Biomolecules 12 01087 v2

biomolecules
Review
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
Krystian Miazek 1, *, Karolina Beton 1 , Agnieszka Śliwińska 2 and Beata Brożek-Płuska 1
1 Laboratory of Laser Molecular Spectroscopy, Institute of Applied Radiation Chemistry,

Lodz University of Technology, Wroblewskiego 15, 93-590 Lodz, Poland
2 Department of Nucleic Acid Biochemistry, Medical University of Lodz, 251 Pomorska Str.,
92-213 Lodz, Poland
* Correspondence: krystian.miazek@dokt.p.lodz.pl
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
Citation: Miazek, K.; Beton, K.; effectiveness of β-carotene, tocopherols and ascorbic acid as anti-oxidants are summarized, based on
Śliwińska, A.; Brożek-Płuska, B. The studies where different ROS-generating (oxidizing) agents are used. Oxidative stress biomarkers,
Effect of β-Carotene, Tocopherols as indicators on OS level and prevention by anti-oxidant supplementation, are presented with a
and Ascorbic Acid as Anti-Oxidant
focus on the methods (spectrophotometric, fluorometric, chromatographic, immuno-enzymatic) of
Molecules on Human and Animal In
their detection. Finally, the application of Raman spectroscopy and imaging as a tool for monitoring
Vitro/In Vivo Studies: A Review of
the effect of anti-oxidant (β-carotene, ascorbic acid) on cell structure and metabolism, is proposed.
Research Design and Analytical
Literature data gathered suggest that β-carotene, tocopherols and ascorbic acid possess potential to
Techniques Used. Biomolecules 2022,
12, 1087. https://doi.org/10.3390/
mitigate oxidative stress in various biological systems. Moreover, Raman spectroscopy and imaging
biom12081087 can be a valuable technique to study the effect of oxidative stress and anti-oxidant molecules in
cell studies.
Academic Editor: Vito Verardo
Received: 9 June 2022 Keywords: oxidative stress; reactive oxygen species; antioxidants; biomarkers; raman spectroscopy
Accepted: 2 August 2022 and imaging
Published: 7 August 2022
Publisher’s Note: MDPI stays neutral

with regard to jurisdictional claims in
published maps and institutional affil-
1. Introduction
iations. Oxidative stress (OS) is defined by the production and accumulation of Reactive Oxy-
gen Species (ROS) in biological systems above the capacity of cells and tissues to neutralize
ROS presence to a safe level [1]. Reactive Oxygen Species (ROS) are molecular oxygen
(O2 )-derived characterized by high reactivity towards other molecules. ROS comprise free
Copyright: © 2022 by the authors. radicals such as superoxide anion radical (O2 •− ) and hydroxyl radical (•OH), and nonradi-
Licensee MDPI, Basel, Switzerland. cal molecules such as singlet oxygen (1 O2 ) and hydrogen peroxide (H2 O2 ). Singlet oxygen
This article is an open access article
(1 O2 ) is the exited state of ground state triplet molecular oxygen (3 O2 ), and is generated via
distributed under the terms and
absorption of energy sufficient to reverse the spin of one of its unpaired electrons what leads
conditions of the Creative Commons
to the formation of singlet state where two electrons possess opposite spin [2]. Superoxide
Attribution (CC BY) license (https://
anion radical (O2 •− ) is generated by one-electron reduction of molecular oxygen (O2 ), what
creativecommons.org/licenses/by/
results in the production of a charged ionic species (O2 •− ) possessing a single unpaired
4.0/).
Biomolecules 2022, 12, 1087. https://doi.org/10.3390/biom12081087 https://www.mdpi.com/journal/biomolecules

Biomolecules 2022, 12, 1087 2 of 45
electron and a negative charge [3]. Hydrogen peroxide (H2 O2 ), a compound possessing an
oxygen-oxygen single bond [4], is generated from dismutation of O2 •− [3]. H2 O2 can be
decomposed via Fenton reaction to OH− and •OH with Fe2+ oxidation, or can react with
O2 •− to form •OH, OH− and O2 [2]. Hydroxyl radical (•OH) possesses a single unpaired
electron and is generated by oxidation of water (H2 O) or hydroxide ions (OH− ), or by
decomposition of hydrogen peroxide (H2 O2 ) via Fenton reaction [5,6].
In living organisms, ROS are produced during indigenous metabolism to serve as
signalling molecules, but can be also generated at high concentrations due to exposure to en-
vironmental stress factors (radiation, pollution, pathogens, etc.) [7]. Generated ROS include
singlet oxygen (1 O2 ), superoxide anion radical (O2 •− ), hydrogen peroxide (H2 O2 ) and hy-
droxyl radical (•OH), and are characterized by different reactivity. 1 O2 is a highly reactive
form of oxygen possessing two electrons with opposite spins, and oxidizing molecules via
reaction with double bonds [8,9]. Although O2 •− is not a strong oxidant, it can undergo
dismutation to H2 O2 that can decompose to form a •OH, one of the strongest oxidants.
O2 •− can also react with nitric oxide radical to form a peroxynitrite, also considered a
powerful oxidant [8]. The elevated production of ROS and oxidative stress cause detrimen-
tal effect on organisms due to the damage of cellular macromolecules, with oxidation of
lipids, proteins and nucleic acids. In human, the occurrence and development of many
diseases such as obesity, atherosclerosis, diabetes mellitus type 2 (DMT2), rheumatoid
arthritis, cancer and neurodegenerative diseases are associated with oxidative stress [10].
To cope with oxidative stress, organisms developed defence mechanisms (Figure 1) involv-
ing anti-oxidant enzymes, as well as non-enzymatic molecules. The human enzymatic
anti-oxidant mechanisms include enzymes such as superoxide dismutase (SOD), cata-
lase (CAT), glutathione peroxidase (GPx), glutathione reductase (GRd) and thioredoxins
(Trx), with glutathione (GSH) serving as electron donor and used in reactions catalysed
by GPx. The non-enzymatic anti-oxidant system in human body is composed of anti-
oxidant proteins, such as proline-rich proteins and ferritin, and low-weight anti-oxidant
molecules synthesized indigenously, such as coenzyme Q10 , α-lipoic acid, melatonin and
bilirubin. Moreover, addition of external low-weight anti-oxidant molecules, such as N-
acetylcysteine, resveratrol, β-carotene, tocopherols and ascorbic acid, can strengthen the
capability of indigenous defence system against OS.
The mitigating effect of β-carotene, tocopherols and ascorbic acid against oxidative
stress is presented in this article. β-carotene classified as provitamin A, ascorbic acid
named as vitamin C and tocopherols possessing activity of vitamin E, are exogenous
anti-oxidant molecules with ROS-scavenging properties that are considered useful in pre-
venting oxidative stress in mammalian cells and organisms. In this review, anti-oxidant
properties of β-carotene, tocopherols and ascorbic acid to alleviate oxidative stress based
on in vitro, in vivo as well as populational studies, are described. Moreover, different ana-
lytical techniques (spectrophotometric, fluorometric, chromatographic, immune-enzymatic,
spectroscopic) used commonly to assess the OS presence and anti-oxidant properties of
tested molecules, are compared and discussed.
Overall, this review provides a broad description regarding the effect of β-carotene,
ascorbic acid and tocopherols as potential antioxidants to mitigate oxidative stress in human
and animal studies, in terms of research designs, experimental outcomes and analytical
techniques applied.
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.
2. Environmental Factors as Inducers of Oxidative Stress

Environmental factors, such as radiation (ultraviolet, ionizing), tobacco smoke, xeno-
biotics and other pollutants as well as infections, are inducers of ROS generation and
oxidative stress (Figure 1).
Ultraviolet (UV) light, a part of electromagnetic radiation spectrum with wavelength
range of 100 nm to 400 nm, is divided into UVA (320–400 nm), UVB (290–320 nm) and UVC
(220–290 nm) light. The sources of UV light can be natural (sun) or artificial (phototherapy,
sunbeds, arc welding, germicidal lamps). UV radiation can be absorbed by cellular com-
ponents resulting in their conversion into the exited state, performing chemical reactions
with ROS generation. UV rays can be also absorbed by photosensitizers (endogenous and
exogenous), which in their exited state, can abstract hydrogen to form free radicals, or can
transfer energy with O2 to form 1 O2 [11]. UV-induced reactive oxygen species contribute to
the damaging effect of UV towards the skin [12].
Ionizing radiation (IR), including gamma and X rays, is the radiation capable of induc-
ing atom ionization via reactions of electron ejection [13]. The sources of IR are radiotherapy,
nuclear accidents, atomic bombing, soil radioisotopes and cosmic rays [14,15]. Ionizing
radiation causes radiolysis of water and linear energy transfer (LET)-mediated generation
of reactive chemical species (•OH, H2 O2 , O2 •− ) that can damage macromolecules (nucleic
acids, proteins, lipids) [16]. IR-generated reactive species cause cellular damage with
detrimental effect on living organisms [13].
Xenobiotics, represented by polycyclic aromatic hydrocarbons and pesticides, are
organic substances being foreign to living organisms and possessing structural abilities to
induce oxidative stress in organs and tissues. Polycyclic aromatic hydrocarbons (PAHs) are
formed during incomplete combustion processes of fuels and from traffic emissions [17].
PAHs undergo oxidation into phenolic intermediates which are converted via semiquinone
anion radicals into quinones, with generation of superoxide anion radicals and H2 O2 [18,19].
Pesticides (herbicides, insecticides, fungicides, etc.), used in agriculture for crop protection,
can be found as contaminants in air, water and food. The mode of oxidative action of
pesticides, with herbicide paraquat as an example, is the induction of mitochondrial damage
and the redox cycle involving quaternary ammonium nitrogen atoms and a bipirydyl ring
in paraquat structure, what leads to production of ROS and paraquat radicals [20].
Heavy metals (HMs), such as Pb, Cd, Cr, Hg and As, are contaminants released from
industry (effluents, waste product storage) to the surroundings (atmosphere, soil, water)
and affecting human beings [21]. Accumulation of HMs ions in body induces oxidative
stress within a range of mechanisms, including inhibition of antioxidant enzyme expression
(by Cd), interaction with cofactors and/or disulphide bonds in antioxidant enzymes (e.g.,
Pb, Hg), haemoglobin autooxidation (by Pb2+ ), binding to sulfhydryl groups (−SH) and
reducing thiol pools (through Cd2+ or Hg2+ ), generating glutathione-thiyl radicals (via
Cr(VI)), changing the oxidation state of HMs with formation of H2 O2 and hydroxyl radicals
(through Cr or As), affecting calcium homeostasis and stimulating oxidative enzymes (by
Hg), cytokine-mediated ROS generation (via Cd2+ ), and others [18,22,23]. Heavy metal-
induced oxidative stress and macromolecules modification/degradation are the cause of
many diseases, amongst which are cancer, cardiovascular disease, neurological disorders
and chronic inflammation [24].
Drugs used for illness treatment are also the source of ROS generation in human
body [25]. Anti-neoplastic agents, such as doxorubicin and cisplatin, are used for the
treatment of different types of cancer. Doxorubicin, a representative of anthracycline
antibiotics, generates ROS by undergoing mitochondrial reductase-mediated one-electron
reduction to anthracycline semiquinone free radicals, that can react with O2 to form O2 •−
or H2 O2 . Doxorubicin can also interact with Fe3+ to form Fe2+ -doxorubicin free radical,
that can reduce oxygen. Oxidative stress induction is the mechanism of doxorubicin
cardiotoxicity [25]. Cisplatin, a platinum containing drug, was reported to increase the ROS
level, via NAPDH oxidase or xanthine oxidase [25,26]. Oxidative stress induction in the
proposed mechanism of cisplatin nephrotoxicity and ototoxicity [26,27].
Smoking, with cigarette smoke containing nicotine, ammonia, acrolein, phenols, ac-
etaldehyde, polycyclic aromatic hydrocarbons, polyphenols, hydrogen cyanide, heavy
metals, etc., is another source of ROS production and oxidative stress occurrence [28,29].
The tobacco smoke contains gas phase and tar phase. The gas phase contains short-lived
radicals, superoxide anion and nitric oxide, which react together to form highly reactive per-
oxynitrite. The tar phase contains stable semiquinone radicals and iron (Fe2+ ). Semiquinone
radicals reduce O2 to O2 •− , that can dismutate into H2 O2 , which in turn can react with Fe2+
via Fenton reaction to form •OH [29,30]. Oxidative stress is considered as a crucial factor
in the pathogenesis of smoking-related disorders, such as lung cancer, chronic obstructive
pulmonary disease and atherosclerosis [31].
Ozone (O3 ) is a gaseous tropospheric pollutant, generated through reactions between
intense solar radiation and pollutants (nitric oxides, sulphur oxides, carbon oxides, volatile
organic compounds) produced from combustion of fossil fuels [32,33]. Contact of O3 with
biological matrix results in creation of H2 O2 and lipids oxidation products [34]. Ozone
exposure-induced oxidative stress is associated with neurodegenerative diseases [33,35].
The infections by viruses or bacteria can be also the cause of oxidative stress in human
body. The body can be infected by viruses, such as DNA and RNA viruses, which enter and
replicate inside host cells [36,37]. ROS are generated during viral infection via inducing
activation of phagocytes [36] or via mediation of viral proteins expressed in host cells
to support viral life cycle [37]. Oxidative stress occurring during bacterial infection is
described for Helicobacter pylori, a gram negative, stomach-infecting bacterium. H. pylori
infection results in oxidative stress via the immune and gastric epithelial cells producing
ROS in an attempt to kill the bacteria, and via bacterial virulence factors inducing epithelium
cellular responses and ROS generation [38].
Unhealthy dietary patterns, based on overconsumption of high-carbohydrate and

high-fat food, are associated with increased risk of overweight and obesity occurrence and
development of diabetes mellitus type 2 (DMT2) and cardiovascular diseases. High-fat or
high-carbohydrate diets results in the elevated influx of substrates into mitochondrial respi-
ration and increased donation of electrons to electron transport chain, leading to the electron
leakage at complex III and elevated superoxide (O2 •− ) generation. [39]. NADPH oxidase
(NOX), an enzyme converting molecular oxygen to its superoxide radical, is also involved
in nutrient-based ROS generation [40]. High-calorie diets may alter oxygen metabolism
and are considered as one of the main factors leading to excessive ROS production [41].
3. Metabolic Pathways as Sources of ROS Generation in Cells

The main endogenous sources of ROS are enzymes of mitochondrial respiratory chain
and nicotinamide adenine dinucleotide phosphate (NADPH) oxidase enzymes, whereas
other sources of ROS are cytochromes P450, lipoxygenases and peroxisomal enzymes
(Figure 1).
Electron transport chain (ETC), embedded within mitochondrial inner membranes, are
sources of ROS production. The ETC includes transmembrane protein complexes NADH:
ubiquinone oxidoreductase (Complex I), succinate dehydrogenase (Complex II), ubiquinol:
cytochrome c oxidoreductase (Complex III) and cytochrome c oxidase (Complex IV), as well
as mobile electron transporters (ubiquinone, cytochrome c). ETC participates in the process
of oxidative phosphorylation (OXPHOS), where O2 is converted to H2 O and adenosine
triphosphate (ATP) is produced. In brief, electrons from mitochondrial matrix tricarboxylic
acid (TCA) cycle are donated via NADH to Complex I and via FADH2 to Complex II,
and are transferred from Complex I and II to ubiquinone (Q) that undergoes reduction
to ubiquinol (QH2 ). Ubiquinol, carrying electrons, becomes re-oxidized via Q-cycle in
Complex III, that passes electrons via cytochrome c (CytC) to Complex IV. Subsequently,
complex IV transfers electrons to O2 as electron acceptor with generation of H2 O. The
electron flow (CI&CII→QH2 →CIII→CytC→CIV→H2 O) is coupled with the pumping of
protons (H+ ) from mitochondrial matrix into the intermembrane space by complexes I, III
and IV. The proton gradient formed across the mitochondrial inner membrane is used by
complex V (ATP synthase) to produce (ATP). ETC can produce reactive oxygen species due
to the leakage of electrons from Complex I, II and III, resulting in one-electron reduction of
oxygen to O2 •− . O2 •− , formed via Complex I and II, occur in the matrix, whereas Complex
III O2 •− is released into both the matrix and intermembrane space. O2 •− is dismutated to
H2 O2 by SOD2 in the matrix and by SOD1 in the intermembrane space [7,42,43]. Complex
IV is not directly involved in ROS production, but its activity can affect the overall electron
flow, with an impact on the electron leakage by previous complexes [44]. It is estimated
that 0.2–2% of the electrons passing through the ETC leak out and interact with oxygen to
produce ROS [43].
The NADPH oxidases (NOX) are multi-subunit enzymes localized in membranes.
NOXs transfer electrons across biological membranes to oxygen, thereby generating su-
peroxide anion radical and or hydrogen peroxide. The mechanism of catalysis involves
transfer of two electrons from NADPH through FAD domain and two heme prosthetic
groups in enzyme structure to O2 [45]. The NOX family comprises of seven isoforms
(NOX1, NOX2, NOX3, NOX4, NOX5, DUOX1 and DUOX2) [46,47] expressed in differ-
ent tissues throughout the body. NOX1 is highly expressed in the epithelial cells of the
gastrointestinal tract [48], and is also present in prostate, uterus, vascular cells [49] and
erythrocytes [50]. NOX2 is typically expressed in phagocytic cells, but also in endothelial
cells, cardiomyocytes, hematopoietic stem cells and platelets [51]. NOX3 is expressed
in the inner ear [48], and is also found in low abundance in the brain, lung and in fetal
tissue [49]. NOX4 is commonly distributed in human tissues, and highly expressed in
kidney, osteoclasts, fibroblasts and endothelial cells [47,52]. NOX5 is expressed in testis and
lymphoid tissue, but also in placenta, uterus, stomach, skeletal muscle, hepatocytes, cells
of the cardiovascular system (cardiomyocytes, endothelial and vascular smooth muscle
cells) [49] and erythrocytes [50]. DUOX1 and DUOX2 are highly expressed in thyroid
gland [49]. O2 •− is generated by NOX1-3 and NOX5, while H2 O2 is produced by DUOX1-2
and NOX4 [53].
Cytochromes P450 (CYPs) are enzymes localized primarily in the endoplasmic reticu-
lum and found mostly in liver and intestinal tissues [54]. CYPs are enzymes containing
a heme prosthetic group in the form of iron protoporphyrin IX and participating in the
metabolism of xenobiotics and endogenous compounds. CYPs possess monooxygenase
activity and catalyse the incorporation of one atom of oxygen from O2 to organic substrate
and reduction of the second oxygen atom to water. The catalytic mechanism of CYPs relies
on redox reaction cycle of cysteine-bound iron atom involving substrate binding to ferric
iron and reduction of Fe3+ to Fe2+ , followed by O2 binding to iron and formation of complex
(Fe2+ -O2 ) that undergoes one-electron reduction (Fe2+ -O2 − ) and protonation to cleave O-O
bond and release H2 O, and the transfer of oxygen atom from FeO3+ complex to substrate,
with the formation of mono-oxygenated substrate and regeneration of Fe3+ in CYP. During
CYP monooxygenase cycle, reactive oxygen species can be produced either via releasing of
superoxide radical with its dismutation to H2 O2 or direct releasing H2 O2 [55,56].
Lipoxygenases (LOXs) are nonheme, iron-containing enzymes that catalyse oxygena-
tion of arachidonic acid (AA) to hydroperoxyeicosatetraenoic acids (HPETEs), which can
be further converted to hydroxyeicosatetraenoic acids (HETEs), leukotrienes, lipoxins and
hepoxilins [57,58]. In human, there are 5-LOX, 12-LOX, and 15-LOX, that catalyse the inser-
tion of oxygen molecule at C-5, C-12 and C-15 of AA, respectively [59,60]. LOX-generated
AA metabolites can induce ROS generation in various cells via NOX upregulation [59].
Peroxisomes are single-membrane subcellular organelles, found in all eukaryotic
cells, participating in the range of metabolic pathways such as α- and β-oxidation of
very long fatty acids, the synthesis of bile acids, detoxification of glyoxylate and H2 O2
metabolism [61,62]. Peroxisomal enzymes, such as xanthine oxidase and acyl-CoA oxidases,
are involved in ROS generation [63]. Xanthine oxidase (XO) participates in the catabolism
of purine nucleic acids [64]. XO catalyses the oxidation of hypoxanthine and xanthine with
generation of superoxide anion or hydrogen peroxide, respectively, via the monovalent and
divalent electron transfer to O2 [65]. Acyl-CoA oxidases are enzymes involved in the first
step of β-oxidation of different substances, such as CoA-esters of very long-chain fatty acids,
branched-chain fatty acids and C27-bile acid intermediates [66]. In human peroxisomes,
straight-chain acyl-CoA oxidase, branched-chain acyl-CoA oxidase and pristanoyl-CoA
oxidase, are available. Acyl-CoA oxidases catalyse dehydrogenation of acyl-CoA esters,
with the Flavin Adenine Dinucleotide (FAD)-mediated transfer of the protons from the
β-carbon bond of an acyl-CoA, resulting in generation of trans-2-enoyl-CoA esters and
FADH2 . Subsequently, FADH2 is regenerated to FAD and hydrogen atoms are transferred
to O2 to form H2 O2 [67,68].
4. Effect of ROS on Cellular Macromolecules

Reactive oxygen species can react with lipids, proteins and nucleic acids (Table 1),
the primal macromolecules in cellular structures. ROS cause peroxidation of polyunsat-
urated fatty acids (PUFAs) with the formation of lipid hydroperoxides, that undergo the
cleavage to dialdehydes, such as malondialdehyde (MDA) and glyoxal, and a range of
unsaturated aldehydes including 4-hydroxynonenal (4-HNE), crotonaldehyde, 2-propenal
(acrolein) and 2-hexenal [69]. ROS can react with amino-acids in protein structure, causing
conversion of phenylalanine to o- or m-tyrosine, tyrosine to di-tyrosine, tryptophan to
N-formylkynurenine, leucine to 4- or 5-hydroxyleucine, valine to 3- or 4-hydroxyvaline, as
well as oxidation of thiol groups in cysteine and methionine. Aldehydes (e.g., 4-HNE), gen-
erated from PUFAs peroxidation, can cause the carbonylation of proteins via addition with
S atom of cysteine, N in the imidizole of histidine and N in the amine of lysine [69,70]. ROS,
such as hydroxyl radical, can also react with DNA bases (G, T, C, A) and sugar moieties. Hy-
droxyl radical reacts with C-8 of guanine (G) to generate an 8-hydroxy-7,8-dihydroguanyl
radical, that can undergo the oxidation to 8-oxo-7,8-dihydroguanine (8-oxoG), or the re-
duction to ring-opened 2,6-diamino-4-hydroxy-5-formamidopyrimidine. The products

of adenine modifications were reported to be 8-oxo-7,8-dihydroadenine (8-oxoA) and
4,6-diamino-5-formamidopyrimidine. Hydroxyl radical also reacts with C-5 and C-6 in
pyrimidines of thymine (T) and cytosine (C), forming 5,6-dihydroxy-5,6-dihydrothymine (T
glycol) and 5,6-dihydroxy-5,6-dihydrocytosine (C glycol) [25,71]. Hydroxyl radical can also
react with deoxyribose, via abstracting H-atom from C-5 of sugar moiety in DNA, resulting
in the creation of C-5-centered radical that forms a bond with C-8 of purine ring in the
same nucleotide, thereby yielding 8,5-cyclopurine-2-deoxynucleoside [71]. ROS-mediated
oxidation of macromolecules leads to degradation of cellular structures which is associated
with metabolic complications and diseases development.
Table 1. Exemplary structures of oxidation products of lipids, proteins and nucleic acids.
Exemplary Structures of Oxidation Products

Name Structure Description Mechanism of Formation
PUFA peroxyl radical undergoes
Malondialdehyde (MDA) intramolecular cyclization to endoperoxide
with further breakdown to MDA
4-hydroxy-2-nonenal Peroxidation of n-6 PUFAs and the

(4-HNE; HNE) generation of α,β unsaturated aldehydes
Generation of a tyrosyl radical, radical

Dityrosine isomerization, diradical reaction, and
enolization
HNE-Lys adduct (protein Michael addition of the HNE double bond to

carbonyl product) NH2 -group of lysine (Lys)
HNE-Cys adduct (protein Michael addition of the HNE double bond to

carbonyl product) SH-group of cysteine (Cys)
Table 1. Cont.
Exemplary Structures of Oxidation Products

Name Structure Description Mechanism of Formation
HNE-His adduct (protein Michael addition of the HNE double bond to

carbonyl product) NH in an imidazole of histidine (His)
8-oxo-20 -deoxyguanosine Reaction between C-8 of guanine (G) and

nucleotide hydroxyl radical (•OH)
8-oxo-20 -deoxyadenosine Reaction between C-8 of adenine (A) and

nucleotide hydroxyl radical (•OH)
5,6-dihydroxy-5,6-
Reaction of hydroxyl radical (•OH) with C-5
dihydrothymidine
and C-6 of thymine (T)
nucleotide
5,6-dihydroxy-5,6-
Reaction of hydroxyl radical (•OH) with C-5
dihydrocytidine
and C-6 of cytosine (C)
nucleotide
5. Oxidative Stress and Diseases Development

Oxidative stress is involved in the occurrence and physiopathology of chronic diseases,
such as obesity, atherosclerosis, diabetes mellitus type 2 (DMT2), rheumatoid arthritis,
cancer and neurodegenerative diseases [10].
Obesity, characterized by an increase in body weight resulting in excessive fat accumu-
lation, represents a public health problem with increasing worldwide prevalence [72,73].
Oxidative stress performs a role in the pathogenesis of obesity by stimulating the deposition
of adipose tissue and altering food intake [74]. Oxidative stress is also induced by obesity,
as the excess of free fatty acids (FFA) leads to increased FFA oxidation and mitochondrial
ROS overproduction. Moreover, the increased release of FA from over-accumulated fat
can result in NOX activation and progressed ROS generation [40]. In obesity, the num-
ber and size of adipocytes are increased, and secretion of pro-inflammatory molecules
is promoted [73]. Oxidative stress occurring in obesity is linked with inflammation and
can contribute to the obesity-associated development of diseases, such as atherosclerosis,
diabetes and cancer [40].
Atherosclerosis is a chronic inflammatory condition, characterized by gradual accu-
mulation of plaques, composed of fibrous cap, lipid-rich core and calcium, within the
artery wall [75]. The rupture of atherosclerotic plaques leads to thrombosis which is the
reason of myocardial infarction or stroke [76]. The origin of atherosclerosis is ascribed to
hypertension, hypercholesterolaemia, smoking or hyperglycemia that cause the damage of
endothelium and the entry of cholesterol-carrying low-density lipoproteins (LDLs) from
the blood stream into the epithelium intima [77]. The endothelial cells, smooth muscle cells
(SMCs) and macrophages are the source of oxidative stress, that cause the oxidation of
LDL particles [78]. Oxidized LDL (Ox-LDL) particles are taken up by macrophages, which
are converted into foam cells. The death of foam cells results in the accumulation of cells
debris and lipids, and release of proinflammatory cytokines that cause SMCs migration
and proliferation. The plaque is progressively formed by agglomerating calcium deposits,
SMCs, collagen and foam cells [75,77,79].
Diabetes mellitus type 2 (DMT2) is a disease characterized by tissue resistance to in-
sulin, hyperglycemia and decreased secretion of insulin by pancreatic β-cells [80]. In DMT2,
the occurrence of OS is ascribed to frequent hyperglycemia, mitochondrial dysfunction
and endoplasmic reticulum (ER) stress in β-cells [81], and OS-induced complications of
diabetes may include macrovascular (coronary heart diseases, stroke) and microvascular
(neuropathy, retinopathy, nephropathy) complications [80].
Rheumatoid arthritis (RA) is an autoimmune disease characterized by joint destruction.
Oxidative stress is involved in the pathogenesis of RA, by stimulating inflammation and
being stimulated by inflammation, what results in the establishment of synovitis, which
causes cartilage and bone damage [82].
Cancer is a disease characterized by transformation of normal cells into malignant
cells that proliferate in an uncontrolled manner and invade normal tissues and organs,
eventually spreading throughout the body. Cancer is a major disease and the second
leading cause of mortality worldwide, with more than 277 different types of cancer diseases
affecting different parts of body (colon, breast, lung, liver, prostate, brain, skin, bladder,
renal, stomach etc.) [83–86]. Oxidative damage of DNA leads to disruption of genome
function, distribution of mutation, selective clonal expansion of the mutated cell and further
cancer progression [87,88]. Oxidative stress may be an initiating factor in carcinogenesis
and can be also the consequence of cancer development [89].
Neurodegenerative diseases, characterized by progressive dysfunction of neural cells
and losses of neurons, include Alzheimer’s disease, Parkinson’s disease and amyotrophic
lateral sclerosis [90]. Oxidative stress is one of factors involved in the pathogenesis of
neurodegenerative disorders [91]. Neural microenvironment is susceptible to oxidative
stress due to high oxygen demand, the abundant presence of redox-active metals, high
level of cellular membrane PUFAs and low levels of GSH in the brain [92].
Biomolecules 2022, 12, 1087 10 of 45
6. Anti-Oxidant Mechanisms as a Protection against Oxidative-Stress

Cells possess antioxidant mechanisms, such as enzymes and other proteins, as well
as endogenous and exogenous low molecular weight anti-oxidant molecules, to protect
cellular structures from oxidative stress and damage (Figure 1).
6.1. Superoxide Dismutase (SOD) and Catalase (CAT)

Superoxide dismutase (SOD) is a metalloenzyme that prevents intracellular O2 •−
accumulation by catalysing dismutation of two molecules of O2 •− to O2 and H2 O2 . The
mechanism of SOD catalytic action is based on the redox cycle of metal ion in the active site,
involving metal reduction and oxidation of first O2 •− to O2 , followed by metal oxidation
and reduction of a second O2 •− to H2 O2 . Three isoforms of SOD: Cu,Zn-SOD (SOD1)
homodimer distributed in the cytosol and the mitochondrial intermembrane, Mn-SOD
(SOD2) homotetramer located in the mitochondrial matrix and inner membrane, and Cu,Zn-
SOD (SOD3) homotetramer anchored to the extracellular matrix, are known to be present
in human [93,94].
Catalase (CAT) is a tetrameric protein with 4 similar subunits, each containing a
ferriprotoporphyrin. CAT catalyses the reduction of 2 molecules of H2 O2 to 2 molecules
of H2 O and one O2 , within a two-step reaction mode. The first step involves reduction of
one H2 O2 molecule with oxidation of heme to an oxyferryl species [porphyrin Fe(IV)-O],
having a porphyrin π-cation radical. In the second step, a porphyrin radical is reduced
by a two-electron transfer from the second H2 O2 molecule to generate the enzyme at
resting state [porphyrin Fe(III)] and produce water and oxygen. In mammalian cells, CAT
is primarily present in peroxisomes, and its absence in mitochondria is compensated by
glutathione peroxidase [95,96].
6.2. Glutathione (GSH), Glutathione Peroxidase (GPx) and Glutathione Reductase (GRd)
Glutathione (GSH) is a tripeptide, possessing L-γ-glutamyl-L-cysteinyl-glycine struc-
ture, present in most cells. GSH is synthetized de novo in the cytosol through a two-step
process starting from L-glutamate and cysteine to form γ-glutamylcysteine intermediate
via the enzyme glutamate cysteine ligase (γ-glutamylcysteine synthetase). Subsequently,
L -glycine is added to the C-terminus of γ-glutamylcysteine via the enzyme glutathione
synthetase to form glutathione. GSH is maintained at high concentrations in cells, where
it performs a role as an antioxidant. GSH serves as electron donor to H2 O2 and lipid
peroxides, that are reduced to water and lipid alcohols, respectively, in reactions catalysed
by glutathione peroxidase (GPx) [97–99].
Glutathione peroxidase (GPx), present in the cytosol and in mitochondria, is a tetrameric
enzyme containing seleno-cysteine (SeC) in the active site [99,100]. The SeC active site
(Se-H) reacts with peroxide to form a selenenic acid (Se-OH), which is reduced by GSH
molecule, resulted in the formation of a glutathiolated selenol (Se-SG) intermediate. Sub-
sequently, the Se-SG bond is reduced by second GSH molecule, leading to the restoration
of the GPx active site and the formation of oxidized glutathione (GSSG). GSSG, formed
via creating a disulphide bridge between two glutathione molecules, is a product of GPx-
catalysed reduction of peroxides [99]. GSSG can be reduced to GSH in reaction catalysed
by glutathione reductase (GRd).
Glutathione reductase (GRd), a homodimer containing one flavine adenine dinu-
cleotide (FAD) per subunit, catalyses the conversion of GSSG to GSH via using the reduced
form of nicotinamide adenine dinucleotide phosphate (NADPH). Mechanism of GSH
restoration from GSSG involves GPx reduction by NADPH and transfer of electrons to
GSSG, facilitated by several key residues in GPx active site [101]. The high availability of
GSH is important for scavenging oxidants and maintaining redox-state balance in healthy
cells [98]. The intracellular concentrations of GSH can be increased via exogenous sources,
such as N-acetylcysteine.
Biomolecules 2022, 12, 1087 11 of 45
6.3. Thioredoxins
Thioredoxins (Trx) are proteins performing important role as endogenous antioxidant
system against oxidative stress. The Trx antioxidant system is composed by thioredoxin
(Trx), NADPH and thioredoxin reductase (TrxR). Trx, an enzyme with cys-gly-pro-cys
in its active site, can be present in either oxidized disulphide form (Trx-S2 ) or reduced
dithiol form [Trx-(SH)2 ]. The reduced form of Trx can act as a reductase towards disulphide
bonds in oxidatively damaged proteins, via a disulphide–dithiol exchange mechanism
resulting in thiol restoration in targeted protein and Trx oxidation [7,102]. The disulphides
in oxidized Trxs are converted to dithiol form in reaction involving the presence of NADPH
and catalysed by thioredoxin reductase (TrxR), a selenocysteine and FAD-containing pro-
tein [103,104]. The TrxR/Trx system can also catalyse reduction of dehydroascorbate [104]
and reduction of GSSG to GSH [101].
Glutaredoxins (Grx) are intracellular redox enzymes belonging to the Trx protein
family. Grx can catalyse reduction of disulphide bonds in substrates via dithiol and/or
monothiol mechanism. In the first reaction mode, dithiols in Grx [Grx-(SH)2 ] reduce disul-
phide in target proteins via disulphide–dithiol exchange mechanism, with the formation
of oxidized Grx (Grx-S2 ) [102]. The active site of oxidized Grx can be reduced back to
dithiol form via GSH. In the monothiol reaction mode, Grx-SH catalyses the reduction
of the disulphide bond (protein-S-SG) between GSH and target protein, resulting in the
release of protein-SH and formation of a new disulphide (Grx-S-SG) between Grx and
GSH. Subsequently, the Grx-S-SG disulphide is reduced by another GSH molecule, yielding
Grx-SH and GSSG [105,106].
6.4. Other Antioxidant Proteins

Except for enzymes, there are other anti-oxidant proteins such as small proline-rich
proteins and ferritin, that participate in anti-oxidant system in human body.
Small proline-rich proteins (SPRRs) are structural components of cornified envelope
in corneocytes, localized in the outermost layer of skin known as stratum corneum [107].
In the structure of cornified cell envelope (CE), SPRRs serve as crosslinking proteins via
transglutaminase-mediated formation of ε-(γ-glutamyl) lysine cross-linkages with other
proteins such as loricrin, thereby increasing the rigidity of CE [107–109]. The structure
of SPRRs, composed of repeating β-turns, is determined by proline content, while high
number of cysteine (-SH) residues in SPRRs are responsible for ROS quenching, with
formation of inter- and intramolecular disulphide (S–S) bonds [110]. Therefore, SPRRs
are part of defence mechanism protecting epidermis from ROS damage induced by UV,
xenobiotics and pollutants [111].
Ferritin is a ubiquitous protein, distributed in the serum and in the cytoplasm, nu-
cleus and mitochondria of cells, with a principal function for iron storage. The ferritin
molecule consists of 24 subunits, organised within two distinct subunit types, heavy (H)
and light (L), forming together a spherical structure [112–114]. Ferritin possesses antioxi-
dant properties by sequestering iron, which in free form (Fe2+ ) can catalyse reduction of
H2 O2 and production of highly reactive •OH via Fenton chemistry [115]. The H subunit of
ferritin possesses ferroxidase activity catalysing oxidation of Fe2+ to stable Fe3+ , while the
L subunit stabilizes protein structure and facilitates the uptake of iron, stored inside the
ferritin shell. Even 4500 iron atoms can be sequestered by ferritin, in the balance between
ferritin-bound iron (Fe3+ ) and Fe2+ pool in the cells, thereby preventing ROS generation via
Fenton reaction [112–114]. It was suggested that increase in ferritin synthesis can serve as
the defence mechanism against oxidative stress [116,117], and overexpression of ferritin H
or L subunits diminished ROS formation in HeLa cells exposed to H2 O2 [117].
6.5. Antioxidant Low Molecular Weight Molecules

Except for enzymes and other proteins, there are anti-oxidant low molecular weight
molecules, of endogenous and exogenous origin, participating in anti-oxidant system in
human body.
Biomolecules 2022, 12, 1087 12 of 45
Coenzyme Q10 (CoQ10 , ubiquinone), with a structure of 2,3-dimethoxy-5-methyl-

6-decaprenyl-1,4-benzoquinone, is a lipophilic molecule synthetized in human and an-
imal cells [118]. In CoQ10 , a benzoquinone ring is synthetized from tyrosine, with 4-
hydroxybenzoate (4HB) as a precursor of CoQ, while an isoprenoid side chain of CoQ10
is obtained from farnesyl pyrophosphate, a product of the mevalonate pathway [119].
CoQ10 , as a component of the mitochondrial respiratory chain, accepts electrons from
complex I (NADH: coenzyme Q reductase) and complex II (succinate: coenzyme Q re-
ductase) and transfers (as CoQ10 H2 ) electrons to complex III (coenzyme Q: cytochrome
c reductase) [42]. The reduced form of CoQ10 (CoQ10 H2 , ubiquinol) acts as a phenolic
antioxidant, protecting DNA, membrane phospholipids and mitochondrial membrane
phospholipids from free-radical-induced oxidative damage [120]. Coenzyme Q10 can be
also obtained exogenously via supplementation. A meta-analysis comprising multiple
clinical trials concluded that CoQ10 administration dosages resulted in the decrease in
MDA levels amongst participants [121].
Melatonin, also named as N-acetyl-5-methoxy-tryptamine, is an endogenous indole
hormone controlling physiologic processes such as sleep and circadian rhythm [122]. Mela-
tonin is secreted by the pineal gland, where it is synthetized via a pathway involving
tryptophane and serotonin, but its synthesis also occurs in brain, lens, skin, retina, lym-
phocytes and bone marrow [123]. Melatonin is synthetized, taken up by, and concentrated
in mitochondria [122]. Melatonin functions as an antioxidant by directly scavenging free
radicals, stimulating anti-oxidant enzymes (SOD, CAT, GPx, GRd), as well as improv-
ing mitochondrial OXPHOS efficiency [124]. Melatonin is a direct ROS scavenger (1 O2 ,
O2 •− ,•OH) by donating electron(s) to the ROS, with the formation of products such as
cyclic 3-hydroxymelatonin or indolyl radical cation of low reactivity [123]. Melatonin can
be also supplied exogenously from diet or supplements [125] to heal jet lag, insomnia,
narcolepsy and other sleep disorders, and is considered as a potential cardioprotective,
anti-inflammatory and anti-cancer agent [126].
Bilirubin (BR) is a tetrapyrrole pigment, composed of two rigid dipyrroles joined by a
methylene bridge at carbon 10, present in the plasma as a form bound to albumin, while
a form conjugated with glucuronic acid is excreted into the intestine with bile [127,128].
Synthesis of bilirubin includes two steps, the cleavage of heme IX via heme-oxygenase
(HO) into biliverdin IXα, carbon monoxide and free ferrous iron, and subsequent conver-
sion of biliverdin (BV) into bilirubin IXα via biliverdin reductase [128,129]. Bilirubin is
a strong endogenous anti-oxidant cytoprotectant that interacts with free oxygen radicals
resulting in the oxidation of BR to BV and immediate reduction to bilirubin via biliverdin
reductase [130]. Bilirubin was reported to neutralize free radicals, providing efficient
protection against 10,000-fold higher concentration of H2 O2 [128,130], and prevent peroxi-
dation of lipids [131]. The anti-oxidant properties of bilirubin also stem from its ability to
inhibit NADPH oxidase [132]. Bilirubin is considered as a crucial substance acting as an
antioxidant substance in serum of human beings [131].
α-Lipoic acid (α-LA), also named as 1,2-dithiolane-3-pentanoic acid, is an organosulfur
compound synthetized by plants, animals and humans [133]. α-LA is synthetized in
mitochondria from octanoic acid and cysteine, and play a crucial role in mitochondrial
bioenergetic reactions. α-LA can be reduced by NADH/NADPH to dihydrolipoic acid
(DHLA), and both forms possess anti-oxidant activity. α-LA and DHLA are capable of
scavenging hydroxyl radicals and preventing protein carbonyl formation, whereas DHLA
can also reduce the oxidized forms of vitamin C and E, and GSH [134]. α-LA is synthetized
endogenously, but is also considered as a valuable supplement with beneficial therapeutic
effects [135–137].
N-acetylcysteine (NAC), a derivative of amino acid L-cysteine, is used as a drug for
the treatment of acetaminophen overdose and as a mucolytic agent in respiratory diseases.
NAC, administered orally, intravenously or by inhalation, is metabolized into cysteine,
cystine, inorganic sulfate and glutathione [138]. N-acetylcysteine possesses direct and
indirect antioxidant activity. In a direct mode, sulfhydryl group (-SH) in the NAC reduces
Biomolecules 2022, 12, 1087 13 of 45
radical species via electron donation. In an indirect antioxidant action, NAC undergoes
deacetylation via acylase to cysteine, the building block in glutathione synthesis. NAC
can also break S-S bonds via thiol-disulphide interchange reaction, thereby restoring thiol
pools involved in redox state regulation [139]. NAC can also bind to metal ions, such as
copper (Cu2+ ), iron (Fe3+ ), cadmium (Cd2+ ), mercury (Hg2+ ) and lead (Pb2+ ), via forming
complexes which are excreted from the body [138]. N-acetylcysteine is considered as a
valuable supplement for conventional treatment of oxidative stress-linked diseases [140].
Resveratrol (RV) is a plant-derived stilbene polyphenol (3,5,40 -trihydroxystilbene)
compound, found in numerous food sources including grapes, wine, blueberry, bilberry,
cranberry and peanuts [141]. RV is administered orally, absorbed via passive diffusion
or in complex with membrane transporters and released into the bloodstream. In liver,
RV is converted to conjugated glucuronides and sulfate metabolites possessing biological
activity [142]. Resveratrol possesses a wide range of biological properties, such as car-
dioprotective, neuroprotective, anti-inflammatory, anticancer, antidiabetic, antimicrobial
and antioxidant activities [143]. Resveratrol showed hydrogen peroxide and superoxide
radical scavenging activities, as well as ferrous ion (Fe2+ ) chelating activity [144]. The ROS-
scavenging activity of resveratrol depends on hydrogen donation via hydrogen abstraction
from para-OH (40 -OH) group or from meta-OH (3-OH or 5-OH) group, leading to generation
of resveratrol phenoxyl radicals, which structures can undergo further reorganization to
form semiquinones [145]. Resveratrol can chelate the ferrous ion with its two hydroxyl
groups (3-OH and 5-OH), forming a complex composed of one Fe2+ and two resveratrol
molecules [144]. Resveratrol also possesses indirect antioxidant activity as a gene regu-
lator. Resveratrol was reported to increase the expression of antioxidant enzymes (SOD,
GPx, CAT), down-regulate the expression and activity of NADPH oxidase, and reduce
mitochondrial superoxide generation via stimulating mitochondria biogenesis [146].
Vitamins A, C and E are important constituents of anti-oxidant barrier against oxida-
tive stress in human body.
7. Vitamins as Antioxidants
β-Carotene (as precursor of vitamin A), tocopherols (as vitamin E forms) and ascorbate
(vitamin C) are described in terms of their structures and anti-oxidant activities.
7.1. Structural and Antioxidant Characteristics of β-Carotene, Tocopherols and Ascorbate

7.1.1. β-Carotene: Structure and Anti-Oxidant Property
β-carotene (β-C) is a non-oxygenated carotenoid molecule, with 40 carbons, 11 con-
jugated double bonds and 2 β-ionone rings (Figure 2), possessing provitamin A activ-
ity [147,148]. β-carotene is an antioxidant molecule serving as a quencher of singlet oxygen
and a scavenger of peroxyl radicals. The physical quenching of singlet oxygen is carried
out by transferring excitation energy from singlet oxygen to carotenoid molecule, to gen-
erate excited triplet-state β-carotene and ground-state oxygen. Subsequently, the energy
is dissipated between the excited β-C and the surroundings to yield normal energy state
carotenoid and thermal energy [147,148]. The chemical quenching of singlet oxygen is car-
ried out by oxygenation of β-carotene with the formation of β-C endoperoxides [149,150].
The scavenging of peroxyl radicals is carried out by addition of peroxyl radical to a suitable
double bond in provitamin A/vitamin A molecules resulting in formation of carbon radical
that can further undergo the conversion into epoxides or react with new peroxyl radical
to form bis-peroxyl products [71,151,152]. β-carotene also showed scavenging activity
towards superoxide anion radical, hydroxyl radical and hydrogen peroxide [153], and
reaction of carotenoids with superoxide anion or hydroxyl radical results in formation of
carotenoid epoxides [154,155].
Biomolecules 2022, 12, 1087 14 of 45
Figure 2. The scavenging mechanism of provitamin A and vitamin A.
7.1.2. Tocopherols: Structure and Anti-Oxidant Property

Tocopherols are plant-derived lipid-soluble molecules, possessing a chromanol ring
and a saturated 16-carbon atom phytyl chain, that belong together with tocotrienols to
the group of vitamin E. There are four isoforms of tocopherol (α-, β-, γ-, δ-), differing
in the number of CH3 − group substitutions on chromanol ring. Tocopherols can be
distinguished from tocotrienols, which possess an unsaturated phytyl (farnesyl) chain.
α-Tocopherol (α-T) (Figure 3) is an antioxidant that functions as a “chain breaker” during
lipid peroxidation in cell membranes and various lipid particles [71]. α-T terminates
the lipid peroxidation chain reactions by donating its phenolic hydrogen atom to a lipid
peroxyl radical (LOO•), leading to the formation of lipid hydroperoxide (LOOH) and
α-tocopheroxyl radical [71,156]. The formed tocopheroxyl radical, insufficiently reactive to
initiate lipid peroxidation, can react with another lipid peroxyl radical (LOO•) to yield a
non-radical product α-tocopherylquinone [152,156]. The tocopheroxyl radical can be also
regenerated to tocopherol via reduction by ascorbate and glutathione [71] or ubiquinol [157].
α-T can also react with hydroxyl radical to form the tocopheroxy radical [158], and react
with superoxide [159] or singlet oxygen [160] to form α-tocopheryl quinones, including
α-tocopheryl quinone epoxide [161,162].
Biomolecules 2022, 12, 1087 15 of 45
Figure 3. The scavenging mechanism of α-tocopherol.
7.1.3. Ascorbic Acid: Structure and Anti-Oxidant Property

Ascorbic acid (AscA), also called vitamin C, is a water-soluble ketolactone with two
ionizable hydroxyl groups. AscA exists in a form of monoanion ascorbate or can undergo
oxidation to dehydroascorbic acid (DHAscA) [163,164]. Ascorbic acid is a potent antioxi-
dant by scavenging oxygen radicals (Figure 4). Ascorbate, a prominent form of vitamin
C at physiological pH, reduces radicals (superoxide, hydroxyl radical, alkoxyl radical,
peroxyl radicals, tocopheroxyl radicals) by donating an electron, and in turn forming the
ascorbate radical [163]. Ascorbate can also donate two electrons to singlet oxygen, resulting
in the formation of DHAscA and H2 O2 [165]. Ascorbate radical can undergo reaction
with another ascorbate radical to yield ascorbate and DHAscA [71]. Ascorbate can be also
regenerated from ascorbate radical via NADH/NADPH-dependent reductases, and from
DHAscA by glutathione, thioredoxin reductase and glutaredoxin [163,166]. Dihydrolipoic
acid is capable of reducing dehydroascorbic acid to ascorbate, and can also reduce ascorbate
radical [167].
Biomolecules 2022, 12, 1087 16 of 45
Figure 4. The scavenging mechanism of ascorbate.
7.2. In Vivo and In Vitro Antioxidant Characteristics of β-Carotene, Tocopherols and Ascorbate
7.2.1. Oxidative Stress Inducers Used during In Vitro and In Vivo Studies
Artificial stress inducers are conditions or compounds applied in order to induce
oxidative stress in cells, tissues or organisms. Stress inducers are used to study the counter,
anti-oxidant effect of β-carotene, tocopherols and ascorbic acid during in vitro and in vivo
studies. For in vitro tests, conditions such as irradiation (UVA, UVB) and molecules such as
H2 O2 are used to procure oxidative stress in target cells. Except for H2 O2 , other inorganic
and organic molecules are used during anti-oxidant studies of β-carotene, tocopherols
and ascorbic acid. These compounds possess different chemical structures (Figure 5) and
induce oxidative stress in cells/organisms via different mechanisms. Tert-butyl hydroper-
oxide (t-BuOOH) can be metabolized by cytochrome P450 with generation of peroxyl and
alkoxyl radicals, or can be converted by the glutathione system to tert-butyl alcohol and
GSSG, causing glutathione (GSH) depletion [168]. Glycochenodeoxycholic acid (GCDCA)
increases mitochondrial oxidative stress through the up-regulation of acetylated SOD2
and inhibition of SOD2 activity [169]. Aristolochic acid (AA) exerts oxidative stress via
depleting glutathione pool, and also induces genotoxicity via formation of AA-derived
DNA adducts or possibly by down-regulating the expression of DNA repair genes [170].
Dichlorvos affect metabolism through inhibiting carboxyl ester hydrolases, causing DNA
alkylation and interfering with mitochondrial bioenergetics [171]. Homocysteine (Hcy)
can affect metabolism via inhibiting Na+ /K+ -ATPase, and can also generate oxidative
stress through auto-oxidation [172]. Sodium selenite can be converted by the glutathione
system to selenium, with ROS generation [168]. Advanced glycation end products (AGEs)
bind to RAGE receptor initiating a range of downstream effects such as the activation
of NADPH oxidase [173]. Ethanol is converted by alcohol dehydrogenase to acetalde-
Biomolecules 2022, 12, 1087 17 of 45
hyde, which subsequently oxidized via aldehyde dehydrogenase to acetate, the latter
possessing toxic activity [168]. For in vivo test, conditions such as heat stress and surgery
injury and compounds such as heavy metals, doxorubicin, sodium azide and quinalphos
were used to evaluate anti-oxidant activity of β-carotene, tocopherols and ascorbate. Cad-
mium (Cd2+ ) presence can decrease glutathione pool and Cd2+ can also bind to protein
thiols in mitochondrial membrane, thereby affecting mitochondrial permeability transition,
and can inhibit mitochondrial complex III, resulting in accumulation of semiubiquinones
and superoxide anion generation [174]. Sodium azide (NaN3 ) inhibits the activity of
mitochondrial cytochrome oxidase resulting in ROS overproduction [175]. Doxorubicin
(DOX) is an anthracycline that within mitochondria undergoes conversion to semiquinone
via one-electron reduction of the quinone moiety, and reacts with oxygen to generate
superoxide anion [176]. Methotrexate (MTX) toxicity is due to inhibition of DNA syn-
thesis [177], reduction in methionine synthesis, decrease of S-Adenosyl Methionine and
affecting methylation reactions [177,178]. MTX also inhibits NADP-dehydrogenases and
NADP malic enzyme [178], leading to the decreased availability of NADPH used by glu-
tathione reductase, consequently resulting in the diminished pool of reduced glutathione
(GSH) [179]. Organophosphate pesticides, such as dichlorvos and quinalphos, inhibit
activity of acetylcholinesterase (AChE) leading to accumulation of acetylcholine [180].
β-cyfluthrin/cyfluthrin are synthetic fluorinated pyrethroid insecticides that alter the per-
meability of sodium channels, inhibit calcium transport enzymes and also generate ROS
via quick metabolism of synthetic pyrethroids [181,182].
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).
Biomolecules 2022, 12, 1087 18 of 45
7.2.2. β-Carotene: Antioxidant Activity In Vitro

β-carotene has been tested in numerous in vitro studies in terms of its ability to prevent
or diminish oxidative stress in human or animal cells (Table 2). In one study, β-carotene
was reported to reduce ROS accumulation in undifferentiated rat pheochromocytoma
(PC12) cells exposed to H2 O2 [153]. In another study, β-C pre-treatment of rat hepatocytes
reduced the level of oxidative stress induced as a result of hepatocyte exposure to gly-
cochenodeoxycholic acid [183]. Furthermore, β-carotene was reported to reduce the level
of ROS produced in the oocytes upon Rosup exposure [184]. Furthermore, treatment of
human erythrocytes with β-carotene prevented the increase in lipid peroxidation caused by
exposure to H2 O2 . Moreover, the activities of SOD and CAT in H2 O2 -exposed erythrocytes
were restored in the presence of β-C [185]. Moreover, pre-incubation of mice-derived
erythrocytes with β-carotene reduced the level of MDA, increased during erythrocytes
incubation in the presence of H2 O2 [186]. β-carotene also suppressed oxidative stress in
cardiomyocyte cells (H9c2), induced as a consequence of advanced glycation end (AGEs)
products exposure. β-C treatment decreased ROS production and MDA content, and re-
stored GPx and SOD activity in AGEs-treated H9c2 cells [187]. Murine normal and tumour
thymocytes cultivated in the presence of t-BuOOH showed increased MDA content. Enrich-
ment of cells with β-C alleviated the t-BuOOH-induced MDA increase during cultivation
in both cell types [188].
Table 2. Antioxidant activity of β-carotene towards oxidative stress (OS) induced in animal and
human cells due to exposure to various stress agents.
Antioxidant Effect of β-Carotene Towards OS-Induced Cells

Oxidative Stress
Cell Type Exposure Mode Intracellular Effect Ref.
Inducer
Incubation: 12 h without β-C and then 12 h
Rat ↑1 ROS
pheochromocytoma with 40 µM H2 O2
H2 O2 [153]
(PC12) cells Incubation: 12 h with 0.5–10 µM β-C and
(undifferentiated)
↓2 ROS
then 12 h with 40 µM H2 O2
Incubation: 0.5 h without β-C and then 4 h
Glycocheno- ↑1 ROS
with 100 µM GCDC
Rat hepatocytes deoxycholic acid [183]
(GCDC) Incubation: 0.5 h with 50 µM β-C and then
↓2 ROS
4 h with 100 µM GCDC
Incubation: 4 h with 33.3 µg/mL Rosup ↑1 ROS
Oocytes Rosup [184]
Incubation: 4 h with 10 µM β-C and 33.3
↓2 ROS
µg/mL Rosup
↑1 lipid peroxidation,
Incubation: 0.5 h with 20 µM H2 O2
↓1 SOD, ↓1 CAT
Human erythrocytes H2 O2 [185]
Incubation: 0.5 h with 3 µM β-C and 20 µM ↓2 lipid peroxidation,
H2 O2 ↑2 SOD, ↑2 CAT
Incubation: 2 mM H2 O2 ↑1 MDA
Mice erythrocytes H2 O2 [186]
Incubation: 10 µg/mL β-C and 2 mM H2 O2 ↓2 MDA
↑1 ROS, ↑1 MDA, ↓1
Incubation: 24 h with 200 µg/mL AGEs
Cardiomyocyte cells GPx, ↓1 SOD
AGEs [187]
(H9c2) Incubation: 24 h with 200 µg/mL AGEs + ↓2 ROS, ↓2 MDA, ↑2
24 h with 40 µM β-C GPx, ↑2 SOD
Incubation: 2 h with 0.5 mM t-BuOOH ↑1 MDA
Normal and tumor
t-BuOOH Incubation: 0.5 h with β-C (2.8 nmol/mg [188]
thymocytes ↓2 MDA
dry wt) and 2 h with 0.5 mM t-BuOOH
1 —if
compared to control without OS inducer used; 2 —if compared to profile with OS inducer used; Measurement
methods of ROS/oxidative stress markers: ROS: DCFA-DA; Lipid peroxidation: TBARS; MDA:TBA.
Biomolecules 2022, 12, 1087 19 of 45
7.2.3. Tocopherols: Antioxidant Activity In Vitro

The ability of tocopherols to decrease oxidative stress has been proved in numerous
studies (Table 3). α-T showed hydrogen peroxide and superoxide radical scavenging
activities, as well as ferrous ion (Fe2+ ) chelating activity [144]. Pre-treatment of human ker-
atinocyte cells with α-T decreased the level of MDA and ROS, induced due to keratinocyte
exposure to ultraviolet A radiation [189]. α-T suppressed formation of lipid peroxidation
and protein carbonyl products in human neuroblastoma (SH-SY5Y) cells exposed to ad-
vanced glycation end products (AGEs) [173]. In another study, vitamin E supplementation
decreased ROS and MDA levels promoted in human umbilical vein endothelial cells after
exposure to homocysteine (Hcy) [172]. Treatment of human erythrocytes with α-T dimin-
ished the increase in MDA content exerted by pesticide dichlorvos [190]. Furthermore,
treatment of human colorectal adenocarcinoma cell line (Caco-2) with vitamin E reduced
the level of MDA, increased in Caco-2 due to exposure to H2 O2 [191]. Furthermore, pre-
treatment of rat hepatocytes with α-T reduced the level of oxidative stress induced as a
result of hepatocyte exposure to glycochenodeoxycholic acid [183]. α-T also attenuated the
H2 O2 level in rat renal tubular epithelial cells (NRK-52E), increased in NRK-52E cells due
to exposure to aristolochic acid [192].
Table 3. Antioxidant activity of tocopherols/vitamin E towards oxidative stress (OS) induced in

animal and human cells due to exposure to various stress agents.
Antioxidant Effect of Vitamin E towards OS-Induced Cells

Oxidative Stress
Cell Type Exposure Mode Exposure Effect Ref.
Inducer
Irradiation (UVA, 8 J/cm2 ) + incubation for 24 h ↑1 ROS, ↑1 MDA
Human keratinocyte [189]
UVA Incubation for 24 h with α-T (2.9–14.7 IU/mL),
cells ↓2 ROS, ↓2 MDA
irradiation (UVA, 8 J/cm2 ) + incubation for 24 h
Incubation for 24 h without α-T, then incubation ↑1 lipid peroxidation, ↑1
Human neuroblastoma with 1.5 mg/mL AGEs for 72 h protein carbonyls
AGEs [173]
(SH-SY5Y) cells
Incubation for 24 h with α-T (200 µM), then ↓2 lipid peroxidation, ↓2
incubation with 1.5 mg/mL AGEs for 72 h protein carbonyls
Human umbilical vein Incubation with Hcy (1 mM) ↑1 ROS, ↑1 MDA

Hcy [172]
endothelial cells Incubation with Hcy (1 mM) and VitE (50 µM) ↓2 ROS, ↓2 MDA
↑1 MDA, ↓1 SOD, ↓1
Incubation with DDVP (10 µM)
CAT, ↓1 GPx
Human erythrocytes Dichlorvos (DDVP) [190]
↓2 MDA, ↑2 SOD, ↑2
Incubation with DDVP (10 µM) and Vit E (30 µM)
CAT, ↑2 GPx
Incubation for 24 h, incubation for 48 h with
Human colorectal ↑1 MDA
H2 O2 (250 µM)
adenocarcinoma cell line H2 O2 [191]
(Caco-2) Incubation for 24 h, incubation for 48 h with
↓2 MDA
H2 O2 (250 µM) and VitE (10 µM)
Incubation: 0.5 h without α-T and then 4 h with
Glycocheno- ↑1 ROS
100 µM GCDC
Rat hepatocytes deoxycholic [183]
acid Incubation: 0.5 h with 100 µM α-T and then 4 h
↓2 ROS
with 100 µM GCDC
Rat renal tubular Incubation: AA (10 µM) ↑1 H2 O2
epithelial cells Aristolochic acid (AA) [192]
(NRK-52E) Incubation: AA (10 µM) + α-T (5–100 µM) ↓2 H2 O2
1 —ifcompared to control without OS inducer used; 2 —if compared to profile with OS inducer used; Measurement
methods of ROS/oxidative stress markers: ROS: DCFA-DA; Lipid peroxidation: TBARS; MDA: TBA; Protein
carbonyls: Immunoblotting; H2 O2 : Chemiluminescence.
Biomolecules 2022, 12, 1087 20 of 45
7.2.4. Ascorbic Acid: Antioxidant Activity In Vitro

Antioxidant activity of vitamin C has been confirmed in different reports (Table 4). In
one study, pre-treatment of human embryonic kidney (HEK293) cells with ascorbic acid
caused the decrease in levels of ROS production, MDA, protein carbonyl and 8-hydroxy-2-
deoxy guanosine (8-OHdG), accumulated in HEK293 cells due to exposure to H2 O2 [193].
In another study, pre-treatment of human lens epithelial cells (LEC) with vitamin C caused
the reduction in ROS activity, increased in H2 O2 -exposed LEC cells [194]. In another study,
ascorbate effectively reduced ROS level in human retinal pigment epithelial (ARPE-19)
cells, increased as a result of cells exposure to H2 O2 or UVB irradiation [195]. Moreover,
treatment of human erythrocytes with ascorbic acid diminished the increase in MDA
content induced by pesticide dichlorvos [190]. Moreover, Vit C also reduced the H2 O2 level
in rat renal tubular epithelial cells (NRK-52E), increased in NRK-52E cells due to exposure
to aristolochic acid [196]. Furthermore, the presence of ascorbic acid in human hepatoma
(HepG2) cells alleviated MDA induction, caused by exposure of HepG2 cells to ethanol,
sodium selenite or t-BuOOH. Ascorbic acid presence also caused the restoration in SOD
activity, CAT activity and GSH content in oxidative stress-induced HepG2 cells [168]. In one
more study, UV radiation or H2 O2 exposure induced oxidative stress (ROS, MDA, 4-HNE,
Isoprostanes) in human skin fibroblasts (CCD 1112Sk), and incubation with ascorbic acid
led to reduction in OS level in CCD 1112Sk cells [197].
Table 4. Antioxidant activity of ascorbic acid towards oxidative stress (OS) induced in animal and
human cells due to exposure to various stress agents.
Antioxidant Effect of Ascorbic Acid towards OS-Induced Cells

Oxidative Stress
Inducer
↑1 ROS, ↑1 MDA, ↓1 CAT,
Incubation: 4 h with H2 O2
↓1 SOD, ↓1 GPx, ↑1 protein
(400 µM)
Human embryonic carbonyl, ↑1 8-OHdG
H2 O2 [193]
kidney (HEK293) cells Incubation: 24 h with Vit C ↓2 ROS, ↓2 MDA, ↑2 CAT,
(1–20 µM), and then 4 h with ↑2 SOD, ↑2 GPx, ↓2 protein
H2 O2 (400 µM) carbonyl, ↓2 8-OHdG
Incubation: 24 h without VitC,
↑1 ROS
and 0.5 h with H2 O2 (0.2 mM)
Human lens epithelial
H2 O2 Incubation: 24 h with VitC [194]
cells (LEC)
(1 mM), and 0.5 h with H2 O2 ↓2 ROS
(0.2 mM)
Incubation: UVB irradiation
(20–100 mJ/cm2 )
↑1 ROS
Incubation: 24 h with H2 O2
(0.2 mM)
Human retinal pigment
epithelial (ARPE-19) H2 O2 or UVB Incubation: 6 h with VitC [195]
cells (500 µM), then UVB irradiation
(100 mJ/cm2 )
↓2 ROS
Incubation: 6 h with VitC
(20 µM), then 24 h with H2 O2
(0.2 mM)
Incubation with DDVP ↑1 MDA, ↓1 SOD, ↓1 CAT,
(10 µM) ↓1 GPx
Human erythrocytes Dichlorvos [190]
Incubation with DDVP (10 µM) ↓2 MDA, ↑2 SOD, ↑2 CAT,
and Vit C (10 µM) ↑2 GPx
Biomolecules 2022, 12, 1087 21 of 45
Table 4. Cont.
Antioxidant Effect of Ascorbic Acid towards OS-Induced Cells

Oxidative Stress
Inducer
Incubation with ethanol
(10–500 µM) or sodium ↑1 MDA ↓1 SOD, ↓1 CAT,
selenite (1–10 µM) or t-BuOOH ↓1 GSH
Human hepatoma Ethanol, sodium (20–200 µM) for 24 h [168]
(HepG2) cells selenite or t-BuOOH
Cotreatment with Vit C
↓2 MDA, ↑2 SOD, ↑2 CAT,
(25–100 µM) and one of OS
↑2 GSH
inducer for 24 h
For irradiation treatment: ↑1 ROS, ↑1 MDA, ↑1 4-HNE,
20 J/cm2 (UVA) or 200 mJ/cm2 ↑1 Carbonyl groups (for all
(UVB) + 24 h incubation; For stress inducers);
H2 O2 treatment: incubation ↑1 Isoprostanes (for
Human skin fibroblasts with 200 µM H2 O2 for 24 h UVA, H2 O2 )
UVA, UVB or H2 O2 [197]
(CCD 1112Sk) ↓2 ROS, ↓2 MDA, ↓2 4-HNE,
Stress induction treatment + ↓2 Carbonyl groups (for all
24 h incubation with 100 µM stress inducers);
ascorbic acid ↓2 Isoprostanes (for
UVA, H2 O2 )
Rat renal tubular Incubation: AA (10 µM) ↑1 H2 O2
epithelial cells Aristolochic acid (AA) Incubation: AA (10 µM) + Vit [196]
(NRK-52E) ↓2 H2 O2
C (5 µM)
1 —if
compared to control without OS inducer used; 2 —if compared to profile with OS inducer used; Measurement
methods of ROS/oxidative stress markers: ROS: DCFA-DA, ESR [197]; MDA:TBA, GC-MS [197]; 4-HNE: GC-
MS [197]; Isoprostanes: LC-MS [197]; Protein carbonyls: DNPH; 8-OHdG: ELISA [193], LC-MS [197]; H2 O2 :
Chemiluminescence.
7.2.5. β-Carotene: Antioxidant Activity In Vivo

Carotenoids, such as β-carotene, are lipid soluble tetraterpenoid molecules naturally
found in plants and microorganisms (algae, yeast and some bacteria), and available in
food. In mammals, lipid-soluble β-carotene is absorbed in small intestine and delivered
to the peripheral tissues (liver, adipose tissue, kidney, skin, lungs) via various lipoprotein
particles. In one of the possible mechanisms for vitamin A synthesis (Figure 6), all trans β-
carotene, the predominant form of β-carotene found in nature, is symmetrically cleaved by
the enzyme β-carotene-15,150 -oxygenase (CMOI) to yield two molecules of retinaldehyde.
Ingested β-C can be cleaved via CMOI in intestine and in various tissues within the body.
Retinaldehyde can be reduced via alcohol dehydrogenase or retinol dehydrogenase to
retinol, a vitamin A. Retinol can be also esterified via lecithin:retinol acyltransferase (LRAT)
to retinyl esters, which constitute vitamin A reserves. Retinaldehyde can be also oxidized,
by enzymes (ALDH 1 or RALDH) from the aldehyde dehydrogenase 1 family, into all-trans
retinoic acid which constitutes the biologically active form of vitamin A, responsible for
transcriptional regulation [198,199].
Biomolecules 2022, 12, 1087 22 of 45
Figure 6. The structures of provitamin A (β-carotene) and vitamin A constituents (retinaldehyde,

retinol, retinoic acid).
The antioxidant activity of β-carotene was evaluated in vivo with different animal
species (Table 5). Wistar rats were characterized by increased MDA level and decreased
SOD and CAT activity, as a result of 2-week high-fat diet (HFD), if compared to control
where normal diet was administered. The addition of β-carotene for two weeks, before or
after 12-week HFD, resulted in reduction of MDA level and restoration in SOD and CAT
activity, in investigated rats [200]. Wistar albino rats were administered methotrexate (MTX)
what resulted in the increased MDA level and reduced activities of SOD, CAT and GPx in
the livers of rats exposed to MTX. Co-administration of MTX with β-carotene diminished
hepatic MDA level and restored activities of SOD, CAT and GPx in rat livers [201]. β-
carotene was reported to attenuate oxidative stress in the spinal cord of rats with spinal cord
injury (SCI). Decreased ROS production and MDA level, and restored SOD activity were
detected in spinal cord tissues of SCI rats fed with β-C [202]. β-carotene also diminished
oxidative stress in mice with traumatic brain injury (TBI). Decreased MDA content and
restored SOD activity were detected in brain tissue of TBI-mice, when β-C doses were
administered [203]. In one more study, incubation of Drosophila melanogaster larvae with
β-carotene diminished the OS level induced as a consequence of larvae exposure to γ-
irradiation [204].
7.2.6. Tocopherols: Antioxidant Activity In Vivo

Vitamin E is found in vegetable oils and seeds, with α-tocopherol being commonly
available in wheat germ, olive, and sunflower oil, and γ-tocopherol being prominent in
soybean, corn, and cottonseed oil. Lipid-containing food intake is the source of vitamin E
in human body [157,205,206]. During the digestion process, triacylglycerols and other ester-
ified fat-soluble compounds are partially processed in the stomach by gastric lipase [207],
and absorption of vitamin E occurs in the small intestine [157]. Vitamin E along with other
Biomolecules 2022, 12, 1087 23 of 45
lipids is incorporated into mixed micelles, the process aided by pancreatic lipases and bile
salts, in the duodenum [206,207]. In the intestine lumen, micelles solubilize hydrophobic
components and diffuse into the glycocalix to approach the brush border membrane of the
enterocytes [157,208]. Vitamin E transport across the enterocyte membrane occurs via pas-
sive diffusion and/or is also mediated by membrane proteins (NPC1L1, SR-BI, CD36) [208].
In enterocytes, vitamin E is incorporated into chylomicrons, which are secreted into the in-
testinal lymph system and released into the bloodstream [208,209]. Vitamin E, transported
in blood via different lipoproteins, is distributed within liver and extrahepatic tissues (adi-
pose tissue, muscle, adrenal glands) [206,209]. In liver, RRR α-tocopherol selectively binds
to the α-tocopherol transfer protein (α-TTP), while other isoforms of vitamin E are excreted
in the bile [206]. α-Tocopherol, incorporated via α-TTP into lipoproteins and re-secreted to
the circulation [206], is the major tocopherol found in human blood and tissues [205].
The antioxidant effect of α-tocopherol has been tested in vivo on animal species
(Table 5). A 15-day intake of α-T by Wistar rats led to reduction in lipoperoxide concen-
trations, increased as a consequence of intraperitoneal injection of Cd2+ ions [210]. In
another study, vitamin E administration reduced cardiac MDA level in rats, increased
due to doxorubicin (DOX) injection. Additionally, cardiac GSH level in DOX-injected
rats fed with vitamin E were higher, if compared to DOX-injected group without vitamin
administered [211]. Further, administration of Vit E (α-tocopheryl acetate) to female white
mice treated with cyfluthrin resulted in the reduction in plasma MDA level and elevation
in erythrocytes CAT activity, if compared to group where cyfluthrin was administered, but
without Vit E [181]. In another study, α-T decreased ischemia/reperfusion injury-induced
ROS production and oxidative modification of phospholipids in mice [212]. Vitamin E
was also tested in terms of its cytoprotective effect towards cardiovascular system in mice
subjected to heat stress. Heart tissue of mice exposed to heat stress (HS) conditions showed
increased ROS levels, but administration of vitamin E possessed ameliorating effect. More-
over, cardiomyocytes of mice subjected to heat stress and fed with vitamin E showed
decreased MDA levels and restored SOD and GSH levels, if compared to HS conditions but
without vitamin administration [213]. Supplementation of VitE (α-tocopheryl acetate) to rat
diet reduced plasma lipid peroxidation and restored plasma SOD and GPx activity, affected
due to rats feeding on high-fat diet [214]. Moreover, supplementation of VitE (α-tocopheryl
acetate) reduced MDA serum concentration and MDA breast muscle content in chicken
broilers, fed on linseed oil-enriched diet [215].
7.2.7. Ascorbic Acid: Antioxidant Activity In Vivo

Vitamin C is naturally present in fruits (kiwifruit, orange, lemon, black currant, rasp-
berry, strawberry, grapes etc.) and vegetables (broccoli, cabbage, spinach, tomato, potato,
pepper etc.) [216,217], but can be also manufactured industrially in reactions involving
various intermediates such as D-sorbitol, L-sorbose and 2-keto-L-gulonic acid [218]. In
animals, L-ascorbic acid is synthetized through glucuronic acid pathway in liver (mam-
mals) or kidney (reptiles, birds) [219]. Humans, apes, guinea pigs and fruit-eating bats
cannot synthetize ascorbic acid due to the lack of enzyme L-gulonolactone oxidase [220]. In
humans, ascorbate and DHAscA (vitamin C equivalents) are absorbed from ingested food
by enterocytes of the small intestine. Ascorbate is absorbed via Na+ -dependent vitamin C
transporters (SVCTs) while DHAscA is absorbed via Na+ -independent facilitative glucose
transporters (GLUTs) and reduced to ascorbate. Vitamin C, absorbed from the intestinal
lumen, is transported with the blood to various peripheral organs that differ in tissue
ascorbate content [163,164,221].
Biomolecules 2022, 12, 1087 24 of 45
Table 5. Antioxidant activity of β-carotene, vitamin E and/or vitamin C based on animal in vivo
studies with different oxidative stress inducers applied.
Antioxidant Effect of β-Carotene, Vitamin E and/or Vitamin C In Vivo

Oxidative Stress
Species/Tissue Exposure Mode Exposure Effect Ref.
Inducer
Single MTX dose (20 mg/kg) on day 21 of ↑1 MDA, ↓1 SOD,
Wistar rats experiment (24 days) ↓1 CAT, ↓1 GPx
n = 24 (total)/Liver Methotrexate (MTX) [201]
tissue β-C dose (10 mg/kg/day) for 24 days + ↓2 MDA, ↑2 SOD,
MTX dose (20 mg/kg) on day 21 ↑2 CAT, ↑2 GPx
High-fat diet (HFD) HFD for 14 weeks + 24 h starving ↑1 MDA
Wistar rats (Mixing of cow fat
β-C administration (300 mg/kg body [200]
n = 30 (total)/Blood (60%) with normal rat
weight) for 2 weeks before or after ↓2 MDA
chow (40%))
12-week HFD + 24 h starving
Male Sprague–Dawley SCI surgery + 72 h ↑1 MDA, ↓1 SOD

rats n = 299 Spinal Cord Injury SCI surgery + β-C (20–80 mg/kg) [202]
(total)/Spinal Cord (SCI) administered intraperitoneally once ↓2 MDA, ↑2 SOD
tissue immediately after the surgery + 72 h
TBI surgery + 7 days ↑1 MDA, ↓1 SOD
Male C57BL/6 mice
Traumatic Brain
n = 108 TBI surgery + β-C (30 mg/kg) [203]
Injury (TBI)
(total)/Brain tissue administered 3 h after the surgery and ↓2 MDA, ↑2 SOD
then every day during 7 days
Exposure to 10 Gy γ-irradiation ↑1 Lipid peroxidation
Drosophila melanogaster
Gamma irradiation Larvae feeding on β-C before exposure to [204]
larvae ↓2 Lipid peroxidation
10 Gy γ-irradiation
Single intraperitoneal injection of Cd2+ ↑1 Lipoperoxide,
ions (2 mg Kg−1 ) ↓1 SOD
Wistar male rats n = 60
Cd2+ (CdCl2 ·H2 O) Single intraperitoneal injection of Cd2+ [210]
(total)/Serum
ions (2 mg/Kg) + administration of drink ↓2 Lipoperoxide,
aqueous solutions of α-T (40 mg/L) for ↑2 SOD
15 days
Intraperitoneal injection (4 mg DOX/
kg body weight) three times per week for ↑1 MDA, ↑1 CAT
Male Sprague–Dawley 2 weeks
rats n = 70 Doxorubicin (DOX) Intra-gastric administration (100 mg [211]
(total)/Heart tissue VitE/kg body weight), two times per
↓2 MDA, ↓2 CAT
week for 3 weeks, started 1 week before
DOX injection
Oral administration of cyfluthrin (a single
↑1 MDA, ↓1 CAT
dose of 100 mg/kg/body weight)
Female white mice
Cyfluthrin Oral administration of cyfluthrin (a dose [181]
n = 160 (total)/Blood
of 100 mg/kg/body weight) followed by
↓2 MDA, ↑2 CAT
intramuscular injection of VitE (a dose of
100 mg/kg/body weight, for 7 days)
I/R injury + 3 days ↑1 Oxidized lipids
C57BL/6 Ischemia/Reperfusion Intraperitoneal injection of α-TOH
[212]
mice/Infarcted tissue (I/R) injury (2.5 mg/kg BW) 2 h before surgery,
↓2 Oxidized lipids
immediately after reperfusion and twice
per day for 3 days
Biomolecules 2022, 12, 1087 25 of 45
Table 5. Cont.
Antioxidant Effect of β-Carotene, Vitamin E and/or Vitamin C In Vivo

Oxidative Stress
Species/Tissue Exposure Mode Exposure Effect Ref.
Inducer
HS conditions (temperature: 40 ◦ C;
humidity: 60%) for 4 h per day during a ↑1 MDA, ↓1 SOD
BALB/c mice n = 40
Heat stress (HS) 4-week period [213]
(total)/Heart tissue
Oral administration of VitE (500 mg/kg)
↓2 MDA, ↑2 SOD
2 h before the initiation of HS
↑1 Lipid peroxidation,
Sprague-Dawley male A 10-week feeding on high-fat diet
↓1 SOD, ↓1 GPx
rats n = 30 High-fat diet [214]
(total)/Blood A 10-week feeding on high-fat diet ↓2 Lipid peroxidation,
supplemented with VitE (350 mg/kg diet) ↑2 SOD, ↑2 GPx
Chickens fed with commercial starter diet
Ross 308 male broilers (1–12 days), commercial grower diet
(21-day old) n = 400 High n-3 dietary (13–20 days), finisher diet enriched with
↓3 MDA [215]
(total)/Blood, Breast PUFAs intake 5% cold-pressed linseed oil and
muscle supplemented with VitE (200 IU/kg)
(21–40 days)
Oral administration of NaN3 (20 mg/kg ↑1 MDA, ↑1 Protein
Wistar rats n = 28
BW) for 9 days Carbonyls
(total)/Stomach, Colon, Sodium azide (NaN3 ) [222]
Kidney tissue Oral administration of NaN3 and VitC ↓2 MDA, ↓2 Protein
(200 mg/kg BW) for 9 days Carbonyls
↑1 Superoxide anion,
Six intraperitoneal DOX injections (2.5
↑1 Lipid peroxidation,
mg/kg body wt) over 3 weeks
↑1 Protein Carbonyls
Wistar male rats
Doxorubicin (DOX) [223]
n = 46/Heart tissue Oral daily administration of VitC (50
↓2 Superoxide anion,
mg/kg) started 1 week before the start of
↓2 Lipid peroxidation,
DOX administration and continued for 2
↓2 Protein Carbonyls
weeks after the last DOX injection
Oral dose of QP (14 mg/kg), daily for ↑1 MDA, ↓1 CAT,
Sprague–Dawley male 10 days ↓1 GPx
rats n = 18 Quinalphos (QP) [224]
Oral administration of VitC (20 mg/kg)
(total)/Heart tissue ↓2 MDA, ↑2 CAT,
daily, 4 h after QP administration, for
↑2 GPx
10 days
1 —if compared to control without OS applied; 2 —if compared to profile with OS applied; 3 —if compared to
profile without anti-oxidant applied; Measurement methods of ROS/oxidative stress markers: Lipid peroxidation:
TBARS; MDA: TBA; Protein carbonyls: DNPH, Oxidized lipids: LC-MS; Superoxide anion: Adrenaline assay.
The antioxidant effect of ascorbic acid has been tested in vivo on animal species
(Table 5). In a study comprising Wistar rats, a 9-day supplementation of ascorbic acid re-
sulted in the decreased levels of malondialdehyde (MDA) and protein carbonyl, increased
in the stomach, colon and kidneys of rats feeding with sodium azide (NaN3 ), as an oxida-
tive stress inducer [222]. In another study, the antioxidant activity of ascorbate was tested
in doxorubicin-administered male Wistar rats. The cardiac tissue of DOX-administered
rats fed with ascorbate, showed reduced levels of superoxide anion, lipid peroxidation
and protein carbonyl products, if compared to the group subjected to DOX administration
but without Vit C treatment. Moreover, ascorbate treatment restored SOD and GPx activ-
ities, decreased in rat cardiac tissue due to DOX administration [223]. In another study,
insecticide quinalphos (QP) administered to Sprague–Dawley rats caused induction of
oxidative stress in rat cardiac tissue, indicated by reduced GPx activity and CAT activity,
and increased MDA content. Co-administration of Vit C to investigated rat group resulted
Biomolecules 2022, 12, 1087 26 of 45
in the alleviation of oxidative stress, expressed by restored GPx activity and CAT activity,
and reduced MDA content in QP-exposed rat cardiac tissue [224].
7.2.8. Antioxidant Characteristics of β-Carotene, Tocopherols and Ascorbate Based on

Populational/Clinical Human Studies
The characteristics of β-carotene, tocopherols and ascorbate were also characterized
in terms of their antioxidant activities in human studies (Table 6). Antioxidant activities
of β-carotene were proved in exemplary populational studies. In one study, including
85 healthy male 22–58 age-volunteers exposed to Pb for 4 to 38 years, the anti-oxidant
effect of β-carotene was evaluated. The oral administration of β-carotene (10 mg/day) to
35 participants for 3 months, resulted in reduced concentration of lipid hydroperoxides
(LHP) in serum and decreased malondialdehyde (MDA) level in erythrocytes and leuko-
cytes, if compared to a group (50 participant) without β-C administered [225]. In another
study, a 3 week-supplementation of dietary (9-cis, all-trans) β-carotene (60 mg/day) to
patients (n = 20) with long-standing non-insulin-dependent diabetes mellitus (NIDDM)
showed that low density lipoproteins of β-C administered NIDDM-patients possessed
increased resistance to oxidation and reduced level of MDA [226]. Antioxidant activities
of tocopherol and ascorbate were also proved in some populational studies. In a study
with participants (n = 35) with polygenic hypercholesterolemia and enhanced oxidative
stress, the 16-week administration of natural α-tocopherol (1600–3200 I.U.) to participants
resulted in a observed reduction of plasma F2 -isoprostane concentrations [227]. In a study,
including 8 healthy white volunteers whose skin was exposed for 6 h to UV radiation, the
8-week supplementation with α-tocopherol (400 IU/day) caused the reduction in tissue
MDA content, induced as a consequence of skin exposure to UVR [228]. Moreover in the
study, comprising totally 704 participants at age of 77, the dietary intake of ascorbic acid
and β-carotene resulted in the reduced urinary level of F2 -isoprostanes, the biomarker of
ROS-mediated peroxidation of arachidonic acid [229].
Table 6. Antioxidant activity of β-carotene, vitamin E and/or vitamin C based on popula-

tional/clinical studies.
Antioxidant Effect of β-Carotene, Vitamin E and/or Vitamin C in Human Studies

Origin of Oxidative
Patients/Participants Treatment Mode Treatment Effect Ref.
Stress
No administration of antioxidants
-
Male workers exposed to for 12 weeks
Pb [225]
lead, n = 85 (total)/Blood Oral administration of β-carotene
↓1,2 MDA, ↓1,2 LHP
(10 mg/day) for 12 weeks
Patients with NIDDM, Supplementation with β-carotene
NIDDM ↓C1 MDA [226]
n = 20 (total)/Blood (60 mg/day) for 3 weeks
Supplementation with placebo for
Participants with polygenic -
Enhanced oxidative 16 weeks
hypercholesterolemia, [227]
stress Supplementation with VitE
n = 35/Blood ↓2 F2 -isoprostane
(1600–3200 I.U.) for 16 weeks
Supplementation with
α-tocopherol (400 IU/day) for
White volunteers, n = 8
UVR 8 weeks, skin exposure to UVR ↓2 MDA [228]
(total)/Skin
(120 mJ/cm2 ), skin biopsy 6 h after
UVR exposure
Participants at age of 77, Dietary intake of ascorbic acid and
n.d. ↓I F2 -isoprostane [229]
n = 704/Urine β-carotene
Biomolecules 2022, 12, 1087 27 of 45
Table 6. Cont.
Antioxidant Effect of β-Carotene, Vitamin E and/or Vitamin C in Human Studies

Origin of Oxidative
Patients/Participants Treatment Mode Treatment Effect Ref.
Stress
Oral administration of placebo
-
Female participants with (800 mg/day) for 6 weeks
type 2 diabetes, n = 34 Type 2 diabetes [230]
Oral administration of
(total)/Blood
α-tocopherol (800 IU/day) for ↓1,2 MDA
6 weeks
Participants with DMT2, Supplementation with VitC ↓1 MDA
DMT2 [231]
n = 5 or 8 (total)/Blood (1000 mg/day) for 6 weeks ↓1 F2 -isoprostane
No supplementation of VitE
Participants with DMT2 ↑1,C2 MDA
and administered with within 3 months
DMT2 [232]
hypoglycemic drug, n = 80 Supplementation of VitE
↓1,2 MDA
(total)/Blood (400 mg/day) for 3 months
Patients with late-stage Oral administration of placebo

-
knee osteoarthritis, n = 72 Enhanced oxidative once daily for 2 months
[233]
(total)/Blood, stress Oral administration of VitE
↓2 MDA
synovial fluid (400 IU/day) for 2 months
1 —ifcompared to the beginning of the study; 2 —if compared to profile without antioxidant used; C1 —if compared
to control: normoglycemic profile; C2 —if compared to control: healthy subjects; I —if compared to lower intake of
antioxidants; Measurement methods of oxidative stress biomarkers: MDA: TBA; LHP: FOX assay; F2 -Isoprostanes:
GC-MS [227], Radioimmunoassay [229], ELISA [231].
In a study consisting of 34 female participants with type 2 diabetes, the oral sup-
plementation of α-tocopherol (800 IU/day) to 13 participants for 6 weeks resulted in the
decrease in MDA erythrocyte level, if compared to a group (n = 21) without α-tocopherol
administration [230]. In another study, 5 participants with diabetes mellitus type 2 who
received vitamin C (1000 mg/day) for 6 weeks, showed decreased plasma levels of MDA
and F2 -isoprostanes, if compared to results before supplementation [231]. In a group of
40 participants with clinically diagnosed DMT2, hypoglycemic drug-administered diabetic
(27 macrovascular, 13 microvascular) patients supplemented for 3 months with vitamin
E (400 mg/day) showed decreased MDA serum concentration and increased serum SOD
activity and erythrocyte GSH content, if compared to the beginning of study, or when com-
pared with 40 DMT2 patients treated for 3 months with hypoglycemic drugs but without
VitE supplementation [232]. The antioxidant activity of VitE has been also evaluated in
a study comprising 72 patients with late-stage knee osteoarthritis (OA) and concluding
that a two-month intake of VitE (400 IU) could reduce oxidative stress conditions (MDA in
plasma and synovial fluid) in patients with OA [233].
7.2.9. Antioxidant Characteristics of β-Carotene, Tocopherols and Ascorbate: A Summary

The literature data gathered show that β-carotene, tocopherols and ascorbic acid act
as efficient antioxidants towards human and animal cells in vitro, under stress conditions.
Supplementation with β-carotene, tocopherols or ascorbic acid, as free-radical quenchers,
decreased the level of OS (bio)markers (MDA, ROS, Protein carbonyls, Isoprostanes, 8-
OHdG) in numerous types of cells exposed to stress conditions. Conducted in vitro studies
varied in terms of not only tested cell lines, but also research conditions including methods
of oxidative stress induction, the form and concentration of antioxidant and the order
of culture supplementation. These differences amongst studies can create limitations
for results comparison and interpretations. Different methods, such as physicochemical
factors as well as inorganic and organic oxidizing agents, were used to induce oxidative
stress (OA) in investigated cell cultures. These stress inducers can cause oxidative stress via
different mechanisms, and were tested towards different human and animal cells. Moreover,
Biomolecules 2022, 12, 1087 28 of 45
different stress inducers used for the same cell line can result in the different OS extent [186].
The concentrations of in vitro tested antioxidants differ amongst studies (Tables 2–4), but
the anti-oxidant concentrations strictly influences the mitigation of oxidative stress [190].
Another important barrier for interpretation of anti-oxidative efficiency is the chemical
form of anti-oxidant tested. Amongst tocopherols, α-T is commonly used during in vitro
tests and its antioxidant activity was confirmed. However, the ability of α-T again to
mitigate OS is not always proved and different tocopherol forms (α-T, γ-T) can possess
different anti-oxidant activity [234]. Furthermore, antioxidant evaluation in vitro includes
their different modes: cell pre-treatment with a selected antioxidant prior to oxidizing
agent (OA)-exposure, co-treatment of cells with antioxidant and OA, or the subject of cells
to oxidizing agent before adding the selected antioxidant. The differences in oxidizing
agent/anti-oxidant supplementation order can also have influence of the outcome of
research studies. Nevertheless, β-carotene, tocopherols and ascorbic acid proved to act as
efficient antioxidants, despite differences between conducted studies.
Animal in vivo studies (Table 5) show that β-carotene, tocopherols and ascorbic acid
can be efficient compounds to mitigate oxidative stress, as indicated by decreased levels
of OS biomarkers (MDA, Lipid peroxidation, Protein carbonyls) in investigated species.
As in case of in vitro research, the problems with interpretation of different experimental
conditions (OS generation method, supplementation mode) also occur, and further discrep-
ancies (single cells vs. whole organism) between in vitro and in vivo appear. Nevertheless,
the antioxidant properties of β-carotene, tocopherols and ascorbic acid was shown, despite
differences between conducted in vivo studies.
Some human populational/clinical studies (Table 6) show that β-carotene, α-tocopherol
or ascorbic acid can improve anti-oxidant status in a selected group of participants. How-
ever, due to a scarce number of studies and a variety of conditions (different number of
participants and health condition status, various time span and biomarkers) influencing
studies outcome, the effectiveness of β-carotene, α-tocopherol or ascorbic acid as effective
anti-oxidants, is not certain. Moreover, other reports do not confirm improvements in anti-
oxidant level upon β-carotene [235], vitamin E [236] or ascorbic acid [237] administration.
Moreover, the connection between free-radical scavenging ability of anti-oxidants and the
use of antioxidants for prevention of human diseases has not been established. Although
α-tocopherol exhibits anti-oxidant activity and inhibits oxidation of low-density lipoprotein
cholesterol, α-T supplements failed to reduce atherosclerosis-related events [78]. What
is more, beneficial effect of vitamin C in reference to human diseases (cancer, atheroscle-
rosis, diabetes, neurodegenerative disease) remains equivocal [217], and a shortage of
clinical trials impedes drawing clear conclusions about therapeutic role of ascorbic acid
administration [238]. Furthermore, it was also reported that supplementation with anti-
oxidant nutrients (vitamin C, vitamin E or β-carotene) offered no prevention against
cancer incidence [239], and there is no certainty that possible cancer-preventive ability of
anti-oxidant molecules, such as β-carotene, can be strictly due to free radical quenching
mechanism [240]. In addition to mentioned discrepancies, it should be also noted that
β-carotene and vitamin C can possess pro-oxidant activity [217,240]. Therefore, further
studies are required to determine the effect of β-carotene, α-tocopherol or ascorbic acid on
the level of oxidative stress and the antioxidant status in broader groups within healthy
and disease-diagnosed populations.
Exposure to oxidative stress causes the alteration in anti-oxidant mechanisms, with
the change in the activity of SOD, CAT and/or GPx (Tables 2–6). The exposure of cells to
different OS inducers resulted in decrease in SOD, CAT and GPx activity. SOD, CAT and
GPx are enzymes participating together to prevent oxidative damage to cells. Saturation of
SOD and CAT during ROS conversion, and decreased availability of GSH for GPx can lead
to decreased activities of these enzymes [190]. Reduced GPx activity can result in decreased
SOD and CAT activities [179], and activity of SOD and CAT can be also inhibited by ROS,
such as H2 O2 , [181] via affecting enzyme active site or via mechanism of cell regulation
(de-/phosphorylation) [185]. Moreover, oxidizing agent tested can be directly involved
Biomolecules 2022, 12, 1087 29 of 45
in inhibition of anti-oxidant enzymes [181]. Overproduction of ROS is accompanied

by inactivation of detoxification systems, consumptions of antioxidants and insufficient
replenishment of antioxidants in cells/tissues [179]. However, the opposite trend with
increased CAT activity is response to oxidizing agent exposure, was observed [211], and
VitE doses were reported to decrease levels of erythrocyte antioxidant enzymes in human
and animal studies [230]. Moreover, increased expression and activity of antioxidant
enzymes was concluded to be a result of compensatory defence mechanisms against
oxidative stress [225]. Nevertheless, the majority of literature data show that the presence
of anti-oxidants (β-C, Vit E, Vit C) increased SOD, CAT and GPx activity in stressed cells,
proving the ROS-scavenging properties of tested molecules.
7.3. Measurement of the Oxidative Stress in Cells and Tissues

The induction of oxidative stress in cells/tissues is typically measured by monitoring
the cellular level of ROS (direct techniques) or the level of specific markers of oxidative
stress, such as lipid peroxidation, protein modification and DNA oxidation products
(indirect techniques). These techniques are used (or can be used) to evaluate the efficacy
of various anti-oxidant molecules, such as β-carotene, vitamin E and ascorbic acid, on
cells/tissues exposed to oxidative stress.
7.3.1. Measurement of ROS Level: ESR Spectroscopy

Electron spin resonance (ESR) spectroscopy is a technique based on the absorption
of microwave radiation by unpaired electron-possessing molecules (radicals) situated
in the applied static magnetic field [241]. ESR spectroscopy is able to monitor the real-
time generation of short-lived ROS radicals (O2 •− , •OH) when coupled with spin traps.
Spin-trapping reagents (nitrone and nitroso compounds) react with ROS radicals to yield
long-lived radicals called spin-adducts [242]. 5,5-dimethyl-1-pyrroline N-oxide (DMPO),
a commonly used nitrone, reacts with O2 •− or •OH to form DMPO-OOH or DMPO-OH
radicals, respectively, while DMPO-OOH further decomposes to DMPO-OH [243]. Free
radicals can be determined by analysing the distinct ESR spectrum of a spin adduct [242].
Apart from spin traps, ESR exploits spin probes (such as cyclic hydroxylamines) that
undergo one-electron oxidation to form nitroxide radical detected by ESR, with higher
sensitivity than spin traps [244]. ESR technique was used to measure ROS generation in
oxidative stress-exposed human skin fibroblasts (CCD 1112Sk). 1-hydroxy-3-methoxy-
carbonyl-2,2,5,5-tetramethyl pyrrolidine (CMH), as a spin probe, was used to react with
ROS and to yield a stable nitroxide CM-radical, while ROS formation was measured
according to the rate of nitroxide accumulation [197].
7.3.2. Measurement of ROS Level: Fluorescent Method (DCFH-DA Probe Assay)

The intracellular level of ROS can be measured by means of the DCFH-DA probe assay.
20 ,70 -dichlorodihydrofluorescein diacetate (DCFH-DA) (Figure S1) is a non-fluorescent
compound that diffuses through the cellular membrane and undergoes the cleavage at
the two ester bonds via intracellular esterase to 20 ,70 -dichlorodihydrofluorescein (DCFH)
which accumulates in cells. The non-fluorescent DCFH undergoes oxidation by ROS, such
as H2 O2 , hydroxyl radicals and peroxyl radicals, to fluorescent 20 ,70 -dichlorofluorescein
(DCF). The presence of DCF can be detected fluorometrically with λexcitation = 485 nm and
λemission = 525 nm [245,246]. ROS detection with DCFH-DA faces possible limitations such
as insufficient accessibility of esterases sequestered within cells, oxidation reaction with
other cellular components, a lack of ability to react with O2 •− or the leakage of DCF out of
the cells [245,246]. DCFH-DA probe assay is commonly used to evaluate the anti-oxidant
effect of vitamins towards OS-induced cells (Tables 2–4).
7.3.3. Measurement of ROS Level: Other Methods

O2 •− and H2 O2 can be measured by means of chemiluminescence method, where
light is emitted as a result of chemical reactions. H2 O2 reacts in the presence of catalyst
Biomolecules 2022, 12, 1087 30 of 45
with luminol (3-aminophthalhydrazide) to produce the excited 3-aminophthalate anion,

that emits light when relaxed to the ground state [247]. O2 •− reaction with lucigenin (N,N0 -
dimethyl-9,90 -biacridinium dinitrate, Luc2+ ) includes one-electron reduction of Luc2+ to
lucigenin cation radical (Luc•+ ) that reacts with O2 •− to form lucigenin dioxetane (LucO2 )
which decomposes to N-methylacridone (NMA) with light emission [248].
O2 •− can be detected via oxidation of adrenaline to adrenochrome, that can be deter-
mined spectrophotometrically at 480 nm [249].
7.3.4. Measurement of Lipid Peroxidation Products

Lipid peroxidation (LPO), the structural degradation of lipids occurring as a conse-
quence of oxidative damage, is a widely used marker for OS presence. LPO is determined
by measuring the level of malondialdehyde (MDA), 4-hydroxynonenal (4-HNE) or F2 -
isoprostanes (F2 -IsoPs).
Measurement of Lipid Peroxidation: MDA, 4-HNE

MDA, a three-carbon dialdehyde, and 4-HNE, an unsaturated aldehyde, are products
of oxidative degradation of polyunsaturated fatty acid (PUFAs), where double bonds in
PUFAs structures are the target for ROS attack to form unstable fatty acid peroxides.
The MDA level is measured spectrophotometrically, and the principle of method is
the reaction (Figure S2) between MDA and 4,6-dihydroxypyrimidine-2-thiol (thiobarbituric
acid; TBA) to form MDA-TBA2 adduct, that absorbs strongly at λ = 532 nm. Alterna-
tively, MDA-TBA2 adduct can be measured spectrofluorometrically (λexcitation = 515 nm/
λemission = 553 nm) [250]. MDA assay is the most common method used to monitor OS
level and evaluate the anti-oxidant effect of β-carotene, vitamin E and ascorbic acid on
cells/tissues subjected to oxidative stress (Tables 2–5). However, MDA determination based
on TBA assay, lacks selectivity due to dependence on reaction conditions (pH, temperature)
as well as reaction of TBA with other components of biological samples such as sugars,
amino acids, bilirubin, albumin and other aldehydes, known as thiobarbituric acid reactive
substances (TBARS) [250,251].
Improvement in MDA detection is achieved by using techniques of Chromatography
and Mass Spectrometry, where molecules are analysed based on chromatogram retention
time and mass to charge (m/z) ratio spectrum. MDA, after reaction with TBA, was reported
to be analysed by HPLC-UV-Vis [252] or HPLC coupled with fluorescence detection [253].
MDA also undergoes derivatization with 2,4-dinitrophenylhydrazine (DNPH) to yield
hydrazone and pyrazole (Figure S3) derivatives (MDA-DNPH) that can be analysed by
HPLC-UV [254] or LC-MS [255]. Analysis of MDA by GC-MS requires derivatization
of MDA with pentafluorobenzyl bromide (PFBB) or pentafluorobenzyl hydroxylamine
(PFBHA) to form MDA-PFB2 (Figure S4) or MDA-PFB2-oxime (Figure S5) derivatives,
respectively [256].
Low density lipoproteins (LDL) modified by MDA (LDL-MDA), markers of atheroscle-
rosis development, can be determined by the enzyme-linked immunosorbent assay (ELISA).
The principle of this assay is the reaction of the specific monoclonal antibody 3B2 with
MDA-LDL in plasma, the capture of the complex by anti-IgM antibody coated to an ELISA
plate, the binding of polyclonal anti-(MDA-LDL) antibody, the binding of anti-rabbit IgG
(secondary antibody) conjugated with horseradish peroxidase (HRP) and the reaction
of HRP with o-phenylenediamine (OPD) to yield a product detected spectrophotometri-
cally [257].
4-HNE can be analysed by means of GC-MS, upon derivatization (Figure S6) with
PFBHA to yield 4-HNE-PFB-oxime product, that subsequently undergoes silylation by
N,O-bis(trimethylsilyl)trifluoroacetamide (BSTFA) in trimethylchlorosilane (TMCS) to form
4-HNE-PFB-oxime-TMS derivative [197,256].
4-HNE protein adducts can be analysed by ELISA, involving the anti-HNE antibody
and HRP-labelled secondary antibody (indirect ELISA) or HRP-labelled anti-HNE antibody
(Sandwich ELISA), and tetramethylbenzidine (TMB) as a HRP-specific substrate [258].
Biomolecules 2022, 12, 1087 31 of 45
Measurement of Lipid Peroxidation: F2 -Isoprostanes (F2 -IsoPs)

Isoprostanes (IsoPs) are prostaglandin (PG)-like compounds formed by non-enzymatic
free-radical mediated peroxidation of arachidonic acid. The 11-, 9-, 12- or 8-peroxidation
of arachidonic acid leads to formations of F2 -isoprostanes classified as 15-series, 5-series,
8-series or 12-series F2 -IsoPs, respectively [251].
F2 -IsoPs are considered as reliable biomarkers of oxidative stress and can be mea-
sured by liquid chromatography-mass spectrometry (LC-MS), where F2 -IsoPs molecules
are detected based on chromatogram retention time and mass to charge (m/z) ratio spec-
trum [259]. F2 -IsoPs can be also measured by gas chromatography-mass spectrometry
(GC-MS), and F2 -isoprostanes undergo reaction (Figure S7) with pentafluorobenzyl bro-
mide (PFBB) to form pentafluorobenzyl (PFB) esters, and react with N,O-bis(trimethylsilyl)
trifluoroacetamide (BSTFA) to form trimethylsilyl (TMS) ether derivatives of F2 -IsoPs PFB
esters, prior to GC-MS analysis [260].
F2 -IsoPs can be also determined by means of commercially available enzyme-linked
immunosorbent assay (ELISA) kit [253], or by radioimmunoassay technique [229,261,262].
Detection of isoprostanes were applied during in vitro (Table 4) and populational
(Table 6) OS studies.
Measurement of Lipid Peroxidation: Lipid Hydroperoxides

Lipid hydroperoxides (LOOHs) are the initial products formed as a result of the
peroxidation of unsaturated fatty acids. Hydroperoxides can be measured via ferrous
oxidation in xylenol orange (FOX) assay, which is based on oxidation of Fe2+ to Fe3+ by
hydroperoxides under acidic conditions and complexation of Fe3+ by xylenol orange (XO)
to form a blue-purple complex with an absorbance maximum at 550–600 nm. For plasma
samples, FOX assay is authenticated by triphenylphosphine (TPP) which selectively reduces
the hydroperoxides into their corresponding alcohols, whereby providing a control and
eliminating the interference from plasma components (Fe3+ ) [263,264].
7.3.5. Measurement of Protein Oxidation: Protein Carbonyls

Protein-bound carbonyls are the most commonly used biomarker for protein oxidation.
Protein carbonyls originate from oxidative cleavage of the protein structure, direct oxidation
of amino acids (lysine, arginine, histidine, proline, glutamic acid, threonine), or introducing
carbonyl groups via reaction of lipid-oxidation derived-aldehydes with cysteine (Cys),
histidine (His), arginine (Arg) and lysine (Lys) residues.
The principle of protein carbonyl measurement is the use of 2,4-dinitrophenylhydrazine
(DNPH) that reacts with protein carbonyl groups producing a protein carbonyl-DNP hydra-
zone (Figure S8), which can be detected spectrophotometrically at λ = 360–390 nm [265,266]
or by HPLC-UV [252]. DNPH-derivatized protein carbonyls can be also detected by im-
munoblotting technique, with antibodies specific to the DNP moiety of the proteins [70,173].
Protein carbonyls were determined during in vitro (Tables 3 and 4) and in vivo (Table 5)
OS studies.
7.3.6. Measurement of DNA Oxidation: 8-OHdG

Oxidation of DNA is commonly measured based on biomarkers such as 8-hydroxydeo
xyguanosine (8-OHdG), the major form of oxidative deoxyribonucleic acid (DNA) dam-
age [267].
The standard technique to detect 8-OHdG is the enzyme-linked immunosorbent assay
(ELISA). The principle of this assay (Figure S9) comprises binding of antibody to 8-OHdG,
subsequent binding of horse radish peroxidase (HRP)-conjugated secondary antibody to
the anti(8-OHdG) antibody, conversion of tetramethylbenzidine (TMB) via HRP in the
presence of H2 O2 to a product and termination of reaction with the use of phosphoric acid.
The product formed as a result of TMB conversion can be quantified spectrophotometrically
at λ = 450 nm [268]. Such a technique was used to monitor oxidative stress in HEK293 cells
exposed to H2 O2 and ascorbic acid [193].
Biomolecules 2022, 12, 1087 32 of 45
8-OHdG can be also detected by means of HPLC with electrochemical detection

(HPLC-ECD) [269], or by LS-MS techniques [270]. A technique of LC-MS was used to detect
and quantify 8-OHdG in OS-exposed human skin fibroblasts (CCD 1112Sk) [197].
7.3.7. Analysis of Oxidative Stress and Vitamin Effect by Raman Spectroscopy and Imaging
The effect of oxidative stress and vitamin supplementation in cells can be also evalu-
ated by means of Raman spectroscopy and imaging. Raman spectroscopy, is a technique
exploiting the ability of a molecule to vibrate and to inelastically scatter (Raman effect)
absorbed light [271]. A source of light to be absorbed by molecules are lasers emitting
monochromatic light from ultraviolet (355 nm), green (532 nm), red (633 nm) or near-
infrared (785 nm, 1064 nm) regions of electromagnetic radiation [271]. The principle of
Raman spectroscopy technique is to analyse the Raman scattered light for chemical and
structural characterization of investigated samples [201]. It is based on the fact that chemical
bonds (C-H, C-C, C=C, C-N, C=O, N-H, -O-P-O-, etc.) vibrate in their specific modes and,
when interacting with light, produce specific spectral bands with different Raman shift. Ra-
man spectroscopy enables structural description of single molecules and polymers as well
as higher macroscopic structures, based on spectral profiles in fingerprint (500–1800 cm− 1 )
and high wavenumber (2700–3100 cm−1 ) region [272]. Raman spectroscopy can be coupled
with optical confocal microscopy, enabling to conduct high-resolution chemical imag-
ing. Raman spectroscopy and imaging have been used for structural investigation of
different types of cells, including brain [273], breast [274] and colon [275] cells. Raman
spectroscopy and imaging were used by our team for structural characterization of human
fibroblast colon (CCD-18Co) cells (Figure 7). Raman spectra contain bands assigned to
specific chemical structures [276], based on vibrational features of molecules within an anal-
ysed cell. The presence of nucleic acids is detected due to Raman bands at 716–723 cm−1
(nucleotides, DNA), 781–787 cm− 1 (nucleotides, DNA, RNA), 1070–1093 cm−1 (PO2 − ,
PO4 3− ). Protein presence can be concluded based on Raman bands at 748–757 cm−1 (tryp-
tophan), 852–858 cm−1 (proline, hydroxyproline, tyrosine), 992–1010 cm−1 (phenylalanine),
1263–1272 cm−1 (Amide III) and 1658–1664 cm−1 (Amide I), and 2926 cm−1 (CH3 ). The
presence of lipids is determined according to Raman bands at 1127–1133 cm−1 (acyl back-
bone, fatty acids), 1299–1305 cm−1 (acyl chains, fatty acids), 1440–1444 cm−1 (CH2 , CH3 ,
fatty acids, cholesterol), 2854 cm−1 (CH2 ) and 3009 cm−1 (=CH). Particular signals in
spectra are ascribed to nucleic acids, proteins and/or lipids, thereby providing structural
characterization of CCD-18Co cells. Furthermore, Raman images of individual cells can be
constructed with the use of Cluster Analysis (CA) method, where Raman images of all clus-
ters identified by CA are assigned to: nucleus, mitochondria, lipid-rich regions, membrane,
cytoplasm, and cell surroundings, within spectral wavenumber (500–3100 cm−1 ) region.
Biomolecules 2022, 12, 1087 33 of 45
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].
Raman spectroscopy and imaging can be further used to monitor structural and
metabolic alterations in cells exposed to oxidative stress (t-BuOOH) and/or treatment with
antioxidants (β-carotene, ascorbic acid) (Figure 8). Raman spectroscopy measurement of
such investigated cells provide spectral pattern with specific intensities of different bans
in the spectrum. A ratio of intensities from different bands within spectrum can provide
further information regarding molecular characterization of cells. Different ratios, for
selected Raman band intensities corresponding to 1004/1254 (phenylalanine/amide III
proteins), 1254/1656 (amide III/amide I proteins), 1004/1078 (phenylalanine/nucleic acids
and phospholipids) and 1004/1658 (phenylalanine/amide I proteins), were determined
by our group to monitor metabolic alterations in cells. In one study, CCD-18Co cells were
subjected to t-BuOOH as OS inducer and β-carotene as an antioxidant [278]. Results showed
that Raman I1004/1254 and I1254/1656 values in CCD-18Co cells changed due to exposure to
t-BuOOH or co-treatment with t-BuOOH and β-C, if compared to control CCD-18Co cells.
In another study, CCD-18Co cells were exposed to t-BuOOH for oxidative stress induction
and ascorbic acid to provide antioxidant protection [277]. Results showed that Raman
I1004/1078 and I1004/1658 values in CCD-18Co cells altered due to exposure to t-BuOOH
or co-treatment with t-BuOOH and AscA, if compared to control CCD-18Co cells [277].
Therefore, intensities of different bands within average spectra compared as ratios can
provide structural and molecular characterization of cells subjected to oxidative/anti-
oxidative treatment.
Biomolecules 2022, 12, 1087 34 of 45
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 I1004/1254 (C) and Raman I1254/1656 (D) graph values with bars: control, t-BuOOH, t-BuOOH
+ β-C, Raman I1004/1078 (E) and Raman I1004/1658 (F) graph values with bars: control, t-BuOOH,
t-BuOOH + Vit C. Reprinted and adapted with permission from [277,278].
7.3.8. Measurement of the Oxidative Stress: A Summary

OS increase during in vitro, in vivo and populational studies can be characterized by
elevated level of direct and indirect specific (bio)markers, such as intracellular ROS level,
fatty acids (lipids) oxidation products (MDA, 4-HNE, F2 -isoprostanes), protein oxidation
products (protein carbonyls) and DNA oxidation products (8-hydroxydeoxyguanosine).
The measurements of biomarkers usually depend on the use of spectrophotometric, flu-
orometric, chromatographic techniques also coupled with mass spectrometry (LC-MS,
GC-MS), as well as ELISA assay. However, the use of these techniques involves a series
of cumbersome steps including cell disruption and extract preparation, its purification
and separation into specific fractions and derivatization of target molecules with specific
compounds prior to analysis. The alternative and/or support to these techniques could be
Raman spectroscopy and imaging that enable the whole-cell characterization of different
chemical structures (proteins, lipids, nucleic acids), without the necessity of cell disrup-
tion, and further fractionization and derivatization. Raman spectroscopy measurement
can provide the monitoring of the alteration of cellular chemical profile, based on Raman
spectra analysis, when cells are exposed to stress inducer and/or antioxidant treatment.
Indeed, results published by our group show that chemical changes in colon CCD-18Co
cells exposed to stress conditions and treated with anti-oxidant (β-carotene, ascorbate) can
be monitored by Raman spectroscopy and imaging. The use of this technique can be further
extended for characterization of other cells and studying the effect of new anti-oxidants.
8. Conclusions
In this review, antioxidant properties of β-carotene, tocopherols and ascorbic acid
are presented based on in vitro, in vivo and populational/clinical studies. Literature data
gathered suggests that β-carotene, tocopherols and ascorbic acid possess the potential to
mitigate oxidative stress in various biological systems. However, interpretation of gathered
results faces possible limitations due to different cells, animal species and OS-inducing
agents used, discrepancies between in vitro and in vivo studies, as well as insufficient
number of participants and various origins of oxidative stress in populational/clinical
Biomolecules 2022, 12, 1087 35 of 45
studies. Therefore, further studies are required to determine the effect of β-carotene,
tocopherols or ascorbic acid on oxidative stress level and the efficacy of tested antioxidants
in different biological systems (in vitro, in vivo animals, populational/clinical). Oxidative
stress (bio)markers are measured by a range of different techniques (spectrophotometric,
fluorometric, chromatographic, immuno-enzymatic). Apart from commonly used analytical
methods, Raman spectroscopy and imaging can be a useful technique to study the effect of
oxidative stress and anti-oxidant molecules in cell studies.
Supplementary Materials: The following are available online at https://www.mdpi.com/article/

10.3390/biom12081087/s1, Figure S1: The mechanism of DCFH-DA conversion to DCFH and DCF;
Figure S2: The mechanism of reaction between MDA and TBA to form MDA-TBA2; Figure S3:
The mechanism of reaction between MDA and DNPH to form MDA-DNPH; Figure S4: The mech-
anism of reaction between MDA and PFBB to form MDA-PFB2; Figure S5: The mechanism of
reaction between MDA and PFBHA to form MDA-PFB2-oxime; Figure S6: The mechanism of 4-HNE-
PFB-oxime-TMS formation and analysis. PFBHA: pentafluorobenzyl hydroxylamine; BSTFA: N,O-
bis(trimethylsilyl)trifluoroacetamide; TMCS: trimethylchlorosilane; TMS: trimethylsilyl; Figure S7:
The mechanism of F2 -isoprostane (F2 -IsoPs) formation and analysis. PFBB: pentafluorobenzyl bro-
mide; BSTFA: N,O-bis(trimethylsilyl)trifluoroacetamide; TMS: trimethylsilyl; Figure S8: The mecha-
nism of protein carbonyl reaction with dinitrophenylhydrazine (DNPH). Figure S9. The mechanism of
deoxyguanosine (dG) oxidation into 8-hydroxydeoxyguanosine (8-OHdG) and 8-oxodeoxyguanosine
(8-OxodG), and the mechanism of 8-OHdG measurement with ELISA.
Author Contributions: Literature research, K.M.; writing—original draft preparation, K.M.; visual-
ization, K.M.; writing—review and editing, K.M.; conceptualization, K.B.; validation and reviewing,
A.Ś.; funding acquisition, B.B.-P. All authors have read and agreed to the published version of
the manuscript.
Funding: This research received no external funding.
Conflicts of Interest: The authors declare no conflict of interest.
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biomolecules

1 Laboratory of Laser Molecular Spectroscopy, Institute of Applied Radiation Chemistry,

Publisher’s Note: MDPI stays neutral

Biomolecules 2022, 12, 1087. https://doi.org/10.3390/biom12081087 https://www.mdpi.com/journal/biomolecules

2. Environmental Factors as Inducers of Oxidative Stress

Unhealthy dietary patterns, based on overconsumption of high-carbohydrate and

3. Metabolic Pathways as Sources of ROS Generation in Cells

4. Effect of ROS on Cellular Macromolecules

duction to ring-opened 2,6-diamino-4-hydroxy-5-formamidopyrimidine. The products

Exemplary Structures of Oxidation Products

4-hydroxy-2-nonenal Peroxidation of n-6 PUFAs and the

Generation of a tyrosyl radical, radical

HNE-Lys adduct (protein Michael addition of the HNE double bond to

HNE-Cys adduct (protein Michael addition of the HNE double bond to

Exemplary Structures of Oxidation Products

HNE-His adduct (protein Michael addition of the HNE double bond to

8-oxo-20 -deoxyguanosine Reaction between C-8 of guanine (G) and

8-oxo-20 -deoxyadenosine Reaction between C-8 of adenine (A) and

5. Oxidative Stress and Diseases Development

6. Anti-Oxidant Mechanisms as a Protection against Oxidative-Stress

6.1. Superoxide Dismutase (SOD) and Catalase (CAT)

6.4. Other Antioxidant Proteins

6.5. Antioxidant Low Molecular Weight Molecules

Coenzyme Q10 (CoQ10 , ubiquinone), with a structure of 2,3-dimethoxy-5-methyl-

7.1. Structural and Antioxidant Characteristics of β-Carotene, Tocopherols and Ascorbate

Figure 2. The scavenging mechanism of provitamin A and vitamin A.

7.1.2. Tocopherols: Structure and Anti-Oxidant Property

Figure 3. The scavenging mechanism of α-tocopherol.

7.1.3. Ascorbic Acid: Structure and Anti-Oxidant Property

Figure 4. The scavenging mechanism of ascorbate.

7.2.2. β-Carotene: Antioxidant Activity In Vitro

Antioxidant Effect of β-Carotene Towards OS-Induced Cells

7.2.3. Tocopherols: Antioxidant Activity In Vitro

Table 3. Antioxidant activity of tocopherols/vitamin E towards oxidative stress (OS) induced in

Antioxidant Effect of Vitamin E towards OS-Induced Cells

Human umbilical vein Incubation with Hcy (1 mM) ↑1 ROS, ↑1 MDA

7.2.4. Ascorbic Acid: Antioxidant Activity In Vitro

Antioxidant Effect of Ascorbic Acid towards OS-Induced Cells

Antioxidant Effect of Ascorbic Acid towards OS-Induced Cells

7.2.5. β-Carotene: Antioxidant Activity In Vivo

Figure 6. The structures of provitamin A (β-carotene) and vitamin A constituents (retinaldehyde,

7.2.6. Tocopherols: Antioxidant Activity In Vivo

7.2.7. Ascorbic Acid: Antioxidant Activity In Vivo

Antioxidant Effect of β-Carotene, Vitamin E and/or Vitamin C In Vivo

Male Sprague–Dawley SCI surgery + 72 h ↑1 MDA, ↓1 SOD

Antioxidant Effect of β-Carotene, Vitamin E and/or Vitamin C In Vivo

7.2.8. Antioxidant Characteristics of β-Carotene, Tocopherols and Ascorbate Based on

Table 6. Antioxidant activity of β-carotene, vitamin E and/or vitamin C based on popula-

Antioxidant Effect of β-Carotene, Vitamin E and/or Vitamin C in Human Studies

Antioxidant Effect of β-Carotene, Vitamin E and/or Vitamin C in Human Studies

Patients with late-stage Oral administration of placebo

7.2.9. Antioxidant Characteristics of β-Carotene, Tocopherols and Ascorbate: A Summary

in inhibition of anti-oxidant enzymes [181]. Overproduction of ROS is accompanied

7.3. Measurement of the Oxidative Stress in Cells and Tissues

7.3.1. Measurement of ROS Level: ESR Spectroscopy

7.3.2. Measurement of ROS Level: Fluorescent Method (DCFH-DA Probe Assay)

7.3.3. Measurement of ROS Level: Other Methods

with luminol (3-aminophthalhydrazide) to produce the excited 3-aminophthalate anion,

7.3.4. Measurement of Lipid Peroxidation Products