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Bisphenol A (BPA) is used as intermediate in production of plastic-based materials. Since plastic materials are widely used in daily life, the amount of BPA in the environment is increasing. Therefore, it is necessary to investigate the effects of BPA on environment, especially on terrestrial plants [1]. The aim of this study was to test whether oxidative stress is mechanism of BPA toxicity. Onion (Allium cepa L.) was used as plant model. Onion roots were treated with BPA at concentrations of 1, 5, 10, 25 and 50 mg/L for three days. After treatment, onion bulbs were collected, oven dried (at 60 °C), and homogenized in PBS buffer (50 mg/mL). The homogenates were centrifuged (10000 g for 10 min) and oxidative stress parameters were measured in supernatant. The level of malondialdehyde (MDA), as a marker of lipid peroxidation, was determined spectrophotometrically with thiobarbituric acid and reactive oxygen species (ROS) were followed with fluorescent probes: H2O2 with fluorescent probe 2’, 7’-dichlorofluorescin diacetate and O2 with fluorescent probe dihydroethidium. Antioxidative defense was determined by assessing superoxide dismutase (SOD) activity using commercial kit (Cayman Chemical, Ann Arbor, MI, USA). All measurements were performed on microplate reader (SpectraMax i3x, Molecular Devices, San Jose, CA, USA). Significant differences between treatments were examined using a one-way test (ANOVA) followed by Duncan's multiple range test. In onion bulbs significant increase in MDA content was observed already after the treatment with BPA at concentration of 5 mg/L. The same BPA concentration (5 mg/L) resulted in a significant increase in H2O2 and O2 level. The decrease in SOD activity was recorded after treatment with the lowest BPA concentration (1 mg/L) indicating that even the lowest applied BPA concentration causes a change in antioxidative defense. From the obtained results it can be concluded that BPA induces changes in oxidative stress parameters, suggesting that oxidative stress is mechanism of BPA phytotoxicity. Funding: Bilateral collaboration projects between Republic of Slovenia and Republic of Croatia (BI- HR/18-19-003 ; BI-HR/20-21-031) ; University of Zagreb ; European Regional Development Fund project FarmInova (KK.01.1.1.02.0021). [1] V. Vujčić Bok, M. Gerić, G. Gajski, S, Gagić and A.-M. Domijan. Phytotoxicity of Bisphenol A to Allium cepa Root Cells Is Mediated through Growth Hormone Gibberellic Acid and Reactive Oxygen Species. Molecules. Vol. 28, 2023, 2046, 1-15.

The 2-methoxy-1, 4-naphthoquinone (2-MNQ, lawsone methyl ether) is chemical compound found in plants and microorganisms with diverse biological activities (antibacterial, antifungal, antiviral, and antitumor) [1]. The aim of this study was to investigate the impact of 2-MNQ on germination and oxidative stress parameters of white mustard (Sinapis alba L.). The seeds of white mustard (n = 25) were placed in Petri dishes on filter paper impregnated with 2-MNQ at the following concentrations: 0, 1, 5, 10, 20, 30 and 40 µg/mL and germinated in dark at room temperature for three days. Each concentration was tested in triplicate and 0.02 M CuSO4 was used as positive control. After germination, the seedlings were weighed, and roots length were measured. For spectrophotometric assessment of oxidative stress parameters: superoxide dismutase (SOD), glutathione (GSH) and malondialdehyde (MDA) plant tissue homogenates were prepared. Significant differences between treatments were tested by one- way ANOVA followed by Duncan's multiple range test. Germination of white mustard seeds was significantly reduced after exposure to 5 µg/mL of 2-MNQ while exposure to 2-MNQ at concentrations 30 and 40 µg/mL, and positive control complete ceased germination of white mustard. The reduction in fresh tissue weight and root length of white mustard seedlings was observed already after exposure to 1 µg/mL of 2-MNQ. Decrease in MDA content was detected in seedlings exposed to 10 and 20 µg/mL of 2-MNQ. The lowest 2-MNQ concentration (1 µg/mL) significantly reduced GSH level of white mustard seedlings, while 2-MNQ at concentrations 5-20 µg/mL significantly increased GSH levels. Significant increase in SOD activity was detected in white mustard seedlings exposed to 1 and 10 µg/mL of 2-MNQ. It can be concluded that 2-MNQ is phytotoxic to white mustard and that oxidative stress is involved in its phytotoxic effect. [1] Ž. Maleš, A.-M. Domijan, I. Duka, T. Marić, M. Bojić, H. Rimac, B. Mitić, D. Hruševar, K. Barišić and D. Verbanac. Determination of naphthoquinones in invasive alien plants Impatiens glandulifera Royle and I. balfourii Hook. from Croatia. Croatica Chemica Acta, Vol. 95, 2022, 1-5.

Konzumacija svježih klijanaca kupusnjača značajno je porasla posljednjih godina, stoga je njihova nutritivna karakterizacija nužna. U ovom radu spektrofotometrijski je izmjeren udio ukupnih fenola, flavonola i hidroksicimetnih kiselina, te su novorazvijenom metodom tekućinske kromatografije visoke razlučivosti analizirani pojedinačni flavonoidi, fenolne kiseline i vitamin C. Određen je i antioksidacijski potencijal pet razvojnih stadija kineskog kupusa. Razvojni stadiji sadržavali su: 1) samo kotiledone (klijanci), 2) kotiledone i dva lista, 3) četiri lista, 4) šest listova ili 5) deset listova. Analiza glavnih komponenata (PCA) i hijerarhijsko grupiranje (HC) korišteni su za vizualizaciju trenda klasifikacije između faza. Klijanci su sadržavali više sinapinske kiseline i vitamina C od starijih biljaka. Biljke koje su sadržavale šest ili deset listova imale su više ferulične kiseline i izoramnetina od onih u ranijim fazama razvoja. Ukupni fenoli, flavonoli, hidroksicimetne kiseline, kvercetin i antioksidacijski potencijal nisu se statistički razlikovali između klijanaca i stadija sa šest ili deset listova, te su njihove koncentracije bile značajno veće nego u stadijima s dva ili četiri lista. PCA i HC potvrdili su veću fitokemijsku sličnost između klijanaca i biljaka sa šest ili deset listova, nego biljaka s dva ili četiri lista. Na temelju navedenih parametara, klijanci kineskog kupusa i biljke sa šest ili deset listova bile bi bolji odabir od biljaka s dva ili četiri lista.

The goal of our study was to evaluate the effect of high (HT: 38°C day/33°C night) and regular (RT: 23°C day/18°C night) growing temperature on the bioavailability of total glucosinolates and phenolics from broccoli (Brassica oleracea L. convar. botrytis (L.) Alef. var. cymosa Duch) sprouts upon final, intestinal, phase of in vitro simulated human digestion. The extracts were prepared from freeze-dried plant material using boiling (100°C) water followed by incubation on rotary extractor for 1 hour at 23°C. The results showed that HT negatively affected the intestinal bioavailability of total glucosinolates from broccoli sprouts, i.e. their concentration was decreased to 47 % of the initial concentration (non-digested extract). On the other hand, RT did not change the bioavailability of total glucosinolates significantly (p≤0.05). Total phenolics bioavailability was not affected neither by HT, nor by RT condition ; in both cases it was very high (>90 %). These results show that glucosinolates of broccoli sprouts are significantly more susceptible to HT than total phenolics, and suggests future more detailed analyses of Brassicaceae microgreens phytochemical temperature susceptibility/resistance.

Plants challenged with high temperature (HT) undergo many adaptive mechanisms at molecular levels to keep normal physiological function. Such adaptations consequently influence their bioactivity. In this work, within the scope of the TEMPHYS project (IP-2020-02- 7585), we investigated the effect of HT on the antidiabetic activity of broccoli (Brassica oleracea L. convar. botrytis (L.) Alef. var. cymosa Duch.) seedlings. The activity was spectrophotometrically assessed via the percentage of inhibition of α-amylase and α-glucosidase, enzymes required for carbohydrate digestion. Enzyme inhibitory activity was calculated from the equation: % inhibition = 100 – [(At – Atb / Ac – Acb) x 100], where At was absorbance of test, Atb was absorbance of test blank, Ac was absorbance of control, and Acb was absorbance of control blank. Seedlings were grown in a climatic chamber with temperature range 23°C/16h, 18°C/8h for control group, and 38°C/16h, 33°C/8h for HT group. The extracts were prepared from freeze- dried plant material using boiling (100°C) water followed by incubation on rotary extractor for 1 hour at 23°C. The results showed that broccoli seedlings aqueous extracts were more efficient in the inhibition of α-amylase (88% relative to the standard acarbose), than α-glucosidase (67% relative to the standard acarbose). HT significantly (p≤0.05) decreased the potential to inhibit α-amylase (80%), however it improved the potential to inhibit α-glucosidase (70%). These results show that high growing temperature significantly changes the biological effects of plants ; moreover, their activity toward different enzymes is specifically affected. Further more detailed analyses into the effect of global warming on cruciferous (Brassicaceae) vegetables bioactivity are needed.

The high demand for healthy food in recent years led to an increasing need for highly bioactive plant materials. One simple method to improve the nutritional properties of plants could be the treatment of plants of known bioactive potential (acceptor) with extracts of different plant species (donors) that contain additional high bioactive compounds, so-called interspecies source-sink phytochemical transfer. The aim of the present study was to evaluate the effect of interspecies transfer of metabolites on plant phenolic and vitamin C profile to improve their gastrointestinal bioavailability, hypoglycemic potential, and antioxidant capacity as well as cytotoxicity. Additionally, it was tested whether there is an effect of phytochemical transfer on the expression of marker genes in phenolic biosynthetic pathways. Chinese cabbage (Brassica rapa ssp. pekinensis) sprouts were chosen as a model of plant acceptor, while the inflorescences of St. John`s-wort (Hypericum perforatum) and chamomile (Matricaria chamomilla), leaves of rose (Rosa sp.) and shoots of black bryony (Tamus communis) were used as plant donors. Even though the chamomile extract increased the highest number of individually identified compounds in Chinese cabbage sprouts, that was not crucial for the improvement of their bioactivity. Rather, black bryony application induced more biopotential parameters in the sprouts such as significantly improved bioavailability of vitamin C, kaempferol and total phenolics both before and during gastrointestinal digestion of sprouts, increased hypoglycemic activity of sprouts, and their antioxidant capacity during digestion. For an increased cytotoxicity against MCF cells, St. John`s-wort, rose and chamomille extracts could be applied to Chinese cabbage sprouts. The results suggest that interspecies phytochemical transfer could enhance the phytochemical profile and the bioactive properties of the acceptor plant.

Arctostaphylos uva-ursi je tradicionalna biljka koja se koristi u terapeutske svrhe za liječenje akutnih urinarnih infekcija, a njezina antiseptička svojstva mogu se pripisati hidrokinonu koji nastaje hidrolizom arbutina. Cilj ove studije bio je odrediti toksikološki profil hidrokinona na stanicama mokraćnog mjehura (T24). Kvantitativna i kvalitativna analiza ekstrakta i digestivna stabilnosti te bioraspoloživosti arbutina i hidrokinona provedena je HPLC testom i simuliranom in vitro probavom. Citotoksični učinak, indukcija reaktivnih kisikovih vrsta (ROS) i promjene u proteomu utvrđeni su Neutral red i DCFH-DA testom te spektrometrijom masa. Tijekom in vitro probave, arbutin je bio stabilan, a koncentracija hidrokinona se povećala nakon želučane probave pepsinom (109, 6%), a zatim se smanjila nakon faze tankog crijeva (65, 4%). Preporučene doze napitka imale su citotoksični učinak kada su svi konjugati hidrokinona bili prevedeni u slobodni hidrokinon (320 i 900 μg/mL), a toksični učinak je bio pojačan 24-satnim oporavkom. Jedna šalica terapijske doze imala je prooksidativni učinak nakon 4 h inkubacije, a kraće vrijeme tretmana (2 h) nije imalo utjecaja na indukciju ROS-ova. Proteomska analiza otkrila je 17 statistički znatno više eksprimiranih proteina u odnosu na kontrolu povezanih s odgovorom na oksidativni stres, signalizaciju prilagođenog odgovora na stres, „heat-shock“ odgovorom i inicijacijom translacije. Dobiveni rezultati su dokaz da biljni spojevi, iako iz prirodnog izvora, mogu pokazati negativna svojstva.

Biljke se visokoj temperaturi (VT) prilagođavaju promjenom transkriptoma, proteoma i metaboloma. Takva prilagodljivost posljedica je njihovih intenzivnih promjena u biokemijskim putevima, što može značajno promijeniti i njihove biološke učinke. U ovom radu istražili smo učinak VT na antiglikacijski potencijal vodenog ekstrakta klijanaca brokule (Brassica oleracea L. convar. botrytis (L.) Alef. var. cymosa Duch.), te njihovu mogućnost inhibicije α-lipaze, enzima potrebnog za hidrolizu prehrambenih lipida. Potencijal antiglikacije procijenjen je postotkom inhibicije stvaranja krajnjih produkata glikacije (AGEs, advanced glycation end products), a potencijal hidrolize prehrambenih lipida putem postotka inhibicije α-lipaze. Klijanci su uzgajani u klima komori s temperaturnim rasponom 23°C/16h, 18°C/8h - kontrolnu skupina i 38°C/16h, 33°C/8h - VT ). Ekstrakti su pripremljeni iz liofiliziranog biljnog materijala u kipućoj (100°C) vodi nakon čega je slijedila inkubacija na rotacijskom ekstraktoru 1 sat/23°C. Rezultati su pokazali da su vodeni ekstrakti HT-klijanaca brokule učinkovitiji u inhibiciji stvaranja AGEs od kontrolne skupine. Tijekom in vitro simulirane ljudske probave, potencijal inhibicije je povećan, ali je razlika između HT- i kontrolne skupine smanjena. S druge strane, aktivnost α-lipaze nije se značajno razlikovala (p≤0, 05) između HT- i kontrole, a potencijal inhibicije tijekom probave se smanjio. Dodatna identifikacija proteina, čija se količina promjenila, je trenutno u procesu. Ovi rezultati pokazuju da HT značajno mijenja biopotencijal biljaka.

The main goal of our study was to evaluate the effect of proteins (40 g/L casein), carbohydrates (25 g/L glucose) and oils (5% olive oil) on phytochemical bioavailability, antioxidant, antidiabetic and weight loss activity of gingko leaves standardized extracts after in vitro gastrointestinal digestion. Spectrophotometric methods were used to determine the concentration of total phytochemicals (TP: total phenols, TF: total flavonoids, TPA: total phenolic acids, TFLA: total flavanols, TFLO: total flavonols, TPAN: total proanthocyanidins and TTL: total triterpene lactones), antioxidant (DPPH and FRAP), antidiabetic (inhibition of α-glucosidase and BSA glycation) and weight loss (inhibition of lipase) activity. Ginkgo olive oil formulation had the highest TFLO, TFLA, TPAN and TTL bioavailability after in vitro gastrointestinal digestion. The highest TF and TPA bioavailability had gingko casein formulation. In the gingko water formulations, the highest TP bioavailability was detected. All gingko formulations had high (>70%) antioxidant activity (DPPH and FRAP), high (>70%) enzyme (α-glucosidase and lipase) inhibitory activity and high (>70%) inhibition of BSA glycation after in vitro digestion. According to our results, ginkgo formulation with olive oil has proved to be the formulation with the highest bioavailability of polyphenolic groups and terpene lactones after in vitro gastrointestinal digestion.

The high demand for healthy food in recent years led to an increasing need for highly bioactive plant materials. One simple method to improve the nutritional properties of plants could be the treatment of plants of known bioactive potential (acceptor) with extracts of different plant species (donors) that contain additional high bioactive compounds, socalled interspecies source-sink phytochemical transfer. The aim of the present study was to evaluate the effect of interspecies transfer of metabolites on plant phenolic and vitamin C profile to improve their gastrointestinal bioavailability, hypoglycemic potential, and antioxidant capacity as well as cytotoxicity. Additionally, it was tested whether there is an effect of phytochemical transfer on the expression of marker genes in phenolic biosynthetic pathways. Chinese cabbage (Brassica rapa ssp. pekinensis) sprouts were chosen as a model of plant acceptor, while the inflorescences of St. John`s-wort (Hypericum perforatum) and chamomile (Matricaria chamomilla), leaves of rose (Rosa sp.) and shoots of black bryony (Tamus communis) were used as plant donors. Even though the chamomile extract increased the highest number of individually identified compounds in Chinese cabbage sprouts, that was not crucial for the improvement of their bioactivity. Rather, black bryony application induced more biopotential parameters in the sprouts such as significantly improved bioavailability of vitamin C, kaempferol and total phenolics both before and during gastrointestinal digestion of sprouts, increased hypoglycemic activity of sprouts, and their antioxidant capacity during digestion. For an increased cytotoxicity against MCF cells, St. John`s-wort, rose and chamomille extracts could be applied to Chinese cabbage sprouts. The results suggest that interspecies phytochemical transfer could enhance the phytochemical profile and the bioactive properties of the acceptor plant.

Exposure of plants to extreme temperatures present challenges for global food security because it could cause changes in metabolite profile. Such changes could have a significant impact on the nutritional and health properties of plants, and indirectly (via plant-based-diet) on animal health. This particularly applies to the widely cultivated and consumed Brassicaceae family, including crops of great economical importance, such as Brassica oleracea and B. rapa, which present a major part of human diet. The aim of this preliminary study was to differentiate high temperature (HT) susceptible from resistable metabolic parameters of broccoli (Brassica oleracea L. convar. botrytis) seedlings before and during in vitro simulated human digestion. Methods included in vitro simulated human digestion, spectrophotometric quantification of total phenolics, flavonoids, flavonols, anthocyanins, hydroxycinnamic acids and antioxidant capacity by the methods ABTS, FRAP and DPPH. Using a RP-HPLC method, vitamin C, kaempferol, quercetin, tiliroside, gallic, p-coumaric, benzoic, caffeic, ferulic and sinapic acid were identified and quantified. Before enzymatic digestion, the highest number of parameters were resistable to HT, however total phenolics, flavonols, benzoic acid and kaempferol were increased, while ABTS, vitamin C, caffeic and ferulic acid were decreased. Both before and after digestion, benzoic acid was increased, total flavonoids resistable, while vitamin C and caffeic acid reduced. During digestion, total hydroxycinnamic acids, flavonols, benzoic and sinapic acid were increased, total phenolics and flavonoids resistable, and caffeic acid was decreased. The results indicate plasticity of brocolli seedlings metabolism under the HT influence, and provide a basis for further research of this effect.

Zbog povećane težnje zdravijoj prehrani, interes za svježu, funkcionalnu biljnu hranu raste. Povrće iz porodice kupusnjača (Brassicaceae) bogato je fenolnim spojevima, vitaminima i glukozinolatima, te posjeduje snažan antioksidacijski potencijal što doprinosi pozitivnom djelovanju na ljudski organizam. U posljednje vrijeme sve je učestalija konzumacija klica ove porodice. Klice su mlado tkivo koje je biokemijski vrlo aktivno i u mnogo slučajeva su bogatije bioaktivnim spojevima od zrelih jedinki. Sadržaj tih tvari varira od varijeteta do varijeteta pa su potrebne usporedne analize kako bi se utvrdilo klice kojih varijeteta su bolji izvor određenog bioaktivnog spoja ili biološkog učinka. Cilj ovog rada bio je usporediti koncentracije fenolnih spojeva klica korabice (Brassica oleracea var. acephala gongylodes), kelja (Brassica oleracea sabauda), prokulice (Brassica oleracea gemmifera), cvjetače (Brassica oleracea botrytis), rotkvice (Raphanus sativus) i kreše (Lepidium sativum), te njihov antioksidacijski potencijal kako bi se za svaki od navedenih parametara moglo izdvojiti klice većeg potencijala. Klice su uzgojene u klima komori pri sobnoj temperaturi na vlažnom filter papiru i sakupljene kada su dosegle stadij s dva prava lista. Koncentracija ukupnih fenolnih spojeva nije se značajno razlikovala između analiziranih varijeteta klica. Najbogatije ukupnim flavonoidima bile su klice cvjetače (10, 7 mg ekvivalenata kvercetina/g suhe mase (SM)). Najbogatije ukupnim hidroksicimetnim kiselinama bile su klice kelja (20, 7 mg ekvivalenata cimetne kiseline/g SM) i rotkvice (20, 5 mg ekvivalenata cimetne kiseline/g SM). Najviša koncentracija ukupnih tanina zabilježena je kod klica korabice (4, 0 mg ekvivalenata taninske kiseline/g SM), a najniža kod klica kelja (2, 3 mg ekvivalenata taninske kiseline/g SM). Prema metodi ABTS, sve klice, osim onih rotkvice, imale su veći antioksidacijski potencijal od standardnog antioksidacijskog spoja troloksa koncentracije 7, 5 mg/mL i 50 mg/mL. Prema metodi DPPH, klice kelja, korabice, prokulice i cvjetače imale su antioksidacijski potencijal sličan onome standarda troloksa spomenutih koncentracija. Prema metodi FRAP, sve testirane klice imale su gotovo identičan antioksidacijski potencijal kao standard troloks. S obzirom na rezultate, zaključujemo da klice kupusnjača predstavljaju vrijedan biomaterijal velikog potencijala za ljudsku prehranu i zdravlje, te su daljnje analize njihova biopotencijala itekako potrebne i poželjne. / Due to the increased desire for a healthier diet, interest in fresh, functional plant foods is growing. Vegetables from the cabbage family (Brassicaceae) are rich in phenolic compounds, vitamins and glucosinolates, and have a strong antioxidant potential that contributes to their positive effects on human organism. Lately, the consumption of seedlings of this family has become more frequent. Seedlings are young tissue that is biochemically very active and in many cases are richer in bioactive compounds than mature individuals. The content of these substances varies from variety to variety, so comparative analyzes are needed to determine which seedlings are better source of a particular bioactive compound or biological effect. The aim of this study was to compare the concentrations of phenolic compounds between the seedlings of kohlrabi (Brassica oleracea var. acephala gongylodes), kale (Brassica oleracea sabauda), Brussels sprouts (Brassica oleracea gemmifera), cauliflower (Brassica oleracea botrytis), radish (Raphanus sativus) and cress (Lepidium sativum), and their antioxidant potential so that the seedlings of higher potential could be isolated for each of the analyzed parameter. Seedlings were grown in an air chamber at room temperature on moist filter paper and collected when they reached the two-leaf stage. The concentration of total phenolics did not differ significantly between the analyzed seedling varieties. The highest concentration of total flavonoids had cauliflower seedlings (10, 7 mg quercetin equivalents/g dry weight (DW)). The richest in total hydroxycinnamic acids were seedlings of kale (20, 7 mg of cinnamic acid equivalents/g DW) and radish (20, 5 mg of cinnamic acid equivalents/g DW). The highest concentration of total tannins was recorded in kohlrabi seedlings (4, 0 mg of tannic acid equivalents/g DW), and the lowest in kale seedlings (2, 3 mg of tannin acid equivalents/g DW). According to the ABTS method, all seedlings, except those of radish, had a higher antioxidant potential than the standard antioxidant compound trolox at the concentration of 7, 5 mg/mL and 50 mg/mL. According to the DPPH method, seedlings of cabbage, kohlrabi, Brussels sprouts and cauliflower had an antioxidant potential similar to that of the trolox standard. According to the FRAP method, all tested seedlings had almost identical antioxidant potential as the trolox standard. Given the results, we conclude that Brassicaceae seedlings represent a valuable biomaterial of great potential for human nutrition and health, and further analysis of their biopotential is necessary and desirable.

The principal component analysis (PCA) was used to gain insight into the relationship between phytochemicals (total phenols - TP, total phenolic acid - TPA, total flavonoids -TF, total anthocyanidins - TA ; total hydroxycinnamic acids - THA, and total flavanols - TFL, glucosinolates – Gls, sugar – TS) and antioxidant, antidiabetic and anti-obesity activity in three developmental stages of broccoli after in vitro digestion. Different developmental stages of broccoli (1st stage: seedlings, 2nd stage: plants with 2 real leaves, and 3rd stage: plants with 4-6 real leaves) were collected in July and August 2021, and extracts were prepared in hot 70% ethanol. In PCA the first and the second principal components described 64.62% and 35.38% of the variance after in vitro digestion, respectively. Together, the first two PCs represent 100% of the total variability. After digestion, 1st developmental stage of broccoli had strong loadings with most of the tested chemicals (TA, Gls, TS), DPPH and inhibition of alpha-amylase. Broccoli 2nd developmental stage had strong loadings with TPA and inhibition of lipase, while 3rd developmental stage had strong loadings with TF and inhibition of alpha-glucosidase. Pearson’s correlation coefficient between phytochemicals, antioxidant (ABTS, DPPH and FRAP), antidiabetic (inhibition of alpha-amylase and alpha-glucosidase) and anti-obesity activity (lipase inhibition) was also performed. After digestion, TP correlated very strongly (0.999) with THA, TPA correlated very strongly with antioxidant (FRAP: 0.998) and anti-obesity activity (inhibition of lipase: 1.000), TA correlated very strongly alpha1.000) with antidiabetic activity (inhibition of alpha-amylase), Gls correlated very strongly with antioxidant activity (DPPH: 0.999), and DPPH correlated very strongly (0.997) with alpha-amylase. According to the results after in vitro digestion, 1st developmental stage of broccoli was the best source of TA, Gls and TS, that are closely related with antioxidant activity (DPPH) and key enzyme linked to type-2 diabetes (alpha-amylase). 2nd developmental stage of broccoli was the best source of TPA, that are closely related with inhibition of key enzyme linked to obesity (lipase) ; and 3rd developmental stage was the best source of TF, that are closely related with inhibition of key enzyme linked to type-2 diabetes (alpha-glucosidase).

Brokula Brassica oleracea botrytis var cymosa Brassicacae je bogata polifenolima proteinima, vitaminima i mineralima, te glukozinolatima spojevima specifičnima za porodicu kupusnjača U prehrani su najzastupljeniji njeni zreli zeleni cvijetni izdanci dok su ostali razvojni stadiji zanemareni Budući da je biopotencijal različitih razvojnih stadija brokule n eistražen cilj ovog rada bio je analizirati biodostupnost specijaliziranih metabolita TP ukupnih fenola TPA ukupnih fenolnih kiselina TF ukupnih flavonoida TA ukupnih antocijana THA ukupnih hidroksicimetnih kiselina TFL ukupnih flavonola i Gls glukozinolata i šećera ( te antioksidacijski kapacitet ( DPPH i FRAP) ekstrakata različitih razvojnih stadija brokule prije i nakon pojedine faze in vitro modela ljudske probave Određen je i stupanj inhibicije enzima α amilaze i α glukozidaze povezanih s dijabetesom tipa II) i lipaze povezanog s pretilošću spomenutim ekstraktima prije i nakon probave.

Cilj ovog rada je ispitati sadržaj polifenola i antioksidacijsku aktivnost metanolnih ekstrakata (80%) bijele imele sakupljene u travnju 2017. na Lašćini i na području Maksimira u Zagrebu (Hrvatska) sa nekoliko različitih domadara (lipa - Tilia platyphyllos Scop. , glog - Crataegus monogyna Jacq. i javor - Acer campestre L). Tekućinskom kromatografijom visoke djelotvornosti s obrnutim fazama (RP-HPLC) detektirani su pojedinačni polifenoli, a količina ukupnih fenolnih spojeva određena je pomoću Folin- Ciocalteu metode. Tri različite metode korištene su za određivanje antioksidacijske aktivnosti ABTS (2, 2 –azinobis (3- ethylbenzothiazoline-6-sulfonic acid), DPPH (1, 1-diphenyl-2-picrylhydrazyl) i FRAP (Ferric Reducing/Antioxidant Power Assay). Dio listova imele sa peteljkama je liofiliziran dok je drugi dio sušen u sjeni i na sobnoj temperaturi. Osušeni biljni materijal usitnjen je u komadiće promjera približno 5 mm i ekstrahiran u 80 % etanolu u staklenoj koloni dimenzije 1×10 cm u trajanju od 24h. Perkolat je ispušten iz kolone brzinom od oko 4 kapi u minuti te uparen na rotavaporu na temperaturi od 30 °C . Metanol (80 %) je dodan kako bi se dobio omjer suhe biljne mase i otapala 1:1. HPLC analizom uzoraka listova imele detektirano je ukupno 8-10 fenolnih spojeva, od toga 5-7 fenolnih kiselina (p-kumarinska, galna, klorogenska, kavena, ferulična, sinapilna i salicilna kiselina u uzorcima imele koja je rasla na glogu, klorogenska kiselina nije detektirana u uzorcima imele koja je rasla na javoru i lipi, a galna kiselina nije detektirana u imeli koja je rasla na lipi) i 3 flavonoida (kvercetin, kempferol i isoramentin). Fenolne kiseline p- kumarinska i sinapilna te antioksidacijska aktivnost mjerena sa ABTS i DPPH metodom statistički značajno je bili viša u skoro svim liofiliziranim uzorcima u odnosu na uzorke imele sušene na sobnoj temperaturi. Najviša koncentracija ukupnih i individualnih fenola i generalno najviša antioksidacijska aktivnost detektirana je u uzorcima imele koja je rasla na glogu. Većina uzoraka imele imala je jaku antioksidacijsku aktivnost (>70%) u odnosu na pozitivnu kontrolu Trolox. Na temelju rezultata možemo zaključiti da uzorci imele koji su rasli na glogu predstavljaju najbolji izvor polifenola i antioksidanta koji se mogu primijeniti u fitoterapiji za liječenje različitih oboljenja. Također, liofilizacija ima pozitivan učinak na antioksidacijsku aktivnost (ABTS i DPPH) te na očuvanje koncentracije p-kumarinske i sinapilne kiseline u uzorcima imele./The aim of this study is investigated polyphenol content and antioxidant activity in methanol extract (80%) of common mistletoe (Viscum album L.) collected in April 2017 in Lašćina and Maksimir area in Zagreb (Croatia) from different plant host (linden - Tilia platyphyllos Scop., hawthorn - Crataegus monogyna Jacq. and maple - Acer campestre L.). To achieved that we performed reversed phase high- performance liquid chromatography (RP-HPLC) for individual detection of polyphenols and spectrophotometric Folin-Ciocalteu method for detection of total polyphenol content. Three different spectrophotometric methods for antioxidant activity determination ABTS (2, 2 – azinobis (3- ethylbenzothiazoline-6-sulfonic acid), DPPH (1, 1-diphenyl-2-picrylhydrazyl) and FRAP (Ferric Reducing/Antioxidant Power Assay) were also performed. One part of mistletoes leaves with their petioles were freeze-dried while other half was dried in shadow at room temperature (RT). Dried plant material was chopped into pieces with a diameter of approximately 5 mm end extract with 80% methanol in glass column (1 × 10 cm) for 24 hours. Percolate was discharged from the column at a rate of about 4 drops per minute and evaporated to dryness by rotavapor at 30 ° C. Methanol (80%) was added to give dry matter to solvent ratio of 1:1. With HPLC analysis of mistletoe samples was detected 8-10 phenolic compounds, of which 5-7 phenolic acids (p-coumaric, gallic, caffeic, chlorogenic, ferulic, synapyl and salicylic acid in mistletoe that grew on hawthorn, chlorogenic acid was not detected in mistletoe that grew on linden and maple and gallic acid was not detected in mistletoe that grew on linden) and 3 flavonoids (quercetin, kaempferol and isoramentin). Phenolic acids p-coumaric acid and sinapinic acid and antioxidant activity measured with ABTS and DPPH were statistically higher in freeze-dried than RT- dried plant material in almost all samples. The highest concentration of total and individual phenolics and generally highest antioxidant activity were detected in mistletoe samples that grew on hawthorn. Most mistletoe samples had strong antioxidant activity (> 70%) in relation to positive control Trolox. According to results, mistletoe that grew on hawthorn is the best source of polyphenols and antioxidants that can be use in phytotherapy for treatment number of different ailments. Also, freeze-drying has positive effect on antioxidant activity and preservation of p- coumaric acid and sinapinic acid in mistletoe samples.

Bearberry extract has been used for centuries in traditional and modern medicine to treat and alleviate the problems caused by urinary infections. Nevertheless, the exact mechanism of action of biologically active compounds from bearberry extract to date has not been fully defined. In this thesis the cytotoxicity effect of bearberry water extract, including the comparison between fresh and freeze-dried extract effects on bacteria Escherichia coli, Staphylococcus aureus, Lactobacillus plantarum and Lactobacillus fermentum was studied. Furthermore, the influence of the extract on adhesion of aforementioned bacteria to human epithelial gastrointestinal tract cells in the in vitro system was studied. Before the adhesion tests, cytotoxicity tests using Neutral red method were carried out together with the tests of potential antioxidant activity of the extract using DCHF-DA method on human tongue squamous cell carcinoma cell line (CAL 27) and human epithelial colorectal adenocarcinoma cells (Caco-2). The results obtained show a strong antimicrobial effect of this extract towards Gram-positive bacteria and also its ability to cause changes in the adhesion of bacterial cells to the epithelial cells of the digestive system. Antioxidant activity of the extract was established and stimulation of human cell proliferation was determined, which depends on the concentration of the extract and the type of treated cells. No significant difference was observed by comparing the biological activity of freshly prepared and freeze-dried bearberry water extract.

Cilj ovih preliminarnih analiza bio je procijeniti učinak visoke temperature (VT) na bioraspoloživost fenolnih spojeva i antioksidacijski potencijal klica brokule (Brassica oleracea L. convar. botrytis) tijekom in vitro simulirane probave čovjeka. Udio ukupnih flavonola bio je značajno viši u VT-klica nego u klica uzgojenih pri sobnoj temperaturi (ST). Ukupni fenoli, flavonoidi, antocijani, fenolne i hidroksicimetne kiseline nisu se razlikovali između VT-i ST-klica. VT nije značajno utjecala na koncentraciju vitamina C, kao ni na koncentraciju galne, benzojeve, kavene, p- kumarinske i sinapinske kiseline, tilirozida, kvercetina i kempferola. Jedini spoj na koji je VT utjecala bila je ferulična kiselina, snizila joj se koncentracija. Antioksidacijski potencijal izmjeren metodom ABTS bio je niži kod VT-klica, međutim mjereno metodama DPPH i FRAP nije se razlikovao između VT- i ST-klica. Tijekom probave, VT-klice imale su viši udio ukupnih fenolnih spojeva, međutim niži ukupnih antocijana u želučanoj fazi, u ostalim fazama nije bilo razlike između dvije skupine klica. Iako se ukupni flavonoidi nisu razlikovali između dvije skupine klica, ukupni flavonoli bili su povećani kod VT- klica u svakoj od faza probave. Ukupne fenolne kiseline nisu se razlikovale među klicama na početku probave, međutim u crijevnoj fazi VT-klice imale su značajno više ukupnih fenolnih kiselina. Ukupne hidroksicimetne kiseline bile su značajno veće u VT-klica nakon svake faze probave. Na koncentraciju vitamina C, tilirozida i kempferola VT nije značajno utjecala ni u jednoj od faza probave. U svakoj od faza VT-klice imale su povećanu koncentraciju benzojeve, a smanjenu kavene kiseline. Koncentracija galne kiseline u VT-klica bila je viša u želučanoj i crijevnoj fazi, ferulične je smanjena u svakoj od faza, osim u crijevnoj. Sinapinska kiselina u VT-klica je povišena u crijevnoj fazi, dok p-kumarinska uopće nije detektirana tijekom probave. Kvercetin je povišen u fazi usta i želučanoj, no nije zabilježen u crijevnoj. Prema metodama ABTS i DPPH, antioksidacijski potencijal bio je veći kod VT-klica nakon faze usta, međutim metodom FRAP zabilježen je niži potencijal. Metodom ABTS nisu primijećene razlike u antioksidacijskom potencijalu između VT- i ST-klica nakon želučane i crijevne faze, dok je metodama DPPH i FRAP zabilježen niži potencijal VT-klica nakon probave u crijevima. Metodom DPPH zabilježen je niži potencijal i nakon faze želuca. Rezultati ovih analiza pokazuju termoosjetljive i termostabilne fenolne spojeve u klicama brokule, njihovu bioraspoloživost tijekom simulirane probave i otkrivaju relevantnost VT za antioksidacijski potencijal klica prije i tijekom probave. / The aim of this preliminary study was to assess the effect of high temperature (HT) on the bioaccessibility of phenolic compounds and antioxidant potential of broccoli (Brassica oleracea L. convar. botrytis) seedlings during in vitro simulated human gastrointestinal digestion. Total flavonols were significantly higher in HT- seedlings than in seedlings grown under the room temperature (RT). Total phenolics, flavonoids, anthocyanins, phenolic and hydroxycinnamic acids did not differ between HT- and RT-seedlings. Neither the concentration of vitamin C, nor that of gallic, benzoic, caffeic, p-coumaric and sinapic acid, tiliroside, quercetin and kaempferol was significantly affected by HT. The only compound that was affected was ferulic acid, it was decreased. Antioxidant potential measured by ABTS method was lower in HT-seedlings, however measured by DPPH and FRAP methods it did not differ between HT- and RT-seedlings. During gastrointestinal digestion, HT-seedlings had increased total phenolics, however decreased total anthocyanins in gastric phase, in other phases there was no difference between the two groups of seedlings. While total flavonoids did not differ between the two groups of seedlings, total flavonols were increased by HT in each of digestion phases. Total phenolic acids did not differ between the seedlings at the beginning of digestion, however upon intestinal phase HT- seedlings had significantly more total phenolic acids. Total hydroxycinnamic acids were significantly higher ih HT seedlings after each of digestion phases. Vitamin C, tiliroside and kaempferol concentration was not significantly affected by HT in either of digestion phases. In each of the phases benzoic acid was increased, while caffeic was decreased. Gallic acid was higher in gastric and intestinal phase, ferulic was decreased in each of the phases, except intestinal. Sinapic acid was increased in intestinal phase, while p-coumaric was not detected during digestion at all. Quercetin was increased in salivary and gastric phase, however not detected in intestinal. According to ABTS and DPPH method, antioxidant potential was higher in HT-seedlings after salivary phase of digestion, however FRAP method revealed a lower potential. No changes in antioxidant potency were detected after gastric and intestinal phase with ABTS method, while DPPH and FRAP method showed a lower potency of HT-seedlings after intestinal digestion. With DPPH method a lower potency was detected after gastric phase as well. These results offer differentiation between thermosensitive and thermostable bioactive compounds in broccoli seedlings, their bioaccessibility during simulated digestion, and reveal relevance of HT for seedlings` antioxidant potential before and during digestion.

Climate changes and their negative impacts on environment are in the focus of scientific community for the last decades. Big interest is pointed towards plant stress physiology as plant’s growth and development are highly affected upon presence of stressors. Salinity and drought are considered the major abiotic stresses resulting in decreased crop’s yields and quality. Knowledge about survival and sustained growth of agronomical important crops during severe environmental conditions presents valuable tool to cope with these issues. Within this purpose, three important Brassica cultivars (Chinese cabbage, white cabbage and kale) were selected and exposed to drought and salinity treatments. Using the interdisciplinary approach, we wanted to underline the response of selected cultivars by observation of salt ions uptake, osmotic adjustment, oxidative stress defense mechanisms and impairment of photosynthesis and biomass production. Furthermore, the fluctuations in phytohormones levels and hormonal cross-talk were the focus of the research due their role in environment sensing and mediating physiological processes. Response mediated by abscisic acid (ABA), salicylic acid (SA) and jasmonates (JAs) together with the altered levels of growth promoting hormones: indole-3- acetic acid (IAA), cytokinins (CKs) and brassinosteroids (BRs) revealed differences in tolerance of Brassicas to drought and salinity. Research conducted on different response levels enabled us to characterize selected crops from the most tolerant to the most sensitive in following order: kale, white cabbage, Chinese cabbage and conclude that drought and salinity tolerance is mostly determined by changes in endogenous levels of phytohormones. However, as the hormonal cross-talk and defense mechanism are very complex, additional research at the molecular level is necessary to shed light on the hormonal players and the interactions responsible for drought and salinity tolerance in Brassicaceae.

Global warming and climate changes have enormous negative impact on crop production in affected areas. Increased soil salinity and drought are abiotic stresses particularly pronounced in Mediterranean, semi-arid, and arid climates. The abiotic stress tolerance of Brassica crops correlated with certain changes in plant hormones such as salicylic acid (SA), abscisic acid (ABA), auxins (IAA), cytokinins (CS) and brassinosteroids (BR). The effect of salinity stress and drought was evaluated on selected Brassica crops: Chinese cabbage (Brassica rapa ssp. pekinensis), white cabbage (B. oleracea var. capitata forma alba) and kale (B. oleracea var. acephala) with particular focus on the role of plant hormones in abiotic stress tolerance. Based on the physiological and biochemical markers the Chinese cabbage exhibited the lowest tolerance, followed by the white cabbage, while the kale appeared to be the most tolerant to drought and salinity stress in aplied experimental conditions.

Biochemical response of Arabidopsis thaliana mutants with impaired function of the enzyme auxin amidohydolase (single ilr1, iar3, and ill2 ; double iar3 ill2 and triple ilr1 iar3 ill2) and the corresponding wild type (wt) of the Wassilewskii ecotype to high salinity (100 mM NaCl) was evaluated after period of 7 days. Auxin amidohydrolases are enzymes that participate in homeostasis of plant hormone auxin by hydrolyzing auxin amide-conjugates and releasing active auxins. The contents of malondialdehyde, proline and ascorbate were determined as well as content of reactive oxygen species (superoxide radical SOˑ and hydrogen peroxide -H2O2) and glutathione using fluorescent probes dihydroethidium (DHE), dichlorodihydrofluorescein diacetate (DCFH-DA) and monochlorobimane (MCB). Salt stress induced significant decrease of ascorbate in ill2 and increase in iar3, iar3 ill2 and iar1 iar3 ill2. Accumulation of proline was significant not only in wt but also in all mutants exposed to salt stress. There was a statistically significant increase in the content of MDA (ilr1, iar3, iar3 ill2 and ilr1 iar3 ill2), glutathione (wt, ill2 and ilr1) and reactive oxygen species (SOˑ ; ilr1 and H2O2 ; ill2) upon salt stress. The highest content of ascorbate, proline and MDA after exposure to salt stress was measured in triple mutant. Based on the obtained results we can conclude that the triple mutant, which has the lowest ability to hydrolyze auxin conjugates, is the most sensitive to salt stress.

Brassicaceae crops are among the oldest known cultivars and their use is widespread. Abiotic stress, such as increased salinity, and oxidative stress are serious threats to agriculture and the natural status of the environment. Increased salinity may cause nutrient deficiencies or imbalances, as well as reduced yield, growth and development of plants depending on their ability to overcome the effects of stress. The ability of plants to tolerate salt is determined by multiple biochemical pathways, those that lead to synthesis of specialized metabolites and support scavenging of reactive oxygen species (ROS). For this reason it is important to understand how Brassicaceae crops struggling against increased salinity. The aim of our research was to evaluate the short-term stress responses of three Brassica species/varieties (Brassica rapa spp. pekinensis, B. oleracea var. capitata, and B.oleracea var. acephala) exposed to saline stress. In order to determine the impact of the salt stress, one-day old seedlings were treated with NaCl (in range of conc. 50- 200 mM), in comparison to corresponding controls, during 24h. The physiological effect of treatments was determined in vivo using the rooth growth assay. The amount of ROS (SO, H2O2) and GSH in seedlings is determined in vivo using a fluorescent microscope connected to the camera after the seedlings are stained with specific dies (dihydroethidium (DHE), dichlorodihydrofluorescein diacetate (DCFH-DA), and monochlorobimane (MCB)). Proline and specialized metabolites were determined used UV/VIS spectrophotometry, while phenolic acids profiles were determined by UHPLC-MS/MS. Ultimately, results indicate that stress response is species specific and depends on the salt concentration. The treatment of seedlings with different concentrations of NaCl cause gradual inhibition of biomass accumulation and root growth where B. rapa spp. pekinensis proved to be the most sensitive species while B. oleracea var. acephala the most tolerant one. Seedlings which are under stress conditions produce higher levels of ROS molecules (SO, H2O2) in comparison to the control, mostly in a dose dependent manner. Stressful conditions also affect at the level of osmoprotectant proline, GSH as well as phenolic antioxidants. Profile of phenolic acids were determined and their fluctuations upon salinity stress were disscussed in comparison to seedlings growth inhibitions and stress tolerance of three Brassica varieties.

2-metoksi-1,4-naftokinon (2-MNQ) je prirodni derivat 1,4-naftokinona koji je u dosad provedenim ispitivanjima pokazao antipruritično, protuupalno, antialergijsko, antimikrobno i antikancerogeno djelovanje. U ovom istraživanju ispitan je utjecaj 2-MNQ na klijanje sjemenki i razinu oksidacijskog stresa u klijanaca. Kao biljni model korištene su sjemenke dviju biljnih vrsta: bijele gorušice (Sinapis alba L.) i rotkvice (Raphanus sativus L.). Sjemenke su bile izložene 2-MNQ u koncentracijama 1, 5, 10, 20, 30 i 40 μg/ml kroz 3 dana. U pokus su bile uključene negativna kontrola (sjemenke izložene 3 dana destiliranoj vodi) i pozitivna kontrola (sjemenke izložene 3 dana 0,02M otopini CuSO₄). Nakon 3-dnevne izloženosti, za obje biljne vrste određena je klijavost (%) i duljina korjenčića (mm) te je u homogenatu klijanaca određena katalitička aktivnost SOD (U/ml). Podaci su statistički obrađeni pomoću jednosmjerne analize varijance (ANOVA test) te primjenom Duncan's New Multiple Range Test-a. Također, određena je korelacija između mjerenih parametara te analiza glavnih komponenti. Statistički značajnim smatrane su vrijednosti koje se razlikuju na razini p 0,05. Povećanjem koncentracije 2-MNQ opao je broj proklijalih sjemenki bijele gorušice i sukladno tome padala je duljina korjenčića, a 2-MNQ u koncentraciji 30 i 40 μg/ml u potpunosti je inhibirao klijavost sjemenki. To ukazuje da 2-MNQ inhibira klijanje sjemenki bijele gorušice. S druge strane 2-MNQ nije imao utjecaja na klijanje rotkvice. U klijanaca bijele gorušice s koncentracijom 2-MNQ rasla je aktivnost SOD-a te je najveća aktivnost SOD-a izmjerena pri koncentracijama 2-MNQ od 1 i 10 μg/ml. Iz navedenog se može zaključiti kako je biljka tada bila izložena najvećem oksidacijskom stresu te je bilo potrebno proizvesti veliku količinu SOD-a kako bi se od njega obranila i samim time omogućila sjemenkama da prokliju. Katalitička aktivnost SOD-a u rotkvici pri svim koncentracijama otopine 2-MNQ približno je bila ista te nisu uočena odstupanja. Utjecaj 2-MNQ na klijavost sjemenki je različit te ovisi i o koncentraciji 2-MNQ i o samoj biljnoj vrsti. Bijela gorušica osjetljivija je na djelovanje 2-MNQ, dok 2-MNQ u ispitivanim koncentracijama nije imao utjecaja na klijavost rotkvice. Mehanizam toksičnog učinka 2-MNQ može se povezati s oksidacijskim stresom s obzirom da je tretman s 2-MNQ doveo do promjene aktivnosti SOD-a u bijeloj gorušici. 2-metoxy-1,4-naphtoquinone (2-MNQ) is a natural derivate of 1,4-naphthoquinone which has shown antipruritic, anti-inflammatory, anti-allergic, antimicrobial and anti-cancer effects.In this study, the influence of 2-MNQ on seed germination and the level of oxidative stress was investigated. As a plant model the seeds of two plant species were used: white mustard (Sinapis alba L.) and radish (Raphanus sativus L.). The seeds were exposed to 2-MNQ in concentrations of 1, 5, 10, 20, 30 and 40 μg/ml for 3 days. The study included a negative control (seeds exposed to distilled water for 3 days) and a positive control (seeds exposed to 0.02M CuSO₄ solution for 3 days). After a 3-day exposure, germination (%) and root length (mm) were measured, and in seedlings homogenate catalytic activity of SOD (U/ml) was determined. Data were statistically processed using one-way analysis of variance (ANOVA test) and Duncan's New Multiple Range Test as well as tested for correlation and Principal Component Analysis. Significance level was set at p ≤ 0.05. With the increase of 2-MNQ concentration the number of germinated seeds of white mustard decreased and 2-MNQ in concentrations of 30 and 40 μg/ml completely ceased germination of white mustard. In parallel the decrease in the length of the radicle with the increase of 2-MNQ concentration was recorded. Thus, 2-MNQ inhibits the germination of white mustard seeds. On contrary, 2-MNQ had no impact on germination of radish. In white mustard seedlings with the increase of 2-MNQ concentration the increase of SOD activity is observed, and the highest SOD activity is recorded at 2-MNQ concentrations 1 and 10 μg/ml. Therefore, it can be concluded that the plant was then exposed to the greatest oxidative stress, and it was necessary to produce a large amount of SOD in order to defend against it and at the same time enable germination. In radish seedlings the catalytic activity of SOD at all concentrations of the 2-MNQ was approximately the same, with no deviations. The impact of 2-MNQ on the seed germination is different and depends on the concentration of 2-MNQ and the plant species itself. White mustard is more sensitive to 2-MNQ, while 2-MNQ in concentrations tested had no impact on radish. The mechanism of the toxic effect of 2-MNQ can be related to oxidative stress, considering that treatment with 2-MNQ led to a change in SOD activity in white mustard.