Učinak 2-metoksi-1,4-naftokinona na parametre oksidacijskog stresa u klijanaca obične rotkvice (Raphanus sativus L.)

master's thesis

• Title (english): Impact of 2-methoxy-1,4-naphtokinone on oxidative stress parameters of radish (Raphanus sativus L.)
• Author: Sven Kraljević
• Mentor: Ana-Marija Domijan (mentor)
• Committee member: Ana-Marija Domijan (predsjednik povjerenstva)
• Committee member: Hrvoje Rimac (član povjerenstva)
• Committee member: Marijan Marijan (član povjerenstva); MBZ: 10976
• Granter: University of Zagreb; Faculty of Pharmacy and Biochemistry; (Department of pharmaceutical botany); Zagreb
• Defense date and country: 2022-07-11, Croatia
• Scientific / art field, discipline and subdiscipline: BIOMEDICINE AND HEALTHCARE; Pharmacy; Pharmacy

Abstract

2-metoksi-1,4-naftokinon (2-MNQ) je sekundarni metabolit biljaka, gljiva i mikroorganizama. Zbog svojih antikancerogenih, antimetastatskih, antimikrobnih i drugih ljekovitih svojstava predmet je brojnih znanstevnih istraživanja. Cilj ovog diplomskog rada bio je ispitati utjecaj 2-MNQ na fitotoksičnost i parametre oksidacijskog stresa u klijanaca obične rotkvice (Raphanus sativus L.). Sjemenke obične rotkvice bile su izložene tri dana 2-MNQ u koncentracijama 1, 5, 10, 20 i 30 μg/mL. U ispitivanje su bile uključene sjemenke rotkvice izložene tri dana destiliranoj vodi (negativna kontrola) i sjemenke rotkvice izložene tri dana 0,02 M otopini CuSO4 (pozitivna kontrola). Modelni organizam tretiran je u triplikatu. Nakon trodnevna izlaganja izvagana je masa svježega tkiva te su pripremljeni homogenati klijanaca tkiva u trikloroctenoj kiselini (100 mg tkiva/0,5 mL). U supernatantu homogenata određeni su parametri oksidacijskog stresa: glutation (GSH), antocijanini i malondialdehid (MDA). Sadržaj GSH određen je pomoću DTNB reagensa, antocijanina pH diferencijalnom metodom, a MDA pomoću TBA testa spektrofotometrijski. Dobiveni rezultati su statistički obrađeni t-testom (P ≤ 0,05). Trodnevna izloženost 2-MNQ u koncentracijskom rasponu (1-30 μg/mL) nije dovela do statistički značajnog smanjenja mase klijanaca rotkvice u odnosu na negativnu kontrolu, što znači da ispitivane koncentracije 2-MNQ nisu imale fitotoksičan učinak na modelni organizam. Izloženost 2-MNQ u koncentracijama 1-20 μg/mL kroz tri dana dovela je do statistički značajnog pad sadržaja GSH u odnosu na negativnu kontrolu (P < 0,05), dok je 2-MNQ u koncentraciji od 30 μg/mL doveo do porasta sadržaja GSH. To se može objasniti potrošnjom zaliha GSH nakon trodnevne izloženosti nižim koncentracijama 2-MNQ u svrhu obrane od ROS-ova te de novo sintezom GSH kao odgovorom na oksidacijski stres nakon izloženosti višoj koncentraciji 2-MNQ. Značajnija promjena sadržaja antocijanina nakon trodnevne izloženosti 2-MNQ u ispitivanom koncentracijskom rasponu u odnosu na negativnu kontrolu nije zabilježena. Trodnevna izloženost najvišoj koncentraciji 2-MNQ (30 μg/mL) uzrokovala je blagi porast sadržaja MDA u odnosu na negativnu kontrolu, dok je izloženost nižim koncentracijama 2-MNQ (1-20 μg/mL) uzrokovala statistički značajan pad sadržaja MDA. S obzirom da je izloženost nižim koncentracijama 2-MNQ uzrokovala pad sadržaja i GSH i MDA, a viša koncentracija rast njihovog sadržaja, može se pretpostaviti da je rotkvica koristila GSH kao obrambeni sustav te se potrošnjom sadržaja GSH smanjivao i sadržaj MDA. Iz dobivenih rezultata može se zaključiti da ispitivane koncentracije 2-MNQ (1-30 μg/mL) nisu fitotoksične za rotkvicu te da je jedan od mehanizama djelovanja 2-MNQ oksidacijski stres.

Abstract (english)

2-methoxy-1,4-naphthoquinone (2-MNQ) is as secondary metabolite of plants, fungi, and microorganisms. Due to its anticancer, antimetastatic, antimicrobic and other beneficial properties, it has been subject of number of scientific studies. The aim of this study was to explore impact of 2-MNQ on phytotoxicity and parameters of oxidative stress in a plant model, radish (Raphanus sativus L.). Radish seeds were exposed to 2-MNQ at concentrations 1, 5, 10, 20 and 30 μg/mL for 3 days. In the experiment a negative control (radish seeds exposed to distilled water for 3 days) and a positive control (radish seeds exposed to 0.02 M CuSO4 solution for 3 days) were included. The model organism was treated in triplicate. After 3 days of exposure, fresh tissue weight was determined, and plant tissue homogenate was prepared using trichloroacetic acid (100 mg tissue/0.5 mL). As parameters of oxidative stress glutathione (GSH), anthocyanins and malondialdehyde (MDA) were assessed. GSH level was determined using DTNB reagent, anthocyanin level by pH differential method, and MDA with TBA assay, spectrophotometrically. The obtained results were statistically analysed by use of t-test (P <0.05). After 3-day exposure of radish seedlings to 2-MNQ (1-30 μg/mL) no significant reduction in fresh tissue weight was observed, suggesting that 2-MNQ in concentrations applied, is not phytotoxic to the model organism. A significant reduction of GSH level in comparison to the negative control was observed after exposure to 2-MNQ in concentrations 1-20 μg/mL. However, exposure to 2-MNQ at 30 μg/mL for 3-days slightly increase GSH levels. A decrease in GSH level after exposure to lower 2-MNQ concentrations can be explained by its consumption by ROS, while an increase of GSH level after exposure to higher concentrations of 2-MNQ can be explained by its de novo synthesis in response to oxidative stress. No significant change in the level anthocyanins after 3-days exposure to 2-MNQ compared to the negative control was recorded. 3-days exposure to the highest 2-MNQ concentration (30 μg/mL) slightly increased MDA level comparing to negative control, while 2-MNQ at lower concentrations (1-20 μg/mL) significantly reduced MDA level. Similar pattern in the level of GSH and MDA in the radish cells after 3-days exposure to 2-MNQ could be due to utilization of GSH as an antioxidant in order to protect cells form oxidative stress that leads to decrease of MDA level. In conclusion, 2-MNQ in tested concentration range was not phytotoxic to radish, and one of its mechanisms of toxicity is oxidative stress.

• Keywords: naftokinoni; 2-MNQ; rotkvica; Raphanus sativus L.; oksidacijski stres; GSH; MDA; antocijanini
• Keywords (english): naphtoquinone; 2-MNQ; radish; Raphanus sativus L.; oxidative stress; GSH; MDA; anthocyanins
• Language: croatian
• URN:NBN: urn:nbn:hr:163:778143
• Study programme: Title: Pharmacy; Study programme type: university; Study level: integrated undergraduate and graduate; Academic / professional title: magistar/magistra farmacije (magistar/magistra farmacije)
• Type of resource: Text
• File origin: Born digital
• Access conditions: Open access
• Created on: 2022-09-15 13:26:37