The Effect of β-Carotene, Tocopherols and Ascorbic Acid as Anti-Oxidant Molecules on Human and Animal In Vitro/In Vivo Studies: A Review of Research Design and Analytical Techniques Used

Abstract

Prolonged elevated oxidative stress (OS) possesses negative effect on cell structure and functioning, and is associated with the development of numerous disorders. Naturally occurred anti-oxidant compounds reduce the oxidative stress in living organisms. In this review, antioxidant properties of β-carotene, tocopherols and ascorbic acid are presented based on in vitro, in vivo and populational studies. Firstly, environmental factors contributing to the OS occurrence and intracellular sources of Reactive Oxygen Species (ROS) generation, as well as ROS-mediated cellular structure degradation, are introduced. Secondly, enzymatic and non-enzymatic mechanism of anti-oxidant defence against OS development, is presented. Furthermore, ROS-preventing mechanisms and effectiveness of β-carotene, tocopherols and ascorbic acid as anti-oxidants are summarized, based on studies where different ROS-generating (oxidizing) agents are used. Oxidative stress biomarkers, as indicators on OS level and prevention by anti-oxidant supplementation, are presented with a focus on the methods (spectrophotometric, fluorometric, chromatographic, immuno-enzymatic) of their detection. Finally, the application of Raman spectroscopy and imaging as a tool for monitoring the effect of anti-oxidant (β-carotene, ascorbic acid) on cell structure and metabolism, is proposed. Literature data gathered suggest that β-carotene, tocopherols and ascorbic acid possess potential to mitigate oxidative stress in various biological systems. Moreover, Raman spectroscopy and imaging can be a valuable technique to study the effect of oxidative stress and anti-oxidant molecules in cell studies.

Keywords: antioxidants; biomarkers; oxidative stress; raman spectroscopy and imaging; reactive oxygen species.

Figure 1

The overview of oxidative stress (OS) generation in human, with various environmental factors as the cause of OS, with different ROS types and different intracellular sites for ROS generation, OS relation with the disease’s occurrence, and anti-oxidant mechanisms involving enzymatic and non-enzymatic molecules.

Figure 2

The scavenging mechanism of provitamin A and vitamin A.

Figure 3

The scavenging mechanism of α-tocopherol.

Figure 4

The scavenging mechanism of ascorbate.

Figure 5

Exemplary organic stress inducers used during in vitro and/or in vivo tests. Structures of glycochenodeoxycholic acid (A), aristolochic acid (B), t-BuOOH (C), dichlorvos (D), homocysteine (E), doxorubicin (F), methotrexate (G) and quinalphos (H).

Figure 6

The structures of provitamin A (β-carotene) and vitamin A constituents (retinaldehyde, retinol, retinoic acid).

Figure 7

The microscopy image of exemplary CCD-18Co cell (A), Raman image constructed based on Cluster Analysis (CA) method (B), Raman images of all clusters identified by CA assigned to: nucleus (red), mitochondria (magenta), lipid-rich regions (blue, orange), membrane (light grey), cytoplasm (green), and cell environment (dark grey) (C), average Raman spectra typical for all clusters identified by CA in a 500–1800 cm⁻¹ (D) and a 2700–3100 cm⁻¹ (E) wavenumber region, average Raman spectrum for the whole cell within 500–3100 cm⁻¹ (F); cells measured in PBS, excitation wavelength: 532 nm. Reprinted with permission from [277].

Figure 8

Raman spectroscopy analysis of cells exposed to t-BuOOH, β-carotene and/or Vit C; Raman image of CCD-18Co exemplary cells (A), average Raman spectra of exemplary CCD-18Co cell (B), Raman I_1004/1254 (C) and Raman I_1254/1656 (D) graph values with bars: control, t-BuOOH, t-BuOOH + β-C, Raman I_1004/1078 (E) and Raman I_1004/1658 (F) graph values with bars: control, t-BuOOH, t-BuOOH + Vit C. Reprinted and adapted with permission from [277,278].