| Abstract | SAŽETAK Raspodjela glukozinolata unutar biljnih porodica reda Brassicales istražena je UHPLC- DAD-MS/MS analizom desulfoglukozinolata na temelju određenih kvalitativnih i kvantitativnih informacija i to u različitim biljnim tkivima četrdeset osam (48) biljaka iz pet (5) biljnih porodica (Brassicaceae, Capparidaceae, Moringaceae, Resedaceae, Tropaeolaceae) koje rastu u Republici Hrvatskoj i šire. Razvijena je baza MS2 spektara desulfoglukozinolata korištenjem komercijalno dostupnih standarda kao i glukozinolata izoliranih iz prirodnih izvora. Biljna porodica Brassicaceae je sadržavala najraznolikije glukozinolate koje potječu od sedam (7) aminokiselinskih prekursora, i to najčešće od metionina, praćeno s glukozinolatima koji potječu od triptopfana te fenilalanina i/ili tirozina, dok su razgranati alifatski glukozinolati koji potječu od valina, leucina i izoleucina zastupljeni tek u rijetkim vrstama. Jedini poznati glukozinolat, glukokaparin, koji potječe od aminokiseline alanina identificiran je samo u porodici Capparidaceae i to kao glavni u svim istraženim vrstama, dok su ostali glukozinolati u većoj mjeri potjecali od triptofana. Moringa oleifera (porodica Moringaceae) sadržavala je 4-(α-L- ramnopiranoziloksi)benzil-glukozinolat te u manjim količinama tri (3) O-acetilirana izomera istog glukozinolata čiji aminokiselinski prekursor je fenilalanin i/ili tirozin. U vrstama Reseda lutea i R. phyteuma (porodica Resedaceae) identificiran je izomer 4-(α-L-ramnopiranoziloksi)benzil-glukozinolata, 2-(α-L-ramnopiranoziloksi)benzil- glukozinolat karakterističan za biljnu porodicu Resedaceae, koji potječe od fenilalanina, dok je u R. alba kao glavni glukozinolat identificiran 2-hidroksi-2-metilpropil- glukozinolat koji potječe od izoleucina. Tropaeolum majus je sadržavao arilalifatski benzil-glukozinolat kao glavni, praćen sa 3-metoksibenzil-glukozinolatom, a oba potječu od fenilalanina, jedinog poznatog aminokiselinskog prekursora glukozinolata u porodici Tropaeolaceae. Uvidom u kvantitativni sastav glukozinolata odabrane su biljne vrste kao dobri izvori glukozinolata koji su izolirani u obliku desulfoglukozinolata i potvrđeni spektroskopskim tehnikama (MS2, 1 H, 13 C, COSY i HSQC NMR). 3-(Metilsulfinil)propil-glukozinolat je izoliran iz biljke Anastatica hierochuntica (porodica Brassicaceae), 4-hidroksibenzil-glukozinolat iz vrste Sinapis alba (porodica Brassicaceae), metil-glukozinolat iz Capparis orientalis (porodica Capparidaceae), 2-hidroksi-2-metilpropil-glukozinolat iz biljke Reseda alba (porodica Resedaceae) te benzil-glukozinolat iz Tropaeolum majus (porodica Tropaeolaceae). Također, na temelju MS2 spektara i biosintetskih karakteristika identificirana su 4 nova glukozinolata: orto ili meta hidroksi-4-(α-L-ramnopiranoziloksi)benzil-glukozinolat (Moringa oleifera), 4´-O- (β-D-apiofuranoziloksi)benzil-glukozinolat (Hesperis laciniata i Thlaspi perfoliatum), 4- hidroksi-1-metoksiindol-3-ilmetil-glukozinolat (Thlaspi perfoliatum) te 3-hidroksi-(4- metilsulfonil)butil-glukozinolat (Barbarea vulgaris) čije strukture trebaju dodatnu potvrdu NMR-om. Stabilnost desulfo-4-(metilsulfinil)but-3-enil-glukozinolata izoliranog iz biljke Matthiola incana ispitivana je tijekom jednomjesečnog razdoblja te je utvrđena njegova nestabilnost u vodenoj otopini, odnosno drastičan pad sadržaja (do 80 %) praćen spontanom konverzijom u cikličke tioimidatne N-okside. Korištenjem konvencionalnih (enzimska hidroliza i hidrodestilacija) te modernih metoda (mikrovalna destilacija te hidrodifuzija i gravitacija) ispitan je enzimski i termički utjecaj na razgradnju glukozinolata. U uvjetima termičke razgradnje potpomognute mikrovalovima dobiveni su manji prinosi razgradnih produkata u odnosu na termičku razgradnju tijekom hidrodestilacije te enzimsku hidrolizu. Izotiocijanati kao razgradni produkti glukozinolata su se pokazali nestabilnim pri uvjetima GC-MS mjerenja i to za ω-metilsulfinilne C3, C5 i C6 izotiocijanate čiji je raspad rezultirao nastankom odgovarajućih olefinskih izotiocijanata. Dodatno, zbog prisutnosti elektron-donirajuće hidroksilne skupine u para položaju na benzenskom prstenu, kod 4-hidroksibenzil- izotiocijanata i 4-hidroksi-3,5-dimetoksibenzil-izotiocijanata, utvrđena je nestabilnost koja vodi ka nastanku artefakata tijekom GC-MS mjerenja. Također, razgradni produkti indolnih glukozinolata nisu identificirani. Ovime je istaknut nedostatak neizravne metode identifikacije glukozinolata preko razgradnih produkata u biljnim vrstama. U biljci Eruca vesicaria biofortificiranoj selenijem iz natrijevog selenata identificiran je 4-(metilseleno)butil-glukozinolat izveden iz selenometionina na temelju MS2 spektra koji je po prvi puta prikazan. Utvrđeno je da hidroponski uzgojene klice pet (5) odabranih biljaka iz porodice Brassicaceae imaju visoki sadržaj karakterističnih glukozinolata zbog čega predstavljaju dobar izvor za njihovu izolaciju. |
| Abstract (english) | ABSTRACT Distribution of glucosinolates within plant families of the order Brassicales was investigated using qualitative and quantitative UHPLC-DAD-MS/MS analysis of desulfoglucosinolates in different plant tissues of forty-eight (48) plants from five (5) plant families (Brassicaceae, Capparidaceceae, Moringaceae, Resedaceae, Tropaeolaceae) growing in the Republic of Croatia and worldwide. A database of MS2 spectra of desulfoglucosinolates was developed using commercially available standards as well as glucosinolates isolated from natural sources. The Brassicaceae plant family contained a wide variety of glucosinolates derived from seven (7) different amino acid precursors, most commonly from methionine, followed by glucosinolates derived from tryptophan, phenylalanine and/or tyrosine, while branched aliphatic glucosinolates deriving from valine, leucine and isoleucine were scarce. Glucocapparin, the only known glucosinolate derived from the amino acid alanine, was identified solely in the Capparidaceae family as the main one in all the species studied, while the other glucosinolates largely derived from tryptophan. Moringa oleifera (family Moringaceae) contained 4-(α-L-rhamnopyranosyloxy)benzyl glucosinolate and, in less amount, three O-acetylated isomers of the same glucosinolate derived from phenylalanine and/or tyrosine. In the species Reseda lutea and R. phyteuma (family Resedaceae) an isomer of 4-(α-L-rhamnopyranosyloxy)benzyl glucosinolate, 2- (α-L-rhamnopyranosyloxy)benzyl glucosinolate, was identified characteristic for the plant family Resedaceae, originating from phenylalanine. On the other hand, 2-hydroxy- 2-methylpropyl glucosinolate derived from isoleucine was the major glucosinolate identified in R. alba. The main glucosinolate in Tropaeolum majus was arylaliphatic benzyl glucosinolate followed by 3-methoxybenzyl glucosinolate, both derived from phenylalanine, the only known amino acid precursor of glucosinolates in the Tropaeolaceae family. Insight into the quantitative composition of glucosinolates enabled the selection of the plant species as the sources of glucosinolates for their isolation in the form of desulfoglucosinolates and confirmation by spectroscopic techniques (MS2, 1 H, 13 C, COSY and HSQC NMR). 3-(Methylsulfinyl)propyl glucosinolate was isolated from Anastatica hierochuntica (family Brassicaceae), 4-hydroxybenzyl glucosinolate from Sinapis alba (family Brassicaceae), methyl glucosinolate from Capparis orientalis (family Capparidaceae), 2-hydroxy-2-methylpropyl glucosinolate from Reseda alba (family Resedaceae) and benzyl glucosinolate from Tropaeolum majus (family Tropaeolaceae). Also, based on MS2 spectra and biosynthetic characteristics, four (4) new glucosinolates were identified: ortho or meta hydroxy-4-(α-L- rhamnopyranosyloxy)benzyl glucosinolate (Moringa oleifera), 4´-O-(β-D- apiofuranosyloxy)benzyl glucosinolate (Hesperis laciniata and Thlaspi perfoliatum), 4- hydroxy-1-methoxyindol-3-ylmethyl glucosinolate (Thlaspi perfoliatum) and 3-hydroxy- (4-methylsulfonyl)butyl glucosinolate (Barbarea vulgaris) whose structures need further confirmation by NMR. The stability of desulfo-4-(methylsulfinyl)but-3-enyl glucosinolate isolated from Matthiola incana was studied over a one-month period and its instability in aqueous solution was observed, showing a significant drop in the content (up to 80%) followed by spontaneous conversion to cyclic thioimidate N-oxides. The enzymatic and thermal degradation of glucosinolates was studied by using conventional (enzymatic hydrolysis and hydrodistillation) and modern methods (microwave distillation and hydrodiffusion and gravity). Under microwave-assisted thermal decomposition conditions, lower yields of decomposition products were obtained compared to thermal decomposition during hydrodistillation and enzymatic hydrolysis. Isothiocyanates as degradation products of glucosinolates showed to be unstable at GC-MS conditions for ω-methylsulfinyl C3, C5 and C6 isothiocyanates, which decomposition resulted in the formation of the corresponding olefinic isothiocyanates. Additionally, due to the presence of an electron- donating hydroxyl group in the para position on the benzene ring, in 4-hydroxybenzyl isothiocyanate and 4-hydroxy-3,5-dimethoxybenzyl isothiocyanate, instability lead to formation of artifacts found during GC-MS analysis. Also, degradation products of indole glucosinolates have not been identified. This highlights the disadvantage of an indirect method for the identification of glucosinolates via their degradation products in plant species. In the Eruca vesicaria biofortified by selenium from sodium selenate, 4- (methylseleno)butyl glucosinolate derived from selenomethionine was identified based on the MS2 spectrum shown for the first time. Hydroponically grown sprouts of the five (5) selected plants of Brassicaceae family were found to have a high content of characteristic glucosinolates presenting a good source for their isolation. |
