Aktivnost superoksid dismutase u klijanaca bijele gorušice (Siapis alba L.) i rotkvice (Raphanus sativus L.) nakon izloženosti žljezdastom nedirku (Impatiens glandulifera Royle)

master's thesis

• Title (english): Activity of superoxide dismutase in seedlings of white mustard (Sinapis alba L.) and radish (Raphanus sativus L.) after exposure to himalayan balsam (Impatiens glandulifera Royle)
• Author: Ivor Borković
• Mentor: Ana-Marija Domijan (mentor)
• Committee member: Ana-Marija Domijan (predsjednik povjerenstva)
• Committee member: Suzana Inić (član povjerenstva)
• Committee member: Mirela Matić (član povjerenstva)
• Granter: University of Zagreb; Faculty of Pharmacy and Biochemistry; (Department of pharmaceutical botany); Zagreb
• Defense date and country: 2021-09-14, Croatia
• Scientific / art field, discipline and subdiscipline: BIOMEDICINE AND HEALTHCARE; Pharmacy; Pharmacy

Abstract

U ovom istraživanju ispitan je oksidacijski stres kao mehanizam alelopatskog djelovanja žljezdastog nedirka (Impatiens glandulifera Royle, Balsaminaceae). Razina oksidacijskog stresa praćena je mjerenjem aktivnosti antioksidacijskog enzima superoksid dismutaze (SOD) u klijanaca bijele gorušice (Sinapis alba L., Brassicaceae) i rotkvice (Raphanus sativus L., Brassicaceae) nakon izloženosti ekstraktu žljezdastog nedirka. Sjemenke bijele gorušice i rotkvice bile su izložene 3 dana metanolnom ekstraktu žljezdastog nedirka u tri doze (1,5; 3 i 6 ml). U pokus je bila uključena pozitivna kontrola (PK, tretman s 0,02 M CuSO4) te negativna kontrola (NK, tretman destiliranom vodom). Svi tretmani provedeni su u triplikatu. Nakon 3-dnevnog klijanja, pripremljen je homogenat biljnoga tkiva, a u supernatantu homogenata izmjerena je aktivnost antioksidacijskog enzima SOD pomoću komercijalnog kita kolorimetrijskom metodom. Prije samog mjerenja aktivnosti SOD u uzorcima, metoda je optimizirana ispitivanjem njene linearnosti i načina pripreme uzorka. Dobiveni rezultati aktivnosti SOD obrađeni su statistički (t-test, Excel; P ≤ 0,05).
Utvrđeno je da je metode za određivanje aktivnosti SOD linearna (koeficijent linearnosti, R2 = 0,9406) s jednadžbom pravca y = 69,302x + 1,1463. Za određivanje aktivnosti SOD u homogenatu biljnoga tkiva optimalnim se pokazalo razrijeđenje supernatanta homogenata uzorka 2x s pojačivačem za uzorke. U klijanaca bijele gorušice nakon 3-dnevnog tretmana s 1,5 ml ekstrakta žljezdastog nedirka aktivnost SOD značajno je pala (52%) u odnosu na NK (100 %, P ≤ 0,05). S povećanjem doze ekstrakta, u klijanaca bijele gorušice zabilježen je porast aktivnosti SOD (3 ml: 73 %, 6 ml: 80 %). U klijanaca rotkvice 3-dnevni tretmana s ekstraktom žljezdastog nedirka uzrokovao je postepeni pad aktivnosti SOD te je u odnosu na NK (100 %) najniža aktivnost SOD (86 %) bila nakon tretmana s 3 ml ekstrakta. Porast aktivnosti SOD nakon 3-dnevnog tretmana s višim dozama ekstrakta žljezdastog nedirka (3 ml i 6 ml zabilježen u klijanaca bijele gorušice i 6 ml zabilježen u klijanaca rotkvice) može se objasniti odgovorom biljke na vanjski utjecaj, odnosno povećanoj sintezi enzima SOD kao odgovor na stres. S obzirom da je značajniji pad aktivnosti SOD zabilježen u klijanaca bijele gorušice u odnosu na klijance rotkvice može se zaključiti da rotkvica ima razvijeniji antioksidacijski sustav. Dobiveni rezultati pokazuju da je oksidacijski stres mehanizam alelopatskog djelovanja žljezdastog nedirka.

Abstract (english)

In this research, oxidative stress, as an allelopathic mechanism of himalayan balsam (Impatiens glandulifera Royle, Balsaminaceae) was examined. The level of oxidative stress was monitored by measuring the activity of antioxidative enzyme superoxide dismutase (SOD) in seedlings of white mustard (Sinapis alba L., Brassicaceae) and radish (Raphanus sativus L., Brassicaceae) exposed to the extract of himalayan balsam. Seeds from both plants, white mustard and radish, were exposed to a methanol extract of himalayan balsam in three doses (1.5, 3 and 6 mL). In experiment, positive control (PC, treatment with 0.02 M CuSO4) and negative control (NC, treatment with destiled water) were also included. All treatments were done in a triplicate. After 3 days of treatment, plant tissue homogenate was prepared and in its supernatant SOD activity was determined via comercial assay kit based on colorimetry. Before determining SOD activity in seedlings, the method was optimised by testing its linearity and sample preparation procedure. Obtained data on SOD activity was processed statisticaly (t-test, Excel; P ≤ 0.05).
The method for assessment SOD activity was linear (R2 = 0.9406; y = 69,302x + 1,1463). Several sample dilutions were tested, and the optimal was dilution of samples 2x with sample buffer. In seedlings of white mustard after 3-days exposure to 1.5 mL of methanol extract of himalayan balsam SOD activity significantly decreased in comparison to NC (52% vs. 100%, P ≤ 0.05). With the increase of the dose of the extract in seedlings of white mustard an increase of SOD activity was recorded (3 mL: 73 %, 6 mL: 80 %). In radish seedlings 3-days exposure to methanol extract of himalayan balsam resulted with slow decrease of SOD activity and the lowest SOD activity in comparison to NC was after exposure to 3 mL of extract (86 % vs. 100 %). An increase of SOD activity after exposure to higher doses of extract of himalayan balsam (3 mL and 6 mL for white mustard and 6 mL for radish) can be explained with the synthesis of SOD as a response of seedlings to stress. Since significant decrease of SOD activity was observed in sedlings of white mustard in comparison to seedlings of radish, can be concluded that seedlings of radish has better antioxidative defence system. The obtained results indicate that oxidative stress is the mechanism of himalayan balsam allelopathy.

• Keywords: žljezdasti nedirak; alelopatija; oksidacijski stres; superoksid dismutaza
• Keywords (english): himalayan balsam; allelopathy; oxidative stress; superoxide dismutase
• Language: croatian
• URN:NBN: urn:nbn:hr:163:084888
• 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: 2022-02-09 16:34:50