Uvod: Doksorubicin se smatra jednim od najpotentnijih hemioterapeutika čija je klinička upotreba ograničena neželjenim dejstvima (ND). Najčešće hronično ND je kardiotoksičnost izazvana doksorubicinom (KID). Mehanizam nastanka KID je multifaktorijalan (oksidativni stres, oštećenje topoizomeraze II i mitohondrija, regulisana ćelijska smrt). Prema smernicama Evropskog kardiološkog društva kod ovih pacijenata prate se elektrokardiografske i ehokardiografske promene, nivoi biohemijskih markera, dok je za dijagnozu poželjna patohistološka potvrda. U cilju ublažavanja i prevencije KID, ispitivani su brojni lekovi i suplementi, a sve više nade se polaže u bioaktivne komponente biljnog porekla i biljne ekstrakte. Karotenoidi su liposolubilni biljni pigmenti za koje se veruje da mogu blokirati nekoliko glavnih mehanizama nastanka KID. NJihova resorpcija zavisi od vrste rastvarača, te je u posebnom fokusu istraživanja najnovija generacija „zelenih“ rastvarača – prirodni eutektički rastvarači (engl. natural deep eutectic solvents – NADES), jer za razliku od konvencionalnih, organskih rastvarača nisu toksični. Ciljevi: Doktorska disertacija je koncipirana sa ciljem da se na prvom mestu odredi kvalitativni i kvantitativni sadržaj karotenoida u ekstraktu, a potom utvrde efekti ekstrakta karotenoida pulpe bundeve na KID analizirajući uticaj na opšte stanje životinja, telesnu masu, masu srca, nivoe srčanih biomarkera (srčani troponin (cTns), srčana kreatin kinaza (CK- MB) i N-terminalproBNP (NT-proBNP), aktivnost antioksidativnih enzima, aktivnost Kompleksa elektron transportnog sistema mitohondrija, histološke znakove oštećenja miokarda, kao i stepen ekspresije Anti-Cardiac Troponin I, Anti-Nrf2, Anti-Bcl-2, Anti- Casp3, Anti-COX2 i Anti-Ki67 u tkivu srca. Materijal i metode: Za ekstrakciju karotenoida korišćena je prirodna eutektička smeša NADES rastvarač, a da bi se dodatno poboljšala efikasnost ekstrakcije i povećao prinos β- karotena iz bundeve (C. moschata), korišćena je kombinacija NADES rastvarača i ultrazvučne ekstrakcione tehnike. In vivo ispitivanja su vršena na 60 pacova soja Wistar koji su metodom slučajnog izbora podeljeni u 6 grupa. Analizirani su parametri opšteg stanja životinja, telesna masa, masa srca, a iz homogenata srca određen je intenzitet lipidne peroksidacije i aktivnost antioksidativnih enzima (SOD, GSH-Px, GR i GST). Analizirani su i pokazatelji srčane funkcije (cTns, CK-MB i NT-proBNP), jetrene funckije (AST, ALT i ukupni bilirubin) i bubrežne funkcije (urea, kreatinin i mokraćna kiselina), kao i parametri mitohondrijske respiracije. Isečci tkiva srca su histološki obrađeni i bojeni hematoksilin-eozin metodom, kao i specijalnim bojenjima (Masson’s trihromno i Periodic Acid Schiff bojenje (PAS)). Za imunohistohemijsko bojenje tkiva koristila su se primarna antitela i to Anti-Cardiac Troponin I, Anti- Nrf2, Anti-Bcl-2, Anti-Casp3, Anti-COX2 i Anti-Ki67. Rezultati: Ekstrakcijom karotenoida pulpe bundeve detektovana dominantna jedinjenja su β- karoten i α- karoten. Daljom analizom dobijena je zastupljenost β- karotena – 114.54 μg/ml i α- karotena – 147.91 μg/ml. Rezultati in vivo istraživanja pokazuju da ekstrakt karotenoida pulpe bundeve, u hroničnom modelu kardiotoksičnosti izazvane doksorubicinom kod pacova soja Wistar, utiče povoljno na ošte stanje, pomaže u očuvanju telesne mase i statistički značajno povećava masu srca. Antioksidativno delovanje karotenoida ispoljava se statistički značajnim povećanjem antioksidativnih enzima i smanjenjem intenziteta lipidne peroksidacije. Ekstrakt statistički značajno smanjuje serumske vrednosti NT-proBNP, AST-a, kreatinina, uree i mokraćne kiseline. Karotenoidi u ovom istraživanju stimulišu maksimalni kapacitet elektron transportnog sistema, kao i aktivnost Kompleksa I, II i IV. Primena karotenoida ublažava stepen oštećenja miokarda (statistički značajno niži DDS score), statistički značajno povećava ekspresiju protektivnih markera (Anti-Cardiac Troponin I, Anti-Nrf2 i Anti-Bcl2 antitela), dok statistički značajno smanjuje broj imunopozitivnih ćelija i ekspresiju Anti-casp3, Anti-COX2 i Anti-Ki67 antitela. Zaključak: Rezultati dobijeni u okviru ove doktorske disertacije snažno sugerišu da ekstrakt karotenoida pulpe bundeve ima kardioprotektivan efekat. S obzirom na to da nije iskazao negativne efekte ni u jednom ispitivanom aspektu stiče se utisak da je upravo on potencijalna terapija u prevenciji KID. Takođe, utvrđeno je da je NADES (C8:C10) rastvarač, korišćen za ekstrakciju karotenoida, bezbedan za primenu kod životinja.Introduction: Doxorubicin is considered one of the most potent chemotherapeutics whose clinical use is limited by adverse effects (AE). The most common chronic AE is doxorubicin-induced cardiotoxicity (DIC). The mechanism of DIC is multifactorial (oxidative stress, damage to topoisomerase II and mitochondria, initiation of regulated cell death). According to the guidelines of the European Society of Cardiology, electrocardiographic and echocardiographic changes, and levels of biochemical markers are monitored in these patients, while pathohistological confirmation is preferred for diagnosis. To alleviate and prevent DIC, numerous drugs and supplements have been tested, and nowdays bioactive components of plant origin are being extracted. Carotenoids are liposoluble plant pigments that are believed to be able to block several major mechanisms of DIC formation. Their resorption depends on the type of solvent, so the latest generation of "green" solvents - natural deep eutectic solvents - NADES - are the special focus of research because, unlike conventional organic solvents, they are not toxic. The aim: The doctoral dissertation aimed to determine the qualitative and quantitative content of carotenoids in the extract, and then determine the effects of the pumpkin pulp carotenoid extract on DIC by analyzing the impact on the general condition of the animals, body weight, heart weight, levels of cardiac biomarkers (cardiac troponin (cTns), cardiac creatine kinase (CK-MB) and N-terminalproBNP (NT- proBNP), the activity of antioxidant enzymes, the activity of the Mitochondrial Electron Transport System Complex, histological signs of myocardial damage, as well as the level of expression of Anti-Cardiac Troponin I, Anti-Nrf2, Anti-Bcl-2, Anti-Casp3, Anti-COX2 and Anti-Ki67 in heart tissue. Material and methods: The natural eutectic mixture NADES solvent was used for carotenoid extraction, and to further improve the extraction efficiency and increase the yield of β-carotene from pumpkin (C. moschata), a combination of NADES solvent and Ultrasound-Assisted Extraction technique was used. In vivo tests were performed on 60 Wistar rats, which were randomly divided into 6 groups. The parameters of the general state of the animals, body mass, and heart mass were analyzed, and the intensity of lipid peroxidation and the activity of antioxidant enzymes (SOD, GSH-Px, GR, and GST) were determined from the heart homogenate. Indicators of cardiac function (cTns, CK-MB, and NT-proBNP), liver function (AST, ALT, and total bilirubin), and kidney function (urea, creatinine, and uric acid), as well as parameters of mitochondrial respiration, were also analyzed. Heart tissue sections were histologically processed and stained with the hematoxylin-eosin method, as well as with special stains (Masson's trichrome and Periodic Acid Schiff staining (PAS)). Primary antibodies Anti-Cardiac Troponin I, Anti-Nrf2, Anti-Bcl-2, Anti-Casp3, Anti-COX2, and Anti-Ki67 were used for immunohistochemical tissue staining. Results: The dominant compounds detected by the extraction of carotenoids from pumpkin pulp are β-carotene and α-carotene. Further analysis revealed the presence of β-carotene - 114.54 μg/ml and α- carotene - 147.91 μg/ml. The results of in vivo research show that the carotenoid extract of pumpkin pulp, in a chronic model of doxorubicin- induced cardiotoxicity in Wistar rats, has a favorable effect on the general condition of animals, helps preserve body mass and statistically significantly increases heart mass. The antioxidant action of carotenoids is manifested by a statistically significant increase in antioxidant enzymes and a decrease in the intensity of lipid peroxidation. The extract statistically significantly reduces the serum values of NT-proBNP, AST, creatinine, urea, and uric acid. Carotenoids in this research stimulate the maximum capacity of the electron transport system, as well as the activity of Complexes I, II, and IV. The use of carotenoids alleviates the degree of myocardial damage (statistically significantly lower DDS score), statistically significantly increases the expression of protective markers (Anti-Cardiac Troponin I, Anti-Nrf2, and Anti-Bcl2 antibodies), while statistically significantly decreases the number of immunopositive cells and the expression of Anti-casp3, Anti- COX2 and Anti-Ki67 antibodies. Conclusion: The results obtained in this doctoral dissertation strongly suggest that the carotenoid extract of pumpkin pulp has a cardioprotective effect. Considering the fact that it did not show negative effects in any of the examined aspects, one gets the impression that it is a potential therapy in the prevention of DIC. Also, the NADES (C8:C10) solvent used for carotenoid extraction was found to be safe for use in animals
