| Abstract | Cilj ovog rada bio je ispitati učinak metanolnog ekstrakta žljezdastog nedirka (Impatiens glandulifera R., Balsaminaceae) na klijavost sjemenki i na razinu oksidacijskog stresa u klijanaca rotkvice (Raphanus sativus L., Brassicaceae). Sjemenke vrste R. sativus tri su dana bile izložene metanolnom ekstraktu lista vrste I. glandulifera kojim se impregnirao filtar papir trima različitim dozama (1,5 ml; 3,0 ml i 6,0 ml). Za pozitivnu kontrolu filtar papir je impregniran s 0,02 M otopinom CuSO4, a negativna kontrola bio je tretman deioniziranom vodom. Modelni organizam (R. sativus) tretiran je u triplikatu. Nakon trodnevne izloženosti, izvagana je masa svježeg tkiva klijanaca rotkvice te je potom pripremljen homogenat biljnog tkiva u trikloroctenoj kiselini (100 mg tkiva/0,5 mL TCA). U supernatantu homogenata određeni su parametri oksidacijskog stresa: sadržaj reduciranog glutationa (GSH), antocijanina, malondialdehida (MDA) i oksidiranih proteina. Metodom prema Ellmanu (1959) određen je sadržaj GSH, antocijanini su pH-diferencijalnom metodom mjereni prema Giusti i Wrolstadu (2001), tiobarbituratnim testom prema Heathu i Packeru (1968) određen je MDA, a derivatizacijom karbonilne skupine proteina s DNPH prema Levine i sur. (1990) određen je sadržaj oksidiranih proteina. Rezultati su statistički obrađeni t-testom (program Excel, P < 0,05). Masa svježeg tkiva klijanaca rotkvice tretiranih ekstraktom žljezdastog nedirka bila je niža u odnosu na negativnu kontrolu. Značajnija promjena u sadržaju neenzimskih antioksidansa, GSH i antocijanina u odnosu na negativnu kontrolu nije zabilježena. Sadržaj MDA u klijanaca rotkvice tretiranih s najvišom dozom (6,0 ml) ekstrakta bio je viši u usporedbi s negativnom kontrolom. Sadržaj oksidiranih proteina klijanaca tretiranih s ekstraktom žljezdastog nedirka bio je viši od negativne kontrole, a statistički značajno viši bio je nakon tretmana s najvišom dozom ekstrakta, 6,0 ml (negativna kontrola vs. najviša doza ekstrakta; 7,553 ± 0,906 μM vs. 9,720 ± 0,548 μM, P = 0,012). Prema dobivenim rezultatima može se zaključiti kako ekstrakt biljne vrste I. glandulifera fitotoksično djeluje na klijanje vrste R. sativus, ali se u modelnoj biljci, u eksperimentalnim uvjetima, nije aktivirao adekvatan odgovor na oksidacijski stres (porast sadržaja GSH i antocijanina). To je rezultiralo porastom sadržaja MDA i oksidiranih proteina, odnosno došlo je do oksidacijskog stresa. Dobiveni rezultati jasno pokazuju da je oksidacijski stres mehanizam alelopatskog djelovanja biljne vrste I. glandulifera. |
| Abstract (english) | The aim of this study was to explore the effect of methanol extract of himalayan balsam (Impatiens glandulifera R., Balsaminaceae) on seed germination and on the level of oxidative stress in radish seedlings (Raphanus sativus L., Brassicaceae). Seeds of R. sativus were treated for three days with methanol extract prepared from I. glandulifera leaves (filter papers were impregnated with three doses of methanol extract: 1.5, 3 and 6 mL). As positive control 0.02 M CuSO4 solution was used, and negative control was treatment with deionized water. The model organism (R. sativus) was treated in triplicate. After three days of exposure, the fresh mass of radish seedlings was weighed. Plant tissue homogenate was prepared using trichloroacetic acid (100 mg tissue/0.5 mL TCA). As parameters of oxidative stress, the content of reduced glutathione (GSH), anthocyanin, malondialdehyde (MDA) and oxidized proteins was determined in the supernatant of the homogenates. The GSH content was determined using the Ellman method (1959), anthocyanins were measured by the pH-differential method according to Giusti and Wrolstad (2001), the MDA was determined by using the thiobarbituric test according to Heath and Packer (1968) and the content of oxidized proteins was determined by derivatization of the carbonyl group of proteins with DNPH according to Levine et al. (1990). The obtained results were statistically analysed (t-test, Excel; P <0.05).The fresh mass weight of radish seedlings treated with 1.5 – 6 ml of I. glandulifera extract was lower compared to the negative control. There was no significant change in the content of non-enzymatic antioxidants, GSH and anthocyanins compared to the negative control. The MDA content was higher in radish seedlings treated with the highest dose of extract (6 ml) compared to the negative control. The content of oxidized proteins was higher in treated seedlings, and treatment with the highest dose of I. glandulifera extract (6 mL) induced a statistically significant increase of oxidized proteins content (negative control vs. the highest extract dose; 7.553 ± 0.906 μM vs. 9.720 ± 0.548 μM, P = 0.012). According to the results, it can be concluded that the I. glandulifera extract has a phytotoxic effect on R. sativus germination, but in the model plant, in experimental conditions, an adequate response to oxidative stress (increase in GSH and anthocyanin content) was not activated. This resulted in an increase in the content of MDA and oxidized proteins, which means that oxidative stress has occurred. Therefore, it can be concluded that one of the mechanisms of allelopathic effect I. glandulifera is oxidative stress. |
