, respectively. Volatiles from horseradish leaves and roots were isolated using microwave assisted-distillation (MAD), microwave hydrodiffusion and gravity (MHG) and hydrodistillation (HD). Allyl ITC was predominant in the leaves regardless of the isolation method while MAD, MHG, and HD of the roots resulted in different yields of allyl ITC, 2-phenylethyl ITC, and their nitriles. The antimicrobial potential of roots volatiles and their main compounds was assessed against sixteen emerging food spoilage and opportunistic pathogens. The MHG isolate was the most active, inhibiting bacteria at minimal inhibitory concentrations (MICs) from only 3.75 to 30 &micro, g/mL, and fungi at MIC50 between &lt, 0.12 and 0.47 &micro, g/mL. Cytotoxic activity of volatile isolates and their main compounds were tested against two human cancer cell lines using MTT assay after 72 h. The roots volatiles showed best cytotoxic activity (HD, IC50 = 2.62 &mu, g/mL) against human lung A549 and human bladder T24 cancer cell lines (HD, IC50 = 0.57 &mu, g/mL). Generally, 2-phenylethyl ITC, which was tested for its antimicrobial and cytotoxic activities along with two other major components allyl ITC and 3-phenylpropanenitrile, showed the best biological activities.

The glucosinolates (GSLs) is a well-defined group of plant metabolites characterized by having an S-β- d -glucopyrano unit anomerically connected to an O-sulfated (Z)-thiohydroximate function. After enzymatic hydrolysis, the sulfated aglucone can undergo rearrangement to an isothiocyanate, or form a nitrile or other products. The number of GSLs known from plants, satisfactorily characterized by modern spectroscopic methods (NMR and MS) by mid-2018, is 88. In addition, a group of partially characterized structures with highly variable evidence counts for approximately a further 49. This means that the total number of characterized GSLs from plants is somewhere between 88 and 137. The diversity of GSLs in plants is critically reviewed here, resulting in significant discrepancies with previous reviews. In general, the well-characterized GSLs show resemblance to C-skeletons of the amino acids Ala, Val, Leu, Trp, Ile, Phe/Tyr and Met, or to homologs of Ile, Phe/Tyr or Met. Insufficiently characterized, still hypothetic GSLs include straight-chain alkyl GSLs and chain-elongated GSLs derived from Leu. Additional reports (since 2011) of insufficiently characterized GSLs are reviewed. Usually the crucial missing information is correctly interpreted NMR, which is the most effective tool for GSL identification. Hence, modern use of NMR for GSL identification is also reviewed and exemplified. Apart from isolation, GSLs may be obtained by organic synthesis, allowing isotopically labeled GSLs and any kind of side chain. Enzymatic turnover of GSLs in plants depends on a considerable number of enzymes and other protein factors and furthermore depends on GSL structure. Identification of GSLs must be presented transparently and live up to standard requirements in natural product chemistry. Unfortunately, many recent reports fail in these respects, including reports based on chromatography hyphenated to MS. In particular, the possibility of isomers and isobaric structures is frequently ignored. Recent reports are re-evaluated and interpreted as evidence of the existence of “isoGSLs”, i.e. non-GSL isomers of GSLs in plants. For GSL analysis, also with MS-detection, we stress the importance of using authentic standards.

The genus Lepidium (Brassicaceae) includes about 175 different plant species strikingly characterized by glucosinolates (GLs) as molecular tags [1]. The cultivated Lepidium latifolium L. (perennial pepperweed) was investigated to decipher its GL profile using 1) an indirect method with GC-MS analysis of the GL degradation volatile products (mostly isothiocyanates and nitriles) obtained by hydrodistillation and organic solvent extraction, and 2) a direct method with LC-MS analysis of intact GLs. GC-MS analysis showed that the most abundant GLs were sinigrin and glucotropaeolin, and a minor one was glucocochlearin. LC-MS analysis confirmed the presence of the above-mentioned GLs but also detected 4-methoxyglucobrassicin. In recent years, studies have been directed towards isothiocyanates due to the broad range of their biological effects [1-3]. Taking that into account, L. latifolium leaf extract and hydrodistillate containing GL degradation products were evaluated for their anticarcinogenic activity against two human tumor cell lines - glioblastoma cell line LN-229 and bladder cancer cell line UM-UC-3. Their antimicrobial activity against pathogenic and food spoilage bacteria and fungi was also assessed. The extract and distillate exhibited very similar anticarcinogenic and antibacterial activities, but with a somewhat better effect for the extract. On the other hand, both extract and distillate were found to exert very strong antifungal activity against Candida albicans (MIC50 8 and 16 µg/mL).

The genus Diplotaxis contains around 34 different plant species and represents one of the flowering genera of the Brassicaceae family. Glucosinalates (GLs), naturally occurring compounds in this family, with isothiocyanates (ITCs) and nitriles as their typical degradation products, make them interesting for the research. ITCs and nitriles can be formed enzymatically, chemically and/or thermally. The goal of the present study was the isolation of the volatiles from wild-growing Diplotaxis tenuifolia (L.) DC. (Split). Two different approaches of isolation of volatiles from fresh plant material include thermal degradation of GLs at 100 °C in both cases: a) conventional hydrodistillation using a Clevenger type apparatus for 2.5 hours ; b) microwave-assisted extraction (MAE) using power of 500W for 35 min. MAE isolation represents new “green” technique without adding any solvents and two fractions were obtained i.e. the fragrance (by distillation) and flavour extraction (by gravity). The resulting volatiles analysed by GC-MS indicated that the main GL was 4-(methylsulfanyl)butyl GL (glucoerucin) which degraded differently i.e. 4-(methylsulfanyl)butyl ITC was dominant degradation product obtained by hydrodistillation, while 5-(methylsulfanyl)pentanenitrile was dominant degradation product obtained by MAE. Flavour fraction had greater yield (0.014%) in comparison to fragrance fraction (0.001%) as well as to hydrodistillation (0.003%).

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 (Sušac 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 (Palagruža 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.

Sea fennel, a rediscovered star of the coastal cuisine, has been investigated for its phytochemical profile and biological potential. Sea fennel flowers, stems and leaves were analyzed for essential oils (EOs) isolated by hydrodistillation, as well as non-volatiles obtained by ethanolic extraction. Limonene were found to be a dominant compound in EOs and ethanolic extracts; ranging from 57.5–74.2 % and 0.7–8.1 mg/g dry plant material, respectively. In addition total phenolic content was determined for ethanolic extracts. All samples and their main phytochemicals were tested for various methods. EO and extract obtained from flowers were tested for vasodilatory activity on rat aortic rings. Antioxidant activity of EOs was extremely low in comparison to extracts, on the contrary to cholinesterase inhibition where EOs showed better activity than extracts. Flower extract and chlorogenic acid showed stronger vasodilators in comparison to EO and limonene. The obtained results point out the potential impact of the dominant compounds from EO and extract on the biological properties of the sea fennel.

The purpose of this study was to compare the essential oil profiles of four Croatian Teucrium species (Lamiaceae), as determined by GC and GC/MS, with their antiphytoviral efficiency. A phytochemical analysis showed that T. polium, T. flavum, T. montanum and T. chamaedrys are characterized by similar essential oil compositions. The investigated oils are characterized by a high proportion of the sesquiterpene hydrocarbons β-caryophyllene (7.1–52.0%) and germacrene D (8.7–17.0%). Other important components were β-pinene from T. montanum and α-pinene from T. flavum. The investigated essential oils were proved to reduce lesion number in the local host Chenopodium quinoa Willd. infected with Cucumber Mosaic Virus (CMV), with reductions of 41.4%, 22.9%, 44.3% and 25.7%, respectively.

Two different volatile isolates from the aerial parts of Cardaria draba (L.) Desv., obtained either by hydrodistillation (Extract I) or by CH(2) Cl(2) extraction subsequent to hydrolysis by exogenous myrosinase (Extract II), were characterized by GC-FID and GC/MS analyses. The main volatiles obtained by hydrodistillation, i.e., 4-(methylsulfanyl)butyl isothiocyanate (1; 28.0%) and 5-(methylsulfanyl)pentanenitrile (2; 13.8%), originated from the degradation of glucoerucin. In Extract I, also volatiles without sulfur and/or nitrogen were identified. These were mostly hexadecanoic acid (10.8%), phytol (10.2%), dibutyl phthalate (4.5%), and some other compounds in smaller percentages. Extract II contained mostly glucosinolate degradation products. They originated from glucoraphanin, viz., 4-(methylsulfinyl)butyl isothiocyanate (3; 69.2%) and 5-(methylsulfinyl)pentanenitrile (4; 4.5%), glucosinalbin, viz., 2-(4-hydroxyphenyl)acetonitrile (5; 7.2%), and glucoerysolin, viz., 4-(methylsulfonyl)butyl isothiocyanate (6; 5.0%). Moreover, the volatile samples were evaluated for their antimicrobial activity using the disc-diffusion method and determining minimum inhibitory concentrations (MIC). All volatile isolates expressed a wide range of growth inhibition activity against both Gram-positive and Gram-negative bacteria and fungi. The MIC values varied between 4 and 128 μg/ml.

In this study, essential oils from wild-grown caper (Capparis spinosa L.) and sea fennel (Crithmum maritimum L.) from Dalmatia (Croatia) were iso- lated by hydrodistillation and analyzed by gas chromatography with mass spectrometry. Methyl isothiocyanate (92.06%) was detected as major compo- nent of essential oil from caper leaves and flower buds, while limonene (58.37%), sabinene (26.46%), terpinene-4-ol (5.59%) and g-terpinene (2.81%) were identified as dominant compounds in sea fennel essential oil. Tested essential oils did not exhibit radical-scavenging activity by 1,1- diphenyl-2-picrylhydrazyl hydrate method, while they showed the antioxidant activity by b-carotene bleaching method and thiobarbituric acid reactive species assay. In addition, sea fennel essential oil exhibited very high inhibi- tory effect on the oxidation of human low-density lipoprotein.

The volatile constituents from the leaves and roots of Raphanus sativus var. black, white and red were isolated by hydrodistillation in Clevenger-type apparatus and were analyzed by gas chromatography and gas chromatography – mass spectrometry (GC, GC-MS). The major components found in the oil of all leaves were: phytol (65.3 – 69.7 %), hexadecanoic acid (2.5 – 14.3 %), methyl linolenate (2.1 – 11.1 %), (Z)-3-hexene-1-ol (0.7 – 6.9 %). Corresponding aliphatic or aromatic glucosinolates compounds identified in the leaves constituted only 0.3 – 5.7 % of the isolated volatiles. They were: 5-methylthio-4-pentenenitrile (0.3 - 2.2 %), 4-methylthiobutyl isothiocyanate (0.3 - 1.7 %), 2-phenylethyl isothiocyanate (0.4 %), 4-methylthio-3-butenyl isothiocyanate, 4-methylpentyl isothiocyanate, benzyl isothiocyanate and benzenepropanenitrile. The major components in the oil of all roots were: hexadecanoic acid (30.3 – 49.9 %), 4-methylthiobutyl isothiocyanate (17.9 - 25.7 %), methyl linolenate (8.5 – 21.7 %), 5-methylthio-4-pentenenitrile (1.5 – 6.9 %), dimethyltrisulfide (1.1 - 3.8 %), 5-methylthiopentyl isothiocyanate (0.5 - 2.0 %), 4-methylthio-3-butenyl isothiocyanate (0.2 - 1.8 %) and 2-phenylethyl isothiocyanate (0.1 - 1.5 %). O-Glycosidically bound volatiles were isolated and hydrolyzed by  -glucosidase from almonds, nineteen volatile aglycons were identified for the first time in all the leaves of radish. The main aglycones were: eugenol (29.2 – 37.1 %), 2-phenylethanol (2.0 - 23.5 %), 4-vinyl-2-methoxyphenol (2.4 – 12.4 %), (Z)-3-hexen-1-ol (2.6 – 10.4 %), methyl vanillate (6.3 – 10.0 %), 1H-indole (0.7 – 6.8 %), benzyl alcohol (1.4 – 4.2 %), vanillin (3.5 – 6.3 %) and methyl salicylate (0.8 – 3.4 %). However, no volatile aglycons were detected after isolation in different varieties of radish roots.

The extracts obtained by 80% EtOH from some Asteraceae plants ( Calendula officinalis, Inula helenium, Arctium lappa, Artemisia absinthium and Achillea millefolium) were studied. Rosmarinic acid, one of the main compounds identified in all extracts, was determined quantitatively by using HPLC. In addition, spectrophotometric methods were evaluated as an alternative for rosmarinic acid content determination. Total phenolic content was also established for all extracts. A. millefolium extract was found to have the highest content of rosmarinic acid as well as total phenols. All extracts were tested for antioxidant and acetylcholinesterase inhibitory activity. A. millefolium was shown to possess the best antioxidant activity (for all tested methods) as well as acetylcholinesterase inhibitory activity. Highly positive linear relationships were obtained between antioxidant/acetylcholinesterase inhibitory activity and the determined rosmarinic acid content indicating its significance for the observed activities.