Hydrophobic NADES-Derived Pumpkin Carotenoid Extract Attenuates Oxidative Stress and Mitochondrial Dysfunction in a Rat Model of Doxorubicin-Induced Cardiotoxicity

Figures

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  Figure 1. Representative HPLC-DAD chromatogram of the NADES-derived pumpkin extract. Major carotenoids identified include (a) α-carotene and (b) β-carotene (red line—standard; blue line tested sample). Peak identity was confirmed by both retention time matching and UV–Vis spectral comparison with reference standards. The overlaid UV–Vis spectra of the standards and corresponding peaks in the extract show high spectral similarity, confirming compound identity. No interfering signals from the NADES matrix were observed at the retention times of target analytes.

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  Figure 2. Experimental design. Created in BioRender. Andrejic Visnjic, B. (2026) https://BioRender.com/nvmv0at (accessed on 1 May 2026).

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  Figure 3. Preparation of myocardium muscle fibers: (A) The heart is placed in a Petri dish with ice-cold medium; (B,C) Sampling tissue pieces; (D) A pair of muscle bundles was cut from the myocardium of the left heart chamber; (E,F) Individual muscle fibers (obtained through careful mechanical dissection) were used for experiments.

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  Figure 4. Antioxidative potential of pumpkin pulp carotenoid extract: (A) Glutathione peroxidase activity (GSH-Px; nmol/mg proteins, X ± SD); (B) Glutathione reductase activity (GR; nmol/mg proteins, X ± SD); (C) Glutathione-S-transferase activity (GST; nmol/mg proteins, X ± SD); (D) superoxide dismutase activity (SOD; U/mg proteins, X ± SD) in rat heart tissue. Each group consisted of 5 animals, while treatments include saline (C), NADES (C8:C10) solvent used for carotenoid extraction (N), pumpkin pulp carotenoid extract corresponding to 900 µg/kg total carotenoids (P), doxorubicin (D), co-treatment with NADES (C8:C10) solvent and doxorubicin (ND), and co-treatment of pumpkin pulp extract corresponding to 900 µg/kg total carotenoids and doxorubicin (PD). *—p < 0.001.

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  Figure 5. The protective role of pumpkin pulp carotenoid extract corresponding to 900 µg/kg total carotenoids in chronic cardiomyopathy induced by DOX and the effect of NADES solution on mitochondrial respiratory parameters. Oxygen consumption analysis was performed in saponin-permeabilized myocardium fibers of the left heart chamber. (A) Mitochondrial activities of complex I (CI) after the addition of pyruvate, malate, and glutamate as specific substrates, respectively. (B) Oxygen consumption rate under ADP phosphorylation conditions (state 3) in the presence of pyruvate, malate, glutamate, and ADP, respectively. (C) FCCP-stimulated maximal respiratory capacity in the presence of pyruvate, malate, glutamate, succinate, ADP, and FCCP, respectively. (D) Complex II-linked respiration after inhibition of Complex I with rotenone and addition of succinate. (E) Activity of complex IV (CIV) measured after inhibition of complex III with antimycin A and the addition of artificial substrates TMPD and ascorbate. Data are presented as mean ± SD; n = 5 animals per group. For mitochondrial respiration, one or two permeabilized fiber preparations were analyzed per animal, and animal means were used for…